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L_9(3~4)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R^2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。
[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 L 9 (3^4) 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 R^2 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.
作者
张恺恺
吕星
杨立莹
陈段芬
邱德有
杨艳芳
ZHANG Kai-kai;LV Xing;YANG Li-ying;CHEN Duan-fen;QIU De-you;YANG Yan-fang(Research Institute of Forestry, Chinese Academy of Forestry, State Key Laboratory of Tree Genetics and Breeding, Key Laboratory ofTree Breeding and Cultivation,National Forestry and Grassland Administration, Beijing 100091, China;College of Horticulture,Hebei Agricultural University, Baoding 071001, Hebei, China)
出处
《林业科学研究》
CSCD
北大核心
2019年第1期39-46,共8页
Forest Research
基金
国家自然科学基金项目(31570675
31670676)
中国林业科学研究院基本科研业务费专项资金(CAFYBB2014QB001)
