低温沼气功能菌群的产甲烷菌分析

2011－12－082012－01－161955－。“”“”2007-YGY-04。30120122JIANGXISCIENCEVol．30No．1Feb．20121001－3679201201－0036－0433002916srDNAPCRqPCR。3320。。。16srDNAPCRS216．4AMethanogensConsortiumAnalysisinLow-temperatureBiogasFunctionalMicrofloraDINGJian-nanTIANXiao-juanZHANGGuo-huaHUANGHuangHUANGJiang-liBiologicalResourcesInstituteJiangxiAcademyofSciencesJiangxiNanchang330029PRCAbstractMethanogenspopulationsinlowandnormaltemperaturebiogasfunctionalmicrofloraswererespectivelyinvestigatedusing16srDNAPCRandreal-timeqPCRtechniques．The16srDNAclonelibraryofmethanogensshowedagreatdifferenceinmethanogensconsortiumdiversityandstructurebetweenthetwomicrofloras．Atotalof33OUTsofmethanogenswereidentifiedincluding20inlow-temperaturemicrofloraand13innormaltemperaturemicroflora．Methanogensrequiringlow-mediumtemperaturewerefoundprevalentinlow-temperaturemicrofloraandthoseactiveatmoderate-hightemperatureoccurredmainlyinnormaltemperaturemicroflora．Howevertheresultsofreal-timeqPCRshowedthattherewasnosignificantdifferenceinmethanogenquantitybetweenthetwostud-iedmicroflorasindicatingastablemethanogeniccommunityhadbeenbuiltinbothmicrofloras．KeywordsLow-temperaturebiogasfunctionalmicrofloraMethanogensPopulationanalysis16srD-NAReal-timePCR012。313℃0．15m3/d·m3。4。。16srRNAPCR。11．1。13℃28℃22。20%w/v10%w/v1%w/v69%pH7．5、1。1．21．2．1DNA210min12000gDNA100mMTris-HCl100mMEDTA1．5MNaCl1%CTABpH8．065℃。5min65℃3。65℃30min1mg/mL37℃180r/min30min10%SDS2%58℃2h15～20min。6000g10min5。//25∶24∶1DNA。12000g10min0．62h4℃12000g10min70%3TEpH=8．00．8%DNA。DNAEZSpinColumnDNAgelExtractionKit5μLDNA0．8%。1．2．216srDNADNA16srDNA5'-ACKGCTCAGTAACACGT-3'5'-GTGCTCCCCCGCCAATTCCT-3'。PCR94℃5min94℃30s53℃1min72℃2min35cycles72℃6min。DNA0．5μL10M1μL2×EasyTaqPCRSupermix12．5μLddH2025μL6。PCRTpMD19-TsimpleTaKaRaDH5α37℃12h。HinPIMspI1OUT1DNAMAN、Genetool、GenBankGeneBankBlastMEGA3．17。1．2．3qPCR5'-TTCGGTGGATCDCARAGRGC-3'5'-GBARGTCGWAWCCGTAGAATCC-3'。qPCRSYBRGreen。PCR10real-timePCR、。IQ5PCRBIO-RAD96。TakaraSYBRPremixExTaqII95℃30s95℃5s60℃20s4095℃5s55℃10s0．5℃10s80。PCRCt。8～10。22．116srRNA·73·1DNA16srRNA171。HinPIMspI17133OUTOUT3316srRNAGenBankEU662665-EU662697。Blast33OUT416srRNA97%F2MethanocorpusculumlabreanumS3Me-thanoculleusmarisnigriP17Methanothrixsoeh-ngeniiT4Methanocorpusculumparvum29。2．2MEGA3．116srRNA。。。33OUT20T501。。。2．3。F2Methanocorpusculumlabreanum33%T4Methanocorpusculumparvum25%OUT58%1842%。Methanocorpusculum20℃30℃。。116SrRNAS2Uniden-tifiedarchaeon38%、S3Methanoculleusmarisnig-ri12%S7Unidentifiedarchaeon10%OUT60%40%。Methanoculleusmarisnig-ri30～45℃。2．4Real-TimePCR≥0．99PCR80%～120%。Ct。Real-TimePCR。Ct22．8122．771．37E+051．94E+05113℃28℃。43·83·20123011．J．20012330116－118．12．J．20066173－175．13YangDYLiXJGaoZJetal．Improvingbiogaspro-ductionofcornstalkthroughchemicalandbiologicapretreatmentapreliminarycomparisonstudyJ．TransactionsoftheCSAE2003195210－212．14．3J．2007195119－12．15．J．200739922－25．16．J．2007255檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶檶8－10．381/℃0．5μL0．5μLCt131．37E+0522．81281．44E+0522．773133OUT4。23320。3。4、Ct。1．J．201143629－32．2．J．2010285132－133138．3．J．2003144604－608．4．J．2007261335－338．5．J．2010301150－55．6JiannanDingRuiyongZhangYizunYuetal．ANovelAcidophilicThermophilicIronAndSulfur-－OxidizngArchaeonisolatedFromAhotspringofTengchongYunnanChinaJ．BrazilianJournalofMicrobiology201142514－525．7YinHQCaoLQiuGetal．Moleculardiversityof16SrRNAandgyrBgenesincopperminesJ．ArchMi-crobiol2008189101－110．8．．PCRJ．2010316－7467－469．9．PORJ．2006391161－169．10．J．201131144126－4134．·34·1