Knocking Out Seed TFs Genes

7/31/2019 Knocking Out Seed TFs Genes

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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

wt

lec1

wt

lec1

wt

lec1

wt

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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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

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Knocking Out Seed TFs Genes

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