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1、作物學報ACTAAGRONOMICASINICA2013,39(9):1594—1601http://zwxb.chinacrops.org/ISSN0496-3490;CODENTSHPA9E—mail:xbzw@chinajouma1.net.caDoI:l0.3724/SP.J.1006.2013.01594農桿菌介導的RNAiCP基因在大豆中的轉化章潔瓊李紅艷胡小南單志慧,唐桂香1,浙江大學農業(yè)與生物技術學院,浙江杭州310058;中國農業(yè)科學院油料作物研究所,湖北武漢430062摘要:花葉病毒(soybeanmosaicvirus,SMV)病
2、是大豆主要病害之一,生產上常采用種植抗性品種方法來防治。本研究以RNA干擾花葉病毒衣殼蛋(coatprotein,CP)基因為表達載體,Bar基因作為篩選標記基因,成熟子葉節(jié)為外植體,采用農桿菌介導法獲得了22株T0代轉基因大豆生根苗,經草丁膦涂抹、Bar試紙條和PeR法鑒定,獲得RNAiCP轉基因植株18株;對轉基因植株T1代的遺傳分析表明,外源基因能夠穩(wěn)定遺傳到下一代且符合孟德爾遺傳規(guī)律;T1代Southern雜交表明,導入的干擾片段為單拷貝;花葉病毒摩擦接種表明,RNAicP轉基因大豆植株具有抗花葉病毒特性;摩擦接種后3周,DASEL1SA檢測進
3、一步表明,RNAiCP轉基因植株花葉病毒檢出率僅為7.69%,而非轉基因植株為100%。這表明RNAi花葉病毒CP基因可用于抗大豆花葉病毒的研究。關鍵詞:大豆;RNAiCP;遺傳轉化;大豆花葉病毒;病毒鑒定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