the trihelix transcription factor family heather hernandez

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!