多重实时荧光定量PCR分析转基因烟草外源基因拷贝数

摘要

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.%[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.