多重实时荧光定量PCR分析转基因烟草外源基因拷贝数

发布时间：2018-01-27 16:41

本文关键词： 转基因烟草多重实时荧光定量PCR 外源基因拷贝数　出处：《中国烟草学报》2017年04期 　论文类型：期刊论文

【摘要】：【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。
[Abstract]:[objective] to detect the exogenous gene 35SNs in transgenic tobacco simultaneously by multiplex real-time fluorescent quantitative PCR (PCR) in a single reaction. Copy number of NPT II. [methods] the genomic DNA of transgenic reference tobacco was diluted by gradient, and the endogenous reference gene NR was obtained simultaneously by multiplex fluorescence quantitative PCR reaction. The standard curve equation of the correlation of the exogenous gene 35SnSN NPT II. The Ct value of the foreign gene was substituted into the standard curve equation and the copy number was estimated. [results] the standard curves of the four genes were obtained, and the R2 of the four genes was close to 1. Among the six transgenic lines tested, three of them were single copy lines. [conclusion] this method can be used to estimate the number of inserted copies of exogenous genes in transgenic tobacco. In order to obtain stable genetic materials to provide a preliminary screening basis.
【作者单位】：贵州省烟草科学研究院烟草行业烟草分子遗传重点实验室;
【基金】：中国烟草总公司贵州省公司科技项目(201604) 贵州省科技支撑计划农业攻关科技计划项目(黔科合NY[2013]3020号) 贵州省科学技术基金(黔科合J字[2014]2117号)
【分类号】：S572
【正文快照】： 1983年,第一株转基因烟草问世[1],烟草作为植物分子生物学研究的模式植物,30多年来,人们对烟草的转基因研究从未停歇,包括将烟草自身基因进行超量表达以验证其基因功能[2-4],外源基因在烟草中表达后的功能验证[5-7]等,这些研究均需使用转基因技术。然而,无论是内源基因的过表

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