药学学报, 2016, 51(10): 1643-1650
引用本文:
姚娜, 郑汉, 荆礼, 马利刚, 申业, 陈敏. 丹参茉莉酸甲基转移酶蛋白表达和纯化的研究[J]. 药学学报, 2016, 51(10): 1643-1650.
YAO Na, ZHENG Han, JING Li, MA Li-gang, SHEN Ye, CHEN Min. Expression and purification of a jasmonic acid carboxyl methyltransferase from Salvia miltiorrhiza in E.coli[J]. Acta Pharmaceutica Sinica, 2016, 51(10): 1643-1650.

丹参茉莉酸甲基转移酶蛋白表达和纯化的研究
姚娜1, 郑汉1,2, 荆礼1, 马利刚3, 申业1, 陈敏1
1. 中国中医科学院中药资源中心, 道地药材国家重点实验室培育基地, 北京 100700;
2. 安徽中医药大学药学院, 安徽 合肥 230038;
3. 河南中医学院, 河南 郑州 450016
摘要:
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)cDNA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-1IPTG,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。
关键词:    丹参      茉莉酸甲基转移酶      蛋白表达纯化      蛋白质印迹      Q-TOF     
Expression and purification of a jasmonic acid carboxyl methyltransferase from Salvia miltiorrhiza in E.coli
YAO Na1, ZHENG Han1,2, JING Li1, MA Li-gang3, SHEN Ye1, CHEN Min1
1. State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China;
2. College of Pharmacy, Anhui University of Traditional Chinese Medicine, Hefei 230038, China;
3. Henan University of Traditional Chinese Medicine, Zhengzhou 450016, China
Abstract:
Jasmonic acid carboxyl methyltransferase (JMT), a key enzyme for jasmonate (JA) biosynthesis, catalyzes the methylation of JA to form MeJA. To characterize the function of JMT, a plasmid pGEX-4T-SmJMT1 harboring JMT1 (SmJMT1) gene from Salvia miltiorrhiza was successfully transformed into E.coli BL21 (DE3) for protein expression. The recombination SmJMT1 was separated using SDS-PAGE and the size of expressed SmJMT1 protein was consistent with the prediction. The bacterial growth conditions were determined for optimal expression, which include growth temperature, incubation time, IPTG concentrations and culture density. The optimal growth conditions for SmJMT1 were that the bacterial cultures were grown to an A600 of 0.8, and induced with IPTG at a final concentration of 0.4 mmol·L-1, and then incubated for 8 h at 20℃. The expression of SmJMT1 in E.coli was confirmed by Western blotting, and mass spectrometry analysis of methyltransferase family. The successful expression and purification of JMT in this study provide the basis for more study of JA biosynthetic pathway and JA-regulated secondary metabolism of medicinal plants.
Key words:    Salvia miltiorrhiza    jasmonic acid carboxyl methyltransferase    protein expression and purification    Western blotting    Q-TOF   
收稿日期: 2016-04-29
DOI: 10.16438/j.0513-4870.2016-0414
基金项目: 中央级公益性科研院所基本科研业务费专项资金资助项目(ZZ0808018);国家自然科学基金资助项目(81173490,81573533).
通讯作者: 申业, 陈敏
Email: shenye70@hotmail.com;cm315keke@163.com
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