|
|
|
| Construction and Optimization of TcERF Gene qRT-PCR System in Taxus sp. |
| JIANG Lu-Yuan1,2, WANG Xu3, ZHANG Kai-Kai1,2, CHEN Duan-Fen1, QIU De-You2, YANG Yan-Fang2,* |
1 College of Horticulture, Hebei Agricultural University, Baoding 071001, China;
2 State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of State Forestry Administration, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China;
3 College of Horticulture Science and Technology, Hebei Normal University of Science & Technology, Qinhuangdao 066004, China |
|
|
|
|
Abstract Taxus sp. can produce a natural diterpenoid anticancer compound-taxol, which has good curative effect on cancers of breast, lung, ovarian, endometrial, and cervical carcinoma. However, both the growth of yew trees and the content of taxol are very low. The conflict between the protection of yew trees and utilization of taxol is serious. Previous studies reported that AP2/ERF (APETALA2/ethylene-responsive factor) transcription factors involved in regulating the biosynthesis of many secondary metabolites, such as tanshiones. AP2/ERF transcription factors also play an important role in the regulation of taxol biosynthesis in Taxus. To better study the function of TcERF gene that had been isolated by our group previously, this study established 3 stable and suitable real-time fluorescent quantitative PCR (qRT-PCR) experiment systems. With the sequence of TcERF gene, three primer pairs were designed and synthesized with the TBC41 (3,5-epimerase-4-reductase) gene as the housekeeping gene. According to the relationship between the upstream and downstream of primers, three primer combinations were obtained. Then the best primer combination F1R2 was screened by PCR and qRT-PCR. The orthogonal test L9(34) method was used to design the test scheme. Under the 3 test schemes with the highest amplification efficiency (A1B3C3, A2B2C3 and A3B1C3), the amplification efficiency of the housekeeping gene were 70%~80%, and the amplification efficiency of F1R2 was 102% and 91% in A1B3C3 and A2B2C3, respectively, but only 72% in A3B1C3. The amplification efficiency of housekeeping gene and target gene did not reach 90%~110% at the same time. Therefore, the qRT-PCR experiment system had to be further optimized. Finally, the best combination of 5 µL small reaction system, 10 and 20 µL common system were screened out. The results indicated that in the optimized 5 µL system which include 2.5 µL Master Mix, 1.7 µL cDNA template, and 0.8 µL primer, the amplification efficiency of TBC41 and TcERF were 94% and 90%; in the optimized 10 µL system which include 5 µL Master Mix, 1.2 µL cDNA template, 1.3 µL primer and 2.5 µL ddH2O, the amplification efficiency of TBC41 and TcERF were 95% and 94%, respectively; in the optimized 20 µL system which include 10 µL Master Mix, 0.5 µL cDNA template, 1.5 µL primer and 8 µL ddH2O, the amplification efficiency of TBC41 and TcERF was 93% and 102%, respectively. All the mentioned amplification system regression coefficient R2 were greater than 0.980. The expression level of TcERF in Tm3 cells that treated with methyl jasmonate for 24 h was detected by 10 µL reaction systems of before and after optimization. A significant difference was existed between the values of TcERF expression obtained from the optimized and unoptimized qRT-PCR composition, which indicated that unreliable results may be obtained by using the unoptimized qRT-PCR system. All the above described 3 reaction systems showed that the amplification efficiency of TBC41 and TcERF closed to 100%, which indicated that these detection programs were suitable to investigate the TcERF gene expression by qRT-PCR method. It provides experimental conditions and technical support for the subsequent transcriptional expression detection and functional research of TcERF gene.
|
|
Received: 06 November 2019
|
|
|
|
Corresponding Authors:
*, echoyyf@caf.ac.cn
|
|
|
|
[1] 酆孟洁, 邱晨, 刘雯雯. 2012. 实时荧光定量PCR的三种数据分析方法比较[J]. 热带病与寄生虫学, 10(3): 128-131.
(Feng M J, Qiu C, Liu W W.2012. Comparison of three data analysis methods for real-time fluorescence quantitative PCR[J]. Tropical Diseases and Parasitology, 10(3): 128-131.)
