PCRSSCP技术用于脱臭微生物群落结构的研究

PCR-SSCP,*,(,200062):PCR-SSCP().,50%89%.2,,16~192;2,,,;,2220,,2.SSCP,333%;444%;,,.:PCR-SSCP;;;;Shannon-Wiener:X172:A:0250-3301(2008)07-1992-06:2007-07-12;:2007-08-28:(51X1408A):(1981~),,,.*,E-mail:[e-mail]munityinaDeodorizationBiofilterbyPCR-SSCPMethodMIWen-xiu,XIEBing,XUYa-tong(TiantongNationalStationofForestEcosystem,KeyLaboratoryofUrbanizationandEcologicalRestorationofShanghai,DepartmentofEnvironmentScience,EastChinaNormalUniversity,Shanghai200062,China)Abstract:MicrobialcommunityofbiofilminabiofiltrationwasinvestigatedusingPCR-SSCP(singlestrandconformationpolymorphism)techniqueinthispaper.Theresultsindicatedtheremovalrateofodorpollutantsimprovedwiththeacclimation,from50%to89%,andthemicrobialdiversityofbiofilterdecreasedatthefirstmonthandthenincreased(diversityindexHfrom16-19to20)whilethesimilaritygraduallyincreasedduringtheoperationtime.Highermicrobialdiversity(H=22)incortexindicatedthemicroorganismswereeasilyattachedtothemediacomparedtothestraw(H=20).DominantbacteriawereBacillusfoundinthebiofilmusingSSCPmethod,andtherateis333%.444%ofthetotalbandsrepresentedtheunculturedbacteria.Thesebacteriaarewidelyexistedinsoil,waterandnatureenvironment,theyhavegoodacclimatizationtoenvironmentandplayedimportantroleintreatingodors.Thebiofilmdevelopmentwasidentifiedbythescanningelectronmicroscopy(SEM),whichsuggestedthatthemicrobialcommunityinbiofiltercouldgrowbyutilizingpollutantsandbecomerichandstablewithrunningtime.Keywords:PCR-SSCP;biodeodorization;biofilm;microbialcommunity;Shannon-WienerdiversityindexPCR-SSCP(singlestandconformationalpolymorphism,SSCP,),,[1,2],,[3~5],SSCP.,SSCP,,,[6,7].,,,.,[8,9].,,,,,[10].PCR-SSCP,,.29720087ENVIRONMENTALSCIENCEVol.29,No.7July,2008111,.:..14812,4,(gp),.4:g1p1g2p2g3p3g4p4.12DNA121DNA--DNA.,,,2g5mL,2~3mLTENS.1min,10000rmin10min,,1.2~3mLTE,.50L10%SDS20L20mgmLK,,37,170rmin1h.[5].DNA-20.122DNA,PCR,PCR,,PCR,DNA.[11]DNA.123DNA5L1L,,(1%),80V1h,EB15min,Smartview(ShanghaiFuRiCo.),DNAMarkerDNA.DNA.DNAc(molmL)=A26050.260nm,DNA.13DNAPCR131PCRSRV3-1SRV3-2[12],SRV3-25,E.coil16SrRNA330~348bp533~515bp..SRV3-15-3:CGG(CT)CCAGACTCCTACGGG,SRV3-25-3:TTACCGCGGCTGCTGGCA.200bp.132PCRPCR:50L,10Buffer(Mg2+)5L,10mmolLdNTP1L,20pmolL1L,1L(50ng),Taq05L,ddH2O50L.PCR:955min;9440s5030s7240s,30;7210min.PCR5L1L,,1%.80V1h,EB15min,Smartview,.14PCR,,PCR[13].60L:(5UL)6L;10buffer6L;PCR30L.374h,,7210min.15SSCP10%(491),5%,SSCP[14].40mL1%(APS)450mL,TEMED40L,30min,150V30min.10L10L(95%,10mmolLNaOH,20mmolLEDTA,002%002%FF),9510min,,5min.4,150V,12h.,..16Smartview,SSCP,1,1,,,Shannon-Wiener[15]:H=-Si=1pilnpi19937:PCR-SSCP,HShannon-Wiener;pii,i;S,SSCP.