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The Maize ropD Gene Christine Neou Dr. John Fowler Botany and Plant Pathology. Why use corn?. Better understanding of how corn and other plants grow and develop. Why use corn?. Better understanding of how corn and other plants grow and develop - PowerPoint PPT Presentation
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The Maize ropD GeneChristine Neou
Dr. John FowlerBotany and Plant Pathology
Why use corn?
Better understanding of how corn and other plants grow and develop
Why use corn?
Better understanding of how corn and other plants grow and develop
Learn mechanisms by which plants signal a response to stress or respond to disease
Why use corn?
Better understanding of how corn and other plants grow and develop
Learn mechanisms by which plants signal a response to stress or respond to disease
Use what we learn to perhaps breed plants that are better equipped to respond against stressors
G proteins - signaling molecules that bind GTP
Family
Ras
Rho
Rab
Arf
Ran
G proteins - signaling molecules that bind GTP
Family
Ras
Rho
Rab
Arf
Ran
Subfamily
Rho Rac Cdc42 Rop
G proteins - signaling molecules that bind GTP
Family
Ras
Rho
Rab
Arf
Ran
Subfamily
Rho Rac Cdc42 Rop(Rho of Plants)
Rop GTPases in Signaling Pathways
Rop
Rop
GDP
GTP
INACTIVE
ACTIVE
Rop GTPases in Signal Pathways
Binding of effector molecule
Rop
Rop
GDP
GTP
INACTIVE
ACTIVE
Rop GTPases in Signal Pathways
Binding of effector molecule
Signal for growth, differentiation or survival
Rop
Rop
GDP
GTP
INACTIVE
ACTIVE
The Role of Rops in Corn
???Function not known
Question: What is the role of Rops in plant growth and development?
At least 9 rops in corn
The ropD genetic map
Mutator Transposons
IR IR
Exons and Introns
Exons - coding region
Intron - sequences that are spliced out
Goals
Identify plants homozygous for the five alleles
Goals
Identify plants homozygous for the five alleles
Characterize the five identified alleles by linking to a phenotype
Goals
Identify plants homozygous for the five alleles
Characterize the five identified alleles by linking to a phenotype
Why homozygous plants?
They are the only plants that will exhibit a mutant phenotype.
Genotyping by PCR
DNA extraction
Polymerase Chain Reaction (PCR)
3 primers used: 2 gene specific primers
(GSP) Mu primer
Genotyping by PCR
GSP DF3 located upstream
of mutation
Genotyping by PCR
GSP DF3 located upstream
of mutation DR5 located
downstream of mutation
Genotyping by PCR
GSP DF3 located upstream
of mutation DR5 located
downstream of mutation
Mu anneals to inverted repeats of transposon
Example: Genotyping of mc3 mutation
1 2 3 4Lanes1. DNA ladder2. Wild type3. Homozygote4. Heterozygote
Agarose gel of genotyping PCR Wild type
Homozygote
Heterozygote
Example: Genotyping of mc3 mutation
1 2 3 4Lanes1. DNA ladder2. Wild type3. Homozygote4. Heterozygote
Agarose gel of genotyping PCR Wild type
Homozygote
Heterozygote
Example: Genotyping of mc3 mutation
1 2 3 4Lanes1. DNA ladder2. Wild type3. Homozygote4. Heterozygote
Agarose gel of genotyping PCR Wild type
Homozygote
Heterozygote
Results of Genotyping
Mutation # genotyped # of homozygotes
m1 52 0
m2 15 1
mc2 10 1
mc3 37 8
mc4 9 1
Example Phenotypes
Epidermal cells of leaf tissue
Wild type cells - mostly straight rows of cells with stomata spread evenly
Epidermal cells of leaf tissue
Wild type - mostly straight rows, very few areas of disorganization
Homozygote - larger areas of disorganization
Epidermal cells at high magnificationWild type Homozygote
RNA
Mature RNA contains only exons
RNA cDNA
Successful extraction of RNA from one sample
Conclusions
Observations have yielded no obvious mutant organismal phenotype
Epidermal cell experiments suggest a cell phenotype for homozygous plants
Preliminary data from RNA experiments are promising, experiments are still ongoing
The future…
Continue the experiments through the rest of the program and through the fall Continue looking for mutant phenotypes for
homozygous plants Use a computer program to analyze
epidermal cells from more plants Get more data from RNA
experiments
Special Thanks to
Howard Hughes Medical Institute
National Science Foundation
John Fowler and Lab
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