利用TaqMan定量PCR技术不依赖参比内源基因测定转基因植物外源基因拷贝数

A method freed from endogenous reference gene for estimating copy number of transgenes in genetically modified plants by TaqMan quantitative PCR

目的:建立一种利用TaqMan荧光PCR定量技术不依赖参比内源基因测定转基因植物外源基因拷贝数的分析方法。方法:以转基因大豆RRS和转基因玉米Bt176为材料,通过转基因植物外源基因与品系特异序列拷贝数的比较测定外源基因整合拷贝数。结果:3次测得的Bt176玉米中Cry1A(b)基因的整合拷贝数分别为2.69,3.43和2.83,平均为2.98,标准偏差(SD)为0.40,相对标准偏差(RSD)为13.28%,即Cry1A(b)基因在Bt176玉米中的整合拷贝数为3;3次测得的RRS大豆中EPSPS基因拷贝数分别为1.08,1.01,0.96,平均为1.01,SD为0.06,RSD为6.06%,即RRS大豆中EPSPS基因拷贝数的测定结果是单拷贝。结论:本研究建立的转基因植物外源基因拷贝数测定方法可准确测定转基因植物外源基因的拷贝数。与现有的通过外源基因与参比内源基因拷贝数比较测定转基因植物外源基因拷贝数的方法相比,不但避免了选择物种特异性的看家基因及测定看家基因拷贝数等大量工作,而且应用范围更广泛。

Objectlve：To develop a method freed from endogenous reference gene for estimating copy number of transgenes in genetically modified plants by TaqMan quantitative PCR. Methods： The system uses TaqMan quantitative PCR and comparison with event - specific sequences of Roundup Ready soybean （RRS） and Bt176 maize, to respectively determine the copy number of exogenous EPSPS and CrylA（b） genes. Results：The copy number of integrated CrylA（b） gene in Bt176 maize was 2. 98, with SD value of 0. 40 and RSD value of 13.28%. The copy number of integrated EPSPS gene in RRS was 1.01, with SD value of 0. 06 and RSD value of 6.06%. Conclusion：The newly developed method, with more convenient process and wider application, can accurately determine copy number of transgenes in genetically modified plants.