酵母的基因工程课件

酵母的基因工程1.BiologicalCharacteristicsoftheBaker’sYeastTheygrowquickly,doublinginabout2hours(16-24hoursinmammaliancells).Theyeastgenomeisverysmall.Yeastisespeciallysuitedforgeneticanalysis(eitherashaploidordiploid).Aseukaryotes,theyareorganizedmuchlikecellsinmorecomplexorganisms.酵母的基因工程Thesizeofthegenome

inselectedorganisms酵母的基因工程2.ClassesofyeastvectorsI=integratingE=episomalR=replicatingC=centromere酵母的基因工程Shuttlevector酵母的基因工程3.Yeastselectablemarkers酵母的基因工程GeneEnzymeSelectionHIS3ImidazoleglycerolphosphatedehydratasehistidineLEU2β-IsopropylmalatedehydragenaseleucineLYS2α-AminoadipatereductaselysineTRP1N-(5’-phosphoribosyl)-anthranilatetryptophanisomerasetryptophanURA3Orotidine-5’-phosphatedecarboxylaseuracilCloningofyeastbiosyntheticgenesbycomplementation

inE.coli4.Genecloning酵母的基因工程Cloningayeastgenebygenetic

complementation酵母的基因工程5.GenetargetingReplicaplating酵母的基因工程Genetargetingbyhomologousrecombination酵母的基因工程Replacingachromosomal

genebytransplacement酵母的基因工程6.Yeastasaeukaryoticmodel

a.Tetradanalysis酵母的基因工程Tetradanalysis酵母的基因工程b.Thepheromonesignalingpathwayacell,afactorαcell,αfactorSTE–sterileComplementation酵母的基因工程CloningoftheGPA1genesupersensitive(hypersensitive)酵母的基因工程c.ThereceptorswapexperimentSTE2-αfactorreceptorSTE3-afactorreceptor酵母的基因工程d.GeneticdemonstrationthatU2RNA

base-pairswithanintronsequence酵母的基因工程e.Ageneticassayfor

protein-proteininteractionTheGAL4transcriptionfactor:DB---DNA-bindingdomain-snf1-beitAD---activationdomain-snf4-preyortargetproteinlacZreportergeneX-galUAS---upstream-activatingsequenceSUC2---SNF1andSNF4酵母的基因工程酵母双杂交系统

酵母激活因子GAL4：N端：147个氨基酸组成的DNA结合域(DNAbindingdomain,BD)，C端：113个氨基酸组成的转录激活域(transcriptionactivationdomain,AD)。GAL4分子的DNA结合域可以和上游激活序列(upstreamactivatingsequence，UAS)结合，而转录激活域则能激活UAS下游的基因进行转录。组成部分：（1）与BD融合的蛋白表达载体，被表达的蛋白称诱饵蛋白（bait）；（2）与AD融合的蛋白表达载体，被其表达的蛋白称靶蛋白（prey）；（3）由一个或多个报告基因的宿主菌株。酵母的基因工程YeastTwoHybrid原理BDXCOOHADcDNANH2

COOH共转化+BDX?ADGal4Gal4PromoterReporterTrp-Leu-cDNAlibraryNH2Gal4Gal4酵母的基因工程protein-protein