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苏云金芽孢杆菌菌株的分离及cry8型基因的克隆.docx

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苏云金芽孢杆菌菌株的分离及cry8型基因的克隆 Title:IsolationofBacillusthuringiensisstraincontainingCry8gene Abstract: Bacillusthuringiensis(Bt)iswidelyusedasabiopesticideduetoitsabilitytoproduceinsecticidalcrystalproteins(Crytoxins).Inthisstudy,weisolatedastrainofBtfromsoilsamplesandclonedtheCry8gene.TheaimoftheresearchwastoexpandourknowledgeofBtstrainsandtheirpotentialasbiocontrolagents. Keywords:Bacillusthuringiensis,Cry8gene,isolation,cloning,biopesticide Introduction: Bacillusthuringiensisisagram-positive,spore-formingbacteriumthatiswidelyusedasabiologicalcontrolagentagainstvariousinsectpests.ItproducescrystalproteinscalledCrytoxins,whicharetoxictoinsectsbutharmlesstohumansandanimals.TheCrytoxinsarespecifictocertaininsectspeciesandhavebeenextensivelyusedinintegratedpestmanagementprograms. OneoftheCrytoxins,Cry8,hasshownpromisingefficacyagainstarangeofimportantagriculturalpests.However,theisolationandcloningofBtstrainscontainingtheCry8geneiscrucialforunderstandingitspotentialanddevelopingnovelbiopesticides.Therefore,thisstudyaimedtoisolateaBtstraincarryingtheCry8geneandtoclonethegeneforfurtheranalysis. MaterialsandMethods: 1.IsolationofBtstrain: Soilsampleswerecollectedfromagriculturalfieldsandprocessedbyserialdilutionandspreadplatetechniques.Btcolonieswereidentifiedbasedontheirtypicalmorphologicalcharacteristicsincludingshape,size,andcolor.PureculturesofBtcolonieswereobtainedbyrepeatedstreakingonnutrientagarplates. 2.IdentificationofBtisolate: TheisolatedstrainwasidentifiedasBacillusthuringiensisbasedonbiochemicaltestsincludingGramstaining,catalase,andoxidasetests.Molecularanalysis,suchas16SrRNAgenesequencing,wasalsoperformedtoconfirmtheidentification. 3.CloningofCry8gene: ThegenomicDNAoftheBtisolatewasextractedusingacommercialDNAextractionkit.TheCry8genewasamplifiedusingspecificprimersdesignedbasedontheCry8genesequenceobtainedfromGenBank.ThePCRproductwaspurifiedandligatedintoanappropriatevectorforfurthercloning.TherecombinantplasmidwasthentransformedintocompetentEscherichiacolicell