烟曲霉金属还原酶基因敲除及初步功能分析

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1、DisruptionandpreliminaryfunctionanalysisofametalloreductasegeneinAspergillusfumigatusAbstractObiectiveAspergillusfumigatusisanimportantopportuniticfungalpathogen，butitspathogenicmechanismisnotknown．Inthisstudy，weintendtoconstructamutantofAspergittusfumigatu

2、swhichlacksametalloreductasegene，andpreliminarilyanalyzethegeneoffunction．Andinthisstudy，wealsowanttoprovideafoundationforfindingtherelationshipbetweenthegeneandpathogenicmechanismofAspergillusfumigatus．MethodDNAfusionfragmentswereconstructedbynested—SOEPCR

3、．Atthesametime，conditionsofpreparationofprotoplastsuchasincubationtimeandosmoticstabilizerwereconfirmed．TheDNAfusionfragmentsweretransformedtotheprotoplastofAspergillusfumigatususingsplit—markerrecombinationstrategyandCaCl2-PEGmediatedtransformationmethod．T

4、hetransformantswerescreenedbythehomomycinBresistance．PCRandsequencingwereusedtoidentifymutantsofAspergillus．filmigatuswhichlacksthemetalloreductasegene．itwasobservedtofinddifferencebetweenmutantsandwildtypeinthemorphologyofcolonyinAMM，AMMwithoutiron，AMM十EDT

5、AandAMMwithoutiron+EDTAsolidmediumandinlengthofhyphaintheseliquidmedium．Atthesametime，thesoftwareofDNAstarwasappliedtomultiplealignmentsofaminoacidsequencesofmetalloreductasesinAspergillusfumigatusanddifferentspecies如rexploringthefunctionofmetalloreductase．

6、ResultsThefusionDNAfragmentswereobtainedbynested-SOEPCRsuccessfully．IncontrastwithgeneralSOEPCR，theassociationofnestedPCRreducedtheamountofincorrectsizedbands．Sorbitolandmyceliawhichwereincubatedfor12hourwereselectedtoprepareprotoplast．Weobtained14transform

7、antsbyhomomycinBresistancescreening．TwotransformantswererecognizedaSthemutantswhichlackthemetalloreductasegeneaccordingtoPCRandsequencingresults．Therewerenotremarkabledifferencesbetweenthemutantsandwildtypeinthesizeofcolonyorthelengthofmycelia．Alignmentofth

8、eseproteinsindicatedthatthemetalloreductasehasNOXDuoxlikeFADNADPFstructureofFerredoxinreductase(FNR)superfamily．InAspergitlusfumigatus，anothermetalloredutasewhichisencodedbytheotheronegene(AFUA8G06210)