Post on 01-Jan-2016
description
transcript
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