【摘要】 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。更多还原

【Abstract】 To quantitatively detect and analyze transgenes in genetically modified(GM) crops, development of endogenous reference genes with taxon-specificity, low copy number and no allelic variation in varieties is prerequisite. In this study, bioinformatic analyses of genes from database and literatures revealed that one pollen- specific protein gene, PSG719(Gen Bank accession: FJ497025.1), from hexaploid wheat genome had high specificity for wheat and low copy number; based on its gene sequence specific primers were designed.These primers were used for qualitative PCR detection of 16 plant species including durum wheat and other closely related cereal species, and no PCR products were obtained from all the plant species except that from common wheat indicating a good taxon-specificity. And the homogeneity was verified on 16 widely different eco- systems wheat varieties by quantitative real- time PCRs. This gene was then used for subsequent qualitative and quantitative PCRs and relevant studies. In conventional qualitative PCRs, the limit of detection(LOD) was about 2 copies per wheat haploid genome per reaction. In the quantitative Real-time PCR assays,LOD and limit of quantitation(LOQ) were about 2 and 5 copies haploid genome, respectively, the latter of which was lower than other reported wheat endogenous reference genes. Furthermore, standard curves through five- fold serial dilutions corresponding to 10 000, 2 500, 625, 156 and 39 copies wheat haploid genome per reaction were performed by the above- developed PSG719 quantitative PCR system, and the results showed ideal slope(- 3.395) and highly linear co- efficiency(R2=0.999) between Cross point(Cp)values and the amounts of DNA, indicating that an appropriate calibration curve could be constructed for quantitative detection of GM wheat. Taken together, these results indicated that the PSG719 gene was a new endogenous reference gene suitable for the specific detection and quantification of genetically modified materials in common wheat. The developed PSG719 system will facilitate the enforcement of genetically modified organism labeling for wheat in the near future.更多还原