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中国农学通报 ›› 2013, Vol. 29 ›› Issue (21): 131-136.doi: 10.11924/j.issn.1000-6850.2012-3125

所属专题: 生物技术 油料作物

• 生物技术科学 • 上一篇    下一篇

大豆FAD2-1b 基因沉默载体的构建

杜景红 刘丽君   

  • 收稿日期:2012-09-13 修回日期:2012-10-25 出版日期:2013-07-25 发布日期:2013-07-25

Construction of Soybean FAD2-1b Gene Silencing Vector

  • Received:2012-09-13 Revised:2012-10-25 Online:2013-07-25 Published:2013-07-25

摘要: 利用RT-PCR方法从转基因大豆中克隆出FAD2-1b基因,构建大豆FAD2-1b基因的沉默载体,目的是培育生物安全的转基因大豆。设计引物、采用RT-PCR从高蛋白大豆品种‘黑农35’中克隆大豆种子特异表达启动子GY1;从高油大豆‘黑农37’中克隆FAD2-1b基因内含子1;从5个转基因受体大豆品种中克隆FAD2-1b基因片段,作为正向臂和反向臂。连接这些目的片段,构建筛选标记基因为bar,启动子为GY1,内含子为FAD2-1b基因内含子1的双T-DNA载体。将双T-DNA载体转化大肠杆菌DH5α,扩增得到的重组质粒经酶切和序列分析鉴定正确,命名为pDT-FAD2I。本研究构建了针对大豆FAD2-1b基因的沉默载体,为进一步获得FAD2-1b基因沉默大豆,培育转基因安全的高品质大豆奠定了基础。

关键词: 环境因子, 环境因子

Abstract: The soybean FAD2-1b gene was amplified by RT-PCR from transgenic soybeans, and soybean FAD2-1b gene silencing vector was constructed for breeding transgenic soybeans with biosafety. Seed specific promoter GY1 of soybean was amplified from high protein soybean ‘Heinong 35 ’; FAD2-1b gene introns 1 was amplified from high oil soybean ‘Heinong 37’; FAD2-1b gene fragment was amplified from five transgenic acceptors soybean, it was used as forward arm and reverse arm. These target fragments were ligated to construct a double T-DNA with bar gene selection marker, promoter GY1 and FAD2-1b gene intron 1. Then double T-DNA was introduced into E. coli DH5α. The cloning of recombination plasmids was identified correctly by enzymes digestion and sequence analysis. The recombination plasmid was named pDT-FAD2I. FAD2-1b gene silencing vector has been constructed. This will constitute an important foundation for further development of FAD2-1b gene silencing soybeans and high quality soybean transgenic security.

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