第十章 基因和发育
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第十章 基因和发育. The Genetic Basis of Development. By Hongwei Guo, Peking University, 2008.12. 概述. 遗传信息的载体 —DNA 和基因 遗传信息的传递 — 中心法则 遗传信息的调控 — 基因表达 基因表达调控的事例 — 疾病和 发育 拟南芥花发育的基因调控 果蝇胚胎发育的基因调控. Eye. Antenna. Leg. Wild type. Mutant. Use mutants to deduce developmental pathways. - PowerPoint PPT PresentationTRANSCRIPT
The Genetic Basis of Development
第十章 基因和发育
By Hongwei Guo, Peking University, 2008.12
概述• 遗传信息的载体— DNA 和基因
• 遗传信息的传递—中心法则
• 遗传信息的调控—基因表达
• 基因表达调控的事例—疾病和发育1. 拟南芥花发育的基因调控2. 果蝇胚胎发育的基因调控
Common MethodologyUse mutants to deduce developmental pathways
Drosophila
Arabidopsis
Eye
Antenna Leg
Wild type Mutant
Model organismsThe organism chosen for understanding broad
biological principles is called a model organism.DROSOPHILA MELANOGASTER
(Fruit fly)CAENORHABDITIS ELEGANS
(Nematode)
MUS MUSCULUS(Mouse)DANIO RERIO
(Zebrafish)
ARABIDOPSIS THAMANA(Arabidopsis)
No human, why?
Arabidopsis thaliana ( 拟南芥 ): a genetic model plant
The advantages of using
Arabidopsis as a model :
1. a small genome size: 125 Mb
2. a short generation time: 6-8
weeks
3. easy to grow, very small, and
produces a lot of seeds
4. self-pollination, easy to cross
5. can be easily transformed
• Transition from the vegetative to the reproductive phase.
Flower Development
• Controlled by developmental and
environmental signals.
• This transition is called flowering ( 开花 ) or
bolting ( 抽苔 ) and results in the formation
of the inflorescence meristem ( 花序分生组织 ), which produces floral meristem ( 花分生组织 ).
Transition from Vegetative toReproductive Development
Shoot Apical Meristem (SAM)
9
Flower development in Arabidopsis
Vegetative meristem
Inflorescence meristem
Floral meristem
Flower: sepals, petals, stamens,
and carpels
Transition to reproduction: Genes & other factors
Flower organ development:Organ identity genes
Flower Organs
拟南芥花的构成
The floral meristem produces 4 sets of floral organ primordia in concentric rings (called whorls)– Sepals - outer ring, 1st whorl– Petals - interior to the sepals, 2nd whorl– Stamens - interior to the petals, 3rd whorl– Carpels - inner ring, 4th whorl
Genetic Approach to Flower Development
• Look for mutants
• Analyze mutant phenotypes
• Characterize genetic interaction
• Clone genes – analyze gene expression
• Sequence – biochemical properties
• Transgenic plant – gain of function
Organ Identity Mutants
ap2
ap1
pi
ap3 ag
Wild-type
class Genes
A
B
C
APETALA1 (AP1)APETALA2 (AP2)
APETALA3 (AP3)PISTILLATA (PI)
AGAMOUS (AG)
mutants
ap1ap2
ap3 pi
ag
phenotype
Eliott Meyerowitz group, Caltech, 1980s-90s
Genetic Approach to Flower Development
• Look for mutants
• Analyze mutant phenotypes
• Characterize genetic interaction
• Clone genes – analyze gene expression
• Sequence – biochemical properties
• Transgenic plant – gain of function
The ABC Model for Floral Organ Identity
A A
B B
C
Sp St CP St P Sp
C
C
(Sp)
(P)
(St)
(C)
The ABC Model• Three classes of gene products
• Combinatorial interactions to give rise to the four types of floral organs
– A = sepals identity
– A+B = petals identity
– B+C = stamens identity
– C = carpels identity
– A and C mutually repress each other
Floral homeotic genes in Arabidopsis
Function Gene products
A
B
C
APETALA1 (AP1)APETALA2 (AP2)
APETALA3 (AP3)PISTILLATA (PI)
AGAMOUS (AG)
Loss of A function- ap1, ap2 mutants
A A
B B
C C
Sp P St C C St P Sp
B B
C C
St C C St St CC St
ap1
ap2
Loss of B function - apetala3/pistillata mutants
ap3
pi
A A
B B
C C
Sp P St C C St P Sp
A AC C
Sp C C SpSp C C Sp
Loss of C function – agamous mutant
A A
B B
C C
Sp P St C C St P Sp
A A
B B
Sp P P SpSp PP Sp
ag
Genetic Approach to Flower Development
• Look for mutants
• Analyze mutant phenotypes
• Characterize genetic interaction
• Clone genes – analyze gene expression
• Sequence – biochemical properties
• Transgenic plant – gain of function
Loss of B and C Functions-- Results in all Sepals
A A
B B
C C
Sp P St C C St P Sp
A A
Sp SpSpSpSpSp SpSp
ap3 ag
Loss of A,B, C Functions results in a ‘flower’ with leaves in place of
floral organs
A A
B B
C C
Sp P St C C St P Sp
L L L L L L L L
Thus, leaves are default structures
--- 花是由叶变态而成的最直接的证据 ---
ap1 ap3 ag
Genetic Approach to Flower Development
• Look for mutants
• Analyze mutant phenotypes
• Characterize genetic interaction
• Clone genes – analyze gene expression
• Sequence – biochemical properties
• Transgenic plant – gain of function
A A
B B
C C
Sp P St C C St P Sp
A, B, C gene mRNA expression pattern revealed by in situ hybridization
AP1 AP3 AG
