Transcript Is My Gene important for seed development in plants?

Is My Gene Important for Seed
Development in Plants??
Gene: AT3G53370
Jonathan Milgrom Spring 2004
What Does My Gene Do?
Transcription Factor*
• Repressor of Spinach gene rps1
• Active in roots
• Highly conserved in plant kingdom
Transcription factor
*Dao-Xiu Zhou, Cordelia Bisanz Seyer
Knockout the gene
and find out!
• Engineer agrobacteria
to insert tDNA
• Grow mutant seeds
• Genotype
tDNA
AT3G53370
Is This Function Integral to Seed
Development ?
Madison & Salk Provide the
Knockouts
Madison
SP
#4
Salk
Exon
SP
#1
5’
3’
AT3G53370
Knockout Analysis
Madison project
began with…
Superpool
PCR products amplified with RV (gene
specific) and tdna primers 4/15/04
Superpool
tDNA line/
Mutant Seed
Plant
Genotype
Sp #4
(small length suggests insertion)
Salk project
began with…
Mutant seeds
Madison Results
Identified Madison
DNA Pool
tDNA
S
P
#
4
Madison PCR
products amplified
with RV (gene
specific) & tdna
primers 5/18/04
5’
SP #4
DNA pool
#6
AT3G53370
RV
33’
SALK Results
145678
W
C
9t
W
1456789t C
7 Plants Genotyped…
Plant #
genotype
1
Homozygous
tDNA
Heterozygous
Wild Type
4
5 6
7
8 9
* * *
*
*
* *
PCR products of various SALK lines amplified with
Tubulin
• Homozygous tDNA exist
• Knockout is not embryo
lethal
PCR products of various SALK lines amplified with
Does My Gene Play Some Role in
Seed Development?
Gene activity/mRNA accumulation patterns
Scarlet Runner Bean equivalent
Arabidopsis
Gene Chip Activity AT3G53370
5000
RT-PCR of 6 organs
Signal Detection Strength
4500
4000
3500
3000
2500
Wild Type
2000
Lec1
mutant
1500
Mutant
1000
500
Organ
14 day old
embryo
Wild Type
Floral Buds
Wild Type
Stem
Wild Type
Leaves
Wild Type
PostMaturation
Wild Type
MidMaturation
Wild Type
Ovules
0
Next…?
• Study rsp1 spinach gene equivalent in
Arabidopsis
• Study lec1 gene
• Identify Madison tDNA line and Genotype