第十章 基因和发育 - PowerPoint PPT Presentation

第十章 基因和发育

The Genetic Basis of Development

By Hongwei Guo, Peking University, 2008.12

• 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

Common Methodology

Arabidopsis

Drosophila

DROSOPHILA MELANOGASTER

(Fruit fly)

CAENORHABDITIS ELEGANS

(Nematode)

ARABIDOPSIS THAMANA

(Arabidopsis)

The organism chosen for understanding broad biological principles is called a model organism.

MUS MUSCULUS

(Mouse)

DANIO RERIO

(Zebrafish)

No human,

why?

• The advantages of using Arabidopsis as a model :
• a small genome size: 125 Mb
• a short generation time: 6-8 weeks
• easy to grow, very small, and produces a lot of seeds
• self-pollination, easy to cross
• can be easily transformed

Flower Development

• Transition from the vegetative to the reproductive phase.

• 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).

Shoot Apical Meristem (SAM)

Flower development in Arabidopsis

Vegetative meristem

Inflorescence meristem

Transition to reproduction:

Genes & other factors

Floral meristem

Flower:

sepals, petals, stamens,

and carpels

Flower organ development:

Organ identity genes

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

拟南芥花的构成

• Look for mutants
• Analyze mutant phenotypes
• Characterize genetic interaction
• Clone genes – analyze gene expression
• Sequence – biochemical properties
• Transgenic plant – gain of function

Wild-type

ap2

pi

ap1

ap3

ag

class

Genes

mutants

phenotype

APETALA1 (AP1)

APETALA2 (AP2)

ap1

ap2

A

APETALA3 (AP3)

PISTILLATA (PI)

ap3

pi

B

C

AGAMOUS (AG)

ag

Eliott Meyerowitz group, Caltech, 1980s-90s

• Look for mutants
• Analyze mutant phenotypes
• Characterize genetic interaction
• Clone genes – analyze gene expression
• Sequence – biochemical properties
• Transgenic plant – gain of function

B

B

A

C

C

A

Sp

P

St

C

C

St

P

Sp

The ABC Modelfor Floral Organ Identity

(Sp)

(P)

(St)

(C)

• 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

Function

Gene products

APETALA1 (AP1)

APETALA2 (AP2)

A

APETALA3 (AP3)

PISTILLATA (PI)

B

C

AGAMOUS (AG)

B

B

A

A

C

C

Sp

P

St

C

C

St

P

Sp

B

B

C

C

St

C

C

C

St

St

St

C

ap2

ap1

B

B

A

A

C

C

Sp

P

St

C

C

St

P

Sp

A

A

C

C

Sp

Sp

C

C

C

C

Sp

Sp

pi

ap3

B

B

A

A

C

C

Sp

P

St

C

C

St

P

Sp

B

B

A

A

Sp

P

P

Sp

Sp

P

P

Sp

ag

• Look for mutants
• Analyze mutant phenotypes
• Characterize genetic interaction
• Clone genes – analyze gene expression
• Sequence – biochemical properties
• Transgenic plant – gain of function

B

B

A

A

C

C

Sp

P

St

C

C

St

P

Sp

A

A

Sp

Sp

Sp

Sp

Sp

Sp

Sp

Sp

ap3 ag

B

B

A

A

C

C

Sp

P

St

C

C

St

P

Sp

Thus, leaves are default structures

---花是由叶变态而成的最直接的证据---

ap1 ap3 ag

L

L

L

L

L

L

L

L

• Look for mutants
• Analyze mutant phenotypes
• Characterize genetic interaction
• Clone genes – analyze gene expression
• Sequence – biochemical properties
• Transgenic plant – gain of function

B

B

A

A

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

A

B

C

Exception: AP2 is expressed in all four whorls

• Look for mutants
• Analyze mutant phenotypes
• Characterize genetic interaction
• Clone genes – analyze gene expression
• Sequence – biochemical properties
• Transgenic plant – gain of function

• 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

• Look for mutants
• Analyze mutant phenotypes
• Characterize genetic interaction
• Clone genes – analyze gene expression
• Sequence – biochemical properties
• Transgenic plant – gain of function

B

B

A

A

C

C

Sp

P

St

C

C

St

P

Sp

B

B

A

A

C

C

P

P

St

St

St

St

P

P

PI/AP3：B function

35S::PI

35S::AP3

c mutant

35S::PI

35S::AP3

a mutant

35S::PI

35S::AP3

E class: SEP1, SEP2, and SEP3

are required for B and C functions

pi ag (BC)

double mutant

sep1 sep2 sep3

triple mutant

wild type

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)

B

A

C

sepal

petall

stamen

carpel

E

• 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

Wild-

type

lfy

ap1

ap1 + cal

lfy + ap1

• 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

• 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?)

FM precursor

FM

Sepal

St + Ca

Pe + St

IM

IM

IM

IM

LFY Expression

LFY and AP1 Expression

LFY and AG Expression

LFY, AP3, and PI Expression

IM: Inflorescence Meristem

FM: Floral Meristem

How Does LFY Activate ABC Genes?

• 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.

LFY

Gene X

Protein X

LFY

AP1

AP1

Activates Transcription

Activates Transcription

• 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

H

B

X

H

S

E3

E2

2.98kb

GUS

• 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

The AG cis-regulatory elements reside in the second intron，~200 bp fragment

Transition to reproduction

Flower

?

Inflorescence

Vegetative phase

Reproductive phase

Factors regulating the transitions

• Genes (flowering-time genes and floral identity genes)
• Day length (photoperiod)
• Temperature (vernalization)
• Hormones (GA, etc)

Vegetative meristem

Inflorescence meristem

Floral meristem

（春化）

（光周期）

LFY

A, B, C, E genes

Take home questions:

• What are the common criteria for those model organisms?
• If you want to make a plant flower early, what gene(s) will you overexpress in that species?
• Can you turn a leaf into a floral organ? how?
• Can you get a plant producing flowers with stamens and carpels outside while sepals and petals inside?
• If you think ABC model is not correct or complete, what could be your evidence?