Transcript PowerPoint Presentation - AGRI-MIS

เทคนิคทางชีวโมเลกลุ กับ
การศึกษาฮอร์ โมน
Why at Molecular Level?
1 hormone
 many responses
 different responses
Different hormones  same responses
Interaction / Induction / Inhibition
Molecular strategies for better understanding
Hormone Studies
physiology
biochemistry
molecular biology
Molecular Studies
gene
gene product
gene expression
control of gene expression
molecular biology & hormone study
synthesis
signal transduction
action/response
Approach for molecular study
genetics
reverse genetics
Advantages/Disadvantages
Genetic Approach
Mutagenesis:making mutants
Mutant: organisms with
altered arrangement or
altered amount of genetic materials
Phenotypic changes: yes / no
Genetic Approach
Mutation:
inversion
translocation
deletion
duplication
point
Point mutation
base substitution
Ethylmethane sulfonate (EMS)
GC to AT transition
Sodium azide (NaN3)
AT to GC transition
Point mutation
base substitution
Results: no change (GGG/GGA = glycine)
amino acid substitution
(effects on function: yes/no)
stop codon (TGG tryp  TGA stop)
Deletion mutation
(ionizing radiation)
Reading frame of Genetic codes: Codon
NNN NNN NNN NNN
NN- N NNN NNN NN
Frame shift mutation
resulting in nonsense peptide or
premature stop codon
Deletion mutation
(ionizing radiation)
Large scale deletion
resulting in loss of
entire coding sequence
or chromosome rearrangement
Insertion mutation
Duplication / Translocation
Transposon (Ac, Ds, etc.)
T-DNA
Results:
+/- Functional recombinant protein
(in-frame insertion)
No/nonsense protein
(off-frame or promoter insertion)
Effect of Mutation
Mostly recessive to wild type allele
Heterozygote with normal phenotype
Homozygous mutant  effect of mutation
Effect of Mutation
Loss of function mutation:
inadequate gene product
of mutant allele
usually Recessive
Effect of Mutation
Gain of function mutation:
overproduction of
normal gene product
production of
novel/toxic gene product
usually Dominant
Mutant Screen
Visible screen:
morphology
anatomy
development
Biochemical screen:
hormone precursor
intermediate
Mutant check
Heritability (selfing):
mutation of germ cells
Pattern of inheritance (crossing):
dominant/recessive trait
single/multiple gene
Allelic test:
complementation group
phenotypic epistasis
Allelic test
Crossing of homozygous mutants
Phenotypes of F1 compared to parents
Mutation at the same locus
same complementation group
phenotype of F1 = ?
xx
x
xx

xx
Allelic test
Mutation at different loci
Different complementation group
dominant x dominant  F1?
recessive x recessive  F1?
dominant x recessive  F1?
Allelic test
dominant x dominant  F1?
xx
x
xx

