16SrDNA克隆文库方法分析MDATIAT同步脱氮除磷系统细菌多样性研究

26620066ActaScientiaeCircumstantiaeVol.26,No.6Jun.,2006:863(No.2002AA601320B)SupportedbytheHi2TechResearchandDevelopmentProgramofChina(863)(No.2002AA601320B):(1976),,(),Tel:[phone],E2mail:[e-mail];3(),E2mail:[e-mail]:WANGHaiyan(1976),female,AssociateProfessor(Ph.D.);3Correspondingauthor,E2mail:[e-mail],,,.2006.16SrDNAMDAT2IAT[J].,26(6):903-911WangHY,ZhouYX,DaiX,etal.2006.Bacterialdiversitystudyforthesimultaneousnitrogenandphosphorusremovalsystem(MDAT2IAT)by16SrDNAcloningmethod[J].ActaScientiaeCircumstantiae,26(6):903-91116SrDNAMDAT2IAT1,1,3,2,1,1,21.,1000122.,100080:2005208211:2006203216:2006203224:16SrDNAMDAT2IAT(modifieddemandaerationtank2intermitaerationtank)IAT.16SrDNA59(500bp),BLAST.,MDAT2IATIAT,556,3,Proteobacteria(),55.17%;2Proteobacteria(34.48%)Bacteroidetes(,20.69%)2Proteobacteria(12.07%)CandidatedivisionTM7(12.07%)2Proteobacteria(5.17%)2Proteobacteria(3145%)Firmicutes(,3.45%)CandidatedivisionOP11(1.72%)Planctomycetes(,1.72%).clustalx5832OTU,IAT.:MDAT2IAT;;;16SrDNA;Proteobacteria:025322468(2006)0620903209:X172:ABacterialdiversitystudyforthesimultaneousnitrogenandphosphorusremovalsystem(MDAT2IAT)by16SrDNAcloningmethodWANGHaiyan1,ZHOUYuexi1,3,DAIXin2,CHAIYanli1,JIANGJinyuan1,LIUShuangjiang21.ChineseResearchAcademyofEnvironmentalSciences,Beijing1000122.InstituteofMicrobiology,ChineseAcademyofSciences,Beijing100080Received11August2005;receivedinrevisedform16March2006;accepted24March2006Abstract:ThebacterialdiversityintheIATtankoftheModifiedDemandAerationTank2IntermitAerationTanksystem(MDAT2IAT),whichwasofhighefficiencyforthesimultaneousnitrogenandphosphorusremoval,wasstudiedbythe16SrDNAcloningandsequencingmethod.59cloneswererandomlyselected,andtheirpartial16SrDNAgene(ca.500bp)wassequencedandblasted.TheresultsindicatedthatthebacterialdiversityintheIATtankoftheMDAT2IATwasveryhigh,where55clonesbelongto6differentpublishedphyla,while3clonesbelongtounknownphylum.ThedominantbacterialcommunityintheIATtankwasProteobacteria,whichaccountedfor55.17%.Thebacterialcommunitysuccessionwereasfollows:the2Proteobacteria(34.48%),Bacteroidetes(20.69%),2Proteobacteria(12.07%),CandidatedivisionTM7(12.07%),2Proteobacteria(5.17%),2Proteobacteria(3.45%),Firmicute(3.45%),CandidatedivisionOP11(1.72%),Planctomycetes(1.72%).32OUTphylotypeswereselectedfromthe58sequencedclonesbyClustralxSoftware,andthephylogeneticanalysisresultsalsodemonstratedhighbacterialdiversityintheIATtank.Keywords:MDAT2IAT;nitrogenandphosphorusremoval;bacterialdiversity