26876.橙色紅曲菌中基于pyrg標(biāo)記的同源轉(zhuǎn)化系統(tǒng)的建立

1、南昌大學(xué)博士學(xué)位論文橙色紅曲菌中基于pyrG標(biāo)記的同源轉(zhuǎn)化系統(tǒng)的建立姓名：王伯華申請學(xué)位級別：博士專業(yè)：食品科學(xué)指導(dǎo)教師：許楊20100601AbstractInthispaper，pyrGgenefragmentwasclonedfromMonascusaurantiacusgenomicDNAusingdegenerateprimersdesignedwithCODEHOPstrategyAnditscompletesequence

2、wasobtainedbyaPCRbasedmethodforscreeningfosmidlibraryUridineauxotrophystrainsfrom彪aurantiacusAS34384wereisolatedbyresistanceto5FOAafterUVmutagenesisOnepositivecolonywasobtainedandnamedUM28，itwasfurtherconfirmedbysequenci

3、ngandtransformationTheformationofprotoplastconditionandtheregenerationofprotoplastconditionofUM28strainwereoptimized，andahomologoustransformationsystemusingpyrGasaselectionmarkerofUM28isestablishedAplasmidpGFPpyrGcontain

4、ingtheGFPexpressioncasseReWasconstructedandtransformedintoUM28protoplast，expressionoftransformedgenewasevaluatedusingtheGFPgeneThemajorfindingswereexpoundedasfollows：1AccordingtotheknownOrotidine5’phosphatedecarboxylase，

5、pyrGgenefragmentwasclonedfromMonascusaurantiacusgenomicDNAusingdegenerateprimersdesignedwithCODEHOPstrategyAnditscompletesequenceWasobtainedbyaPCR—basedmethodforscreeningfosmidlibrary(AccessionNoGU723506)Aftertheblastxco

6、mparison，theclonedgenewasconfirmedthatitwasMonascuspyrGgenewith81％sequenceidentitytothatofAspergillusflavusNRRL3357ItCanbeusedasaselectivemarkerforMonascushomologustransformationsystem；2ApyrGmutantstrainfrom肱aurantiacusA

7、S34384，namedUM28，wasisolatedbyresistanceto5FOAafterUVmutagenesisSequenceanalysisofthismutatedgenerevealedthatitcontainedapointmutationatnucleotideposition220causedsubstitutionofPr0一Ser，withinactivationofOMPdecasefunction

8、Complementationof膨aurantiacuspyrGmutantwasperformedbytransformingAspergillusoryzaepyrGgeneintoUM28，andittransformedUM28intouridineprototrophyUM28wasauridineauxotrophwimafrequencyofreversemutmionoflessthan10一，anditcouldbe

9、usedintheMonascushomologoustransformationsystem；3TheformationofprotoplastconditionandtheregenerationofprotoplastconditionofUM28strainwereoptimizedTwocomplementationplasmids，pAOPandpMAPwererespectivelytransformedintoproto