变性梯度凝胶电泳(DGGE)在微生物生态学中的应用

23820038ACTAECOLOGICASINICAVol.23,No.8Aug.,2003(DGGE)1,2,CarolaHolmstrm2,JeremyWebb2,StaffanKjelleberg2(1.,116023;2.SchoolofBiotechnologyandBiomolecularSciences,TheUniversityofNewSouthWales,Sydney2052,Australia):;(DP0211584):2002208217;:2003204220:(1963),,,,E2mail:[e-mail]:ChinaScholarshipCouncil,theCentreforMarineBiofoulingandBio2innovationandtheAustralianResearchCouncilgrant(No.DP0211584)Receiveddate:2002208217;Accepteddate:2003204220Biography:MAYue2Xin,Associateprofessor,mainlyengagedinmarinemicrobialecology.:,DNA,PCR,,,,DGGE,,,DGGE:,16SrRNA,18SrRNA(mmoX)A2(amoA),DGGEPCRDGGE,PCRPCRDNADNA[5]DNA,DGGE16SrRNA18SrRNA,DGGE,,,,,16SrDNA,DGGEDGGE,DGGE:DGGE;;PCR;rRNAApplicationofdenaturinggradientgelelectrophoresis(DGGE)inmicrobialecologyMAYue2Xin1,2,CarolaHolmstrm2,JeremyWebb2,StaffanKjelleberg2(1.SchoolofLife'1995-2006TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.ScienceandTechnology,DalianFisheryUniversity116023,China;2.SchoolofBiotechnologyandBiomolecularSciences,TheUniversityofNewSouthWales,Sydney2052,Australia).ActaEcologicaSinica,2003,23(8):15611569.Abstract:Becauseofthedifficultyassociatedwithisolatingandculturingbacteriafromenvironmentalsamples,alternativemethodsbasedonmoleculartechniqueshavebeendevelopedtodescribeandidentifymicrobialcommunities.RecentyearshavewitnessedarapiddevelopmentofDNA2basedmethodsforcommunityanalysissuchasPCRamplification,clonelibraries,fluorescentin2situhybridisation,restrictionfragmentlengthpolymorphism,denaturingandtemperaturegradientgelelectrophoresis.DGGEhasbeenwidelyusedinanalyzingthebiodiversityofbacterial,cyanobacterial,archaeal,picoeukaryotic,eukaryoticandviralcommunitiesinnaturalhabitats.Thistechniquecanprovideinformationonthepredominantspeciesinacommunityandanalyzemultiplesamplessimultaneously.Thereproducibilityandease2ofuseofthistechniquepermitinvestigationofthespatialandtemporalvariabilityofthepopulationandidentificationofcommunitymembersbysequencingofexcisedbandsorbyhybridizationanalysiswithspecificprobes.ThegeneralprocedureforDGGEanalysisofcommunitiesofmicroorganismsisasfollows:First,nucleicacidextraction;Second,amplificationofgenesencodingthe16SrRNA,18SrRNAorfunctionalgenessuchasmmoX,amoA;Third,analyzePCRproductsbyDGGE.DGGEemploysapolyacrylamidegelwithachemicaldenaturinggradientthatdifferentiallymeltsthePCRamplifiedproducts.ThedifferentDNAfragmentsgeneratedbyPCRareofthesamelengthbutdifferinthenucleotidesequence.Therefore,thedifferentdoublestrandedDNAfragmentswillstopmigratingatdifferentpositionintheDGGEgelduetotheirdifferentmeltingbehavioralongthechemicalgradient.ThedifferencesinthemeltingbehavioroftheDNAleadtoabandingpattern,whichisaprofileofthepredominantspeciespresentinthecommunity.DGGEutilizesagenefragmentconservedamongallorganismsforexample16SrRNAgenefragmentforbacteriaand18SrRNAforfungi.Itmustbeemphasizedthat,aswithothermolecularmethods,DGGEisnotfreeofbiases.OneofthesebiasesisthatDGGEallowsseparationonlyofsmallfragments,whichlimitstheamountofsequenceinformationforphylogeneticcomparisonaswellasprobedesign.Insomecircumstances,identificationofcommunitystructuretothespecieslevelisproblematicduetomultiplecopiesofthegeneofinterestresultinginmorethanonebandinasinglespecies.Inaddition,thistechniquecarriestheinherentproblemofheterogeneitybetweencopiesof,forexample,the16SrDNAinasinglebacterialspecies,whichleadstoanoverestimationofthenumberofmicrobeswithinnaturalcommunities.DGGEisapowerfultoolfortheanalysisofmicrobialcommunities.However,inordertoreducepotentialbiasesandlimitationsofDGGEandothertechniques,itissuggestedthatresearcherscombineDGGEwithothermolecularandmicrobiologicaltechniquestoobtainamoredetailedviewofmicrobialcommunitystructureandfunction.Keywords:DGGE;microbialecology;PCR;rRNA:100020933(2003)0821561209:Q938:A,,;,[1],,16SrDNA,,[2],16SrRNA265123©1995-2006TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.[3];,,,,[4]3,,,DGGEPCRDGGE(),PCRPCRDNADNA[5]DNA,DGGE16SrRNA18SrRNA1993DGGE[6],[7]DGGE,,1DGGEKawaiPCR16SrDNADGGE[8]PCRDGGE,PCR[9]Nbel16SrDNA,DGGE[10]Salles(Burkholderik)PCR2DGGEBurkholderik[11]DGGE16SrDNAMariagerFjord[12],DNAPCR,rRNADGGEPCRRT2PCRrRNADGGEDNADGGE,,[13]DGGE,DGGEPCR2DGGEpH[14]DGGE,DGGE,C2[15]PCR2DGGEVybiral[16]DGGE3(,012Lm012Lm),,012LmDGGEDGGE012LmDGGEDNA012Lm,A2B222(CFB)DGGE,15d,,É,Ë,ÓVa[17],,DGGE,DGGE36518:(DGGE)©1995-2006TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,,A2,B2C2(Firmicutes),,[18]HoldRFLPPCR16SrRNADGGE16SrRNAAlexandriumspp.Scrippsiellatrochoider,,,,[19],PCR2DGGE,,DGGECitrussinesis[20]Ávreas[21]rDNADGGE,meromictic,,,DGGE,PCR2DGGE,[22]PCR2DGGEPCR18SrDNADGGEAmmophilaarenaria,DGGEDNA[23]18SrDNADGGE,,[24]DGGEPCR18SrDNA,,DGGE[25][26]Dez[27]DGGE,PCR18SrDNADGGE,,DGGE,,33PCRShort[28]PCRDNA(pol)DGGE2DGGE,Schauer[29]PCR2DGGECatalan(NWMediterranean),DGGE,Barcelona,PCR2DGGE,DGGE,[30]PCR2DGGE,,[31]Duarte[32]PCR2DGGE(DBT),,,DBT,PCR2DGGE,[33]Lapara[34]465123©1995-2006TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,DGGEPCR2DGGE4,[35]Norris[36]PCR2DGGE(UV),404713,,Schafer[37]PCR2DGGE,DNARNADGGE,RNADGG