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利用PCR的方法克隆豇豆胰蛋白酶基因CPTI、基因表达调控元件35S启动子、NOS终止子以及棉花内标基因Sad1,连接到克隆载体上,构建成质粒标准分子pGB。PCR检测过程中,质粒标准分子和转基因棉花中能扩增出4个目的条带,而非转基因棉花中不能扩增出相应的条带,证明构建好的质粒标准分子能用于转基因棉花的定性检测。荧光定量PCR绘制4个目的基因片段的标准曲线,各标准曲线的相关系数均达到0.985以上,说明建立的PCR具有很好的Ct值-初始浓度相关性,达到定量分析的需求,而且荧光定量PCR反应具有很好的重复性和稳定性,可用于实际样品的定量检测。
PCR was used to clone cowpea trypsin gene CPTI, gene expression regulatory element 35S promoter, NOS terminator and cotton internal standard Sad1, and ligated into the cloning vector to construct the plasmid pGB. During the PCR detection, four bands could be amplified in the standard plasmid and transgenic cotton, but not in non-transgenic cotton, which proved that the constructed standard plasmid could be used to characterize the transgenic cotton Detection. Fluorescent quantitative PCR was used to draw the standard curve of four target gene fragments. The correlation coefficients of each standard curve were all above 0.985, which indicated that the established PCR had a good Ct value-initial concentration correlation to meet the needs of quantitative analysis. Furthermore, The reaction has good repeatability and stability, can be used for quantitative detection of actual samples.