1. Arabidopsis Experiments
Developmental Screen and Phase Changes
Reverse Genetics
PCR Genotyping

2. Phase Changes zygote to embryo germination vegetative to reproductive
gametophyte development
flower development
zygote to embryo
germination
vegetative to reproductive
juvenile to adult

3. Phase Change Studies Genetic and molecular genetic approaches,
isolate mutants that fail in some way to change phase properly,
study genes, gene products and associated molecules, and resulting structures.

5. Forward vs. Reverse Genetics
Treat thousands of organisms with a mutagen,
random mutagenesis,
Identify an individual with a phenotype of interest,
Identify the gene.
Forward
Treat thousands of organisms with a mutagen (usually),
random mutagenesis,
Identify an individual with a genotype of interest,
Identify the phenotype.
Reverse

6. Proton Pumps in planta Arabidopsis Pollen tip growth Anthers
cell elongation
Stems
transport;
sucrose
hormones
Leaves
stomata (gas exchange)
sucrose transport
Embryo/Seeds
loading
Roots
root hair growth
mineral uptake
Arabidopsis

7. H+ (protons) ATP synthase ATP hydrolase (ATPase) Transporters
- carriers,
- channels.
ATP hydrolase (ATPase)
Adapted from Biochemistry and Molecular Biology of Plants, pp. 115

8. Arabidopsis Genome ~125 Mb (Megabases, million base pairs),
Rice: 420 Mb, Human: 3 Gb,
25,498 genes from 11,000 gene families,
Rice: 32, ,000, Human: 25, ,000.

9. Phylogenetic Family Tree
(ClustalW --> Phylip: protdist, fitch)
Baxter et al. , Plant Physiol, 123, (2003)
Arabidopsis H+-ATPase Gene Family

10. Reverse Genetics Functional Genomics
Gene DNA
Sequence
Phenotype
Analysis
Function
Gene Disruption
Mutate
DNA Sequence
Genetically Link
Development
Physiology
Cell Biology

11. Out: Ti genes, opine genes,
Ti-Plasmid
T-DNA
Plant Cells
Nature
Hormones
Opines
Agrobacterium
Lab
Out: Ti genes, opine genes,
In: DNA of choice.
T-DNA
Selectable Markers
Reporter Genes
Genes

12. Mother Nature Agro food Agrobacterium tumefaciens
Ti Plasmid (Tumor inducing)
Mother Nature wt
plant
chromosome
hormone genes (i.e. auxins)
opaline
Ti Plasmid
(from agro)
virulence
genes
Agro
food
nopaline
neoplastic transformation
virulence
genes
hormone genes
opaline, nopaline

13. …if the T-DNA lands in a gene, the gene is disrupted.
T-DNA (Transfer DNA)
Laboratory
selection genes
…can put other genes.
virulence
genes
Construct T-DNA
transform, select for agro with T-DNA
Agrobacterium
infect plant, select for plants with T-DNA
…if the T-DNA lands in a gene, the gene is disrupted.

14. Probability of Finding an Insert in a Specific Gene
p = 1-(1-f)n
p = probability of insertion event
f = 1-(Genome/Size of Gene)
n = number of T-DNA inserts
thousands of inserts

15. Knockology
Plants/Pools
DNA/Pools

16. Set-Up DNA Pooling PCR Screen Maintain lines as pools of seed.
Seeds (9)
Germinate and grow seeds in liquid culture.
Seedlings (225)
Extract DNA,
DNA (225)
Super Pool DNA,
Super
Pools
(2025) 1 2 3 4 5 6
…30
PCR Screen

18. Denature Step 94o Synthesis 72o PCR ~65o Annealing Step ~1 minute/kb
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
5’--GCATGCATTAT
CTGATCGTGAC--5’
Denature Step
~30 seconds
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’
94o
Synthesis
~1 minute/kb
72o
PCR
~65o
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
5’--GCATGCATTAT
CTGATCGTGAC--5’
Annealing Step
~30 seconds
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
5’--GCATGCATTAT
CTGATCGTGAC--5’

19. PCR Strategy Polymerase Chain Reaction (PCR),
with oligonucleotide primers with homology to the 5’ and 3’ ends of your gene, amplify the DNA sequence between the primers.
Reaction: 5’ 3’
Your gene
Product:
Your gene amplified

20. Reverse Genetic PCR Strategy
Reaction:
Product: none.
Reaction:
T-DNA
Product:

21. PCR Screens for Mutants

22. PCR Strategy
T-DNA
Reaction:
Product:
T-DNA
Reaction:
Product:

23. Find the Plant
You are ~here.

24. T-DNA Mutants Genetic Analysis
tagged
seed line 2x
T-DNA
Segregation
tt x TT (wt)
isolate
homozygous
mutant Tt TT Tt tt T t F2
backcross
to wildtype
phenotype
analysis

25. PCR Genotyping L t T 5’ 3’ L t T 5’ 3’ L t T 5’ 3’ homozygote wt
heterozygote L t T 5’ 3’
homozygote
mutant

26. Genetic Analysis F2 Segregation
1 : 2 : 1 TT Tt tt T t
Not Lethal
1 wt : 2 het TT Tt tt T t
Lethal
1 wt : 1 het TT Tt tt T t
Gametophyte
Lethal

27. To Do Tuesday: Thursday: Extract Plant DNA PCR,
Continue Developmental Screen,
Thursday:
Run PCR fragments on gels,
Thin Developmental sceen plants.