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Annual Report 2004(Apr.2003 – Mar.2004)

The buildings of NIAS in the cherry blossoms seasonThe buildings of NIAS in the cherry blossoms season

Message from the President

The objective of the National Institute of AgrobiologicalSciences aims is to develop advanced technologies foragriculture and life science industries based on genomeresearch. We aim to be recognized as a Center ofExcellence for plants, animal, insects and microorganismsciences. In our endeavor we seek cooperation withuniversities, private, and public research institutions.

This year’s accomplishments of our institute includethe clarification of the base sequence of the rice centromere,determination of the base sequence of the silk wormgenome by the whole genome shotgun method, developmentof a new tissue culturing method using animal tissuesections as functional culture matrices, and cultivationof mutant rose varieties with novel flower shape andcolor by chronic γ-ray irradiation.

In order to improve our performance it is necessaryto have our research activities and results reviewed bythe widest possible audience. We would greatlyappreciate comments from the international communityon the activities reported here.

Masaki Iwabuchi, Ph. D.PresidentNational Institute of Agrobiological Sciences

Message from the PresidentOrganization

Topics of Research in This Year

Genome and Biodiversity Research Division¡A draft sequence of the silkworm –A genomic basis for the promotion of insect industries– ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 1¡Fkbp6: a newly identified gene responsible for meiosis in spermatogenesis ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 2¡A breakthrough for centromere biology –The first complete sequence of

a plant centromere coming out from the rice genome ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 3¡First report of a gene expression analysis in specific plant cells

using laser capture microdissection ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 5¡A genomic island involved in flagellin glycosylation and pathogenicity

in Pseudomonas syringae ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 6

Insect and Animal Sciences Division¡A new physiological function of insect neuropeptide, corazonin, in the

silkworm, Bombyx mori ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 8¡Functions of Bombyx mori paralytic peptide in defense reaction and

hematopoiesis ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 9¡A novel insect antifungal peptide, which inhibits the growth

of phytopathogenic fungi ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 10¡A novel cell culture method utilizing animal tissue sections

as functional culture substrata ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 11¡Papain protects papaya trees from herbivorous insects: role of

cysteine proteases in plant latex ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 13¡The chemical factors affecting the host range of Ophraella communa LeSaga,

an intruding leaf beetle ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 14¡Translation initiation with elongator tRNAs ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 16¡New marker gene for the detection of transgenic silkworms

using white egg 1 mutant and kynurenine oxygenase gene ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 17¡Efficient foreign gene expression method using GAL4/UAS system

in transgenic silkworms ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 18¡Denim fabric using ‘Net raw silk’ ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 20

Plant Science Division¡Molecular cloning of the quantitative trait locus, qUVR-10,

conferring resistance to ultraviolet-B radiation in rice ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 21¡Collection, mapping and functional annotation of over 32,000

full-length cDNA clones from japonica rice ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 23¡Rice Proteome Database: A step toward functional analysis of the rice genome ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 24¡Hydrogen and hydration structures in protein ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 26¡Crystal structure of rice dwarf virus ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 27¡Overexpression of ZPT2-3, a zinc-finger transcription factor of petunia,

enhances drought tolerance in transgenic petunia ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 28¡Isolation and identification of a natural substance that induces resistance

to tobacco mosaic virus infection ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 29¡Overexpression of a rice vacuolar Na+/H+ antiporter confers salt tolerance to rice ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 30¡The complete nucleotide sequence of the mitochondrial genome

of rapeseed (Brassica napus L.) and comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 32

¡Five mutant varieties induced by chronic γ-ray irradiation in rose ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 34¡A mutant variety, “Pineapple Okinawa No. 16” induced

by chronic γ-rays irradiation in gamma greenhouse ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 35

Contents

Research Activities

Genome and Biodiversity Research Division¡Genome Research Department ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 38¡Genetic Diversity Department ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 45¡Genebank ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 51

Insect and Animal Sciences Division¡Developmental Biology Department ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 55¡Molecular Biology and Immunology Department ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 60¡Physiology and Genetic Regulation Department ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 64¡Insect Genetics and Evolution Department ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 69¡Insect Biomaterial and Technology Department ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 73¡Insect Biotechnology and Sericology Department ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 76

Plant Science Division¡Molecular Genetics Department ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 82¡Biochemistry Department ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 88¡Plant Physiology Department ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 91¡Plant Biotechnology Department ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 97¡Institute of Radiation Breeding ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 103

List of Publications¡Original papers ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 106

(Author Index) ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 123¡Reviews ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 127¡Monographs ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 128

International Meetings and Foreign Visitors ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 129

Executive Members and Research Staff Members ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 134

Members of NIAS Evaluation Comittee ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅ 140

Financial overviewLocation and How to access

The color on respective divisions is the same color on pages of “Topics of Researchin This Year” and “Research Activities” for each division in Annual Report 2004.

Organization

PresidentVice President (Insect and Animal Sciences)Vice President (Plant Sciences)AuditorDepartment of Research Planning and

CoordinationResearch Planning SectionResearch Evaluation SectionResearch Coordination SectionOffice for GMO Research and DevelopmentTechnology Transfer SectionAdministration SectionInformation and Public Relation SectionField Management Section

Department of AdministrationGeneral Affairs SectionAccounting SectionFacility Management Section

Genome and BiodiversityResearch DivisionDirector of Genome and Biodiversity Research Division

Genome Research DepartmentPlant Genome LaboratoryAnimal Genome LaboratoryInsect Genome LaboratoryBioinformatics LaboratoryDNA BankRice Genome Resource Center

Genetic Diversity DepartmentMolecular Biodiversity LaboratoryBiosystematics LaboratoryEvolutionary Dynamics LaboratoryGerm Cell Conservation LaboratoryApplied Microbiology LaboratoryAdaptation Systems LaboratoryBiometrics Laboratory

GenebankPlant Genetic Resources LabolatoryMicroorganism Genetic Resources LaboratoryAnimal Genetic Resources LaboratoryGenetic Resources Management Section

Insect and Animal SciencesDivision

Developmental Biology DepartmentDevelopmental Mechanisms LaboratoryDevelopmental and Differentiation LaboratoryAnimal Genetic Engineering LaboratoryEmbryonic Technology LaboratoryInsect Growth Regulation LaboratoryReproductive Biology and Technology LaboratoryMolecular Biology and Immunolory LaboratoryMolecular Immunolory LaboratoryInnate Immunity LaboratoryExperimental Animals Laboratory

Physiology and Genetic Regulation DepartmentInsect Life-Cycles and Physiology LaboratoryInsect Nutrition and Metabolism LaboratoryInsect Neurobiology LaboratoryInsect Behavior LaboratoryAnimal Gene Function LaboratoryAnimal Cell Biology LaboratoryAnimal Neurophysiolory LaboratoryAnimal Neuroendocrinolory Laboratory

Insect Genetics and Evolution DepartmentInsect-Plant Interactions LaboratoryNatural Enemies LaboratorySymbiosis LaboratoryInsect Pathology LaboratoryInsect Genetics LaboratoryInsect Molecular Evolution Laboratory

Insect Biomaterial and Technology DepartmentBiopolymer Characterization LaboratoryBiomaterial Development LaboratoryBiomimetic LaboratoryInsect Products Utilization Laboratory

Insect Biotechnology and Sericology DepartmentInsect Cell Engineering LaboratoryInsect Gene Engineering LaboratoryMass Production System LaboratoryNew Silk Materials LaboratorySericultural Science Laboratory

Plant Science DivisionMolecular Genetics Department

Functional Genomics LaboratoryApplied Genomics LaboratoryEpigenetics LaboratoryGene Expression LaboratoryGene Regulation Laboratory

Biochemistry DepartmentCrystallography LaboratoryBiophysics LaboratoryGlycobiology LaboratoryMembrane Biology Laboratory

Plant Physiology DepartmentPhotosynthesis LaboratoryCarbon Metabolism Laboratory Development Biology LaboratoryEnvironmental Physiology LaboratoryDisease Physiology LaboratoryNitrogen Fixation Laboratory

Plant Biotechnology DepartmentGene Design LaboratoryPlant Gene Engineering LaboratoryPlant Cell Engineering LaboratoryMolecular Breeding LaboratoryBiosystems Laboratory

Institute of Radiation BreedingMutation Genetics LaboratoryRadiation Technology LaboratoryMutation Breeding Laboratory

Annual Report 2004 1

In Japan, the silkworm has long beenused as a model system and provides anumber of mutants and genetically improvedstrains. We performed three-fold wholegenome shotgun (WGS) sequencing of thesilkworm to obtain a silkworm draftsequence, which will provide new genome-based approaches for insect industries suchas sericulture, insecticides and efficientproductions of useful materials usingsilkworm.

The WGS method has been established asthe most powerful tool available for generatinga draft genome sequence efficiently andquickly with much less cost. In this study,the genome DNA was extracted fromsilkglands of silkworm of an inbred strain,p50T, followed by random fragmentation.About 500-base nucleotide sequences fromboth ends of about 1.4 million DNA fragmentswere sequenced, resulting in 2.8 millionnucleotide sequences that correspond tothree-fold of the genome size, which were

assembled with the “RAMEN assembler” newlydeveloped by the University of Tokyo (Fig.).

Analysis of the silkworm WGS data usingthe RAMEN assembler program resulted in213 k sequence contigs. The total length ofthe sequence contigs was 390 Mb, suggestingthat 80% of the genome was sequenced(Table). Homology searches using 50characteristic Bombyx genes and 11,202 non-redundant ESTs indicate the validity of theWGS sequence data for identifying Bombyxgenes.

The silkworm genome informationobtained by the WGS method will be madepublic in the HP of NIAS (http://sgp.dna.affrc.go.jp/), and every researcher can utilize theinformation for functional studies. Thus, astrong impact on insect science andindustries will be expected. We will facilitateinterchanges of information with researchersof other countries, promote internationalcollaborations, and play a leading role in theinternational silkworm genome works.

Topics of Research in This Year

A draft sequence of the silkworm ––A genomic basis for the promotion of insectindustries––

Kazuei Mita, Yuji Yasukochi, Kimiko Yamamoto, Keiko Kadono-OkudaGenome Research Department

Silkworm genome size 490 M bp

Total number of reads 2,843 K readsTotal number of sequenced nucleotides 1,600 M bpNumber of sequence contigs 213,289Total length of sequence contigs 387 M bp

Table Statistics of Bombyx mori sequence assembly.

2 Annual Report 2004

Fig.Outline of whole genome shotgun (WGS) sequencing

1.4 M DNA fragments

About 500-base sequencefrom both ends of eachfragment were sequenced.

RAMEN assembler

2.8 million sequences wereassembled

Assembled sequences (Sequence contigs)

Silkworm genome DNA (490 Mb)

Meiosis is an essential process to producegametes with different genetic information,which conserves genetic diversity in species.During meiosis, homologous chromosomepairs are in alignment. A zipper-like structure,the symaptonemal complex, formed betweenthe chromosomes, is a fundamental structureto promote homologous recombination.

Male rats possessing as mutant genehomologously showed arrested spermatogenesisat meiotic prophase. Linkage analysisclarified a responsible region of chromosomewhere the causative gene localized. A geneFkbp6, coding FK506 binding protein(FKBP6), located in the region. Wheninvestigating Fkbp6 expression, the mutanttestis showed a shorter fragment of the 3’

region than normal by RT-PCR and lackedprotein expression by both Western blot andimmuno-histochemistry. Genomic DNAanalysis unveiled deletion of about 1,000 bpsincluding exon 8, therefore we concluded thatas is a mutant gene of Fkbp6.

FK506 binding protein (FKBP6) localizedto meiotic chromosome cores and regions ofhomologous chromosome synapsis. Targetedinactivation of Fkbp6 in mice as well ashomozygous bearing of as in rats resulted inaspermic males accompanying abnormalpachytene spermatocytes (Fig.A). Preciseanalysis of the mutant mice revealed that theloss of FKBP6 resulted in abnormal pairingand misalignments between homologouschromosomes, non-homologous partner

Fkbp6: a newly identified gene responsible for meiosis in spermatogenesis

Junko NoguchiGenetic Diversity Department


switches, and formation of autosynapsis of Xchromosome cores in meiotic spermatocytes(Fig.B,C). Despite similar localization of theprotein in oocytes, chromosome pairing wasunaffected in Fkbp6-null females. Fertilityand meiosis in females were normal in bothFkbp6 mutant animals. Thus, FKBP6 is anovel component of the synaptonemalcomplex, essential for sex-specific fertilityand the fidelity of homologous chromosomepairing in meiosis.

Fig.Spermatogenesis in as homozygous mutant rats (A) and abnormal chromosome pairing in Fkbp6-null male mice (B andillustrated in C). Normal testis completed meiosis and contained resultant spermatids and sperms (arrows in left panel ofA), but mutant testis arrested spermatogenesis at the stage of pachytene spermatocytes, showing an abnormalmorphological feature (arrowheads in right panel of A). A pair of chromosomes (arrow in B and C) partly paired with non-homologous chromosomes.

Understanding the organization ofeukaryotic centromeres has both fundamentaland applied importance because of their rolesin chromosome segregation, karyotypicstability, and artificial chromosome-basedcloning and expression vectors. Because ofthe highly heterochromatic structure ofcentromeres (Fig. 1), however, completelycloning, sequencing, and assembling theirgenomic components has remained asignificant challenge. In the Rice GenomeResearch Program, we have focused on thecentromeric region of rice chromosome 8 withefforts to obtain the complete genomicsequence of this important chromosomecomponent, using clone-by-clone sequencing

methodology. Rice PAC/BAC clones weremapped to the centromeric region (54.3 cM)of rice chromosome 8 by using sequences of11 STS/EST markers to conduct the in silicomapping of Monsanto BAC clones and PCRscreening of RGP PAC/BAC libraries. Gapson the physical map were closed by screeningand choosing the bridge clones from eitherCUGI-assembled BAC contigs or RGPPAC/BAC libraries based on the sequence-tagged connector (STC) or chromosomalwalking methods. As a result, 18 clones werefinally selected that covered the entirecentromeric region. We sequenced these PACand BAC clones using a shotgun approach(two shotgun libraries with different insert

A breakthrough for centromere biology —The first complete sequence of a plant centromere coming out from the rice genome

Jianzhong Wu, Yuichi KatayoseHiroshi Mizuno, Takashi MatsumotoGenome Research Department


sizes, average 2-kb and 5-kb, for each clone).Each PAC or BAC clone was assembled fromthe shotgun sequences by PHRED/PHRAPsoftware. Assembled sequence contigs wereviewed and edited by CONSED. Sequencegaps between the assembled contigs withineach PAC/BAC clone were generally filled bysequencing the bridge sub-clones withcustom primers. Regions with low quality

scores were improved by resequencing withcustom primers or by alternative chemistries.

A transposon insertion/sequencing system(Genome Priming System GPS-1) was usedfor complete sequencing of the subclones inthe problematic regions that containedrepeats such as the rice centromere-specificsatellite DNA (CentO).

Through assembling the PAC/BAC clonesequences, 1, 972, 546-bp of the contiguousnucleotide sequence was obtained. Finishedassemblies of the sequences were confirmedby comparing sizes of the fragments fromrestriction enzyme digests of each PAC/BACclone with those of the virtual fragment sizesof the finished sequence. Analysis of this1.97-Mb of genomic sequence revealed threelarge clusters of CentO satellite repeats (68.5kb of 155-bp repeats) and >220 TE(transposable element)-related sequences;together these account for nearly 60% of thewhole genomic region (Fig. 2). The total

Fig. 1FISH analysis of rice mitotic chromosomes using CentO repeats as probes.

Fig. 2Annotation map of the 1.97-Mb centromeric region of rice chromosome 8. Orange: genetic markers. White boxes: predicted genes. Greenand gray boxes: transposable elements. Arrow lines: LTR sequences and orientations. Arched black lines with arrows: TE sequenceinterrupted by DNA insertions. Red bars: rice centromere-specific element RCE1. Yellow boxes with red arrows: CentO clusters andorientations.


length of CentO repeat sequences coincidesvery well with the CentO amount andlocation pattern detected by fiber-FISHanalysis of this chromosome and weredemonstrated at this study to be tandemlyarrayed head-to-tail within the clusters,which had different orientations andinterruption of TE-related sequences. Theindividual 155-bp CentO satellite repeatsshowed frequent transitions and transversionsat eight nucleotide positions. The 40 TEelements with highly conserved sequenceswere mostly gypsy-type retrotransposons.Gene prediction for the 1.97-Mb sequencerevealed the presence of 201 opening readingframes (excluding those of DNA transposons

and retrotransposons), within them 48 genesshowed high BLAST homology to knownproteins or to rice full-length cDNAs; somewere close to the CentO clusters. Our studyis the first to provide the complete sequenceof a centromeric region from either plants oranimals and will likely provide insight intothe evolutionary and functional analysis ofplant centromeres. The next challenge is toextensively investigate in vivo the roles ofthe centromere-specific sequences, especiallythe CentO satellite repeats and someretroelements like the RIRE7 and RIRE8families, in both karyotypic stability andchromosome segregation.

Investigation of gene expression profilesin individual cells is an effective approach foridentifying and characterizing the genesinvolved in cell-specific biological processes.The laser capture microdissection (LCM)system, which allows the isolation ofselectively targeted cells from a tissue sectionwithout contamination from adjacentnontarget cells, was developed as an effectivetool for analysis. The technique has beensuccessfully used for the isolation of specificmammalian cells, mainly cancer cells.However, the application of LCM to the geneexpression analysis of plant to the geneexpression analysis of plant cells has neverbeen reported.

We used the LCM system and isolatedonly phloem cells of rice leaf tissues whose

morphology was apparently different fromthe surrounding cells (Fig. 1). Total RNA wasextracted from the captured phloem cells, theisolated RNA was amplified, and theresultant RNAs subjected to synthesis ofdouble-stranded cDNA.

First report of a gene expression analysis in specific plant cells using laser capture microdissection

Takayuki Asano*, Koh-ichi KadowakiGenetic Diversity Department*Present address: Molecular Genetics Department

Fig. 1Laser capture microdissection from rice leaf sections(A) Before microdissection(B) After microdissectionArrowheads indicate a captured location of rice phloem


We constructed a phloem-specific cDNAlibrary using the obtained cDNAs andsubsequently determined the DNA sequencesof 413 clones at random from the library. A

sequence analysis revealed thatapproximately 37% of the cDNA clones hadnovel components. In situ hybridizationrevealed that putative amino acid permease,one of the cDNA clones, was specificallyexpressed in the phloem (Fig. 2). The resultsproved the effectiveness of constructing aspecialized cDNA library from specific plantcells.

Further investigation of the clonesobtained from a phloem-specific cDNAlibrary might provide new insights into themechanisms underlying phloem functions.Furthermore, the combination of ourtechnique and DNA microarray analyses willbe a useful tool for understanding gene-expression profiles in target plant cells, suchas with/without environmental stress orplant/microbe interactions.

Fig. 2Localization of mRNA of putative amino acid permease in stems by in situhybridization(A) Antisense riboprobe, stem cross section(C) Sense riboprobe, stem cross section(B) and (D) Magnification of Figures A and C, respectively

The isolates of the phytopathogenicbacterium Pseudomonas syringae can beclassified into more than 50 pathovars by itsvirulence toward respective host plantspecies. Its flagellin, an essential componentof flagella filament of bacteria, has beenfound to be an elicitor that causes hypersensitivereactions (HR) in its nonhost plants. Thedifference of the ability to induce HR hasbeen thought to depend on the post-translational modification of the flagellins.

To investigate the role of this post-translational modification in the interactionsbetween plants and bacterial pathogens, weisolated genes which are potentially involved

A genomic island involved in flagellin glycosylationand pathogenicity in Pseudomonas syringae

Kasumi Takeuchi1), Fumiko Taguchi2),Yuki Ichinose2)

1) Genebank2) Faculty of Agriculture, Okayama University

Fig. 1Schematic representation of the ORFs in the regionupstream of the flagellin gene (fliC) from the genomesequence of P. s. pv. glycinea and its comparison withthe flagell in glycosylation island in Pseudomonasaeruginosa strain PAK. Red pentagons and yellowpentagons are genes homologous to putativeglycosyltransferases and 3-oxoacyl-[acyl-carrier-protein]-synthase III, respectively.


in the post-translational modification offlagellin in P. syringae pv. glycinea (glycosylationisland). Their defective mutants were thengenerated. There are three orfs, orf1, orf2and orf3, in the glycosylation island. Orf1and orf2 encode putative glycosyltransferases(Fig. 1), and their defective mutants, ∆orf1and ∆orf2 , secreted nonglycosylated orslightly glycosylated flagellins, respectively(Fig. 2). On the other hand, orf3 showedsignificant homology to putative 3-oxoacyl-[acyl-carrier-protein]-synthase III (Fig. 1).There was no significant difference in theglycosylation status of the flagellins from the

wild-type strain and the ∆orf3 mutant (Fig.2). An inoculation test of these mutants onoriginal nonhost tobacco leaves revealed that∆orf1 and ∆orf2 could grow on tobacco leavesand caused symptom-like change (Fig. 3A).In contrast, these mutants failed to causesymptoms on original host soybean leaves(Fig. 3B). These data indicate that putativeglycosyltransferases in flagellin glycosylationisland are strongly involved in therecognition by the plant and could be thespecific determinant of compatibility betweenphytopathogenic bacteria and plant species.

Fig. 2Glycosylation of flagellins from the wild type (WT), ∆orf1,∆orf2 and ∆orf3 mutants of P. syringae pv. glycinea. (A)Western blot analysis of the flagellin protein in the totalproteins. (B) Staining of the glycosyl moiety in purifiedflagellin.

Fig. 3Inoculation test with nonhost tobacco leaves (A) and hostsoybean leaves (B). Leaves were inoculated with eachbacterium by spraying. Tobacco and soybean leaveswere photographed 10 and 13 days after inoculation,respectively.


A single peptide sometimes displaysdiverse physiological functions in differentspecies of animals. An insect neuropeptide,corazonin, is a good example of such aversatile function in insect species.Corazonin was first isolated from theAmerican cockroach as a cardiostimulatorypeptide but recently, it was found thatcorazonin induces the dark-color of themigratory locust, Locusta migratoria byusing an albino strain of L. migratoria.However, the physiological effect of thispeptide had been unknown in the otherinsects although the same structure ofcorazonin has been found in many insectspecies and crustaceans. We identifiedcorazonin in the silkworm, Bombyx mori, byamino acid sequencing and cloned the cDNAencoding its precursor, but corazonin showedno effect on the body-color in B. mori .However, we found a new physiological effectof this peptide in B. mori. When corazoninwas injected into the fourth instar larvae, we

could not observe any apparent effect on thelarval development until the initiation ofspinning, but pupal ecdysis was delayed andsome larvae died before pupation (Fig.).Corazonin-injected larvae spun for a longerperiod but made thinner cocoons as comparedwith normal larvae. We also found a similareffect of corazonin in the wild silkmoth,Samia cynthia ricini, but it did not have anyeffect on the development of a non-cocooninginsect, the army worm Pseudaletia separate.This suggests that the effect of corazonin isrelated to spinning in cocooning insects. Thestructure-activity relations of corazoninusing different bioassay systems, the effecton spinning rate in B. mori , and thedarkening in albino nymphs of the locustexhibited similar structure-activity relationships.This suggests that they might have similarreceptor systems despite the differentphysiological affects.

Corazonin or factors similar to corazoninare found to be present in the brain-

A new physiological function of insect neuropeptide, corazonin, in thesilkworm, Bombyx mori

Yoshiaki TanakaDevelopmental Biology Department

Fig.Effect of corazonin injection on the development of Bombyx mori.Fifty pmol of corazonin was injected into 1-day-old 4th instar larva.


retrocerebral neuroendocrine system andventral nerve cord of at least 10 insect ordersby immunohistochemistry and bioassay ofalbino strain of L. migratoria. As most ofthese insects neither spin as much as thesilkworm nor show body-color polymorphism,

corazonin may have other functions in thoseinsects. It is important to identify thesefunctions and understand how the samepeptide or peptide with similar structure hasdeveloped multiple functions in differentinsect species.

The silkworm paralytic peptide (BmPP) isa 23 amino acid peptide belonging to theENF peptide family. We have already shownthat BmPP has paralytic activity and growthblocking activity against the silkwormlarvae, and mitogenic activity in severalsilkworm cell lines. Now we studied thefunctions of BmPP in the defense reactionand hematopoiesis of the silkworm larvae.

BmPP treatment stimulated spreading ofplasmatocytes that circulated in larvalhemolymph, but the percentage of spreadplasmatocytes was only 20%. BmPP,however, elicited prominent spreading in70% of young plasmatocytes discharged fromcultured hematopoietic organs (Fig.1). Cellsin hematopoietic organs that wereenzymatically dispersed did not spread even

after adding BmPP, indicating that plasma-tocytes acquired BmPP-sensitivity immediatelyafter discharge. When cultured in a mediumcontaining larval plasma, hematopoieticorgans grew markedly and discharged a largenumber of hemocytes, over 95% of whichwere morphologically plasmatocytes. Thehemocyte discharge was blocked in themedium containing BmPP dose-dependently,although hematopoietic organ growth wasnot suppressed (Fig.2). These results suggestthat BmPP plays important roles both inhematopoietic regulation and in thehemocyte immune reaction of the silkworm.

Functions of Bombyx mori paralytic peptidein defense reaction and hematopoiesis

Manabu Kamimura, Yuichi Nakahara,Yasushi Kanamori, Makoto KiuchiDevelopmental Biology Department

Fig.1 Spreading reaction of newly-born plasmatocytes inducedby BmPP. (A) Control, (B) 100 nM of BmPP was added.

Discharged cells

Cells in organ

Cel

ls

60000

40000

20000

0BmPP (M) Before

culture Without larval plasma With larvalplasma (10%)

Fig. 2 Effect of BmPP on the hematopoietic process. Larval hematopoieticorgans were cultured in medium containing various concentrations ofBmPP and larval plasma for 48 hours and then numbers of dischargedhemocytes and cells remaining in organs were counted.


Insect immune systems contain a batteryof potent antibacterial and antifungalpeptides/polypeptides. Although numerousantibacterial peptides have been identified,only a small number of antifungal peptides/polypeptides have been reported frominsects. Insect immune peptides/polypeptidesthat show antifungal activity are drosomycin,metchinikowin, cecropin A and B, andandropin from Drosophila melanogaster,heliomicin from Heliothis virescens, cecropinA from Hyalophora cecropia, thanatin fromPodisus maculiventris, antifungal protein

(AFP) from Sarcophaga peregrina, holotricin3 from Holotrichia diomphalia, and termicinand spinigerin from Pseudacanthotermesspiniger . Of these antifungal peptides/polypeptides, drosomycin, thanatin, heliomicin,and termicin are known to contain cysteineresidues and form disulfide bridges.

In our study of insect antifungal peptidesas genetic resources to establish transgenicplants, we isolated a novel cysteine-containing antifungal peptide from thecoconut rhinoceros beetle, Oryctes rhinoceros.

cDNA of the antifungal peptide designatedscarabaecin was cloned by reversetranscriptase-polymerase chain reactions(RT-PCR) using a primer based on the N-terminal amino acid sequence. The aminoacid sequence deduced from scarabaecincDNA showed no significant similarity tothose of reported proteins. Chemically

A novel insect antifungal peptide, which inhibits the growth of phytopathogenic fungi

Jun Ishibashi, Hiromitsu Tanaka, MinoruYamakawaMolecular Biology and Immunology Department

00 10 20 30 40

20

40

60

80

100

Fig.Scarabaecin-chitin binding. Chitin was mixed with the indicated amount ofscarabaecin and incubated at room temperature for15min. One hundred percent denotes scarabaecin boundto chitin in 32 µM of scarabaecin. Data are means fromthree experiments.

Scarabaecin

IC50 (mM)

Amphotericin B

FungiP. oryzaeR. solania

S. tritici R. solanib

P. aphanidermatumP. infestanceB. cinerea

BacteriaA. tumefaciens N1001C.michiganese pv. michiganeseE. carotovora subsp. carotovora P. cichori NL7630P. glumae N1169P. syringae pv. mori S6804P. syringae pv. japonicaX. compestris pv. compestris N1076X. campestris pv. campestris N1086

16.032.016.0

128>250>250

4.0

>100>100>100>100>100

25>100>100>100

20.618.2

1.46.7

>250>250

2.7

Table Antimicrobial activity of synthetic scarabaecin againstphytopathogenic fungi and bacteria

Amphotericin B, a fungicide, was used as a positive control.aRhizoctonia solani was isolated from rice.bRhizoctonia solani was isolated from bentgrass.


synthesized scarabaecin indicated antifungalactivity against phytopathogenic fungi suchas Pyricularia oryzae, Rhizoctonia solani,and Botrytis cinerea , but not againstphytopathogenic bacterium (Table). Scarabaecinshowed a chitin binding property and its Kd

was 1.315µM (Fig.). A comparison of putativechitin-binding domains among scarabaecin,invertebrate, and plant chitin- bindingproteins suggests that scarabaecin is a newmember of the chitin-binding antimicrobialproteins.

Thin tissue sections commonly preparedon a glass slide for histopathology retainmany in vivo biochemical attributes relatednot only to structure but also function. Wehypothesized that such tissue sections mightserve as novel cell culture substrata thatwould reflect tissue conditions in vivo anddeveloped a breakthrough technology ofculturing animal cells on a substratum madeof tissue/organ sections for histopathology(TOSHI-substratum). The advantages of theTOSHI-substratum prepared from a bovineplacenta for cell differentiation, a serum-freeculture, and tissue reconstruction wasdemonstrated by utilizing four different typesof cells: BeWo (human choriocarcinoma cellline), CPAE (bovine pulmonary arteryendothelial cell line), PC-12 (ratpheochromocytoma cell line), and NHDFs(normal human dermal fibroblasts). Thelabyrinth region of the substratum inducedunique cell behaviors to form multicellularspheroids of BeWo cells, a capillary network-like structure for CPAE cells, and a neuronalnetwork-like structure for PC-12 cells. Thesubstratum provided a microenvironment inwhich the viability of PC-12 cells maintainedeven in a serum-free condition. Also, a

multicellular mass of NHDFs involvingacellularized section-derived components wassuccessfully prepared. The multicellular

A novel cell culture method utilizinganimal tissue sections as functionalculture substrata

Toshiaki TakezawaPhysiology and Genetic Regulation Department

Fig. 1Serum-free culture of PC-12 cells on placenta cryosectionsubstrata. Cells were suspended in a serum-free mediumand seeded at a density of 4.6x104/cm2 and cultured on aglass slide substratum as well as on 5 µm-section or 5µm-acellularized section substrata. Following HEstaining, the morphology of cells cultured on the glassslide (A), acellularized basal lamina (B), labyrinth (C, D),basal lamina (E), and glandular zone (F) for 2 days (A-C)or 5 days (D-F) was observed by optical microscopy.Scale bars, 100 µm.


mass incorporating the section componentsderived from the TOSHI-substratum mayhave important clinical applications as animplant because it is easily prepared usingboth cryosections and cells derived frompatient biopsies. Further, TOSHI-substratacan be prepared not only from all animaltissues/organs of any age regardless ofpathology, but also from the entire bodies ofsmall animals. These substrata retain theoriginal microarchitecture and component ofthe tissue and conserve many of thebiochemical factors that serve as signalingcues for inducing cell behavior.

Most of these factors are easily detectedby ordinary techniques such as immuno-histochemistry or in situ hybridization.Considering advantages, the analysis ofinteractions between different cell types andvarious TOSHI-substrata will play animportant role as a novel approach in thestudy of cellomics. In the near future,arranging the proper cell-substratumcombination will promote effective cellomicsstudies such as the exploration andproduction of bioactive molecules, analysis ofcell characteristics, estimation of genefunction, and reconstruction of tissues/organs.

Fig. 2Formation of a three-dimensional multicellular aggregate of normal human dermal fibroblasts containing the acellularizedplacenta cryosection substratum-derived components. Cells were seeded at a density of 3.3x104/cm2 and cultured on a20 µm-acellularized section substratum. Proliferating cells cultured on the acellularized section substratum for 7 daysbegan to detach from the glass slide (A) and a cell sheet containing the acellularized section substratum could becompletely detached by pipetting the culture medium (B). A three-dimensional multicellular aggregate containing theacellularized section substratum-derived components was formed after an additional 2 days of culture on a non-adhesivesubstratum (C). The morphology of cells in culture was observed by phase-contrast microscopy (A-C). HE-stained crosssections of the aggregate were observed by optical microscopy (D, E). Scale bars, 200 µm.


Many plants contain latex that exudeswhen leaves are damaged, and a number ofproteins and enzymes have been found in it.For example, large amounts of cysteineprotease are known to exist in the latex ofseveral plants such as papaya and fig trees.The roles of those latex proteins andenzymes, however, are as yet poorlyunderstood. We found that papain, a cysteineprotease in latex of the papaya tree (Caricapapaya, Caricaceae), is a crucial factor in thedefense of the papaya tree againstlepidopteran larvae such as oligophagousSamia ricini (Saturniidae, Eri silkworm) andtwo notorious polyphagous pests, Mamestrabrassicae (Noctuidae) and Spodoptera litura(Noctuidae). Papaya leaves showed strongtoxicity and growth inhibition against theseinsects. S. ricini larvae (Fig. 1A) and M.brassicae larvae (Fig. 1B, Fig2) died by Day4, and S. litura larvae (Fig. 1C) did not growat all when papaya leaves were fed to theselarvae. In contrast, papaya leaves losttoxicity when the leaves were cut into narrowleaf strips and washed to eliminate latex;larvae fed washed leaves grew very wellwithout dying (Figs. 1, 2). Latex of papayacontained a large titer of cysteine-proteaseactivity in a very condensed manner. E-64, acysteine protease-specific inhibitor,completely deprived the leaves of toxicitywhen painted on the surface of papaya andfig leaves. Larvae of all three lepidopteranspecies fed papaya leaves painted with E-64grew very rapidly without showing anysymptoms of toxicity (Figs. 1, 2). Cysteine

proteases, such as papain, ficin, andbromelain, all showed toxicity when added to

A

Control papaya leaf +E-64 Washed papayaleaf strips

Castor oil plant leaf

Samia ricini 2nd instar larvae Day4

2cm

Mamestra brassicae 2nd instar larvae Day4

Spodoptera litura 2nd instar larvae Day4

Control papaya leaf

Control papaya leaf

+E-64

+E-64

Washed papayaleaf strips


2cm

2cm

B

C

Fig. 1Defensive effects of cysteine proteases in latex of papayaleaves and effects of E-64 (cysteine protease-specificinhibitor) painting and latex elimination. Newly hatched 1st

instar larvae of Samia ricini (Eri silkworm) (A), newlymolted 2nd instar larvae of Mamestra brassicae (B) andSpodoptera litura (C) were fed control papaya leaf,papaya leaf painted with E-64 (0.36 mg/ g fresh leaf), orwashed papaya leaf strips, and reared at 25 ˚C for 4 days.S. ricini larvae were also fed their natural host plant,castor oil plant leaves, as a control.

Papain protects papaya trees from herbivorous insects:role of cysteine proteases in plant latexKotaro Konno1), Yasumori Tamura1), Makoto Hattori1),Chikara Hirayama2), Masatoshi Nakamura2), KenTateishi3), Katsuyuki Kohno4)

1) Insect Genetics and Evolution Department2) Physiology and Genetic Regulation Department3) Molecular Biology and Immunology Department4) National Institute of Vegetable and Tea Sciences


an artificial diet and fed to the larvae (Fig.3). These results suggested that cysteineproteases in plant latex provide plants(papaya and probably other plants withcysteine protease-containing latex) with ageneral defense mechanism against

herbivorous insects. Our results furtherimply that many other latex enzymes andproteins in various plant species may alsohave similar defensive activities that wouldbe applicable to future plant protectionmethods.

0 2 40

5

10

15

Larv

al m

ass

(mg)

Days after molting

Control papayaleaf (+glycerol)

+E-64


a 15%

b 5%

100%

a 10%b 5%c 80%

M. brassicae 2nd instar larvae

Fig. 2Growth and mortality of Mamestra brassicae larvae fedvariously treated papaya leaves. Newly molted 2nd instarlarvae of M. brassicae were fed control papaya leaf,papaya leaf painted with E-64 (0.36 mg/ g fresh leaf), orwashed papaya leaf strips, reared at 25 ˚C for 4 days, andlarval mass (mg) and mortality (%) were observed. Valuesnot followed by the same letters at the same time pointare significantly different (P < 0.01 Tukey’s test formultiple comparison, average ± SD, n = 20).

S. ricini 1st instar larvae

Control(L4M diet)

PapainA low

PapainB high

Ficin Bromelain

**0%

0%

**80%

**80%

**45%

0

2

4

6

8

10

12

14

16

Larv

al m

ass

on

Day

2 (m

g)

Fig. 3Effects of papain, ficin, and bromelain on the growth andmortality of Samia ricini (Eri silkworm). First instar larvae(1.58 mg) were fed an artificial diet (L4M) containingcysteine proteases (papain, ficin, bromelain) at 25 ˚C, andlarval mass (mg) and mortality (%) were observed on Day2 (48hr). Error bars indicate SD. ** Values significantlysmaller than the value observed in the control (P< 0.01, t-test, n=20).

Ophraella communa LeSage, originallydistributed in the Nearctic region, is anoligophagous leaf beetle that feeds onAsteraceae plant species including ragweed,A. artemisiifolia. This beetle seems to havebeen accidentally introduced into Japan and

was discovered for the first time in ChibaPrefecture in 1996. O. communa has rapidlyspread and caused severe injury to ragweed,A. artemisiifolia in many areas. The pollen ofA. artemisiifolia causes hay fever. Therefore,O. communa may be useful as a biological

The chemical factors affecting the host range ofOphraella communa LeSaga, an intruding leaf beetle

Yasumori Tamura, Makoto Hattori,Kotaro KonnoInsect Genetics and Evolution Department


control agent for A. artemisiifolia. However,this beetle is regarded as a pest because italso feeds on sunflowers in Japan. AlthoughO. communa prefers A. artemisiifolia exclusivelyamong Asteraceae plants in Japan, thecompounds inducing the feeding behavior ofO. communa have not been reported. Revealingfeeding stimulants in specific host plants issignificant for predicting crops and weeds aspotential host plants of this insect.