(SorensonPairwiseSimilarityCoefficient).CS=2j(a+b)100,CS;aPCR-SSCP;bPCR-SSCP;j2.2Sorenson0,2DNASorenson100%.MSVPSSCP.17ddH2O,1~902mLPCR,.(-20,45)3,.6L(10~20ng),SRV3-1SRV3-2,PCR.PCRSSCP,.NCBI,.18,(11)cm2,3%,4,25nmJSM-5610LV().221,,1.588,11885,147,719%,890%.1,,,50%,8,80%,,.16,,,.1Fig.1Odorconcentrationofinletandoutletanditsremovalratewithrunningtime22DNAPCRDNA.--21kbDNA.DNA,,DNA(),,,PCR.SRV3-1SRV3-2g1~g4p1~p4DNAPCR,200bp,.23PCR-SSCPPCRSSCP,2.,SSCP,,10.1,1,,,.2,5789,,,9.,,1~4..162,Shannon-Wiener,3.3,15~25.,,.,,1994292PCRSSCPFig.2ResultsofPCR-SSCPofmicrobialcommunityinbiofiltersamples3Shannon-WienerFig.3Shannon-Wienerdiversityindexofdifferentbiofiltermicrobes,,1(g1p1)Shannon-Wiener,22;2(4)g2p2,H16~19,,,,,,;,,,,,8H2,12g4p4H22231,,.,(p)(g),H2220,,.2Smartview,MVSP,UPGMA44UPGMAFig.4UPGMASorensonsimilarityofdifferentbiofiltersamples4,,g2g4,85%,60%,p1p270%~85%,60%~81%,,,,.,p3p4g3g4p1p2g1g2,,,,.,,,,12,1,(56),,.24PCR-SSCP2SSCP,,1~9,DNA,,NCBI,1.,,333%.19957:PCR-SSCP5SEMFig.5SEMphotosofstrawsurface6SEMFig.6SEMphotosofcortexsurface1Table1Resultofbandsblasting%1Bankit922930Bacillussp.YACN-998,2Bankit922932Unculturedbacteriumclonesv21d596,3Bankit922189Bacillusmycoides96,4Bankit922193UnculturedBacillussp.955Bankit922960Pseudomonassp.Hugh2768936Bankit922964Pseudomonasplecoglossicida977Bankit922966UnculturedEnterobacteriaceaebacterium988Bankit922199UnculturedbacteriumcloneP6D1-496989Bankit922970Mesorhizobiumsp.100,,[16~18],.,,444%.,.3(1),50%89%199629.,2,,,..2,.(2),444%.,.(3)PCR-SSCP,.:[1]OritaM,IwahanaH,KanazawaH,etal.DetectionofpolymorphismsofhumanDNAbygetelectrophoresisassinglestrandconformationpolymorphisms[J].ProceedingsoftheNationalAcademyofScience,1989,86:2766-2770.[2]FrankS,ChristophCTA.NewapproachtoutilizePCR-Signa-lStrand-Conformationpolymorphismfor16SrRNAGene-BasedMicrobialCommunityAnalysis[J].AppliedandEnvironmentalMicrobiology,1998,64:4870-4876.[3]SilwinsklMK,GoodmanRM.SpatialheterogeneityofcrearchaealassemblageswithinmesophilicsoilecosystemsasrevealedbyPCRsinglestrandedconformationpolymorphismprofiling[J].AppliedandEnvironmentalMicrobiology,2004,70:1811-1820.[4]DuthoitF,GodonJ,MontelM.Bacterialcommunitydynamicsduringproductionofregistereddesignationoforiginsalerscheeseasevaluatedby16SRNAgenesingle-strandconformationpolymorphismanalysis[J].AppliedandEnvironmentalMicrobiology,2003,69:3840-3848.[5]SabineP,StefanieK,FrankS.Successionofmicrobialcommunitiesduringh