茶树α-tubulin基因实时荧光定量RT-PCR方法的建立

Establishment of Real-Time Fluorescence Quantitative RT-PCR Method for α-Tubulin Gene of Camellia sinensis

目的：建立茶树仅-tubulin基因实时荧光定量RT-PCR方法。方法：根据GenBank中茶树d-tubulin基因保守区域设计-对特异性引物，将PCR扩增得到的a-tubulin基因克隆到pTGl9-T载体上，构建的重组质粒标准品经1／10梯度稀释后，用SYBRGreenI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±0．3C。结论：建立了茶树d-tu．bulin基因实时荧光定量RT-PCR法，为以oL-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。

Objective： To construct a realtime fluorescence quantitative RTPCR for the xtubulin gene of Ca mellia sinensis. Methods： According to the cxtubulin gene sequence of C.sinensis available in GenBank, a pair of primers was designed and the amplified fragment of cttubulin gene were linked with pTG19T 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 0.99l. The melting curve showed a single peak with the temperature of 81±0.3C. Conclusion： A realtime RTPCR method for cttubulin gene of C.sinensis was constructed successfully and it provided the basis for use of the ottubulin gene of C.sinensis as a reference gene in quantitative analysis of differences in functional gene expression.