TGGE分析焦化废水处理系统活性污泥细菌种群动态变化及多样性高平平

2310200310　　　　　　ACTAECOLOGICASINICAVol.23,No.10Oct.,2003TGGE高平平1,晁群芳2*,张学礼2,王凌华2,赵立平2**(1.,　030006;2.,　200240):“863”(SZ-03-01-04,2001AA214131):2003-07-08;:2003-08-20:(1957～),,,,。E-mail:[e-mail]**Authorforcorrespondence,E-mail:[e-mail]*Foundationitem:ChineseNationalProgramesforHighResearchandDevelopment(No.SZ-03-01-04,2001AA214131)Receiveddate:2003-07-08;Accepteddate:2003-08-20Biography:GAOPing-Ping,Ph.D.candidate,mainlyengagedinthemolecularmicrobialecology.E-mail:[e-mail]:TGGE。3d1,816SrDNAV3-PCRTGGE,(Cs)100%。TGGE。TGGE,Cs83.3%。TJ1TGGE9、、,4,TGGE。3297%16(OTU),14OTUGenBank≥97%,2OTU95%94%。10OTU,8OTU。:;;;16SrDNA;TGGE;Populationdynamicsandcommunitystructureofbacteriainanin-dustrialphenol-degradingprocessanalyzedwithTemperatureGra-dientGelElectrophoresis(TGGE)GAOPing-Ping1,CHAOQun-Fang2,ZHANGXue-Li2,WANGLing-Hua2,ZHAOLi-Ping2　(1.ShanxiKeyLab.ofBiotechnology,ShanxiUniversity,Taiyuan030006,China;2.SchoolofLifeScienceandBiotechnology,ShanghaiJiaoTongUniversity,Shanghai200240,China).ActaEcologicaSinica,2003,23(10):1963～1969.Abstract:TGGEanalysisof16SrDNAgeneV3regionwasusedtostudythepopulationdynamicsandbac-terialdiversityintwofunctionallydifferentaerationtanks(TJ1andTJ2)withinaphenol-degradingacti-vatedsludgeprocess.Thebandpatternsproducedfromsamplescollectedat3-dayintervalsover8timepointsfromeachaerationtankdemonstratedahighdegreeofsimilarity,withCsvaluesat100%.TGGEfingerprintingforactivatedsludgesamplesfromdifferentsitesinonetankshowedidenticalpatterns,butthatfromfunctionallydifferenttankexhibitedbothcommonbandsanduniquebands,withaCsvalueof83.3%.NinemajorbandsfromTGGEprofilesofTJ1samplewaselutedfromgel,reamplified,clonedandalibraryestablished.Fourclonesfromeachbandweresequencedandmorethanonesequencetypewasfoundineachbanddemonstratingtheco-migrationofDNAfragmentswithsubstantialsequencevaria-tioninTGGEbands.The32sequencesforwhichtherewasnoindicationofchimerismwerealignedwiththesequencesofclosestrelativesinGenBank.Theresultssuggestedthattheybelongedto16OTUsat97%homologycutoff.14OTUsshared97%～100%homologywithknownsequencesinthedatabasewhile2OTUshad94%～95%homologywithknownsequences.AmongalltheOTUs,10weresimilartobacteriaindatabasethatwerereportedtohavebeenisolatedfromdifferentactivatedsludgesystemsandcontaminatedenvironments.Theisolatesfrom8OTUsgroupedtogetherwithsomeunculturedeubacteriainthedatabase.Aphylogeneticanalysisof16OTUsclassifiedthemintotwomajorgroups:theOTU13lineageandtheother15OTUslineage,thelaterwasclassifiedfurtherinto4subgroups.Thesequencedi-versityfromTJ1revealedthathighdiversityofbacterialpopulationsexistedintheindustrialphenolbiore-mediationsystemandthecommunitystructurewasrelativelystableovertime.Keywords:phenol-degradingactivatedsludge;communitystructure;populationdynamics;16SrDNA;TGGE;sequence:1000-0933(2003)10-1963-07　:Q938.1　:A　　,,、。