茉莉酸羧基甲基转移酶基因的克隆及其高效表达载体的构建

以拟南芥花为材料,应用RT-PCR方法克隆拟南芥茉莉酸甲酯合成途径中重要的限速酶--茉莉酸羧基甲基转移酶基因,并进行生物信息学分析,以pCAMBIA1304+为基本载体构建其植物高效表达载体pCAM-BIA1304-AtJMT克隆到的AtJMT编码区序列为1 170 bp,编码389个氨基酸的多肽.生物信息学分析该蛋白理论分子量为43.37 kD,等电点为5.18.三级结构预测表明其具有典型的羧基甲基转移酶蛋白功能域甲基供体的结合位点,蛋白质三维模型具有典型的甲基转移酶的蛋白立体结构.获得的工程菌可用于遗传转化长春花等,为萜类吲哚生物碱代谢工程新型策略奠定基础.

Abstract：

Jasmonic acid carboxyl methyltransferase (JMT) is one of the most important key enzymes involved in the biosynthetic pathway of methyl jasmonate (MeJA) in Arabidopsis thaliana. The JMT gene of A. thaliana (AtJMT) was isolated through RT-PCR with a pair of specific primers that were designed according to the sequences of the JMT reported. We characterized it by bioinformatic analysis and constructed its over-expression vector. The computational analysis of the coding sequence showed that it had 1170 bp nucleic acids, encoding 389 amino acids (aa) polypeptide with a predicted molecular weight of 43. 37kD and an isoelectric point of 5.18. Bioinformatic analysis demonstrated that AtJMT contained the typical functional domains of JMT, methyl donor binding sites. The 3-D structure of AtJMT had the typical structure of known methyltransferase protein. The engineered bacteria can be used to genetically transform Catharanthus roseus, to promote the bioaccumulation of TIAS. The cloning, characterization and construction of plant expression vector will be a fundamental work to facilitate new strategies of the metabolic engineering of TIAS.

Cloning of the Jasmonic Acid Carboxyl Methyltransferase JMT Gene and Constructing of Its Over-expression Vector