Transcript At5g65640 - A mutant phenotype?

At5g65640 - A mutant
phenotype?
Emily Eder
HC70AL - Spring 2005
Arabidopsis Gene At5g65640
• Plant genotyping
• Sequencing and T-DNA
insert
• First observation of mutant
phenotypes
• Nomarski
• RNA extraction –
microarray – expression
Genotyping
4 Homozygous, 0 Heterozygous, and 8
Wild Type Plants
Homozygous
Sequencing the Mutant Band
• Why is it necessary to sequence the
band?
• We must verify the T-DNA insertion site
AT5g65640
2026 Base Pairs in Length
Codes for basic helix-loop-helix (bHLH) protein
146
Mutant Phenotypes
Mutant Phenotypes cont.
Using Microscope to
Examine Phenotypes
Microscope Examination cont.
Microscope Examination cont.
Microscope Examination cont.
Microscope Examination cont.
Mutant Nomarski
Wild Type Nomarski
Using Gel Electrophoresis to
Determine RNA Expression
Gel Electrophoresis to
Determine RNA Expression
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T
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(3
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)
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Using a Genechip (Microarray) to
Determine RNA Expression
2000
1500
1000
500
0
Level of Gene Chip
Expression
Arabidopsis Gene
AT5g65640
• 4 Homozygous Mutant Plants - all with
visible phenotype
• 1 T-DNA insert - forward orientation
• RNA expression revealed a large
amount of RNA present in the seed
What’s next?
• Can we be sure that
this is an actual
mutant phenotype?
• More research is
necessary