A filter paper bioassay showed that thefeeding of adult O. communa is stronglystimulated by methanolic extracts of A.artemisiifolia . Hence, the leaves of A.artemisiifolia were considered to containfeeding stimulants of this beetle. Fourfeeding stimulants for O. communa havebeen isolated from methanolic extracts of A.artemisiifolia by using silica gel 60 CC, silicagel 60 HPTLC and reversed phase HPLC.From spectroscopic and chemical evidence,

the feeding stimulants for O. communa in A.artemisiifolia have been identified as a-amyrin acetate (α-AA), β-amyrin acetate (β-AA), 5-caffeoylquinic acid (5-CQA) and 3, 5-dicaffeoylquinic acid (3,5-DCQA) (Fig.1).Triterpenoid derivatives (α-AA or β-AA) andcaffeic acid derivatives (5-CQA or 3, 5-DCQA)showed strong feeding stimulant activity onlywhen mixed together.

The distribution of the four feedingstimulants for O. communa was analyzed in11 genera and 17 species of asteraceousplants. These compounds were found inmany species including nonhost plants,however, amounts and compositions differed.Even when the methanolic extracts of A.artemisiifolia were added to extracts of someasteraceous species which did not elicitfeeding of O. communa, the feeding of O.communa was not induced by the inactiveplants. Feeding deterrents as well as feedingstimulants in the leaves of asteraceousplants are likely to determine the feedingpreference of O. communa.

Japanese asteraceous crops, such as alettuce, Lactuca sativa , a crown daisy,Chrysanthemum coronarium, and a burdock,Arctium lappa, were found to also containthe feeding stimulants of O. communa.Hence, we inferred that O. communa couldinfest these crops. Host suitability tests to O.communa were carried out by giving thoseasteraceous crop cultivars. Although adultbeetles could feed on all plants tested, theynever laid eggs. Almost all newly hatchedlarvae died in a short time and neverpupated when they fed on these crops. Theseresults show that there is little possibility forO. communa to utilize these crops as host,even though adult beetles can tentativelyinfest C. coronarium, A. lappa, and L. sativa.We would like to thank Mrs. Hitomi Oguchi(pictured right) for her invaluable help inbreeding plants.

Fig. 1Feeding stimulants of Ophraella communa from Ambrosiaartemisiifolia(1: β-amyrin acetate, 2: α -amyrin acetate, 3: 5-caffeoylquinic acid (chlorogenic acid),4: 3, 5-dicaffeoylquinic acid).


Initiator methionyl tRNA (Met-tRNAi) isused for the initiation of protein synthesis ineukaryotes. This tRNA is discriminated fromelongator tRNAs by eukaryotic initiationfactor 2 and/or ribosomal 40S subunit, thuselongator tRNAs are never used forinitiation. This means that the N-terminal

amino acid of polypeptides which aregenerated from ribosomes is alwaysmethionine. This frequently cause troublewhen we intend to produce functionalpolypeptides in laboratories by a cell-freetranslation system combined with recombinantDNA techniques. Because many functionalproteins have been processed by peptidasesor proteases, such proteins do not have N-terminal methionine. Additional N-terminalmethionine corresponding to AUG initiationcodon sometimes changes the functionality ofrecombinant proteins from authentic ones.

Some groups of viruses have special RNAelements which recruit ribosomes ontomRNAs, termed internal ribosome entry site

Translation initiation with elongator tRNAs

Nobuhiko NakashimaInsect Genetics and Evolution Department

Fig. 1Translation initiation with various codons.

Fig. 2(A) Structure of plasmids used for in vitro translationanalysis. Each number of plasmids corresponds to thelane numbers in the panel (B). (B) Translation productsfrom RNAs lacking AUG initiation codons.


(IRES) elements. We found a unique IRESlocated in the intergenic region (IGR) in theRNA genome of Plautia stali intestine virus(PSIV), which was recently classified into anew family, Dicistroviridae. The IGR-IRES ofPSIV consists of about 200 nucleotides andinitiate translation using glutamine in nativeconditions. Our analysis showed that thestructure of the IRES (Fig. 1) is responsiblefor translation activity and this IRES canmediate translation initiation using varioustriplets encoding 20 amino acids, namelyusing elongator tRNAs (Fig. 1). To examine

the requirements of the viral capsid codingsequence (CP in Fig. 1) for the translation,we replaced the CP with the initiator AUG-deleted coding sequences of firefly luciferase(Fluc), Renilla luciferase (Rluc), and greenfluorescent protein (GFP). Althoughtranslation efficiency of the three codingsequences differed, their translation productswere observed (Fig. 2). This indicates thatdirect synthesis of polypeptides lacking N-terminal methionine is possible in vitro usingIGR-IRES elements.

A germ-line transformation method insilkworm has been developed using DNAtype transposon piggyBac and greenfluorescent (GFP) gene as a marker. Thedeveloped method can construct thetransgenic silkworms very efficiently and isused for the production of recombinantproteins, new fiber and resistant strainsagainst nuclear poly hedrosis virus.

However, the fluorescent marker genesused for the screening of transgenic silkwormare under the control of patents. In addition,expensive fluorescent binoculars have to beused for the detection of the transgenicsilkworms. Thus, the development of new

marker gene which is not restricted by apatient and can be applied without expensivetools has been desired.

A vector plasmid inserted silkwormkynurenine oxygenase gene under the controlof silkworm cytoplasmic actin promoter wasconstructed (Fig.1). The vector and helperplasmids were injected into the eggs of whiteegg 1 mutant strain and three transgeniclines were successfully obtained. Theanalysis of the transgenic silkworms intowhich was inserted the wild type kynueninegene showed that the ocelli of embryos andlarvae, skin of young larvae, and compoundeyes of late pupa and adults were turned to

New marker gene for the detection of transgenic silkworms using white egg 1mutant and kynurenine oxygenase gene

Isao Kobayashi, Guoxing Quan,Katsura Kojima, Toshio Kanda,Toshiki TamuraInsect Biotechnology and Sericology Department

Fig. 1 Physical map of vector plasmid.KMO, wild type kynureine oxigenase gene;A3p, actin promoter; R and L arms, right and left inverted terminal repeatsequences of transposon piggyBac.


brown. (Fig 2). The strength of the browncolor directly related to the expression levelof the inserted kynurenine gene. The resultindicates that the kynurenine oxygenasegene can be used as a marker gene for thetransgnesis in the silkworm when the whiteegg 1 mutant is used as a host strain.

The marker gene obtained in theexperiment can be used without a patentregulation and applied to obtain anindependent patent as a new marker gene forthe transgenesis in the silkworm. Using themarker gene, it becomes possible to screenthe transgenic silkworms without usingfluorescent binoculars.

Fig. 2Eyes and eggs in the transgenic silkworms w-1, white egg 1 mutant used as a host strain. 50, 66 and129, transgenic strains.Eye, eye color of newly immerged moth; egg, eggs 3 daysafter oviposition.

The method for making a transgenicsilkworm have been developed and manysilkworms with various foreign genes havebeen constructed. However, the expression ofintroduced gene was unstable. It was difficultto control the expression level in desiredtissues and stages. Therefore, a new methodthat gives more stable and controlledexpression of introduced genes was desired.In this study, we applied a binary expressionsystem of yeast GAL4/UAS for the transgenicsilkworms and confirmed that the system isuseful for the control of the expression ofintroduced genes.

We first constructed a vector with

UASGFP gene, GFP gene, under the controlof the target sequence (UAS) of transcriptionalregulation factor GAL4 (Fig.1). Then, twovector plasmids that possessed GAL4 genesunder the control of the promoters of A3(with the activity to express the gene inwhole tissues) and 3XP3 (with the characterof eye specific expression) were constructed.Using the two vectors, the silkworms withthe two genes, UASGFP and GAL4, werecreated. The silkworms with the two genes,GAL4 and UASGFP , showed the tissuespecificity of their promoters, A3 or 3XP3, inthe expression of GFP, suggesting that theGAL4/UAS system works in the transgenic

Efficient foreign gene expression methodusing GAL4/UAS systemin transgenic silkworms

Morikazu Imamura, Junichi Nakai,Satoshi Inoue, Guoxing Quan,Toshio Kanda and Toshiki TamuraInsect Biotechnology and Sericology Department


silkworm.Then, the new GAL4 vector plasmid,

pBacLchain (FibL)GAL4/3XP3CFP, wasconstructed. The vector possessed GAL4 geneunder the control of fibroin L chain genepromoter. The transgenic silkworm to whichthe GAL4 was introduced was mated withUASGFP strain and an expression of GFPwas investigated. The silkworm with theGAL4 gene was identified by 3XP3CFPmarker gene (Fig. 2 A and B). The young

larva with the two genes had a silk glandexpressing GFP and it can be detectedwithout dissecting the larva (Fig. 2 C and E).The expression of GFP was much stronger inthe last instar larvae (Fig. 2 E) and wasidentified under visible light (Fig.2 F)

The result indicates that the GAL4/UASsystem from the yeast works well in thetransgenic silkworms and is useful fordeveloping the production system of therecombinant proteins in the silk gland.

Fig. 1Structure of vector plasmids for the expression of GFP in the posterior division of the silk gland.pBacUAS-GFP, physical map of vector plasmid possessed GFP gene under the control of target sequence (UAS) ofGAL4; IVR, inverted terminal repeat sequence of a transposon piggyBac; pBacLchain(FibL)GAL4/3XP3CFP, physicalmap of vector plasmid possessed GAL4 gene under the control of fibroin L chain promoter; 3XP3CFP, marker gene thatexpresses CFP in ocellus and compound eye.

Fig. 2Expression of GFP in the transgenic silkworm possessed GAL4 and UASGFP genes.A, embryo inserted 3XP3CFP gene, showing the ocelli with the expression of CFP gene; B, embryo of non-transgenicsilkworm as a control, showing no fluorescence; C, expression of GFP in young larva of the transgenic silkworm (top,silkworm that possessed GAL4 and UASGFP genes ; middle, larvae that possessed only GAL4 gene; bottom, non-transgenic silkworm as a control); D, the same larva of C under visible light; E, fluorescence of the silk gland of thetransgenic silkworm that possessed LchainGAL4 and UASGFP genes; F, the same silk gland under visible light; G,fluorescence of the silk gland of non-transgenic silkworm; H, The same silk gland of G under visible light.


We have used silk for casual clothing(especially ‘jeans’), for which silk has hardlybeen used, to make use of indigenous andhigh-quality Japanese materials. We usedsilk from various silkworm species in Japan.

We produced silk denim using ‘Net rawsilk’, a bulky silk yarn, as weft and twillweaving (Fig. 1). Consequently, silk wasexpressed in the reverse (the side contactingskin) of the denim. We found that silk retainsbody heat well.

When we reeled ‘Net raw silk’, the napwas restrained by covering the raw silk tocreate ‘Net raw silk’. Its tenacity, elongationand Young’s modulus showed high valueswhen compared with physical properties ofcotton yarn that is typically used for denim.

Comparison of fabric performance of thesilk denim regarding strength in the weftdirection of the silk denim showed that it was1.5 times stronger than cotton denim and itselongation was three times greater.Moreover, bending resistance showed thatthe silk denim was soft in comparison withcotton denim (Fig. 2). Jeans were producedusing denim woven by this method and afitting examination was done. The jeans werelight in comparison with the cotton jeans.They felt smooth and were evaluated aswarm.

Denim fabric using ‘Net raw silk’

Kenichi Nakajima, Chiyuki Takabayashi,Hidetoshi TeramotoInsect Biomaterial and Technology Department

Fig. 1Silk denim jeans. A: reverse, B: obverse.

Fig. 2Performance comparison of silk and cotton denims


The depletion of the stratospheric ozonelayer by chlorofluoromethanes and othergasses has resulted in increases in solarultraviolet-B (UVB) radiation. Future, theincreases of harmful UVB radiation mightaffect the growth and yield of crops in thefield. Wide variation in UVB resistance isobserved among rice varieties. Threequantitative trait loci (QTLs) controllingUVB resistance were detected by QTLanalysis using backcross inbred lines (BILs)derived from a cross between an japonicavariety, Nipponbare (UVB-resistant), and anindica variety, Kasalath (UVB-sensitive)(Fig. 1A). Among them, qUVR-10, a QTL forUVB resistance on chromosome 10 showedthe largest effect.

To verify the genetic effect of the Kasalathallele of qUVR-10, we produced a nearlyisogenic line [NIL(qUVR-10)] in which achromosomal segment including Kasalathallele at the qUVR-10 was substituted intothe Nipponbare genetic background (Fig. 1A).Kasalath and NIL(qUVR-10) showed a largenumber of necrotic lesions on the leaves afterthree weeks of 1.0 Wm-2 UVB radiation. Incontrast, Nipponnbare showed no or a smallnumber of necrotic lesions (Fig. 1B).

In order to understand the molecularmechanism of the UVB-resistance, In thisstudy, we performed a map-based cloning ofqUVR-10. High-resolution mapping using1,850 segregants enabled us to delimit acandidate genomic region of qUVR-10 to lessthan <27-kb genomic region. Based on thegene annotation of the region, we identified a

candidate gene encoding cyclobutylpyrimidine dimer (CPD) specific photolyase,which is an enzyme that efficiently removesUV-induced CPDs from DNA in a light-dependent manner. Analysis of the CPDsphotorepair activity in Nipponbare andNIL(qUVR-10) showed that NIL(qUVR-10)contained defective photolyase. For geneticcomplementation, we transformed introducedNipponbare genomic fragment containingCPD photolyase gene into NIL(qUVR-10) andselected the plants, which carriedhomozygous of one copy transgene from T2

populations. UV-resistance of these T2 plantshomozygous of the transgene exhibited ahigh-level of UVB-resistance as same as thatof Nipponbare (Fig. 1B). Thus we concludedthat qUVR-10 encodes CPD photolyase.

A sequence comparison between Nipponbareand Kasalath allele revealed many sequencevariations in the no non-coding region and asingle-base substitution caused an aminoacid change in exon 4: 296th glutamine (CAG)in Nipponbare and 296th histidine (CAC) inKasalath (Fig. 2A). In the process ofdelimitation of the qUVR-10 locus, we foundan informative plant with a recombinationbreakpoint in CPD photolyase gene itself(line RS24-23). The region covering promoterand exon 1 was homozygous for Kasalathallele and the downstream region washomozygous for the Nipponbare allele in lineRS24-23 and it showed the same phenotypeas Nipponbare in the degree of necrotic lesion(Fig 2B) and the capacity of CPDsphotorepair. These results suggested that the

Molecular cloning of the quantitative trait locus, qUVR-10, conferring resistance to ultraviolet-B radiation in rice

Tadamasa Ueda, Takeshi Izawa,Shin’ichi Yamamoto, Masahiro YanoMolecular Genetics Department


allelic difference of qUVR-10 betweenNipponbare and Kasalath is caused by a

single-base substitution occurred in exon 4.Interestingly, one transgenic plants (T2-

22-9) carrying multi-copy of Nipponbare CPDphotolyase gene in the NIL(qUVR-10) geneticbackground, showed a higher level of UVB-resistance compared with not only that ofNIL(qUVR-10) with empty vector, but alsothat of Nipponbare. In this plant, a higherlevel of CPD photolyase transcripts was alsoobserved. These results clearly demonstratethat a higher-level of UVB-resistance can beachieved by increasing the expression level ofCPD photolyase. We could produce UV-hyperresistance by enhancement of CPDphotolyase expression.

Fig. 1A. Chromosomal location of QTLs for UVB-resistanceand graphical genotype of NIL(qUVR-10) Black and white regions represent the segment ofthechromosome derived from Kasalath and Nipponbarerespectively. TheCircles indicate QTLs detected usingBILs.B. Necrotic lesion on leaf after 3 weeks 1W/m2 UVBradiation. Non transgenic control: (A)Nipponbare, (B)Kasalathand(C) NIL(qUVR-10). Transgenic plant: (D)NIL(qUVR-10) with single copy of Nipponbaregenomic fragment ofCPD photolyase and(E) NIL(qUVR-10) with the emptyvector

NipponbareCAG (Gln)

RS24-23

number ofnecroticlegion

Nipponbare

NIL(qUVR-10)

KasalathCAC (His)

S

SL

A

B

Fig. 2A. Comparison of genomic sequence of Nipponbareand Kasalath CPD photolyase geneBoxes indicate the ORF region. Vertical lines representsingle-base substitutions between Nipponbare andKasalath. Arrowheads indicate the insertions. B. Graphical genotype of CPD photolyase region ineach line. Black and white regions represent the segment of thechromosome derived from Kasalath and Nipponbare,respectively. Number of necrotic legion is indicated by S(small number) and L (large number).


Three Japanese institutes, the NationalInstitute of Agrobiological Sciences (NIAS),the Foundation for the Advancement ofInternational Science (FAIS), and theInstitute of Physical and Chemical Research(RIKEN) collaborated with the Bio-orientedTechnology Research Advancement Institute(BRAIN) to compile a complete set of full-length cDNAs from japonica rice. In the firstphase of collection, 32,127 clones werecompletely sequenced and they correspondedto about 20.6K Transcription Units (TU) onthe rice genome (Ref. 1, Fig. 1 and KOMEWEB site Fig. 2 http://cdna01/dna.affrc.go.jp/cDNA/). In the second phase of collection,180K clones were picked up from the newcDNA libraries and sequenced from bothends. Alignment analyses of the full-lengthcDNA clones to the rice genome sequencerevealed the following interesting transcriptstructures. Of around 700 pairs of sense-antisense transcript pairs (Ref. 2), more than1500TUs have the alternatively splicedtranscripts and some trans-splicing clones.

Using the sequence information of phase Iclones, the 22K rice oligo microarray systemhas been commercialized since November2003.

References1. Collection, Mapping, and Annotation ofOver 28,000 cDNA Clones from japonica RiceScience (2003) 301, 376-379 Kikuchi et al. 2.KOME: Knowledge-based Oryza Molecularbiological Encyclopedia http://cdna01.dna.affrc.go.jp/cDNA/2. Antisense transcripts with rice full-length

cDNAs Genome Biology 5:R5 (2003) N Osato,H Yamada, K Satoh, H Ooka, M Yamamoto,K Suzuki, J Kawai, P Carninci, Y Ohtomo, KMurakami, K Matsubara, S Kikuchi, YHayashizaki

Collection, mapping and functional annotationof over 32,000 full-length cDNA clonesfrom japonica rice

Shoshi KikuchiMolecular Genetics Department

Fig. 1Mapping results of 32,127 full-length cDNA clones to japonica rice genomesequence

Fig. 2The front page of the KOME site


The technique of proteome analysis usingtwo-dimensional polyacrylamide gelelectrophoresis (2D-PAGE) has the power tomonitor global changes that occur in theprotein complement of tissues and subcellularcompartments. In this study, a RiceProteome Database was constructed, proteinsof rice were catalogued and a functionalcharacterization of some of the identifiedproteins was undertaken. Proteins extracted

from tissues and subcellular compartmentsin rice were separated by 2D-PAGE and animage analyzer was used to construct adisplay of the proteins. The Rice ProteomeDatabase contains 23 reference maps basedon 2D-PAGE of proteins from rice tissues andsubcellular compartments. These referencemaps comprise 13,129 identified proteinsfrom various tissue and subcellular locations,and the amino acid sequences of 5,676

Rice Proteome Database: A step towardfunctional analysis of the rice genome

Setsuko Komatsu, Keiichi Kojima,Koji Suzuki, Kenichi HigoMolecular Genetics Department

Fig. 1Example of the Rice Proteome Database compartment browser.A) Example of a 2D-PAGE reference map on the Rice Proteome Database. This figure shows the 2D-PAGE referencemap of cultured suspension cells. B) Example of a protein entry from the Rice Proteome Database. This figure showsthe spot information for PMT00132, which is spot number 132 for mitochondria in rice. C) Example of a result frommass spectrometry on the Rice Proteome Database. This figure shows the Mascot Search Results for PVM00012.


proteins are entered in the database.The Rice Proteome Database contains the

calculated properties of each protein such asmolecular weight, isoelectric point, andexpression, experimentally determinedproperties such as amino acid sequencesobtained using protein sequencers and massspectrometry, and the results of databasesearches such as sequence homologies. Thedatabase is searchable by keyword, accessionnumber, protein name, isoelectric point andmolecular weight, amino acid sequence, or byselection of a spot on one of the 2D-PAGEreference maps (Fig. 1). Cross-references areprovided to tools for proteomics and to other2D-PAGE databases, which in turn providemany links to other molecular databases(Fig. 2). The information on the RiceProteome Database is updated frequently,and is available on the World Wide Web athttp://gene64.dna.affrc.go.jp/RPD/.

One major advantage of the Rice

Proteome Database, in which known proteinsare recorded along with where and whenthey are expressed, is the wealth of newlyidentified proteins on which furtherexperiments can be conducted at thebiochemical and molecular levels. Majorproteins involved in growth or stressresponses were identified using the proteomeapproach and some of these proteins haveunique functions. In addition to facilitatingthe identification of known proteins,sequences in the database can be used toprepare oligodeoxyribonucleotides, which areessential for cloning the correspondingcDNAs. Finally, an attempt is also made tostudy the physiological significance of someproteins thus identified from rice. Theinformation obtained from the Rice ProteomeDatabase will aid in their molecular cloningand will be helpful in predicting the functionof unknown proteins.

Fig. 2Overview of the Rice Proteome Database


Hydrogen contains half of the atoms in aprotein molecule and is of great importancestructurally and functionally. Neutroncrystallography is capable of directlyrevealing the positions of hydrogen atoms.However, this method requires very largesize of protein crystals, and it is generallydifficult to prepare such crystals. On theother hand, X-ray crystallography allows usto detect a part of hydrogen atom positions, if

the protein structure is solved at atomicresolution. We have determined the crystalstructure of BGTI at 0.93 Å resolution, andfound some hydrogen atom positionsparticularly attached to Cα atoms and in theplace involving hydrogen bonds. Informationabout accurate hydrogen atom positionsbecomes more important for understandingbiological function and three-dimensionalarchitecture of protein molecules.

On the other hand, recent studies showedthat hydration in protein contributes not onlyto protein stabilization but also to biologicalfunction. We found that water moleculeslying in the protein-protein interface play animportant role in stabilization and molecularrecognition. A total of 181 water moleculeswere observed in an asymmetric unit of thecrystal structure of the complex betweenanticoagulant protein (hetero dimmer ofsubunits A and B) and Gla domain of factorX. Fig. shows the hydrated structure.Twenty-two water molecules were located inthe interface between two protein molecules.These water molecules form a hydrogenbonding network involving water-mediatedinteraction between proteins, contributing tostabilization of the complex structure andwater-mediated molecular recognition.

Hydrogen and hydration structuresin protein

Hiroshi MizunoBiochemistry Department

Fig.Stereo view of ribbon model of the complex between anticoagulant protein(subunit A, pink, and subunit B, green) and the Gla domain of factor X(yellow). Water molecules and Ca2+ ions are shown in blue spheres andorange spheres.


Rice dwarf virus (RDV), the causal agentof rice dwarf disease, is a member of thegenus Phytoreovirus in the familyReoviridae. RDV is an icosahedral double-shelled particle with a total molecular massof approximately 70 million Dalton, andcontains 12 segments of double-strandedRNA as the genome.

Recently we succeeded in the crystalstructure analysis of RDV at 3.5 Å resolution.The double-shelled structure consists of twodifferent proteins, the core protein P3 (114kDa) and the outer shell protein P8 (46kDa).The outer shell of RDV has a T=13 symmetryformed by 780 copies of P8. P8 protein forms

a trimer structure, so that the outer shell isfilled by 260 trimers of P8 as shown in Fig. 1.The core protein forms a dimer structure,having a T=1 symmetry formed by 120 copiesof P3 (or 60 copies of P3 dimer, see Fig. 2). P8has positively charged patches on the innersurface that contact the concentration ofnegative charge on the outer shell of P3,located at the icosahedral threefold axes ofthe core particles. Such configurations of theheterologous core P3 and outer P8 proteinsare considered to guide the assembly of theouter shell, be aided by lateral interactionsby the P8 trimer, and finally to complete theparticle construction.

Crystal structure of rice dwarf virus

Hiroshi MizunoBiochemistry Department

Fig. 1.Cα trace of the outer shell protein P8 of RDV. Triangularshapes drawn in different colors are P8 trimer, so that theouter shell is formed by 260 copies of the P8 trimer.

Fig. 2Cα trace of the core P3 of RDV. The core shell is filled upby 60 copies of dimer unit (red and green).


Environmental stresses can causeprofound damage to the growth anddevelopment of plants. Recent studies haveshown that transcription factors playimportant roles in stress responses. ZPT2-3is a Cys2/His2-type zinc finger protein ofpetunia (renamed from EPF2-7) that isspecifically expressed in petals and sepalsunder unstressed conditions1). Here we showsome evidences indicating that ZPT2-3 isresponsive to various stresses and plays arole in plant tolerance to dehydration2).

Drought treatment induced theaccumulation of ZPT2-3 transcripts in

petunia (Fig. A). Real-time monitoring ofZPT2-3::luciferase activity in transgenicpetunia seedlings with a 2D luminometershowed that the upregulation was conferredby the 1,668-bp ZPT2-3 upstream sequence(Fig. B). ZPT2-3 expression was also inducedby wounding, cold, and heavy metaltreatments, demonstrating responsiveness ofthis gene to multiple stresses. The sameZPT2-3 promoter sequence showed a similarresponsiveness to wounding, cold, droughttreatments in Arabidopsis when investigatedusing a β-glucuronidase reporter gene,indicating conservation of similar signalingpathways between the two plant species.ZPT2-3 was constitutively overexpressed intransgenic petunia plants under the controlof the CaMV 35S promoter and the plantswere tested for stress tolerance. As shown inTable, the tolerance of the plants to dehydrationwas dramatically increased. In our hands,however, no difference was found in thetolerance to other stresses. In a transientassay using Arabidopsis leaves, ZPT2-3functioned as an active repressor and atranscriptional repression domain waslocated near the C-terminus of the protein.These results demonstrate the involvementof ZPT2-3 in plant response to variousstresses, and suggest its potential utility toimprove drought tolerance.1) Takatsuji, H. et al. (1994) Plant Cell, 6:

947-958.2) Sugano, S. et al. (2003) Plant J., 36: 830-

841.

Overexpression of ZPT2-3, a zinc-fingertranscription factor of petunia, enhancesdrought tolerance in transgenic petunia

Shoji Sugano and Hiroshi TakatsujiPlant Physiology Department

Line Survived Total %

Experiment 1 Control 5 20 25.0#34 19 20 95.0#35 17 20 85.0

Experiment 2 Control 0 18 0#34 17 18 94.4#35 18 18 100

Table Drought tolerance of wild-type and 35S::ZPT2-3 plants

Fig.Drought response of ZPT2-3.A. Northern blot analysis. B. ZPT2-3::luciferase (LUC) activity in transgenicpetunia seedlings. Two-week-old petunia seedlings transformed with ZPT2-3::LUC were subjected to drought treatment for the indicated periods.

Four-week-old soil-grown plants were not watered for 30 days, wateredagain for a week, and scored for survival. Results of two independentexperiments are shown.


Mitogen-activated protein kinases(MAPKs) are Ser/Thr protein kinases thathave been conserved evolutionarily ineukaryotes. They are activated by diverseextracellular stimuli, thereby regulatingmultiple intracellular responses. In tobacco,activation of a wound-induced protein kinase(WIPK), a MAPK, has been implicated in thedefense response to pathogen infection.Despite the important role of WIPK in thepathogen signal transduction pathway, noendogenous signal responsible for itsactivation has been identified. To identify theendogenous signal(s) responsible foractivating WIPK and understand themolecular mechanism of the WIPK-mediatedsignal transduction, we isolated a WIPK-activating substance from TMV-infectedtobacco leaves and identified it as (11E,13E)-labda-11,13-diene-8a,15-diol, designatedWAF-1 (WIPK-activating factor1) (Fig. 1). Atnanomolar levels, either natural WAF-1 orchemically synthesized WAF-1 activatedWIPK as well as SIPK, a defense-relatedtobacco MAPK (Fig. 2), and enhanced theaccumulation of transcripts of defense-related genes (Fig. 3). Quantitative analysisof endogenous WAF-1 revealed that levelsincreased rapidly in leaves during ahypersensitive response to TMV. Furthermore,treatment of leaves with WAF-1 resulted inenhanced resistance to TMV infection (Fig.4). These results suggest that WAF-1, a novelditerpene, functions as an endogenous signalmediating the defense response of tobaccoplants to TMV infection.

Isolation and identification of a natural substancethat induces resistance totobacco mosaic virus infection

Shigemi SeoPlant Physiology Department

Fig.1Chemical structure of WAF-1.

Fig. 2Activation of MAPKs by exogenous WAF-1.


Rice, one of the most important crops inthe world, has low salt tolerance, and itsproduction and planting area are greatlyaffected by soil salinity. Therefore, it is ofagricultural importance to analyze andimprove the salt tolerance of rice.

Na+/H+ antiporters, which catalyze theexchange of Na+ for H+ across membranes,contribute to the regulation of internal pH,cell volume, and the sodium level in thecytoplasm. The antiporters are wide-spreadmembrane proteins found in animals, yeasts,bacteria, and plants. In particular, vacuolarNa+/H+ antiporters, which compartmentalizeNa+ into the vacuoles for detoxification, havebeen investigated as the key to salt tolerance

Overexpression of a rice vacuolar Na+/H+

antiporter conferssalt tolerance to rice

Atsunori Fukuda and Yoshiyuki TanakaPlant Physiology Department

Fig. 1Localization of OsNHX1-sGFP fusion protein expressed inrice cells. Green fluorescence of sGFP in the cells wasimaged under a laser-scanning confocal microscope (a).Tonoplasts are stained red with FM4-64 (b). The twoimages of a and b are overlaid in c. Differentialinterference images of the cells are shown in d.

Fig. 4Effect of exogenous WAF-1 on necrotic lesion development.

Fig. 3Effect of exogenous WAF-1 on the expression of defense-related genes.


in plants.We have cloned a vacuolar Na+/H+

antiporter gene (OsNHX1) from rice (Oryzasativa). We examined the function andintracellular localization of the product ofOsNHX1 gene. OsNHX1 has the ability tosuppress Na+, Li+, K+, and hygromycinsensitivity of yeast scnhx1 mutants. Analysisusing rice cells expressing a fusion protein ofOsNHX1 and green fluorescent protein (Fig.1) and western blot analysis using antibodiesspecific for OsNHX1 confirmed thelocalization of OsNHX1 on the tonoplasts.These results indicate that the OsNHX1 geneencodes a vacuolar (Na+, K+)/H+ antiporter. Inaddition, treatment with high concentrationsof NaCl and KCl increased the transcriptlevels of OsNHX1 in rice roots and shoots.

We assessed whether the overexpressionof OsNHX1 allowed rice cells and plants togrow in the presence of high NaCl. Thegrowth of the transgenic rice cells

overexpressing OsNHX1 in the presence of0.1 and 0.2 M NaCl was improved relative tothat of the control cells (vector control) incorrelation with the increase in the level ofOsNHX1. In addition, the Na+ content of thetransgenic cells grown in the presence of ahigh NaCl concentration was higher thanthat of the control cells, and correlated withthe increase in the level of OsNHX1. Thegrowth of the control rice plants was severelyinhibited in the presence of high NaClconcentrations, whereas that of thetransgenic plants overexpressing OsNHX1was improved relative to that of the controlplants (Fig. 2). These results suggest thatOsNHX1 on the tonoplasts plays importantroles in the compartmentation of Na+ highlyaccumulated in the cytoplasm into thevacuoles, and the amount of the antiporter isone of the most important factorsdetermining salt tolerance in rice.

Fig. 2Salt tolerance of transgenic rice plants grown at various salt concentrations. Control plants transformed with empty vectorand transgenic plants overexpressing OsNHX1 were grown under normal conditions for 18 days, and then treated with 0(a), 0.05 (b), or 0.1 (c) M NaCl for 7 weeks.


The sizes of mitochondrial genomes varywidely even among higher plants. With 208kb, Brassica hirta has the smallestmitochondrial genome in higher plants, whilethe muskmelon mitochondrial genome isestimated to be over 2,400 kb. This sizevariation can occur relatively rapidly inevolution, and also the plant mitochondrialgenome organization is known to be verydynamic. It is of great interest to understandsuch rapid evolutionary changes in size andstructure, and their consequences withrespect to gene content. To date, the completemitochondrial genome sequences in higherplants have been determined in threespecies, Arabidopsis thaliana, sugar beet andrice. Although significant structuraldifferences are observed among these plants,most of the genes are conserved aside fromthe occasional transfer of a mitochondrialgene to the nuclear genome in oneangiosperm clade or the other. However,these plants are only distantly related toeach other, and are not suitable forinvestigating rapid evolutionary changesover the entire mitochondrial genome.

In this study, rapeseed was chosen for theanalysis of the tempo and mode ofevolutionary changes for several reasons.First, rapeseed belongs to Cruciferae, thesame family as Arabidopsis. It occupies agood phylogenetic position for makingcomparisons with the already sequencedArabidopsis mitochondrial genome. Second,physical mapping revealed that the rapeseedmitochondrial genome is about 220 kb in size,

which is only two-thirds of that inArabidopsis (367 kb). This implies that thesetwo plants can be used to study the evolutionof genome size. Third, it is well known thatBrassica species, including rapeseed, havethe smallest mitochondrial genomes amonghigher plants. Investigation of the rapeseedmitochondrial genome can thus reveal theminimum sequence requirement for thehigher plant mitochondrial genome.

The entire mitochondrial genome ofrapeseed was sequenced and compared withthat of Arabidopsis. The 221,853-bp genomecontains 34 protein-coding genes, 3 rRNAgenes, and 17 tRNA genes (Fig.). This genecontent is almost identical to that ofArabidopsis . RNA editing events weresystematically investigated on the basis ofthe sequence of the rapeseed mitochondrialgenome. A total of 427 C to U conversionswere identified in ORFs, which is nearlyidentical to the number in Arabidopsis (441sites). The gene sequences and intronstructures are mostly conserved (more than99% similarity for protein-coding regions);however, only 358 editing sites (83% of total)are shared by rapeseed and Arabidopsis.Non-coding regions are mostly divergentbetween the two plants. One-third (about78.7 kb) and two-thirds (about 223.8 kb) ofthe rapeseed and Arabidopsis mitochondrialgenomes, respectively, cannot be alignedwith each other, and most of these regions donot show any homology to sequences inpublic DNA databases. The results of thecomparative analysis between the rapeseed

The complete nucleotide sequence of the mitochondrialgenome of rapeseed (Brassica napus L.) and comparative analysis of themitochondrial genomes of rapeseedand Arabidopsis thalianaHirokazu HandaPlant Biotechnology Department


and Arabidopsis mitochondrial genomessuggest that higher plant mitochondria areextremely conservative with respect to codingsequences, but that non-coding parts of plant

mitochondrial DNA are extraordinarilydynamic with respect to structural changes,sequence acquisition and/or sequence loss.

Fig.Gene organization of the rapeseed mitochondrial genome. Genes homologous to known protein-coding genes areindicated by red boxes. The blue boxes represent rRNA genes. Pink boxes represent unidentified ORFs longer than 150amino acids. tRNA genes are represented by yellow boxes. Pseudo genes including plastid gene segments are shown inpale green. orf222, a cms-related gene, is shown by a green box. Arrowheads indicate the direction of reading frames.Dark green boxes located inside the circle represent 2 kb repeat regions.


Throughout the world, the rose (Roseaceaessp.) is one of the most popular flowersproduced by the floral industry and theproduction of fresh cut stem roses is aprofitable economic industry throughoutJapan. Typically, during the summermonths, high temperatures result in areduction of stem length. A reduction in thestem length of cut roses is reflected by alower quality and grade of the final product.However, the cultivar “Samantha” does notexhibit this tendency and long stem roses canbe produced throughout the growing season.

In collaboration with the OkinawaPrefectural Agricultural Experiment Station(OAES), the Institute of Radiation Breeding(IRB) has induced mutations in the cultivarSamantha via chronic irradiation to generatenew varieties possessing various color andflower shapes.

Vegetatively propagated small plants of“Samantha” were planted in a gamma field

in IRB at a position where the irradiationdose rate ranged 0.25 ~ 1.5 Gy/day in termsof 20 hours per day, that is, these plantswere irradiated at a total exposure of 98.4 ~590.6 Gy for 16 months. During chronicirradiation, shoots were cut back repeatedlyto enlarge the potential mutant sectors andevaluate the color of the flower petals. In thismanner, mutants in flower color wereisolated. Those were propogated by cuttingsand the mature plants were checked forchimera. Individuals exhibiting non-chimeral mutations were retained. Followingthe evaluation of these characteristics in thenursery at OAES, five mutant lines wereselected and were applied for registration asnew varieties. The characteristics of eachvariety differ from the original in thefollowing points:IRB90-1: Strong red flower color and lowertree height than the original variety. Usefulas a garden rose.

Five mutant varieties induced by chronic γ-rayirradiation in rose

Hiroyasu Yamaguchi, Shigeki Nagatomi,Masao Kawakatsu, Konosuke DegiInstitute of Radiation Breeding

IRB90–7 IRB90–8 IRB90–9

IRB90–1 IRB90–4LSamantha

Fig.Five mutant varieties induced from rose variety “Samantha” by chronic gamma ray irradiation


IRB90-4L: Bright purplish red flower colorand unique flower shape due to double ortriple whirls of flower center. Useful for cutflower production and as a garden rose.IRB90-7: Bright purplish red flower color.Useful for cut flower production and as a

garden rose.IRB90-8: Strong pink flower color. Useful forcut flower production and a garden rose.IRB90-9: Strong red flower color, roundpetal and folding flower type. Useful for cutflower production and a garden rose.

Variety Dose Rate Stem Length Flower Type Flower Color Petal Type(Gy/day) (cm)

IRB90-1 1.50 50 - 60 High- centered Strong red Slightly tippedIRB90-4L 1.25 70 - 80 High- centered Bright purplish red Slightly tippedIRB90-7 1.00 70 - 80 High- centered Bright purplish red Slightly tippedIRB90-8 1.00 70 - 80 High- centered Strong red Slightly tippedIRB90-9 0.75 70 - 80 Folding Strong red RoundSamantha 70 - 80 High- centered Deep red Slightly tipped

Table Characteristics of five mutant varieties induced from the rose variety “Samantha” by chronic γ-rayirradiation

A pineapple wild species, “Ananasananasoides” has a small fruit on a slenderpeduncle, and has been cultivated as anornamental plant such as a cut flower.Mutation breeding on pineapple had beencarried out to improve the admiration valueof “Ananas ananasoides” in the Institute ofRadiation Breeding (IRB) and the NagoBranch, the Okinawa Prefectural Agri-cultural Experiment Station (OPAES). Amutant variety “Pineapple Okinawa No. 16”has been raised and characterized as tri-colorvariegation on the whole leaves and asmaller fruit than the original plant.

