PCRDGGE技术在农村户用沼气发酵微生物研究中的初步应用师晓爽

20076222()JournalofShandongNormalUniversity(NaturalScience)Jun.2007Vol.22No.2PCR-DGGE*师晓爽1),2)刘德立1)郎志宏2)黄大2)陆伟2)李敏2)**(1),430079,;2),100081,24,,)PCR-DGGE,,1345,DNA,16SrDNAV3,DGGE(),,DGGE.DGGE16SrDNAV3,NCBI,.:,,,,,DGGE16SrDNA.(DGGE);;;Q938.1,,.,,,,,.,99%,[1].(denaturinggradientgelelectrophoresis,DGGE)[2]2080,16SrDNA,,.[3],.PCR-DGGE,,,.11.1,.20061345,4,12h.1.21.2.1DNA提取DNA[4],.(V-genePCRClean-upKIT).1.2.216SrDNAV3可变区的扩增16SrDNAV3[2]:P1:5-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3P2:5-ATTACCGCGGCTGCTGG-3,P1GC,DGGE,.PCRPCR,:2.5l10PCRbuffer(Mg2+free),2.0lMgCl2(25mmolL),2.0ldNTPs(2.5mmolL),0.1l(50pmolL),1UExTag(5U/lTakaRa),DNA1l(1ng),25l.945min,9430s,65~5530s(6556,1,555),721min25,726min,1.2%.PCR,ReconditioningPCRPCR[5,6]::5l10buffer(Mg2+free),4lMg2+(25mmolL),4ldNTPs(25mmolL),0.2l(50pmolL),ExTag2U(5UlTaKaRa),PCR5l,50l.:945min;941min,551min,721min,5;7210min.1.2%.1.2.3DGGEReconditioningPCRDGGE(DcodeTMSystem(bio-RadLaboratories),*(30671209);**::E-mail:[e-mail]:2007-01-268%,35%~56%,60,150V,1TAE,4h30min.DGGEBassam[7],.1.2.4DGGE条带回收及序列分析DGGE,100l,10min,7210min,PCR,GC,1.2.2.DNA,pMD18-T(TakaRa),E.coliDH5.,GenBank.1.2.5DGGE图谱的相似性分析ImageJ,MathworksMatlabv7.1PASTDGGE.ImageJDGGE;MathworksMatlabv7.1;PAST.22.1DNADNA0.8%,DNA20kb,.,DNAPCR-DGGE.2.216SrDNAV3DNA,16SrDNAV3PCRRecondition-ingPCR,PCR200bp,.2.3DGGEDGGE,,.(1),40,,,;().,,;,1,1ab;c145,d.,225,1DGGE2DGGE;,3032,.,.DGGE(2),460%.65%,80%.2.4DGGEDGGE,NCBI,(2a-i),ciP8-GENWN-FWB-12595%89%,797%,1212,:PCR-DGGE.,DGGE.3PCR-DGGE,.,,,,,.,DGGE.99%[8].,.PCR-DGGE,,.,DGGE[9],.,DGGE,DGGEPCR,.4[1]AmannRI,LudwigW,SchleiferKH.Schleifer,Phylogeneticidentificationandinsitudetectionofindividualmicrobialcellswithoutcultivation[J].Microb-iolRev,1995,59:143~169[2]MuyzerG,deWaalEC,UitterlindenAG.Profilingofcomplexmicrobialpopulationsbydenaturinggradientgelelectrophoresisanalysisofpolymerasechainreaction-amplifiedgenescodingfor16SrRNA[J].ApplEnvironMicrobiol,1993,59:695~700[3],.[J].(),2005,20(3):69~71[4],.DNA[J].2002,22(11):2015~2019[5]ThompsonJR,MarcelinoLA,PolzMF.Heteroduplexesinmixed-templateamplifications:formation,consequenceandeliminationbyreconditioningPCR[J].NucleicAcideRes,2002,30:2083~2088[6]VonWintzingerodeF,GobelUB,StackebrandtE.Determinationofmicrobialdiversityinenvironmentalsamples:pitfallsofPCR-basedrRNAanalysis[J].FEMSMicrobiolRev,1997,21:213~229[7]BassamBJ,Caetano-AnollesG,GresshoffPM.FastandsensitivesilverstainingofDNAinpolyacrylamidegels[J].AnalBiochem.,1991,196:80~83[8]AmannRI,LudwingW,SchleiferKH.Phylogeneticidentificationandinsitudetectionofindividualmicrobialcellswithoutcultivation[J].MicrobiolRev,1995,59:143~169[9]SekiguchiH,TomiokaN,NakaharaT,etal.Asinglebanddoesnotalwaysrepresentsinglebacterialstrainsindenaturinggradientgelelectrophoresisanalysis[J].BiotechnolLett,2001,23:1205~1208APRELIMINARYAPPLICATIONOFPCR-DGGEINMICROBIALCOMMUNITYSTRUCTUREANALYSISOFRURALHOUSEHOLDBIOGASDIGESTERSShiXiaoshuang1),2)LiuDeli1)LangZhihong2)HuangDafang2)LuWei2)LiMin2)(1)CollegeofLifeSciences,HuazhongNormalUniversity,430079,Wuhan,China;2)BiotechnologyResearchInstitute,ChineseAcademyofAgriculturalSciences,100081,Beijing,China)AbstractBasedonPCR-DGGEtechnology,thebacterialcommunityofruralhouseholdbiogasdigesterswasobservedandmonitored.GenomicDNAwasextracteddirectlyfromtheactivatedsludgeswhichweresampledinJanuary,March,AprilandMayof2006.The16SrDNAgenesV3regionwereamplifiedbyusingthebacterialspecificprimers,andseparatedbyDGGE.ThenDGGEpatternswereanalyzedwithassociatedsoftwares.Thesequencesof16SrDNADGGEpredominantbandsfragmentsweredeterminedbycomparisonwithNCBIdatabase.Theresultsshowedthatthebacterialdiversitywasabundantandrelativelystableinruralhouseholdbiogasdigesters,butthebacterialcommunitystructurewaschangedvariouslyindifferentmonths.Andmostofthepredominantbacteriawereunculturedbacteria.KeywordsDGGE;ruralhouseholdbiogasdigesters;bacteria;dynamics122()22