the trihelix transcription factor family
DESCRIPTION
The Trihelix Transcription Factor Family. Heather Hernandez. What is a Trihelix Transcription Factor?. Also called GT factors. DNA binding proteins. Bind to GT elements in promoters. Bind to promoter elements required for light responsiveness. Regulate plant-specific processes. - PowerPoint PPT PresentationTRANSCRIPT
The Trihelix Transcription Factor Family
Heather Hernandez
•Also called GT factors.•DNA binding proteins.•Bind to GT elements in promoters.•Bind to promoter elements required for light responsiveness.•Regulate plant-specific processes.•Specific regulatory functions largely unknown.
What is a Trihelix Transcription Factor?
“Conserved Domain” found in all
trihelix proteins.
Trihelix Transcription Factor GenesGene name: At3g14180
Location: Chromosome 3
Gene size: 1736 Base Pairs
Protein size: 444 amino acids
Protein encoded: expressed protein similar to 6b-interacting protein 1 (found in Tobacco)
UTR UTR
REVERSE ORIENTATION
5’ 3’ EXON
Where in Arabidopsis thaliana is At3g14180 active?
RT-PCR 05/23/06
1.5% agarose gel
1.5 hours @ 90v
10 uL EtBr
Ladd
er
INF
RT
+IN
F R
T-
SIL
RT
+
SIL
RT
-
+ C
ontr
ol
- C
ontr
ol
UCLA Genechip Data+
Con
trol
- C
ontr
ol
What genotypes are present in my plants?
Ladd
erP
lant
1P
lant
2
Pla
nt 3
Pla
nt 4
Pla
nt 5
Wild
type
+ C
ontr
ol-
Con
trol
At3g14180 Genotyping
1% Agarose gel
1.5 hrs @ 105 v
10 uL EtBr
T-DNA “bottom”T-DNA “top”
Wildtype band
Ladd
er
Pla
nt 6
Pla
nt 7
Pla
nt 8
Pla
nt 9
Pla
nt 1
0
Pla
nt 1
1P
lant
12
Pla
nt 1
3
Pla
nt 1
4
Pla
nt 1
5
Pla
nt 1
6
Wild
type
+ C
ontr
ol
- C
ontr
ol
1% Agarose gel
1.5 hrs @ 105 v
10 uL EtBr
At3g14180 Genotyping
Wildtype: 1
Heterozygous mutant: 4
Homozygous knockout: 11
Where did the two T-DNA bands insert?
Ladd
er
LBb1LBb1
B T
5’ 3’
BP 1 BP 1736830 846
Location from SALK matches my sequencing results. (SALK predicted 844 BP into the gene.)
My sequencing results:
What is the impact of the T-DNA inserts?
• No visible phenotypical differences seen in homozygous knockout plants vs. wildtype plants.
What is the impact of the T-DNA inserts on seed development?
Wildtype heart stage
Mutant heart stage
Wildtype late torpedo
Mutant torpedo Mutant fully developed
Wildtype fully developed
suspensor
Trihelix Transcription Factor Gene 2
• Gene name: At1g54060• Location: Chromosome 1• Gene size: 1646 Base Pairs• Protein size: 384 amino acids• Protein encoded: expressed protein similar to 6b-interacting protein 1 (found in
Tobacco)
UTR
293 BP
UTR
200 BP5’ 3’
Exon (BP 1-1646)
Forward Orientation
Where in Arabidopsis thaliana is At1g54060 active?
Ladd
er
Ladd
er
INF
+R
T
INF
-R
T
SIL
RT
+S
IL R
T -
+ C
ontr
ol
- C
ontr
ol
1.5 % Agarose gel
1.5 hrs @ 105 v
10 uL EtBr
RT-PCR At1g54060 Genechip Data
What genotypes are present in my plants?
Ladd
erP
lant
1
Pla
nt 2
Pla
nt 3
Pla
nt 4
Pla
nt 5
Pla
nt 6
Pla
nt 7
Pla
nt 8
Pla
nt 9
Pla
nt 1
0P
lant
12
Pla
nt 1
3
Pla
nt 1
4
Pla
nt 1
5W
ildty
pe
+ C
ontr
ol
- Con
trol
Genotyping At1g54060
1% Agarose gel
1.5 hrs @ 105 v
10 uL EtBr
Wildtype: 6
Heterozygous T-DNA: 3
Homozygous knockout: 5
Where did the T-DNA insert?
LBb1
BP 635
5’ 3’BP 1 BP
1646
My sequencing results:
My results matched the SALK institute expected insert site of 640 BP into the gene.
What is the impact of the T-DNA inserts on phenotype?
Plant 6 Silique 1Plant 6, Silique 1, Top
Plant 6, Silique 1, Bottom Plant 6, Silique 1, Dissected
T-DNA impact continued…
Plant 6, Silique 2, Dissected
Plant 6, Silique 1, White seeds
Plant 6, Silique 2, Bottom
Plant 6, Silique 2, 12.5x
How does upstream regulatory activity show where the protein is
translated?
iProof PCR At3g14180
Ladd
er“P
rom
oter
Reg
ion”
+ C
ontr
ol-
Con
trol
1% Agarose gel
1.5 hrs @ 105 v
10 uL EtBr
EcoRI Digest At1g54060
Ladd
er
Sam
ple
1
Sam
ple
2
Sam
ple
3
Sam
ple
4
Acknowledgement s
• Dr. Robert Goldberg• Dr. Anthu Bui• Mike Gavino• Jonathan Russell• Garima Yagnik• Tomo Kawashima• Jessica Luke• Dr. Xingjun Wang• Lab members!