一种基于PCR技术鉴定单拷贝转基因烟草的方法

中国生物工程杂志

2010, Vol. 30

Issue (04): 83-88

技术与方法

一种基于PCR技术鉴定单拷贝转基因烟草的方法

宋锋,孙敏^**,罗克明^**

西南大学生命科学学院三峡库区生态环境教育部重点实验室重庆 400715

A Simple and Rapid PCR-based Method for Identifying Transgenic Tobacco Plants Carrying a Single Copy of the Integrated Gene

SONG Feng,SUN Min,LUO Ke-ming

Key Laboratory of Ecoenvironments of Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Southwest University, Chongqing 400715, China

全文: PDF(498 KB) HTML

摘要：

为了鉴定携带单拷贝外源基因的转基因烟草植株，以烟草核基因组上已知的单拷贝内源基因（RNR2）为内参，转基因烟草植株基因组DNA为模板，在同一PCR反应体系中扩增内源基因（RNR2）和外源目的基因（NPTⅡ）。反应产物在琼脂糖凝胶上电泳，获得了预期大小的两条特异性扩增条带。经ImageJ软件捕捉分析两条目的条带的灰度比，当T1代转基因烟草植株中外源基因与内源基因的扩增条带灰度比为1时，所检测植株即为单拷贝外源基因的转基因烟草植株。孟德尔经典遗传学方法证实了上述检测结果高度可信。

关键词： 转基因烟草; 单拷贝外源基因; 聚合酶链式反应

Abstract:

A simple, rapid, and low-cost method to identify transgenic tobacco plants carrying a single copy of integrated DNA by one-step genomic polymerase chain reaction was described. The reaction employs one set of primer pairs that amplify the target gene (NPTⅡ) derived from the integrated T-DNA together with the know endogenous single-copy gene (RNR2) as a reference under the same melting temperature in a single PCR. After PCR amplification of the genomic DNA from transgenic tobacco plants, two expected bands were observed. When the band intensity ratio is one, this means that the transformants are carrying a single copy of the integrated gene. It is further observed that the expected 3∶1 Mendelian ratio was obtained in all single-copy T-DNA transgenic lines.

Key words: Transgenic tobacco plants Single-copy integrated gene Polymerase chain reaction (PCR)

收稿日期: 2009-11-16 出版日期: 2010-04-29

基金资助:

国家转基因重大专项（2008ZX0801003）、国家自然科学基金（30871576）、重庆市自然科学基金重点项目（CSTC，2009BA1004）资助项目

通讯作者: 孙敏,罗克明 E-mail: kemingl@swu.edu.cn;jwcsm@swu.edu.cn

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引用本文:

宋锋孙敏罗克明. 一种基于PCR技术鉴定单拷贝转基因烟草的方法[J]. 中国生物工程杂志, 2010, 30(04): 83-88.

SONG Feng, SUN Min, LUO Ke-Meng. A Simple and Rapid PCR-based Method for Identifying Transgenic Tobacco Plants Carrying a Single Copy of the Integrated Gene. China Biotechnology, 2010, 30(04): 83-88.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I04/83

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