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一种基于PCR技术鉴定单拷贝转基因烟草的方法
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(4):83-88.
作者姓名:宋锋  孙敏  罗克明
作者单位: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
基金项目: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
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  
收稿时间:2009-11-16
修稿时间:2010-01-18

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.A Simple and Rapid PCR-based Method for Identifying Transgenic Tobacco Plants Carrying a Single Copy of the Integrated Gene[J].China Biotechnology,2010,30(4):83-88.
Authors:SONG Feng  SUN Min  LUO Ke-ming
Institution:Key Laboratory of Eco environments of Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Southwest University, Chongqing 400715, China
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(RNR_2) 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.
Keywords:Transgenic tobacco plants Single-copy integrated gene Polymerase chain reaction (PCR)  
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