文献类型：期刊文献

中文题名：红豆杉TbAP2基因荧光定量PCR体系的建立及优化

英文题名：Optimization of Fluorescent Quantitative Real-time PCR System of Taxus chinensis var. mairei

作者：张恺恺[1,2] 吕星[2] 杨立莹[2] 陈段芬[2] 邱德有[1] 杨艳芳[1]

第一作者：张恺恺

机构：[1]中国林业科学研究院林业研究所林木遗传育种国家重点实验室国家林业和草原局林木培育重点实验室;[2]河北农业大学园艺学院

年份：2019

卷号：32

期号：1

起止页码：39-46

中文期刊名：林业科学研究

外文期刊名：Forest Research

收录：CSTPCD;;Scopus;北大核心:【北大核心2017】；CSCD:【CSCD2019_2020】；

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)

语种：中文

中文关键词：红豆杉;AP2;正交试验;实时荧光定量PCR

外文关键词：Taxus L.;AP2;orthogonal test;qRT-PCR

分类号：S718.46

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.