Magnetospirillum gryphiswaldense MSR-1鐵吸收調節(jié)蛋白fur-like基因克隆及功能分析.pdf

1、中國農業(yè)大學博士學位論文MagospirillumgryphiswaldenseMSR1鐵吸收調節(jié)蛋白furlike基因克隆及功能分析姓名：黃亦鈞申請學位級別：博士專業(yè)：微生物學指導教師：李穎20070601AbstractThesocalledmagnetotscticbacteriaareprokaryoteswhichcansynthesizemagneticparticles(magnetosomes)incytoplasmAt

2、presentthebiomineralizationprocessandpotentialUSeSofmagnetosomehaveintriguedscientiststoelucidatethemechanismofmagnetosomeformationandtodevelopthebatchculturetechniqueThereisupto3％intracellularironcontentinmagneticbacter

3、ia，100foldmorethaninEcoil，whichindicatesthereforethataneffectivestrategyhasbeeninvolvedincaptureingironaswellasrestrictingthetoxiceffectsofironFerricuptakeregulator(Fur)isaglobalcontrolfactortokeepironhomeostasisinotherb

4、acteriasuch∞EcoilInvestigationofmagnetotscticbacteria’FurfunctionisexpectedtoclearoutthemechanismofironuptakeandmagnetosomesynthesisInthisstudiestwogenomiclibrariesofMagnetospirillumgryphiswaldenseMSR一1Wereconqructedusin

5、g^EMBL3andcosmidpKC505勰vectorsrespectivelyA3633bpofdesiredfragmentwasisolatedfromtheCOSlibrarywith丘rneclcotidesprobeofMmagnetotacticumMS1(namedDlOMmfur)4ORFsweretentativelyidentifiedbyORFFindingsandBLASTalignmentOnNCBLOR

6、F2consistsof420bpandcodesFurprotein(MgFur)containin9139aminoacidresiduesORFIislocatedOiltheupstreamoffurgeile，whichencodesahypothetical乏2nitropropanedioxygenasegune(npd)containing406aminoacidresiduesSequenceanalysisshowe

7、dthatFurproteinofMSR1containedahighlyconservedHDHDmotifandamodifiedFeZ／Zn“bindingsiteHterminusstructureofMgFurwaspredicatedtobecloselysimilartothatofPseudomonasaureglylOSa(PaFUObySWISSMODELwhichacts勰theDNAbindingmotifeve

8、ilthoughsequenceofMgFurisalignedtobe273％identitywithPaFurHoweverstructureofCterminuswasunabletobeshownduetothelowsimilarityThefurgeneofMSR1wasexpressedsuccessfullymEcoliandgelretardationassaysuggestedthatMg—Furwereableto

9、bindtoFurboxofEcolibutitdidn’tcomplementEcoilfurmutant(H1681)TodeterminedMgFurfunction，asuicideplasmid(pSUP202containingthedisruptedfur：：kan)wasconstructedandafu，mutantofMgryphiswaMenseWasobtainedbyhiconjugationrecombina

10、tiondesignatedDFH1Nomagnetosomebutmorc8polyhydmxybutyrate(P耶)particlescouldbeobservedinDFH1underthetransmissionelectronmicroscope，comparedwiththewildtypestrainMSR1Analysisofironcontentindicatedthatitwaskeptconstantlyabou

11、t0037％levelinDFII一1，grownintheregardlessofloworhighironconditions，whichwas10foldlessthanthatofMSR1HoweverDHt1wereable10resistlmMhydrogenperoxide，5foldmoretolerancethantheWildtypestrainMSR1Inthesestudiesafurgenewascloneda