生物技术通报 ›› 2018, Vol. 34 ›› Issue (3): 53-58.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0756

• 综述与专论 • 上一篇    下一篇

数字PCR在转基因水稻拷贝数鉴定中的应用

王永1,2, 兰青阔2, 赵新2, 陈锐2, 沈晓玲2, 李文3, 张耀中3, 李亮4, 王勤英1   

  1. 1. 河北农业大学植物保护学院,河北 保定 071001;
    2. 天津市农业质量标准与检测技术研究所,天津 300381;
    3. 天津农学院,天津 300382;
    4. 中国农业科学院生物技术研究所,北京 100081
  • 收稿日期:2017-09-12 出版日期:2018-03-20 发布日期:2018-04-10
  • 作者简介:王永,男,博士,研究方向农产品质量安全分子检测技术;E-mailwytaas@126.com
  • 基金资助:
    国家自然科学基金面上项目(31671964),转基因重大专项(2016ZX08012003)

Estimation of the Copy Number of Exogenous Genes in Genetically Modified Rice by Droplet Digital PCR

WANG Yong1,2, LAN Qing-kuo2, ZHAO Xin2, CHEN Rui2, SHEN Xiao-ling2, LI Wen3, ZHANG Yao-zhong3, LI Liang4, WANG Qin-ying1   

  1. 1. College of Plant Protection,Hebei Agricultural University,Baoding 071001;
    2. Tianjin Institute of Agricultural Quality Standard and Testing Technology,Tianjin Academy of Agricultural Sciences,Tianjin 300381;
    3. Tianjin Agricultural University,Tianjin 300384;
    4. Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081
  • Received:2017-09-12 Published:2018-03-20 Online:2018-04-10

摘要: 转基因作物中插入外源基因拷贝数等分子特征是转基因生物安全评价的重要内容,但是以往的拷贝数鉴定方法存在操作性差等不足,因此亟需操作简单且准确的鉴定方法。应用微流滴数字PCR方法鉴定转基因水稻中插入目的基因的拷贝数,测试方法的可行性。实验结果表明,4个重复样品的目的/内标准基因的比值分别为674/662、516/541、737/759、528/571,均值为0.97,因此验证的目的基因拷贝数为1,与Southern 杂交结果一致。微流滴数字PCR作为插入外源基因拷贝数鉴定的新方法,具有操作简单、准确度高、设计灵活等优点。

关键词: 转基因水稻, 目的基因拷贝数, 微流滴数字PCR

Abstract: Copy number of inserted exogenous gene is an important index of assessing the safety of genetically modified organisms. However,there are issues such as poor maneuverability in previous methods for identifying the copy number of gene,therefore,a simple-operation and accurate identification method is urgently needed. This study testes the feasibility of this method while droplet digital PCR was applied to identify the copy number of target gene in transgenic rice. The experimental results showed that the Cry1Ab/PLD ratio of four samples was 674/662,516/541,737/759 and 516/541,respectively,with 0.97 in average. In other words,the copy number of target gene was 1 by droplet digital PCR,which was consistent with the results from Southern blotting. Conclusively,as a novel method to identify the copy number of inserted exogenous gene,droplet digital PCR has some advantages such as simple operation,high accuracy,and flexible design.

Key words: transgenic rice, copy number of target gene, droplet digital PCR(ddPCR)