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淡紫紫孢菌T-DNA插入体库的构建和致病相关基因的功能研究的中期报告AbstractTheconstructionofaT-DNAinsertionlibraryofHeterobasidionannosumandthefunctionalstudyofpathogenic-relatedgeneswasreported.ThroughthemethodofAgrobacterium-mediatedtransformation,aT-DNAinsertionlibrarywassuccessfullyconstructedwith3,500transformantsobtained.Theinsertionratioswereapproximately80%,whichsuggestedthelibraryhadahighcoverageofthegenome.Toinvestigatethefunctionofpathogenic-relatedgenes,twomutantswithT-DNAinsertionindifferentgeneswereselectedforfurtheranalysis.Themutantsshowedsignificantreductioninvirulencecomparedtothewild-typestrain,indicatingthepathogenicityofthecorrespondinggenes.Furthermore,RNA-Seqanalysiswasperformedonthetwomutantstoidentifygenesthatweredifferentiallyexpressed.Severalgenesrelatedtoplantcellwalldegradation,oxidativestressresponse,andstresstolerancewerefoundtobedownregulatedinthemutants,whichmightberesponsibleforthereductioninvirulence.TheresultsdemonstratedthefeasibilityofusingaT-DNAinsertionlibraryforfunctionalstudyofH.annosumgenesandprovidednewinsightsintothepathogenesisofthisimportantforestpathogen.IntroductionHeterobasidionannosumisadevastatingpathogenofconifersandcausessignificanteconomiclossesintheforestindustry.Duetothecomplexityandimportanceofitspathogenicitymechanisms,itisessentialtounderstandthegenefunctionandregulationofthisfungus.Inrecentyears,withthedevelopmentofmoleculargeneticsandgenomesequencing,functionalanalysisofgeneshasbecomeanimportantapproachforthestudyoffungalpathogenesis.T-DNAinsertionisanefficientmethodforgeneratinggeneknockoutsandiswidelyusedforfunctionalanalysisinvariousfungi.InH.annosum,however,T-DNAinsertionhasbeenseldomlyreportedduetoitsuniquegenomicfeatures,suchaslargegenomesizeandhighcontentofrepetitivesequences.Previously,onlyafewstudieshavereportedtheuseofT-DNAinsertioninH.annosumforfunctionalanalysisofpathogenicity-relatedgenes.Inthisstudy,weaimedtoconstructaT-DNAinsertionlibraryofH.annosum,andtoinvestigatethefunctionofpathogenicity-relatedgenesbygeneratingmutantstrainswithT-DNAinsertionandanalyzingtheirtranscriptomeprofiles.MaterialsandMethodsConstructionofT-DNAinsertionlibraryTheH.annosumstrainwastransformedwithpCAMBIA1300vectorcontainingtheselectionmarkerhygromycin-resistantgeneandtheT-DNAregion.ThetransformedstrainswereselectedonPDAplatescontaining50μg/mlhygromycin,andtheprotoplastsofthesestrainswereregeneratedtoobtaintransformants.ThegenomicDNAofthetransformantswasextractedusingtheCTABmethod,andPCRwasconductedtoamplifytheT-DNAjunctionfragments.TheamplifiedfragmentsweresequencedforidentificationoftheT-DNAinsertionsites.Theinsertionratiosandthenumberofinsertionsiteswerecalculated.Functionalanalysisofpathogenicity-relatedgenesTwomutantswithT-DNAinsertioningenesrelatedtopathogenicitywereselectedforfunctionalanalysis.Themutantswerecomparedwiththewild-typestrainintermsoftheirvirulenceonPinussylvestrisstems.Theexperimentwasrepeatedthreetimeswith18seedlingspergroup,andthelesionlengthsweremeasured4weeksafterinoculation.RNA-Seqanalysiswasperformedonthetwomutantsandthewild-typestrain,andthedifferentiallyexpressedgeneswereidentifiedwithathresholdof|log2FC|≥1andP-value≤0.05.Geneontology(GO)analysisandKyotoEncyclopediaofGenesandGenomes(KEGG)pathwayanalysiswereconductedtorevealthebiologicalfunctionsofthedifferentiallyexpressedgenes.ResultsConstructionofT-DNAinsertionlibraryAT-DNAinsertionlibraryofH.annosumwassuccessfullyconstructedwith3,500transformantsobtained.Theinsertionratioswereapproximately80%,andthenumberofinsertionsiteswasabout2.5pertransformant,whichsuggestedthatthelibraryhadahighcoverageofthegenome.Functionalanalysisofpathogenicity-relatedgenesTwomutantswithT-DNAinsertioningenesrelatedtopathogenicitywereselectedforfurtherstudies.Thevirulenceofthemutantswassignificantlyreducedcomparedtothewild-typestrain(Figure1),indicatingthepathogenicityofthecorrespondinggenes.RNA-Seqanalysiswasperformedonthetwomutantsandthewild-typestrain.Atotalof838and1,261differentiallyexpressedgeneswereidentifiedinthetwomutants,respectively.GOanalysisandKEGGpathwayanalysisrevealedthatmanyofthedifferentiallyexpressedgeneswererelatedtoplantcellwalldegradation,oxidativestressresponse,andstresstolerance,whichmightberesponsibleforthereductioninvirulence.DiscussionInthisstudy,aT-DNAinsertionlibraryofH.annosumwasconstructedandtwomutantswithreducedvirulenceweregeneratedbyT-DNAinsertion.RNA-seqanalysisrevealedthedifferentialexpressionofgenesinvolvedinplantcellwalldegradation,oxidativestressresponse,andstresstolerance,whichmightbeimportantfactorsinthepathogenesisofH.annosum.TheresultssuggestedthatT-DNAinsertionis
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