《农杆菌介导的RNAi CP基因在大豆中的转化 Agrobacterium tumefaciens Mediated Transformation of RNAi CP Gene into Soybean (Glycine max L.)》.pdfVIP

AGRONOMICASINICA 作物学报ACTA 2013，39 9 ：1594—1601 ISSN TSFIPA9 0496—3490；CODEN DoI：10．37241SP．J．1006．2013．01594 农杆菌介导的RNAiCP基因在大豆中的转化 章洁琼1 李红艳1 胡小南1 单志慧2，4 唐桂香 1．奉 浙江大学农业与生物技术学院，浙江杭州310058；2中国农业科学院油料作物研究所，湖北武汉430062 摘 mosaic 要：花叶病毒 soybeanvirus，SMV 病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白 coat protein，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To代转基因大豆生根苗，经草丁膦涂抹、Bar试纸条和PCR法鉴定，获得RNAi CP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； CP转基因大豆植株具有抗花叶病 Tl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，RNAi 毒特性；摩擦接种后3周，DAS．ELISA检测进一步表明，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；RNAi CP；遗传转化；大豆花叶病毒；病毒鉴定 RNAiCPGeneinto MediatedTransformationof Agrobacteriumtumefaciens max L． Soybean Glycine ZHANG Xiao．Nan TANG 1，LI 1，SHANZhi—Hui2+，and Jie—QiongHong—Yanl，HU Gui．Xian91，+ 1 Instituteof of and 3 Oil Research AgricultureBiotechnology,Zhejiang 10058，China；2Crops Institute， CropScience，College University,Hangzhou Chinese Sciences，Wuhan430062，China AcademyofAgricultural mosaic aseverediseasein canbe by resis- Abstract：Soybeanvirus SMV causes soybean，whichefficientlypreventedplanting rantcultivars．Inorderto theSMVresistanceof mediatedtransformation improve soybean。anagrobacterium．tumefaciensgene in RNAi mosaicvirescoat asthe asthe wasconductedthis vector,bar studybyusing soybcan protein CPlgene expression gene selectivemarker asthe to transformationresults，22 trans— gene．andcotyledonary．nodeexplantget融qAi soybean．As putative wereobtainedand18 wereidentified withleavesherbicide． geniesoybcanplants positivetransgenicsoybeanplants bycoating bar stickandPCR ratioof showedthatthetransformed TI usingproteinquickdip analysis．Thesegregation transgenicprogeny couldbeinherited totheMendel’slaw,111eT1southernblot showedthatthe interference gene according analysis imported frag— CP showedresistancetoSMV． mentwasone SMVfrictioninoculation．theRNAi copy．After transgenicsoybeanplants good SMV that1 the linesSMVwhile DAS．ELIS