遗传 ›› 2018, Vol. 40 ›› Issue (8): 676-682.doi: 10.16288/j.yczz.18-080

• 技术与方法 • 上一篇    下一篇

利用重测序技术获取转基因植物T-DNA插入位点

徐纪明,胡晗,毛文轩,毛传澡()   

  1. 浙江大学生命科学学院植物生物学研究所,杭州 310058
  • 收稿日期:2018-04-02 修回日期:2018-06-11 出版日期:2018-08-16 发布日期:2018-07-25
  • 通讯作者: 毛传澡 E-mail:mcz@zju.edu.cn
  • 作者简介:徐纪明,博士,助理研究员,研究方向：作物磷高效分子机制。E-mail: xujiming@zju.edu.cn
  • 基金资助:
    国家转基因生物新品种培育科技重大专项(2016ZX08001003-009);国家自然科学基金项目资助(31701983)

Identifying T-DNA insertion site(s) of transgenic plants by whole-genome resequencing

Jiming Xu,Han Hu,Wenxuan Mao,Chuanzao Mao()   

  1. Institute of Plant Biology, College of Life Science, Zhejiang University, Hangzhou 310058, China
  • Received:2018-04-02 Revised:2018-06-11 Online:2018-08-16 Published:2018-07-25
  • Contact: Mao Chuanzao E-mail:mcz@zju.edu.cn
  • Supported by:
    Supported by the National Transgenic Project(2016ZX08001003-009);the National Natural Science Foundation of China(31701983)

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摘要：

T-DNA插入位点的获得对于植物功能基因组学研究及转基因植物的筛选鉴定非常重要,但是目前常用的方法如反向PCR、半随机引物PCR等,除了操作复杂、消耗时间长外,特异性较差,效率也很低。本研究利用全基因组重测序技术,将3份转基因材料基因组DNA打包后进行重测序,利用转基因载体序列作为参考序列进行比对分析,得到4个T-DNA插入位点。对3份转基因材料进行PCR和Southern blot验证分析,成功获得了3份转基因材料全部T-DNA插入位点,其中1份材料为2拷贝插入。本文利用重测序技术建立了一种简单、可靠、高效的获取转基因植物T-DNA插入位点的方法,以期为植物功能基因组学及转基因研究奠定基础。

关键词: T-DNA插入位点, 重测序技术, 转基因植物

Abstract:

The availability of T-DNA insertion sites is very important for plant functional genomics research and the screening and identification of transgenic plants. However, the present protocols for identifying T-DNA insertion sites, like reverse PCR and semi-random primer PCR, are not only complex and time-consuming, but also inefficient. In this paper, a DNA pool of three transgenic plants was sequenced by whole-genome resequencing, and four T-DNA insertion sites were identified by blasting using transgenic T-DNA sequences. After PCR and Southern blot analysis, the T-DNA insertion sites of the three transgenic plants were successfully confirmed, and one of the transgenic plants showed two insertion sites. In conclusion, this study established a simple, reliable and efficient method for obtaining T-DNA insertion sites in transgenic plants.

Key words: T-DNA insertion site, whole-genome resequencing, transgenic plant