抗草甘膦基因cp4-epsps表达载体构建及对甘薯的遗传转化研究

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1、ExpressionVectorConstructionandTransformationofGlyphosateResistanceGeneCP4-EPSPSinSweetPotatoAbstractSweetpotato[Ipomoeabatatas(L．)Lam】isoneofimportantfoodcropsandindustrialcrops．Itsproductionissufferringalotfromweedsdamage，eachyearitcanbecutdown5％一15％byweeds．Theglyphosateresistantvarietieswasconta

2、inedwiththegeneCP4-EPSPStransformedintosweetpotato．Inthisstudy,theplantexpressionvectorpCAMBIAl301--CP4··EPSPSwassuccessfullyconstructedbyconnectingthecloningvectorpMD18·-T-CP4·—EPSPSwiththeplantexpressionvectorpCAMBIAl301containingCaMV35Spromoter,NOSterminator,basedontheamplificationofgeneCP4一EPSP

3、Sfromasoybeanvarietywithresistancetoglyphosate．ThenthestemsectionofsweetpotatowasinfectedwithAgrobacteriumtumefaciensharboringpCAMBIAl301·-CP4--EPSPSrecombinantvectorassistedbyvacuuminfiltrationmethod．ItttLmedoutthat5ofthe72sampleswereidentifiedtohepositivebyPCRverifivation，including1individualofGu

4、angshu87and4individualsofFushu18．Glyphosatetoleranceidentificationresultsshowedthat3individualsofFushu18couldtolerateglyphosateof0．12％concentration．Theaboveresultswasrespectedtofacilitatedbreedingvarieties、Ⅳitllresistancetoglyphosateandcommercialproductionofsweetpotatoandprovedthatitwasfeasibletotr

5、ansferthestemsectionwithvacuuminfiltrationassistedAgrobacteriumtumefaciensmediatedtransformationmethod．KeyWords：SweetPotato，Glyphosate，Vectorconstruction，TransformationII万方数据目录摘要IAbstract．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．III前言I1．I甘薯基因工程育种11．2甘薯遗传转化研究进展21．2．1

6、基因枪法．21．2．2电击法．21．2．3发根农杆菌介导法．21．2．4根癌农杆菌介导法．2I．3转基因研究现状31．3．1转基因作物．31．3．2抗除草剂转基因作物．51．3．3抗除草剂基因．51．4草甘膦及抗草甘膦转基因研究进展61．4．1草甘膦概述．61．4．2草甘膦作用机理．71．4．3抗草甘膦作物的获得策略．．71．4．4抗草甘膦作物研究．91．5本研究的目的和意义91．6本研究的创新点92材料和方法．102．I材料．．102．1．1菌株和载体102．1．2植物受体102．1．3主要试剂102．2TA克隆一102．2．1引物设计与合成102．2．2CP4一剧强附基因扩增112．2．

7、3CP4一剧)．哪基因目的片段回收112．2．4CP4．剧强盼基因片段和pMDl8．T连接．112．2．5pMDl8．T-CP4．EPSPS克隆载体鉴定一122-3pCAMBIAl301．CP4．EPSPS表达载体构建122．3．1pMDl8-T-CP4-EPSPS载体和pCAMBIAl301载体酶切．．12万方数据2．3．2酶切后CP4一剧l哪基因和pCAMBIAl301载体回收．122．3．3CP4．剧峪