农杆菌介导的RNAiCP基因在大豆中的转化-论文.pdf

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1、作物学报ACTAAGRONOMICASINICA2013，39(9)：1594—1601http：／／zwxb．chinacrops．org／ISSN0496-3490；CODENTSHPA9E—mail：xbzw@chinajouma1．net．caDoI：l0．3724／SP．J．1006．2013．01594农杆菌介导的RNAiCP基因在大豆中的转化章洁琼李红艳胡小南单志慧，唐桂香1，浙江大学农业与生物技术学院，浙江杭州310058；中国农业科学院油料作物研究所，湖北武汉430062摘要：花叶病毒(soybeanmosaicvirus，SMV)病

2、是大豆主要病害之一，生产上常采用种植抗性品种方法来防治。本研究以RNA干扰花叶病毒衣壳蛋(coatprotein，CP)基因为表达载体，Bar基因作为筛选标记基因，成熟子叶节为外植体，采用农杆菌介导法获得了22株T0代转基因大豆生根苗，经草丁膦涂抹、Bar试纸条和PeR法鉴定，获得RNAiCP转基因植株18株；对转基因植株T1代的遗传分析表明，外源基因能够稳定遗传到下一代且符合孟德尔遗传规律；T1代Southern杂交表明，导入的干扰片段为单拷贝；花叶病毒摩擦接种表明，RNAicP转基因大豆植株具有抗花叶病毒特性；摩擦接种后3周，DASEL1SA检测进

3、一步表明，RNAiCP转基因植株花叶病毒检出率仅为7．69％，而非转基因植株为100％。这表明RNAi花叶病毒CP基因可用于抗大豆花叶病毒的研究。关键词：大豆；RNAiCP；遗传转化；大豆花叶病毒；病毒鉴定AgrobacteriumtumefaciensMediatedTransformationofRNAiCPGeneintoSoybean(GlycinemaxL．)ZHANGJie．Qiong，LIHong—Yan，HUXiao．Nan，SHANZhi—Hui，，andTANGGui—Xiang，InstituteofCropScience，Col

4、legeofAgricultureandBiotechnology,ZhejiangUniversity,Hangzhou310058，China；OilCropsResearchInstituteChineseAcademyofAgriculturalSciences，Wuhan430062，ChinaAbstraet：SoybeanmosaicvirusfSMV)causesaseverediseaseinsoybean，whichcanbeefficientlypreventedbyplantingresis．tantcultivars．Inor

5、dertoireprovetheSMVresistanceofsoybean．anagrobacterium．tumqfaciensmediatedgenetransformationwasconductedinthisstudybyusingRNAisoybeanruesaicviruscoatprotein(CP)geneastheexpressionvector，bargeneastheselectivemarkergene．andcotyledonary-nodeastheexplanttogetRNAitransformationsoybea

6、n．Asresults．22putativetrans．geniesoybeanplantswereobtainedand18positivetransgenicsoybeanplantswereidentifiedbycoatingwithleavesherbicide．usingbarproteinquickdipstickandPeRanalysis．ThesegregationratioofT1transgenicprogenyshowedthatthetransformedgenecouldbeinheritedaccordingtotheM

7、endel’slaw．TheT1southemblotanalysisshowedthattheimportedinterferencefrag．meritwasonecopy．AfterSMVfrictioninoculation．theRNAiCPtransgenicsoybeanplantsshowedgoodresistancetoSMVDAS—ELISAanalysisatthreeweeksafterSMVinoculationrevealedthat100％ofthenon．transgeniclinesSMVwhileonly7．69％

8、OftheRNAiplantswereinfectedbySMVTheresultsdemon