摘 要 ：快捷、准确地检测转基因植物中外源基因的拷贝数,是转基因生物育种的重要研究内容,具有较大的应用前景。本研究以7个抗虫BT(Bacillus thuringiensis)和抗草甘膦EPSPS-G10转基因大豆事件为材料,以SYBR Green I为荧光染料,利用实时荧光定量PCR方法,根据PCR反应获得的Ct值与起始模板数的对数值存在线性反比关系这一原理,建立了相关性系数在0.99以上的模板定量标线。通过目的基因与内参基因——大豆肌动蛋白编码基因ACTIN的起始模板量比较,估算出各株系的目的基因拷贝数。数据显示,株系1、2、3和4的2个基因均为单拷贝,株系6的2个基因均有2个拷贝,株系5和7的2个基因的拷贝数为3。利用Southern 印迹方法对材料1~4的拷贝数进行验证,结果表明,两者的检测结果基本一致,证明实时荧光PCR检测法是大豆外源基因拷贝数检测中快速有效的方法。实时荧光PCR高效快速检测基因拷贝数,对于大规模转基因株系的外源基因拷贝数检测应用具有重大意义。
Abstract:A robust method to rapidly and accurately determine the copy number of the target transgene is essential for the genetic engineering, thus has great potential for the application. In the study, we used seven transgenic soybean events harboring insect resistant gene BT and herbicide tolerance gene EPSPS-G10. The BT and EPSPS-G10 gene are the target genes, with ACTIN gene as the endogenous reference gene. Based on the theory that Cycle Threshold (Ct) value is linearly correlated with the logarithm of the amount of the initial DNA template, the standard curves of all three genes were constructed with the correlation coefficiency greater than 0.99. Using the standard curves and the Ct value of the samples, we calculated the amounts of ACTIN and target gene templates. As ACTIN has two copies in soybean genome, the ratio of target gene and ACTIN can be used to estimate the copy number of the transgenes. Result showed that line 1, 2, 3 and 4 contain a single copy of the BT and EPSPS-G10 gene. Line 5, 6, 7 have 3, 2, and 3 copies for both BT and EPSPS-G10 genes, respectively. To verify the accuracy of the estimation, a Southern blot analysis was conducted using transgenic line 1-4. The results showed that the transgene copy number detected by Southern analysis in these lines matched that estimated by the real-time fluorescence quantitative PCR. Therefore, the real-time fluorescence quantitative PCR method is a rapid, reproducible and effective method to estimate the transgene copy number, which is of great significance for high throughput analysis of transgene copy number.