rice hirschmaniella molecular interaction
DESCRIPTION
TRANSCRIPT
CLONING AND EXPRESSION ANALYSIS OF RICE GENES
INVOLVED IN INFECTION WITH HIRSCHMANNIELLA ORYZAE REGMI, HOMAN (S1, ENTOMOLOGY)
Promoter
Prof. Dr Godelieve Gheysen
Faculty of Bio-Science Engineering
Gent University, Belgium
Supervisors
Dr Tina Kyndt and Lander Bauters
Faculty of Bio-Science Engineering
Gent University, Belgium
INTRODUCTION
Rice
major staple food of nearly half of the world’s population
model monocot plant
Genome size: 430 Mb
around 57 thousands sequenced rice genes available in rice genome database for free
Hirschmaniella oryzae
migratory endo-parasite
well adapted in deep water (anaerobic condition)
irrigated rice covers about 72% of total rice production area
the most prevailing nematode in rice world wide
Rice nematode molecular interaction
defense response genes believed to be involved in the
resistance mechanism
very less work done to know about rice and H. oryzae
molecular interaction
RICE GENES SELECTED FOR THE STUDY Locus Gene Expression under H.
oryzae infection
(Kyndt, unpublished)
Other information
LOC_Os08g09080 Cupin domain
containing protein
Up-regulated upregulated upon blast (Swarbrick et
al.,2008) and Striga infection (Marcel
et al., 2010)
LOC_Os11g44910 DEAD-box ATP-
dependent RNA
helicase
Down-regulated down-regulated at early phase of rice
germination (Huang et al., 2009)
LOC_Os03g08880 purine permease Up-regulated upregulated upon M. oryze infection
(Marcel et al., 2010)
LOC_Os04g33830 membrane protein Down-regulated down-regualted in resistant Nipponbare
under Striga hermonthica infection
(Swarbrick et al.,2008)
LOC_Os04g50100 RING-H2 finger
protein
Down-regulated ATL5G has been suggested to be
involved in the early steps of the plant
defense signaling pathway
LOC_Os02g39360 B-box zinc finger
family protein
Down-regulated down-regulated under Xanthomonas
oryzae pv. oryzicola infection (Zhou et
al., 2010)
OBJECTIVES
cloning of the rice genes of interest for rice
transformation
expression analysis of the rice genes under
Hirschmaniella oryzae infection by q-PCR
MATERIALS AND METHOD
Cloning
RNA extraction from 2 week old rice roots and cDNA
synthesized
amplification of concerned genes by PCR
purified PCR products cloned and sequenced
Sequences confirmed by
http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.h
tml
http://rice.plantbiology.msu.edu/index.shtml
MATERIALS AND METHODS…
Infection Experiment
80 well grown 16 days old rice seedlings
40 plants infected by nematode @ 205/plants
40 plants for control(not to be infected)
MATERIALS AND METHODS…
5 plants considered as 1 pool
32 total samples of root and shoot tissues collected
from infected & uninfected plants at 1, 3, 5 and 7 dpi
RNA extracted, cDNA synthesized
Quality check of cDNA sample and qPCR primers
q-PCR
data obtained were analysed by REST-2009 software
RESULTS AND DISCUSSION
CLONING
Amplification of DEAD, purine and Cupin
Amplification of rice genes with 52ºC
annealing temperature for 40 cycles Amplification of rice genes with 45ºC
annealing temperature for 5 cycles
followed by 52ºC for 35 cycles
Cupin was successfully cloned
Rice genes expression pattern
Relative expression level of three rice genes in root tissues at different
time points after H. oryzae infection (infected vs non-infected plants)
Cupin
Cupin highly up regulated at 3 dpi in root tissue under
H. oryzae infection (Kyndt, 2011. unpublished data)
Cupin up-regulated at 2 dpi in Nipponbare rice under
Striga hermonthica infection (Swarbrick et al.,2008) and also
under infection with Magnaporthe oryzae (Marcel et al.,
2010).