x
x
Check for phenotypic epistasis
Hormone mutant
With hormone
no response
Without hormone
responsive phenotype
Hormone mutant
changes in
Synthesis: synthetic pathway
Sensitivity: perception / signaling
Regulation: responsive phenotype
Synthesis mutant
Analysis of hormone level
Use of hormone
hormone inhibitor
Rescue WT phenotypes
Synthesis mutant
Reproducible
Clear
Complete penetrance
Ethylene mutants
Triple response
Ethylene overproduction:
ctr1 and eto2
chromosome 5
GA mutants
seed germination
stem elongation
flowering
GA-deficient mutants
Seeds unable to germinate
on basal medium
Germinated after being transferred
to medium with GA
5 complementation groups: all recessive
ga1, ga2, ga3, ga4, and ga5
GA-deficient mutants
Mutant phenotypes complemented
by chemical compounds
ga1, ga2, and ga3
dwarf plants
tall with exogenous/supplied GA
ABA mutants
Seed dormancy
Stomatal closure
ABA mutants
ABA-deficient mutants:
aba1, aba2, and aba3
precocious germination
viviparous
germinate in the presence of
paclobutrazol (GA inhibitor)
high zeaxanthin (ABA precursor)
ABA-deficient mutants:
aba1
droopy stem under low humidity
lack of stomatal aperture control
Auxin mutants
IAA synthesis:
Trp-independent pathway
Little labeled trp converted to Iabeled IAA
Trp-deficient mutant accumulated IAA
Several pathways for IAA biosynthesis
Signal transduction
Effects of hormone depending on
type
concentration
mode of application
developmental stage
Specific information pathway
From extrinsic signal to specific response
Signal transduction
Specific receptor:
membrane protein
with high affinity binding
upon binding with hormone
conformation change
Activated receptor to Signal cascade
intermediate steps
Signaling molecule
Positive or Negative regulator
Phosphorylation
Dephosphorylation
Hydrolysis of guanine nucleotide
Rapid and Reversible
Redundancy
Signaling mutant
Mutation to signaling components
Gain of Function
Loss of Function
Signaling molecule
Positive regulator
without hormone
no/inactivated signaling molecule
no response
Signaling molecule
Positive regulator
with hormone
activated signaling molecule
response
Mutation?
Signaling molecule
Negative regulator
without hormone
activated signaling molecule
no response
Signaling molecule
Negative regulator
with hormone
deactivated signaling molecule
response
Mutation?
Identification of signaling mutant
Basic mutant screen
Rule out synthesis mutant
Define complementation groups
Epistatic analysis of components
Identification of signaling mutant
With hormone application:
(+) oversensitive response
(0) insensitive response
Without hormone application:
(-) auxotrophic / deficient phenotype
GA insensitive mutants
gai: fail to respond to applied GA
elevated endogenous GA
normal seed germination
poor stem elongation
delayed flowering
GA insensitive mutants
gai: gai mutation on chromosome1
semidominant
GAI gene product
inhibits stem elongation
GA de-represses GAI action
GA oversensitive mutants
spy mutants:
longer hypocotyl
spindly
early flowering
resistant to paclobutrazol
GA oversensitive mutants
spy mutants:
normal GA synthesis
Resistant to ga1 mutation
Suppress all phenotypes
associated with GA deficiency
GA oversensitive mutants
Wild type SPY gene product
a negative regulator of
GA signaling flux
Mutation affects GA signal
transduction pathway
in a GA-independent manner
ABA mutants
ABA insensitive mutants:
abi1, abi2, abi3, abi4, and abi5
Germination with
exogenous ABA application
Decreased seed dormancy
Elevated endogenous ABA level
ABA mutants
ABA insensitive mutants:
Other phenotypes
similar to aba mutants
abi1 and abi2:
wilty and viviparous
abi3: viviparous
seed maturation processes
Auxin insensitive mutants:
resistant to
toxic precursor (2,4-D)
toxic level of applied auxin
Auxin insensitive mutants:
axr: reduced hypocotyl elongation
reduced fertility
increased lateral branch
reduced root gravitropism
aux: not responsive to gravitropism
Signaling component interaction
Epistatic analysis of components
shared or separate pathway
acting order of gene products
Epistasis of phenotypes
Double mutant: combined mutant loci
Signaling component interaction
Consider 2 loci: A and B
A activate / inhibit B
B activate / inhibit A
Single mutant a:
aa BB
Single mutant b:
AA bb
Double mutant ab: aa bb
Signaling component interaction
Opposite responses
Epistatic phenotype:
downstream component
Same responses
Epistatic phenotype:
upstream component
Signaling component interaction
eg. Opposite responses
activated A component = + response
activated B component = - response
Hormone A  B  response
double mutant phenotype:
b mutant (downstream)
B epistatic to A
Signaling component interaction
Suppression analysis
mutation of mutant
Intragenic suppressor
same gene / new allele
Extragenic suppressor
second site revertant
Transgene technology
Breakdown species barrier
Transfer genes
from any source
to plants
Transgene technology
Initiate changes in
specific Tissues
certain developmental stage
Manipulate hormone level or
hormone sensitivity
Using transgenes
Transgenic plants for hormone study
Mechanism of hormone biosynthesis
Role of hormone in development
Control of hormone function
and monitor responses
Gene structure
Coding sequence
Regulatory sequence
promoter
terminator
Agrobacterium
Soil bacteria
A. tumefaciens: crown gall tumor
A. rhizogenes: hairy root disease
Chromosomal DNA
Plasmid DNA
Agrobacterium
Tumor-inducing plasmid
Ti plasmid
Transferred DNA
T-DNA
genes involved in
synthesis of amino acid derivatives
tumor formation
Agrobacterium
Gene transfer
Activated by plant substances
phenolics acetosyringone
coniferyl alcohol
from wound
For production of amino acid
derivatives : opine
octopine and nopaline
Agrobacterium
Opines synthesized only in plant cells
Used by bacteria
For transformation:
replace bacterial DNA
between T-DNA borders
with desired genes
System selection for gene transfer
Transformation technique
established
easy
available
System selection for gene transfer
Hormone-related mutants
Background information
on hormone level
Physiological work
on hormone effect
growth & development
Transgenic plants
Effect of gene expression on
overall development
Interaction among various hormones
Auxin/CK
Auxin/C2H4
GA/ABA
Apical dominance
High auxin inhibits
dormancy release of
lateral buds
High CK stimulates
lateral bud growth
Apical dominance
Transgenic plant with
auxin overproduction
CK application
to a dormant lateral bud
branching
Apical dominance
High auxin activates C2H4 production
Transgenics w/ iaaM:
reduced internode elongation
Transgenics w/ iaaM and ACCD:
normal height
No difference on apical dominance
Vascular differentiation
Auxin and xylem formation
quality and quantity
Petunia w/ high auxin:
xylem cells w/ increased numbers
smaller and more lignified
Tobacco w/ low auxin:
larger and less-lignified xylem cells
Vascular differentiation
Auxin:
cell division in vascular cambium
secondary wall formation
lignin synthesis
Auxin content:
rate of cell division
Fruit ripening
C2H4 as a catalyst:
involved in some aspects of ripening
Ripening occurs with lower than
a significant level (slow but not delay)
chlorophyll breakdown
fruit softening
Fruit ripening
C2H4 as a coordinator:
activates a large number of genes
for rapid and uniform ripening
***************
Senescence
Transgenic tomato (P35S:ACCS)
Vegetative tissues remain green
until setting fruit
Most flowers abort prior to fertilization
due to premature induction
of abscission in pedicel
Senescence
Transgenic tomato with
antisense ACCS
antisense ACCO
expressing ACCD
Delayed senescence