In the gamma greenhouse at IRB, thepotted plants of “A. ananasoides” wereirradiated at dose rate 0.75Gy/day, total

exposure 169 Gy in 225 days. The stems ofirradiated plants were vertically divided into6 to 8 portions so that axillary buds arose.Some of the plants grown from the axillarybuds, showing chimerical variegated colorleaves, were selected in OPAES. From them,the plant with tri-color variegation on theleaf was isolated, and a new variety,“Pineapple Okinawa No.16” was raised.

The new variety has variegation of thevertical stripe with yellow on green leaf.When the anthocyan is produced on surfaceof leaves of whole-plant, the crowns andslips turn to beautiful vertical stripes withyellow, pink and green in color. Though thecolor of the pericarp is a vivid pink whenflowering, it changes with the growth of the

A mutant variety, “Pineapple Okinawa No. 16” inducedby chronic γ-ray irradiationin the gamma greenhouse

Konosuke Degi, Shigeki Nagatomi,Moriyuki Shoda, Hidekazu IkemiyaInstitute of Radiation Breeding


fruit, and becomes yellow white inmaturation. And, the variety produces a lotof fruits on slender peduncles from the oneplant as well as the original variety throughout the several years after planting. Thevariety keeps good quality for a long timeafter the flowering therefore it can berecommended as a cut flower, potted plant orinterior plant.

Since a fruit of the new variety is smallerin size and low in sugar content, it is notsuitable to eat . It is adaptable to acidic soilin subtropical zones, and it may be producedfor cut flowers and potted plant, etc., at anytime by the flower induction treatment. It isnecessary that the plant are kept in warmcondition for maintaining leaf color.

Plate 1The variegated crown and slips attached on the fruit ofPineapple Okinawa No.16.

Plate 2The Pineapple Okinawa No.16


The National Institute of AgrobiologicalSciences (NIAS) is composed of threedivisions: (1) the Genome and BiodiversityResearch Division, (2) the Insect and AnimalSciences Division, and (3) the Plant ScienceDivision. The research field of the Genomeand Biodiversity Division networks with theInsect and Animal Science Division andPlant Science Division. Therefore, researchdata is shared to maximize researchefficiency.

In the Genome and Biodiversity Division,there are 3 departments: (1) the GenomeResearch Department, (2) the GeneticDiversity Department and (3) the Genebank.The overall research output of these threedepartments is transmitted to the basic andapplied scientific community. As a result, theGenome and Biodiversity Research Divisioncontributes to science, agriculture and sociallife by distributing useful bio-resources andscientific results internationally.

In the Genome Research Department,genome analyses of rice, pig and silkworm isongoing. Our institute played a leading rolein the International Rice Genome SequencingProject (IRGSP) to attain the complete ricegenome sequence and also published thecomplete rice mitochondrial genomesequence. Thus, all three genomes of rice, thenuclear, chloroplast and mitochondrialgenomes, have been sequenced. Theseresearch outputs are tightly linked to severalimportant projects on rice functionalgenomics. In order to provide otherresearchers with access to our usefulresources, NIAS established the Rice GenomeResource Center (RGRC) in 2003. Detailed

information can be obtained via the web at(http://rgp.dna.affrc.go.jp/ and http://rgrc.dna.affrc.go.jp/).

Genetic erosion caused by variousenvironmental, socio-economical andecological changes necessitates research ongenetic resources to maintain the geneticdiversity of the biological world. In theGenetic Diversity Department, basicresearch on plant, microbe and animaldiversity is conducted. The results of theresearch have contributed to rational andefficient methods for the classification,characterization, preservation and discoveryof new biological resources.

To accelerate research on geneticresources, NIAS also is the center of theMAFF Genebank system for agriculturalgenetic resources in Japan. The genebankhas the responsibility of conserving andutilizing plant, microorganism and animalgenetic resources and their DNA. TheGenebank system involved the close linkagebetween NIAS and other sub-genebanksacross Japan. Distributed genetic resourcesare used for research and breeding purposesunder the regulations of the Convention onBiological Diversity and InternationalTreaty. Information related to genebankmaterials can be found at (http://www.gene.affrc.go.jp/ and http://www.dna.affrc.go.jp).

An international workshop on geneticresources was held in Tsukuba in 2003. Thetheme of this year’s workshop was “the genusOryza” or the genus of rice. Rice is the mainstaple food for half the world’s population.Due to population increases, environmentaldegradation and stagnating yield levels in

Research Activities

Genome and Biodiversity Research Division

IntroductionSignificant progress in the analysis of the

genomes of three major agriculturalorganisms, namely, rice, pig and silkworm,which is the main thrust of the GenomeResearch Department, has been made. TheRice Genome Research Program (RGP) isnow at the final stage of sequencing and thehigh-quality sequence of the entire genome isexpected to be completed by the end of 2004.The Animal Genome Research Program(AGP) has continued to develop various toolsfor porcine genome analysis and a databaseof porcine ESTs. The Silkworm GenomeResearch Program (SGP) has released thesequence of almost 80% of the silkwormgenome, which has been generated using thewhole genome shotgun (WGS) sequencingstrategy. The tasks ahead consist ofsynthesizing as much biological informationfrom the sequence data as possible andcontributing to the development of agriculturalproductivity. In an effort to provide access tobiological materials for functional genomicsresearch, the Rice Genome Resource Center(RGRC) was established in April 2003 andbegan the distribution of rice full-lengthcDNA clones, Tos17 mutant lines and geneticpopulations to researchers all over the world.The following reports summarize the majorresearch topics and achievements of the sixlaboratories of this department.

The Finishing Phase of RiceGenome Sequencing

The high-quality draft sequence of the ricegenome was completed in December 2002 by

the International Rice Genome SequencingProject (IRGSP). The Rice Genome ResearchProgram (RGP), a joint collaboration of NIASand STAFF Institute, is responsible for thesequencing of chromosomes 1, 2, 6, 7, 8 and9, which correspond to almost half of thegenome. To facilitate a more efficient andcomprehensive analysis of the genome, allPAC/BAC sequence data were combined andvirtual sequences or pseudomolecules of the12 rice chromosomes were constructed. Thesesequences with a total length of about 400Mb were analyzed by the IRGSP membersand other specialists in various areas of riceresearch. A detailed structural and functionalcharacterization of the entire genome basedon the high-quality draft sequence will bepublished soon. The sequencing effort hasthen shifted to the “finishing” phase with theaim of closing the remaining gaps and raisingthe sequence quality to phase 3 finished-quality level. The IRGSP has set thecompletion of sequencing at the end of year2004. We have already saturated thephysical map with PAC/BAC clones from theIRGSP genomic libraries, BAC clones fromMonsanto, and sequences from the whole-genome shotgun assembly of Syngenta. Tofacilitate gap-filling, two additional libraries,namely, a 10-kb plasmid library and a fosmidlibrary with 40-kb average insert constructedby the Arizona Genomics Institute (AGI),have been used for physical mapping. Thefosmid library has been found to be effectivein extending the PAC/BAC contigs in the gapregions. Among 37 overgo probes designedbased on the unique sequences in contig


rice new approaches and innovations tosustain and increase rice production areneeded. The wider genepool of rice, the genusOryza, includes genetic resources that maybe useful in overcoming constraints to

increasing rice production.The research activities of the Genome

Research Department, the Genetic DiversityDepartment and Genebank are describedbelow.

G enome Research Department


ends, 24 could screen positive fosmid clonesby colony hybridization. As a result, the mostrecent physical map of the 6 chromosomesassigned to RGP has about 95% genomecoverage (Fig.1). Sequencing of these positivefosmid clones is currently underway. Wehave also accelerated the completion of thegenome sequence by systematizing the wholeexperimental step for sequence gap-filling,sequence quality improvement by re-sequencing, and the strategies for resolvingmisassembled repeats. Our “finishing” rate isabout 80-90 clones per month, which isalmost the same as the speed of high-throughput sequencing that led to the earlycompletion of the draft sequence. As of March2004, we have completed the sequence of1,567 PAC/BAC clones out of a total of 1,865minimum tiling clones. This corresponds to148.6 Mb sequence or 72.6% of the sixchromosomes (Table 1). At present, the entiresequences of chromosomes 1, 7 and 8, morethan half of chromosomes 2 and 9, and asignificant portion of chromosome 6 havebeen completed already. With the rest of the6 rice chromosomes, it is expected that the

Chr 1 Chr 2 Chr 6 Chr 7 Chr 8 Chr 9

Fig. 1Construction of PAC/BAC clone contigs in the 6 chromosomes assigned to RGP.Overall sizes of these physical maps are estimated to be 194Mb, which corresponds to 95% of the total chromosomes.

For IRGSP

base pair chr.length(Mb) completeness(%)

For RGP

base pair

chr1chr2chr3chr4chr5chr6chr7chr8chr9chr10chr11chr12total

94.779.786.191.638.813.294.090.054.594.230.080.571.4

72.6

45.238.739.336.031.834.331.230.724.623.832.230.5

398.3

204.7

42824365308327633383281632985741123500004538138

293196042764220213400828224304499673973

24546789284377668

chr1chr2chr6chr7chr8chr9total

42824365308327634538138

293196042764220213400828

148557900

Table 1 Non-overlapping, completed nucleotide length of the ricegenome sequence(March, 2004)

whole genome will be completely sequencedas scheduled.

A New Database of ThreeDimensional Structures of NaturalMetabolites

It is well known that many proteinsfunction by interacting with small moleculessuch as substrates or ligands. We haveutilized a method called docking simulationto predict the mode of interaction based onthree dimensional protein structures. For thelast several years, this method has beenwidely applied to design new medicine andagro-chemicals with specific compoundsfitted to a pocket which appears in apredicted or experimentally identified threedimensional protein structure. Prediction ofthe three dimensional structure of a proteinis also now possible using the homologymodelling method. These techniques havebeen applied mainly in designing new drugs

but have not yet been tried in searchingligands of completely new proteins. In orderto apply the docking simulation method tothe prediction of protein function, weconstructed a database of natural metaboliteswith their three-dimensional structures. Thisdatabase not only collects the three-dimensionalstructures of natural metabolites but alsocalculates their molecular properties. Atpresent, the database has more than 5,000entries with energy minimized 3D structuresstored as two kinds of formats of molecularfiles. A total of 17 properties characterizingthe entries (entry code, name, formula,volume, weight etc.) are incorporated to serveas indeces of the ligand molecules in thedatabase. A searching system of appropriateentries has been developed as well. The webinterface of this database is shown in Fig. 2.Using this system, we can easily obtain adataset of 3D structures of metabolites forparticular requirements.


Fig. 2The web interface of 3DMET provides a search function based on several properties of natural metabolites.


The complete nucleotide sequenceof Numida meleagris (HelmetedGuineafowl) mitochondrial genomeand its utilization in phylogeneticanalysis

Guineafowl has been classified into theorder Galliformes. However, little geneticinformation is available to establish thephylogenetic position of the Guineafowl. Inthe present study, we subjected themitochondrial DNA (mtDNA) of Numidameleagris , a representative member ofGuineafowl, to complete sequencing. It wasrevealed that the mtDNA is a circular DNAof 16,726 bp with similar genomic structureas the mtDNA of Gallus gallus var.domesticus and Coturnix japonica mtDNAs,although it is 62 bp smaller than G.g.domesticus mtDNA and 29 bp larger than C.japonica mtDNA. Similarities of the 13 genesand two ribosomal RNAs except D-loop andtransfer RNAs between N. meleagris and G.g.domesticus and between N. meleagris and C.japonica ranged from 77.0% to 88.8% andfrom 76.2% to 88.4%, respectively. As thesequences of NADH dehydrogenase subunit 2and cytochrome b genes have been reportedfor nine species (Bambusicola thoracis,Coturnix chinensis, C .japonica, G.g. domesticus,Gallus varius, Pavo cristatus, Perdix perdix,Phasianus colchicus, Tympanchus phasianellus)in the order Galliformes, the concatenatednucleotide sequences and amino-acidsequences of these two genes were subjectedto phylogenetic analysis of N. meleagrisagainst these nine species with Ayathyaamericana (Anseriformes) as an outgroupusing a maximum likelihood (ML) method.The ML analyses of the first/second bases ofcodons, the third base of codons, and theamino-acid sequence consistently demonstratedthat N. meleagris did not form clades withother species in the order but stayed in aremote position in the tree.

Construction of a database for ESTsderived from porcine full-lengthcDNA libraries

Expressed sequence tag (EST) analysis is

considered to be an effective method forcollecting expressed genes, which can beuseful for livestock breeding and studies onregenerative medicine. However, expressedmRNA sequences that encode full-lengthcoding sequences (CDS) can rarely beobtained by usual cDNA synthesis methodsin ordinary EST analysis, and assembledEST sequences have revealed relatively fewfull-length protein sequences expressed inpigs. In contrast, cDNA clones containingfull-length CDS are very beneficial foranalyses using protein products translatedfrom clones, such as the preparation ofantibodies against porcine antigens. Tocatalogue the full-length mRNA sequencesexpressed in pigs, full-length cDNA librarieswere constructed from various porcinetissues such as thymus, spleen, ovary, liver,peripheral blood mononuclear cells, uterus,lung, mesenteric lymph nodes and tracheaderived from western crossbred pigs andMeishan pigs. A large-scale EST analysisfrom 5’-end was performed and the resulting107,068 reads were clustered and assembledinto 6,655 contigs and 41,487 singlets. These

Fig. 3The Pig EST Data Explorer (PEDE) provides a search function usingkeywords or gene symbols and a BLAST search tool against the assembledsequences.


sequences were subjected to a BLAST searchagainst the sequences of mammalian genes(UniGene and RefSeq in NCBI) and thehuman genome. The assembled sequencesand BLAST results were incorporated into adatabase, PEDE (Pig EST Data Explorer,http://pede.dna.affrc.go.jp/), equipped withweb interfaces for various kinds of searches.The PEDE database provides porcine cDNAclones, the inserts of which are estimated toinclude the full-length sequence of at least6,000 different genes. Furthermore, PEDEalso contains more than 1,200 SNPsextracted in the contigs, which are classifiedby their origin and effect on the coding aminoacids. The SNPs can be utilized for linkageanalysis in experimental resource families.The PEDE database will help users toexplore genes that may be responsible fortraits like disease susceptibility.

Construction of an EST database forBombyx mori and its application

To build a foundation for the completegenome analysis of Bombyx mori, we have

constructed an EST database. Because thegene expression patterns highly depend ontissues as well as developmental stages, weanalyzed many cDNA libraries prepared fromvarious tissues and different developmentalstages to cover the entire set of Bombyxgenes. So far, the Bombyx EST databasecontains 35,000 ESTs from 36 cDNAlibraries, which are grouped into about11,000 nonredundant ESTs with the averagelength of 1.25 kb. All sequenced ESTs arecompiled into the Bombyx EST database,named KAIKObase, which can be accessed athttp://sgp.dna.affrc.go.jp. The comparisonwith FlyBase suggests that the present ESTdatabase covers >55% of all genes of Bombyx(Table 2). We have constructed ESTmicroarrays containing 6,000 nonredundantESTs for many functional studies as well asfor genome analysis as a major application ofthe EST database. We designed andsynthesized 6,000 specific primers locatedapproximately 500 base pairs downstreamfrom the 5’ end of each cDNA to removerepetitive sequences from DNAs to be used

No. of categorizedgenes in KAIKObase

Gene Ontology termNo. of categorizedgenes in FlyBase

Ratio of KAIKObase vs FlyBase (%)

Ribosomal proteinTranslation initiation factorElongation factorActin bindingTubulin bindingRNA binding1)

Chaperone/heat shockProteasomeTranscription factorDevelopmental proteinCell cycleCell adhesionAxon/neurotransmitterSignal transductionChannelProtein kinase/phosphataseEnzyme2)

MotorTransportTotal

97209

10928

27416057

28071

1103798

10440

2221,270

63391

3,440

10421

912739

35914814348515019347

163246136370

2,59598

8036,236

939510086727695405847577960422960496449

55.2

Table 2 Silkworm ESTs categorized by gene ontology terms.

1) Includes “RNA processing” and “snRNP”; 2) Does not include “protein kinase/phosphatase”


for the microarray. A total of 6,000 DNAswere amplified by PCR using specificprimers, followed by spotting on glass slides.EST microarray technology was successfullyused to identify and isolate ecdysone-responsive genes, and to detect the changesof gene expression in wing discs duringmetamorphosis as a model to describe thegene cascade triggered by ecdysteroidhormone (Fig. 4).

A continuing commitment to provideaccess to genomics resources andinformation

The DNA bank was established in 1994and is now on its 10th year of providingaccess to DNA materials, protein and genomeinformation to researchers within andoutside Japan. Its major activities are: 1)distribution of DNA materials such as cDNAclones, RFLP markers, YAC/PAC/BAC clonesfrom various genome projects of MAFF; 2)opening of databases derived from theseprojects to the public domain; and 3)collecting nucleotide sequence and proteininformation from publicly available databasesas well as developing tools for homologysearch and large-scale genome analysis. Inthe last 10 years, the DNA Bank receivedabout 1,840 requests for DNA materials from33 countries, which resulted in thedistribution of almost 23,788 clones. The

DNA Bank homepage at http://www.dna.affrc.go.jp/ registered a total of 385,685,084access hits for various similarity search toolsincluding 6,727,542 hits for BLAST, 103,210for FASTA, and 96,878 for Smith-Waterman.For the year 2003, a total of 86 requests forDNA materials from 12 countries have beenprocessed, corresponding to 603 rice clonesand 213 pig clones. As to the mirrored datafrom the public databases, 32,697,630 entriescorresponding to 38,021,600,000 nucleotidebases and 1,411,310 entries corresponding to460,216,453 protein sequences have beenaccumulated so far. The access hits in theperiod from April 2003 to March 2004 forhomology search systems using thesesequence data has increased by 1,468,016access hits for BLAST, 8,725 for FASTA and1,712 for Smith-Waterman. The automatedannotation tool RiceGAAS has recorded atotal of 23,697 access hits as a result of theacceleration of the rice genome sequencingefforts this year (Fig. 5). The RiceBLAST, ahomology search tool against rice specificsequences, has recorded a total of 88,156access hits since it was opened last year. Aspart of the current progress of the SilkwormGenome Research Program (SGP), a three-fold shotgun sequence of the silkwormgenome has been released, and analysis toolssuch as KAIKO-BLAST, a homology searchsystem against silkworm nucleotide and

Fig.4Developmental profiles of gene expression in wing discs during metamorphosis by EST microarray experiment. Stages:V4, fifth-instar day 4; S0, beginning of spinning; S1, 1 day after spinning; S2, 2 days after spinning; S3, 3 days afterspinning; S4, 4 days after spinning; P0, beginning of pupation. In this experiment, the cDNAs from reference RNAsextracted from wing discs of stage V4 were labeled with Cy5, while cDNAs from the RNAs of wing discs at each timepoint were labeled with Cy3.


protein sequences, and KAIKOGAAS, anautomated annotation system for silkwormspecific sequences, have been constructed.The genome sequence data and other detailsof the silkworm genome can be accessed athttp://sgp.dna.affrc.go.jp/. To foster moreefficient access to all genome information,the DNA Bank homepage has been renewedand upgraded (Fig. 6). The NIAS alsoestablished the Rice Genome ResourceCenter (RGRC) in April 2003 as a newsupport section of the agrobiological resources

initiative with the aim of providing access toadditional research materials from the ricegenome project and promoting advanceresearches in functional and appliedgenomics (Fig. 7). The biological materialscurrently distributed through RGRC include:1) 32,172 full-length cDNA clones, 2) geneticanalysis materials such as backcross inbredlines, chromosome segment substitution linesand doubled-haploid lines, and 3) Tos17insertion mutant lines. Details are availableat http://rgrc.dna. affrc.go.jp/.

Fig. 5Utilization of RiceGAAS

Fig. 6New NIAS DNA Bank Homepage

Fig. 7Website access to biological materials from the Rice Genome ResourceCenter.


The objectives of the Genetic DiversityDepartment are to conduct basic researchinto plant, animal and microorganismdiversity from the molecular to thepopulation level. Such research contributesto the development of new and improvedmethods for classification, characterizationand preservation of germplasm and discoveryof new biological resources for use inagriculture and other industries.

Current plant research includes molecularand biological characterization andevaluation of the genera Hordeum, Oryza,Saccharum, Triticum, and Vigna. Mechanismsin plants that confer cold hardiness are alsobeing investigated. Microbiological researchincludes genome analysis of plant pathogens,biosystematics and functional proteomics ofsuch organisms as fungi, yeasts and bacteria.Animal research includes the development ofthe methods to utilize various types ofgermplasm from domestic animals. Inaddition, to support the MAFF Genebankproject, database management systems are afocus of research and development. Majorresearch outputs of the department exceptfor ‘Topics of Research’ during fiscal 2003 aredescribed below.

Distinction in chromosome structurebetween two subspecies ofcultivated rice, japonica and indicaby two-colored FISH

Intra-species cytological diversity ofcultivated rice Oryza sativa was shown bydifferent numbers of loci of two tandemrepetitive sequences, 45S ribosomal RNAgenes (rDNA) and Os48. Two-colored FISH ofOs48 and rDNA was carried out to determinethe correlation between their loci and thedifference in chromosome structure betweenjaponica and indica varieties (Fig.1). In allindica varieties, rDNA loci were mapped to

the distal regions of the chromosomesopposite to the Os48 location. But suchlocalization was not detected in any japonicavarieties examined, even in the temperateupland and tropical varieties having tworDNA loci as indica varieties. These resultsrevealed that chromosomal structure differeddistinctly between two subspecies, japonicaand indica.

Development of simple sequencerepeat (SSR) markers by a newmethod in plants and theirapplication to azuki bean

Azuki bean, after soybean, is the mostimportant grain legume in Japan. Inaddition, azuki bean forms a crop complexwith its wild and weedy relatives acrossmuch of Japan. To better understand theevolutionary interactions between thisindigenous crop and its wild relatives wehave developed SSR markers from an SSRenriched library based on a new method forplant material. We found in the azuki beangenome (AG)n motifs are a rich source ofmarkers and thus constructed an (AG)n-SSRenriched library. The SSR library constructioninvolved an oligo-primed second-strandsynthesis enrichment procedure of the singlestrand genomic library. The enriched libraryenabled primer pairs to be designed and

G enetic Diversity Department

Fig. 1Two-colored FISH of Os48 (red) and rDNA (green) oncultivated rice, Oryza sativa, temperate upland japonicavariety (A) and indica variety (B). Bar indicates 5µm.


these primers had a high percentage (98%) ofsuccessful single-locus amplification.

The SSR markers have been used for thefirst time in developing a genome map of anAsian Vigna species and resolved the 11linkage groups equivalent to the haploidchromosome number of diploid species in thissubgenus. The mapping population wasdeveloped from a cross between an azukibean landrace from Japan and a wild relativeof azuki bean from Nepal called Vignanepalensis. In a BC1F1 population of 187individuals from this cross a genome maphas been constructed using 205 SSRmarkers, 93 RFLP markers and 187 AFLPmarkers (Fig. 2). The populations developedfrom this cross are enabling domesticationrelated traits and other useful traits to belocated on the azuki genome map.

Many traits associated withdomestication in azuki bean foundon linkage group 1.

The traits associated with domesticationare the most important in determining cropyield. These traits include seed size and podsize. We have analysed a wide range ofcharacteristics related to domestication inazuki bean and found their association withdifferent locations on the genome map ofazuki bean. This revealed that one part of the

azuki bean genome appears to be particularlyimportant in relation to domesticationrelated traits and this is the distal end oflinkage group one (Fig. 3). In this regionQTL’s associated with seed size, pod size, lossof dormancy, stem width and length, andprimary leaf size have been found. Theseresults agree with results from other cropsthat human selection favours themaintenance of co-adapted character setswithin specific genome regions. By a fullerunderstanding of the domesticationsyndrome we expect to be able to identifyapproaches to enhancing yield in grainlegumes.

In situ localization of transcripts forrice catalase gene, CatB

Catalase is one of the enzymes needed todecompose harmful hydrogen peroxide intooxygen and water. We investigated thespatial patterns of the mRNA for catalasegene, CatB in the shoot and root apices ofrice seedlings by in situ hybridization.Although the antisense RNA probes for CatBshowed weak hybridization in shoot apical

Fig.2The linkage map of azuki bean constructed by SSR (red), RFLP (blue) andAFLP (black) markers using BC1F1 population derived from Vigna nepalensisX V. angularis.

Fig. 3QTL associated with domestication in azuki bean foundon linkage group 1. ↑ : positive effect of allele from cultivated parent; ↓ :negative effect of allele from cultivated parent. Bar: LOD1support interval.


meristems strong signals were detected inthe branch root primordial (Fig. 4A). In theroot tips, CatB mRNA was detectedabundantly in the root apices, including theroot apical meristems and root caps (Fig. 4C).Fusion of the GUS gene to the promoterregion (–329 to +298, counting the cDNAstart site as +1) of CatB conferred strongexpression in the same areas (Figs. 4B and4D), indicating that the presence of thisregion was sufficient to express CatBspecifically in the roots. The –329 to +298region of CatB could be useful for expressingtransgenes preferentially in the roots of rice.

Characterization of deepsupercooling in cold hardy bambooshoots

Most cold hardy species in Graminae,such as wheat, rye and orchard grass toleratefreezing temperatures by undergoingextracellular freezing. Our research on woody

Graminae plants revealed that cold hardybamboo employs deep supercooling as themechanism to survive freezing temperaturesin most of their tissues. Cold hardiness of 30bamboo species was determined to rangefrom –3 to –25 ˚C, which corresponded well totheir ability to deep-supercool. Deepsupercooling in leaf blades of Sasa senanensiswas further characterized. Deep supercoolingability increased from August to Decemberby cold-acclimatization and it varieddepending on the leaf order within a shoot.Structural studies revealed that smallcompartments surrounded by the verticaland transverse veins were important as theunits of supercooling (Fig. 5). In themeantime, boiled or frozen (–40 ˚C) leavesshowed decreased supercooling ability, whichimplies the involvement of some biochemicalmechanisms as well. These results indicatediversity in the evolution of cold hardinessmechanisms in Graminae plants.

Genetic and physical mapping ofavirulence gene AvrPit inMagnaporthe grisea with AFLPmarkers selected by bulkedsegregant analysis

The interaction of blast fungus Magnaporthe

Fig. 4Localization of CatB (A and C) and chimeric GUS (B andD) mRNAs by in situ hybridization. A and B: Longitudinal sections from the shoot apex. RP:branch root primordia. SAM: shoot apical meristem. C andD: Longitudinal sections from the root tip region. RC: rootcap. RAM: root apical meristem.

Fig. 5Small compartments surrounded by cross and verticalveins in the leaf blades of Sasa senanensis. These unitsfroze one after another when the temperature decreasedfrom –15 ˚C (a) to –20 ˚C (b) and –22 ˚C (c), which couldbeen recognized by darkening in their color.


grisea with rice cultivars is dependent ongene-for-gene specific manner. Unravelingthe molecular basis of such interactionsrequires cloning of both avirulence genes inthe fungus and corresponding resistancegenes in rice. In this study, we have identified,genetically mapped and constructed a partialphysical map of a new avirulence gene AvrPitin the fungus M. grisea corresponding to theresistance gene Pit in the rice cultivar K59.We constructed a high-resolution mapping ofAvrPit locus using 730 progeny derived fromthree different crosses paired with threedifferent parent pair strains with AFLPmarkers identified through bulked segregantanalysis and identified 16 AFLP markersclosely linked to the AvrPit locus (0 to 31.25cM) segregating in three different crosses(Fig. 6). As a part of positional cloning, wephysically mapped the AvrPit locus usingAFLP markers by screening an M. griseaBAC library.

Search of HrpX regulons fromgenome database of Xanthomonasoryzae pv. oryzae (Xoo)

Of the genes related to the pathogenicityof plant-pathogenic bacteria, a cluster of hrpgenes encoding a type III secretion system(TTSS) is essential. The TTSS is conservedamong both plant and animal pathogens andinjects effector proteins into host cells. Inxanthomonads, expression of genes of the

TTSS and some effector genes are regulatedby hrpG and hrpX gene products. Some genesincluding hrp genes regulated by HrpXpossess the consensus nucleotide sequencesTTCGC⋅⋅⋅N15⋅⋅⋅TTCGC designated as theplant-inducible-promoter (PIP) box. Wesearched for genes with the PIP box-likesequence in our Xoo geneome database inorder to identify candidate genes in HrpXregulons and found 37 genes containing aputative PIP box in their promoter regions.Six of these genes, including a novel hrpH1,are located in the hrp genes cluster andothers are scattered on the genome. Of the 37genes containing PIP box, one was an outermembrane receptor protein, mostly Fetransport (TonB dependent receptor), andtwo were regulatory proteins containing DNAbinding motifs. Functional analysis of eachgene is in progress.

Five species of Fusarium have beenidentified among pathogens ofsoybean SDS and related dry-beanroot rot.

Soybean sudden death syndrome (SDS)pathogens isolated from USA, Argentina andBrazil and related dry-bean root rotpathogens from USA and Japan weretaxonomically and phylogenetically examinedin NIAS, Japan and NCAUR, USDA in a

Fig. 6Comparative HEGS/AFLP maps around AvrPit locus of M. grisea, derivedfrom three different crosses. Cr. 4120/4986: 590 progeny isolates, Cr. 4000: 80 progeny isolates, Cr.5058: 60 progeny isolates.

Fig. 7Morphology of newly described species of Fusarium, F.virguliforme O’Donnell et T. Aoki.


joint study of both institutes. Comparativephenotypic examination of macro- andmicroscopic features and phylogeneticanalyses of multilocus DNA sequence dataindicated that they are consisted of fivedistinct species. Soybean SDS in North andSouth America was caused by four distinctspecies: Fusarium virguliforme, F. tucumaniaeand two undescribed species of Fusarium.Further, dry bean root-rot in North Americaand Japan was caused by F. phaseoli and oneof the undescribed species of Fusariummentioned above. Among them, F. virguliforme(Fig. 7) and F. tucumaniae were reported as newspecies and F. phaseoli as a new combinationwithin this study. Formal new speciesdescriptions for the two undescribed speciesare in preparation.

Killer mechanisms of yeast revealedby yeast-genome set screen

Antagonistic interactions between yeastsby secreted proteinaceous toxins appear tooccur quite often in natural habitats. Tounderstand the molecular mechanisms ofyeast killer function, we screened a set ofSaccharomyces cerevisiae mutants, individuallydeleted for 4901 yeast genes, for alteredsensitivity against purified killer proteins ofWilliopsis mrakii (HMK) and Kluyveromyceslactis (KlKP) (Fig 8). The data werecompared with the results with S. cerevisiae

K1 killer protein. Cell wall receptors of K1and KlKP are reported as β-1,6-glucan andchitin, respectively. The anti-killer spectrumof HMK on the gene disruptants of N-glycanbiosynthesis is similar to that of K1. Thesemutants are hyper-sensitive to KlKP.However, deletion of the gene for RNApolymerase II machinery made yeast cellsresistant to HMK, KlKP and K1. The resultssuggest that these killer proteins share theintracellular killer mechanisms despite thedifference in the docking sites on the cellsurface.

Piglet production byintractyoplasmic sperm injection(ICSI)

The viability or motility of thespermatozoa after thawing is quite variabledepending on the individuals or the lot,especially in pigs. ICSI of a nonmotile cellinto the ooplasm for assisted fertilization is ahighly specialized procedure for producingthe next generation. The production of pigletsby ICSI has succeeded only when in vivomatured oocytes were used as recipients. Inthe present study, the in vitro maturedoocytes were injected with a sperm head,stimulated with an electrical pulse and thentransferred to a total of 7 estrous-synchronizedrecipients (55–150 oocytes per recipient). Tworecipients became pregnant and one of thesefarrowed three (a male and two female)healthy piglets (Fig. 9). The resultsdemonstrated that in vitro matured oocytesafter ICSI are developmentally competentand can generate viable piglets.

Fig. 8Determination of killer activities with yeast-genome setscreen.Some gene-disruptants survived in the presence of killerprotein (clone 1, 2, 3 etc.). WT: wild type yeast.

Fig. 9Piglets produced by intractyoplasmic sperm injection intoin vitro matured oocytes


Improvement of genetic gain inswine by including an additionalsystematic environmental effect inthe evaluation model

The advantage of mixed-model techniquesover a selection index under differentmagnitudes of an additional systematicenvironmental effect (ASEE) in terms ofexpected genetic gain was evaluated. Thedata attempted to simulate a closed herd in aswine breeding program. Additional systematicenvironmental constants with four levels in ageneration were assigned from a uniformdistribution at different ranges. Breedingvalues of animals were estimated on thebasis of an index of individual phenotype (SI-U), SI-U adjusted for ASEE using a least-squares mean (SI-A), best linear unbiasedprediction (BLUP) using an animal model

excluding ASEE (AM-E), and an animalmodel including ASEE (AM-I). Expectedgenetic gain was larger by the animal modelthan by the selection index, even if ASEEwas set to zero (Table 1). The differences inexpected gain between SI-U and SI-A, andbetween AM-I and AM-E increased as therange of ASEE increased. It was concludedthat ASEE should be always included in anevaluation model, however slight anysystematic environmental effect may be in aherd.

Development of managementsystem for plant image data

Image data of genetic resources allows usto totally understand the morphologicalcharacteristics which are difficult to representwith words or numbers. We have developedthe management system for image data ofthe plant genetic resources in the MAFFGenebank in order to expand the characteristicsdatabase linked to the existing passport data.At the same time, we have also developed aprogram to input image data into themanagement system from local Sub-Banksthrough the internet. The accumulated datawill be published on the MAFF Genebankhome page in the near future by thedevelopment of an application program.

Range of systematic Method of selection2)

environmental effect1) SI-U SI-A AM-E AM-I±0.00 0.56 0.56 0.80 0.79±0.25 0.56 0.56 0.79 0.79±0.50 0.53 0.56 0.74 0.79±0.75 0.49 0.56 0.69 0.79±1.00 0.48 0.56 0.65 0.79

Table 1. Expected genetic gain from different selection methods withdifferent ranges of additional systematic environmentaleffect.

1) Unit is phenotypic standard deviation.2) See in the text.


Genebank ProjectThe genebank project on plant,

microorganism and animal genetic resourceshas been implemented in national andinternational collaboration with a largenumber of organizations in public andprivate sectors. NIAS coordinates as a centerof the project activities including theexploration, collection, characterization,evaluation, preservation, multiplication andinformation management of geneticresources.

In fiscal year 2003, five missions werecarried out to collect genetic resources of beetin Russia, apples in Turkey, mulberries inPakistan, persimmons in Korea and tea inVietnam. One mission was also dispatchedto Mongolia to collect goat DNA. All theexploration missions have collaborated withthe research institutes in each of thecountries. Joint field survey programs havebeen undertaken on sweet potato inIndonesia and perilla in Korea. Internalexploration missions were carried out withinJapan. The total number of genetic resourcespreserved at the NIAS genebank and partnerinstitutions is presently 230,337 accessions ofplant genetic resources, 20,472 accessions ofmicroorganism genetic resources and 896accessions of animal genetic resources. In2003, 12,292 accessions of plant geneticresources, 816 accessions of microorganismgenetic resources and 43 accessions of animalgenetic resources were distributed to internaland external users for basic and appliedresearches. About 10 to 20 % of geneticresources were distributed overseas.Passport and characteristics data ofindividual accessions are provided on thewebsite, (http://www.gene.affrc.go.jp/).

The international genetic resourcesworkshop was held to discuss geneticdiversity and evolution of the genus Oryza inSeptember, 2003 in Tsukuba. The

international meeting was held to evaluatethe final report of a 3-year collaborativeresearch program between Japan andIndonesia on “in situ conservation of sweetpotato in Indonesia” in December, 2003.

Genetic variation and geographicaldistribution of GBSS 1 gene infoxtail millet, Setaria italica (L.) P.Beauv.

Foxtail millet , Setaria italica (L.) P.Beauv., is one of the world’s oldest cultivatedcrops. There are a non-waxy endospermtype, waxy type, and intermediate (lowamylose) type of this millet. Waxyendosperm arises through the disruptedexpression or loss of function of GBSS 1 genethat encodes starch granule-bound starchsynthase 1 (GBSS 1). Waxy type ischaracterized by little or no starch amylose,which constitutes about 20% or more of thetotal starch in the non-waxy endosperm.Waxy and low amylose types are cultivatedrather limitedly in E Asia and SE Asia. Wehave studied the structure of the GBSS 1gene and have revealed different andcomplicated crop evolution pathways inwhich waxy and low amylose endospermvariants were generated by varioustransposable elements (TEs). We surveyed870 landraces and identified 12 allele (2 non-waxy, 3 low amylose, and 7 waxy) types ofthe GBSS 1 gene generated by 11 differentTEs. This indicates that the TE-inducedmutants have been selected under theinfluence of ethnobotanical preferencetoward sticky foods in E Asia and SE Asia,while non-waxy type is commonly grownthroughout Asia and Europe.

Cryopreservation of plant geneticresources

In our genebank project, sub-banks areresponsible for the conservation of

G enebank


vegetatively propagated crops such aspotatoes, sweet potatoes, fruits, tea and othercrops. In principle, these field collectionshave been maintained by the duplicatedconservation system. However, there aresome exceptions involving mulberry, rushand taro which have been maintained at onlyone sub-bank. Cryopreservation should beconsidered as a backup to field collections toinsure against loss. The cryopreservationtechniques are now advanced to the stagewhere they can be implemented for usefulstorage of germplasm systematically. By this,we mean that the priority of collections to becryopreserved should be given to the “at risk”collections that have an increased chance ofbeing lost from a collection. In our genebank,the Morus germplasm collection in Tsukuba,representing about 756 lines within severalspecies, is going to be cryopreserved in threeyears. We started cryo-storage of winterbuds of about 101 lines of mulberry from thefiscal year of 2003. The national cryo-genebank in Japan is expected to beestablished for the germplasm of vegetativelypropagated crops, plants with recalcitrantseeds and valuable culture materials.

