红豆杉TbAP2基因荧光定量PCR体系的建立及优化
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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.
[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.
引文
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[7] Liao W F, Zhao S Y, Zhang M, et al. Transcriptome assembly and systematic identification of novel cytochrome P450s in Taxus chinensis[J]. Front Plant Sci, 2017, 8: 1468.
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[9] Ramirez-Estrada K, Osuna L, Moyano E, et al. Changes in gene transcription and taxane production in elicited cell cultures of Taxus × media and Taxus globosa[J]. Phytochemistry, 2015, 117:174-184.
[10] Nolan T, Hands R E, Bustin S A. Quantification of mRNA using real-time PCR[J]. Nat Protoc, 2006, 1(3): 1559-1582.
[11] Udvardi M K, Czechowski T, Scheible W R. Eleven golden rules of quantitative RT-PCR[J]. Plant Cell, 2008, 20(7): 1736-1737.
[12] Baker M. qPCR: quicker and easier but don’t be sloppy [J]. Nat Methods, 2011, 8(3): 207-212.
[13] 王效维,刘海霞,杨颖丽,等.低表达甘蓝花药发育相关基因小体积荧光定量反应体系的简历和可靠性分析[J]. 西北农业学报,2011, 20(4): 111-115.
[14] 李婷,韩莹琰,范双喜,等.叶用莴苣荧光定量PCR体系的优化[J]. 中国农学通报,2013, 29(13): 106-113.
[2] 刘正霞,徐阳,徐进梅,等.不同引物及数据分析方法对定量PCR结果的影响[J]. 南京医科大学学报:自然科学版, 2009, 29(8): 1112-1117.
[3] Croteau R, Ketchum R E, Long R M, et al. Taxol biosynthesis and molecular genetics [J]. Phytochem Rev, 2006, 5(1): 75-97.
[4] 杨艳芳, 刘洪伟, 邱德有.欧洲红豆杉AP2/ERF转录因子基因序列分析[J]. 生物资源,2014, 36(4):54-59.
[5] 李艳艳,杨艳芳,王俊青,等.红豆杉TcLBDs基因的克隆与表达分析[J]. 林业科学,2015, 51(10): 126-133.
[6] Nasiri J, Naghavi M R, Alizadeh H, et al. Seasonal-based temporal changes fluctuate expression patterns of TXS, DBAT, BAPT and DBTNBT genes alongside production of associated taxanes in Taxus baccata[J]. Plant Cell Rep, 2016, 35(5):1103-1119.
[7] Liao W F, Zhao S Y, Zhang M, et al. Transcriptome assembly and systematic identification of novel cytochrome P450s in Taxus chinensis[J]. Front Plant Sci, 2017, 8: 1468.
[8] Zhang M, Chen Y, Nie L, et al. Molecular, structural, and phylogenetic analyses of Taxus chinensis JAZs[J]. Gene, 2017, 620: 66-74.
[9] Ramirez-Estrada K, Osuna L, Moyano E, et al. Changes in gene transcription and taxane production in elicited cell cultures of Taxus × media and Taxus globosa[J]. Phytochemistry, 2015, 117:174-184.
[10] Nolan T, Hands R E, Bustin S A. Quantification of mRNA using real-time PCR[J]. Nat Protoc, 2006, 1(3): 1559-1582.
[11] Udvardi M K, Czechowski T, Scheible W R. Eleven golden rules of quantitative RT-PCR[J]. Plant Cell, 2008, 20(7): 1736-1737.
[12] Baker M. qPCR: quicker and easier but don’t be sloppy [J]. Nat Methods, 2011, 8(3): 207-212.
[13] 王效维,刘海霞,杨颖丽,等.低表达甘蓝花药发育相关基因小体积荧光定量反应体系的简历和可靠性分析[J]. 西北农业学报,2011, 20(4): 111-115.
[14] 李婷,韩莹琰,范双喜,等.叶用莴苣荧光定量PCR体系的优化[J]. 中国农学通报,2013, 29(13): 106-113.