油菜菌病菌pg的分离纯化、理化性质与pg基因的克隆与表达

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1、2扬州大学硕士学位论文Isolation，purification，characterizationofpolygalacturonaseofSclerotiniasclerotiorumandcloning，expressionotthepathogenpolygalacturonasegenesI-1一⋯一Abstract：FivecellwalldegradingenzymessuchaSpolygalacturonase(PG)，polymethylgalacturonase(PMG)，cellulase(Cx)，polyga

2、lacturonicacidtrails-eliminase(PGTE)andpectinmethyltrans-eliminase6,MTE)couldbeproducedintheimprovedMarcus’SmediumbySclerotiniasclerotiorumcausingrapestemrot．Amongthem，PGhadthehi曲estenzymeactivity、)l，itll249．11U／mL．ThecrudePGintheculturefiltratecouldbeprecipitatedwithac

3、etoneandobtainedbycentrifugation(8000r／rain)at4"Cfor15min，andsuccessivelypurifiedbyDEAESepharoseFastFlowionexchangecolumn,Phenyl-Sepharose6FastFlowhydrophobiccolumnandSephadexG一75gelcolumn．ThemolecularweightofthepurifiedPGwas37．17kDbySDS-PAGEandthepIvalueWas6．14byIEF—PA

4、GE．ThePGWasdemonstratedasakindofglycoproteinthatcontained2．51％saccharide，butnOlipidandaromaticaminoacids．ThePGactivityWaSobservedintherangeofpHvaluefrom3to11，andthestrongestoneWasatpH5．ThePGwasnotstableinthehighertemperature(>40℃)andlostitsactivityinthetreatmentof100。Cf

5、or20min．ThePGWasalsosensitivetoultravioletradiation,chloroform，trypsinandproteinaseK．TwopairsofprimerswasdesignedtoamplifythecompletesequencesaccordingtothepathogenPGgenenueleotidesinGenBank(AF501307andAY312510)bypolymerasechainreaction(PcR)．TwoPGgenesinthepathogenwerec

6、lonedandsequenced．Ofthem，Sspgldconsistedof1084bp，coding326deducedaminoacidsandSsp93consistedof1701bp，coding482deducedaminoacids．Whenbeingblasted,SspgIdandSsp93showeda100％similaritytopgldandtop93inthesequences，repectively．ThePGgenesSspgldandSsp93wereconstructedintheexpre

7、ssionvectorpET-28a(+)andresultedintherecombinantplasmidspETSspgldandpETSsp93，repecfively．Theyweretransferredin五coliBL21(DE3)andexpressedbyIPTGinduction．ThePGactivityof764．35U／mLWasdetectedwhenBL21containing刘振：油菜菌核病菌PG的分离纯化、理化性质及PG基因的克隆与表达3pETSspgldwasculturedinLBmediuma

8、ndinducedwith0．4mMIPTG．WhenBL21containingpETSsp93wasculturedinthesamecondition,thePGexpressedwasdetectedwimthe