Pathogenic fungi causing newdiseases of oncidium and crinum

Pathogenic fungi causing new diseases oftwo flowering plants were found. One is ananamorphic fungus causing a disease ononcidium (Oncidium sphacelatum) ofOrchidaceae, and the other is that on crinum(Crinum asiaticum var. sinicum) ofAmaryllidaceae. The former fungus bringsbulb rot, leaf rot, defoliation or whole plantblight of oncidium (Fig. 1-A). Mycelia andsclerotia of the causal fungus frequentlyappeared on their lesions. In culture of thefungal isolates on water agar in the dark,hyaline hyphae with clamp connectionsdeveloped to form sclerotia as same as thoseon host lesions (Fig. 1-B, C). Primary hyphalcells were < 450 µm in length and 4–6 µm inwidth. A secondary hypha was branchingjust beneath a primary hyphal septum, 1–2µm in width and elongating along the

primary hypha in the early times. Maturesclerotia were subglobose to ovoid, 0.7–1.5mm in diameter. The fungus was identifiedas Sclerotium rolfsii based on itsmorphological character, and the disease wasnamed southern blight, shirakinu-byô inJapanese. The latter fungus causes leaf spotor leaf blight of crinum, on which lesionsbecome uniquely red in color (Fig.2-A).Pycnidia of the causal fungus frequentlyappeared on their lesions (Fig.2-B). Inculture of the fungal isolates on potatodextrose agar under black light, pycnidiawhich resemble to those on host lesions wereformed. They were subglobose, 200–560 µmin height and 192–460 µm in width. Conidiawere hyaline to pale brown, ellipsoid tocylindrical, 0–3 septate and 5–24 × 3–7 µm in

Fig. 1Southern blight of oncidium.A: Natural symptom. B, C: The causal fungus, Sclerotiumrolfsii. B: Hypha with clamp connection; C: Sclerotium.

Fig. 2Red leaf spot of crinum.A: Natural symptom. B, C: The causal fungus, Stagonosporacurtisii. B: Pycnidium; C: Conidium.


size (Fig. 2-C). The fungus was identified asStagonospora curtisii based on itsmorphological and cultural character, andthe disease was named red leaf spot,sekihan-byô in Japanese. The fungal isolateswith information about the two new diseasesare important as reference isolates fordiagnosis of the diseases, and moreoveruseful for construction of their preventionmeasures and development of mycologicalstudies from the viewpoints of taxonomy,distribution, bionomics and parasitism.

Genetic analysis of Japanese nativecattle breeds and populations

Genetic variability of three breeds,Japanese Black, Japanese Brown andJapanese Shorthorn and two pure nativecattle populations, Kuchinoshima feral cattleand Mishima cattle, was studied using 23microsatellite DNA markers. Ppoly, averageheterozygosity and the average number ofeffective alleles in each breed and population

are shown (Table 1). Genetic variability inthe three breeds, Japanese Black, JapaneseBrown and Japanese Shorthorn, is at almostthe same level as the indices estimated bymicrosatellite DNA polymorphisms. On theother hand, Mishima cattle andKuchinoshima feral cattle revealed lowgenetic variability, equal to almost half thatof the other breeds. The genetic distanceestimated from DNA polymorphism showsthat the Japanese native cattle, Mishima andKuchinoshima, are relatively more distantfrom Wagyu breeds than those within theirgroup. The genetic distance betweenMishima and Kuchinoshima is larger thanthat to Japanese Black and Japanese Brown(Table 2). The large distance between the twoJapanese native populations is thought to bedue partially to genetic drift in differentdirections after the introduction onto bothislands and also suggests the existence ofgeographical differentiation in the pastJapanese cattle population.

No. ofanimals

P. poly Average numberof alelles

Average numberof effective alelles

Average observedheterozygosity

KuchinoshimaSE

MishimaSE

Japanese BlackSE

Japanese ShorthornSE

Japanese BrownSE

31

62

60

30

31

56.5%

52.2%

87.0%

95.7%

95.7%

1.78±0.85

1.78±0.90

4.09±2.21

4.13±1.74

4.48±2.11

1.48±0.59

1.40±0.48

2.28±1.09

2.51±1.17

2.90±1.35

0.242±0.248

0.209±0.236

0.446±0.281

0.516±0.234

0.560±0.267

Table 1 Genetic variability of five Japanese native cattle breeds and populations

KS

Kuchinoshima (KS)Mishima (MS)

Japanese Black (JBL)Japanese Shorthorn (JS)Japanese Brown (JBR)

*0.3490.2490.3820.251

MS

*0.2250.3830.254

JBL

*0.1960.104

JS

*0.183

JBR

*

Table 2 Genetic distance among Japanese native cattle breedsand populations


The Insect and Animal Sciences Divisionconsists of the following six departments.

The Developmental Biology Department isconducting researches to elucidate thephysiological functions of germ cell lines andthe hormonal effect of growth regulation, anddevelops embryonic technology as well. Theseresearches focus on several invertebrates(nematodes, annelids, sawfly, fruit fly andsilkworm) and vertebrates (mouse, chickenand pig).

The Molecular Biology and ImmunologyDepartment is concerned with the molecularevents involved in the signaling forantibacterial peptide synthesis in insects andsignaling of cytokines and T cell receptors inmammals, the functional analysis ofmacrophage/microglia, hematopoietic stemcells and T cell subsets, the geneticmechanisms underlying complex diseasetraits, and the development and improvementof transgenic animal technology. Majorresearches on cytokine signaling in themammalian immune system, innate immunesystem in insect, the transformation ofspermatogenic cells in vivo and geneticanalysis of multi-factorial are beingconducted.

The Physiology and Genetic RegulationDepartment aims to elucidate the mechanismsof unique physiological phenomena observedin insects and animals. The studies includeanalyses of neural functions including brain,metabolic regulation, physiological bases for

adaptation to environment, perception andfunction of behavior regulating chemicals,cell differentiation and the genes regulatingthem, in both invertebrates and vertebrates.

The research activities of the InsectGenetics and Evolution Department aremainly focused on molecular and conventionalgenetics of insects including silkworm,biochemical analyses of insect-plantinteraction, characterization and breeding ofnatural enemies, and basic and appliedstudies on insect-associated microbes includingpathogens and symbiotes.

The Insect Biomaterial and TechnologyDepartment is carrying out researches whichaims to develop the biomimetic techniques ofinsects and their utilization such as thesugar reception of the flesh fly, and to clarifythe characteristics of insect-born biomaterialsfor the development of new technologies.

The major targets of the Insect Biotechnologyand Sericology Department are the developmentof functional insect cell lines and transgenicinsects, large-scale production of usefulsubstances using the transgenic insectsystems, and the development of new silkmaterials which are good for healthful andwealthy human life and which are based onthe development of new sericultural technologiesincluding breeding of characteristic newvarieties of silkworms.

The major research topics for 2004 fromthese six departments in the Insect andAnimal Sciences Division are as follows:

Insect and Animal Sciences Division


Signal transduction regulatingdevelopment/immunity in nematodes

Toll membrane receptor regulatesdevelopment and immunity in variousanimals. Previously, we reported that RNAinterference (RNAi) knock-down of nematodeToll inhibited normal morphogenesis andearly development. Since some essentialfactors in the typical Toll signaling pathwayare not found in the nematode genome, weexpect that a novel signaling pathway(s) maybe involved in nematode development andimmunity. To elucidate the pathway(s), todate, we have isolated one candidatemolecule that interacts directly with theintracellular domain of nematode Toll andtwo distinct groups of immune-inducibleantimicrobial peptides (ASABF andnematode cecropins) (Fig. 1).

Identification of genes involved inannelid regeneration

The fragmenting oligochaete, Enchytraeusjaponensis can regenerate a completeindividual from a small body fragment within4-5 days. To isolate genes involved in theregeneration mechanisms, we performedcDNA subtraction between regeneratingfragments and intact worms. Based on theresults of membrane array screening, virtualnorthern analysis and the abundance of

expression, six clones were chosen for full-length sequencing. Data-base homologysearches of these clones revealed that threeof them were novel genes and the others weresimilar to genes of glutamine synthetase,unc-119 and flotillin-2, respectively.

Involvement of the MAP kinasecascade in the meiotic cell cyclearrest in the sawfly, Athalia rosae

In most animals, the meiosis arrests at acertain stage during egg maturation. Themolecular mechanisms of the meiotic arrestremain still un-clear in invertebrates. Thesawfly serves as a good model systembecause artificial egg activation is practicaland it enables one to examine eventsoccurring at and around resumption ofmeiosis. We demonstrated that the MAPkinase cascade participated in egg maturationin the sawfly as reported in vertebrates, andthe Mos-like protein that functions as acytostatic factor to regulate the meioticarrest was involved in the cascade.

Real-time observation ofprogrammed cell death ofDrosophila salivary glands

In insect metamorphosis, the steroidhormone 20-hydroxyecdysone activatesautophagic programmed cell death (PCD) toeliminate larval structures that are no longerneeded. Autophagy is a form of PCD that ismorphologically distinguished from apoptosis,and is as prevalent as apoptosis, at leastduring development. In comparison withapoptosis, however, there are not manystudies on the regulation mechanisms ofautophagy. To provide a useful model forstudying autophagic PCD, we established anin vitro culture system that enables real–time observation of autophagic cell destructionof Drosophila salivary glands (Fig. 2). Thenew system revealed that de novo gene

D evelopmental Biology Department

Fig. 1Elucidation of nematode Toll signaling pathways.


expression was still required for thedestruction of salivary glands dissected frompupae after head eversion.

Expression profiling for three sub-populations of ES cell which weresorted by flowcytometry

Last year, we found that the expressionsof Platelet Endothelial Cell AdhesionMolecule (PECAM)-1 and Stage SpecificEmbryonic Antigen (SSEA)-1 in ES cellswere greatly varied and ES cells were sortedinto three sub-populations; PECAM-1-SSEA-1- (NN), PECAM-1+SSEA-1- (PN) andPECAM-1+SSEA-1+ (PP) by FACS. Each sub-population had a different developmentalability in chimera formation. These resultssuggest that the expression levels of PECAM-1 and SSEA-1 in ES cells are closelycorrelated with the pluripotent differentiation

capability and/or the competency of incorporationinto epiblast of chimeric embryos. In thecurrent fiscal year, to identify novel geneexpressions that are correlated with thestemness of ES cells, we have comparedexpression profiles among three sub-populations (NN vs PN and PN vs PP) of EScell (Fig. 3). Agilent 60-mer Oligo Microarreies(Mouse and Mouse (Development)) were usedto screen up to 34,000 genes. Scatter plots oftwo comparisons are shown in Fig. 4.Changes of gene expression in NN vs PNwere larger than PN vs PP. The number of

Fig. 3Three sub-populations of ES cell

Fig. 4Scatter plots of two comparisons

Fig. 2Time-lapse photography of a fixed portion of a live salivary gland dissected from a Drosophila pupa immediately afterhead eversion and cultured in vitro for 0-6 hours; changes in cellular morphology including bleb formation (arrows),shrinkage, and fragmentation are shown. Scale bar, 50 µm.


genes with a significant difference of morethan twice was about 2,000 in NN vs PN andabout 700 in PN vs PP. In this analysis, wehave detected some known genes that arerelated to stemness, such as oct4 and nanog.Now we are going to screen novel genes andto investigate their function in thedevelopment and differentiation.

A simple method for typing singlenucleotide polymorphisms (SNPs) inchicken mt-DNA

A simple method for typing singlenucleotide polymorphisms (SNPs) in thechicken is required to analyze the chimeras.

We have searched for SNPs in the D-loopregion of chicken mitochondrial DNA(mtDNA), which is about 1.2 kb, and morepolymorphic than genomic or other mitochondrialregions. The D-loop region in mtDNA wassequenced and compared among six chickens,and 11 SNPs were observed. For six of theSNP sites, PCR primers were producedhaving a base matched for one allele at the 3’end and with each base (A, G, C and T) asthe penultimate (N2) base (Fig. 5). For eachSNP site, primers corresponding to bothstrands (5’ -> 3’ and 3’ <-5’), terminating withthe allele-specific base at the 3’ end, andhaving four kinds of N2 base were produced,

Fig. 5PCR product is amplified only from the specific allele using N2 mismatched primer.


resulting in 16 primers. Thus, consideringthat the N2 base in primers corresponded toone strand and one allele, one primer had anN2 base that matched the template (N2-matched primer), whereas the other threeprimers had mismatched bases (N2-mismatched primer). Twenty-one out of 96primers succeeded in distinguishing theSNPs by the presence or absence of PCRproduct. Though this method requires theinitial use of several primers to identify theappropriate primers, after determining theseprimers, only one PCR step (including twoPCR with each allele specific primer) cantype the SNP in each individual. This methodwill provide an easy way to type SNPs inmany individuals and be useful todistinguish the chimeric chicken.

Regulation of Insect DevelopmentThe regulatory mechanisms on insect

development, such as ecdysis andmetamorphosis are being studied on thesilkworm at the Insect Growth RegulationLaboratory. Recent progress as follows: (1)Affinity ligands with partial structure ofinsect growth control agents (IGRs), JHanalog and KK-42 which induces a precociousmetamorphosis were prepared. The ligandswere coupled with reporter enzyme for thedetection of proteins with high affinity, andcoupled with resin to obtain affinity gel for apurification of the protein, and the conditionfor the purification was optimized. (2) It wasfound that phosphorylation cascades are notinvolved in an action of ecdysone on midgutbecause the action was not inhibited byinhibitors of protein phosphorylation.Corazonin had an effect on the regulation ofspinning in Bombyx. (3) It was shown thatBR-C transcription factor regulated threegenes specifically expressed at pupation withreporter gene assay. It was stronglysuggested that Bombyx EGFR was a receptorfor Bombyx paralytic peptide since the EGFRexpressed in COS cells bound with 125I-labeled GFP. (4) The main organs weredisected from both male female individualsat the entire growing stage of B. mori (from

egg to adult), and applied for 2-D PAGEprotein analyses. Most of the proteinsisolated on the 2-D gels have been spottedand digested by tripsin after reductivealkylation. (5) The cell number and polyploidlevel in dorsal epidermis of 5th segment from3rd instar to pupa were measured byflowcytometer. The 8C cells became the mostabundant in the 3rd and 4th instar but the16C cells became major in the 5th instar (Fig.6). The cell number increased graduallyduring feeding period and decreased duringmolting. This decrease of the epidermal cellnumber during molting might not be causedby apoptosis because of little caspase-3activity.

EGFP transgenic cloned pigAlthough pronuclea microinjection has

been used for many years to producetransgenic pigs, the efficiency of transferringgenes into pigs is quite low, under 1%. Eventhough transgenic pigs were produced bymicroinjection method, variable transgeneexpression patterns and uncertain transmissioninto germ line cannot be avoided. Theproduction of somatic cell clones derived fromcultured cells opens new horizons fortransgenic animals. The integration of geneand level of gene expression can be confirmedand screened in the cultured cells after genetransfer, and it will improve the efficiency oftransgenic animals through nuclear transferusing such genetically modified cells. One of

0

10

20

30

4050

60

70

80

90

0 24 48 72 0 24 48 72 96 0 24 48 72 96 120 144

time after each ecdysis (hr)

%

2n 4n 8n 16n 32n 64n 128n

3rd 4th 5th

Fig. 6Developmental changes in ratio of ploidity of Bombyxepidarmal cells.


the important factors for successful cloningdepends on the period of cell culture. A longcell culture decreases the cell viability andcauses chromosomal abnormalities. Usualgene transfection and selection proceduresrequire a prolonged culture period and it willlead to an insufficient condition of cells fornuclear transfer.

In the present study, we transfectedEGFP reporter gene immediately afterisolation of fetal cells and selected themunder puromicine for 5 days. Because almostall the colonies that appeared after selectionwere confirmed EGFP-positive under fluorescentmicroscopy, we did not isolate each colonyand continued to culture heterogeneouslyuntil expansion for subsequent nucleartransfer. By means of this procedure, theperiod of cell culture for the establishment ofEGFP expressed cells was shortened at leastfor 10 days.

Fifty-nine-point-three percent(1791/3020)of nuclear transferred embryos derived fromthe EGFP expressed cells were developed tothe 2-8 cell stage and transferred to 14synchronized recipient pigs. Four recipientswere pregnant and delivered 9 piglets. Thepresence of EGFP was confirmed by PCR intissues and primary cell culture derived fromoffspring. The expression of EGFP was alsodetected by exposure of fluorescence (Fig. 7).All the piglets were EGFP-positive but onlyone piglet survived and matured. Thetransmission of EGFP transgene will beexamined later.

Fig. 7Transgenic cloned pig showing EGFP expression in the snout. Conventional light (A) and fluorescence (B)

Fig. 8Hierarchical clustering analysis of gene expressionprofiles in bovine folliclesFollicular size in diameter, S : 40-100mm, M : 3-5mm, L :10mm�


Transcriptome analysis duringfollicular development in the cow

Mammalian follicular development initiatesin the fetal life and a pool of primordialfollicles (approximately 1 million) is formedduring the fetal age or soon after birth.Thereafter a ple-thora of primordial folliclesbegins to grow continuously throughout thelife or until the pool is exhausted. However,cattle ovulate a single follicle per estrouscycle, other numerous numbers of folliclesundergo atresia during any stages of thedevelopment. How the follicles are recruitedand selected for final maturation leading tothe ovulation an intriguing mechanism. Sincedynamic changes of follicular morphologysuggest the involvement of specific genes inthe follicular growth, a numbers of genesresponsible for the follicular growth havebeen enumerated. However, a bird’s-eye viewof follicular gene expression has not beenelucidated. In the present study folliculargene expression profiles were analyzed acrossthe folliculogenesis using a custom DNAmicroarray. The microarray, containing

approximately 1800 individual genes, wasdeveloped from the bovine utero-placentalcDNA library. The ovarian follicles werecollected at the local slaughterhouse andclassified into three categories with their sizein diameter, 40-100 µm, (small: S), 3-5 mm(medium: M) and over 10 mm (large: L). Thetotal RNA was isolated and utilized for theT7-based linear amplification procedure togenerate antisense RNA (aRNA). AmplifiedaRNA was reverse-transcribed with Cy3- andCy5-dUTP for hybridization. Approximately1500 genes were differentially expressedbetween the M and L. This finding suggeststhat the microarray derived from bovineutero-placental cDNA library is successfullyutilized for transcription analysis in ovarianfollicles (Fig. 8). Some genes, e.g. collagens,cathepsin L, IGFBP-3, MMP-2, FGFs andinhibin, were deferentially expresseddepending on the stage of folliculardevelopment. These genes were suggested tobe a member of the candidates for folliculardevelopment in the cow.

The research activities of our departmentinclude the molecular analyses of defensemechanisms in mammalians and in insects,and the development of model animals usefulfor studies on diseases.

Cytokine signaling in themammalian immune system

T cell receptor (TCR) signaling inducesproduction of interleukin 2 (IL-2), accompaniedby cytoskeletal rearrangement in these cells.Wiskott-Aldrich syndrome protein (WASP),the gene product responsible for the Wiskott-Aldrich immunodeficiency syndrome, acts asan adaptor molecule in TCR signaling. Infact, T cells from WASP-deficient miceexhibited neither cytoskeletal rearrangement

nor IL-2 production on TCR stimulation. Incontrast, we demonstrated that T cells fromWASP-transgenic mice overexpressing N-terminal EVH1 domain, did not produce IL-2,but cytoskeletal rearrangements in thesecells were normal. To further clarify thefunctions of the EVH1 domain in thesignaling pathway for IL-2 synthesis, weemploy an intrabody strategy in whichfunctions of the WASP-EVH1 domain areknocked down by single-chain antibodies(scFvs). Anti-WASP-EVH1 intrabody wasexpressed in the scFv vector transfected Tcells, and the binding of the scFv intrabody toWASP was confirmed. Further evaluation ofthe intrabody strategy for WASP-EVH1domain in TCR signaling is now in progress

M olecular Biology and Immunology Department


in transgenic mice.Maintenance of immunological tolerance

is essential to prevent unwanted inflammationsuch as allergy, autoimmune diseases, andthat upon organ transplantation. As theimmunological homeostasis largely dependson the existence of regulatory cells, which arepreferentially induced in the intestine, wecloned antigen-specific CD4+CD25+ regulatoryT cells from Peyer’s patches. They wereindeed effective to ameliorate detrimentalimmune responses in a food allergy model(DO11.10 mice: Fig. 1). We found thatregulatory cytokines such as TGF-β and IL-10 are important for the suppressivemechanism of intestinal regulatory T cells,and inflammatory cytokines such as IL-18are essential for their functional maturation.It has thus become important to search forthe efficient immune-modifier to induceregulatory cells. Food components andcommensal bacteria make the largest pool ofimmune-modifier for intestine where in themajority of immune cells reside. Weidentified Lactococcus lactis strains(probiotics) which induce high IL-10 productionfrom dendritic cells which may contribute tomaintain immunological homeostasis andprevent inflammatory diseases.

Finally, we have established immortalizedmicroglial cell lines from WASP-transgenic,prion-transgenic, and prion-deficient mice bya retroviral vector containing human c-myc.

These cell lines provide useful in vitro assaysystems to evaluate the immunological rolesof microglia in neurological disorders, such asprion diseases.

Innate Immune System in InsectA protein showing strong antiviral activity

against Bombyx mori nucleopolyhedrovirus(BmNPV) was purified from the digestivejuice of B. mori larvae (Fig. 2). A homologysearch of the deduced amino acid sequence ofthe protein cDNA revealed 56% homologywith Drosophyla melanogaster lipase and21% homology with human lipase. As lipase

Fig. 1In vivo transfer of antigen-induced regulatory T cell clone derived from Peyer’s patches ameliorated the phenotype ofmurine model for food allergy.

Fig. 2Effect of Bmlipase-1 on BmNPV-ODV infectivity. ODVinfectivity was examined after treatment of ODV (860ng/larva) with different concentrations of Bmlipase-1. Datashown are means±standard deviations of results from fiveexperiments. Luciferase activity was measured at 136 hpi.Note that luciferase activity of hemolymph samples fromnontreated B. mori larvae was also measured todetermine the background level, and the level of relativelight units (RLU) per 10µl of hemolymph was 120±3.4.


activity of the protein was confirmed in vitro,this protein was designated Bmlipase-1.Northern blot analysis showed that theBmlipse-1 gene was shown not to beexpressed in the molting and wanderingstages, indicating that the gene is hormonallyregulated. Our results suggest that an insectdigestive enzyme has potential as aphysiological barrier against BmNPV at theinitial site of viral infection.

Spontaneous system of cecropin B, anantibacterial peptide, was observed in themolting stage in two lepidopteran insects,Bombyx mori and Antheraea pernyi, byimmunohistochemical staining. The naturalinduction of cecropin B gene expression wasalso observed in artificially induced moltingof B. mori larvae, suggesting that the naturalinduction is tightly linked to ecdysis. Asinsect molting is strictly controlled byecdysteroids, a possibility that the hormoneinduces antibacterial peptide gene expressionwas tested by using a fat body primaryculture of B. mori. The results showed that20-hydroxyecdysone failed to induce geneexpression of three antibacterial peptides,cecropin B, attacin, and lebocin, suggestingthat an unknown endogenous trigger(s) isinvolved in the natural induction. The resultssuggest that insects contain a novel ecdysis-linked immune system, which is strikinglydifferent from that of vertebrates.

Ticks have an efficient defense system forpreventing microbial infection. The anti-microbial peptide defensin is one effectivemolecule in this system. Here we investigatedimmune competence and the involvement ofdefensin in the humoral defense of the softtick, Ornithodoros moubata. Semiquantitativereverse transcriptase-polymerase chainreaction (RT-PCR) revealed that geneexpression of all four defensin isoforms wasup-regulated by bacteria or bacterialcomponents. Defensin gene up-regulation byhemocoelic inoculation of bacteria involvesthe midgut and granulocytes. In immunode-tection analysis, immunization by bacterialinjection increases the relative concentrationof defensin-like material in the hemolymph

plasma. Furthermore, elevated antibacterialactivity against Gram-positive bacteria butnot against Gram-negative bacteria wasobserved after immunization by a liquidgrowth inhibition assay. Therefore, enhancedanti-Gram-positive bacterial activity appearsto be partially dependent on the release ofdefensin into the hemolymph. These findingsdemonstrate that defensin plays animportant role in the up-regulated humoralresponse of O. moubata.

Transformation of spermatogeniccells in vivo

We are trying to develop a convenient andefficient transgenic technology to producemodel animals. We have already found thatmicroinjection of DNA solution into seminiferoustubules of the cryptorchid testis successfullytransformed mouse spermatogenic cells invivo. This year, we also found that applicationof the same technology to the infant mousetestis gave the same results (Fig. 3).However, we experienced loss of animalsduring surgical operation using the populargeneral anesthetic procedures. Thoseprocedures were found to be inadequate forthe infant animals. Because a safe andadequate procedure for general anesthesia ofinfant mice has not been reported, we testedisoflurane inhalation anesthesia. Six- to ten-day-old infant C57BL/6JJcl male mice wereused. We assessed the safety and efficacy ofinhalation anesthesia by isoflurane deliveredthrough a Univentor 400 anesthesia unit,and found the procedure safely providedsufficient anesthesia for medium-duration

Fig. 3Expression of GFP gene by injecting the DNA intoseminiferous tubules of testis.A; normal control testis. B; a testis three months afterinjection of GFP gene.


surgery of infant mice.

Genetic analysis of multi-factorialdiseases

For the QTL study, we focused on abnormallipid metabolism as the phenotype ofinterest. We have already identified that theinbred mouse strain KK show hyper-lipidemia. In the present study, KK and RRstrains of mice were used for geneticalanalysis. Genes which control the plasmalevels of cholesterol and triglyceride wereanalyzed by QTL method. We identified asignificant cholesterol QTL (Cq5, lod score5.6) on chromosome 9, and a significanttriglyceride QTL (Tgq2, lod score 4.7) onchromosome 8. Compared to our previousstudies using different strains of mice, bothQTLs were found to locate in the samechromosome, but the precise maps of thegenes were different. Identification ofdistinct, but related traits in an identicalchromosomal region will facilitate revealingthe responsible gene. Another QTL study wason genetic control of the pleiotropic effects ofanti-inflamatory peptide action. In thestudy, C3H/HeJ and C57BL/6J-Ay strains ofmice were used. The QTL analysis identifiedtwo modifier genes (Dmyaq1 and Dmyaq2) onchromosome 1 and one modifier gene(Dmyaq3) on chromosome 15 in mice. As thepleiotropic effects of the various combinationsof the multigenic alleles, mice with a series ofdifferent coat colors were obtained in the F2

generation (Fig. 4).

EphA9, the latest member of the Ephtyrosine kinase receptor family

Receptor-type protein tyrosine kinases(RTKs) play pivotal roles in the regulation of

cell growth, differentiation, and directed cellmovement. In particular, RTKs of the Ephfamily act as crucial signaling molecules thatregulate the migratory behavior of neurons,and are implicated in tissue patterning andblood vessel formation. On the basis ofstructural similarities and ligand-bindingspecificites, a total of 14 receptors of the Ephfamily are thus far divided into EphA andEphB subgroups. In a search for proteintyrosine kinase (TK) genes expressed inchicken primordial germ cells, we foundcDNA fragments that could represent a novelRTK gene. We then obtained the full-lengthcDNA by the RACE method. The cDNA wasshown to encode a novel RTK, designated asEphA9, of the Eph family (Fig. 5). The aminoacid sequence of EphA9 was most homologousto human EphA1 (amino acid sequenceidentity between these proteins was 63.0%).The ephA9 transcripts were present in thekidney, lung, testis, and thymus, but not inthe brain or liver of adult chickens. The TKenzymatic activity of EphA9 was demonstratedby expressing the intracellular domain ofEphA9 in bacterial cells.

The observations in this study suggestthat EphA9 plays an important role(s) invarious non-neuronal tissues as anenzymatically active TK. It would be ofinterest to examine the biological role ofEphA9, the latest member of the Eph family.

Fig. 4Comparison of graded yellow coat colors in six F2-Ay

females.Fig. 5Phylogenic tree of the EphA receptors The entire amino acid sequence of EphA9 (cEphA9) iscompared with those of other human (h) EphA receptors.Holizontal lines represent the numbers of differences perresidue. The boostrap values are indicated at thebranches.


Hormonal Regulation of LarvalDiapause of Scarabaeid beetle,Anomala cuprea Hope(Coleoptera:Scarabeidae)

Scarabaeid beetle, Anomala cuprea, has alarval diapause at the stage of third instar.

Injection of 20-hydroxy ecdysone, moultinghormone , has induced the termination of thelarval diapause. Implanting operation of thepupal brain to the diapausing larvae hardlyinduced definite positive effect on thepupatiom. Injection of the extracts of thepupal brain to the diapausing larvae inducedno effect. Application of the distilled water asthe control, however, induced the terminationof the larval diapause. The result suggeststhe direct dose of water to the haemolymphhas a role as a trigger on the hormonalregulation of the larval diapause. Distilledwater was injected to determine the timingof susceptibility of the distilled water on thediapausing larvae. A relatively significanteffect was proven on the diapausing larvaeafter one and two months. Positive clearsusceptible timing of the effect on thediapausing larvae was not shown.

Synchronized neuronal activities ininsect brain.

The representation and discrimination ofthe sensory information in the insect brain isthought to be encoded by synchronizedspiking across an ensemble of neuronsdistributed in neuronal networks.

In our study, by using a multiple electrode,spike activities were simultaneously recordedfrom multiple neurons in the antennal lobe(AL) of the adult male cockroach (P.americana) during antennal stimulationswith test odor. Odor evoked extracellularpotentials and spike activities of AL neuronsrecorded from same multiple-electrode oftenrevealed phase-locking of the oscillations tothese spikes. This suggests synchrony offiring the AL neurons. A cross-correlationanalysis showed correlations among theneurons recorded with odor stimuli (Fig.2).Most pairs of the AL neurons showedcorrelations of conjoint excitation and apositive correlation occurred with no delay. AAmong neurons, those pair of the neuronsshowed functional overlapping and correlatedactivity with different odor stimuli.

P hysiology and Genetic Regulation Department

Fig. 1Metamorphosis from diapausing larvae to pulpal stage in Anomala cuprea1: third instar larva in diapause; 2: prepupa terminating diapause; 3: pupaeafter diapause

Fig.2Cross-correlogram of the spikes of a pair of antennal lobeneuron showing a pattern with a central peak which issuggestive common input to these neurons.


Cryptobiosis in Polypedilumvanderplanki: an internal factortriggering trehalose synthesis

Larvae of P. vanderplanki are completelydehydrated when the pools dry up andundergo anhydrobiosis until the next raincomes. During the dehydration processlarvae accumulate large amounts of trehalosewhich provide effective protection againstdesiccation because of its high capacity forwater-replacement and vitrification. However,what factor(s) trigger explosive synthesis oftrehalose during desiccation remainsunknown in this species.

As the occurrence of drastic trehalosesynthesis coincided with loss of body water,changes of osmolarity in the body werethought to be a cue for trehalose synthesis.Indeed, exposure to high salinity triggeredrapid and efficient accumulation of trehaloseeven without desiccation treatment (Fig.3).As the explosive production occurred mainlyin high concentrations of salt solutions, it isconcluded that an increase of internal ionconcentration triggers trehalose synthesisassociated with cryptobiosis in this species.

Metabolism and Nutrition ofSilkworms

Ornithine, which is one of the major aminoacids in larval hemolymph of the silkworm,Bombyx mori, decreases during the larval -pupal metamorphosis. Activity of ornitine

Fig. 3Effect of various salt solutions on trehalose content of P. vanderplanki larvae. Larvae were incubated for 1 day in thesame osmotic pressure (342 mOs) of each solution. Control shows the content of untreated larvae. Numbers to the rightof SE bar indicated larval activity after 1-day treatment of each solution.

Fig. 4SDS-PAGE analysis of purified ornitin aminotransferasefrom pupal fat body of the silkworm Bombyx mori.lane 1: molecular weight maker, lane 2: purified ornitinaminotransferase


aminotranferase(OAT) was not detectableduring the larval stage, but appeared in thepupal fat body. As OAT is thought to beresponsible for the decrease in hemolymphornitine during the larval pupal metamorphosis,characterization of OAT was carried out.First, OAT was purified by ammonium sulfatefractionation and the combination of severalcolumn chromatographys from pupal fatbody. Specific activity was raised to 800 foldand SDS-PAGE analysis showed homogeneityof the prified enzyme protein (Fig. 4). Then,the following characteristics were obtained:optimum pH was 7.8, Km for ornithine was2.6 mM, Km for a-ketogultarete was 4.2mM.

A novel C-type lectin in mouthpartsof the flesh fly

We have isolated a novel gene, CLEM 36,of the flesh fly Sarcophaga peregrina, whichshows significant homology to the C-typelectin family. CLEM 36 mRNA was transcribedexcessively from the second day after eclosiononly in the tip of mouthparts, and wasexpressed in the C-type lectin-producingtissue (CLPT) located at the entrance of thefood canal and between the labellum andhaustellum. To determine localization ofCLEM 36 protein, we prepared proteins ofthe labellum excluding CLPT, CLPT,hemolymph, and vomited fluid containingsaliva. We found that antibody raisedagainst CLEM 36 recognized a 19 kDa bandin both the CLPT and vomited fluid, but notin the labellum or hemolymph (Fig.5). NoCLEM 36 band was detected by preimmuneserum in any protein samples (data notshown). The molecular mass of 19 kDa waslarger than expected(18.1 kDa), suggestingthat the CLEM 36 may be glycosylated at thepotential N-glycosylation site. Since theCLEM 36 gene was expressed only in CLPTand homolymph did not contain the encodedprotein, CLEM 36 protein detected in thevomited fluid is considered to be deliveredfrom saliva. These results showed that themature form of CLEM 36 was synthesized inthe CLPT, and then secreted into saliva.CLEM 36 may play an important role in the

biological defense against pathogens duringthe food intake of this insect.

Sexually differentiated functions offemale-produced free amino-acidpheromone in the black chafer

Ether extract of the abdominal gland offemale black chafers Holotrichia loochooanaloochooana (Sawada) (“Ryukyu kurokogane”in Japanese: Coleoptera: Scarabaeidae)induced a series of pre-mating behaviors thatincluded short-distance orientation,mounting and abdominal bending towardfemales in the laboratory. GC-MS analysesof the extract revealed that active fractionscontained anthranilic acid (2-aminobenzoicacid) as a major compound (Fig.6). Whenpartitioned to basic, acidic and amphotericfractions, pheromonal activity was observedwith the latter two fractions and anthranilicacid was detected in these fractions by HPLCanalyses. The amount of anthranilic acid inthe female extract was estimated to be ca. 1.3µg/female. Authentic anthranilic acid inducedpre-mating behavior in males of at levelsequal to that elicited by the compound in theextract. In the field, both males and femaleswere attracted with authentic anthranilicacid. Male chafers were observed to apparentlydirectly locate cotton balls impregnated with1 to 10 mg of pheromone. In contrast, femalesnever directly oriented to the treated ballsbut landed 0.2 - 1.5 m away and exposedtheir abdominal glands in a calling posture,

Fig. 5Immunoblot analysis of CLEM 36 proteinProtein samples of the labellum (La), CLPT (CL), vomitedfluid (VF), and hemolymph (HL) were separated by 15%SDS-PAGE and immunoblotted using anti-CLEM 36antiserum. The arrow shows the expected migrationposition of the CLEM 36 protein (molecular mass: 18.1kDa).


which occasionally resulted in aggregation ofboth females and males. This suggested themating aggregation of this species could beprimarily induced by pheromone released byfemales.

A Novel Collagen Scaffold Useful forthe Reconstruction of an Epithelial-Mesenchymal Model

We applied the vitrification technology ofheat-denatured proteins to a traditionalopaque and soft collagen gel prepared byincubating a cold and clear neutral saltsolution containing type-I collagen at 37˚C,and converted it into a rigid material likeglass. Then, we attempted to rehydrate theglass-like material and succeeded inpreparing a novel collagen scaffold possessingvaluable physical properties, that is, a thinand transparent membrane with enhancedgel strength. Further, the framework-embedded thin collagen gel membranescaffold that can be easily reversed by forcepswas prepared by inserting a nylon membranering in the collagen solution prior to thegelation (Fig. 7a). Anchorage-dependent cellscan be cultured on the both surfaces of thescaffold by the manipulation of two-dimensional cultures and consequently it

resulted in the reconstruction of a three-dimensional organoid. An intestinal epithelial-mesenchymal model was reconstructed by co-culturing fibroblasts on the opposite side ofthe monolayered Caco-2 cells on the scaffold(Fig. 7b). These results demonstrate that theframework-embedded thin collagen gelmembrane scaffold can provide an excellentthree-dimensional culture system forreconstructing epithelial-mesenchymalmodels (Fig. 7c).

AFLP analysis reveals a locusresponsible for specific diseaseresistance of chicken

One of the DNA finger printing methods,AFLP (Amplified fragment length poly-morphisms), has the potential to physically“catch” loci influencing a phenotypic traitunder study. It is an efficient method ofgenome scanning when defined sample poolscan be generated for alternative phenotypes(e.g. resistant and susceptible).

One strain of chicken was identifiedwhere blood plasma showed almost noimmune reaction to Mycoplasma sinoviae(MS) antigen, yet many other strains in thesame chicken barn showed strong reaction.We therefore expected that this strain is non-

Fig. 6Mass spectrum of the compoundin fractions that evoked matingbehavior of H. l. loochooanamales.

:Epit helial cells:Thin collagen gelmembrane:Mesechymal cells

Fig.7Thin collagen gel membrane scaffold useful for reconstructing epithelial-mesenchymal models.

sensitive, or has resistance to MS. Weconstructed several experimental families.The F2 generation of one family was derivedfrom the mating of one non-sensitive femaleand one sensitive male of the grandparentalgeneration. Six females in the F2 did notshow any reaction and one male and sevenfemales showed strong reaction. We collectedDNA samples of these chickens and submittedthem to AFLP analysis.

All six non-sensitive chickens and thenon-sensitive parent (P1) did not have aspecific AFLP band. Five of the eightsensitive chickens had the specific AFLPband (green arrow) that the sensitive grandparent (P2) had. Cloning of the band revealedthat a polymorphic site in the sequencerelating to the band were visible(C) orinvisible(T). The sensitive grand parent hadheterozygous (T/C) and the sequence havingC was visible. The three sensitive F2individuals which had an invisible band (T/T)might have inherited the correspondinggenomic locus from the sensitive P2. Thecausative locus is expected to reside veryclose to the gene that was identified by AFLPand subsequently sequenced.