[2] 欧阳松应, 杨冬, 欧阳红生, 等. 2004. 实时荧光定量PCR技术及其应用[J]. 生命的化学, 24(1): 74-76.
(Ouyang S Y, Yang D, Ouyang H S, et al.2004. Real-time fluorescence quantitative PCR and its application[J]. Chemistry of Life, 24(1): 74-76.)
[3] 杨艳芳, 刘洪伟, 邱德有. 2014. 欧洲红豆杉AP2/ERF转录因子基因序列分析[J]. 氨基酸和生物资源, 36(4): 54-59.
(Yang Y F, Liu H W, Qiu D Y.2014. Sequence analysis of AP2/ERF transcription factor gene in Taxus chinensis[J]. Amino Acids and Biotic Resources, 36(4): 54-59.)
[4] 张恺恺, 吕星, 杨立莹, 等. 2019. 红豆杉TbAP2基因荧光定量PCR体系的建立及优化[J]. 林业科学研究, (1): 39-46. (Zhang K K, Lv X, Yang L Y, et al. 2019. Establishment and optimization of fluorescence quantitative PCR system for Taxus chinensis TbAP2 gene[J]. Forestry Scientific Research, (1): 39-46.)
[5] 张计育, 王庆菊, 郭忠仁. 2012. 植物AP2/ERF类转录因子研究进展[J]. 遗传, 34(7): 835-847.
(Zhang J Y, Wang Q J, Guo Z R.2012. Advances in the study of AP2/ERF transcription factors in plants[J]. Heredity, 34(7): 835-847.)
[6] 赵锐, 赵玮玮. 2009.抗癌植物药紫杉醇研究进展与动态[J]. 中草药, 40(7): 1172-附2 (Zhao R, Zhao W W. 2009. Advances and developments in the study of paclitaxel in anti-cancer plants[J]. Chinese Herbal Medicine, 40(7):1172-attach 2.)
[7] Dai Y L, Qin Q L, Dai D L, et al.2009. Isolation and characterization of a novel cDNA encoding methyl jasmonate-responsive transcription factor TcAP2 from Taxus cuspidata[J]. Biotechnology Letters, 31(11): 1801-1809.
[8] Jofuku K D, den Boer B G, van Montagu M, et al.1994. Control of Arabidopsis flower and seed development by the homeotic gene APETALA2[J]. The Plant Cell, 6(9): 1211-1225.
[9] Liao W F, Zhao S Y, Zhang M, et al.2017. Transcriptome assembly and systematic identification of novel cytochrome P450s in Taxus chinensis[J]. Frontiers in Plant Science, 8: 1468.
[10] Nakano T, Suzuki K, Fujimura T, et al.2006. Genome-wide analysis of the ERF gene family in Arabidopsis and rice[J]. Plant Physiology, 140(2): 411-432.
[11] Nasiri J, Naghavi M R, Alizadeh H, et al.2016. 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 Reports, 35(5): 1103-1119.
[12] Nolan T, Hands R E, Bustin S A.2006. Quantification of mRNA using real-time PCR[J]. Nature Protocols, 1(3): 1559-1582.
[13] Ramirez-Estrada K, Osuna L, Moyano E, et al.2015. Changes in gene transcription and taxane production in elicited cell cultures of Taxus×media and Taxus globosa[J]. Phytochemistry, 117: 174-184.
[14] Sun G L, Yang Y F, Xie F L, et al.2013. Deep sequencing reveals transcriptome re-programming of Taxus×media cells to the elicitation with methyl jasmonate[J]. PLOS ONE, 8(4): e62865.
[15] Udvardi M K, Czechowski T, Scheible W R.2008. Eleven golden rules of quantitative RT-PCR[J]. The Plant Cell, 20(7): 1736-1737.
[16] Yang Y F, Zhang K K, Yang L Y, et al.2018. Identification and characterization of MYC transcription factors in Taxus sp.[J]. Gene, 675: 1-8.
[17] Zhang M, Li S T, Nie L, et al.2015. Two jasmonate-responsive factors, TcERF12 and TcERF15, respectively act as repressor and activator of tasy gene of taxol biosynthesis in Taxus chinensis[J]. Plant Molecular Biology, 89(4-5): 463-473. |
|
|
|
