利用双标准曲线法检测刺萼龙葵SrDOG1基因的表达

摘要

Double standard curve method is a relative quantification method widely used in quantitative detection of gene expressions.Combined with the principle and method of absolute quantification,the traditional double standard curve method was improved.The fragment of target gene DELAY OF GERMINATION 1 (SrDOG1) and reference gene β-actin (SrACT) were cloned into pEASY-Blunt Zero vector to make the standard plasmid,respectively.The total RNA were extracted from Solanum rostratum seeds and the standard curves were achieved by real-time PCR reaction using the dilutions of standard plasmid as the template.According to the standard curve,the relative expression of SrDOG 1 was obtained.The results showed that SrDOG 1 transcript level in seeds under cold storage was significantly higher than that under room temperature,suggesting that double standard curve method has high sensitivity in quantification of expression differences of DOG1 gene.This method is stable,reproducible and simple in data processing.It is suitable for the comparison of relative gene expression levels under various conditions and is useful to further determine the gene expression and gene function of SrDOG1 in the future.%双标准曲线法是基因表达定量研究中应用较广的一种相对定量检测方法.本研究结合绝对定量的原理,对传统双标准曲线法进行了改进.以刺萼龙葵延迟萌发基因DELAYOFGERMINATION1 (DOG1)的表达检测为例,选择β-actin为内参基因,分别将目的基因和内参基因片段插入pEASY-Blunt Zero载体,制作标准质粒.提取冷藏或常温储藏种子的RNA进行荧光扩增,同时将标准质粒梯度稀释液作为模板构建标准曲线,以此计算Sr-DOG1的相对表达量.结果表明,双标准曲线法能够灵敏地检测SrDOG1基因的差异表达,发现冷藏种子中Sr-DOG1基因的表达量较常温储藏显著增高.双标准曲线法检测结果稳定,重现性好,数据处理简单,适用于多种条件下基因表达水平的比较,为进一步系统研究刺萼龙葵DOG1基因的表达特性和基因功能奠定了基础.