转基因烟草中外源基因实时荧光定量PCR检测方法的建立

Detection method of exogenous gene in transgenic tobacco by real-time fluorescence quantitative PCR

【目的】建立SYBR GreenⅠ实时荧光定量PCR检测转基因烟草中外源基因拷贝数的方法,为转基因植物中外源基因拷贝数的检测提供参考。【方法】采用SYBR GreenⅠ实时荧光定量PCR方法,检测转基因烟草中外源绿色荧光蛋白基因(GFP)的拷贝数,以烟草单拷贝的内源核糖核酸还原酶基因(RNR2)作为内参基因,建立相对定量的双标准曲线法检测转基因烟草中外源基因拷贝数的方法。【结果】构建RNR2基因、GFP基因实时荧光定量PCR的标准曲线,分别为y=-0.2858x+5.6695和y=-0.2826x+2.1048,R2分别为0.9994和0.9989,相关性较高。在检测的5株转基因烟草中GFP基因的拷贝数分别为5、8、19、28和45,非转基因烟草植株的GFP基因拷贝数为0。【结论】建立的转基因烟草中外源基因SYBR GreenⅠ实时荧光定量PCR检测方法具有简单、快速、准确等优点,可用于基因工程中对优良转化植株的筛选及外源基因表达量的快速检测和定量分析。

[Objective]The SYBR Green I real-time quantitative PCR method for detecting copy number of exogenous gene in transgenic tobacco was establish to privide reference for detecting copy number of exogenous gene in transgenic plant. [Method]By SYBR Green I real-time fluorescence quantitative PCR technique, the copy number of green-fluores- cent protein gene （ GFP） as exogenous gene in transgenlc tobacco was detected, taking endogenous ribonucleotide reductase gene （RNR2） of tobacco as reference gene. Then two standard curve method of relative quantification was establish to de- tect copy number of exogenous gene in transgenic tobacco. [ Result ]The standard curves of GFP gene and RNR2 gene were constructed,whiCh were y=-0.2826x＋2.1048 and y=-0.2858x＋5.6695, respectively, and the correlation coefficients were 0.9994 and 0.9989. The copy numbers of the five putative transgenic tobaccos were 5, 8, 19, 28 and 45, respec- tively, and that of non-transgenic tobacco was 0. [Conclusion]The SYBR Green I real-time quantitative PCR method for detecting copy number of exogenous gene is rapid, convenience and accurate,which is applied to screening transgenic plant, and rapid detection and quantitative analysis of exogenous gene expression.