丝状真菌目标基因替换过程中的策略与方法

HEREDITAS(Beijing)2007年7月,29(7):898904ISSN0253-9772−12−06;:2007−01−26:(:30370925),(:Y306638)(:2005038279)[SupportedbyNationalNaturalScienceFoundationofChina(No.30370925),ZhejiangNaturalScienceFoundation(No.Y306638)andChinesePostdoctoralScienceFoundation(No.2005038279)]:(1978−),,,,,:Tel:[phone]E-mail:[e-mail]:(1963−),,,,:Tel:[phone]E-mail:[e-mail](1966−),,,,:Tel:[phone]E-mail:[e-mail]:10.1360/yc-007-08981,2,1,1,1,21.,310021;2.,310029摘要:,,,关键词:;;StrategiesoftargetedgenereplacementinfilamentousfungiWANGJiao-Yu1,2,DUXin-Fa1,CHAIRong-Yao1,SUNGuo-Chang1,LINFu-Cheng21.InstituteofPlantProtectionandMicrobiology,ZhejiangAcademyofAgriculturalScience,Hangzhou310021,China;2.ColledageofAgricultureandBiotechnology,ZhejiangUniverstiy,Hangzhou310029,ChinaAbstract:Targetedgenereplacement(TGR)isanimportanttechniqueforgenefunctionanalysis.Withthedevelopmentofgenomesequencingandtransformation,TGRhasbeenappliedwidelytofilamentousfungi,andmanynewsystemsandapproacheshavebeenestablished.Inthispaper,strategiesinvolvedinTGRinfilamentousfungiwerereviewed,includingtransformationsystems,targetingvectorconstruction,mutantselectionandsoon.ComparedwithTGR,RNAiandothertechniquesofgenefunctionanalysiswereintroducedandreviewedaswell.Keywords:targetedgenereplacement;filamentousfungi;genefunctionanalysis(Targetedgenereplacement,TGR),,,,,TGR2080DNA(homologousrecombination,HR),[1],TGR,,,1TGRTGR,[2,3]CaCl2/(Agrobacterium7:899tumefaciensConn.mediatetransformation,AtMT)CaCl2/[4],1050mmol/LCaCl21030min[5](),,DNA,,[6],,,DNA[7,8],,[8][8],,,,[9],(ErysiphegraminisDC.),[10],AtMT[2,11],A.tumefaciensDNA[12]AtMT,AtMT,[12]AtMT,,[2],TGR[13]TGR,,,AtMT[2][7]TGR,AtMT2TGRDNA(targetingvector,TV),TGR500bp[14]2.1基于克隆的传统方法cosmid,,TV,,,PCR,TV(5kb),,(~7kb),,,,TV,(Magnaporthegrisea(Hebert)Barr.)(Gibberellazeae(Schw.)Petch),PCRTV,,2.2基于PCR的高效方法TV,,TVTGRPCR(double-jointedPCR)PCR(fusionPCR)[14]TV(1),3PCRPCR,()5;PCR,,,,PCR;3PCR,(Aspergillusnidulans(Eidam)Win-ter)(A.fumigatusFres.)(FusariumgraminearumSchw.)TV[14],(A.awamoriNakaz.)TGR[15],M.griseaTGR,,TVPCR(ligation-mediatedPCR)[16],,DNA,PCR,900HEREDITAS(Beijing)2007291PCRTGR[14]A:PCR;B:PCR,,PCR;C:PCR;D:TGR(GgprA)(910),,TGR,3.2kb,3.8kbFig.1Constructionofagenereplacementcassetteviadouble-jointedPCR[14]A:FirstroundPCR:amplificationofthecomponentsusingthespecificandchimericprimers.B:SecondroundPCR:theassemblyreactioniscarriedoutwithoutusinganyspecificprimers,astheoverhangingchimericextensionsactasprimers.C:ThirdroundPCR:amplificationofthefinalproductusingnestedprimers.D:Confirmationofgenereplacement(anexampleofdeletionconfirmationofthegprAgeneencodingaputativeGproteincoupledreceptor):transformantswereexaminedbyPCRamplificationofthegprAlocususingaprimerpair(primers9and10)beyondtheflankingregionsincludedinthecassettetoidentifygenedeletionmutantsfromrandominsertiontransformants.Asshown,ampliconsofwildtype(3.2kb)anddeletion(3.8kb)allelesofgprAdifferinsize.,,PCRTV,AtMT[15]2.3高通量的转座子队列基因敲除TGR(Transposonarrayedgeneknockout,TAGKO)[17]cosmid,(),Tn7cosmid,cosmidTV,TAGKOTGR,11TAGKOTV(Mycosphaerellagramini-colaFuck.),TGR15%~28%[17],AtMTTAGKOTGRTV(~40kb)[17]TAGKOTGRTAGKO,BAC[18],TAGKO-AtMTTGRTVTAGKO,GPS-1genome-priming[19],T/ApGEMPYR4pGPS1,TV,3TGRTGRSouthernHR,PCRYu[14]7:901(1),PCR,DNAPCR/(positiveandnegativedualselectionsystem)[13,20]TGR(herpessim-plexvirusthymidinekinase,HSVtk)(5-fluoro-2-deoxyuridine,F2dU),HSVtk,TV(2)[13],F2dU;TGRHRHSVtk,F2dUTGR,AtMT(AtMT-DS)TGR[13],amdS(fluoroacetamide),[15]GUS,,,M.grisea(Ustilaginoidenvirens(Cooke)Tak.)(),TV,4TGRTGR,,[21]4.1转化方法与转化效率TGR,,,AtMT,DNAA.tumefaciensDNA[22],T-DNA,HR[23],AtMTTGR,(Restric-tionEnzymeMediatedIntegration,REMI)[24,25]TGR2AtMT-DS[13]((hygromycinB)HPT,)TGRHSVtk()T-DNA,A.tumefaciens,T-DNAHRHSVtkHPT,HSVtk();HPTHSVtk(),F2dU,,F2dU,TGRFig.2SchematicdiagramofAtMT-DS[13]Abinaryvectorcarryingamutantallele(disruptedbyapositiveselectionmaker,suchassuchasHPTgeneconferringresistancetohygro-mycinB;markedasthefilledbox)andHSVtk(denotedbythediamond)ontheT-DNAistransportedintoA.tumefacienscells.Viaco-cultivationwithfungalcells,theT-DNAistransportedintofungalnuclei.HomologousrecombinationbetweenthenativegeneandthemutantalleleontheT-DNAleadstothelossofHSVtk(left).IftheT-DNAintegratesintoarandomlocationinthefungalgenomevianon-homologousrecombination,bothhptandHSVtkwillbeexpressed(right).Targetedgenereplacementmutantscanbeselectedbysubject-ingtransformantstoboththepositive(hygromycinB)andnegative(F2dU)selectionagents.902HEREDITAS(Beijing)200729[25],TGR,REMI,[25],,TGR,,,4.2HR频率,HRTV(Saccharo-mycescerevisiaeHansen),HR50~100bp,,1kbTGR[14]TGRTV,100%,HR[7]HR,HR[26]TGR[27]S.cerevisiaeHRRAD51[28],A.nidulans,rad51uvsCTGR[26],UVSC,HR4.3多核现象,TGRDNA,,,[29][8,29],,[30],,[31],,[32],RNAiknockdown,mRNA,5TGRRNAiTGR,,TGRHR,,,TGRRNA(RNAinterference,RNAi)(genesilencing),,[33]RNAimRNA,,,TGRTGR,RNAi,RNAi,,,RNAimRNA,,,RNAi,TGR,,,RNAi[34],RNAi,RNA,[35],RNAi,RNAi,,[35],,,RNAiRNAi[35,36],,,,GFP[37],RNAi,RNAi7:903,RNAi,,,RNAi,RNAiRNAi,UTR(untranslatedregion),UT