【摘要】 丁香酚合成酶(eugenol synthase, EGS)是北细辛(Asarum heterotropoides)主要活性成分甲基丁香酚体内合成途径中的关键酶之一。该研究根据北细辛转录组数据库中筛选到的序列设计特异引物,以RT-PCR法扩增获得北细辛丁香酚合成酶基因(AhEGS)的开放阅读框(ORF)序列,进行了相应的生物信息学分析。同时运用实时定量PCR法分析AhEGS在不同发育时期(幼叶期、花期、果期)和不同组织部位(叶、根茎、根)中的表达谱,并进行原核表达分析。结果显示:(1)AhEGS基因ORF序列长为951 bp,编码316个氨基酸,理论分子量为34.93 kD,等电点为6.19,AhEGS蛋白为亲水性蛋白,无跨膜结构,无信号肽序列;同源序列比对发现,北细辛AhEGS与月季RcEGS(AFQ98278.1)同源性最高。(2)实时定量PCR分析表明,AhEGS基因在北细辛幼叶期的根中表达量最高。(3)成功构建了原核表达载体pET28a-AhEGS-BL21,经pET28a-AhEGS重组子转化E.coli BL21(DE3),SDS-PAGE检测显示在大肠杆菌(Escherichia coli,E.coli)中诱导产生了35 kD左右的特异性蛋白,与理论分子量一致;最佳诱导条件为16℃、14 h, IPTG浓度为0.2 mmol/L。该研究首次克隆了北细辛的EGS基因,构建了原核表达载体,筛选出该蛋白最佳诱导条件,研究结果为北细辛甲基丁香酚代谢工程的应用奠定基础。更多还原

【Abstract】 Eugenol synthase was one of the key enzymes in the synthesis pathway of methyl-eugenol, which was the main active constituent in Asarum heterotropoides. Gene clone and functional analysis of AhEGS would lay the foundation for revealing the synthesis pathway of methyl-eugenol and its metabolism regulation mechanism. Specific primers were designed according to the sequence screened in the A. heterotropoides transcriptome database, the ORF sequence of AhEGS was amplified by RT-PCR, and the corresponding bioinformatics analysis was performed. Real-time fluorescent quantitative PCR method was used to analyze the expression of AhEGS in different developmental stages(young leaf period, flower period, fruit period) and different tissue parts(leaf, rhizome, root). Also we performed prokaryotic expression analysis. The results indicated that:(1) the ORF sequence of the AhEGS gene was 951 bp in length, encoding 316 amino acids, with a theoretical molecular weight of 34.93 kD and an isoelectric point of 6.19. It was a hydrophilic protein with no transmembrane structure and no signal peptide sequence. AhEGS had the highest homology with rose RcEGS(AFQ98278.1).(2) Real-time quantitative PCR analysis showed that the expression of AhEGS was highest in roots at young-leaf stage.(3) The prokaryotic expression vector pET28 a-AhEGS-BL21 was successfully constructed and transformated into Escherichia coli(E.coli) BL21(DE3). It was showed that about 35 kD of specific proteins was induced in E.coli by SDS-PAGE, which was consistent with the theoretical molecular weight; the best induction condition was 16 ℃, 14 h, and 0.2 mmol/L IPTG. It was the first time to clone AhEGS gene. In this study, the prokaryotic expression vector for AhEGS was constructed and also the optimal induction conditions were screened out. These results might provide scientific evidence for the application of methyl-eugenol metabolic engineering.更多还原