Exception: AP2 is expressed in all four whorls
A B C
Genetic Approach to Flower Development
• Look for mutants
• Analyze mutant phenotypes
• Characterize genetic interaction
• Clone genes – analyze gene expression
• Sequence – biochemical properties
• Transgenic plant – gain of function
flower homeotic genes
• Floral homeotic genes or floral identity gene encode MADS-domain proteins
• Proteins dimerize and bind to DNA to act as transcription factors
• Are found in plants, fungi, animals
Genetic Approach to Flower Development
• Look for mutants
• Analyze mutant phenotypes
• Characterize genetic interaction
• Clone genes – analyze gene expression
• Sequence – biochemical properties
• Transgenic plant – gain of function
35S::PI35S::AP3 c mutant
35S::PI35S::AP3 a mutant
35S::PI35S::AP3
A A
B B
C C
Sp P St C C St P Sp
PI/AP3 : B function
A A
B B
C C
P St St PP St St P
E class: SEP1, SEP2, and SEP3 are required for B and C functions
wild typesep1 sep2 sep3
triple mutant
pi ag (BC)double mutant
SEP1, SEP2, SEP3 = E class
•MADS box proteins (most similar to AP1)
•Have redundant function
Single mutants show subtle phenotype
Triple mutant similar to bc double mutant
•Interact with B and C proteins
Q: How was this triple mutant obtained?(hint: reverse genetic approach)
“ Revisionist” ABC Model
AB
CE
petallstamencarpelsepal
Meristem Identity Genes
• Floral transition activates genes important for reproductive meristems, called meristem identity genes– Promote the floral meristem identity
• LEAFY (LFY) • APETALA1 (AP1) with CAULIFLOWER (CAL)• APETALA2 (AP2)
– Maintenance of meristem identity • TERMINAL FLOWER -- inflorescence meristem
• AGAMOUS– fully committed floral meristem
Floral Meristem Mutants
lfy
lfy + ap1ap1 ap1 + cal
Wild-type
LFY: meristem identity gene
• In strong lfy mutants, sepals, petals, and stamens are placed by leaf-like organs, bracts, carpels are formed by abnormal
• LFY is expressed in the inflorescence meristem that will form the floral meristem, and in the floral meristem
• The LFY protein is a plant-specific transcription factor
LFY is both necessary and sufficient for ABC gene expression
• In lfy mutants, AP1 expression is delayed and reduced
• In lfy mutants, AP3 and PI expressed is reduced
• In lfy ap1 double mutant, AG expression is abnormal
• Ectopic expression of LFY can cause ectopic AP1, AP3 and AG expression (what could be the phenotype of 35S::LFY?)
A Model for LFY Function in Activating ABC Genes
IM
FM
LFY Expression
IM IM
LFY and AP1 Expression
LFY and AG Expression
IM
LFY, AP3, and PI Expression
FM precursor Sepal St + Ca Pe + St
IM: Inflorescence MeristemFM: Floral Meristem
So, LFY can activate AP1 Expression
• To study LFY activity, a fusion of LFY to a inducible protein, GR, was made.
• In the absence of the glucocorticoid hormone, GR-LFY is inactive;
• When the hormone is present, GR-LFY becomes active.
• When LFY-GR was inactive, AP1 expression was not activated, when LFY-GR was active, AP1 was activated.
How Does LFY Activate ABC Genes?
Is this activation direct regulation?
---test of direct regulation
LFY
AP1
Activates Transcription
LFY
Protein X
Gene X
AP1
Activates Transcription
• LFY binds to cis elements of the AP1 gene– EMSA (Electrophoresis Mobility Shift Assay)– ChIP (Chromatin Immunoprecipitation)
• LFY activates AP1 expression when there is no new protein synthesis– Cycloheximide treatment (inhibit translation)
• Therefore, the activation of AP1 by LFY is direct
Similarly, LFY also directly regulates AG
The AG cis-regulatory elements reside in the second intron , ~200 bp fragment
H S XB HE3
GUS2.98kbE2
• This fragment contains two LFY-binding sites• If the sites are mutated, then LFY does not
bind• These mutant fragments cannot support LFY
induced expression
44
45
Transition to reproduction
Vegetative phaseReproductive phase
Inflorescence
Flower
Factors regulating the transitions
Vegetative meristem
Inflorescence meristem
Floral meristem
• Genes (flowering-time genes and floral identity genes)
• Day length (photoperiod)• Temperature (vernalization)• Hormones (GA, etc)
LFY
A, B, C, E genes
(光周期) (春化)
Take home questions:
1. What are the common criteria for those model organisms?
2. If you want to make a plant flower early, what gene(s) will you overexpress in that species?
3. Can you turn a leaf into a floral organ? how?
4. Can you get a plant producing flowers with stamens and carpels outside while sepals and petals inside?
5. If you think ABC model is not correct or complete, what could be your evidence?