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上海交通大学硕士学位论文DGGE指纹图谱分析太湖富营养化水体中细菌群落结构的变化姓名：吴鑫申请学位级别：硕士专业：环境科学指导教师：杨虹20070101IVDGGEPCR-DGGE()(0649)DNA357F-GC-clamp518RPCRPCRDGGE40%55%DGGEDGGEQuantityOneV17αβγ-398%16SrRNAVICHANGESINBACTERIALCOMMUNITYSTRUCTUREOFAHYPERTROPHICFRESHWATERLAKETAIHU,DETERMINEDBYDENATURINGGRADIENTGELELECTROPHORESISFINGERPRINTABSTRACTLakeTaihu,thethird-largestlakeinPeople’sRepublicofChina,isatypicalshallowfreshwaterlake.Duetoagriculturalintensificationandexcessiveexploitation,itstrophicstatuswasincreasinglygettingaggravated.Waterqualitywasgettingworseandtheecosystemwasdeclining.Innature,thereareextraordinaryrelationshipsamongmicroorganismsandbetweenmicroorganismsandenvironments.Duetothelackofculturedbacteriainaquaticecosystems,thediversityofbacterialcommunitieshasnotbeenextensivelystudied.However,developmentsinmolecularbiologicaltechnologybringanewpathforscientiststoinvestigatebacterialcommunities.Inordertodescribeacompletepictureofthestructureofbacterialcommunitiesinahypertrophicfreshwaterlake,PCR-DGGEmethodwasappliedtostudywatersamplesfromLakeTaihu.Watersampleswerecollectedwithdifferentlocations(Eastern,southernVIIandnorthernpartofLakeTaihu)anddifferentmonths(FromApriltoSeptemberin2006).AfterDNAextraction,theDNAwasusedastemplatetoamplify16SrRNAgenewithuniversalprimers357F-GC-clampand518r.ResultsindicatedthatnonspecificamplificationcouldhaveabadinfluencetoDGGEprocess.Thefactors,whichmightleadtoabadamplification,includedthetypeofprimersandtheirconcentrations,concentrationoftemplate,annealingtemperatureandtotalcycles.Thegradientofdenaturantwasfinallyconfirmedas40%-55%.AnalysisofDGGEfingerprintrevealedthatrichnessofbacteriainLakeTaihuwasrelativelyhigh.Distinctdifferencesweredetectedinfingerprintatbandnumbers,bandpatternsanddensitieswithdifferentsamplingsitesanddifferentsamplingmonths.QuantitativeanalysesbyQuantityOnesoftwarepackageshowedthattherewerefourmajortypesofbacterialcommunitystructureinLakeTaihu,includingwinterperiod,beginningperiod,happeningperiodandheavyperiod.AsanimportanthypertrophicpartofLakeTaihu,MeliangBaywasfounddifferentlytoothertwolocationsinbacterialcommunitycomposition.Seventeentypicalbandswereexcisedandsequenced.ThesequencesobtainedwereaffiliatedwithActinobacteria,α-,β-,γ-Proteobacteria,Bacteriodetes,ThermomicrobiaandCyanobacteria.Remarkably,themostrelativerepresentativesofthreesequencescamefromapreviousstudybasedonclonelibrarymethodtoinvestigatebacterialcommunitycompositionofMeiliangVIIIBay.Thesimilaritywasabove98%.Accordingtophylogeneticanalysis,thesesequencescouldbegroupedtopreviousputativefreshwaterclusters.Twostatisticalmethods,principalcomponentanalysis(PCA)andcanonicalcorrespondenceanalysis(CCA),werecarriedoutforfurtheranalysis.SeasonalchangesofbacterialcommunitystructureinLakeTaihucouldbeconfirmedbasedonPCA.ResultsofCCAindicatedthatbacterialcommunitystructureofLakeTaihuwasprimarilyassociatedwithtemperature,followedbynitrogen,phosphateandcarbon.KEYWORDSbacterialcommunitystructuredenaturinggradientgelelectrophoresis16SrRNALakeTaihustatisticalanalysisII2007131III20071312007131-1-1.130°55’31°34’119°53’120°36’3165km33770381030/km3GDP1/81/61/4201995GDP10%2427.8km32338.1km31.89m53m3224701502.99m44.23m34.65m83m31cm2300m3471123209080ΙIIVVV280-1.525196067(1-1)19900.5m21161.31994-2-1-1NP(mg/l)[1]196019801987198919911992199319941995TN1.892.391.641.942.262.46NO3--N0.021.151.080.6620.4740.7270.4530.764NO2--N0.010.0090.030.0330.0050.0010.0530.013NH4+-N0.020.120.150.360.190.0420.1120.2940.261NTON0.631.091.501.121.10TP0.0080.0310.0750.0620.0710.1530.0510.076PO4--N0.020.0070.0200.0210.0250.0050.003PTOP0.0210.0280.0360.1041-2[1](µg/L)(m)TN(µg/L)TP(mg/L)COD(mg/L)300.664.44.60.080.96504.101.323.00.313.607026.000.40110.01.2014.008064.000.22250.02.3027.00200071.2[2]2025mg/la10mg/l2.0m10%-3-1.2.1[2]••1g/0.21g••••-4-1.2.2•30193319941946041990157•(COD)(BOD)(SS)(THMs)()•-5-1998200029%71%[3]200423%77%1.3(DenaturingGradientGelElectrophoresis)[4](MicrobialEcosystem)0.1%15%[5]DNA[6,7]PCR[8,9][10,11][12](1-1)(DGGE)FischerLerman1979DNA(Agarose)(PAGE)1985Myers[13]DGGEGC1993Muzyer[12]DGGEDGGE-6-•••••1.3.1DGGEDNADNADNAAT2GC3ATGCDNADNA()DNA(1-2)DGGE1-1Figure1-1Pathofmolecularbiologicaltechnology-7-DNADNAPCR(1-3)GCGC(GC-clampGC3050)PCRGC5’DGGEDNA,DNADGGEDNA1-2DNAFigure1-2MeltingprocessofDNAdoublehelix1-3DNAFigure1-3MovementofDNAfragmentingradientgel-8-1.3.2DGGE[14]DGGEa.DGGE0%100%20%70%b.DGGEDNADNADGGEDGGEDNADNADNA1.3.2.1DNADNA()DNADNADNADNADNADNADNADNADNA“S”Tm()DNATmTm101030%1.3.2.2DGGEDNATmDNA5065DGGE-9-DNADNA200bp150V,4hDNADNAWinMelt/MacMelt()TGGE-STAR()Polandanalysis()1.3.2.3“GC”DNAGCATGCGC(GC-clamp)3050bpGCDNADNADGGE500bpDNA50%GC100%1-3GCDGGEGCGCPCR(Proofreadingpolymerase)PCRGCGC1-3“GC”[15](5’→3’)CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCCGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG1.3.2.4(Ethidiumbromide,EB)EB-10-(Ag+)(Ag+)EB200DNASYBRGoldSYBRGreenISYBRGreenIIEB101.3.3DGGE[1