转豇豆胰蛋白酶CPTI基因棉花检测用标准分子的制备  

Construction of a Standard Reference Plasmid for Detecting CPTI Gene in Transgenic Cotton

作　　者：王旭静[1] 董雷[1] 苗猛猛[1] 唐巧玲[1] 王志兴[1] 

机构地区：[1]中国农业科学院生物技术研究所,北京100081

出　　处：《中国生物工程杂志》2011年第8期85-91,共7页China Biotechnology

基　　金：国家"十一五"重大科技专项资助项目(2009ZX08012-008B;2009ZX08012-019B;2008ZX08010-3)

摘　　要：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。The objective gene fragments cowpea trypsin gene CPTI,35S promoter,NOS terminator and cotton endogenous reference gene Sad1were cloned by PCR method and ligated into cloning vector to construct a standard reference plasmid pGB,When pGB,transgenic cotton and non-transgenic cotton DNA were used as template to amplify CPTI,35S,NOS and Sad1 gene,four targets DNA fragments can be amplified from pGB and transgnic cotton,but only SadI can be amplified from non-transgenic cotton.It was draw that quantitative standard curve of CPTI,35S,NOS and Sad1 gene by quantitative PCR method.The correlation coefficient of the standard curve reached more than 0.985,indicating that the quantitative PCR reaction system has a good relativity between Ct value and the initial concentration.Also,quantitative PCR reaction system has reproducibility and stability,can be used for quantitative analysis of real samples.

关 键 词：标准分子 转基因棉花 定性检测 定量检测 

分 类 号：Q819[生物学—生物工程]