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主要内容一、转基因（Transgenesis）技术1、微生物中的基因转移2、植物转基因技术3、动物转基因技术二、基因敲除技术1．利用基因同源重组进行基因敲除2.利用随机插入突变进行基因敲除——基因捕获法3.RNA干扰引起的基因敲除基因敲除技术与诺贝尔奖文献：Constructionofpha-Operon-DefinedKnockoutMutantsofPseudomonasputidaKT2442andtheirApplicationsinPoly(hydroxyalkanoate)ProductionPseudomonasputidaKT2442couldaccumulatemedium-chain-lengthpoly(hydroxyalkanoate)s(PHA)consistingof3-hydroxyhexanoate,3-hydroxyoctanoate,3-hydroxydecanoate,and3-hydroxydodecanoatefromawiderangeofcarbonsources.Inthisstudy,thePHAsynthasephaoperon(phaC1-phaZ-phaC2)wasknockedoutandthevgbgeneencodingvitreoscillahemoglobinprotein(VHb),whichcouldenhanceoxygenuptakerateespeciallyatlowoxygenconcentration,wasintegratedintotheP.putidaKT2442genometoreplacethedeletedfragment.TheresultingmutantP.putidaKTOY01orgene-replacedmutantKTOY02wasusedasthehosttostudyPHAsynthasepropertiesandPHAproduction.DifferentPHApolymerase(PhaC)genes,phaCRefromRastoniaeutrophaH16,phaCAcfromAeromonascavie,andphaC2PsfromPseudomonasstutzeri1317,wereexpressedinthemutantstrainstotestthePhaCenzymesubstratespecificity.TheresultshowedP.putidaKTOY01orKTOY02couldprovidenotonlymclPHAmonomersbutalso3-hydroxybutyratefromfattyacids,whichmayallowtheproductionofcopolyesterspoly(3HB-co-mcl3HA).PlasmidpCJY10containingphaC2Ps,phbA,andphbBgenesencodingPHApolymerase,b-ketothiolase,andacetoacetyl-CoAreductase,respectively,weretransformedintoP.putidaKTOY01andKTOY02.Shake-flaskcultureshowedP.putidaKTOY01orKTOY02(pCJY10)couldaccumulatepoly(3HB-co-mcl3HA)fromglucose.TheaboveresultshowedphaoperonknockoutmutantofP.putidaKT2442wasaveryusefulhostofgreatpotentialnotonlyforstudyingPhaCsynthase,butalsoformicrobialproductionofcopolyesterspoly(3HB-co-mcl3HA),whichisverydifficulttoabtain.RECOMBINATIONaA1A1致谢补充资料
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