Download lecture 5

Arabidopsis Experiments
Forward Genetic Screen (Ethylene Insensitive Mutants)
Reverse Genetic Screen / PCR Genotyping (H+- ATPase Mutants)
Arabidopsis
 Arabidopsis thaliana is the predominant model organism used by plant
biologists today. Considered a “weed" in nature, this small mustard serves as
an experimental subject for everything from root growth to flower
development in the laboratory. Arabidopsis has gained prominence as a model
organism for several reasons:

Generation time. Seed to seed in about 42 days.

Fecund. One Arabidopsis plant yields thousands of seeds.

Genome size. 125 megabases. Maize has 5000 mb, tobacco1500 mb.

Diploid. Relatively simple genome.

Tractable. Easily worked and amenable to genetic, molecular genetic, physiological
and biochemical studies.

Real plant. Roots, leaves, flowers, seeds, and a full component of physiological and
biochemical processes.
Arabidopsis
History
Linnaeus
*Yours Truly
Forward vs. Reverse Genetics
 Treat thousands of organisms with a mutagen,
- random mutagenesis,
 Identify an individual with a phenotype of interest,
Forward
 Identify the gene.
• Treat thousands of organisms with a mutagen (usually),
– random mutagenesis,
• Identify an individual with a genotype of interest,
• Identify the phenotype.
Reverse
First, Forward Genetics
Experiment #1
Conditional Screen for Ethylene Insensitivity
Ethylene
 Egyptians gassed figs in order
to stimulate ripening,
“the gaseous
hormone”
 The ancient Chinese burned
incense in closed rooms to
enhance the ripening of pears.
H2C = CH2
 In 1864, gas leaks from street
lights were observed to stunt
plant growth, twist plants, and
abnormally thicken stems
 Dimitry Neljubow (1901)
showed that the active
component was ethylene.
 R. Gane (1934) reported that
plants synthesize ethylene.
Receptor
enzyme-linked receptor
…found first in bacteria, then in plants,
now in most eukaryotes, including
mammals.
Two-component regulators.
Ethylene
…promotes fruit ripening,
 Ethylene signals the
transition from
unripe to ripe fruits,
 cell wall components
are broken down,
 starches and acids are
broken down
resulting in
“sweetening” and
aromatic compounds ,
 pigmentation may
also be induced.
Ethylene
…promotes the “triple response”,
…in etiolated seedlings,
 reduced stem
elongation,
 thicker stem,
 horizontal growth,
 May provide the plant
with “behavior” that
will provide escape
from soil impediments.
Ethylene
…mutant analysis,
wild type
ein
wild type
ctr
ein (ethylene present),
ctr (ethylene absent),
…ethylene insensitive.
…constitutive triple response.
Ethylene Signal
Transduction
…negative regulation.
Tricky Concept(s)
In the absence of ethylene, the enzyme
receptor activates CTR1,

