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.
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