knocking out seed tfs genes
TRANSCRIPT
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HC70AL Spring 2011Gene Discovery Laboratory
Knocking-Out Seed TranscriptionFactor Genes
4/5/11
inistratorp
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Using Reverse Genetics To FindGenes That Control Seed Development
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lec1
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lec1
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lec1
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lec1
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Recall..Arabidopsis Features That Make It aModel Plant & Crop
1.Fast Generation Time2.Small Size3.Small Genome (~120 Mb & 25,000 Genes)4.Sequenced Genome5.Large Scientific Community-Many Resources For
Genomics & Functional Studies
6.Knock-Out in Every Gene
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Arabidopsis thaliana
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Table 1-1 (part 2 of 2) Molecular Biology of the Cell, Fifth Edition ( Garland Science 2008)
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Figure 1-37 Molecular Biology of the Cell, Fifth Edition ( Garland Science 2008)
Arabidopsis Has Same Number of Genes as Humans
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Recall.Plant Life Cycle
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RecallEmbryos Develop From the Fertilized Egg
Early Embryos Contain Two Regions-Suspensor & EmbryoProper With Different Developmental Fates
The Plant
Degenerates
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RecallEmbryos Undergo a Series of Events Leading
to a Dormant Embryo in a Mature Seed
1.
2.
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RecallThe End Product is Dormant Seed With aMature Embryo!
A Seed Consists of Three Parts With Distinct Genetic Origins1. A Seed Coat From Maternal Floral Tissue2. An Endosperm From Fertilization of Embryo Sac Central Cell3. An Embryo From Fertilization of the EggThe Embryo Becomes the Next Plant Generation
Embryo
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RecallGenome-Wide Profiling of mRNAs DuringArabidopsis Seed Development & Plant Life Cycle
Differentiation Prepare For Dormancy & Germination
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DesiccationTolerance
DesiccationTolerance
Suppression of Suspensor Embryonic Potential Development of Cotyledon Identity Initiation and Maintenance of Seed Maturation Inhibition Germination
Embryo Rescue
SAM
Activated SAM
wt
lec1
Searching For Functions of Seed Transcription Gactor GenesUsing Reverse Genetics (e.g., lec1 Mutants
Disrupt Seed Development)
Lotan et al., Cell,1998
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Reverse Genetics Starts With Gene Sequence and SearchesFor a Mutant Phenotype (i.e., Function)
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Figure 8-56 Molecular Biology of the Cell( Garland Science 2008)
Knock-Out Mutations Are Loss-of-Function
or Null Mutations
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Figure 8-64 Molecular Biology of the Cell( Garland Science 2008)
Knock-Out Mutations Are Loss-of-Functionor Null Mutations
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Inserting Foreign DNA Into the LacZ Gene is aKnock-Out Mutation
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Inserting Foreign DNA Into the LacZGene is a Knock-Out Mutation
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Simple Mendelian Geneticsa Review
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Simple Mendelian Geneticsa Review
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Simple Mendelian Geneticsa Review
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What Happens if the KO Results in a Lethal Phenotype?
Embryo/SeeLethality
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Note Segregation of Wt and Mutant Seeds
Arabidopsis Siliques and Developing Seeds
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Using Agrobacterium T-DNA as a Mutagen
T-DNA Inserts Randomly Into Plant Genome
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Figure 8-67a Molecular Biology of the Cell( Garland Science 2008)
Transforming Plants With T-DNA
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Figure 20-25
Agrobacterium Ti-Plasmid
~5kb For Your Vector
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A Collection of Arabidopsis T-DNA Mutants Exists
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T-DNA Allele Wt Allele
T-DNAAllele
T-DNA/TDNA Wt/T-DNA
Wt Allele T-DNA/Wt Wt/Wt
Segregation of T-DNA Inserts in Knock-Out Lines
Male Gametes
FemaleGametes
If Gene Is Critical For Controlling Seed DevelopmentThe T-DNA/T-DNA Class Does Not Appear!
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Inheritance of the T-DNA Inserts
If Seed Lethal This ClassDoes Not Appear
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Using PCR to Genotype Segregating Plants
Knock Out
What Do You Expect If Using Only Gene Primers?1.Homozygous Wt Plant?2.Heterozygous Plant?3.Homozygous T-DNA Plant?
1.If Lethal?2.If Not Lethal?
Forward Reverse