蘇云金芽胞桿菌活性基因的克隆及其表達.pdf

1、華中農(nóng)業(yè)大學碩士學位論文蘇云金芽胞桿菌活性基因的克隆及其表達姓名：白培勝申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：喻子牛孫明2002.5.18Cloneandexpt’essionofactivegenefromBacillusthurhlgiensis^1)SllaclAgenenamedJ7P，”15J3avebeenobtainedwhjchencodesnematode—activeproteinsfl0111口ci

2、cil／usIlnuingiensisYBT一1518withhomologousprobeItexpressedi11non—cl、satlmutationol’Bacillus砌，，加gfe“s括(Bt)strain171and4Q7andtheoutputislargerthanwhatlOmOIJginalstrainWedelnonstratedpreliminarytheproteinistoxictoplantparasi

3、ticnefnatodeSequencingindicatesthatthesimilarityofneml5anditshomologyachieve98％Iastudiedtherelationofneml5andthesubsequentORF(ORF2)1TheproteinsfromBtstrainYB，f1518whichproducesnematicideproteins13asstudiedYBI“一1518produc

4、es57KDand45KDtwolnainproteinsButthecrystalCallnotbe】lecognizedwithopticalmicrosopeYBT一1518reachsporulationafter30hotvsatNSMc“[tIIelllCdiuln2Ⅵ’eseachtileGenBankBLASTdatabankwithcry6Aalwhichhavebeenreportedencodesnematodea

5、clix7eproteina11ddesignedprimer、、iththe!lOlrtologoussequencesAbout700bp：cgmentamplified、、，iIllthcprinlerbyPCR、、’asusedasprobeSouthernh、，blidizationIndicatedtiletal。getgeneallocateatabout3OkbHindlllsegmentfromCOlnpletedig

6、estionoftotal1)N＼ofYBT1518j11he3Okbsegment、VasrestrivedandligatedwithclonevectorpUC19orslmttlevectorpHT304thenwegotasubgenomiclibraryThepositiveclonewerescreenedwithPCRandspolblotTheyarep40，P102whosevectorispUCl9andp15wh

7、osevetorispHT3044Sequencingindicatedthattheclonesegmentis2941bpTheBLASTresultshowed98％simiarity、、ith6／3’6AalThegeneencodes475aminoacid，whosemolecularweight54KDandisoelectricpoints501GeneScanshowedthereisaORF(ORF2)afterne

8、ml588bpORF2CL1COttcs：lprotein、、7hichhave301aminoacid，uhosemolecularweight35KDandisoelectricpointsS025TheclonesegmentwastransformatedintononcrysatlnmtationofBc|cillustlna‘ingiensis(Bt)strain17land4Q7Theyexpressed54KDprote

9、incorrespondingwithOl’iginalsH‘a(chǎn)inYBT1518FurthermoretheoutputislargerthanYBT一1518Thepromot6r。lelmioumutationp1550、、asconstructedbyselfligationafterdigestionofpl5withSaclrJ]e()ll二inscltnlutatiol3pl512wasconstructedbyinser

10、tionTecresistantgenefromD869att11elUCUSOI、＼mSDSPAGEindicatedthatpl550havenotproducetheconespondin2protelnbutPI5l2expresses54KDproteinoverexceed，thatsuggestionORF2havenegtJveregulatioJltoORFl6Bioassai)’indicatedthemixture

11、ofsporeandcrystalofYBT一1518havenoeffecttoPIzI，P，，(fi兒jwellaandHeliolhLsalllligel’。，Theproteinencodedbyneml5haveequivalencetoxicityfoplantpalasiticnematodewithYBT1518Ke3wOI’ds：骯，c’il／usthlingiensis(Bt1nematodegenecloneexp