active CTR1 inhibits the triple
response,
With ethylene present, or the receptor
“absent”, or the CTR1 or the gene
mutated,
 the triple response is activated.
no ethylene
ethylene,
ein, etr, etc,
…no triple response.
…or ctr mutant,
…blocks pathway.
active
inactive
?
erf: ethylene
response factor.
induces transcription,
Tuesday’s Work
Sterilizing/Planting
Germinating
 70% ETOH/0.1% Triton X
 Breaking Dormancy
 95% ETOH
 H2O/Imbibition,
 Murishige and Skoog
Media (MS),
 O2/Aeration,
 plant minimal medium
 0.5x strength
 With ACC.
 Cold/Prechilling
"stratification”
 Inducing Germination
 Light
ACC: 1-aminocyclopropane-1-carboxylate
Conditional Screen
 Grow on ACC,
…in the dark (etiolated).
 Score for mutants,
Not
this
Class.
 Transfer to 0.5X MS
(Murisige and Skoog)
media (-ACC),
 Grow in light.
What Next?
dominant
Thought Experiments…
 Backcross to wild-type,
 what might the F1 and F2
tell us?
 Complementation tests?
recessive
Second, Reverse Genetics
Experiment #2
PCR genotyping of a t-DNA mutant
Proton Pumps in planta
Pollen
tip growth
Anthers
cell elongation
Stems
transport;
sucrose
hormones
Arabidopsis
Leaves
stomata (gas exchange)
sucrose transport
Roots
root hair
growth
mineral uptake
Embryo/Seeds
loading
+
H (protons)
ATP synthase
Transporters
- carriers,
- channels.
ATP hydrolase (ATPase)
Adapted from Biochemistry and Molecular Biology of Plants, pp. 115
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 - 50,000, Human: 25,000 - 66,000.
Proton Pumps in planta
Pollen
tip growth
Anthers
cell elongation
Stems
transport;
sucrose
hormones
Arabidopsis
Leaves
stomata (gas exchange)
sucrose transport
Roots
root hair
growth
mineral uptake
Embryo/Seeds
loading
Phylogenetic Family Tree
Arabidopsis
H+-ATPase
(ClustalW --> Phylip: protdist, fitch)
Gene Family
Gene
AHA1
AHA2
AHA3
AHA4
AHA5
AHA6
AHA7
AHA8
AHA9
AHA10
AHA11
AHA12
Location
whole plant
root cortex
phloem
root endode rmis
whole plant
anthers
seeds
hypoco tyl
-
Function
?
?
?
nutrient uptake
?
?
?
?
?
?
?
psuedogen e
Baxter et al. , Plant Physiol, 123, (2003)
Reverse Genetics
Functional Genomics
Gene DNA
Sequence
Gene Disruption
Phenotype
Analysis
Function
Mutate
DNA Sequence
Genetically Link
Development
Physiology
Cell Biology
Nature
Ti-Plasmid
T-DNA
Plant Cells
HormonesOpines
Agrobacterium
Lab
T-DNA
Out: Ti genes, opine
genes,
In: DNA of choice.
Selectable Markers
Reporter Genes
Genes
Agrobacterium tumefaciens
Ti Plasmid (Tumor inducing)
Mother Nature
wt
plant
chromosome
hormone genes (i.e. auxins)
Ti Plasmid
(from agro)
opaline
virulence
genes
nopaline
neoplastic transformation
opaline, nopaline
virulence
genes
hormone genes
Agro
food
T-DNA (Transfer DNA)
Laboratory
Construct T-DNA
selection genes
virulence
genes
…can put other genes.
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.
Germination
To Do
 Breaking Dormancy
 H2O/Imbibition,
Surface Sterilize Seeds
Plant on Nutrient Media
Germinate
 O2/Aeration,
 Cold/Prechilling
"scarification”
1. EMS Treated Seeds on
MS/ACC media.
 Inducing Germination
2. aha3-1 on MS media.
 Light
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
Knockology
Plants/Pools
DNA/Pools
Set-Up
DNA Pooling
Maintain lines as pools of seed.
Seeds (9)
Germinate and grow seeds in liquid culture.
Seedlings
(225)
Extract DNA,
DNA (225)
Super Pool DNA,
1
2
3
4
5
PCR Screen
6
…30
Super
Pools
(2025)
5’--GCATGCATTAT
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
CTGATCGTGAC--5’
o
94
Synthesis
~1 minute/kb
72o
Denature Step
~30 seconds
PCR
~65o
Annealing Step
~30 seconds
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
CTGATCGTGAC--5’
5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’
5’--GCATGCATTAT
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
5’--GCATGCATTAT
3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’
CTGATCGTGAC--5’
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:
Product:
5’
Your gene
Your gene amplified
3’
Reverse Genetic PCR Strategy
Reaction:
Product:
Reaction:
Product:
none.
T-DNA
PCR Screens for Mutants
PCR Strategy
Reaction:
T-DNA
Product:
Reaction:
Product:
T-DNA
Find the Plant
You are ~here
T-DNA
Mutants
tagged
seed line
Genetic Analysis
tt x TT (wt)
isolate
homozygous
mutant
2x
backcross
to wildtype
phenotype
analysis
Tt
T-DNA
Segregation
T
t
T
TT
Tt
t
Tt
tt
F2
PCR Genotyping
5’
homozygote
wt
5’
heterozygote
homozygote
mutant
L
t
T
L
t
T
L
t
3’
3’
5’
3’
5’
3’
5’
3’
5’
3’
T
Genetic Analysis
F2 Segregation
T
t
T
TT
Tt
t
Tt
tt
1:2:1
Not Lethal
T
t
T
TT
Tt
t
Tt
tt
1 wt : 2 het
Lethal
T
t
T
TT
Tt
t
Tt
tt
1 wt : 1 het
Gametophyte
Lethal
Midterm
 Review on Wednesday,
 1 hour midterm,
 Look for Ethylene-Insensitive Mutants.