,,[1]。。85%～99%[2]。(DGGE,denaturinggradientgelelectrophoresis)(TGGE,temper-aturegradientgelelectrophoresis)DNA。DNA,。1993Muyzer[3]DGGE,16SrDNA,,、。DGGETGGE,,。,MuyzerDGGE,,。,TGGE/DGGE,[4,5],[1]。16SrDNAV3-PCR/TGGE。、、TGGE,,。1　1.1　1.1.1　　。(AB),(TJTJA)。(TJ1TJA-1),。(TJ2TJA-2)TJ1TJA-1,。TJ,3d1,8,1964　　　　23M1-M8;M1,,TJ1TJA-12,S1、S3S2、S4;,TJ2TJA-21,S5S6。1.1.2　　DH5T(E.coli)MarkerP1-46P1-7。1.1.3　　TaqDNA,T4DNA,pGEMT-EasyPromega,IPTGX-Gal,。PCRMOBIO(SikabaBeach,CA),AgNO3Sigma。1.1.4　PCR　16SrDNA(V3)Watanabe[6],P2P3:5′-ATTACCgCggCTgCTgg-3′5′-CgCCCgCCgCgCgCggCgggCggggCgggggCACggggggCCTACgggAggCAgCAg-3′(40GC)。1.2　1.2.1　DNA　DNADNA[7]。1.2.2　16SrDNAV3　Niemi(2001)[8],5min。:95℃5min,94℃45s,55℃45s,72℃60s,30,72℃6min。50μL,2μLdNTP(2.5mmol/L),5μl10×Buffer,3μlMgCl2(25mM),0.2μlTag(5u/μl)。P2P310pmol。PCRPCRSprint(ThermoHybaid,Ashford,UK)。1.2.3　TGGE　TGGEMaxisystem(Whatman,BiometraGmBH)16SrDNA(V3)。8%8M20%。36～45℃16SrDNA,120V,15h。TGGE,400ml(30%EtOH,10%glacialacid)30min,0.2%AgNO3(30min)(1.5%NaOH;0.5%)。,(10%EtOH,0.5%glacialacid)。(ScanMaker4680,MICROTEK)。1.2.4　TGGE　Sorenson(pairwisesimilaritycoefficient,Cs)TGGE[9]。Cs=2j/(a+b)×100,aTGGE,bTGGE,j。1.2.5　TGGE　TGGE,20μLEP,EP4℃DNA[10]。1min(10,000×g,4℃),3～5μlPCR,。1.2.6　TGGE　DNA、(UltraCleanPCRClean-upDNAPurificationKit,MOBIO,SikabaBeach,CA)。DNApGEM-TEasy,、pGEM-TEasy《》[11]。1.2.7　TGGE　16SrDNA(V3),GenBank,AY351568-AY351583,GenBankBLAST,ClustalX(1.81version)。2　2.1　8TJ1TJ2TGGETJ1TJ2816SrDNAV3,TGGE,。18TJ1TGGE,12(1-A),3,9。TJ2,12(1-B)。8TGGE196510:TGGE　,Cs100%,。1　8TJ1TJ2TGGEFig.1　TGGEprofilesgeneratedfromtheactivatedsludgesamplesofthefirst(TJ1)andthesecond(TJ2)aera-tiontankafterPCRamplificationoftheV3regionofthe16SrDNAgeneover8consecutivesamplingdays(M1-M8)at3daysintervals.Lanes:1-8:M1-M88theactivatedsludgesamplescollectedatM1toM8dates.;MrA:P1-46;MrB:P1-7;A:gelimageofactivatedsludgesamplesformTJ1;B:gelimageofactivatedsludgesamplesformTJ2　2　TGGEFig.2　TGGEprofilesgeneratedfromtheactivatedsludgesamplesofdifferentsitesinonetankandthatindifferenttankafterPCRamplificationoftheV3regionofthe16SrDNAgene.L1-L2(S1-S3):TJ1;activatedsludgesamplesfromTJ1;L3-L4(S2-S4):TJA-1;activatedsludgesamplesfromTJA-1;L5-L6(S5):TJ2;activatedsludgesamplesfromTJ2;L7-L8(S6):TJA-2;ac