转基因水稻检测用阳性质粒分子的构建及应用

生物技术通报 ›› 2013, Vol. 0 ›› Issue (7): 71-77.

转基因水稻检测用阳性质粒分子的构建及应用

李晓飞¹ 谭小力¹ 李俊² 武玉花² 曹应龙²

（1.江苏大学生命科学研究院，镇江 212013；2.中国农业科学研究院油料作物研究所，武汉 430062）

收稿日期:2013-03-28 修回日期:2013-07-19 出版日期:2013-07-19 发布日期:2013-09-02
作者简介:李晓飞，男，硕士研究生，研究方向：基因工程与转基因检测；E-mail：lixiaofei512@126.com
基金资助:
国家转基因生物新品种培育重大专项（2011ZX08012-003，2011ZX08012-005）

Construction and Application of a Plasmid Reference Molecule for Detecting Transgenic Rice

Li Xiaofei¹ Tan Xiaoli¹ Li Jun² Wu Yuhua² Cao Yinglong²

（1. Institute of Life Sciences，Jiangsu University，Zhenjiang 212013；2. Oil Crops Research Institute of Chinese Academy of Agricultural Sciences，Wuhan 430062）

Received:2013-03-28 Revised:2013-07-19 Published:2013-07-19 Online:2013-09-02

摘要/Abstract

摘要： 通过分析转基因水稻（Oryza sativa）中调控元件和功能基因的种类及应用频率，结合水稻基因工程研究中标记基因的使用情况，确定将 CaMV35S 启动子、NOS 终止子，标记基因 Bar、HPT 和 NPTⅡ基因作为转基因水稻的筛查检测靶标。通过重叠延伸 PCR 技术，将水稻内标基因 SPS、筛选靶标（CaMV35S启动子、NOS终止子、Bar基因、HPT基因和NPTⅡ基因）、事件特异性检测靶标（TT51-1、KF-6和KMD1）聚合克隆到质粒载体上，构建质粒分子pBS Rice。应用结果表明，阳性质粒分子pBS Rice既适用于转基因水稻的筛查检测，也适用于抗虫水稻TT51-1、KF-6和KMD1的事件特异性检测。研究结果为转基因水稻安全监管提供了一种不依赖于转基因水稻原材料供应的阳性对照，其对目前国内外批准的转基因水稻检出率达100%，满足了监管过程中转基因水稻的检测需要。

关键词: 转基因水稻, 阳性质粒分子, 定性PCR, 筛查检测, 事件特异性检测

Abstract: Five targets, including the CaMV35S promoter, NOS terminator, Bar gene, HPT gene and NPTⅡ gene were selected to be detecting targets of transgenic rice screening, based on the application frequency of the regulation elements and function genes used in rice（Oryza sativa）genetic engineering research. A rice reference gene SPS, five screening targets（CaMV35S promoter, NOS terminator, Bar gene, HPT gene and NPTⅡ gene）and three event-specific detection fragments（TT51-1, KF-6, KMD1）were fused using a overlap extension PCR technique and cloned into a plasmid vector pBluescript II SK+ to construct pBS Rice. The results showed that the plasmid molecules pBS Rice could be applied not only for the screening of genetically modified rice, but also in the event-specific detections of insect-resistant rice TT51-1, KF-6 and KMD1. This study provides a raw materials independent positive control with a coverage rate of 100% for the worldwide approved rice events, meeting the needs of the transgenic rice security supervision.

Key words: Transgenic rice, Reference plasmid molecule, Qualitative PCR, Screening detection, Event-specific detection

李晓飞,谭小力,李俊,武玉花,曹应龙. 转基因水稻检测用阳性质粒分子的构建及应用[J]. 生物技术通报, 2013, 0(7): 71-77.

Li Xiaofei,Tan Xiaoli,Li Jun,Wu Yuhua,Cao Yinglong. Construction and Application of a Plasmid Reference Molecule for Detecting Transgenic Rice[J]. Biotechnology Bulletin, 2013, 0(7): 71-77.

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