实时荧光PCR检测转基因大豆外源基因的拷贝数

发布时间：2018-04-11 15:44

本文选题：实时荧光定量PCR + 拷贝数　；参考：《核农学报》2016年04期

【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]:Rapid and accurate detection of the copy number of foreign genes in transgenic plants is an important research content in transgenic biological breeding and has a great application prospect.In this study, 7 insect-resistant BT(Bacillus thuringiensis) and glyphosate resistant EPSPS-G10 transgenic soybean events were used as materials, SYBR Green I was used as fluorescent dye, and real-time fluorescence quantitative PCR method was used.Based on the principle that there is a linear inverse relationship between the Ct value and the logarithmic value of the initial template number obtained by PCR reaction, the template quantitative marking with correlation coefficient above 0.99 is established.The copy number of the target gene was estimated by comparing the initial template quantity of the target gene with that of the soybean actin encoding gene (ACTIN).The data showed that the two genes of strain 1, 2, 2 and 4 were all single copies, the 2 genes of line 6 had 2 copies, and the copy numbers of 2 genes of lines 5 and 7 were 3.The Southern blot method was used to verify the copy number of the material 1H4. The results showed that the two methods were basically the same. It was proved that the real-time fluorescent PCR assay was a rapid and effective method for the detection of foreign gene copy number of soybean.High efficiency and fast detection of gene copy number by real-time fluorescent PCR is of great significance for the detection of foreign gene copy number in large-scale transgenic lines.
【作者单位】：浙江大学生命科学学院植物生理与生物化学国家重点实验室;
【基金】：农业部转基因生物新品种培育重大专项基金(2014ZX08004-001,2014ZX0800401B-002)
【分类号】：S565.1

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