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1、東北農(nóng)業(yè)大學(xué)碩士學(xué)位論文枯草芽孢桿菌224yplQ、ytjAa基因缺失菌株的構(gòu)建及對(duì)其溶血性影響姓名：喻江申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：微生物學(xué)指導(dǎo)教師：李景鵬20080620AbstractDelectedMutantConstructionofyplQandytjAageneinBacillussubtilis224andInfluenceonHemolysisAbstractBacillussubtilis224wasanon-path

2、ogenicBacillusthatseparatedfromhumanbodyAccordingtotheMicroecologyprincipleandtheantagonismamongthemicroorganism，WhiteSwanaerosolmadeofliveBS224wasaeffectivemedicineforbumexternalusing，itcouldtreatinfectedburnwoundsandpr

3、omotewoundhealingButbecauseofhemolysis，ithasbeenstoppedusinginmedicinenOWUptonow,noinvestigationwasperformedontheBacillussubtilis224genomesequencing，butBacillussubtilis168genomesequencinghasbeensure，eightgeneswerepossibe

4、relatedtohemolysisyplQ(642bp)、ytjAa(228bp)weretwoofthem，andexpressionproductofytjAawassimilartoalpha-hemolysin。Here，yplQandytjAaDNAfragmentswereamplifiedbyPCRwithchromosomalDNAofBS224asthetemplateandinsertedintocloningve

5、ctorpMDl8TtoconstructtherecombinantplasmidspMDl8一T-yplQ、pMDl8-T-ytjAaTheprimersofPCRamplificationreactionweredesignedaccordingtothegenomesequenceofBacillussubtilis168Therecombinantplasmidsweretransformedintocompetentcell

6、sofEcoliJMl09PositivecloneswerescreenedfromtheLBsolidmediumplatesincludingAmpicillinThepositiverecombinantswereidentifiedbyrestrictionenzymedigestandfinallysequencedTheresultssuggestedthatthehomologybetweenthenucleotides

7、equenceoftheclonedyplQ、ytjAaandthereportedoneswereseparately998％、994％SoitcouldbesurethatthecloningofyplQandytjAagenesisrightinthegenomeofBS224AnintegrationplasmidpMDl8-T-neoyplQfortheyplOgenedisruptioninBacillussubtil婦22

8、4wasconstructedTheplasmidcontainedupstreamregionoftheyplQgene10kbandtheyplQgenedownstream05kbhomologousfragmentswithinsertionoftheexpressionunitoftheneom-ycinresistancegenebetweenthemUpstreamanddownstreamDNAfragmentswere

9、synthesizedbyPCRfromBacillussubtilis224chromosomalDNATheconstructedgenetargetingvectorwaslinearizedwithHindIII，andelectroporatedintocompetentcellsofBS224Positivetransformerswerescreenedfromthe50Pg‘ml～neomycinresistancepl

10、ates36transformerswereanalyzedbyPCRtoidentifyyplQgenedeletionAnintegrationplasmidpMD18-T-neo—ytjAafortheytjAagenedisruptioninBacillussubtilis224wasconstructedTheplasmidcontainedtheytjAagenedownstream05kbandtheytjAageneup

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