microrna in developing and germinating arabidopsis thaliana seeds jing sun mentor: dr. hiro nonogaki
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MicroRNA in Developing and Germinating Arabidopsis
thaliana Seeds
Jing Sun
Mentor: Dr. Hiro Nonogaki
Importance of Seeds• Environment• Protection of the embryo • Dispersal of offspring• Agriculture and food
www.invest.moldova.md/_1economy.htmlwww.psln.com/pete/images.htm
www.julianessamphotography.com/professional/pages/vegetables.htm
http://osulibrary.oregonstate.edu/video/agri55.html
Project Goal
To examine the involvement of microRNA in gene expression in Arabidopsis thaliana
seeds and siliques
www.bpmp.cnrs.fr/
What are MicroRNAs?
• Discovered in 1993 by
Dr. Victor Ambros and colleagues
• Originally for viral studies
• Involved in posttranscriptional gene regulation
Photo by Amanda Weatherman
http://dms.dartmouth.edu/news/2001/25oct2001.shtml
How are MiRNAs Produced?
Single Stranded RNA
Double Stranded Hairpin LoopPri-microRNA
MicroRNA
Pre-microRNA
mRNA cleavage
MiRNA Function in Gene Regulation
DNA RNA Protein
TranslationTranscription
mRNA cleavage
MiRNA Function in Gene Regulation
DNA RNA Protein
TranslationTranscription
Biological Function of MiRNA
• Developmental control
• Environmental response
• Homeostasis
www.britishcouncil.org/coratia-science.htmhttp://www.mun.ca/biochem/course/3107/Topics/Flower_development.html
Methodology Overview
Screening
Extraction, Purification, Separation
Detection & Identification
GUS Expression
Functional Analysis
RNA Extraction
Phenol-SDS Extraction Method
Isopropanol Fractionation
Low Molecular Weight RNA
(5S RNA, tRNA, siRNA, microRNA)
High Molecular Weight RNA
(18S RNA, 28S RNA, rRNA, mRNA)
RNA Purification & Separation
ppt1 ppt3
10
0.25
2.0
1.0
(kb)
28S rRNA18S rRNA
Small RNAs
0% 20 -50%
ppt1 ppt3
10
0.25
2.0
1.0
(kb)
28S rRNA18S rRNA
Small RNAs
0% 20 -50%
RNA Probe Detection
Target miRNA
Antisense probe
Digoxigenin label
C A G C C A A G G A U G A C U U G C C G A
G U C G G U U C C U A C U G A A C G G C U
D D
* * * *
Extra bases
Anti – DIG Ab
Alkaline PhosphataseAPAP AP
Microarrays
But not yet commercially available
for microRNA
Examines the whole genome
www.bpp.msu.edu/ Seed/SeedArray.htm
Membrane
Probe solution (170 µl)
miR
162
miR
161
miR
172
miR
171
miR
170
miR
168
Screening of Expressed miRNAs
MicroRNAs Expressed
Developing siliques
Micro RNA
Developing Silique
0
0.2
0.4
0.6
0.8
1
1.2
MicroRNA
Developing Silique
0
0.2
0.4
0.6
0.8
1
1.2
MicroRNA
15
61
57
15
81
59
16
01
61
16
21
63
16
41
65
16
61
67
16
81
69
17
01
71
17
21
73
31
9
39
03
91
39
33
94
39
53
96
39
73
98
39
9R
ela
tiv
e E
xp
res
sio
n (
%)
100
50
-
-
Germination
15
61
57
15
81
59
16
01
61
16
21
63
16
41
65
16
61
67
16
81
69
17
01
71
17
21
73
31
9
39
03
91
39
33
94
39
53
96
39
73
98
39
9R
ela
tiv
e E
xp
res
sio
n (
%)
100
50
-
-
Germination
microRNA
MicroRNAs Expressed
15
61
57
15
81
59
16
01
61
16
21
63
16
41
65
16
61
67
16
81
69
17
01
71
17
21
73
31
93
90
39
13
93
39
43
95
39
63
97
39
83
99
Re
lati
ve
Ex
pre
ss
ion
(%
)
100
50
-
-
Germination
15
61
57
15
81
59
16
01
61
16
21
63
16
41
65
16
61
67
16
81
69
17
01
71
17
21
73
31
93
90
39
13
93
39
43
95
39
63
97
39
83
99
Re
lati
ve
Ex
pre
ss
ion
(%
)
100
50
-
-
Germination
Target MicroRNAsDeveloping Silique
0
0.2
0.4
0.6
0.8
1
1.2
170 171 172 173 310 390 391 393 394 395 396 397 398 399
MicroRNA
Rel
ativ
e In
ten
sity
miR156: SPL 13
miR157: SPL 5
miR160: ARF
miR393: TIR/F-BOX
miR396: GRF
When Target Gene Expressed
• Reverse Transcription Polymerase Chain Reaction (RT-PCR)
6 hr
12 hr
18 hr
24 hr
• Northern Blotting
6 hr 12 hr 18 hr 24 hr
Where Gene Expressed
Selection Marker Antibiotic resistance
Kanamycin
Reporter Gene GUS
Target Gene Promoter
Localization
GUS Expression – vascular bundles
GUS Expression – stem, flower, silique
Functional Analysis
• Mutate microRNA binding site
• Observe phenotype
(Palatnik et al., 2003; Nature 425: 257-263)
MicroRNA Resistant Target
Wild Type
Wild Type
Control
MicroRNA Resistant Target
Wild Type
Wild Type
MicroRNA Resistant Target
Summary
• Screened for microRNAs in developing and germinating seeds
• GUS expression target genes
• Characterizing mutations target genes
100
50
-
-
Germination
50
-
-
Germination
• Target gene expression
Acknowledgements
Dr. Hiro NonogakiHoward Hughes Medical Institute
Dr. Kevin AhernDr. Chris Mathews
Ernest and Pauline Jaworski Summer Research GrantDr. Jim Carrington
Tanja HomrichhausenPo-Pu (Eric) LiuJessica Hewitt
Nobuya KoizukaRuth Martin