比较基因组学与分子进化3

Why Reverse genetics: From gene to phenotype/function 1. Many unknown genes are sequenced due to the advancement of sequence technology 2. Can control where, when, and how much an active gene product to be made 3. Easy to design specific mutations into the gene product 4. Add TAGs to a gene product to simplify its study within the cell 5. Create mutation on gene that has no available mutation
1.起始步 沉默触发物被裂解，产生小 起始步 沉默触发物被裂解， 干扰性RNA 干扰性 （small interfering RNAs，siRNAs） ， ）
2.第二步：效应步 siRNAs与多种亚 第二步： 第二步 与多种亚 单位形成复合物—RNA诱导的沉默复 单位形成复合物 诱导的沉默复 合物（ ），然后以 合物（RISC），然后以 ），然后以RISC的方式 的方式 降解单链的靶mRNA. 降解单链的靶
比较基因组学与分子进化
Comparative genomics and molecular evolution
11/3/2010
比较基因组学与分子进化在生命科学研究中应用及贡献 1.模式生物测序的意义 • 模式生物 • DNA测序技术 2.新学科的快速诞生 2.新学科的快速诞生 3.推动物种起源和生物进化发展 推动物种起源和生物进化发展 4.研究思路和方法突破 研究思路和方法突破 Genetics VS Reverse genetics
How to Silencing a Gene
5’ control region mRNA
Form double stranded hairpin RNA (dsRNA)
3’ control region
The dsRNA results in formation of random 20-22 nts small RNA, which then triggers the degradation of the mRNA where the homologous derived from.
Four model organisms
Mouse
Drosophila
C.elegans
Arabidopsis
Drosophila mutant affects WING development
Normal (wild type, WT)
Mutant
Arabidopsis seedling development
How to identify Genes critical in a given development process
1. Genetics: What wrong with a mutant lacking a functional gene tells the normal role of the gene. Start with mutants, then the gene & how it works 2. Reverse genetics: Starting with a gene of known identity, modifying its expression or activity to reveal its development function
Worm cadherins: cdh-1, hmr-1a, hmr-1b, cdh-3, cdh-4, cdh-5, fmi-1, cdh-7, cdh-8, cdh-9, cdh-10, casy-1, cdh-12
genomic DNA
PCR
AT G
GFP vector
3-5kb
Unc-4(e120)
3 common ways to control where, when, and how much an active gene product in a cell
1. Utilization of desirable regulatory sequence to drive the expression of your gene product 5’ control region protein coding region 3’ control region
The transgenic kernels contained169-fold the normal amount of -carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate. (Naqvi et al., 12/5/2009, PNAS)
启动子
5‘ 外显子
3‘
2-5kb
25-30bp
内含子
tttcagATC
attcagGAT
GTTAGgt
ATTTCAgt
正确剪接位点
成熟 (mRNA) 结构 起始密码： 起始密码：ATG 外显子 N 5‘ 64 密码子 3‘
终止密码： 终止密码： TAA TAG TGA
C
蛋白质(20 氨基酸)
Promoter::GFP reporters reveal worm Cadherin gene expression
Breakthrough and milestone for genomic era • New fields of biology open up • Genetics VS Reverse genetics
• Case analysis in model organism
Key Point of Understanding Development
Ye X. et al., 2000, Science
Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways
microinjection
with markers
Promoter::GFP reporter construct
transgenic lines
In situ western hybridization of ferritin transgenic rice seeds. The chimeric soybean ferrition gene construct. NT: Non-transformants Soybean ferritin cDNA was fused to rice endosperm-specific Glb-1 promoter. The chimeric FK22: transformants with soybean ferritin gene genes were inserted into the pGPTV-35S-htp binary soyferH-1directed by GluB-1 Vector between the restriction sites SalI and SacI DF: double transformants with soybean ferritin gene GluB-1: glutelin gene promoter Glb-1: globulin gene promoter soyferH-1directed by GluB-1 and Glb-1 promoter. OF: transformants with soybean ferritin gene soyferH-1directed by Glub-1 em embryo, en endosperm, sa subaleurone Qu et al., 2005, Planta
2. Gene Silencing or Antisense approach to control the level of mRNA of a given gene 3. Expression of desirable Dominant Affecting Mutant form
基因结构(DNA) A-T C-G
3.第三步：沉默效应的放大和扩散 第三步： 第三步 整个生物体。 整个生物体。
ຫໍສະໝຸດ Baidu
The mechanisms of RNAi, RNA interference, RNAi
Genetic interference following ingestion of dsRNA-expressing bacteria by C.elegans a.General scheme for dsRNA production, bacterial strain(BL21/DE3). b. C.elegans show high GFP-fluorescence in body muscles c. 12% animals show a dramatic decrease in GFP Fire et al., 1998, Nature
Golden rice - Three β-carotenoid (provitamin A) biosynthetic enzymes (two from plants and one
from bacteria) were engineered for simultaneous expression in rice endosperm. The resulting first generation transgenic rice produced yellow endosperm containing β-carotene at levels that would provide 10% of the recommended daily allowance with an average daily rice intake.
The Nobel Prize in Physiology or Medicine 2006 "for their discovery of RNA interference - gene silencing by double-stranded RNA"
• What Genes involved in a given development process • How those genes (and proteins) work at cellular/molecular level • How those genes (and proteins) activities are regulated
In the light
In darkness
Wild type
Wild type
Mutant
Why Reverse genetics (From gene to phenotype/function)
1. Many undefined genes are sequenced due to the advancement of sequence technology 2. Can control where, when, and how much an active gene product to be made 3. Easy to design specific mutations into the gene product 4. Add TAGs to a gene product to simplify its study within the cell 5. Create mutation on gene that has no available mutation or not feasible to have