Mutations in Arabidopsis Exocyst Gene AtSEC8
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Transcript of Mutations in Arabidopsis Exocyst Gene AtSEC8
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Mutations in Arabidopsis Exocyst Gene AtSEC8
Jennie HinesMentor: John Fowler
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The Big Picture
• Plant morphology
• Cells have the ability to grow in specialized ways
• What the Fowler lab examines is the mechanism behind the way that cells grow
Arabidopsis leaf cells
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The Exocyst• Thought to direct the way in which
cells grow
The exocyst in yeast
• Polarized cell growth in yeast• An 8 protein complex
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Pollen Tubes
• Pollen tubes exhibit polarized growth.
• One question is whether or not the exocyst is important for the pollen tube polarized growth.
Picture thanks to Rex Cole
pollen grain
pollen tube
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How does this apply to what I’m doing?
My hypothesis is that the gene that codes for a part of the exocyst in Arabidopsis, AtSEC8, plays a role in the way pollen tubes grow.
I have several different mutant plants, and the mutations may adversely affect AtSEC8.
Prediction: The AtSEC8 mutant plants will have poorly
functioning pollen.
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Transmission Defects
A mutation that causes pollen to function poorly also causes a transmission defect.
Pollen grain
Pollen tube
Egg sac
The mutation will be present in the next generation at a lower than expected frequency.
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The wild-type gene
T-DNA
T-DNA
Bam!
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F
Mutations
• F and G
G
My mutations No defectTransmission defect
5’ 3’
AtSEC8
• Other mutations previously studied• Transmission defects• F and G are in between
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Part One:Initial Genotyping
DNA was extracted from the plants and PCR was done to find out which plants had the insert
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Primers
From http://signal.salk.edu/tdnaprimers.html
LBb1
R F
T-DNA
Genome
~900 bp
~600 bp
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900 bp
600 bp
Heterozygote Homozygous mutant
Homozygous wildtype
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Results of Initial Genotyping
Homozygous mutants
Heterozygous mutants
Homozygous wild-type
F Plants 5 4 5
G Plants 1 6 10
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Part Two:Crosses
The plants were crossed to see if the next generation had the mutation
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Two Types of Crosses
• Self pollination• Backcrosses with
wildtypeTeeny tiny seeds that were planted one by one
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Pretty Wild
From the AtSec8 “G” mutation self-cross
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A Transmission Defect in the “G” Mutant
Homozygous mutants
Heterozygous mutants
Homozygous wildtype
F expected 5.5 11 5.5F Self-cross 3 11 8G expected 12 24 12G Self-cross 4 15 29
SIGNIFICANTLY DIFFERENT - p < 0.001
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F
Location Matters
G
No defectTransmission defect
5’ 3’
AtSEC8
5’ end have 0% transmission.3’ end have 100% transmission.G is located between with 33% transmission.
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Conclusions
Since there was a transmission defect, it can be concluded that my “G” mutation in AtSEC8 likely causes problems with pollen tube growth.
A New Hypothesis:The 3’ end of the gene (encoding the protein
C-terminal end) is not critical for exocyst function.
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What next?
• Genotype the backcrosses• Study the pollen in media• Look for phenotypic differences
– Roots– Size of plants
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Preliminary Results
Small homozygous mutantG mutant roots 0.3 cm longer
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Special Thanks to…John Fowler, Rex Cole, Zuzana Vejlupkova and Kirstin Arthur.And to the HHMI program for giving me the opportunity to be
a part of this.
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Fin