Arabidopsis Thaliana

A Study of Genes and Embryo

Development

By Garen Polatoglu

A T 1 G 2 2 1 9 0

• Gene• Gene Length: 1511 bp• Translated Region: 786

bp• PCR Product: 1004 bp

•Encoded Protein

•Size: 261 amino acids

•Putative AP2 domain containing transcription factor

Genotyping

• Homozygous Mutants (~1.2kb)– Plants 1, 2, 4,

6, 7

• Wild Type (~1kb)– Plants 3, 5, 8

tDNA Insertion Site

AT1G22190

Excised Band Sequence

Alignment at 477 bp

Does not agree with proposed SALK_139727

Gene Expression

• RT-PCR– Results indicate

that the gene is active in both Leaf and Silique.

– Bands in –RT lanes and Negative Control erroneous

Gene ExpressionAT1G22190 Genechip Data

From the laboratory of Dr. Goldberg

Promoter Region

• Promoter Region is immediately upstream of AT1G22190

• Size: 3002 bp• Restriction Sites: EcoRI

– GAATTC– Promoter cut into 1950 bp + 1052 bp

Promoter Analysis

• The promoter clone of Colony 1 was sequenced for verification purposes

• The sequence did not match exactly with that of TAIR’s

• Entire promoter (~3kb) was not sequenced

EcoRI Promoter Digestion Gel

3.5kb2.0kb

1.0kbPlasmid

Plant Phenotypes

• No variations were observed between the homozygous mutant plants and wild type plants

• Seeds of both genotypes were observed under a Nomarski microscope– No differences were noted

Nomarski Images

Wild Type Seed Mutant Seed

What’s next?

• Causes for no phenotypic effect of knockout gene could be varied– Genetic Redundancy– Phenotype more apparent in other

conditions

• Multiple knockouts of similar genes may induce a varied phenotype