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作物学报 ›› 2013, Vol. 39 ›› Issue (09): 1594-1601.doi: 10.3724/SP.J.1006.2013.01594

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

农杆菌介导的RNAi CP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,*,唐桂香1,*   

  1. 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
  • 收稿日期:2012-11-28 修回日期:2013-05-24 出版日期:2013-09-12 网络出版日期:2013-07-09
  • 通讯作者: 唐桂香, E-mail: tanggx@zju.edu.cn; 单志慧, E-mail: zhihuijimi@163.com
  • 基金资助:

    本研究由农业部转基因生物新品种培育重大专项(20112X08004-004-009)，国家自然科学基金项目(31071443)和浙江省科技厅公益技术项目(2012C32001)资助。

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

ZHANG Jie-Qiong1,LI Hong-Yan1,HU Xiao-Nan1,SHAN Zhi-Hui2,*,TANG Gui-Xiang1,*   

  1. 1 Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China; 2 Oil Crops Research Institute, Chinese Academy of Agriculture Sciences, Wuhan 430062, China
  • Received:2012-11-28 Revised:2013-05-24 Published:2013-09-12 Published online:2013-07-09
  • Contact: 唐桂香, E-mail: tanggx@zju.edu.cn; 单志慧, E-mail: zhihuijimi@163.com

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被引次数

8

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T0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；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。

关键词: 大豆, RNAi CP, 遗传转化, 大豆花叶病毒, 病毒鉴定

Abstract:

 Soybean mosaic virus (SMV) causes a severe disease in soybean, which can be efficiently prevented by planting resistant 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 transgenic 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 fragment was one copy. After SMV friction inoculation, the RNAi CP transgenic soybean plants showed good resistance to SMV. DAS-ELISA analysisat 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.

Key words: Soybean, RNAi CP, Genetic transformation, Soybean mosaic virus, Virus identification

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