Cupin domain gene shows homology with a germin-
like protein from Barley (http://rice.plantbiology.msu.edu/cgi-
bin/ORF_infopage.cgi)
GLPs in basal host resistance against powdery mildew
(Blumeria graminis spp.) in barley and wheat (Christensen et al., 2004; Zimmermann et al, 2006).
GLP governing gene chr8 OsGLP contributes broad
resistance against two rice fungal pathogens which
are causal agent of rice blast and sheath blight
diseases (Manosalva et al., 2009).
Membrane
down-regulation of Membrane at different time points
supports the unpublished finding of Kyndt (2011)
Membrane down-regulated in Nipponbare under
Striga hermonthica infection (Swarbrick et al., 2008)
B-box
B-box was down-regulated under H. oryzae infection
in the research done by Kyndt (2011).
Mukhopadhyay et al. (2003) have reported that
transgenic tobacco over-expressing a zinc-finger
protein gene from rice conferred tolerance to cold,
dehydration, and salt stress.
CONCLUSION AND SUGGESTIONS
Cupin domain gene may have role in basal defense
against H. oryzae
Membrane protein and B-box zinc finger family protein
also may have significant role during rice and the
nematode interaction.
all three genes have systemic activation in whole plant
under H. oryzae infection
these genes can be over-expressed or silenced in rice
and their impact on the nematode population and its
infectivity can be observed.
THANK YOU
FOR YOUR
KIND ATTENTION
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http://rice.plantbiology.msu.edu/cgi-bin/ORF_infopage.cgi
LIST OF PRIMERS USED TO AMPLIFY GENES AND IN
COLONY PCR
Name Locus F-primer R-primer
DEAD LOC_Os11g44910 atggcggggtacgagagg tcaagagggaatctttatgcagttg
Purine LOC_Os03g08880 atggccaccattactgctgc ctaaggcgccgctgactc
Cupin LOC_Os08g09080 atggcttcgtcttccttcc tcagtaatggttgttctcccag
Membrane LOC_Os04g33830.1 atggccgcctccaccgtctc tcacttgtcgaagtagccctc
Ring LOC_Os04g50100 atggcatcctctgctcctgc tcacattggatgatctgaatc
B-box LOC_Os02g39360.1 atgaagatccagtgcgacgcgt tcaaccaagatcagggacga
SP6 taatacgactcactatagggcgaattgg
T7 atttaggtgacactatagaatactcaagc
OligodT tttttttttttttttttttttttttvn
LIST OF Q-PCR PRIMERS
Gene name Locus F-primer R-primer
Cupin LOC_Os08g09080 tgtgttcgtattccctgtgg agggttctggctgctaagtg
Member LOC_Os04g33830.1 gctcttcttctccagcatcg caggaacagcccaaggtg
Ring LOC_Os04g50100 gacgttctgcaggaatccat cccttcccttgctgttctc
B-box LOC_Os02g39360.1 cctccagttctccgactacg ttgtggaacaggtcgatgtc
EXP LOC_Os03g27010 tgtgagcagcttctcgtttg tgttgttgcctgtgagatcg
EXPnarsai LOC_Os11g21990.1 aggaacatggagaagaacaagg cagaggtggtgcagatgaaa
EIF5C LOC_Os07g02340.1 cacgttacggtgacacctttt gacgctctccttcttcctcag
Genes 1 dpi samples 3 dpi samples 5 dpi samples 7 dpi samples
Cupin Bl S1 Ho S2 Bl R1 Ho S2
Membrane Bl R2 Ho R1 Bl S2 Ho R2
Ring Ho R1 Bl S1 Ho S1 Bl R1
B-box Ho S2 Bl R2 Ho R2 Bl S2
LIST OF RANDOMLY SELECTED CDNA SAMPLES TO CHECK
Q-PCR PRIMERS
Bl: uninfected; Ho: infected; S: shoot; R: root; dpi: day/s post infection