| 王新果, 赵丹丹, 黄红娟*, 黄兆峰, 陈景超, 王慧敏, 张朝贤, 魏守辉*.利用双标准曲线法检测刺萼龙葵 SrDOG1基因的表达[J].植物保护,2018,44(1):121-126. |
| 利用双标准曲线法检测刺萼龙葵 SrDOG1基因的表达 |
| Detection of DOG1 gene expression in Solanum rostratum by double standard curve method |
| 投稿时间：2017-04-21 修订日期：2017-05-25 |
| DOI：10.16688/j.zwbh.2017119 |
| 中文关键词: 双标准曲线法 刺萼龙葵 DOG1 基因 相对定量 表达检测 |
| 英文关键词:double standard curve Solanum rostratum DOG 1 relative quantification expression detection |
| 基金项目:国家自然科学基金 (31572022, 31171867) |
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| 中文摘要: |
| 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 DELAY OF GERMINATION 1 ( 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 SrDOG1 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 SrDOG1 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。 |
| 英文摘要: |
| 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 was 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 SrDOG1 was obtained. The results showed that SrDOG1 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. |
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