茶树α-tubulin基因实时荧光定量RT-PCR方法的建立

摘要

Objective: To construct a real-time fluorescence quantitative RT-PCR for the a-tubulin gene of Camellia sinensis. Methods: According to the a-tubulin gene sequence of C.sinensis available in GenBank, a pair of primers was designed and the amplified fragment of a-tubulin gene were linked with pTG19-T vector to construct recombined plasmid. Then the positive plasmid was diluted and the standard curve was established using SYBR Green Ⅰ, and the melting curve was analyzed. Results: The linear range of standard curve Ct was from 14.56 to 27.09, and the correlation coefficient was 0.991. The melting curve showed a single peak with the temperature of 81±0.3℃. Conclusion: A real-time RT-PCR method for a-tubulin gene of C.sinensis was constructed successfully and it provided the basis for use of the a-tubulin gene of C.sinensis as a reference gene in quantitative analysis of differences in functional gene expression.%目的:建立茶树α-tubulin基因实时荧光定量RT-PCR方法.方法:根据GenBank中茶树α-tubulin基因保守区域设计一对特异性引物,将PCR扩增得到的α-tubulin基因克隆到pTG 19-T载体上,构建的重组质粒标准品经1/10梯度稀释后,用SYBR Green Ⅰ染料法绘制标准曲线,并进行融解曲线分析.结果:标准曲线Ct值检测范围为14.56～27.09,相关系数为0.991,溶解曲线分析显示产物为特异的单峰,其Tm值为81±0.3℃.结论:建立了茶树α-tu-bulin基因实时荧光定量RT-PCR法,为以α-tubulin作为茶树内参基因进行功能基因表达差异研究奠定了基础.