Transcript Do AT4G13640 and AT3G24120 Play a Significant Role in Seed

Do AT4G13640 and AT3G24120 Play a
Significant Role in Seed Development
for Arabidopsis Thaliana?
By Jordan Fischer
June 8, 2006
Professor Goldberg
hc70al
What Is a Transcription Factor?
Examples of Transcription Factors
• Transcription factors are
proteins involved in the
regulation of gene expression.
• Bind to the promoter region
upstream of gene and either
facilitate or inhibit
transcription.
• Control and regulate gene
expression.
• Activated by:
– Physiological stimuli.
– Therapeutically.
– Pathological stimuli.
What Is the Significance of the G2-like
Transcription Factor Family?
• Recently categorized GARP super
family of transcription factors defined
by G2 in maize; the Arabidopsis
RESPONSE REGULATOR-B
proteins; and the PHOSPHATE
STARVATION RESPONSE1 protein
of Chlamydomonas.
• G2 function is specifically committed
to the differentiation of bundle sheath
cell chloroplasts in C4 leaf blades.
• Act as transcriptional regulators of celltype differentiation processes.
• Likely that GLK proteins act as
transcriptional regulators of chloroplast
development.
What is the Structure of Gene
AT4G13640?
AT4G13640:
LbB1
Rev.
Fw.
5Õ
UTR
Exon 1
Exon 2
Exon 3
Exon 4 Exon 5
Exon 6
3Õ
UTR
AT3G24120:
LbB1
Rev.
Fw.
5Õ
UTR
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
3Õ
UTR
Rev.
Fw.
5Õ
UTR
What is the Structure of Gene
AT3G24120?
Exon 1
Exon 2
Exon 3
Exon 4 Exon 5
Exon 6
3Õ
UTR
AT3G24120:
LbB1
Rev.
Fw.
5Õ
UTR
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
3Õ
UTR
What Other Information is Important
Pertaining to These Genes?
AT4G13640
AT3G24120
Protein Type
MYB
MYB
Size (bp)
2039
2436
Size (aa)
292
295
# Exons
6
6
# Introns
5
5
Most Active
24-Hr. Seed
Ovule and
24-Hr. Seed
According to Gene Chip, Where are These
Genes Active in the Arabidopsis Plant?
Gene AT3G24120
Gene AT4G13640
Control (Gene AT1G74840)
Are the Results from RT-PCR Consistent with
AT4G13640
Those of Gene Chip?
(+) Control
Silique -RT
Silique +RT
Leaf -RT
Leaf +RT
(+) Control
Silique -RT
Silique +RT
Leaf -RT
Leaf +RT
Yes, according to Gene
Chip and RT-PCR both
genes are active in the leaf
and silique of the
Arabidopsis plant.
AT3G24120
Did My Experiments Agree With Salk’s
Regarding the T-DNA Insert Location?
AT4G13640: YES
LbB1
Rev.
Fw.
5Õ
UTR
Exon 1
Exon 2
Exon 3
Exon 4 Exon 5
Exon 6
3Õ
UTR
AT3G24120: NO
LbB1
LbB1
Rev.
Fw.
5Õ
UTR
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
3Õ
UTR
What Genotyping Data Was Collected For
Gene AT4G13640?
2nd Batch
1st Batch
1 Kb Ladder
Control (-)
Control (+)
Plant #18
Plant #17
Plant #16
Plant #15
Plant #14
Plant #13
Plant #12
Plant #11
Plant #10
Plant #9
Plant #8
Plant # 7
1 Kb Ladder
Control (-)
Control (+)
Plant #6
Plant #5
Plant #4
Plant #3
Plant #2
Plant #1
1 Kb Ladder
Wild Type Band
Mutant Type Band
What Genotyping Data Was Collected For
Gene AT3G24120?
LBb1 Cont.
FW & RV
FW & LBb1
RV & LBb1
1 Kb Ladder
LBb1 Control
Control (-)
Control (+)
Plant #17
Plant #16
Plant #15
Plant #14
Plant #13
Plant #12
Plant #11
Control (-)
Control (+)
Plant #17
Plant #16
Plant #15
Plant #14
Plant #13
Plant #12
Plant #11
Control (-)
Control (+)
Plant #17
Plant #16
Plant #15
Plant #14
Plant #13
Plant #12
Plant #11
1 Kb Ladder
Wild Type Band
Mutant Type Band
What is the Summary of Genotyping Results
for Genes AT4G13640 and AT3G24120?
AT4G13640
AT3G24120
Wild/Wild
5
8
Mutant/Mutant
2
5
Heterozygous
0
5
TOTAL
7
18
How Did the Phenotypes of Homozygous
Mutant and Wild Type Plants Compare?
Mutant
Wild Type
How is Promoter Cloning Used in this
Experiment?
AT4G13640
(-) Control
(+) Control
Amp. Region
1 Kb Ladder
Colony # 4
Colony #3
Colony #1
1 Kb Ladder
AT3G24120
Colony # 4
Colony #3
Colony #2
Colony #1
1 Kb Ladder
(-) Control
(+) Control
Amp. Region
1 Kb Ladder
What Conclusions Can Be Made Based on
these Experiments?
• Genes AT4G13640 and AT3G24120 are not active
in seed development to the extent where a knock
out of either gene will be lethal to seed
development.
• A knockout of these genes causes no observable
phenotypical differences compared to the wild type.
What Further Experiments Should be Done on
Genes AT4G13640 and AT3G24120?
• Use GFP as a marker to help to determine where in the
plant the promoter regions activate transcription.
• Cross homozygous mutant plants from both knock out
lines and determine if a complete knock out of both genes
is fatal to seed development or if this creates any
phenotypic differences.
• Use Nomarksi microscope to check for phenotypical
differences in seed development.