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茉莉酸甲基转移酶(jasmonic acid carboxyl methyltransferase,JMT)是茉莉酸生物合成途径中的关键酶,能够催化茉莉酸甲基化形成茉莉酸甲酯。本研究将丹参茉莉酸甲基转移酶基因(SmJMT1)c DNA构建到原核表达载体pGEX-4T-1上,转化大肠杆菌BL21(DE3)并诱导表达。SDS-PAGE凝胶电泳结果表明该蛋白的大小约66 kDa,与预测的蛋白分子质量相同;同时对影响蛋白表达的4个因素:诱导时间、诱导温度、异丙基硫代半乳糖苷(IPTG)浓度及诱导前菌液的浓度进行优化,结果表明当诱导前菌液的A600在0.8左右时,加入0.4 mmol·L~(-1)IPTG,20℃诱导培养8 h后SmJMT1蛋白的表达量较高。蛋白质印迹检测显示,anti-GST抗体可以特异性地识别SmJMT1蛋白,证实丹参SmJMT1蛋白在大肠杆菌中表达成功;Q-TOF检测数据检索分析表明,SmJMT1蛋白属于甲基转移酶亚家族。丹参中茉莉酸甲基转移酶SmJMT1的成功表达和纯化对研究茉莉酸生物合成途径及茉莉酸对药用植物次生代谢调控奠定了一定基础。
Jasmonic acid methyltransferase (JMT) is a key enzyme in the jasmonic acid biosynthesis pathway, which can catalyze the methylation of jasmonic acid to form methyl jasmonate. In this study, the SmJMT1 c DNA of Salvia miltiorrhiza was cloned into prokaryotic expression vector pGEX-4T-1 and transformed into E.coli BL21 (DE3) for expression. SDS-PAGE gel electrophoresis results showed that the size of the protein was about 66 kDa, which was the same as the predicted molecular weight of the protein. At the same time, four factors affecting the protein expression: induction time, induction temperature, isopropylthiogalactoside (IPTG ) Concentration and the concentration of the pre-induction bacteria solution were optimized. The results showed that when the A600 value of the pre-induction strain was about 0.8, 0.4 mmol·L -1 IPTG was added and the expression of SmJMT1 protein was induced at 20 ℃ for 8 h Higher. Western blotting showed that the anti-GST antibody could specifically recognize SmJMT1 protein and confirmed the successful expression of SmJMT1 protein in E. coli. The Q-TOF detection data showed that the SmJMT1 protein belongs to the methyltransferase subfamily. The successful expression and purification of SmJMT1 from Salvia miltiorrhiza Bunge in jasmonic acid methyltransferase laid a solid foundation for studying the biosynthesis pathway of jasmonic acid and the regulation of secondary metabolism of jasmonic acid on medicinal plants.