人参β-actin基因实时荧光定量PCR方法的建立

Development of real-time fluorescence quantitative RT-PCR assay for β-actin gene of Panax ginseng

目的建立人参β-actin基因实时荧光定量PCR的方法。方法根据GenBank上其他高等植物β-actin基因保守区域设计一对特异性引物,将从人参中PCR扩增得到的β-actin基因克隆到pMD20-T载体上构建重组质粒,经1/10梯度稀释后用于制定标准曲线,并进行反应的灵敏性、特异性和重复性试验。结果该方法检测β-actin基因的最低拷贝数为43拷贝/μL,在较广的范围内（43~4.3×107拷贝/μL）有良好的线性关系（R2=0.995 3）;熔解曲线分析显示扩增产物为特异性单峰,其Tm值为（84.51±0.01）℃;5个不同浓度对照品组内试验变异系数为0.58%~2.79%,组间试验变异系数为2.61%~4.41%。结论该方法具有快速、灵敏、特异、高通量及重复性强等优点,为β-actin基因作为内参基因进行人参功能基因表达的定量分析提供了方法学基础。

Objective To construct a real-time fluorescence quantitative RT-PCR method for the β-actin gene of Panax ginseng. Methods According to the β-actin gene of other higher plants available in Genbank, A pair of primers weredesigned and the amplified fragment of β-actin gene was linked with pMD20-T vector to construct recombinant plasmids. Then the positive plasmids were diluted and the standard curve was established. The sensitivity, specificity, and repeatability were detected. Results The results showed that the lowest copy number for detection of β-actin gene with this method was 43.0 copies/μL, and there was a good linear relationship in a wide range from 43 to 4.3 × 107 copies/μL（R2 = 0.995 3）. The melting curve showed a single peak with the temperature of（84.51 ± 0.01） ℃. The coefficient of variation（CV） of five different concentration of positive plasmids was 0.58% to 2.79% and 2.61% to 4.41% in intra-assay and inter-assay, respectively. Conclusion The method established in this paper has the advantage of rapidity, sensitivity, specificity, high throughput, and good repeatability, which provides a methodological basis for the quantitative analysis on the functional genes of P. ginseng when β-actin gene is taken as a reference gene.