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?

• 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.

Examples of Transcription Factors

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 cell-type differentiation processes.

• Likely that GLK proteins act as transcriptional regulators of chloroplast development.

What is the Structure of Gene AT4G13640?

What is the Structure of Gene AT3G24120?

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 AT4G13640Gene AT3G24120

Control (Gene AT1G74840)

Are the Results from RT-PCR Consistent with

Those of Gene Chip? AT4G13640 AT3G24120

Leaf +

RT

Leaf -R

T

Silique +

RT

Silique -R

T

Leaf +

RT

Leaf -R

T

Silique +

RT

Silique -R

T

(+) C

ontrol

(+) C

ontrol

Yes, according to Gene Chip and RT-PCR both genes are active in the leaf and silique of the Arabidopsis plant.

Did My Experiments Agree With Salk’s Regarding the T-DNA Insert Location?

AT4G13640: YES

AT3G24120: NO

5’UTR

5’UTR

3’UTR

3’UTR

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6

LbB1

LbB1

Rev.Fw.

Rev.Fw.

LbB1

What Genotyping Data Was Collected For Gene AT4G13640?

1st Batch 2nd Batch

1 Kb L

adderC

ontrol (-)C

ontrol (+)

Plant #18Plant #17Plant #16Plant #15Plant #14Plant #13Plant #12Plant #11Plant #10Plant #9Plant #8Plant # 71 K

b Ladder

Control (-)

Control (+

)P

lant #6P

lant #5P

lant #4P

lant #3P

lant #2P

lant #11 K

b Ladder

Wild Type Band

Mutant Type Band

What Genotyping Data Was Collected For Gene AT3G24120?

RV & LBb1 FW & LBb1 FW & RV LBb1 Cont.

Control (-)

Control (+

)P

lant #17P

lant #16P

lant #15P

lant #14P

lant #13P

lant #12P

lant #111 K

b Ladder

Control (-)

Control (+

)P

lant #17P

lant #16P

lant #15P

lant #14P

lant #13P

lant #12P

lant #11

1 Kb L

adderL

Bb1 C

ontrolC

ontrol (-)C

ontrol (+)

Plant #17

Plant #16

Plant #15

Plant #14

Plant #13

Plant #12

Plant #11

Wild Type Band

Mutant Type Band

What is the Summary of Genotyping Results for Genes AT4G13640 and AT3G24120?

AT4G13640 AT3G24120

Wild/Wild

Mutant/Mutant

Heterozygous

TOTAL

5 8

2 5

50

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?

Colony # 4

Colony #3

Colony #1

1 Kb L

adder

(-) Control

(+) C

ontrolA

mp. R

egion1 K

b Ladder

(-) Control

(+) C

ontrolA

mp. R

egion1 K

b Ladder

Colony # 4

Colony #3

Colony #2

Colony #1

1 Kb L

adderAT4G13640

AT3G24120

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.