粘紅酵母乙酰輔酶A羧化酶基因的克隆及表達.pdf

1、華中農業(yè)大學碩士學位論文粘紅酵母乙酰輔酶A羧化酶基因的克隆及表達姓名：李潔瓊申請學位級別：碩士專業(yè)：微生物學指導教師：喻子牛；鄭世學201106華中農業(yè)大學201l屆碩：I：研究生學位論文AbstractAcetyl—CoAcarboxylase(ACC)isakeyenzymeoffattyacidsynthesisItcatalyzesacetyICoAtoformmalonyl—CoA，whichisusedasabuilding

2、blockintwocarbonincreasementstoextendthechainlengthoffattyacidsTherefore，ACCisalsoaratelimitingenzymeoffattyacidsynthesisThemainresultsareasfollowsinthestudy：UsingthemethodsofdegeneratePCRandchromosomewalking，thispaperob

3、tainedthefullsequenceinformationofACCgenefromRhodotorulaglutinisforthefirsttimeSequenceanalysisshowedthatACCgenefromgenomicDNAhavetwointrons，rangingfrom42to147and315to677nucleotides，andthecodingregionis6801bpinlengthSeco

4、ndarystructureanalysisofthededucedaminoacidsequencerevealedthatitcontainsthreetypicaldomainsofACC：biotincarboxylase(BC)，biotincarboxylcarrierprotein(BCCP)andcarboxyltransferase(C1)ACCfromRhodotorulaglutinisisanmulti—doma

5、inenzyme，anditsmonomermolecularweightisabove200kDIt’SverydifficulttoexpressfulllengtheDNAofACC，SOaccordingtomethodsforstudingmulti—subunitACC，thispaperdidresearchonthreedomainsofACCfromRhodotorulaglutinisThisstudyhadused

6、twoprokaryoticexpressionsystemspET28a／BL21(DE3)andpETDuet1／Transetta(DE3)toseparatedlyexpressandcoexpressthreedomaingenesFirstlynineexpreesionvectorsweresuccessfullyconstructedSDSPAGEanalysisrevealedthateachdomaingenewas

7、successfullyexpressedintwoexpressionsystemsMoreoverithadachievdtocoexpressBCandCTdomains，aswellasthreedomainsFattyacidofrecombinantstrail塔wasextractedbybathandanalyzedbyGaschromatographyResultssuggestedthatthefattyacidco

8、ntentofrecombinantstrainsinthesystempETDuet1／Transetta(DE3)，whichhadsepamtedlyexpressedBCorCTcoexpressedBCandCT，aswellasthreedomains，respectivelyincreased154％，2707％，35％，7062％comparedwiththecontrolgroupHoweveritdescreased

9、505％forexpressionofBCCPdomainInpET28a／BL21(DE3)expressionsystem，fattyacidcontentofrecombinantstraiilscompared、析nlthecontrolwithoutanyincreaseItdemonstratedthatthegeneshadachievdfunctionalexpressioninpETDuet1／Transetta(DE