【摘要】 红豆杉(Taxus sp.)能够产生天然抗癌药物紫杉醇(taxol),但含量极低,开发与利用之间存在严重矛盾。乙烯响应因子(APETALA2/ethylene-responsive factor, AP2/ERF)对红豆杉中紫杉醇的生物合成起到重要调控作用。研究组前期获得了1个红豆杉ERF转录因子基因(命名为TcERF),为了更好地研究该基因的功能,本研究以曼地亚红豆杉(Taxus×media)愈伤组织为实验材料,采用正交实验L9(34)方法,根据TcERF基因序列设计、组合出3对引物,从qRT-PCR反应体系、cDNA模板用量和引物用量等方面进行优化,分别筛选出该基因在5μL小反应体系及10、20μL常用体系下的最佳组合。结果表明,在本研究优化后的5μL体系下,TcERF和看家基因:3,5-表异构酶-4-还原酶(3,5-epimerase-4-reductase, TBC41)基因的扩增效率分别为90%和94%;在优化后的10μL下,TcERF和TBC41的扩增效率分别为94%和95%;在优化后的20μL下,TcERF和TBC41的扩增效率分别为102%和93%,以上各扩增体系回归系数R2均大于0.980。以茉莉酸甲酯(methyl jasmonate, MeJA)处理24 h的红豆杉愈伤组织为材料,利用优化前和优化后的10μL体系检测并比较TcERF基因的表达,发现两个体系下该基因表达水平存在显著性差异,充分表明建立并优化qRT-PCR实验体系的必要性和重要性。本研究建立的3个qRT-PCR反应体系均可以保证红豆杉TcERF基因和看家基因具有接近100%的扩增效率,为后续该基因转录水平的表达检测以及功能研究提供了实验条件和技术支撑。更多还原

【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 F1 R2 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（A1 B3 C3, A2 B2 C3 and A3 B1 C3）, the amplification efficiency of the housekeeping gene were 70%～80%, and the amplification efficiency of F1 R2 was 102% and 91% in A1 B3 C3 and A2 B2 C3, respectively, but only 72% in A3 B1 C3. 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 c DNA 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 ddH2 O, 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 ddH2 O, 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 to100%, 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.更多还原