Tumor necrosis factor-α in thecentral nervous system of ChineseMeishan pigs

It has been shown that tumor necrosisfactor-α (TNF-α ), a proinflammatorycytokine, is possibly involved in the centralnervous system (CNS) functions and stress-

induced sickness behavior in pigs. However,the physiological significance and productionof TNF-α in the CNS are not well establishedin this animal. In the study with ChineseMeishan pigs, the abundance of TNF-αmessenger RNA and immunoreactive TNF-αwere observed in brain tissues (hypothalamus,amygdala, or hippocampus) and the pituitary.The concentrations of TNF-α were increased(P < 0.05) in these brain tissues and thepituitary by intramuscular injection oflipopolysaccharide (LPS) (J. Anim. Sci.81:1274-80, 2003; Fig. 9). Plasma concentrationsof TNF-α and cortisol were also increased byintravenous injection of LPS, which causedhyperthermia, inhibition of food intake andincrease in standing rate. These resultssuggest that TNF-α is present in the CNS,and plays some roles in its biologicalregulation in pigs.

Histochemical study of theaccessory olfactory bulb in goats

Pheromone is a chemical signal thatmediates species-specific social interactionbetween individuals such as mating,parenting and aggression. Pheromonalinformation is processed in the vomeronasalsystem, and the accessory olfactory bulb(AOB) is the primary center within thissystem. In the present study, we histochemically


Fig. 8AFLP analysis of chickens which blood serum beingsensitive or non sensitive to Mycoplasma sinoviae antigen

Fig. 9Effects of LPS on TNF-α concentrations in the braintissues and the pituitary. All values are expressed as themean ± S.E.M. (five pigs per treatment). Open columnsshow the values in the control. Different superscript lettersindicate significant differences (P < 0.05 or lower). Hyp =Hypothalamus, Amg = Amygdala, Hip = Hippocampus,and Pit = Pituitary.


examined the AOB in goats, and itsmorphological characteristics were comparedwith those described in rats.

Sections containing the goat AOB werestained with nissl, and appearance, size andthe number of cells were observed. Somesections were processed for lectin histochemistryto examine chemical components in the AOB.The goat AOB was located on the caudal anddorso-medial aspect of the olfactory bulb, andwas comparable to the rat AOB in terms ofthe size and the number of output neurons,suggesting that the goat AOB is able toreceive and process as much pheromonal

information as the rat AOB. On the otherhand, the laminar structure of the AOBmarkedly differed between two species.While the AOB is composed of five layers inrats, it was divided into only three layers ingoats (Fig. 10). Further, the patterns oflectin binding in the goat AOB contrasted tothose reported in rats, indicating thedifferent chemical composition of respectivelayers. These results indicate the diversityin organization of the AOB in mammals,which may relate to particular ecologicaldemands for organization and function of theAOB in each species.

Fig. 10Schematic drawings of the structure of the AOB.

The research activities of our departmentare mainly focused on molecular andconventional genetics of insects including thesilkworm, biochemical analyses of insect-plant interaction, characterization andbreeding of natural enemies, and basic andapplied studies on insect-associated microbes,including pathogens and symbiotes.

Molecular and evolutional analysesof insects

Genetic diversityA possible intact element of Bombyx

MITE-like transposon Organdy wasidentified (Fig.1). This element encodes atransposase-like amino acid sequence similarto that of Pong, which is an active MITE inrice, and is about 3.0 kb long (Organdy3.0),although most Organdy are about 550 bp insize (Organdy0.5). As a different size class ofOrgandy, we identified a 1.4 kb long copy,which is a specific Organdy0.5 in which threeindependent insertions occurred. One ofthese three inserts is a Bm1.2 retroposon,which is most abundant class of repetitiveelements in Bombyx genome.

Silkworms have two xanthine dehydrogenase

I nsect Genetics and Evolution Department

(XDH) genes, BmXDH1 and BmXDH2, andtwo XDH isozymes, XDHalpha and XDHbeta.It has been shown that BmXDH1 encodesXDHalpha but the relationship betweenBmXDH2 and XDHbeta has remainedunclear. It was shown that BmXDH2 encodesXDHbeta by two-dimensional Westernblotting in which the first dimension wasNative PAGE paralleled with XDHzymogram and the second one was SDS-PAGE. A silkworm ABC transporter gene,Bmwh3, was found to be on the w-3 locus,which is responsible for silkworm translucentlarval skin mutation, suggesting that Bmwh3works as a uric acid transporter.

Molecular genetic structure was comparedamong populations of the oriental fruit fly,Bactrocera dorsalis, one of the most destructivefruit pests of tropical Asia and the Pacificregion. The results showed that the Hawaiipopulation was genetically homogeneous anddid not share any common mitochondrialhaplotypes with Asian populations. On theother hand, haplotypic compositions weresimilar among Asian countries and recentlycollected Japanese populations. These resultssuggest that this species has expandedgeographic distribution in several differentways. Similar analyses revealed that aconsiderable genetic differentiation hasoccurred among geographic populations of aheteropteran predatory insect Macrogerris.

Some kind of stimuli make living things

stronger. We tested this relationship withseveral pest insects and insecticides. Then,the brown planthopper, serious pest for riceplant, was revealed to have resistance tosome kind of insecticides when it was treatedwith a subleathal dose of chemicals in onegeneration, not caused by the selection.

Planthopper EST analysis and cDNAmicroarray

A number of cDNA clones made fromvarious tissues of a rice pest, the brownplanthopper Nilaparvata lugens , weresequenced. Thirty thousand ESTs in totalwere obtained and tissue specific genes wereselected from the ESTs. Using cDNA microarrayof the silkworm, we have studied the effect ofagricultural chemicals on the silkworm andsilkworm cell lines. The microarrays werealso used for the analyses of inducedexpression by microorganism infection.

Insect cellulase genesVarieties and phylogenetic origin of insect

cellulase genes were surveyed. Cellulasesbelonged to glycoside-hydrolase family 9(GH9) were primarily found in hemimetabolousinsects like termites, cockroaches andgrasshoppers, while GH45 cellulase geneswere mainly distributed in Coleopteraninsects. GH5 were found only in longicornbeetles. The results showed diversity anddifferent phylogenetic origins of insectcellulase genes. For industrial application,the improvement of the cellulase genes wastried. Four termite endogenous cellulasecDNAs encoding enzymes placed in glycoside-hydrolase family 9 from different hosts werehomogonously recombined by randomdigestion by DNAse I and following primer-less PCR amplification. Some of the obtainedclones produced cellulases, which showedmuch higher activity than that of parentclones.

Genetics and evaluation of silkwormstocks

The morphology of the intersex silkwormmoths from the Isx-2 strain was investigated.


Organdy3.0

Organdy0.5

Organdy1.4

Terminal Inverted Repeat

Fig.1Three classes of transposon Organdy: Organdy3.0 is anelement encoding a possible transposase in ORF2.Organdy0.5 is an element derived from Organdy3.0 by aninternal deletion covering about 2.4 kb. Organdy1.4 is aspecific Organdy0.5 containing three independentinsertions.


Although external sexual organs of theintersex moths were a mixture of both sexes,internal sexual organs of the intersex mothsresembled those of female moths in manyindividuals. As a trial to identify intersexgene, we compared the genome of Isx-2 strainto that of Daizo race by a genomic subtractionmethod. As a result, we obtained one candidateclone that was located on W chromosome.The nucleotide sequence of this clone showedno homology to known sequences. The copynumber of this clone in the genome wasanalyzed by quantitative PCR and Southernhybridization. These results showed that thecopy number of Isx-2 was approximately one-third in comparison to that of the Daizo race.

In addition, a new mutation expressingbrown egg color was named “maternal brownegg of Shimizu” (b-2s), which was located atposition 29.1 on the sixth linkage group.Mapping of homeotic mutant genes wasexamined by three-point tests. Threehomeotic mutant genes belonging to E-pseudoalleles (EMu, ENc and EAl) and Ncwere arranged in order from the proximalside of the sixth linkage group.

A total of 248 silkworm breeding stockswere maintained and characterized. Oncocoon character, 220 items were collected forthe construction of a database.

Insect-plant interactions Based on our findings that papain, a

cysteine protease found in papaya latex,plays a crucial role in the defense of papayatrees against herbivorous lepidopteranlarvae, and that very high protease activityexists in the latex of Ficus virgata, wesuspected that cysteine proteases in latex offig species also have defensive roles similarto that of papain. To investigate thispossibility, we performed E-64-paintingbioassays on F. virgata leaves using 1stinstar Eri silkworms. When control F.virgata leaves were fed to the larvae, all thelarvae died within 4 days (some died within 6hours, and 85% died within 2 days) withoutany increase in mass (Fig. 2A). However,larvae that were fed E-64-painted leaves

(Fig. 2B) or leaves that were cut into narrowstrips and then washed with water toeliminate latex (Fig. 2C) grew far better. Inboth cases, larval mass increased rapidly andmortalities were 10-30% by Day 4. Theseresults indicate that cysteine protease playsa defensive role in F. virgata, and imply ageneral defensive role of cysteine proteasesfound in various plants groups.

The leaf beetle, Ophraella communainfests almost exclusively Ambrosiaartemisiifolia in the field. Four compoundsidentified as the feeding stimulants for O.communa have been isolated from A.artemisiifolia. Triterpenoid derivatives (α-amyrin acetate or β-amyrin acetate) andcaffeic acid derivatives (3, 5-dicaffoylquinicacid or 5-caffeoylquinic acid) showed feedingstimulant activity when mixed together.

Enzymatic activities in the saliva ofNephotettix cincticeps were examined. Thesalivary gland extracts and fed-sucrosesolution hydrolysed p-nitrophenyl β-D-galactoside (NPβGal) better than NPβGlu.The β-galactosidase activity was detected inthe III cells of the principal salivary glandsby X-Gal.

Fig. 2Defensive effects of cysteine proteases in latex of Ficusvirgata leaves on the Eri silkworm, and effects of E-64(cysteine protease-specific inhibitor) painting and latexelimination. Newly hatched 1st instar larvae were fedcontrol papaya leaf, papaya leaf painted with E-64 (0.36mg/g fresh leaf), or washed papaya leaf strips, and rearedat 25C for 4 days.


Natural enemiesPhytoseiid mite, Amblyseius womersleyi,

is one of promising natural enemies againstspider mites on various crops. It is knownthat these mites are attracted by volatilesmediated from plants infested with spidermites. However, there seems to be anintraspecific difference on the olfactoryresponse to those volatiles. Then, first weanalyzed if the olfactory response is relatedto the other traits in A. womersleyi. Althoughthe olfactory response to the volatiles was notrelated to the predation rate, number of eggslaid, and developmental rate, it wasnegatively related to arrestment behavior.Second, we tested the olfactory responses ofvarious populations of A. womersleyi to thosevolatiles in a Y-shaped olfactometer in orderto obtain genetically different predatorstrains. After repeating selections onolfactory response three times, the fast andslow response groups were successfullydivided.

Pirate bug Orius spp. are known as aneffective predator of thrips, and severalspecies are already on the market. To tracethe crosses between commercial and nativestrains in the field and predict the possibleresults, genetic markers are useful. To detectgenetic diversity within and among populationsof Orius strigicollis, eight microsatellitemarkers have been developed. Using 6 of the

8 markers, we compared genetic diversity offive wild populations and two commerciallyavailable strains. The commercially availablestrains of O. strigicollis were fixed on mostloci and far different from wild populations inthe genetic diversity. In wild populations inO. strigicollis, we found a positive relationshipbetween the geographical distance andgenetic distance.

To investigate whether if the release ofcommercial strains of Orius species affectsdiapause of native strains, we conducted thecross experiments between a nativeFukushima strain (diapause) and twocommercial strains of Orius strigicollis. Theobserved incidence of diapause in thecommercial strains was lower than that ofFukushima strain. The observed incidence ofdiapause in F1 between the commercialstrain and Fukushima strain was intermediatebeing between them.

Symbiotes

Endosymbiotes of arthropodsIntracellular bacteria that cause

reproductive alteration on insect and mitehosts were cultivated in the cell lines andwere injected into new hosts. Theperformance of the symbiotic bacteria,Wolbachia, was examined in the new host.CFB (Cytophaga-Flavobacterium-Bacteroides)bacteria (Fig. 3) were detected from severalmites and one of them caused cytoplasmicincompatibility in its host mite. The effects ofthe propagation of CFB bacteria in their hostcells were studied using silkworm cDNAmicroarray.

DicistrovirusesThe translation initiation with elongator

tRNAs in dicistroviruses was investigated.The intergenic internal ribosome entry site(IRES) of dicistroviruses determines thetranslation initiation site by virtue of theirown tertiary structure formation in theabsence of initiator Met-tRNA and AUGtriplets. Although previous deletion analysisshowed that several nucleotides in the viral

Fig.3Phylogenetic position of CFB bacteria: CFB bacteria are intracelluarsymbiotes of a new group. They alter host arthropod reproduction as well asWolbachia.


coding sequence were necessary for theIRES-mediated initiation, recent studiesshowed that the viral coding region was notan absolute requirement for the initiation.This indicates that translation initiation withelongator tRNAs is possible in the case of theIRES-mediated protein synthesis.

Bacteria and phytoplasmaA part of the genomic region of Enterobacter

cloacae, which is related to its colonization ininsect gut, was cloned and sequenced.Homology search of the sequence showedthat unknown genes were coded. A newphytoplasma-vector, leafhopper Erythroneuramori, was identified. Phytoplasma-infectiveleafhoppers caused mulberry dwarf symptomsin approximately 25% of mulberry seedlings.

Insect pathogensWe studied several insect pathogens,

including viruses, bacteria and fungi, andplant pathogenic fungi to clarify biologicalfunction, and applied our findings to thebiological control of pests and other purposes.

Anomala cuptea entomopoxvirus (AcEPV)spindle, paracrystalin proteinaceous bodies,enhance nucleopolyhedrovirus(NPV) infection.

This ability of the spindle did not decreaseafter the heat treatment or dipping togermicidal agent. These properties indicatedthat these bodies are potentially stable co-agents of microbial insecticides.

Each hybrid derived by crossing amongseven silkworm races showing resistance toCry1Ab toxin of Bacillus thuringiensis andone Cry1Ab toxin susceptible race wassusceptible to Cry1Ab toxin. These resultsshowed the Cry1Ab resistant genes of sevenresistant races to be recessive.

The difference in susceptibility againstBeauveria brongniartii between resistant andsusceptible silkworm races was related to thefungal multiplication within the haemocoel.In addition, the resistance of one race wasconsidered to have some relationship withthe phagocytic ability of the larval haemocytes.

Myrothecium verrucaria from a diseasedplant produced toxic substances. The substancesinduced necrosis on the 108 species of plantsand showed toxicity to eight species of yeastand 17 species of fungi, but did not showedtoxicity to 15 bacterial species and twospecies of Myrothecium. These results showthat the substances have potentiality asherbicides or antifungal agents.

Development of biosensors andrelated materials focusing on theimmolization of chemicalrecognition molecules

In order to immobilize liposomecontaining sugar receptor molecules fromlabella of fresh fly to solid substrate,polyhydroxyethylacrylamide as a hydrophiliclayer, and polydimethyaminopropyl-acrylamide as a cationic layer, wereimmobilized onto the surface of quartz glassby the graft polymerization technique. Thegraft polymerization was confirmed by thechange of surface contact angle and surface

zeta potential.We prepared conjugates (Lac-CY-SF) of

silk fibroin and lactose bearing the galactoseresidue, which is recognized by hepatocytes,and studied recognition interactions of Lac-CY-SF with galactose-binding lectins (Ricinuscommunis agglutinin, RCA120). We observedthe RCA120 lectin-induced aggregation of Lac-CY-SF in an aqueous solution and thefixation of fluorescein isothiocyanate-labeledRCA120 (FITC- RCA120) onto the surface ofLac-CY-SF films (Fig.1). These resultsindicated that the galactose residue of Lac-CY-SF was valid as recognition ligands in the

I nsect Biomaterials and Technology Department

interactions with lectins and hepatocytes.N-channel MOSFETs were developed by

thermally impurity diffusion of phosphateinto a P-type silicon wafer. Micro-pit arrayelectrodes were also fabricated on a siliconwafer and we confirmed whether lipidmembranes were made on micro-pit arrays ornot by observation by optical microscopy andthe measurement of membrane capacitance.From these experimental results, it wassuggested that lipid membranes were madeon micro-pit array electrodes.

Development of measurement andrecording methods for obtaining bio-physical information on insects

The average density of 4 varieties ofsilkworms was calculated from the measuredvalue by using an acoustic volumeter. Bothsexes of the density of silkworm larvae were1.08 to 1.09. The density of pupae was 1.06to 1.07 (male) and 1.07 to 1.08 (female).Between the larva of the same variety, andthe sex of pupa, there was no difference indensity (official approval of 1% of rates ofdanger). The density between 4 varietiescompared for every sex was not differentbetween female larvae (1% of rates ofdanger), between male larvae, and betweenpupa of a female and a male, there was adifference at 5% of rates of danger. Thesilkworm aligning method and silkwormsautomatic supply equipment, which areapplied to the silkworm hemolymphcollection system and are needed for

establishment of the production system of theuseful substance using silkworms, weredeveloped.

In order to measure the acceleration of afreely flying hawk moth, Agrius convolvuli, asmall and light circuit was developed using amicro-accelerometer, ADXL202. We couldmeasure the acceleration of moth’s thoraxwhile it warmed up and hovered.

MOSFETs, which were developed bythermally impurity diffusion into a siliconwafer, and micro-probes, which were madeby and reactive ion etching, were fabricatedin order to record insect bio-potentials. Wecould record the muscle potentials fromsilkworm moth’s dorsal longitudinal musclesand its signal-to-noise ratio was not goodbecause of very small recording sites.

Development of new sericologicaltechnology and new materials usingsericulture related products

Development of functional materials suchas fine chemical using chitin and fibroin etc.

For the purpose of the resource recovery ofthe dead silkworm larvae disposed as wastematerial from an insect factory, the wasteselection device which sorted out cuticles,silkglands and residue powders wasimproved. As a result of the improvement,each performance was optimized by unitizingeach part of hopper, crush, sieve, recovery,and the selection efficiency was improved.

The chitin was purified from the waste


Fig. 1Fluorescence images of cast films of Lac-CY-SF and SF after immersion into FITC- RCA120 solution.


cuticles, and the spherical microsphere wasobtained. It was found that the extractionresidue in the sterilized dead body hadinhibitory activity for the α-glucosidase. Inaddition, several unsaturated fatty acidswere able to be extracted from the wastecuticles under the condition approximate toroom temperature, when the supercriticalcarbon dioxide was used.

The biodegradation behaviors of silk filmsby the action of enzymes indicated that theextent of weight loss depended on the type ofenzymes. Protease was more aggressive thana-chymotrypsin or collagenase, and alwaysresulted in higher values of weight loss. Theplot of “I1264/I1231” as a function of the weightloss of silk films suggested the degree ofcrystallinity of biodegraded films increased toan extent directly related to the weight loss,that is, the higher the weight loss the higherthe degree of crystallinity. The breaking loadand elongation at break decreased. Proteasewas more aggressive than collagenase or a-chymotrypsin, resulting in a loss of strengthand elongation of 33% and 45%, respectively,after 17 days of incubation.

Characterization silk fibroin andother biopolymers from chemical andphysical respect and development oftheir applications to wound coveringmaterials and others

In order to attempt the utilization of silkprotein for wound-covering materials, agrowth-promoting effect of silk fibroin L-

chain for the fibroblast was examined. Allthe fractions were obtained by enzymatichydrolysis and by the separation of HPLC,the growth-promoting activity. The aminoacid sequence was examined on two peptidefractions of which the activity was high. Thebiologically active peptide consisted of 10 and8 amino acid residues, respectively. Wesucceeded in making the synthetic fiber inwhich surface the fibroin fine powder withtheir particle size 5µ or less.

As this result, the tensile strength of thishybrid fiber did not lower, and it wasexcellent in dyeing ability and softness.

β-sheet structure in silk fibroin spongewas observed by FTIR measurement and theratio was different by the sponge formingconditions. X-ray diffraction analysis alsoindicated the existence of crystal structure insilk fibroin sponge and the difference of thecrystal morphology by the sponge formingconditions, especially the kind of solvent, wasobserved. Fibroblasts were able to proliferatewell in silk fibrin sponge and we found thegrowth rate depended on the compressivemodules of the silk fibroin sponge in thepreliminary study.

Spider silk and silkworm silk are fibrousproteins, which have become the subject ofextensive study as model polymer for high-performance fibers. In order to analyze themolecular orientation and secondarystructure of these fibrous proteins,attenuated total reflectance (ATR) infraredspectroscopy was applied to investigate themolecular structure of natural silk from thespider Araneus ventricosus and the silkwormBombyx mori. Polarized ATR measurementshowed that most of the amide group inprotein were oriented in the silk fibers.Secondary structures of silk protein wereestimated by Fourier self-deconvolution andcurve-fitting procedures. These datasuggested that the β-sheet structure ispredominantly in spider silk and thecomponent of aggregated strand was rich insilkworm silk. It may be expected that thedifferences of secondary structure betweenspider silk and silkworm silk have an

Fig 2The microspheres prepared from the chitin of wastecuticles derivation.


influence upon the physicochemicalproperties.

We generated transgenic silkworm whichproduces recombinant fibroin L-chain proteinfused to enhanced green fluorescent protein(L-EGFP) in the silk gland by using the germline transformation technique (Fig.3). Wemade a cast film using aqueous solution offibroin extracted from the L-EGFPtransgenic silkworm. We found the surfaceproperties of the film by contact angleagainst water, surface zeta-potential, and celladhesiveness measurements changedcompared with a cast film prepared from wildtype silkworm. Improvement of celladhesiveness on the cast film of L-EGFP silkfibroin was observed.

Fig. 3L-EGFP transgenic silkworm.5th instar larva was dissected from the back andobserved under bright (A) and fluorescent (B) microscope.Silk fibroin in the silk gland shows fluorescence of L-EGFP fusion protein (B).

Analysis of characteristics of silk-worm races maintained as geneticresource

We analyzed the characteristics of Japanese

improved silkworm races by comparing themwith those of Japanese native races (Table 1).

The ratio of the actual cocoon volume tothe theoretical cocoon volume of Japanese

I nsect Biotechnology and Sericology Department

Quantitative characteristics

Japanese native race Japanese improved race

mean coefficient of

variatium

maximum minimum

X1 Number of eggs laid

X2 Last inster days (days)

X3 Total inster days (days)

X4 Susceptibilty (ppm)

X5 Cocoon length (cm)

X6 Cocoon width (cm)

X7 Cocoon weight (g)

X8 Ratio of length and width (%)

X9 Actual cocoon volume (cm3)

X10 Theoretical cocoon volume (cm3)

X11 Volume ratio (X9/X10)

X12 Raw silk percentag (%)

X13 Reelabilty percentag (%)

X14 Length of cocoon filament (m)

X15 Weight of cocoonfilament (cg)

X16 Size of cocoon filament (d)

X17 Neatness (points)

X18 degumming loss percentage (%)

X19 Lousiness (points)

575

5.2

21.6

4954

3.34

1.63

1.47

48.5

5.49

5.83

0.89

10.10

80.5

531.9

15.00

2.51

78.2

23.1

0.577

8.8(%)

9.2(%)

4.9(%)

37.6(%)

6.3(%)

5.5(%)

8.8(%)

8.2(%)

11.5(%)

13.2(%)

9.0(%)

12.7(%)

15.0(%)

12.7(%)

17.9(%)

12.7(%)

13.0(%)

7.7(%)

79.7(%)

675

7

23.9

8920

3.80

1.89

1.76

62.6

6.95

7.33

1.10

11.85

97.0

669

19.4

3.15

98.0

28.2

1.93

417

4.3

19.2

1950

2.79

1.41

1.17

40.0

4.15

3.85

0.70

4.23

37.0

374

5.6

1.30

57.5

19.5

0

mean coefficient of

variatium

maximum minimum

585

6.6

22.7

1765

3.73

1.78

1.60

47.9

6.98

7.99

0.85

14.03

82.3

777.2

22.81

2.63

85.4

24.9

2.05

7.9(%)

14.5(%)

5.9(%)

38.9(%)

6.8(%)

11.4(%)

12.2(%)

9.0(%)

17.7(%)

31.0(%)

14.5(%)

19.3(%)

15.9(%)

22.5(%)

26.2(%)

10.1(%)

16.8(%)

8.1(%)

42.4(%)

718

9

25.9

3280

4.42

2.66

2.08

62.6

10.00

18.57

1.10

20.09

96.0

1200

35.8

3.21

99.0

30.4

3.98

482

5

19.9

320

2.93

1.57

1.22

42.2

4.80

519

0.50

6.95

25.0

478

11.3

1.97

50.0

20.6

0.69

Table 1. Quantitative characteristics of Japanese silkworm races


improved races is 0.85. From this result, thecharacteristics of the cocoon shape of Japaneseimproved races can be determined. Namely,they have a longer major axis than that oftrue ellipse or have a concave part in thecenter of the cocoon body.

Japanese improved races are better thanJapanese native races in the quantitativecharacteristics such as cocoon length, cocoonwidth, cocoon weight, cocoon volume, raw silkpercentage, length of cocoon filament andneatness. On the other hand, the lousiness ofJapanese improved races is worse than thatof the native races. This is because the cocoonshell weight of Japanese improved races islarger than that of the native races. Thus, itis found that there is a correlation betweenthe lousiness and the weight of the cocoonshell. The coefficient variations of susceptibilityto the fungicide(Maneb), the theoreticalvolume and lousiness are 30% or more.

There are some fluffy cocoon races in theJapanese improved races. We found that thesusceptibility to the fungicide (Maneb) andthe development of lousiness were geneticallystable and were not subjected to the rearingconditions. The susceptibility to the fungicideand the development of lousiness areremarkably influenced by the silkworm

varieties.

Improvement and application ofmapping systems in the silkworm,Bombyx mori

The molecular linkage map has beenimproved and finally 280 EST-cDNA cloneshave been mapped on 28 linkage groups.

The molecular markers on this linkagemap have been applied to determine whetherthese markers were effective to makehomozygote of Nid-1 (dominant resistantgene against DNV-I) or not. The most closelylinked clone, m274, revealed to give effectiveRFLP between N150 (+/+) and N203 (Nid-1).Direct screening of homozygote (H) wascarried out using m274’ RFLP as DNAmarker after mating heterozygous Nid-1/+(Fig.1). The segregants crossing between theselected individuals were identified andRFLP of EST-cDNA clone could selecthomozygote of Nid-1 by DNA-tagging (Fig. 2).These selected individuals were identified ashomozygotes of Nid-1 by crossing withsensitive (+/+) individuals. It was shown thatDNA-tagged selection was very much easierthan the methods to make homozygote byvirus infection.

A) Male B) Female

HHHHHH

Fig. 1Direct screening of homozygote (H)Both male and female were selected by homozygote (H) of m274.

Fig. 2Homozygote of Nid-1 by DNA-tagging

Construction of new method in insectcell culture and characterization ofimmune - responsiveness ofLepidopteran cell line.

As an experimental tool, cell line isabsolutely necessary for molecular biologyscience. However, it takes still more severalmonths to establish cell lines with a traditionalcell culture method. Cell lines should beestablished simply and immediately wheneverthey are required. We constructed anexpression vector that integrated SV40-TcDNA of oncogene to induce a hyperplasia ofcells. It was confirmed by Western blotanalysis that this SV40-T cDNA was insertedin genome of Bombyx mori cells. By usingnew culture method that we discovered lastyear, we succeeded in making a Bombyx moricell line that is cultivable by serum freemedium, and we constructed an in vitrolarge-scale production system. Also, we madea cell line that has a responsibility to insecthormone (Fig.3). This can be used as acertification system of biochemical products.In an insect immune response, we clarified

that Epoxy hydrolase gene of Drosophilacould induce a gene transcription by medicaltreatment medicine of hyperlipidemia.

Construction of expression systemfor useful materials by transgenicinsects using modifiedtransposones

Technology related to an application oftransgenic silkworm has been progressedextensively. The relationship between thesize of inserted gene into the plasmid vectorand an efficiency to construct transgenicsilkworms was investigated. The resultssuggest that the efficiency was decreased,accompanied by an increase of insertion genesize (Fig.4). However, the decrease was ratherslight, suggesting that the system usingtransposon piggyBac as a vector for thetransgenesis in the silkworm possesses thecapacity to insert the genes with the sizes of20 to 30 kb into the chromosomal DNA. Thepromoters for the transgenic silkworms havebeen characterized and used for the regulationof the introduced gene. The promoters withspecificity of the expression in posterior silkgland, middle silk gland and heat-treatedtissue were analyzed.

Concerning the virus system, an efficientoral infection method was developed and aninactivation method for the viruses in thehemolymph collected from larvae wereinvestigated. Furthermore, genes controllingcircadian rhythm in Drosophila were studied.The fly genes were over-expressed in the


Fig. 3Filamentous cytoplasmic extensions

Fig. 4Relatioship between the insert size of vector and theefficiency of construction in transgenic silkworm.


timeless neuron and the candidate genesregulating the locomotor rhythm wereselected.

Inhibition of microsporidians byadministrating fumagillin andbenomyl

The efficacy of inhibition of microsporidiansfrom Bombyx mori and Antheraea pernyisilkworms by administrating fumagillin andbenomyl was investigated using differentvariation microsporidians. It was shown thatthe fumagillin was significantly effective atall stages of the prolification of Nosemabombycis, while benomyl was effective onlyat the initial stage of the infection of N.bombycis. Additionally, fumagillin effectivelyinhibited the prolification of variousmicrosporidians. However, benomyl waseffective in eliminating N. bombycis, NIS-402, NIS-408, NIS-520, NIS-611, Nosema sp.NIS-SAKUSAN, Pleistophora sp. NIS-M27and Thelohania sp. NIS-M32. Benomyl hasbeen found to be ineffective in eliminatinginfection by Vairimorpha sp. NIS-M12 andVairimorpha sp. AP.

Promotion of nucleopolyhedrovirusinfection in larvae of the silkworm,Bombyx mori, by chitin synthesisinhibitors

We demonstrated that chitin synthesisinhibitors promote nucleopolyhedrovirus(BmNPV) infection in the silkworm whenthey are fed to the silkworm prior to aninoculation of the virus.

Chitin synthesis inhibitors (Table 2) wereincorporated into the larval diet and fed tothe silkworm larvae for 24 h. Thereafter, thelarvae were inoculated with BmNPVinclusion bodies perorally. The median lethaldose (LD50) of the BmNPV inclusion body inthe silkworm that had ingested thosechemicals was drastically decreased (Table2).

Insect viruses such as NPVs are oftenused as a microbial pesticide that protectsagricultural crops from insect pests. Thus,chitin synthesis inhibitors that promote viral

infection will serve as synergists of themicrobial pesticide.

Breeding of silkworm races havingspecial features

We selected two kinds of silkworm breedssecreting high concentration of sericin withflavonoid or carotenoid pigment for a stableappearance of these colors. The yield of theyellow cocoon “PNY×PCY” and green cocoon“PNG×PCG” strain was also improved to apractical level. The green cocoon showed thehigh anti-oxidation ability while the yellowcocoon had the same ability as ordinarywhite cocoon. Moreover, the soft and lustrousnecktie was woven using the super-thinfilament race “Hakugin”. This necktie wasevaluated as a high quality luxurious good by200 anonymous persons, and has beenreleased as a new silk product at thecommercial level.

The quality of the cocoon filament of the

Concentration of the

inhibitor in a diet

ingested by the

silkworm (ppm)*

LD50

(Inclusion

bodies/larva)

No (control)

Captan

Flufenoxuron

Nikkomycin Z

Polyoxin

2.0x106

2.5x101

1.1x100

0.8x100

2.2x101

—

800

100

10

500

Table 2. Effect of chitin synthesis inhibitors onBmNPV infection in 4th-stadium Bombyxmori larvae

*Concentrations in which the maximum promoting effecton the viral infection is to be obtained.

Fig. 5Effcects of nitrogen fertilizer to size and length of cocoon filament of“Hakugin”

silkworm race “Hakugin” fed by mulberryleaves of different amounts of fertilizer(nitrogen base) was investigated. As a result,the size and length of cocoon filament wassmaller and shorter, respectively, in the plotof the mulberry leaves of poor nitrogen(Fig.5).

Basic study on silkworm racebreeding

Sensory response of the deterrent cell inmedial styloconic sensilla to salicin wasmeasured in the two type of segregantsderived from a backcross population ofpolyphagous strain, Sawa-J, with normalstrain, Daiankyo, by an electrophysiologicaltechnique. The result indicated that the foodpreference gene (pph) is related to therecognition of bitter substances like salicin(Fig.6). The high concentration sericinproducing race, “Sericin-hope” was certifiedas a suitable race for recombinant proteinproduction by the baculovirus systembecause of a high amount of hemolymph andits effective collection. The filament size ofcocoon was affected by eight chromosomesbesides the RFLP linkage group 9. The sixkinds of cDNA clone regions located on thesame linkage group were directly detected ona chromosome by FISH analysis indicatingthe coincidence between the genetical andcytological maps of B. mori.

The chemical modification of silksericin and the development ofcharacteristic silk products

LiCl/DMSO is a good solvent for silksericin. It is quite useful for modification ofsericin hydroxyl groups under nonaqueouscondition. Therefor, some sericin derivativeswere synthesized using the new modificationtechnique. As a result, it is demonstratedthat the chemical modification of theabundant hydroxyl groups in LiCl/DMSO isquite useful for adding new properties tosericin.

Denim fabrics were woven by the net rawsilk, and it was confirmed that the physicalproperties of strength, softness and warmthof fabric were more than the ordinary cottondenim fabrics.

The high bulk silk reeling machine wasreformed by attaching a cocoon feedingmechanism, the size control mechanism andbulkiness controlling mechanism.

The formation of an artificial skein, bloodtube, and tendon by using cocoon filamentswas examined(Fig.7). The artificial skein wasformed by spreading cocoon filaments as aflat state and pressing it. The blood tube andtendon were made by the combination ofwinding and knitting of cocoon filaments.

Potentialities of natural dyestuffs asantifeedants against varied carpetbeetle, Anthrenus verbasci

Woolen fabrics dyed with different naturaldyestuffs were studied on their feeding by


Fig. 6Examples of records in Sawa-J lem, Daiankyo, and BF1,in sensory response of the deterrent cell in the medialstyloconic sensilla to 25mM Salicin + 20mM NaCl.

Fig. 7Artificial tendon model made of cocoon filaments.


the larvae of the Anthrenus verbasci L.. Thefollowing results were obtained.

Most of the natural dyestuffs studied,except for turmeric and lithospermi radixsignificantly suppressed the attack of A.verbasci. The feeding damage by the insectwas not related to the extent of color depth orshade of the dyed fabric. Water-solublesubstances having an absorption peak ataround 280nm, commonly present in thenatural dyestuffs except turmeric, were

believed to be related to antifeeding action bythe harmful insect. The polyphenols, tannicacid and catechin, were closely related to theantifeeding effect of the natural dyestuffs:the more their amount in the dyed fabric, thehigher the antifeedanting effect. Lithospermiradix was very well fed by A. verbasci,however, fabric wool dyed by dialysislithospermi radix strongly suppressed attackof the larvae (Fig. 8).

Fig. 8Result of feeding test of wool fabrics dyed by dialysis lithospermi radix kept with 10 A. verbasci larvae after 4 weeks(Individual feeding tests).


The Plant Science Division consists of thefollowing five departments, and is activelyengaged in multidisciplinary researches.

Scientists at the Molecular GeneticsDepartment are engaged in studies of thestructure and function of plant genomes,genes, their products, and their networks,which are involved in various agriculturallyimportant traits, and also the mechanismsregulating expression of these genes.

Those at the Biochemistry Departmentare involved in researches on the three-dimensional structure of proteins, andstructure-function relationships of proteinsinvolved in response to hormones and otherbiotic signals in plant cells.

The Plant Physiology Department isengaged in analyses of molecular mechanismsof important physiological processes in plantsincluding photosynthesis, morphogenesis

such as leaf and floral organ development,the symbiotic process of nitrogen fixation,mechanisms of defense against plant pathogens,and tolerance against environmental stresses.

The Plant Biotechnology Department isdeveloping new techniques for next-generation plant biotechnology and alsoproducing novel transgenic crops withsuperior traits which conventional breedingtechniques can not produce.

The Institute of Radiation Breeding isdeveloping new technologies utilizingradiation for plant breeding, the creation ofplant genetic resources through mutationinduction, and the elucidation of geneexpression mechanisms in plant mutants.

Major topics in each department aredescribed below.

Plant Science Division

The research activities of this departmentare mainly focused on the analysis of thestructure and function of rice genome, genesand their products, the development of toolsand resources for functional analysis of ricegenes, and the mechanisms regulating geneexpression. Major topics in the fiscal year2003 are described as follows.

In silico analyses of the NAC familytranscription factors in rice andArabidopsis

Over 32,000 cDNA clones were collectedand completely sequenced from japonica ricein the rice full-length cDNA project (year2000-2003). Along with the nearly completegenome sequence of rice, information of the

full-length cDNA clone is very important forthe correct annotation of the gene structure,especially for the initiation and terminationsites of the transcription and the exon-intronboundary structure. Collection of cDNAclones has also been enhanced to analyze thegene families in rice genome. Among manykinds of gene families, we have first focusedon the gene families of the transcriptionfactors. In 28,469 clones, as published in ourkey paper in Science, 1336 clones have theDNA binding domain, which were classifiedinto 18 independent groups. We focused onNAM/NAC-type transcription factor at first,because it belongs to a plant specifictranscription factor family.

The NAC domain was originally charac-

M olecular Genetics Department


terized from consensus sequences frompetunia NAM and from Arabidopsis ATAF1,ATAF2, and CUC2. Genes containing theNAC domain (NAC family genes) are plantspecific transcriptional regulators and areexpressed in various developmental stagesand tissues (Fig. 1). We performed acomprehensive analysis of NAC family genesin rice (a monocot) and Arabidopsis (a dicot).We found 75 predicted NAC proteins in full-length cDNA data sets of rice (28,469 clones)and 105 in putative genes (28,581 sequences)from the Arabidopsis genome. NAC domainsfrom both predicted and known NAC familyproteins were classified into two groups and18 subgroups by sequence similarity asshown in Fig. 2. There were a few differencesin amino acid sequences in the NAC domainsbetween rice and Arabidopsis. In addition,we found 13 common sequence motifs fromtranscriptional activation regions in the C-terminal regions of predicted NAC proteins.These motifs probably diverged havingcorrelations with NAC domain structures.The relationship between the structure andfunction of the NAC family proteins in lightof our results and the published data arediscussed. Details could be seen in our recentpublication (Ooka et al. DNA Research 10:239-247).

