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1、,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3.推动物种起源和生物进化发展4.研究思路和方法突破 Genetics VS Reverse genetics,Breakthrough and milestone for genomic era,New fields of biology open up Genetics VS Reverse genetics,Case analysis in
2、model organism,Key Point of Understanding Development,What Genes involved in a given development processHow those genes (and proteins) work at cellular/molecular levelHow those genes (and proteins) activities are regulated,How to identify Genes critical in a given development process,1. Genetics: Wh
3、at 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,Four model organisms,Arabidopsis,M
4、ouse,Drosophila,C.elegans,Drosophila mutant affects WING development,Normal (wild type, WT),Mutant,Arabidopsis seedling development,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, a
5、nd 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,Why Reverse genetics: From gene to phenotype/
6、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 geneproduct,4. Add TAGs to a gene product to simplify its study within the cell,5. Creat
7、e mutation on gene that has no available mutation,3 common ways to control where, when, and how much an active gene product in a cell,2. Gene Silencing or Antisense approach to control the level of mRNA of a given gene 3. Expression of desirable Dominant Affecting Mutant form,1. Utilization of desir
8、able regulatory sequence to drive the expression of your gene product,基因结构,(,DNA),外,显子,内含子,启动子,25,-,30,bp,A,-,T,C,-,G,tttcag,ATC,GTTAGgt,正确剪接位点,5,3,2-5kb,attcagGAT ATTTCAgt,外显子,成熟,(,mRNA,),结构,蛋白质,(20,氨基酸,),64,密码子,起始密码：,ATG,N,终止密码：,TAA TAG TGA,5,3,C,Promoter:GFP reporters reveal worm Cadherin gene ex
9、pression,The chimeric soybean ferrition gene construct. Soybean ferritin cDNA was fused to rice endosperm-specific Glb-1 promoter. The chimericgenes were inserted into the pGPTV-35S-htp binary Vector between the restriction sites SalI and SacI,In situ western hybridization of ferritin transgenic ric
10、e seeds. NT: Non-transformants FK22: transformants with soybean ferritin gene soyferH-1directed by GluB-1 DF: double transformants with soybean ferritin gene soyferH-1directed by GluB-1 and Glb-1 promoter. OF: transformants with soybean ferritin gene soyferH-1directed by Glub-1,em embryo, en endospe
11、rm, sa subaleurone,Qu et al., 2005, Planta,GluB-1: glutelin gene promoter Glb-1: globulin gene promoter,Ye X. et al., 2000, Science,Golden rice - Three -carotenoid (provitamin A) biosynthetic enzymes (two from plants and one from bacteria) were engineered for simultaneous expression in rice endosper
12、m. The resulting first generation transgenic rice produced yellow endosperm containing -carotene at levels that would provide 10% of the recommended daily allowance with an average daily rice intake.,Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing
13、three distinct metabolic pathways,(Naqvi et al., 12/5/2009, PNAS),The transgenic kernels contained169-fold the normal amount of -carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate.,How to Silencing a Gene,mRNA,Form double stranded hairpin RNA (dsRNA),The dsRNA re
14、sults in formation of random 20-22 nts small RNA, which then triggers the degradation of the mRNA where the homologous derived from.,The mechanisms of RNAi, RNA interference, RNAi,1.起始步 沉默触发物被裂解，产生小干扰性RNA （small interfering RNAs，siRNAs）2.第二步：效应步 siRNAs与多种亚单位形成复合物RNA诱导的沉默复合物（RISC），然后以RISC的方式降解单链的靶mRN
15、A.3.第三步：沉默效应的放大和扩散 整个生物体。,Fire et al., 1998, Nature,Genetic interference following ingestion of dsRNA-expressing bacteria by C.elegansa.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
16、GFP,The Nobel Prize in Physiology or Medicine 2006,“for their discovery of RNA interference - gene silencing by double-stranded RNA“,Case 1: Innate versus learned odour processing in the mouse olfactory bulb,Kobayakawa et al., 2007.Nature.450: 1-8,Mouse kiss Cat,RNAI silencing 16D10 parasitism gene in preparasitic M. incognita J2,Huang et al., PNAS, 2006,Reduction transcript: 93-97%,Reduction protein: 65-69%,
