野生烟草甲羟戊酸激酶NsyMK基因克隆与序列分析

Cloning and Analysis of a Mevalonate Kinase (NsyMK) Gene in Nicotiana sylvestris

甲羟戊酸激酶是植物中控制整个甲羟戊酸途径重要的限速酶,其代谢产物萜类物质是烟草芳香物质的主要来源。本研究采用同源克隆,利用RACE方法获得二倍体林烟草(Nicotiana sylvestris)甲羟戊酸激酶(NsyMK)的开放阅读框序列,对NsyMK序列进行生物信息学分析,预测蛋白理化性质、二级结构、三维结构并推测其结构与功能。结果表明,克隆获得的林烟草NsyMK的开放阅读框全长为1158bp,编码385个氨基酸,预测分子量41.4kDa、等电点为6.05,GenBank登录号KC871597。蛋白质结构分析显示NsyMK存在典型GHMP激酶和甲羟戊酸激酶保守结构域。生物信息学预测NsyMK蛋白不含跨膜区与信号肽。本研究成功克隆并分析了一个未报道的林烟草NsyMK的全长序列,为进一步阐明栽培烟草中萜类代谢途径奠定基础。

Mevalonate kinase （MVK）, a key enzyme in plant mevalonate pathway, participate in biosynthesis of terpenoids ,which is the main aromatic substance in tobacco. We obtained the diploid Nicotiana sylvestris mevalonate kinase （NsyMK）ORF through homology-based cloning combined with RACE, the physical and chemical properties, secondary structure, three-dimensional structure and structure and function of the NsyMK protein were predicted based on the sequence. The sequence contains a 1158 bp open reading frame and encodes a predicted protein of 385 amino acids, with a predicted molecular weight of 41.4kDa and pI of 6.05. The GenBank accession number for this gene is KC871597. No transmembrane region and signal peptide were present in KC871597. The conserved domains of typical GHMP Kinase and mevalonate kinase were found in NsyMK. In this study, we cloned and analyzed the NsyMK gene from Nicotiana sylvestris. Results from this study have set a foundation for future exploration of terpenoid biosynthesis in Nicotiana tabacum.