Large-scale sequence analysis ofrice genes disrupted by the insertionof the retrotransposon Tos17

Functional analysis of genes is animportant target for molecular genetics ofrice. Gene disruption is one of the mostpowerful methods for this purpose. Themutant panel, knock-out mutant lines usingretrotransposon Tos17 has been producedduring the last five years in our department.Tos17 is an endogenous retrotransposon inrice which is only active in cultured rice cells,and becomes inactivated in the regeneratedplant. The transposed Tos17 copies are fixedin the regenerated plants and segregated inthe next generation. Copy number of Tos17 isonly 2 in japonica rice Nipponbare. Anaverage of 10 copies of Tos17 are transposedin each mutant line. Because we haveproduced more than 50,000 lines of Tos17insertion lines, more than 500,000 loci havebeen disrupted.

To identify each insertion point of Tos17in rice genome, each flanking region wasamplified using a TAIL-PCR and suppressionPCR method and then nucleotide sequencefrom each fragment was determined.Currently, 5,000 lines have been analyzedand more than 20,000 insertion loci havebeen assigned to the rice genomic sequences.Tos17 is inserted into all rice chromosomes.However distribution of insertion points isnot random (Fig.3). High frequency insertionsare observed at near the telomeric regions onrice chromosome 1. The insertion is biased tothe genic region. The region annotated withexon or intron is three times more frequentlyinserted than the intergenic region. Thispreference is very suitable for large-scaleanalysis of disrupted genes. On thepericentromeric regions consisting ofheterochromatin, density of Tos17 insertionsis relatively low. Because the gypsy typeretrotransposon makes its cluster on thepericentromeric region, there might be a kindof quota system of retrotransposons on ricegenome.

We have developed flanking sequencedatabase of Tos17 mutant lines. The

Fig. 1NAM and NAC domains and TAR in an NAC familyprotein. The NAC family protein is shown by thetransparent rectangles, and the locations of variousknown and predicted domains are shown as follows. NACdomain: striped region; Activation domain of AtNAM:hatched region. Subdomains A to E are shown by solidlines in the NAC domain. The DBD (DNA-binding domain)of AtNAM is contained within the subdomains D and E.The NAM domain in InterPro consists of subdomains A toD. The subdomain E of 50 aa (amino acids) was added tothe NAM domain in InterPro. The TAR (transcriptionalactivation region) is the C-terminal region of the NACfamily protein.


Fig. 2Phylogenetic tree of all NAC domains. The unrooted phylogenetic tree of NAC domains was depicted by the CLUSTAL Xprogram, and was constructed by the neighbor-joining method.28 The numbers beside the branches represent bootstrapvalues (≥ 500) based on 1000 replications. The NAC domains were classified into two large groups: Groups I and II.Group I was divided into 14 subgroups (TERN, ONAC022, SENU5, NAP, AtNAC3, ATAF, OsNAC3, NAC2, ANAC011,TIP, OsNAC8, OsNAC7, NAC1, and NAM), although two NAC domains (ONAC024, ANAC077) did not belong to anysubgroup. Group II was divided into ANAC001, ONAC003, ONAC001, and ANAC063. Green names beginning with“ONAC” are NAC domains in O. sativa. Magenta names beginning with “ANAC” are NAC domains in A. thaliana. Bluenames are the NAC domains from reported NAC families. Roman numerals (i to xiii) written after names indicate motifs inTARs. In clones marked with an exclamation point (!), TARs were not analyzed because they were too short. Asterisks(*) indicate homologues.


database can be accessed on the Internet.The URL of our database is http://tos.nias.affrc.go.jp/. Users can search nucleotideand/or amino acids sequences of theirinterest against our flanking sequences. Ifthe query sequence hits our flankingsequence, the corresponding line may be amutant which was disrupted with Tos17.When the mutant line is found, users canrequest the mutant lines via our web site.This system is very powerful tool forfunctional genomics.

Generation of activation tagginglines of rice: Isolation andcharacterization of a lesion mimicmutant

A T-DNA vector that contains tetramer ofthe CaMV 35S enhancer followed by itsminimum promoter at its right border was

introduced to rice (Oryza sativa L. cvNipponbare) by the Agrobacterium-mediatedtransformation method. We have generated12,000 transgenic rice lines that carry the T-DNA insertion. Among them, severalmutants including lesion mimic, stripe,dwarf, and short grain were obtained in theT0 generation. A lesion mimic mutant, whichwas designated as Lesion mimic 1 (Lmm1),was further characterized. Lmm1 wasdominant and the mutant phenotype was co-segregated to the T-DNA in the T1

generation. An ORF (LMM1) was locatedabout 500 bp downstream of the T-DNA andthe lesion mimic phenotype correlated withthe enhanced expression of the mRNA.LMM1 protein shows sequence similarity to aprotein whose expression is induced byhypersensitive reaction in tobacco. Inaddition, transcriptional levels of PR proteins

Fig. 3The distribution of Tos17 insertions on rice chromosome 1. Frequency represents number of insertions of Tos17 on each100kb fragment.

Fig. 4Accumulation of rice phytoalexins in the Lmm1 mutant leaf. Momilactone A content. (B) Sakuranetin content. WT1 andWT2: Wild type. 42,44,45: Lmm1. The number of + indicates the severity of the lesion mimic.

were increased, and phytoalexins, bothmomilactone A and sakuranetin, accumulatedin Lmm1 (Fig. 4). Furthermore, T1 plantswhich exhibit lesion mimic were resistant toa rice fungal pathogen, rice blast fungus,which indicates the correlation between theLMM1 protein and defense mechanism.Finally, we confirmed that the overexpressionof LMM1 is sufficient to induce the lesionmimic phenotype by re-transformation.

Ehd1, a B-type response regulator inrice, confers short-day promotion offlowering independently of Hd1

Two evolutionarily distant plant species,rice (Oryza sativa L.), a short-day plant, andArabidopsis thaliana, a long-day plant, sharea conserved genetic network controllingphotoperiodic flowering. The orthologousfloral regulators—rice Heading date 1 (Hd1)and Arabidopsis CONSTANS (CO)—integrate the circadian clock and externallight signals into mRNA expression of theFLOWERING LOCUS T (FT) group floralinducer. According to daily light/dark cycles,the circadian clock expresses Hd1/CO mRNAmainly during the subjective night. In rice,Hd1 inhibits and promotes floral transitionby suppressing and activating FT-like genesdepending on the day-lengths, respectively.In Arabidopsis, CO functions as an activatorof FT under LD conditions. Thus, theregulation of FT group genes by Hd1/COplays a central role in the photoperiodism ofboth SD and LD plants. Regardless of theimportant role of Hd1, this model does notfully explain photoperiodic flowering in rice,

because Hd1-deficient strains exhibit asignificant delay of flowering undercontinuous-light conditions compared withSD conditions.

To better understand the molecularmechanisms of photoperiodic flowering inrice, we studied a japonica cultivar,‘Taichung 65’ (T65), which exhibits a broadregional adaptability because of its reducedresponse to photoperiod. We here show thatT65 contains loss-of-function alleles of Hd1and Ehd1 (Early heading date 1), both ofwhich play important roles in photoperiodicflowering of rice. We first detected two majorQTLs at Hd1 and Ehd1 regions in the T65 ×Nipponbare cross (Fig. 5). This result led usto analyze the genomic DNA sequence of Hd1in T65. We found that a retroelement-likefragment was inserted into the second exon,and the T65 allele of Hd1 seemed to be aloss-of-function allele. To further confirm thedeficiency of Hd1 in T65, we transformed thefunctional Nipponbare allele of Hd1 (Hd1-Nip) into T65 to generate the T65+Hd1transgenic line. Hd1-Nip in T65 clearlypromoted flowering under the SD conditionand delayed flowering dramatically underthe LD condition (Fig. 6).

To reveal the molecular nature of theEhd1 gene, we next performed map-basedcloning and have demonstrated that Ehd1encodes a B-type response regulator whose


Fig. 5Major QTLs identified in 89 recoombinant inbred lines derived from a crossbetween T65 and Nipponbare. F values of single point QTL analyses at theHd1 and Ehd1 loci are shown.

Fig. 6A) Hd1 can promote and repress flowering in T65background under SD and LD conditions, respectively. Incontrast, Ehd1 can promote flowering in T65 under bothSD and LD conditions. Note that floral promotion by Ehd1does not require the Hd1 function since T65 contains anull al lele of Hd1. B) Right; T65+Hd1 under LDconditions, Left; T65 under LD conditions. Around 180days after sowing.


ortholog may not exist in Arabidopsis. Ehd1induces mRNA expression of FT-like genesafter SD treatment, indicating that a noveltwo-component signaling cascade is integratedin the conserved photoperiodic floweringpathway in rice. When the functional allele ofEhd1 was introduced into the Hd1-deficientcultivar, T65, the transgenic rice plantsflowered early , especially under SD, clearlyindicating that Ehd1 can function as a floralinducer independently of Hd1.

Characterization of a xyloglucanendotransglucosylase gene that isup regulated by gibberellin in riceseedlings

Gibberellins (GAs) are a class ofphytohormones that regulate many aspectsof plant growth and development processes,including stem elongation, flowering andseed germination. To elucidate the effect ofGA on these processes, the GA biosynthesisand signaling pathway have been widelystudied in many plants using genetic,molecular and biochemical approaches. Toscreen downstream genes related to shootelongation in GA signaling in rice (Oryzasativa L. cv Nipphonbare), a cDNA microarraycontaining 4,000 cDNA clones randomlyselected from a rice cDNA library, washybridized to the cDNA synthesized fromRNA prepared from the leaf sheath includingapical meristem of rice seedlings treated withGA3 for 24 h. Among 4,000 genes analyzed,28 unique clones showed enhanced expression,while 44 clones were repressed by GA3

treatment when compared to the control.Among the GA up-regulated clones, one clone that shows homology to xyloglucan endo-transglucosylase (XTH) related sequences,was designated as OsXTH8, cloned andcharacterized. OsXTH8 is a single copy geneand its cDNA is 1298 bp encoding a predictedprotein of 290 amino acids. Phylogenicanalysis revealed that OsXTH8 with barleyXTH related genes, HvPM2 and HvPM5,makes a different fourth subfamily in thepreviously reported three subfamilies ofXTHs related genes. OsXTH8 preferentially

expressed in rice leaf sheath in response toGA3. OsXTH8 expressed mainly, as revealedby in situ hybridization and OsXTH8promoter GUS fusion analysis, in vascularbundles of leaf sheath and young nodal rootswhere the cells are actively undergoingelongation and differentiation. OsXTH8transcript was up-regulated by GA3 alone orin combination with brassinolide (BL) andrepressed by abscisic acid, while nosignificant effect of auxin was observed (Fig.7). In two genetic mutants of GA, GAconstitutive response mutant Slr1 and GApartial deficient mutant Tanginbozu, withabnormal heights, the expression of OsXTH8correlated with the height of the mutants.To investigate the function of OsXTH8, RNAiOsXTH8 was placed under the control of 35SCaMV promoter and transferred into rice.Transgenic rice expressing RNAi in OsXTH8exhibited repressed growth and plants wereabout 20-50% shorter compared with that ofvector control at the time of maturity. Theseresults suggest that OsXTH8 is a novel XTHrelated gene that is up-regulated by GA3 andmay be involved in GA-regulated stemelongation processes in rice.

Fig. 7Hormonal regulation of OsXTH8 expression. A and B)Effects of different phytohormones and their combinationon the expression of OsXTH8. C) OsXTH8 expression indifferent GA mutants exhibiting abnormal heights.


Structural biology

X-ray crystallographic analysis ofproteins and their complexes

Crystal structure studies of severalbiologically important proteins have beencarried out. α-Galactosidases catalyze thehydrolysis of α-1,6-linked galactosyl residuesfrom galacto-oligosaccharides and polymericgalacto-(gluco)mannans. The crystal structureof rice α-galactosidase was determined at 1.5Å resolution (Fig. 1). The structure consistedof a catalytic domain and a C-terminaldomain. The catalytic domain had a (β/α)8-barrel structure and the C-terminal domainwas made up of eight β-strands containing aGreek key motif. The structure was solved asa complex with D-galactose, providing a modeof substrate binding in detail. The D-galactosemolecule was found bound in the active sitepocket on the C-terminal side of the centralβ-barrel of the catalytic domain. Structuralcomparisons of rice α -galactosidase withchicken α -N-acetylgalactosaminidase

provided further understanding of thesubstrate recognition mechanism in theseenzymes. The crystal structure of Bacillussubtilis α -amylase, in complex with thepseudotetrasaccharide inhibitor acarbose,was determined at 2.3 Å resolution. Thecomplex structure revealed an hexasaccharidein the active site as a result of transglycosylationof acarbose (Fig. 2). Based on comparisonwith the known structure of the catalytic sitemutant complexed with the native substratemaltopentaose, it is suggested that thepresent structure represents a mimicintermediate in the initial stage of thecatalytic process.

Unique DNA Recognition Mode of thePlant Zinc Finger Protein ZPT2-2

The ZPT family is a group of DNA-bindingproteins in petunia consisting of two to fourCys2/His2 (C2H2) zinc finger motifs that areimplicated in floral-organ specific transcriptionalregulation. In contrast to cluster-type zincfinger proteins observed in animals, theseproteins are characterized by the existence oflong spacers in variable lengths between zincfingers. Moreover, the zinc finger motif itself

B iochemistry Department

Fig. 1The ribbon model of the crystal structure of rice α -galactosidase in complex with D-galactose. Two catalyticresidues are shown in red, and the bound D-galactosemolecule is shown in black ball-and-stick drawings.

Fig. 2The surface potential model of the Bacillus subtilis α-amylase in complex with acarbose. The acarbosemolecule is shown in wire drawing.


contains a highly conserved QALGGHsequence, which has so far not been reportedfor any organisms other than plants. Wehave studied the three-dimensional structureof the DNA-binding domain (ZF1F2) of ZPT2-2, containing two zinc finger motifs (F1 andF2) separated by 44-amino acid lengthspacer, and its interaction with target DNAby NMR spectroscopy. ZF1F2 contains twoββα motifs, typical for the classical C2H2 zincfingers, in the F1 and F2 regions (Fig. 3). NoNOE contacts were observed between F1 andF2, indicating that the two zinc finger motifsof ZF1F2 have no interactions and behaveindependently in the absence of DNA due tohigh mobility of the spacer connecting F1 andF2. This spacer is highly flexible even in thecomplex with the target DNA. This flexibilitymainly provides a unique DNA recognitionmode in which ZPT2-2 can interact withtarget DNAs containing two core sequencesin different arrangements. Chemical shift

perturbation experiments showed that theconserved QALGGH sequence, occupying the+2 to +7 region of the recognition α-helix inboth F1 and F2, interacts with the targetDNA. In addition, the C-terminal region ofthe α-helix (+8 to +16) is also involved inDNA binding. These results prompt us topropose a model of DNA recognition by ZPT2-2 where the single zinc finger motif can bindto the core sequence using nearly the fulllength of the α-helix (Fig. 4a). This model isquite different from DNA recognition modeby the cluster-type C2H2 zinc finger proteinsfrom animals. In the case of the cluster-typeproteins, two consecutive zinc fingerscooperatively bind to the core using only the -1 to +6 region of the recognition α-helix (Fig.4b).

Proteomic analysisBased on the proteomic approach, several

materials such as rice, silkworm, andbacteria, have been analyzed using thecombination of two-dimensional polyacrylamidegel electrophoresis and mass spectrometry.Many genes encoding the proteins weremapped on the gels and several isomers wereidentified. The next paradigm in proteomicsseems to be the identification of proteininteraction, then investigation using non-denatured gel electrophoresis and othertechniques were attempted to introduce theproteomics through the developing newmethods and application for the analysisprotein network. To understand the protein

Fig. 4(A) DNA recognition mode by the second zinc finger of ZPT2-2. The model structure of the complex was constructed bylocating all DNA-interacting residues determined by chemical shift perturbation experiments so as to interact with thetarget DNA core sequence, CAGT. (B) X-ray crystal structure of the TFIIIA F1F2/DNA complex extracted from the six-finger TFIIIA/DNA complex.

Legends to Figures

Fig. 3Ribbon drawings of energy-minimized average NMRstructures of ZPT2-2 F1(Gly96-Ile130) (A) andF2(Pro162-Leu194) (B). Side-chain heavy atoms of zinc-chelating residues and zinc atom are shown as stick andCPK models, respectively. The highly conservedQALGGH sequence in the plant specific ZPT family iscolored in magenta.


interaction, we recently developed the nativeprotein electrophoresis system using plastictube gel for two-dimensional polyacrylamidegel electrophoresis (Fig. 5).

Molecular analysis of signalperception and transduction

Analysis of elicitor- and gibberellin-responsive genes for GRAS familyproteins in rice.

By DNA microarray analysis, weidentified two novel members of the GRASgene family from rice, CIGR1 and CIGR2,inducible by the potent elicitor, N-acetylchitooligosaccharide, and gibberellins.The predicted proteins encoded by CIGR1and CIGR2 share a significant sequencesimilarity with members of the GRAS familypreviously identified, such as ArabidopsisSCARECROW, GAI, RGA, tomato Lateralsuppressor, and rice SLR1, all of which haveVHIID domains, implying that these twogene products play roles in cellular signaling.Fusions of these two proteins with GreenFluorescent Protein were found exclusively inthe nuclei of onion epidermal cells. Theexpression of CIGR1 and CIGR2 wasdependent on the structure of N-acetylchitooligosaccharides and gibberellins,both of which show good correlation with the

biological activity. The results suggestedthat the induction of the genes by N-acetylchitooligosaccharide and gibberellin ismediated by a biological receptor capable ofspecific recognition and signal transductionupon perception of the compounds. ThemRNA accumulation was independent of denovo protein synthesis. Co-cultivation of ricecells with rice blast fungus strongly inducedthe expression of CIGR1 and CIGR2 ,whereas inoculation of suspension cells withphyto-pathogenic bacteria did not. Furtherpharmacological analysis revealed that theCIGR1 and CIGR2 mRNA accumulation bygibberellin is dependent upon proteinphosphorylation/dephosphorylation events ina different manner from that by N-acetylchitooligosaccharide. Lavendustin Aand Okadaic Acid, an inhibitor of tyrosinekinase and protein phosphatase, respectively,strongly blocked the mRNA accumulationinduced by gibberellin but not by N-acetylchitooligosaccharide. Furthermore, inrice calli derived from d1, a mutant that isdeficient in the α -subunit of theheterotrimeric G-protein, CIGR1 and CIGR2were induced by N-acetylchitooligosaccharideelicitor, but not by gibberellin (Fig. 6). Theseresults strongly suggested that CIGR1 andCIGR2 are the crossing point of elicitor andgibberellin signals, and further implied thatCIGR1 and CIGR2 have dual, distinct rolesin defense and development.

Fig. 5The first dimension was native gel electrophoresis ofsilkworm body fluid using plastic tube. The seconddimension was SDS polyacrylamide gel electrophoresis.Oligomeric proteins which were co-migrated in the firstdimension were separated in the second dimension bydenaturation. Those proteins should then be linedvertically in 2D-gel profile.

Fig. 6Dependency on D1 (α-subunit of heterotrimeric G-protein)of the mRNA accumulation for CIGR1 and CIGR2 bytreatment with N-acetylchitoheptaose and GA3.Suspension-cultured cells of rice mutant line, d1 , weretreated with N-acetylchitoheptaose (GN7; 1 µM) or GA3

(50 µM) and incubated for the time indicated. Total RNAswere subjected to northern blot hybridization.


The research activities of our departmentare mainly focused on the elucidation ofmolecular mechanisms of importantphysiological processes in plants. The majortopics in fiscal 2003 are as follows.

Photosynthesis and carbonmetabolism

Identification and expression analyses ofrice three PEPC kinase genes

Phosphoenolpyruvate carboxylase (PEPC),a key enzyme of C4 photosynthesis, performsan important role in the primary carbon andnitrogen metabolism in C3 plants, and itundergoes activity regulation throughreversible phosphorylation. Three PEPCkinase (PPCK) genes, OsPPCK1, OsPPCK2and OsPPCK3, were identified in the ricegenome sequence database, and their fulllength cDNAs were isolated. In vitrotranscription/translation of the cDNAsfollowed by the assay of PPCK activityindicated that all three genes encode afunctional PPCK protein. OsPPCK2 has an

intriguing feature, in that it has twotranscription initiation sites. Transcriptionfrom the second initiation site gives rise to atranslation product similar to those of

Auxin receptor at the plasma membraneAuxin is a plant hormone functioning on a

wide range of growth and developmentalprocesses including cell division, elongationand differentiation. An auxin-binding protein(ABP1) is a candidate for the auxin receptoron the plasma membrane. Several genemanipulation analyses on the ABP1 geneindicate that ABP1 functions on the auxin-induced cell elongation. ABP1 is a solubleprotein, and no transmembrane region hasbeen identified within the ABP1 sequence.Thus, it has been postulated that ABP1 isresident at the plasma membrane by bindingto a transmembrane protein, termed adocking protein. Photoaffinity crosslinkinganalysis demonstrated a membrane protein

interacting with ABP1.

Candidate gene for QTL influencing tissueculture trait using rice DNA microarray

Two candidate genes for quantitative traitloci (QTL) controlling tissue culture traits inrice were screened using DNA microarrayanalysis. The QTL on chromosome 1 wasdetected using a population derived from twovarieties, Koshihikari and Kasalath, andKasalath allele gave improved tissue cultureability in subculture and regeneration. Sincethe candidate genes were suppressed inKasalath callus, now we are trying tosuppress these genes in Koshihikari callus tosee if these genes are related to tissueculture ability.

P lant Physiology Department

237

399

Leaf

Leaf

She

ath

Ste

m

Roo

t

Flo

wer

Glu

me

OsPPCK1

OsPPCK2-1

OsPPCK3

Actin

331

(bp)

799

OsPPCK2-2 435

Day

time

Nig

ht

Leaf

Fig. 1Expression characteristics of three PPCK genes analyzed by RT-PCR.OsPPCK2-1 and OsPPCK2-2 represent the total and long transcripts,respectively, of OsPPCK2.

OsPPCK1 and OsPPCK3, while transcriptionfrom the first site gives rise to a longerprotein with an extension of 13 amino acidresidues at the N terminus. RT-PCR analyses(Fig. 1) indicated that three genes showeddifferent expression characteristics regardingorgan specificity and diurnal change in theleaves. Expression profiles of the twotranscripts of OsPPCK2 were also different.These results suggest that the three PPCKgenes have different functions in rice plantsand that OsPPCK2 might have dualfunctions, mediated through selection of thetranscription initiation site.

Species variation in potentialphotorespiratory capacity of C4 plants

In C4 plants, photorespiration is decreasedrelative to C3 plants. However, it remainsunclear how much photorespiration C4 plantshave. We investigated photorespiratorycapacity in C4 plants by analyzing theaccumulation of glycine decarboxylase (GDC)in leaves. In all species examined, GDC waslocalized in the bundle-sheath (BS)mitochondria, but the amount of GDC variedamong species. Both the NAD-ME and PCKspecies contained more GDC in the BS than

the NADP-ME species. There was a highpositive correlation between the amount ofGDC and the degree of granal developmentin the BS chloroplasts, suggesting that C4

species having high PS II activity in the BSchloroplasts possess high photorespiratorycapacity.

Identification of a new gene controllingplant height in rice by using the candidategene strategy

We identified a gene underlying aquantitative trait loci controlling plantheight on chromosome 1 (QTLph1) in rice byusing the candidate gene strategy (Fig. 2).Physiological analyses of a near isogenic lineexpressing QTLph1 (NIL6) suggested thatthe targeted gene might control plant heightby the high ability to translate sucrose inleaves. The results pointed to the gene forsucrose phosphate synthase (SPS), the majorlimiting enzyme for sucrose synthesis, as acandidate gene for QTLph1 among theannotation of the region of QTLph1. Theeffect of increased SPS activity on plantheight was proven with transgenic riceplants with a maize SPS gene.


Fig. 2Phenotypic Analysis of NIL6 or Nipponbare. a Nipponbare (control plants) and NIL6 plants at 75 days after transplanting.b Change in plant height of Nipponbare and NIL6. c Comparison of the length of panicle and upper 4 internodes of mainculms. Data are presented as the mean of 10 independent plants. *** means significant at T-test (P < 0.001).


Plant development

Possible role of LIF in the regulation oflocal cytokinin metabolism aroundaxillary buds

Shoot branching is among the key factorsthat define the overall architecture of plants.We have previously found that overexpressionof a cDNA for a zinc-finger protein ofpetunia, designated Lateral shoot InducingFactor (LIF), in transgenic petunia plantsresulted in a dramatic increase in lateralshoots. LIF is specifically expressed aroundthe bases of axillary buds and this expressiondeclines after decapitation.

To investigate whether changes ofhormone levels are responsible for the LIF-oxphenotypes, we determined auxin, gibberellin,and cytokinin contents in LIF-ox and wild-type petunia. The levels of auxin andgibberellins were comparable between thetwo plants. By contrast, we found that freecytokinins (presumed active forms), weresignificantly decreased, while their nucleosideand nucleotide forms were increased in theleaves and stems of LIF-ox plants, suggestingthat the final steps of cytokinin metabolismare blocked by LIF-ox (Table I). This finding,taken together with the expression patterns

of LIF, may suggest that LIF plays a role inmaintaining local cytokinin content at lowlevels thereby keeping the axillary budsdormant.

Control of branching is of immenseeconomic importance in horticulture andagriculture. To examine whether LIF couldenhance branching in other plant species, weoverexpressed LIF in tobacco and Arabidopsisand found that LIF-ox resulted in enhancedbranching in both the plants. These resultssuggest that LIF function may be widelyconserved at least among dicotyledonousplants.

Specific roles of phytochromes A, B andC in rice development

We have been studying distinct functionsof rice phytochromes by the isolation andanalysis of phytochromes mutants. Ricephytochromes form a small gene family,PHYA, B and C. We have isolated mutantsfor all phytochromes and also produceddouble and triple mutants.

In the natural day length condition(considered a long day), the flowering time ofa phyA single mutant was the same as thatof the wild type, while phyB and phyCmutants were about 10 days earlier than the

Leaves Stems

WT

Concentration(pmol/g FW)


% WTConcentration(pmol/g FW)


% WT

LIF-ox WT LIF-ox

621761723087814310015516170427

1.03 ± 0.070.95 ± 0.2215.0 ± 5.290.22 ± 0.020.26 ± 0.041.94 ± 0.470.06 ± 0.010.06 ± 0.011.07 ± 0.130.43 ± 0.081.53 ± 0.61142.1 ± 85.6

1.66 ± 0.180.54 ± 0.058.72 ± 0.680.73 ± 0.100.30 ± 0.032.39 ± 0.280.14 ± 0.010.06 ± 0.020.69 ± 0.042.71 ± 0.820.90 ± 0.2133.26 ± 4.61

73280272521359917550030826450170

0.29 ± 0.110.14 ± 0.091.84 ± 1.050.36 ± 0.100.23 ± 0.082.34 ± 0.410.21 ± 0.160.10 ± 0.060.80 ± 0.320.73 ± 0.042.70 ± 2.9192.66 ± 33.2

0.40 ± 0.060.05 ± 0.010.68 ± 0.150.69 ± 0.050.17 ± 0.022.36 ± 0.280.12 ± 0.010.02 ± 0.010.26 ± 0.052.86 ± 0.050.60 ± 0.0654.42 ± 20.3

tZtZRtZMPcZcZRcZMPDHZDHZRDHZMPiPiPRiPMP

Table 1. Cytokinin contents in LIF-ox and wild-type petunia plants.

Abbreviations: tZ, trans-zeatin; tZR trans-zeatin riboside; tZMP, trans-zeatin riboside monophosphate; cZ, cis-zeatin;cZR, cis-zeatin riboside; cZMP, cis-zeatin riboside monophosphate; DHZ, dihydrozeatin; DHZR, dihydrozeatinriboside; DHZMP, dihydrozeatin riboside monophosphate; iP, N6-(∆2-isopentenyl) adenine; iPR, N6- (∆2-isopentenyl)adenine riboside; iPMP, N6-(∆2-isopentenyl) adenine riboside 5’-monophosphate.

wild type. A phyBC double mutant floweredat the same time as the phyB or phyC singlemutant. These results indicate that phyBand phyC have the same effect (delaying theflowering) on the flowering-time determinationunder the long day condition. In contrast,phyAB and phyAC double mutants showedvery early flowering. These observationsmean that the phyA mutation alone does notaffect the flowering time so much, but that inthe phyB or phyC mutant background, phyAmutation has a big effect on the floweringtime in this condition. In order to elucidatethe points where these phytochromes act, weexamined the expression levels of theflowering-related genes, Hd1 and Hd3a,during the growing process up to theflowering. The results of these experimentssuggest that phyB and phyC promote thelong-day signal at the upstream and phyAsuppresses the Hd3a expression in thedownstream.

Disease resistance

Identification of mitogen-activated proteinkinase phosphatase as a novelcalmodulin-binding protein

We have been studying the molecularmechanism of the defense response of tobaccoto pathogen infection and wounding. We haveisolated 13 tobacco calmodulin (CaM) genes,which were subdivided into three types, andhave showed that each type CaM exhibitscharacteristic gene expression profile and

distinctive activation profile for putativetarget proteins. To study the function ofCaMs, we identified mitogen-activatedprotein kinase (MAPK) phosphatase (NtMKP1)as a CaM-binding protein by screening thecDNA expression library. RecombinantNtMKP1 protein physically interacted withthree types of CaM in vitro. Using mutatedNtMKP1 protein, we identified amino acidresidues critical for binding CaM. Over-expression of NtMKP1 in tobacco plantscompromised the wound-induced activationof two defense-related MAPKs, WIPK andSIPK. These results suggest that plant CaMsare involved in these stress-activated MAPKcascades via NtMKP1 (Fig. 3).

Cause of genomic variation of diseaseresistance genes is mainly due toaccumulation of minor nucleotidemutations

Plant’s true disease resistance genesrecognize the infection of correspondingpathogens through perception of theirmolecular components like the immunesystem of higher animals (vertebrates). Inboth cases, how to keep recognizing thepathogen’s components, which are veryrapidly changed by mutations to escape thesehosts’ surveillance system, is the mainproblem. However, although animal’s immunesystems harbor more than 107 variationscorresponding to each lymphocyte, plantshave no circulating system like animals, andthey must keep the variation of theresistance genes in their population and theirlimited size of the genome. We have shownthat these variations are mainly due toaccumulation of minor nucleotide mutations,which occur at a much higher frequency inthe resistance gene regions, including anisolated rice blast resistance gene, Pi-b, andfive other isolated resistance genes, and theiranalogues in Arabidopsis (Fig.4). Moreover,although the major genome variation in mostof the genome in Arabidopsis was said to bedue to the recombination between twoancestral types (biallelic variation), ourinvestigation of the resistance gene


TMV Wounding

MAPKKK

MAPKK

WIPK WIPK SIPK SIPK

MKP1CaM

Fig. 3Possible model for signal transduction in tobacco inresponse to TMV infection and wounding.


variations in 10 ecotypes indicated that atleast in the highly mutating region, it isreally due to the accumulation of novelnucleotide variations.

Salt stress

Membrane transport of cations andanions in the high salinity environment inrice

Vacuolar Na+/H+ antiporter transportsNa+ from the cytoplasm to vacuoles using apH gradient generated by proton pumps. Weidentified three novel members (OsNHX2-4)of the rice NHX-type antiporter gene familyby analysis of the recently completed ricegenome database. The deduced amino acid

sequence of OsNHX1 has a high similaritywith OsNHX2 (75%) and OsNHX3 (71%), andis similar to OsNHX4 with 33% identity.Overexpression of the OsNHX2, 3, and 4genes suppressed NaCl, KCl, and hygromycinsensitivity of the yeast nhx1 mutant. Theexpression of the OsNHX1 to 4 genes showeddifferent patterns in rice treated with salt orosmotic stress.

We hardly know about intercellularbehavior of chloride ions, a counter ion forNa+, under the high salt condition. Weisolated two cDNA clones for the rice chloridechannel genes, OsCLC-1 and -2, and found agenomic sequence in the rice genomedatabase. Both genes have six introns andthe positions of insertion resembled the case

Fig. 4Variation of structure of a resistance gene of Arabidopsis in 8 ecotypes. Non-marked letters indicates the samenucleotides as those of the standard ecotype, Columbia. Yellow: the second type of nucleotides. Green: third type, red:fourth type. Blue and deep green belts over the sequence indicate the regions related to molecular recognition. Only thenucleotides with mutations are indicated.

of AtCLC-a, -b and -c. OsCLC-1 and -2 couldpartially substitute for GEF1 protein, thesole chloride channel in yeast, that is,restored the growth of a strain that this genedisrupted. OsCLC-1 and -2 suppressed theseveral phenotypes of the gef1 mutant.However, the complementation patterns weredifferent from the case of AtCLC-d and -d.We expect that OsCLC-1 and -2 are importantfor the ionic homeostasis in the cell.

Symbiotic nitrogen fixation

The IGN1 gene is required formaintenance of bacterial symbiosis inlegume root nodules

A fix- mutant ign1 (inefficient greenishnodules) of Lotus japonicus forms noduleswith the apparently normal development ofbacterial endosymbiosis, but rapid disintegrationof infected cells occurs just after the onset of

nitrogen fixation (Fig. 5). As a consequence,the nodules exhibit extreme “early senescense”phenotypes and the ign1 plants cannot growwithout an exogenous nitrogen supply. TheIGN1 gene was isolated by a map-basedcloning strategy. The IGN1 locus was locatedin the ca. 70kb region between two dCAPSmarkers on chromosome 5. In this region, atotal of five genes were predicted. Directsequence comparison of them allowed us toidentify a gene that contains a single basedeletion in the ign1 mutant genome, whichresults in an ORF shift. Knock-down of thisgene expression in wild type L. japonicus byRNA silencing technique reproduced the ign1phenotypes, and transfection of this gene intothe mutant line rescued the phenotypes. Wepropose that the IGN1 gene is essential forspecific recognition of endosymbiotic bacteriaat relatively late stage of symbioticinteractions.


Fig. 5Nodule infected cells in wild type “Gifu” plants and ign1 mutant. After 17 days of M. loti inoculation, infected cells of ign1begin to collapse and the bacteroids aggregate within the infected cytoplasm. Bars=10 µm.


The research activities of our departmentare mainly focused on the development ofbasic methods in plant biotechnology as wellas the generation of novel transgenic cropswith superior traits which conventionalbreeding techniques can not produce. Majortopics in 2003-2004 were as follows.

Gene targeting in higher plants

Gene targeting of mutated acetolactatesynthase (ALS) gene in Arabidopsis

Precise modification of a plant genome bygene targeting (GT) provides an importanttool for studies of gene function in vivo. Wepresent a straightforward system for thedetection of GT events in Arabidopsis usingan endogenous nuclear gene encodingacetolactate synthase (ALS) which isinvolved in the biosynthesis of branched-chain amino acids in plants and is the site ofaction for imidazolinone (IM) herbicides.

A single amino-acid change (Ser -> Asn atposition 653) in the ALS protein of Arabidopsisconfers resistance to IM. A truncatednonfunctional form of the ALS gene, carryingthe above S653N mutation specifying IMresistance, was cloned into a binary Tiplasmid. After in planta transformation ofthis construct into Arabidopsis, IM resistantplants were obtained and proved to begenerated by gene targeting.

We are applying a similar strategy to theestablishment of an efficient site specificgene modification system in rice.

Isolation and characterization of Rad52pathway genes in plants

Homologous recombination (HR) is anessential process for the maintenance andvariability of a genome. For better under-standing of the HR in higher plants, we havecloned all members of RAD51 paralogs andRAD54 from Arabidopsis. In addition, RAD51,

RAD54 and DMC1 genes from rice have beenisolated and characterized.

Two genes each for RAD51 and DMC1recombinase genes, eukaryotic homologs ofrecA, have been found in rice. Expression ofOsRAD51-A1 and -A2 genes was observed invarious tissues with higher expression levelsin both shoot and root apical meristems andyoung panicles, and was elevated by γ-rayirradiation with a higher induction level inOsRAD51A2. Expression of OsRad51A2promoter::GUS chimeric gene in rice wasstrongly up-regulated by double-strand break(DSB) inducers, γ-ray and bleomycin.

Antisense expression of OsRad51A1cDNA conferred growth retardation of T2transgenic rice plants in vegetative andreproductive phases. Those transgenic lines

P lant Biotechnology Department

Fig. 1Transgenic rice plants expressing OsRad51A1 antisensegene exhibited hypersensitivity to γ-ray irradiation. Riceseeds from wild type (cv. Kitaake) and OsRad51A1-antisense l ine (A137) were imbibed for 24 hours,irradiated with γ-ray (300 Gy) and grown in vitro for afurther six days.

also displayed hypersensitivity against γ-rayirradiation (Fig. 1). These results indicatedthat OsRad51A2 expression is controlled atthe transcription level by its 5'-upstreamsequence containing the possible DNA-damage responsive element, and thatlowering OsRad51 transcript levels couldraise sensitivity toward DSB inducers byinhibiting the formation of HR-machinerysufficient for DSB repair.

DMC1 is one of the key players in meioticrecombination. Both OsDMC1-A and -Bgenes were expressed not only in anthers,but in cultured cells, suggesting a role inmitotic recombination in plants. Transgenicrice plants bearing antisense expression of35S::OsDMC1A/B genes produced pollenswith abnormal shape and very low fertility,though they did not give any obviousphenotypes in vegetative stages. The resultsrevealed that the OsDMC1 genes are crucialin rice gametegenesis. Rice dmc1-A and -Bmutant lines caused by Tos17 retrotransposoninsertion affected their pollen fertility onlypartially, indicating that Dmc1A and Dmc1Bproteins are not only highly homologous, butfunctionally redundant.

