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利用定量PCR检测和分析转基因作物外源基因时,首先需要一种具有种属特异性、品种间不显示等位基因变化和较低拷贝数的内标准基因作为对照。本研究通过生物信息学方法和现有文献,筛选到小麦PSG719基因(Gen Bank登录号:FJ497025.1)具有特异性强以及拷贝数低的特点;基于该基因特异区段设计的引物,对包括硬粒小麦在内的16个物种的定性PCR鉴定和16个不同生态区小麦品种的定量PCR检测结果表明,该基因具有普通小麦的特异性和品种间的稳定性。该基因的定性PCR检测极限为2个拷贝的普通小麦基因组;定量PCR检测极限为2个拷贝,定量极限为5个拷贝;这些极限值在已有的小麦内标准基因中均处于最低水平,证明该基因PCR反应的灵敏度较高,可以对小麦转基因成分含量较低的样品进行检测和定量。以经梯度稀释的小麦基因组DNA为模板,定量PCR标准曲线斜率-3.395,相关系数R2为0.999,表明该基因的定量PCR体系适用转基因小麦样品的定量分析。这些结果表明,普通小麦内标准基因PSG719是一个理想的内标准基因,可以用于转基因小麦的定性和定量检测。本研究为将来转基因小麦标识制度的建立提供了一种可靠的定量检测体系。
When quantitative PCR is used to detect and analyze exogenous genes in transgenic crops, an internal standard gene that has species specificity and does not show allelic variation and a lower copy number among varieties is needed as a control. In this study, bioinformatics methods and existing literature were selected to screen wheat PSG719 gene (Gen Bank accession number: FJ497025.1) with strong specificity and low copy number; primers designed based on the specific segment of the gene, The qualitative PCR identification of 16 species including Durum wheat and the quantitative PCR of 16 wheat cultivars in different ecological regions showed that this gene has the specificity of common wheat and the stability among varieties. The qualitative PCR detection limit of this gene was two copies of the common wheat genome. The limit of quantitation PCR was 2 copies and the limit of quantification was 5 copies. These limits were the lowest in the existing wheat standard genes, The PCR reaction of this gene has higher sensitivity and can detect and quantify the samples with lower GM content. The gradient of the standard genomic DNA of wheat was used as a template to quantify the slope of the standard curve of PCR-3.955 with a correlation coefficient R2 of 0.999, indicating that the quantitative PCR system of this gene is suitable for the quantitative analysis of transgenic wheat samples. These results indicated that the common wheat internal standard gene PSG719 is an ideal internal standard gene and can be used for the qualitative and quantitative detection of transgenic wheat. This study provides a reliable quantitative detection system for the establishment of the identification system of transgenic wheat in the future.