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多重实时荧光定量PCR分析转基因烟草外源基因拷贝数
引用本文:余婧,邹颉,付强,郭玉双,林世锋,赵杰宏. 多重实时荧光定量PCR分析转基因烟草外源基因拷贝数[J]. 中国烟草学报, 2017, 23(4): 92-97. DOI: 10.16472/j.chinatobacco.2016.021
作者姓名:余婧  邹颉  付强  郭玉双  林世锋  赵杰宏
作者单位:贵州省烟草科学研究院, 烟草行业烟草分子遗传重点实验室, 贵州省贵阳市龙滩坝路29号 550081
基金项目:中国烟草总公司贵州省公司科技项目(201604);贵州省科技支撑计划农业攻关科技计划项目(黔科合NY[2013]3020号);贵州省科学技术基金(黔科合J字[2014]2117号)
摘    要:[目的]采用多重实时荧光定量PCR方法在一个反应内同时检测转基因烟草中插入外源基因35S、NOS、NPT Ⅱ的拷贝数。[方法]将转基因阳性参照烟草基因组DNA经梯度稀释,通过多重荧光定量PCR反应同时获得烟草内源参照基因NR、外源基因35S、NOS、NPT Ⅱ的相关性标准曲线方程,将待测株系外源基因的Ct值代入标准曲线方程后估算其拷贝数。[结果]获得了上述4个基因的标准曲线,其R2均接近于1,相关性较高。在检测的六个转基因株系中初筛到3株3个外源基因均为单拷贝的株系。[结论]可使用该方法对转基因烟草外源基因插入拷贝数进行估算,为获得稳定遗传材料提供初筛依据。 

关 键 词:转基因烟草   多重实时荧光定量PCR   外源基因   拷贝数
收稿时间:2017-03-02

Detecting copy number of exogenous genes in transgenic tobacco by multiplex RT-PCR
Affiliation:Guizhou Academy of Tobacco Science, Key Laboratory of Molecular Genetics, China National Tobacco Corporation, Guiyang 550081, China
Abstract:This paper utilized a multiplex real-time PCR assay to simultaneously detect copy number of inserted exogenous gene 35S, NOS and NPT Ⅱ in transgenic tobacco within one reaction. Gradient dilution was applied to positive reference tobacco genome DNA, and multiplex real-time PCR assay was used to obtain standard curve equation of tobacco endogenuous reference gene NR and exogenous gene 35S, NOS, as well as NPT Ⅱ. Copy number of exogenous genes was estimated after substituting the Ct values of both endogenuous and exogenous genes of the strains to be tested into the standard curve equation. Results showed that standard curves of the 4 genes were obtained, and all R2 values were close to 1 with relatively high correlation. In the tested 6 transgenic strains, preliminarily screened 3 strains with their 3 exogenous genes were all single copy. This method can be used for the estimation of copy number of inserted exogenous genes in transgenic tobacco, so as to provide preliminary screen basis for obtaining stable genetic materials. 
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