Searching for Genes Important in Seed Development

At1g19000

At1g74840

BY: Mike Douglas

MYB-Related

Transcription Factor

At1g19000(GENE MODEL)

Genotyping

Expected Wild Type Band:

1104 bpT-DNA Mutant Band:

1076 bpT-DNA Mutant Band

325 bp

Plant Layout Chart & Genotyping Results

15 : 13 : 3Homozygous Wild-Type : Heterozygous : Homozygous Mutant

Where is AT1g19000 Expressed?

Cloning of Promoter Region in E. Coli Plasmid Vector

ECO-RI Digestion

1 kb

Lad

der

(+) C

olon

y

Col

ony

I

Col

ony

II

Col

ony

III

Col

ony

IV

(+)

(-)1617

1287

3500

Sequence using T7 and

SP6 PrimersBlast: T7 Sequencing vs. Actual Promoter Sequence Identities = 637/637 (100%)

T7 Sequencing

Blast: SP6 Sequencing vs. Actual Promoter Sequence Identities = 708/709 (99%)

1 MUTATION

SP6 Sequencing

Mutant Phenotypes• No Obvious Physical Phenotype• Nomarski Imaging

WILD TYPE EMBRYOS

HOMOZYGOUS MUTANT EMBRYOS

At1g74840(GENE MODEL)

MYB-Related

Transcription Factor

Genotyping1

kb la

dder

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 WIL

D T

YP

E

(+)

(-)

1 kb

Lad

der

(-)

Plant Layout Chart & Genotyping Results

ALL 18 Plants are WILD TYPE

Where is AT1g74840 Expressed?

Expected RT-PCR product: 603 bp

Ladd

er

Leaf

+R

T

Sili

que

+RT

gDN

A

TA L

eaf +

RT

TA S

eed

+RT

H20

Ladd

er

NOTE: No Tubulin Band

Cloning of Promoter Region in E. Coli Plasmid Vector

ECORI Digestion

T7 and SP6 Sequencing

Blast: T7 Sequencing vs. Actual Promoter Sequence Identities = 719/722 (99%) Plus/Plus

Blast: SP6 Sequencing vs. Actual Promoter Sequence Identities = 705/706 (99%) Plus/Minus

4 MUTATIONS

Col

ony

I

Col

ony

II

Col

ony

III

1 kb

Lad

der

1888

3500

Phylogenic Relationship of At1g19000 and At1g74840

Cross to make DOUBLE MUTANT- may eliminate possible redundancy for mutant phenotype