农杆菌介导的RNAiCP基因在大豆中的转化  

Agrobacterium tumefaciens Mediated Transformation of RNAi CP Gene into Soybean (Glycine max L.)

作　　者：章洁琼[1] 李红艳[1] 胡小南[1] 单志慧[2] 唐桂香[1] 

机构地区：[1]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

出　　处：《作物学报》2013年第9期1594-1601,共8页Acta Agronomica Sinica

基　　金：国家转基因生物新品种培育科技重大专项(20112X08004-004-009);国家自然科学基金项目(31071443);浙江省科技厅公益技术项目(2012C32001)资助

摘　　要：花叶病毒(soybean mosaic virus,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T_0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T_1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;T_1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 CP转基因大豆植株具有抗花叶病毒特性;摩擦接种后3周,DAS-ELISA检测进一步表明,RNAi CP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。Soybean mosaic virus （SMV） causes a severe disease in soybean, which can be efficiently prevented by planting resis- tant cultivars. In order to improve the SMV resistance of soybean, an agrobacterium-tumefaciens mediated gene transformation was conducted in this study by using RNAi soybean mosaic virus coat protein （CP） gene as the expression vector, bar gene as the selective marker gene, and cotyledonary-node as the explant to get RNAi transformation soybean. As results, 22 putative trans- genic soybean plants were obtained and 18 positive transgenic soybean plants were identified by coating with leaves herbicide, using bar protein quick dip stick and PCR analysis. The segregation ratio of T1 transgenic progeny showed that the transformed gene could be inherited according to the Mendel＇s law. The T1 southern blot analysis showed that the imported interference frag- ment was one copy. After SMV friction inoculation, the RNAi CP transgenic soybean plants showed good resistance to SMV. DAS-ELISA analysis at three weeks after SMV inoculation revealed that 100% of the non-transgenic lines SMV while only 7.69% of the RNAi plants were infected by SMV. The results demonstrated that the RNAi CP transgenic soybean plants obtained in the study are valuable resources for improving the SMV resistance of soybean.

关 键 词：大豆 RNAI CP 遗传转化 大豆花叶病毒 病毒鉴定 

分 类 号：S565.1[农业科学—作物学]