We have identified and characterized anArabidopsis mutant defective in AtRAD51C,the homologue of RAD51C. Atrad51c plantsare sterile, disrupting both male and femalegametogenesis, while they showed normalvegetative development. The result indicatedan essential role of AtRad51C in plantmeiosis. Furthermore, Atrad51c cells andplants were hypersensitive to γ-ray irradiation,supporting the involvement of thisArabidopsis Rad51-like protein in recombina-tional repair.

Gamma-ray resistant mutantGamma rays strongly penetrate organisms

and damage DNA through DSBs. Repair ofDNA damage is essential for themaintenance of genetic stability. In order tostudy these DNA damage and repairmechanisms in higher plants, we isolatedArabidopsis mutants that showed enhancedresistance against γ-ray irradiation. One of

the isolated mutants (ir1) was extremelyresistant against γ-ray and could surviveafter 800 Gy irradiation, the lethal dosage forthe wild type plant (Fig.2). We have mappedthe mutant phenotype on chromosome 5 nearthe CAPS marker CHS.

Gene engineering

Expression patterns of various promotersfor storage protein accumulation in riceseeds

For the production of recombinant proteinin plants, seed systems have recently beenproven to be more advantageous thansystems using leaf or root. The seed is astorage organ within which a small numberof storage proteins are highly and stablyaccumulated in specific organelles designatedas protein bodies. Taking advantage of theseproperties, the seed has been utilized as anideal bio-reactor for the production ofrecombinant proteins. Recombinant proteinsaccumulated in seeds are highly stable, anddo not require processing or purification,allowing direct oral delivery.

Rice is one of the most important cropsand food resources in the world. Conventionalbreeding has long been employed for improvingseed yield and quality. As a productionsystem, rice seed has several advantagesover other cereal crops such as wheat, barley,


Fig. 2Phenotype of Columbia (WT) and ir1 after 800 Gy γ-rayirradiation. Imbibed seeds were treated with gammaradiation and grown in vitro.


and rye, including easier storage and processing,greater biomass (yield per unit area) andlower production costs. In addition, trans-formation systems have already beenestablished, and whole genome sequenceinformation is now available.

For rice seed to be utilized as a platformfor the production of recombinant proteins, itis important to use promoters tailored to therequirements of each particular protein andbiotechnology application, because thepromoter controls not only the time and placebut also the level of expression. For this

purpose , the promoters of fifteen genesexpressed in rice seed were analyzed for theirspatial- and temporal-expression pattern andtheir potential to promote the expression ofrecombinant proteins in seeds using stabletransgenic rice (Fig.3). The fifteen genesincluded ten seed storage protein genes andfive genes for enzymes involved in carbohydrateand nitrogen metabolism. The promoters forthe glutelins, and the 13 kDa and 16 kDaprolamins, directed endosperm-specificexpression, especially in the outer portion(peripheral region) of endosperm, while theembryo globulin and 18kD oleosin promotersdirected expression in the embryo andaleurone layer. Fusion of the GUS gene tothe 26 kDa globulin promoter resulted inexpression in the inner starchy endospermtissue. It should be noted that the 10 kDaprolamin gene was the only one tested thatrequired both the 5' and 3' flanking regionsfor the intrinsic endosperm-specific expression.

The promoters from the pyruvateorthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) geneswere active not only in the seed, but also inthe phloem of vegetative tissues. Within theseed, the expression from these two promotersdiffered, in that the PPDK gene was onlyexpressed in the endosperm, whereas theAGPase gene was expressed throughout theseed. The GUS reporter gene fused to thealanine aminotransferase (AlaAT) promoterwas expressed in the inner portion of thestarchy endosperm, while the starch branchingenzyme (SBE1) and the glutamate synthase(GOGAT) genes were mainly expressed in thescutellum (between the endosperm andembryo). When promoter activities wereexamined during seed maturation, theglutelin GluB-4, 26 kD globulin, and 10 kDaand 16 kDa prolamin promoters exhibitedmuch higher activities than the others. Theseed promoters analyzed here exhibited awide variety of activities and expressionpatterns, thus providing many choicessuitable for various applications in plantbiotechnology.

a b c d

e

i

m n o p

j k l

f g h

Fig. 3Histochemical analysis of GUS expression directed byvarious seed storage and non-storage protein genepromoters. GUS protein was detected by incubating hand-cut longitudinal sections of storage seeds in X-Glucsolution.(a) 1.3 kb GluB-1 promoter; (b) 2.3 kb GluB-1 promoter;(c)GluB-2 promoter; (d)GluB-4 promoter; (e)10 kDaprolamin; (f) 13 kDa prolamin promoter; (g) 16 kDaprolamin promoter; (h) 26 kDa Glb-1 promoter; (i) REG-2promoter; (j) Ole18 promoter; (k) . –conglycinin promoter;(l) AlaAT promoter: (m) GOGAT promoter; (n) AGPasesmall subunit promoter; (o) PPDK promoter; (p) SBE1promoter. al, aleurone; em, embryo; en, endosperm

Establishment of novel transgeniccrops

Blast disease resistance in transgenicrice plants expressing the chitinase Cgene from S. griseus

In order to obtain transgenic rice plants,which show enhanced disease resistance toblast fungus, we introduced the chitinase C(ChiC) gene under the modified CaMV 35Spromoter. ChiC is the first bacterial family19 chitinase discovered in Streptomycesgriseus HUT6037 and clearly shows higheranti-fungal activity than other testedchitinases (Fig.4). Approximately ninetypercent of the transgenic rice plants (T1

generation) expressing ChiC showed higherresistance to blast disease than non-transgenic plants. The ChiC gene wasintroduced into rice plants with a signalsequence of a rice chitinase gene and ChiCaccumulated intercellularly as expected.Using T2 plants of the selected six promisinglines, we tested the relation between ChiCexpression levels and disease resistance.Enhanced disease resistance of sibling plantsclearly depended on the ChiC expression.Although the transgenic plants weremorphologically normal and fertile in thegreen house, we are concerned that the

constitutive over-expression could have a badinfluence on the growth in the field.Accordingly, we isolated several genes, whoseexpression is induced after blast inoculation,and analyzed expression sites of the threeclones, M8, OsHin2, and PR10 on blast-infected leaves by the in situ RNA hybridizationmethod. They were specifically expressed inthe cells surrounding the brown blast lesionsat 3 and 4 days after inoculation. Since thePR10 gene expression was most remarkable,we isolated the promoter region of PR10 andtested its activity in the stable transformantsusing the GFP reporter gene. GFP wasdetected in roots and in the cells surroundingthe brown blast lesions and mimicry lesionson the uninoculated leaves. Thus, the chiCgene introduced into rice plants with thePR10 promoter is expected to express in thelimited cells after blast infection. This workwas carried out in collaboration with NiigataUniversity and Dainippon Ink and ChemicalsInc.

Transgenic crops expressing P450monooxygenase

The increased use of pesticides andherbicides on the agricultural scene hascaused serious problems for plants, fish,insects, mammals, and sometimes humans.These chemicals are usually removed from theenvironment by natural degradation anddegradation by bacteria and plants. Todecrease the load on the environment, theenhancement of the degradation of thesechemicals by plants should be as effective asthe reduction in their usage.

Cytochrome P450 monooxygenase playsan important role in the oxidative metabolismof xenobiotics in higher plants as well as inmammals. The enzyme system of microsomesconsists of many P450 species and a fewNADPH-cytochrome P450 oxidoreductasemolecules. Agrochemicals including herbicideswere metabolized by P450 species, conjugatedwith glutathione or sugars, and compart-mentalized into vacuoles, or cell walls, inplant body. The oxidation by P450 species isconsidered to be the limiting step of


0

1

2

3

Fig. 4Comparison of inhibited growth of Trichoderma reeseiwith different chitinases.0; distilled water, 1; Streptomyces griseus ChiC, 40ug, 2;rice CHT3(class 1b), 40ug, 3; Bacillus. circulans ChiA1,40ug


metabolism of foreign chemicals.We have introduced mammalian P450

species related to the metabolism ofxenobiotics into the rice variety, Nippnbare.These mammalian P450 species have a highactivity to metabolize various herbicides withdifferent modes of action and in differentchemical functional groups.

These transgenic rice plants expressinghuman P450 species, CYP1A1 or CYP2B6exhibited a remarkable cross-tolerancetoward various herbicides. These transgenicrice plants also had an enhanced ability tometabolize them with different chemicalstructures owing to the introduced P450s.These chemicals were supposed to beabsorbed by the transgenic plants in thefields and metabolized rapidly intononphytotoxic compounds. These transgenicrice plants should prove useful in executingeffective, inexpensive methods of decreasingvarious chemicals that are widespread inagricultural environments.

Development of dwarf rice plant byintroducing gibberellin 2-oxidase gene

To fully control the phenotype of crops isone of the final goals of crop breeding. Thetarget of our study is to obtain the dwarf typeof rice by genetically modifying thegibberellin (GA) metabolic pathway. Thistype of plants are expectedly tolerant tolodging due to extremely windy and rainyweather conditions.

We previously obtained transgenic riceplants harboring rice GA2-oxidase genedriven by the constitutively expressing actinpromoter. Although they showed dwarfphenotype, there was no seed setting. Wedevised a new strategy using rice GA2-oxidase gene together with D18 promoterwhich is derived from rice GA3-oxidase geneand specifically expresses in stems andleaves, instead of actin promoter. Consequently,we obtained transgenic rice plants (Oryzasativa japonica var. Don-to-koi) with dwarfphenotype of about 70% of non-transgeniccontrol rice as well as normal seed setting(Fig. 5).

Using T2 progeny, we assessed some of thepotential impacts of the transgenic rice onthe environment in a screen greenhouse byexamining morphological characteristics,reproductive characteristics, production ofunintentional harmful substances and itseffect on the ecosystem. Table 1 shows one ofthe data about the production of allelochemical-like substances that might affect the seedlinggrowth of other plants. The growth ofmustard was compared between the residualsoil on which transgenic or non-transgenicrice plants had been harvested and we didnot find any significant difference betweenthe two soil conditions.

The dwarf phenotype was inheriteddominantly, so the transgenic dwarf plantscan be used as hopeful breeding parents forproducing hybrid rice, and the introduction of

Fig. 5GA2-oxidase gene introduced rice. Transduced genewas driven by the D18 promoter.

Germination rate (%)

Mean SD Mean SDSoil origin

Plant weight (mg/fw)

Dontokoi (non-transgenic rice)

G-1-1-10 (transgenic rice)

G-3-3-22 (transgenic rice)

Hosetsuwaisei (d-18 mutant rice)

Un-cultivated soil

17.5

23.4

8.7

26.0

58.7

±

±

±

±

±

6.3

10.3

1.8

10.7

11.2**

37.5

26.0

40.0

34.0

70.0

±

±

±

±

±

17.1

5.5

21.2

8.9

12.3**

Table 1. Growth of leaf mustard on transgenic rice - cultivated soil.

Plant weight was determined 2 weeks after sowing. 5 replicates (N=30) . ** significant at 1% level by Dunnet analysis.

GA2-oxidase gene into other crops or fruittrees would be useful to breed new dwarfvarieties.

Regulation of organogenesis andembryogenesis

Improved regeneration ability inrecalcitrant rice cultivars

Totipotency is an important factor forplant transformation. The ability toregenerate is affected by the plant genotype.Konansou, an indica type rice, shows a highregeneration ability of callus. We tried tointroduce this ability into the Japaneseleading variety, Koshihikari, which has apoor regeneration ability. Isogenic lines ofKoshihikari were obtained which have a highregeneration ability. The isogenic linesshowed almost the same characteristics asKoshihikari including plant type, headingdate and eating quality in the field trials forthree years. It is supposed that these isogeniclines are useful for producing high qualitytransgenic rice plants.

We analyzed these Isogenic lines ofKoshihikari by RFLP (restriction fragmentlength polymorphism) mapping so far, andfound that chromosome 1 around thecentromere region had been substituted withKonansou chromosome 1. The length of theregion was about 16 cM.

To find out the gene involved in highregeneration ability, we obtained 7 SNPs(single nucleotide polymorphisms) on thisregion between Koshihikari and Konansou bysequencing PCR fragments using EST dataprovided by the Rice Genome Project. Weinduced callus from these isogenic lines ofKoshihikari (BC7F1) and analyzed thegenotype of the callus with high regenerationability. The genes related with highregeneration ability were supposed to belocated in a 1.6 cM region near the centromereof chromosome 1.

Production of fertile transgenic rice andwheat plants via mature seedelectroporation

We have developed a novel system thatcan directly deliver foreign genes into plantmature seeds using electroporation. Matureseeds of rice and wheat were soaked in waterovernight. Then the seeds were incubated inelectroporation buffer for 1 h under thecondition of reduced air pressure. Theelectroporation buffer contained spermidine,calcium chloride, PVP, Tween20 and 100µg/ml plasmid DNA. Electroporation wascarried out with a CUY-21 (NEPA GENECo., Ltd, Chiba Japan) in a 1.0 cm-widecuvette containing 1.0 ml of electroporationbuffer. Plasmid DNAs were pWI-GUS (uidA)and/or pWI-H5K (npt II) (Ugaki et al. 1991).Fifty to 100 electric pulses (50 V/cm, 50ms/pulse) were applied in one experiment.After eclectroporation the seeds wereincubated in water supplemented with 0.5%PVP for two days, after which they weretransferred to a selection medium containinggeneticin sulfate (GIBCO). GUS assay wascarried out 48 h after electroporation (Fig. 6).Both in rice and wheat, transgenic plantswere regenerated and self-fertilized seedswere obtained (Fig. 6). Transgene integrationwas confirmed by Southern hybridization.Transmission of the transgene to the nextgeneration (T1) was confirmed by PCRanalysis. This method of transformation is


Fig. 6GUS gene expression in wheat mature seeds into whichpWI-GUS Plasmid was introduced (left) and fertiletransgenic plants (right). A: depressurization pretreatmentwas made prior to electroporation. B: depressurizationpretreatment was not made prior to electroporation. C:Non- transformed control.


simpler and more rapid than conventionaltechniques such as particle bombardment,and it can be applied to a wide range ofcommercial rice and wheat varieties. Thewheat transformation procedure presentedhere does not require the establishment ofgenotype-dependent tissue culture of

immature embryos. At present, thistechnique is dependent on a combination offactors including the pretreatment of seeds,plasmid constructs and optimization ofelectroporation conditions. We have alreadyfinished the patent application.

The research activities of Institute ofRadiation Breeding are focused on thedevelopment of new strains of seed-propagated, vegetatively propagated andwoody crops through mutation by theapplication of various forms of irradiation.Mutations are induced by the followingradiation sources: γ-ray irradiated in theGamma Field, the Gamma Greenhouse andthe Gamma Room, and by ion beams fromHe, C and Ne sources. The institute is alsoinvolved in the development of newtechnologies for plant breeding utilizing γ-rayirradiation and ion beams, including theelucidation of gene expression mechanisms inmutants. The institute provides irradiationservice and cooperative research at therequest of universities, private industries,prefectural experiment stations, andincorporated agencies of Ministry ofAgriculture, Forestry and Fisheries. Themajor topics in fiscal 2003 are as follows.

Non-transmissible large deletionsinduced by γ-ray in Arabidopsisthaliana

In spite of recent numerous molecularanalyses of mutants in plants, comprehensivemolecular characterization of γ-ray inducedmutations has not been undertaken. Toanalyze various mutations induced by γ-rayincluding non-transmissible mutations, weemployed the pollen irradiation system. Theexperimental procedure is as follows (Fig. 1):1) Pollen of wild type male plants is

irradiated. 2) The irradiated pollen is appliedto a female plant that is homozygous for arecessive marker mutation. 3) F1 plants are

I nstitute of Radiation Breeding

Fig. 1Principle of the experiment. Pollen of male parent linesare irradiated and crossed with female parent linescarrying a recessive marker mutant gene (a). Theresultant F1 plants are screened for the markerphenotype. Those mutants were analyzed with molecularmarkers around the marker locus, which distinguish Coland Ler genomes. This enables to detect deleted regionsin Col genome in the F1 plants.

screened for the recessive marker phenotype.Because all tissues of an F1 plant are derivedfrom a single fertilized egg, this approachavoids chimeras, which have made themolecular analysis of mutations difficult.

In this study, Arabidopsis thalianaecotype Columbia (Col) was used as the maleparent for mutagenesis and ecotypeLandsberg erecta (Ler) as the female parent.This enabled us to identify radiation-inducedmutations in pollen using molecular markersdistinguishing Col and Ler genomes. As arecessive visible marker mutation, atrichomeless mutation, gl1-1 in the Lerbackground, was used. The male sterilemutation ms1-1 in the Ler background wasalso introduced into the female plant lines tocut the labor of emasculation.

In the gl1 mutants induced by γ-ray, onlythree of 44 retained the GL1 sequence fromthe male parent (Col). Two of them had 1-and 4-bp deletions in the third exon. One hada reciprocal translocation with a complexstructure involving GL1. The CAPs/dCAPsanalysis revealed that all the remainingmutants carried large deletions, rangingfrom 80 kbp to more than 6 Mbp, whichcorrespond to more than 5 % of the wholegenome. The average size of the deletionswas 2.3 Mbp.

Rugged shape of seed pods was observedin majority of the γ-ray-induced gl1 mutants.In those seed pods, only about half of seedshave been developed normally. Interestingly,all of these semi-sterile mutants carried largedeletions. When progenies of the semi-sterilemutants were analyzed, no Col genotypes formarkers closely linked to the GL1 locus weredetected in the F2 progenies. Theseobservations suggest that the large deletionscannot be transmitted through either male orfemale gametes probably because of gametelethality. It suggests that the large deletionsinvolve not only GL1 but also a geneessential for gamete formation or gameteviability. We made a similar observation inanother locus, TTG1, suggesting that suchgenes are not rare and are dispersed inArabidopsis genome.

Dose response and mutants inducedby chronic γ-ray irradiation in rose

The production of fresh cut stem roses is aprofitable economic industry throughoutJapan. Typically, during the summermonths, high temperatures result in areduction of stem length. A reduction in thestem length of cut roses is considered in themarketplace as a rose of lower quality.However, the cultivar “Samantha” does notexhibit this tendency and long stem roses canbe produced throughout the growing season.

Therefore, in collaboration with theOkinawa Prefectural Agricultural Experi-ment Station (OAES), new varieties possessingvarious color and flower shapes have beenproduced by induced mutations in the rosecultivar “Samantha” via chronic irradiation.

Vegetatively propagated small plants of“Samantha” were planted in a gamma fieldat a position where the irradiation dose rateranged 0.25 - 1.5 Gy/day in terms of 20 hoursper day, that is, these plants were irradiatedtotal exposure of 98.4 - 590.6 Gy in 16months. Stem length of the plants reducedwith increasing irradiation dose rate (Fig. 2).It was presumed from the data that theoptimum irradiation dose rate for efficientmutation induction is between 0.25 Gy/dayand 1 Gy/day. During chronic irradiation,shoots were cut back repeatedly to enlargepotential mutant sectors and evaluate thecolor of the flower petals (Fig. 3). In thismanner, mutants in flower color wereisolated. Such mutants were propagated bycuttings and the mature plants were checkedfor chimera. Individuals exhibiting non-


Fig. 2Dose response of shoot elongation to irradiation dose rate


chimeral mutations in flower color wereretained. In some individuals, modificationsof flower shape or in plant height were alsoobserved. Following the evaluation of thesecharacteristics in the nursery at OAES, fivemutant lines were selected and were appliedfor registration as new varieties.

The effects of heavy-ion beamirradiations on apple and Japanesepear

In fruit tree crops, mutation breeding hasplayed an important role in providing specificcharacters such as resistance to toxinsproduced by host-specific disease and fruitmaturity. It has, however, been difficult byartificial induction to obtain mutants havingother important agricultural characteristics.In recent years, the effects of heavy-ion beamirradiation for inducing mutation werereported to be different from those of γ-rayirradiation in ornamental flower crops. Theobjective of this study is to evaluate theeffects of heavy-ion beam irradiation forimproving mutation breeding of tree fruitcrops and the effects of heavy-ion beamirradiations on apple and Japanese pear

were investigated. Dormant scions of apple (cv. Fuji) and

Japanese pear (cv. Kosui) were used as theirradiation materials. Scions were irradiatedwith carbon(12C6+) and neon(20Ne10+) ions atmaximum linear energy transfer(LET), 84.1keV/µm and 42.0 keV/µm, respectively atsurface of the materials in the heavy-ionirradiation room of the RIKEN AcceleratorResearch Facility (RARF). For adjusting theLET of both ions to about 8 mm in depthfrom the surface of scions, an absorber wasinserted between the irradiation source andthe scions. Forty-nine scions were used foreach dose treatment and grafted onto therootstocks. The rate of survived scions whichdevelop new shoot and visible mutation wereinvestigated.

The survival rate decreased with theincrease in exposure dose. The lethal dose 50(LD50) both for apple and for Japanese pearwere estimated in carbon and neon ions,respectively (fig. 4 and 5). Abnormal shape inthe first leaves were observed, but the newleaves following them showed normal shapein most of the surviving plants. Onevariegated leaf (partly chlorophyll deficient)mutant of apple was observed at 7.5Gy ofneon ion irradiation. Further observations,however, are required to determine visiblemutation rate by heavy-ion beam irradiationin fruit tree crops due to possible chimericstructures. Using DNA analysis and othermethods, we are now selecting the mutantsshowing self-compatibility, low tree heighthabit, etc.

Fig. 3Sectors observed in petals

Fig. 4Survival rate curves for ion beams on apple (cv. Fuji)

Fig. 5Survival rate curves for ion beams on Japanese pear (cv.Kosui)


List of Publications

1 Akagi S, Takahashi S, Adachi N, Hasegawa K, Sugawara T, Tozuka Y, Yamamoto E, Shimizu M, Izaike Y (2003)In vitro and in vivo developmental potential of nuclear transfer embryos using bovine cumulus cells prepared in fourdifferent conditions Cloning and Stem Cells 5(2):101-108

2 Akamine S, Nakamori K, Chechetka SA, Banba M, Umehara Y, Kouchi H, Izui K, Hata S (2003) cDNA cloning,mRNA expression, and mutational analysis of the squalene synthase gene of Lotus japonicus Biochimica etBiophysica Acta 1626:97-101

3 Akimoto-Tomiyama C, Sakata K, Yazaki J, Nakamura K, Fujii F, Shimbo K, Yamamoto K, Sasaki T, Kishimoto N,Kikuchi S, Shibuya N, Minami E (2003) Rice gene expression in response to N-acetylchitooligosaccharide elicitor:comprehensive analysis by DNA microarray with randomly selected ESTs Plant Molecular Biology 52:537-551

4 Aoki N, Ono K, Sasaki H, Seneweera PS, Sakai H, Kobayashi K, Ishimaru K (2003) Effects of elevated CO2

concentration on photosynthetic carbon metabolism in flag-leaf blades of rice before and after heading PlantProduction Science 6(1):52-58

5 Aoki T, O'Donnell K, Homma Y, Lattanzi AR (2003) Sudden-death syndrome of soybean is caused by twomorphologically and phylogenetically distinct species within the Fusarium solani species complex–F. virguliforme inNorth America and F. tucumaniae in South America Mycologia 95(4):660-684

6 Arakaki N, Wakamura S, Yasui H, Sadoyama Y, Kishita M (2003) Sexually differentiated functions of female-produced pheromone of the black chafer Holotrichia loochooana loochooana (Sawada) (Coleopotera: Scarabaeidae)Chemoecology 13:183-186

7 Arakawa T (2003) Chitin synthesis inhibiting antifungal agents promote nucleopolyhedrovirus infection in silkworm,Bombyx mori (Lepidoptera: Bombycidae) larvae Journal of Invertebrate Pathology 83:261-263

8 Asaoka K (2003) Ultrastructure of maxillary sensilla in the silkworm, Bombyx mori: differences among strains?Journal of Insect Biotechnology and Sericology 72:117-125

9 Bing YZ, Che LM, Hirao Y, Takenouchi N, Rodriguez-Martinez H, Nagai T (2003) Parthenogenetic activation andsubsequent development of porcine oocytes activated by a combined electric pulse and butyrolactone l treatmentJournal of Reproduction and Development 49(2):159-166

10 Birkett MA, Chamberlain K, Guerrieri E, Pickett JA, Wadhams LJ, Yasuda T (2003) Volatiles from whitefly-infestedplants elicit a host-locating response in the parasitoid, Encarsia formosa Journal of Chemical Ecology29(7):1589-1600

11 Borisov AY, Madsen LH, Tsyganov VE, Umehara Y, Voroshilova VA, Batagov AO, Sandal N, Mortensen A, SchauserL, Ellis N, Tikhonovich IA, Stougaard J (2003) The sym35 gene required for root nodule development in pea is anortholog of nin from Lotus japonicus Plant Physiology 131:1009-1017

12 Bosak N, Faraut T, Mikawa S, Uenishi H, Kiuchi S, Hiraiwa H, Hayashi T, Yasue H (2003) Construction of a high-resolution comparative gene map between swine chromosome region 6q11 → 921 and human chromosome 19q-armby RH mapping of 51 genes Cytogenetic and Genome Research 102:109-115

13 Bosak N, Fujisaki S, Kiuchi S, Hiraiwa H, Yasue H (2003) Assignment1 of DNA cytosine-5-methyltransferase1(DNMT1) gene to porcine chromosome 2q21 → q 22 by somatic cell and radiation hybrid panel mappingCytogenetic and Genome Research 101:178D

14 Bosak N, Fujisaki S, Kiuchi S, Hiraiwa H, Yasue H (2003) Assignment1 of the gene (MBD3) encoding methyl-CpGbinding domain protein 3 to porcine chromosome 2q21 → q 22 by somatic cell and radiation hybrid panel mappingCytogenetic and Genome Research 101:178E

Original Papers


15 Choi P, Mano Y, Ishikawa A, Odashima M, Umezawa T, Fujimura T, Takahata Y, Komatsuda T (2003)Construction of a high-density AFLP and SSR map using recombinant inbred lines of cultivated X weedy soybeanBreeding Science 53:335-344

16 Crackower AM, Kolas KN, Noguchi J, Sarao R, Kikuchi K, Kaneko H, Kobayashi E, Kawai Y, Kozieradzki I, LandersR, Mo R, Hui C-C, Nieves E, Cohen EP, Osborne RL, Wada T, Kunieda T, Moens BP, Penninger MJ (2003)Essential role of Fkbp6 in male fertility and homologous chromosome pairing in meiosis Science 300:1291-1295

17 Daniel X, Sugano S, Tobin EM (2004) CK2 phosphorylation of CCA1 is necessary for its circadian oscillatorfunction in Arabidopsis Proceedings of the National Academy of Science USA 101(9):3292-3297

18 Debi BR, Mushika J, Taketa S, Miyao A, Hirochika H, Ichii M (2003) Isoloation and characterization of a shortlateral root mutant in rice (Oryza sativa L.) Plant Science 165:895-903

19 Dedos SG, Szurdoki F, Szekacs A, Shiotsuki T, Hammock BD, Shimada J, Fugo H (2002) Fenoxycarb levels andtheir effects on general and juvenile hormone esterase activity in the hemolymph of the silkworm, Bombyx moriPesticide Biochemistry and Physiology 73:174-187

20 Demura T, Tashiro G, Horiguchi G, Kishimoto N, Kubo M, Matsuoka N, Minami A, Nagata-Hiwatashi M, Nakamura K,Okamura Y, Sassa N, Suzuki S, Yazaki J, Kikuchi S, Fukuda H (2002) Visualization by comprehensive microarrayanalysis of gene expression programs during transdifferentiation of mesophyll cells into xylem cells Proceedings ofthe National Academy of Sciences USA 99(24):15794-15799

21 Fabre-Nys C, Chesneau D, De La Riva C, Hinton MR, Locatelli A, Ohkura S, Kendrick KM (2003) Biphasic role ofdopamine on female sexual behaviour via D2 receptors in the mediobasal hypothalamus Neuropharmacology44:354-366

22 Federici MT, Vaughan DA, Tomooka N, Kaga A, Wang XW, Doi K, Marta F, Zorrilla G, Saldain N (2001) Analysisof Uruguayan weedy rice genetic diversity using AFLP molecular markers Electronic Journal of Biotechnology4(3):130-145

23 Fichtner L, Jabronowski D, Schierhorn A, Kitamoto-K H, Stark MJR, Schaffrath R (2003) Elongator's toxin-target(TOT) function is nuclear localization sequence dependent and suppressed by post-translational modificationMolecular Microbiology 49(5):1297-1307

24 Fujii Y, Okuda D, Fujimoto Z, Horii K, Morita T, Mizuno H (2003) Crystal structure of trimestatin, a disintegrincontaining a cell adhesion recognition motif RGD Journal of Molecular Biology 332:1115-1122

25 Fujimori S, Washio T, Higo K, Ohtomo Y, Murakami K, Matsubara K, Kawai J, Carninci P, Hayashizaki Y, Kikuchi S,Tomita M (2003) A novel feature of microsatellites in plants: a distribution gradient alongthe direction oftranscription FEBS Letters 554:17-22

26 Fujimoto Z, Kaneko S, Kuno A, Kobayashi H, Kusakabe I, Mizuno H (2004) Crystal structures of decoratedxylooligosaccharides bound to a family 10 xylanase from Streptomyces olivaceoviridis E-86 Journal of BiologicalChemistry 279(10):9606-9614

27 Fujimoto Z, Kaneko S, Momma M, Kobayashi H, Mizuno H (2003) Crystal structure of rice α-galactosidasecomplexed with D-galactose Journal of Biological Chemistry 278(22):20313-20318

28 Fujimoto Z, Kim W-D, Kaneko S, Park G-G, Momma M, Kobayashi H, Mizuno H (2003) Crystallization andpreliminary X-ray crystallographic studies of α-galactosidase I from Mortierella vinacea Acta CrystallographicaSection D 59:2289-2291

29 Fujisaki S, Sugiyama A, Eguchi T, Watanabe Y, Hiraiwa H, Honma D, Saito T, Yasue H (2004) Analysis of a full-length cDNA library constructed from swine olfactory bulb for elucidation of expressed genes and their transcriptioninitiation sites Journal of Veterinary Medical Science 66(1):15-23

30 Fujisawa M, Nakayama S, Nishio T, Fujishita M, Hayashi K, Ishizaki K, Kajikawa M, Yamato TK, Fukuzawa H,Ohyama K (2003) Evolution of ribosomal DNA unit on the X chromosome independent of autosomal units in theliverwort Marchantia polymorpha Chromosome Research 11(7):695-703

31 Fujishima-Kanaya N, Toki D, Suzuki K, Sawazaki T, Hiraiwa H, Iida M, Hayashi T, Uenishi H, Wada Y, Ito Y, Awata T(2003) Develepment of 50 gene-associated microsatellite markers using BAC clones and the construction of alinakge map of swine chromosome 4 Animal Genetics 34:135-141

32 Fukayama H, Hatch MD, Tamai T, Tsuchida H, Sudo S, Furbank RT, Miyao M (2003) Activity regulation andphysiological impacts of maize C4-specific phosphoenolpyruvate carboxylase overproduced in transgenic rice plantsPhotosynthesis Research 77:227-239

33 Fukuda A, Chiba K, Maeda M, Nakamura A, Maeshima M, Tanaka Y (2004) Effect of salt and osmotic stresses onthe expression of genes for the vacuolar H+-pyrophosphatase, H+-ATPase subunit A, and Na+/H+ antiporter frombarley Journal of Experimental Botany 55(397):585-594

34 Fukuda A, Nakamura A, Tagiri A, Tanaka H, Miyao A, Hirochika H, Tanaka Y (2004) Function, intracellularlocalization and the importance in salt tolerance of a vacuolar Na+/H+ antiporter from rice Plant and CellPhysiology 45(2):146-159

35 Fukumoto F, Masuda Y, Hanada K (2003) Pea tissue necrosis induced by Cucumber mosaic virus alone ortogether with Watermelon mosaic virus Plant Disease 87(4):324-328

36 Fukuoka S, Natalya A, Ebana K, Luu NT, Nagamine T (2003) Analysis of Vietnamese rice germplasm provides aninsight into Japonica rice differentiation Plant Breeding 122:497-502

37 Furusawa T, Hosoe M, Ohkoshi K, Takahashi S, Kiyokawa N, Fujimoto J, Amemiya H, Suzuki S, Tokunaga T(2003) Catalytic RAG1 mutants obstruct V(D)J recombination in vitro and in vivo Molecular Immunology 39:871-878

38 Futani A, Tsuge S, Oku T, Tsuno K, Inoue Y, Ochiai H, Kaku H, Kubo Y (2003) Hpa1 secretion via type IIIsecretion system in Xanthomonas oryzae pv. oryzae Journal of General Plant Pathology 69:271-275

39 Goto T, Noda H, Hong X-Y (2003) Wolbachia distribution and cytoplasmic incompatibility based on a survey of 42spider mite species (Acari: Tetranychidae) in Japan Heredity 91:208-216

40 Goto Y, Niimi S, Hayakawa T, Miyashita T (2004) Preparation of lactose-silk fibroin conjugates and theirapplication as a scaffold for hepatocyte attachment Biomaterials 25:1131-1140

41 Gusev O, Ikeda H, Okouchi T, Lee JM, Hatakeyama M, Kobayashi C, Agata K, Yamada H, Saigusa M (2004)Purification and cDNA cloning of the ovigerous-hair stripping substance (OHSS) contained in the hatch water of anestuarine crab Sesarma haematocheir Journal of Experimental Biology 207:621-632

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280 Yamaguchi T, Minami E, Shibuya N (2003) Activation of phospholipases by N-acetylchitooligosaccharide elicitor insuspension-cultured rice cells mediates reactive oxygen generation Physiologia Plantrum 118:361-370

281 Yamaguchi T, Nagasawa N, Kawasaki S, Matsuoka M, Nagato Y, Hirano H (2004) The YABBY gene DROOPINGLEAF regulates carpel specification and midrib development in Oryza sativa Plant Cell 16:500-509

282 Yamakawa H, Kato S, Seo S, Mitsuhara I, Kamada H, Ohashi Y (2004) Plant MAPK phosphatase interacts withcalmodulins Journal of Biological Chemistry 279(2):928-936

283 Yamamoto T, Kimura S, Mori Y, Uchiyama Y, Ishibashi T, Hashimoto J, Sakaguchi K (2003) Interaction betweenproliferating cell nuclear antigen and JUN-activation-domain-binding protein 1 in the meristem of rice, Oryza sativa L.Planta 217:175-183

284 Yamauchi N, Kizaki K, Yamada O, Takahashi T, Herath CB, Hashizume K (2003) Expression of integrin subunitsdepend on bovine endometrial stromal cells cultured in vitro Connective Tissue 35:1-7

285 Yamauchi N, Takezawa T, Kizaki K, Herath CB, Hashizume K (2003) Proliferative potential of endometrial stromalcells, and endometrial and placental expression of cyclin in the bovine Journal of Reproduction andDevelopment 49(6):553-560

286 Yamazaki T, Takaoka M, Kato E, Hanada K, Sakita M, Sakata K, Nishiuchi Y, Hirano H (2003) A possiblephysiological function and the tertiary structure of a 4-kDa peptide in legumes European Journal of Biochemistry270(6):1269-1276

287 Yang G, Komatsu S (2004) Molecular cloning and characterization of a novel brassinolide enhanced gene OsBLE1in Oriza sativa seedlings Plant Physiology and Biochemistry 42:1-6

288 Yang G, Matsuoka M, Iwasaki Y, Komatsu S (2003) A novel brassinolide-enhanced gene identified by cDNAmicroarray is involved in the growth of rice Plant Molecular Biology 52:843-854

289 Yang G, Shen S, Yang S, Komatsu S (2003) OsCDPK13, a calcium-dependent protein kinase gene from rice, isinduced in response to cold and gibberellin Plant Physiology and Biochemistry 41:369-374

290 Yasui H, Akino T, Yasuda T, Fukaya M, Ono H, Wakamura S (2003) Ketone components in the contact sexpheromone of the white-spotted longicorn beetle, Anoplophora malasiaca, and pheromonal activity of syntheticketones Entomologia Experimentalis et Applicata 107:167-176

291 Yasui H, Wakamura S, Arakaki N, Kishita M, Sadoyama Y (2003) Anthranilic acid: a free amino-acid pheromone inthe black chafer, Holotrichia loochooana loochooana Chemoecology 13:75-80

292 Yazaki J, Kishimoto N, Nagata Y, Ishikawa M, Fujii F, Hashimoto A, Shimbo K, Shimatani Z, Kojima K, Suzuki K,Yamamoto M, Honda S, Endo A, Yoshida Y, Sato Y, Takeuchi K, Toyoshima K, Miyamoto C, Wu J, Sasaki T, SakataK, Yamamoto K, Iba K, Oda T, Otomo Y, Murakami K, Matsubara K, Kawai J, Carninci P, Hayashizaki Y, Kikuchi S(2003) Genomics approach to abscisic-acid- and gibberellin-responsive genes in rice DNA Research 10(6):249-261

293 Yazaki J, Kojima K, Suzuki K, Kishimoto N, Kikuchi S (2004) The Rice PIPELINE: a unification tool for plantfunctional genomics Nucleic Acids Research 32(1):383-387

294 Yoshikawa M, Yang G, Kawaguchi K, Komatsu S (2003) Expression analyses of β-tubulin isoytype genes in ricePlant and Cell Physiology 44(11):1202-1207

295 Zhang H, Kato Y (2003) Common structural properties specifically found in the CSαβ-type antimicrobial peptides innematodes and mollusks: evidence for the same evolutionary origin? Developmental and ComparativeImmunology 27:499-503

296 Zhao D-F, Yamashita H, Matsuzaki M, Takano T, Abe S, Naito M, Kuwana T (2003) Genetic factors affect thenumber of circulating primordial germ cells in early chick embryos Journal of Poultry Science 40(2):101-113

297 Zong XX, Kaga A, Tomooka N, Wang XW, Han OK, Vaughan DA (2003) The genetic diversity of the Vignaangularis complex in Asia Genome 46:647-658



