文献类型：期刊文献

中文题名：红豆杉TcERF基因qRT-PCR体系构建与优化

英文题名：Construction and Optimization of TcERF Gene qRT-PCR System in Taxus sp.

作者：蒋路园[1,2] 王旭[3] 张恺恺[1,2] 陈段芬[1] 邱德有[2] 杨艳芳[2]

第一作者：蒋路园

机构：[1]河北农业大学园艺学院,保定071001;[2]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/国家林业局林木培育重点实验室,北京100091;[3]河北科技师范学院园艺科技学院,秦皇岛066004

年份：2020

卷号：28

期号：6

起止页码：1096-1104

中文期刊名：农业生物技术学报

外文期刊名：Journal of Agricultural Biotechnology

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

基金：国家自然科学基金(31570675);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;乙烯响应因子(ERF);正交试验;qRT-PCR

外文关键词：Taxus sp.;APETALA2/ethylene-responsive factor(ERF);Orthogonal test;qRT-PCR

分类号：S718.46

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。
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 5μL small reaction system,10 and 20μL common system were screened out.The results indicated that in the optimized 5μL system which include 2.5μL Master Mix,1.7μL cDNA template,and 0.8μL primer,the amplification efficiency of TBC41 and TcERF were 94%and 90%;in the optimized 10μL system which include 5μL Master Mix,1.2μL cDNA template,1.3μL primer and 2.5μL ddH2O,the amplification efficiency of TBC41 and TcERF were 95%and 94%,respectively;in the optimized 20μL system which include 10μL Master Mix,0.5μL cDNA template,1.5μL primer and 8μL 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 10μL 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.