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1、中國農(nóng)業(yè)大學碩士學位論文黑曲霉外源蛋白表達分泌相關基因功能研究姓名：薛偉申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：趙武玲王敖全20050601AbstractFiLamentousfungiarewidelyusedinproductionofeukaryoticproteinduetotheirfollowingcharacteristics：highsecretorycapabilityposttranslational

2、modificationssimilartothatofmammalandsoonHoweverforproductionofbeterologousproteins，theyieldsarestillpoorProteolyticdegradationandlowsecretionefficiencyarethemajorreasonsTocontrolproteolyticdegradationthustoimproveexpres

3、sionofheterologousprotein，threeproteasegenesdppIVdppVandpepAcwerestudyedtobedisruptedthrou曲genetargetingtofurtherstudytheinfluenceoftheirdisruptiononexpressionofheterologousproteininthisdissertationThedpplVanddppVgenesen

4、codetwotypesofdipeptidylpeptidaseswhichreleasespecificdipeptidesfromtheNterminiofpolypeptidesItwasreportedthatDPPIVandDPPVparticipatedindegradationofextmcellularproteininAoryzaeandAfumigatusThepepAegenewasfoundtobeahomol

5、ogofpepAgeneandwasidentifiednewlyfromthegenomicsequencesofAnigerThisdissertationtriedtostudywhetherPEPACwasalsooneoftheextracellularacidproteasesinvolvedinproteolyticdegradationofbeterologousproteinInaddition，throughgene

6、targetingthemnnlOgenewhichencodedonetypeofmannosyltransferasewasdisruptedinordertostudytheinfluenceofitsdisruptiononexpressionofheterologousproteinandthefunctionofmnnlOgeneinbiosynthesisofhighmalmanchainofglucoproteininA

7、niger：DNAfragmentsofcorrespondinggeneswereamplifiedusingtheprimersdesignedaccordingtothesequencesofdpplVdppVpepAcandmnnlOgeneandligatedwithcloningvectorpBSTorpMWlThenhphexpressionunitoramdSgenewereinsertedintothemiddlepa

8、rtofeachgenetogeneraterespectivegenedisruptionplasmidspBSAdppIVhphpBSAdpplVamd，pBSAdppVhph，pBSApepAchphandpMWtAmnnl0ThedisruptionplasmidspBSAdpplVhph，pBSAdppVhph，pBSApepAchphandpMWlAmnnl0weredigestedwithrespectivecorresp

9、ondingrestrictionenzymesandtransformedintoAnigerGICC2773straininwhichalaccaseexpressionvectorhadbeenintegratedThegeneratingffansformantswerescreenedfordisruptionstrainusingPCRmethodTheresultsshowedthatAdpplV#17，AdppV#26a

10、ndAmnnl0#2weredisruptionstrainsofdpplVdppVandmnnlOgene，respectivelyHowever，nopepAcdisruptionstraincouldbefoundwhereastotal286transformantsfromtransformionofpBSApepAchphwerescreenedUsingsimilarmethod，pBSAdpplVamdwastransf

11、ormedintodppVdisruptionstrainAdppV#26andthedpplVanddppVdoubledisruptionstrainAdppVAdpplV#2—3wasachievedLaeeaseactivitywereanalyzedinthesedisruptionstrainstostudytheinfluenceofeachgenedisruptiononexpressionofheterologousp

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