冰川土壤低温脂肪酶基因多样性研究及其基因克隆

《冰川土壤低温脂肪酶基因多样性研究及其基因克隆》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

1、中国农业科学院博士学位论文冰川土壤低温脂肪酶基因多样性研究及其基因克隆姓名：张宇宏申请学位级别：博士专业：农业微生物学指导教师：姚斌20080601稳定性较差，具有低温脂肪酶的特点。以上结果证明，使用简并引物PCR扩增基因序列的方法能够快速有效地评价复杂环境中脂肪酶基因的多样性。而且在多样性研究中大量脂肪酶基因片段的获得也为将来从环境宏基因组文库中高通量筛选大量新的脂肪酶基因奠定了基础。关键词：脂肪酶，基因多样性，宏基因组文库，冰川，土壤AbstractLipase(EC3．1．1．3)hydrolyzestriacylglycerolsandcatalyz

2、esthereactionsofestersynthesisandtransesterification．Itisaversatilebiocatalystwithgoodstabilityinorganicsolvents，excellentsubstratespecificityandhighstereo—selectivity．Duetothecharacteristicsofgentlereactionconditions，fewerby—productsandlesscontaminationtoenvironment，lipaseshavebee

3、nextensivelyappliedinindustries．Cold—relatedlipasesaleagroupoflipaseswithhighcatalyticefficacyatlowtemperaturesandsensitivitytohightemperature，andhavepotentialapplicationsinfieldsofdetergents，foodindustryandbiodegradationofoil·pollutedsoil．Thepurposeofthisstudywastoinvestigatelipas

4、ediversityinthemicroorganismsfromglaciersoilbasedonculture—independentmetagenomicDNAmethod．ThevalidityofthismethodWasconfirmedbylipasegenecloningandheterogeneousexpressionbasedonthecellcultureapproach．Thelipasefragmentsobtainedindiversitystudywillbeusedforfurtherlipasegenecloningfr

5、omglaciersoil．BasedontheaminoacidsequencealignmentoflowtemperaturelipasesinHormone-sensitiveLipase(HSL)familyobtainedbytheEntrezsearchatNCBI，twoconservedmotifs【V／L]一[F／Y／D]-[F／I]一H—G-G-[G／A】andG一[D／V]-S-[A／V]-G-G-[N／C]一【L／M】werefoundaroundtheoxyanionholeandtheactivesite，respectivel

6、y．Asetofdegenerateprimers(CLFandCLR)Wasdesignedbasedonthesetwoconservedmotifs．MicrobialmetagenomicDNAwasisolatedusingthedirectlysismethodfromNo．1glaciersoilinChina．PrimersCLFandCLRwereusedtodirectlyamplifyfragmentsfromthemetagenomicDNAviaPCRreaction．ThePCRproductswereusedtoconstruc

7、tacold—relatedlipasegenefragmentclonelibrary(GCLP)．Amongthe300clonessequencedforanalysis，201clonesencodingpartiallipasesshared33-82％identitytotheknownlipasesinGenBankbasedonBLASTanalysisandweresubjecttophylogeneticanalysis．TheresultsindicatedthatthelipasesfragmentsinGCLPlibrarywere

8、diverseandhadhighsimilarit