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1、AbstractAspergillusniger13-mannanasegenecloningandexpressionofprokaryoticandeukaryotict；-mannanase(endo-1，4-13·D-mannanase；mannohydrolase；EC3．2．1．78)areakindofclassabletohydrolyzeoligosaccharides，mannosepolysaccharides(includingmannart,galactomammn,glu
2、comannan,etc．)of1,4-／3一D—mannosidaseendobondhydrolaseandasemi-celluloseenzymes．Theapplicationprospectof13-mannanaseisveryextensivestudyofitshi；ghcommercialvalueandscientificsignificance．Now13-mannanasehasbeenwidelyusedinthefood,pharmaceutical，papermaki
3、ng，textilepriminganddyeing，oilexplorationandotheraspectsofbiologicalresearchtechniques．IndustrialproductionofAspergillusnigeristhemainspeciesmatlnanase．However,thecurrentproductionstrainexistsalargenumberofdisadvantages，suchasanumberofsporesduringferme
4、ntation，shortfermentationperiod，lowenzymeproduction．Inthiscase，theapplicationofconstructionofgeneticengineeringbacteriamannanase，simplifiedfermentationconditions，increasedenzymeproductioIl，haveimportantresearchvalue．Inthisresearch,mannanasegeneproducin
5、gstrainofAspe哂llusnigerasagenedonor，clonedmanrlanasegene(MAN)，wereseperatelyconstructedtheE．coliexpressionvector,PichiapastorisexpressionvectorandtheAgrobacterium-mediatedgeneMANhomologousrecombinationvector,respectively,inthemeantime，weretransferredto
6、E．coliBL21，PichiapastorisGS15andglucoamylaseproducingstrainofAspergillusnigerforlayingthefoundationforhi曲lyexpressedgenes-Themainresultsare私follows：Itapplied13-mannanaseproducingstrainofAspergillusnigerasthematerialandobtainedbyRT-PCRamplificationtogai
7、nlB-marmanasegeneMANoftheeDNAfragments．ThoughPCRamplificationofIB-mannanasegenegainedMANoftheDNAfragments，thematerialthatisAspergillusnigerglucoamylaseproductionobtainedbyPCRamplificationGIAgene5。and3’homologousarmoftheDNAfragments．Asaresult，thesequenc
8、eiscorrect．1．MANgeneexpressioninE．coil．Itapplied13-mannanaseproducingstrainofAspergillasnigerasthematerialandobtainedbyRT-PCRamplificationtogainlB-mannanasegeneMANoftheeDNAfragments．MANgeneore．coilwasconstructedtoexpressvectorpET-MAN。Af