;16SrDNA;Proteobacteria2,,26(1%10%);,16SrDNA(Darbyetal.,2001),.16SrDNA,500bp,(Hugenholtzetal.,1998),(Brambillaetal.,2001).16SrDNA-RFLP(restrictionfragmentlengthpolymorphism,),(KawaharasakiM,etal.2002)16SrDNA2DGGE(denaturalization-gradientgelelectrophoresis,),(muyzeretal.,1995)16SrDNA,.,DNA,16SrDNA.16SrDNA,,(Brambillaetal.,2002;Felskeetal.,1999;,2001;Bondetal.,1995;Schuppleretal.,1995;Bakeretal.,2001),,.16SrDNA(Suzukietal.,1996),rRNA(Sonensheinetal.,1993),DNAPCR(Raineyetal.,1994).,16SrDNA,(Brambillaetal.,2001).MDAT2IAT(ModifiedDemandAerationTank-IntermittentAerationTank)SBR(,2005),,,16SrDNAMDAT2IATIAT(),,,,,.1(Materialsandmethods)1.1MDAT2IATIAT100mL(,IAT),1.-20.DNA,DNA.1MDAT2IATTable1TheoperationparametersandresultsoftheMDAT2IATprocessHRT/SRTDO/(mgL-1)/MLSS/(mgL-1)/(kgkg-1d-1)20h/3.5d:0.10.2;2.015.522.71h,1h,2h,4h440070000.47COD/(mgL-1)CODNH+42N/(mgL-1)NH+42NPO3-42P/(mgL-1)PO3-42PSS/(mgL-1)SV/(gL-1)9.230.696.3%98.7%3.27.680.4%92.5%0.20.590.6%95.9%3.09.075%80%14151.2:DNA(Germany,Promega)TaqDNA(Germany,Promega)dNTP(Janpan,Takara)T4DNA(Germany,Promega)().:ThermocyclergradientPCR(Biometra)Q2901Votex(4096:16SrDNAMDAT2IAT)HV23000()(),8682pH(ThermoOrion)Bio2RadDOC1000(Bio2Rad)(LKBBROMMA)(HeraeusSepatech)(NewBrunswickScientific).1.3DNAPCR:MillerDNA(Milleretal.,1999),.DNA,16SrDNA27f:52AGACTTTGATCCTGGCTCAG231492r:52CGGCTACCTTGTTACGACTTC23(16SrDNA82714921512)PCR.PCR:944min,941min;501.5min;722min,277210min.PCR:10LPCR(500mmolL-1KCl,15mmolL-1MgCl2,100mmolL-1Tris2HCl[pH9.0],1%TritonX2100),4L5mmolL-1dNTPs,50molL-127f1492r2L,5UL-1TaqDNA0.4L,ddH2O100L,4,1LDNA.PCRPCRPCR.16SrDNA:PCRQIAquickPCRDNA.pGEM2Teasy,EscherichiacoliJM109(,2003),Amp2Xgal/IPTG16SrDNA.DNA:16SrDNA59,16SrDNA27f.CHECK2CHEMERA.ClustalX118(Thompeonetal.,1994),GenBank,BLAST,ClustalX1.8DNATreeconw(113b).2(Experimentalresults)2.1DNAPCR1DNA.112IATDNAmarker,1DNA,21kb,DNAPCR.2123456DNA100100010000100000PCRmaker,2DNA1000PCR,PCR,DNA1000PCR.2PCRDNA1.5kb,16SrDNA.51.5kbDNA,,PCR16SrDNA.1IATDNA(1.DNA,2.marker)Fig.1TheextractionDNAelectrophoresisofthesludgesamplefromtheIAT(1.theextractionDNA,2.marker)2DNAPCR(1.DNA100,2.DNA1000,3.DNA10000,4.DNA100000,5.,6.marker)Fig.2ThePCRresultselectrophoresisofthedifferentdilutionDNAtemplate(1.100dilution,2.1000dilution,3.10000dilution,4.100000dilution,5.blankcontrol,6.marker)509262.216SrDNADNA16SrDNA:IAT16SrDNA,59(1.5kb),PCR500bpDNA,16SrRNAC1C2D1D2.591,,5816SrDNABLAST2.2IAT16srDNATable2Bacterial16SrDNAcloningresultsoftheIATtankOTUOTU/bpEMBL(NCBI)A123811.72%530AM180046Catellibacteriumne