Author Index

Author Department Paper.No.Akimoto-Tomiyama Biochemistry Department 3

ChiharuAkino Toshiharu Physiology and Genetic Regulation Department 290

Aoki Takayuki Genetic Diversity Department 5

Arakawa Toru Insect Biotechnology and Sericology Department 7

Asaoka Kiyoshi Physiology and Genetic Regulation Department 8

Awata Takashi Genome Research Department 31, 53, 220, 250, 265, 266

Degi Konosuke Institute of Radiation Breeding 136

Ebana Kaoru Genebank 36

Fuchimoto Daiichiro Developmental Biology Department 125, 222

Fujimoto Zui Biochemistry Department 24, 26, 27, 28, 78, 133, 147,

217

Fukayama Hiroshi Plant Physiology Department 32, 129

Fukuda Atsunori Plant Physiology Department 33, 34

Fukuoka Shuuichi Genebank 36

Furusawa Tadashi Developmental Biology Department 37, 63, 172, 173

Goto Yoko Insect Biomaterial and Technology Department 40

Habu Yoshiki Plant Biotechnology Department 254

Hamasima Noriyuki Genome Research Department 43, 138, 265

Hanada Kaoru Department of Research Planning and Coordination 35, 178

Handa Hirokazu Plant Biotechnology Department 45

Hara Wajiro Insect Biotechnology and Sericology Department 171

Harumi Takashi Developmental Biology Department 202, 203

Hashimoto Junji Genetic Diversity Department 68, 94, 114, 240, 283

Hashizume Kazuyoshi Developmental Biology Department 52, 57, 58, 72, 97, 109, 156,

185, 284, 285

Hata Tamako Insect Biomaterial and Technology Department 121

Hatakeyama Masatsugu Developmental Biology Department 41, 226

Hattori Makoto Insect Genetics and Evolution Department 106, 213

Hayasaka Shoji Insect Biotechnology and Sericology Department 47, 96, 127

Hayashi Nagao Genetic Diversity Department 119

Hayashi Takeshi Genome Research Department 12, 31, 53, 250

Higo Ken-ichi Molecular Genetics Department 25, 74, 101

Hinomoto Norihide Insect Genetics and Evolution Department 164, 237

Hirayama Chikara Physiology and Genetic Regulation Department 106

Hirochika Hirohiko Molecular Genetics Department 18, 34, 77, 83, 128, 169, 224,

248

Hirogari Yasuhiro Department of Research Planning and Coordination 59

Hirokawa Masahiko Insect Genetics and Evolution Department 255

Ichikawa Akio Physiology and Genetic Regulation Department 64

Ichikawa Hiroaki Plant Biotechnology Department 65

Imanishi Shigeo Insect Biotechnology and Sericology Department 67, 127, 238

Ishibashi Jun Molecular Biology and Immunology Department 50, 152, 158, 187, 252, 259

Ishikawa Masaya Genetic Diversity Department 210, 247

Ishimaru Ken Plant Physiology Department 4, 70, 71, 84, 120, 180

Ito Yoshiyasu Physiology and Genetic Regulation Department 31, 53

Iwamoto Masao Genome Research Department 74, 125

Izaike Yoshiaki Developmental Biology Department 1, 72, 172, 173, 196, 261

Kadono-Okuda Keiko Genome Research Department 171

Kadowaki Kouichi Genetic Diversity Department 110, 271

Kaga Akito Genetic Diversity Department 22, 278, 297

Author Department Paper.No.Kaku Hisatoshi Genetic Diversity Department 38, 263

Kamimura Manabu Developmental Biology Department 79, 96, 126, 150, 151, 238

Kaneko Hiroyuki Genetic Diversity Department 16, 81, 82

Kaneyama Kanako Developmental Biology Department 242

Kasuya Etsuko Physiology and Genetic Regulation Department 198, 200

Katayose Yuuichi Genome Research Department 277

Katoh Etsuko Biochemistry Department 48, 85, 86, 141, 142, 286

Kato Yusuke Developmental Biology Department 186, 295

Kawagoe Yasushi Plant Biotechnology Department 268

Kawahigashi Hiroyuki Plant Biotechnology Department 87, 88, 184

Kawasaki Shinji Plant Physiology Department 61, 89, 92, 99, 214, 281

Kayano Toshiaki Department of Research Planning and Coordination 44, 184, 199

Kikawada Takahiro Physiology and Genetic Regulation Department 276

Kikuchi Kazuhiro Genetic Diversity Department 16, 81, 82, 222

Kikuchi Shoushi Molecular Genetics Department 3, 20, 25, 91, 92, 160, 181,

182, 183, 218, 275, 292, 293

Kishimoto Naoki Molecular Genetics Department 3, 20, 91, 160, 183, 218, 275,

292, 293

Kitamoto Hiroko Genetic Diversity Department 23, 94

Kitani Hiroshi Molecular Biology and Immunology Department 233

Kiuchi Makoto Department of Research Planning and Coordination 79, 96, 238

Koga-Ban Yasunori Plant Biotechnology Department 44

Komatsu Setsuko Molecular Genetics Department 75, 101, 102, 105, 192, 193,

240, 251, 287, 288, 289, 294

Komatsuda Takao Genetic Diversity Department 15

Komoto Natsuo Insect Genetics and Evolution Department 104

Konno Koutaro Insect Genetics and Evolution Department 106

Kosegawa Eiichi Insect Genetics and Evolution Department 255

Kotaki Toyomi Physiology and Genetic Regulation Department 108

Kouchi Hiroshi Plant Physiology Department 2, 112, 148, 149

Kurisaki Jun-ichi Genetic Diversity Department 93, 131, 264

Kusaba Makoto Institute of Radiation Breeding 113

Machii Hiroaki Insect Biotechnology and Sericology Department 212

Maeda Miki Biochemistry Department 33

Matsubara Yuko Developmental Biology Department 202, 203

Matsumoto Takashi Genome Research Department 277

Meshi Tetsuo Plant Physiology Department 11, 247

Mikawa Satoshi Genome Research Department 103

Minami Eiichi Biochemistry Department 3, 86, 279, 280

Minezawa Mitsuru Genetic Diversity Department 90, 205

Mita Kazuei Genome Research Department 100, 168, 196

Mitsuhara Ichiro Plant Physiology Department 282

Mitsuhashi Tadayoshi Physiology and Genetic Regulation Department 43, 207

Miyahara Kenzo Plant Biotechnology Department 113

Miyao Akio Molecular Genetics Department 18, 34, 77, 83, 128, 169, 248

Miyao Mitsue Plant Physiology Department 32, 129, 132

Miyazawa Mitsuhiro Insect Biomaterial and Technology Department 124, 256

Mizuno Hiroshi Biochemistry Department 24, 26, 27, 28, 62, 78, 122,

123, 133, 147, 177, 217, 277

Momma Mitsuru Biochemistry Department 28, 78, 133

Morishita Toshikazu Institute of Radiation Breeding 135, 136



Author Department Paper.No.Myohara Maroko Developmental Biology Department 143, 144

Nagai Takashi Genetic Diversity Department 9, 56, 76, 125, 222, 229, 230

Nagamine Tsukasa Genetic Diversity Department 36

Nagayasu Ken-ichi Insect Genetics and Evolution Department 171

Naito Mitsuru Developmental Biology Department 146, 202, 203, 204, 296

Nakamura Masatoshi Physiology and Genetic Regulation Department 106

Nakashima Nobuhiko Insect Genetics and Evolution Department 166, 216

Nakayama Shigeki Genetic Diversity Department 30

Nishimura Minoru Institute of Radiation Breeding 113

Nishimura Marie Genetic Diversity Department 163

Nishizawa Yoko Plant Biotechnology Department 73, 95, 130, 219, 262

Noda Hiroaki Insect Genetics and Evolution Department 39, 159, 167

Noguchi Junko Genetic Diversity Department 16, 81, 82

Ochiai Hirokazu Genetic Diversity Department 38, 263

Ohkoshi Katsuhiro Developmental Biology Department 37, 172, 173

Ohkura Satoshi Physiology and Genetic Regulation Department 21, 42

Ohura Masanobu Insect Biotechnology and Sericology Department 174, 175

Okuda Takashi Physiology and Genetic Regulation Department 276

Onishi Akira Developmental Biology Department 125, 222, 242

Saito Toshiyuki Physiology and Genetic Regulation Department 198

Sakumoto Ryousuke Physiology and Genetic Regulation Department 198, 200, 201

Sakurai Michiharu Molecular Biology and Immunology Department 204

Sasaki Takuji Genome Research Department 3, 80, 116, 160, 183, 188,

218, 244, 262, 275, 277, 292

Sato Mamoru Insect Genetics and Evolution Department 206

Sato Toyozou Genetic Diversity Department 137, 145, 260

Sekikawa Kenji Molecular Biology and Immunology Department 227, 228

Seo Shigemi Plant Physiology Department 211, 239, 282

Shimoda Masami Insect Biotechnology and Sericology Department 134, 165

Shimura Sachiko Developmental Biology Department 79

Shiotsuki Takahiro Developmental Biology Department 19, 69

Sugano Shouji Plant Physiology Department 17, 223

Sugimoto Kazuhiko Molecular Genetics Department 224

Suto Jun-ichi Molecular Biology and Immunology Department 227, 228

Suzuki Shun-ichi Developmental Biology Department 232

Tabei Yutaka Plant Biotechnology Department 44, 95

Takahashi Hideaki Genetic Diversity Department 90

Takahashi Toru Developmental Biology Department 52, 54, 72, 97, 156, 185, 284

Takaiwa Fumio Plant Biotechnology Department 176, 189, 190, 234

Takano Makoto Institute of Radiation Breeding 113

Takase Kenji Biochemistry Department 78

Takatsuji Hiroshi Plant Physiology Department 48, 223

Takenouchi Takato Molecular Biology and Immunology Department 46, 219

Takeuchi Kasumi Genetic Diversity Department 243

Takeya Masaru Genetic Diversity Department 245

Takezawa Toshiaki Physiology and Genetic Regulation Department 241, 246, 285

Tamura Toshiki Insect Biotechnology and Sericology Department 66, 231

Tamura Yasumori Insect Genetics and Evolution Department 106

Tanaka Hiromitsu Molecular Biology and Immunology Department 158, 187

Tanaka Hiroshi Plant Biotechnology Department 44, 199

Tanaka Seiji Physiology and Genetic Regulation Department 194, 252

Author Department Paper.No.Tanaka Yoshiyuki Plant Physiology Department 33, 34, 194, 252

Tateishi Ken Department of Research Planning and Coordination 106

Tatematsu Ken-ichirou Insect Genetics and Evolution Department 255

Teramoto Hidetoshi Insect Biotechnology and Sericology Department 256

Tokunaga Tomoyuki Developmental Biology Department 37, 55, 63, 172, 173, 261

Tomita Shuuichiro Insect Genetics and Evolution Department 209

Tomiyama Masamitsu Genetic Diversity Department 170, 219

Tomooka Norihiko Genetic Diversity Department 22, 278, 297

Tsuji Noriko Molecular Biology and Immunology Department 264

Tsukada Masuhiro Insect Biomaterial and Technology Department 235, 236

Ueda Tadamasa Molecular Genetics Department 208

Uenishi Hirohide Genome Research Department 12, 31, 53, 60, 138, 220, 250,

265, 266

Ueno Osamu Plant Physiology Department 267, 274

Umehara Yousuke Plant Physiology Department 2, 11, 148, 191

Vaughan Genetic Diversity Department 22, 269, 270, 271, 278, 297

Duncan AlexanderWada Sanae Insect Genetics and Evolution Department 272

Wakamura Sadao Physiology and Genetic Regulation Department 6, 290, 291

Watanabe Hirofumi Insect Genetics and Evolution Department 117, 118, 225

Watanabe Kenji Insect Genetics and Evolution Department 206

Watanabe Masahiko Physiology and Genetic Regulation Department 276

Yamaguchi Hiroyasu Institute of Radiation Breeding 136

Yamakawa Minoru Molecular Biology and Immunology Department 50, 152, 153, 154, 155, 158,

187, 259

Yamamoto Kimiko Genome Research Department 160, 181, 183, 218, 275

Yamazaki Muneo Molecular Genetics Department 248

Yamazaki Toshimasa Biochemistry Department 48, 51, 85, 86, 179, 286

Yano Masahiro Molecular Genetics Department 49, 115, 116, 161, 188, 208,

244, 277

Yasuda Tetsuya Physiology and Genetic Regulation Department 10, 290

Yasue Hiroshi Genome Research Department 6, 12, 13, 14, 29, 53, 60, 139,

140, 157, 162, 221, 250, 266,

273, 290, 291

Yasukochi Yuji Genome Research Department 196

Yoshikawa Manabu Molecular Genetics Department 294

Yukuhiro Kenji Insect Genetics and Evolution Department 104, 253



1 Sasaki T (2002) Rice genome analysis: Understanding the genetic secrets of the rice plant Breeding Science53:281-289

2 Sasaki T (2003) Complete and accurate sequencing of the entire rice genome of Oryza sativa japonica Nipponbareby the IRGSP Rice Sciences: Innovation and Impact for Livelihood. Proceedings of the International RiceResearch Conference 147-155

3 Sasaki T (2004) Rice Encyclopedia of Plant Crop Science 1102-1105

4 Vaughan DA (2003) Genepools of Genus Oryza Monograph on Genus Oryza 113-138

5 Vaughan DA (2003) Biosystematics of the Genus Oryza RICE - Origin, Histry, Technology, and Production27-65

6 Kikuchi K (2003) Developmental competence of porcine blastocysts produced in vitro Journal of Reproductionand Development 50:21-28

7 Kikuchi K (2003) In vitro embryo production in pigs Hungarian Journal of Animal Production 52:115-122

8 Okuno K, Ebana K (2003) Identification of QTL controlling allelopathic effects in rice: genetic approaches tobiological control of weeds Japan Agricultural Research Quarterly 37(2):77-81

9 Naito M (2003) Development of avian embryo manipulation techniques and their application to germ cellmanipulation Animal Science Journal 74:157-168

10 Naito M (2003) Genetic manipulation in chickens World's Poultry Science Journal 59:361-371

11 Kren R, Kikuchi K, Nakai M, Miyano T, Ogushi S, Nagai T, Suzuki S, Fulka J, Fulka J Jr (2003) Intracytoplasmicsperm injection in the pig : Where is the problem? Journal of Reproduction and Development 49(4):271-273

12 Dinnyes A, Bagis H, JI W, Kikuchi K, Lee JW, LI X, Nagai T, Presicce GA, Somfai T, Si W, Yang X (2003) Genebanking in rare breeds and species whose gametics are difficult to cryopreserrve Allattenyesztes esTakarmanyozas 52:82-91

13 Sun QY, Nagai T (2003) Molecular mechanisms underlying pig oocyte maturation and fertilization Journal ofReproduction and Development 49(5):347-359

14 Fulka Jr. J, Miyashita N, Nagai T, Ogura A (2004) Do cloned mammals skip a reprogramming step? NatureBiotechnology 22(1):25-26

15 Okuda K, Sakumoto R (2003) Multiple roles of TNF super family members in corpus luteum functionReproductive Biology and Endocrinology 1:95

16 Sakumoto R, Okuda K (2004) Possible actions of tumor necrosis factor-α in ovarian function Journal ofReproduction and Development 50(1):39-46

17 Hattori M (2003) Electronic monitoring of feeding and oviposition behavior of rice planthoppers and its applicationin plant resistance study Chinese Journal of Rice Science 17:31-36

18 Izawa T, Takahashi Y, Yano M (2003) Comparative biology comes into bloom: genomic and genetic comparison offlowering pathways in rice and Arabidopsis Current Opinion in Plant Biology 6:113-120

19 Yazaki J, Kishimoto N, Ishikawa M, Kikuchi S (2002) Rice expression database: the gateway to rice functionalgenomics Trends in Plant Science 7:563-564

20 Yang G, Komatsu S (2003) Brassinosteroid signaling: From perception to gene expression Recent ResearchDevelopments in Plant Cell Physiology 1:7-17

21 Sharma A, Komatsu S (2004) Calreticulin: A chaperon sharing multiple functions in rice Recent ResearchBiochemistry 5:103-113

22 Murata K, Kuroki S, Kato E, Ando I (2003) A study of conformational stability of polypeptide blends by solid-stateNMR spectroscopy Annual Reports on NMR Spectroscopy 51:1-57

Reviews


1 Von Bothmer R, Sato K, Komatsuda T, Yasuda S, Fischbeck G (2003) The domestication of cultivated barleyDiversity in Barley (Hordeum vulgare) 9-27

2 Nagai T, Kikuchi K (2002) Current status and perspectives in cloning and related studies Cloning and StemCells 4(3):179

3 Takeda S (2003) Bombyx mori Encyclopedia of Insects 133-135

4 Takeda S (2003) Sericulture Encyclopedia of Insects 1027-1029

Monographs


The 11th NIAS International Workshop on Genetic Resources: “The genusOryza”

With the near completion of the complete sequencing of the rice genome there is increasinginterest in the genomes of the relatives of rice in the genus Oryza. Consequently NIASorganised a workshop on the topic of the genus Oryza bringing together leaders in the fieldfrom all over the world. From the 24th to the 26th of September 2003, 100 participantsattended the workshop 18 from outside Japan. Among the speakers were Dr. Rod Wing andDr. Scott Jackson from the US who are currently involved in a major Oryza project thatincludes development of BAC libraries of all Oryza genomes. Present and former staff of theNational Institute of Genetics, Japan, a lead institute in Japan in Oryza research, deliveredseveral contributions. In addition to the oral sessions a poster session was arranged so thatyoung rice researchers could fully participate in the workshop. It is likely that in the next fewyears wild Oryza will become far more than a novelty value but increasingly used in riceimprovement.

NIAS/COE International Symposium: “Endosymbiosis and MolecularEvolution in Insects”

The NIAS/COE international Symposium entitled “Endosymbiosis and Molecular Evolutionin Insects” was held on October 29-31, 2003, at Ibaraki Prefectural Kennan Life-long LearningCenter in Tsuchiura. Twenty-five scientists, 11 from overseas and 14 from Japan, were invitedas lecturers on three major topics, Insect Receptors, Molecular Evolution and InsectSymbionts. The session “Insect symbionts” was further divided into three sub-sessions“Buchnera & Aphid Symbionts, Wolbachia, and Other Symbionts.” Recent progress of insectreceptors, mainly G-protein coupled receptors (GPCRs), was shown by the invited speakersand how to find ligands for neuropeptide GPCRs was discussed. Molecular evolution of odorantreceptor genes and spider silk genes attracted the audience's the attention. Progress of wholegenome shotgun sequences of Wolbachia was introduced, and newly-found endosymbioticbacteria, which alters host insect reproduction, were presented by two speakers. One hundred-fifty participants including 20 foreigners enjoyed the invited lectures, poster presentations anddiscussion.

The 11th Animal Genome International Workshop: “The Application ofAnimal Genome Research to Industry”

The animal genome international workshop was held under the title “The application ofanimal genome research to industry” on December 6, 2003 at KKR Hotel Tokyo in Tokyo with

International Meetings and ForeignVisitors

International Meetings

134 participants. Six invited speakers (2 from overseas and 4 Japanese) gave presentations ongenome research achievements and their application to industrialization. Main interest post-genome research studies regarding the human genome have key elements of direct connectionto industry such as development of new drugs and establishment of diagnostic and therapeuticmethods. In livestock animals, it has been progressed marker assisted selection for infectiondisease resistant, meat quality, growth rate, etc.

The NIAS-COE International Symposium: “Protein Trafficking Mechanismand its Application to Molecular Farming”

The NIAS-COE International Symposium entitled “Protein Trafficking Mechanism and itsApplication to Molecular Farming” was held on November 11-12th, 2003, at TsukubaInternational Congress Center (Epocal Tsukuba) with about 130 participants. The purpose ofthis symposium was to discuss recent progress in the fields of molecular mechanisms involvedin membrane trafficking, protein targeting to protein bodies, quality control et al. underlyingproduction of recombinant proteins and their application to molecular farming 17 oralpresentations, 8 from overseas and 9 from Japan, were given. This symposium stimulated theactivities of many participants on plant molecular farming and gave several new ideas as tothe next step.

The International Rice Genome Workshop

The International Rice Genome Workshop was held at the Epochal Tsukuba InternationalCongress Center on Feb 4th & 5th, 2004 as part of the annual Rice Genome Meeting. Thisyear’s workshop included a session on rice genome sequencing on the first day andmanagement of sequence data on the second day. As the international sequencingcollaboration focuses on finishing the entire genome before the end of 2004, severalpresentations dealt with a more in-depth analysis of the structure and various features of thegenome. The utilization of the genome sequence in functional genomics and current studies onapplied genomics were also presented. The session on sequence data management includedpresentations on genome annotation, analysis of full-length cDNA sequences, databases andontology. Representatives from NCBI, EMBL-EBI and MIPS provided insights on plantgenomics resources and analytical frameworks for comparative genomics. A poster sessioncovering various aspects of plant genomics was also held. A total of 178 participants fromvarious institutes, research centers and universities attended the workshop.

The 12th International Rice Genome Forum

The 12th International Rice Genome Forum was held on February 6, 2004 at the main hallof the Epochal Tsukuba International Congress Center, following the 2-day Rice GenomeWorkshop. As part of the annual rice genome meeting, the Forum aims to bring together thescientific and non-scientific community, and to present general topics as well currentperspectives on plant genomics. The presentations this year included genome-wide approachesusing Arabidopsis, rice walking into the wheat genome, construction of physical maps for riceand maize sequencing, legume functional genomics and gene targeting / gene taggingstrategies in rice. A total of 229 participants from various institutes, research centers anduniversities attended the workshop.



Signaling to acquired resistance

An international symposium entitled “Plant Immunity: Signaling to acquired resistance”was held on March 3 and 4, 2004, at Tsukuba International Congress Center, Tsukuba, withmore than 260 attendants. The study of plant defense mechanisms has been a central area inplant science in the last few years. The symposium aimed at outlining recent progress anddiscussing future perspectives in the field. The symposium had two oral sessions, “Signalperception and following defense responses” and “Signal networks in acquired resistance”. Wehad 22 speakers including 7 foreign speakers, who gave us exciting talks on their recentresults. In addition, 38 posters were presented. The topics widely covered the mechanisms ofplant-microbe NIAS-COE/PROBRAIN/TOKUTEI International Symposium: “Plant Immunity:interactions including pathogenic and symbiotic ones.

The first International Conference of Bacterial Blight of Rice

The first International Conference of Bacterial Blight of Rice (ICBB) was held from March17 to 19, 2004 at Tsukuba International Congress Center (Epochal Tsukuba), withapproximately 150 participants from eleven countries. The meeting was organized by theinternational committee of ICBB, and was billed as the “first comprehensive event for theworld’s most important disease of rice”. It was also billed as one of the cycle of events for theInternational Year of Rice. A total of 25 invited speakers gave presentations on the genomicsof rice and the pathogen Xanthomonas oryzae pv. oryzae(Xoo), molecular aspect of rice-Xoointeraction, genetic diversity of Xoo, genetics and breeding strategy for resistance, resistancemechanisms and control of the disease. In addition, the serious status of the outbreak of thedisease in Indonesia, Thailand, India and Pakistan was reported in the session of countryreport. The conference was accompanied by two-hour poster sessions (34 presentations) for twodays. Through the conference, the bacterial bight of rice was confirmed to be one of the mostimportant system for analysis of plant-microbe interaction and also to be one of the mostserious pests for rice in Asia. The 2nd meeting of ICBB will be held in Nanjing, China in 2007.


Main Foreign Visitors to NIAS

21 May. : Dr. Thomas Lumpkin

Director General, Asian Vegetable Research and Development Center: AVRDC (Taiwan)

23 May. : Prof. Nguyen Duc Hung

Vice president, Hue University (Viet Nam)

16 Jul. : Dr. Badaruddin Soomro

Chairman, Pakistan Agricultural Research Council, Ministry of Food, Agriculture & Livestock

(Pakistan)

Dr. Rashid Anwar

Deputy Director Deneral, Institute of Agri. Biotechnology and Genetic Resources (Pakistan)

23 Jul. : Dr. Wiliam Dar

Director General, International Crops Research Institute for the Semi-Arid Tropics:ICRISAT

(India)

24 Jul. : Dr. David Ho

Director, Academia Sinica Plant Genomue Center (Taiwan)

6 Aug. : Datuk Abi Musa Ashaari

Secretary General, Ministry of Agriculture (Malaysia)

Datuk Dr. Saharan Hj

Director General, Malaysian Agricultural Research & Development Institute:MARDI (Malaysia)

Dato' Ismail Ibrahim

Director General, Department of Agriculture (Malaysia)

Dato' Dr. Hawari Hussein

Director General, Department of Veterinary Services (Malaysia)

14 Nov. : Dr. Clive James

Chairman, International Service for the Acquisition of Agri-biotech Applications: ISAAA (USA)

22 Jan. : Mr. Simon Moeketsi Modisenyane

Deputy Director, Department of Science and Technology (South Africa)

10 Feb. : Dr. William Erskine

Vice Director, International Center for Agricultural Research in the Dry Areas :ICARDA (Syria)

19 Feb. : Mr. Francisco Lopez Tostado

Vice Minister Agriculture, Secretary of Agriculture, Livestock, Rural, Development,

Fisheries and Food (Mexico)

(April 2003-March 2004)


Mr. Eduardo Benitez Paulin

General Director for Technological Development, Secretary of Agriculture, Livestock, Rural,

Development, Fisheries and Food (Mexico)

12 Mar. : Mr. Marcelo Tolchinsky

Director, National Institute for Agricultural Technology, MarcosJuarez Agricultural Experimental

Station (Argentine)

15 Mar. : Dr. Li Yiren

Director General, The Chinese Academy of Agricultural Sciences, Institute of Sericulture (China)


Executive Members andResearch Staff Members

(as of March 31,2004)

Executive MembersPresident Iwabuchi Masaki

Vice President Kitamura Chikayoshi

Vice President Higo Kenichi

Auditor Motoi Yoshiko

Auditor Matsui Takehisa

Research StaffDepartment of Research Planning and Director Shinbo Hiroshi

CoordinationSenior Research Planner Miyashita Kiyotaka

Research Planner Hirai Kazuo

Research Planner Awata Takashi

Research Planner Kadowaki Kouichi

Research Planning Section Head Kiuchi Makoto

Research Evaluation Section Head Hanada Kaoru

Research Coordination Section Head Shirata Akira

Imai Tsuneo

Office for GMO Research and Development Head Tabei Yutaka

Watanabe Shinichirou

Technology Transfer Section Head Ogawa Masafumi

Kayano Toshiaki

Hirogari Yasuhiro

Field Management Section Head Obo Masahiro

Head Kawauchi Ikuo

Head Kawakatsu Masao

Genome and Biodiversity Research Division Director Obata Taro

Genome Research Department Director Sasaki Takuji

Research Leader Yasue Hiroshi

Plant Genome Laboratory Head Matsumoto Takashi

Katayose Yuuichi

Mizuno Hiroshi

Animal Genome Laboratory Head Awata Takashi

Hamasima Noriyuki

Hayashi Takeshi

Mikawa Satoshi

Uenishi Hirohide

Insect Genome Laboratory Head Mita Kazuei

Kadono Keiko

Yamamoto Kimiko

Yasukochi Yuji

Bioinfomatics Laboratory Maeda Miki

Itou Takeshi

Numa Hisataka

Baba Koutarou

DNA Bank Head Nagamura Yoshiaki

Baltazar Alcaraz Antonio


Iwamoto Masao

Rice Genome Resource Center Nagamura Yoshiaki

Miyao Akio

Genetic Diversity Department Director Kurisaki Junichi

Research Leader Hashimoto Junji

Research Leader Kaku Hisatoshi

Research Leader Umehara Masamichi

Molecular Biodiversity Laboratory Head Kadowaki Kouichi

Nakayama Shigeki

Nishikawa Tomotarou

Takahashi Sakiko

Biosystematics Laboratory Aoki Takayuki

Ochiai Hirokazu

Evolutionary Dynamics Laboratory Head Duncan Alexander Vaughan

Tomooka Norihiko

Kaga Akito

Germ Cell Conservation Laboratory Head Kaneko Hiroyuki

Noguchi Junko

Kikuchi Kazuhiro

Applied Microbiology Laboratory Head Hayashi Nagao

Tomiyama Masamitsu

Kitamoto Hiroko

Nishimura Marie

Adaptation Systems Laboratory Ishikawa Masaya

Komatsuda Takao

Biometrics Laboratory Satou Masahiro

Takeya Masaru

Genebank Director Okuno Kazutoshi

Research Leader Shirata Kazuto

Research Leader Nagamine Tsukasa

Niino Takao

Plant Genetic Resources Laboratory Kawase Makoto

Ebana Kaoru

Fukuoka Shuuichi

Kojima Yoichiro

Microorganism Genetic Resources Laboratory Head Sato Toyozou

Nagai Toshirou

Tomioka Keisuke

Takeuchi Kasumi

Animal Genetic Resources Laboratory Head Minezawa Mitsuru

Takahashi Hideaki

Kawada Masae

Developmental Biology Department Director Izaike Yoshiaki

Research Leader Furutachi Hiroshi

Developmental Mechanisms Laboratory Head Myohara Maroko

Nakao Hajime

Kato Yusuke

Hatakeyama Masatsugu

Development and Differentiation Laboratory Head Tokunaga Tomoyuki

Furusawa Tadashi

Ohkoshi Katsuhiro

Animal Genetic Engineering Laboratory Head Naito Mitsuru

Matsubara Yuko

Harumi Takashi

Embryonic Technology Laboratory Head Nagai Takashi

Onishi Akira

Watanabe Satoshi

Fuchimoto Daiichiro

Suzuki Shunichi

Insect Growth Regulation Laboratory Head Shiotsuki Takahiro

Tanaka Yoshiaki

Tateishi Ken

Kamimura Manabu

Shimura Sachiko

Reproductive Biology and Technology Laboratory Takahashi Toru

Kanayama Kanako

Molecular Biology and Immunology Department Director Sekikawa Kenji

Research Leader Sakurai Michiharu

Molecular Immunology Laboratory Head Kitani Hiroshi

Tsuji Noriko

Takenouchi Takato

Satou Mitsuru

Innate Immunity Laboratory Head Yamakawa Minoru

Ishibashi Jun

Tanaka Hiromitsu

Experimental Animals Laboratory Head Goto Hideo

Suto Junichi

Matsumoto Yukiko

Physiology and Genetic Regulation Department Director Kawasaki Kenjiro

Research Leader Hirai Yoshio

Research Leader Inouchi Jun

Insect Life-Cycles and Physiology Laboratory Head Tanaka Seiji

Okuda Takashi

Kotaki Toyomi

Watanabe Masahiko

Insect Nutrition and Metabolism Laboratory Head Nakamura Masatoshi

Hirayama Chikara

Kikawada Takahiro

Insect Neurobiology Laboratory Head Asaoka Kiyoshi

Inoue Hisashi

Ichikawa Akio

Kihara Mami

Insect Behavior Laboratory Head Wakamura Sadao

Yasuda Tetsuya

Akino Toshiharu

Yasui Hiroe

Animal Gene Function Laboratory Head Mitsuhashi Tadayoshi

Kojima Misaki

Ito Yoshiyasu

Animal Cell Biology Laboratory Takezawa Toshiaki

Miyashita Norikazu

Animal Neurophysiology Laboratory Head Saito Toshiyuki



Kasuya Etsuko

Sakumoto Ryousuke

Animal Neuroendocrinology Laboratory Head Okamura Hiroaki

Okura Satoshi

Yayou Kenichi

Insect Genetics and Evolution Department Director Sato Mamoru

Katou Masao

Insect-Plant Interactions Laboratory Head Hattori Makoto

Konno Koutaro

Tamura Yasumori

Natural Enemies Laboratory Head Noda Takashi

Hinomoto Norihide

Maeda Tarou

Symbiosis Laboratory Head Noda Hiroaki

Watanabe Hirofumi

Nakashima Nobuhiko

Watanabe Kenji

Insect Pathology Laboratory Head Miyamoto Kazuhisa

Mitsuhahi Wataru

Wada Sanae

Murakami Ritsuko

Insect Genetics Laboratory Head Kosegawa Eiichi

Hirokawa Masahiko

Tatematsu Kenichirou

Insect Molecular Evolution Laboratory Head Nagayasu Kenichi

Yukuhiro Kenji

Muraji Masahiko

Tomita Shuuichiro

Hasegawa Tsuyoshi

Komoto Natsuo

Insect Biomaterial and Technology Department Director Takeda Satoshi

Research Leader Tsukada Masuhiro

Biopolymer Characterization Laboratory Head Tsubouchi Kozo

Toshima Yoshiyuki

Hata Tamako

Takasu Youko

Biomaterial Development Laboratory Miyazawa Mitsuhiro

Kameda Tsunenori

Biomimetic Laboratory Head Tamada Yasushi

Kobayashi Toru

Goto Yoko

Kuwana Yoshihiko

Kozima Katura

Insect Products Utilization Laboratory Haga Atsunobu

Insect Biotechnology and Sericology Department Director Machii Hiroaki

Research Leader Kato Hiroshi

Research Leader Hayasaka Shoji

Research Leader Hara Wajiro

Research Leader Kinoshita Haruo

Research Leader Ichihashi Takahisa

Insect Cell Engineering Laboratory Head Imanishi Shigeo

Taniai Kiyoko

Akizuki Gaku

Insect Gene Engineering Laboratory Head Tamura Toshiki

Yonemura Naoyuki

Shimoda Masami

Mass Production System Laboratory Head Ohura Masanobu

Koyama Akio

Arakawa Toru

New Silk Materials Laboratory Head Takabayashi Chiyuki

Nakajima Kenichi

Teramoto Hidetoshi

Sericultural Science Laboratory Head Yamamoto Toshio

Mase Keisuke

Okada Eiji

Fukui Kuniaki

Iizuka Tetsuya

Molecular Genetics Department Director Hirochika Hirohiko

Functional Genomics Laboratory Hagiwara Kiyoshi

Miyao Akio

Yamazaki Muneo

Applied Genomics Laboratory Head Yano Masahiro

Izawa Takeshi

Yamamoto Shinichi

Ueda Tadamasa

Epigenetics Laboratory Okuizumi Hisato

Mochizuki Atsuko

Gene Expression Laboratory Head Kikuchi Shoushi

Mori Masaki

Kishimoto Naoki

Gene Regulation Laboratory Head Komatsu Setsuko

Yoshikawa Manabu

Asano Takayuki

Biochemistry Department Director Kobayashi Mikihiko

Crystallography Laboratory Head Mizuno Hiroshi

Takase Kenji

Momma Mitsuru

Fujimoto Zui

Wako Toshiyuki

Biophysics Laboratory Yamazaki Toshimasa

Katoh Etsuko

Glycobiology Laboratory Head Minami Eiichi

Kaku Hanae

Akimoto Chiharu

Membrane Biology Laboratory Head Shimomura Shouji

Kajiwara Hideyuki

Taguchi Fumio

Plant Physiology Department Director Meshi Tetsuo

Research Leader Tanaka Yoshiyuki

Research Leader Kawasaki Shinji

Photosynthesis Laboratory Head Tokutomi Mitsue

Inagaki Noritoshi



Fukayama Hiroshi

Carbon Metabolism Laboratory Head Ueno Osamu

Ishimaru Ken

Sentoku Naoki

Developmental Biology Laboratory Head Takatsuji Hiroshi

Sugano Shouji

Chang-Jie Jiang

Baba Akiko

Environmental Physiology Laboratory Head Takano Makoto

Takeichi Tetsuo

Yazaki Yoshiaki

Kiyota Seiichirou

Disease Physiology Laboratory Head Ishikawa Masayuki

Mitsuhara Ichiro

Fukuda Atsunori

Seo Shigemi

Nitrogen Fixation Laboratory Head Kouchi Hiroshi

Nakayama Yasuji

Umehara Yousuke

Plant Biotechnology Department Director Oka Seibi

Gene Design Laboratory Head Miyahara Kenzo

Ichikawa Hiroaki

Nishizawa Yoko

Plant Gene Engineering Laboratory Head Takaiwa Fumio

Toki Seiichi

Kawagoe Yasushi

Plant Cell Engineering Laboratory Head Tabei Yutaka

Hagio Takashi

Ogawa Taiichi

Habu Yoshiki

Molecular Breeding Laboratory Koga-Ban Yasunori

Otake Yuko

Biosystems Laboratory Head Handa Hirokazu

Kawahigashi Hiroyuki

Institute of Radiation Breeding Director Nagatomi Shigeki

Mutation Genetics Laboratory Head Nishimura Minoru

Kusaba Makoto

Radiation Technology Laboratory Head Morishita Toshikazu

Degi Konosuke

Yamaguchi Hiroyasu

Mutation Breeding Laboratory Head Ito Yuuji

Takyuu Toshio

Yamanouchi Hiroaki


Members of NIAS EvaluationComittee

(as of March 31,2004)

Ueda Ryu National Institute of Genetics

Uchimiya Hirofumi The University of Tokyo

Katsuki Motoya Okazaki National Research Institutes

Kiguchi Kenji Shinshu University

Kimoto Noriko Journalist

Sakaki Yoshiyuki The University of Tokyo

Takeda Kazuyoshi Okayama University

Nakamura Yasuhiko Journalist


thousands of yen

TOTAL BUDGET 12,786,026

OPERATING COSTS 4,906,842

Personnel (434)* 4,294,432

President (1) Vice President (2)Auditor (2)

Administrators I (98)**Administrators II (44)***Researchers (287)

* Number of persons shown in ( )** General administration*** Field management and transportations

Administrative costs 612,410

RESEARCH PROMOTION COSTS 7,879,184

Research Grant from MAFF 3,076,227Entrusted Research Expenses from MAFF 3,523,945Entrusted Research Expenses from MEXT 506,122Entrusted Research Expenses from others 772,890

Personnel4,294,432 (33.6%)

Entrusted ResearchExpenses from others772,890 (6.0%)Entrusted Research

Expenses from MEXT506,122 (4.0%)

Entrusted Research Expenses from MAFF3,523,945 (27.6%)

Administrative costs612,410 (4.8%)

MAFF : Ministry of Agriculture, Forestry and FishriesMEXT : Ministry of Education, Culture, Sports, Science and technology

Research Grant from MAFF3,076,227 (24.0%)

FINANCIAL OVERVIEWFiscal Year 2003 (April 2003- March 2004)

Annual Report 2004(Apr. 2003 Mar. 2004)

No. 3

Printed by ISEBU Printing Co., Ltd. TEL : 029(851)2515

Published by National Institute of Agrobiological Sciences

2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan

TEL : 029(838)7406

FAX : 029(838)7408

URL : http://www.nias.affrc.go.jp/index_e.html

annual report 2004annual report 2004 1 in japan, the silkworm has long been used as a model system...

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