【摘要】 [目的]建立稳定可靠的、适合检测红豆杉（Taxus L.）TbAP2基因表达量的荧光定量PCR实验体系。对于检测该物种中基因的组织特异性表达具有重要意义。[方法]以曼地亚红豆杉细胞为试材,提取总RNA并反转录为cDNA,根据TbAP2基因序列设计多对引物,合成内参基因TBC41的引物,采用正交试验L₉（3⁴）方法分别筛选以上2个基因5μL和10μL小反应体系及20μL常用体系中的最佳组合,并通过cDNA模板用量和引物用量等方面进行优化,以确保基因扩增效率在90%～105%之间。[结果]本研究建立了TBC41和TbAP2基因在5、10、20μL体系下的荧光定量最佳PCR反应体系,在优化后的5μL体系下,加入Mix（2×） Universal 2.5μL,cDNA模板1.0μL,正反引物共1.5μL,内参基因TBC41和目的基因TbAP2的扩增效率均为94%;在优化后的10μL下,加入Mix（2×） Universal 5μL,cDNA模板1.2μL,正反引物共1.3μL,TBC41和TbAP2的扩增效率分别为95%和94%。在优化后的20μL下,加入Mix（2×） Universal 10μL,cDNA模板0.5μL,正反引物共1.5μL,TBC41和TbAP2的扩增效率分别为93%和99%,以上各扩增体系回归系数R²均大于0.980。[结论]在以上3种反应体系下,内参基因和目的基因均具有接近100%的扩增效率,表明本研究成功建立了适合检测红豆杉TbAP2基因表达量的荧光定量PCR实验体系,并为红豆杉其它基因的表达研究提供参考。更多还原

【Abstract】 [Objective] To establish a stable and suitable real-time fluorescent quantitative PCR（qRT-PCR） experiment system of TbAP2 in Taxus L. [Method]Total RNA was extracted from the cell line of Taxus×media and used to reverse transcript cDNA. According to the sequence of TbAP2 gene obtained previously, 7 primer pairs were designed and synthesized with the TBC41 gene as the housekeeping gene. The orthogonal test L₉（3⁴） method were used to choose the stable and suitable qRT-PCR experiment system with the cDNA as template. The volume of qRT-PCR reaction included 5, 10 and 20 μL. The amplification efficiency would be assured between 90%-105% by adjusting the dosage of cDNA template and primer pairs, respectively. [Result]This study established the optimal qRT-PCR reaction system of TBC41 TbAP2 and gene in 5 μL, 10 μL and 20 μL volume. In the optimized 5 μL system, the amplification efficiency of both TBC41 and TbAP2 were 94%. In the optimized 10 μL system, the amplification efficiency of TBC41 and TbAP2 were 95% and 94%, respectively. In the optimized 20 μL system, the amplification efficiency of TBC41 and TbAP2 were 93% and 99%, respectively. The regression coefficient R² in all the three amplification system were greater than 0.980. [Conclusion]All the reaction systems mentioned above show that the amplification efficiency of TBC41 and TbAP2 close to 100%, indicating that these detection program are suitable to investigate the TbAP2 gene expression by qRT-PCR method.更多还原