丹参4-(5′-二磷酸胞苷)-2-C-甲基-D-赤藓醇激酶的cDNA全长克隆及其诱导表达分析  

A full length cDNA of 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase cloning and analysis of introduced gene expression in Salvia miltiorrhiza

作　　者：王学勇[1,2] 崔光红[2] 黄璐琦[2] 高伟[2] 袁媛[2] 

机构地区：[1]北京中医药大学中药学院,北京100102 [2]中国中医科学院中药研究所,北京100700

出　　处：《药学学报》2008年第12期1251-1257,共7页Acta Pharmaceutica Sinica

基　　金：国家重点基础研究发展计划资助项目(2006CB504700);国家高技术研究发展计划资助项目(2003AA2Z2040)

摘　　要：本文首次从丹参毛状根中克隆得到了丹参4-(5′-二磷酸胞苷)-2-C-甲基-D-赤藓醇激酶(简称为SmCMK)的编码基因的全长cDNA序列,GenBank注册号:EF534309。KEGG软件分析表明:SmCMK位于二萜类化合物生物合成的上游途径——非甲羟戊酸途径(DXP),是DXP途径上唯一的激酶。所克隆的SmCMK基因序列全长1 493 bp,包括完整的开放阅读框(open reading frame,ORF),推测编码396个氨基酸的多肽,分子质量为43.302 kDa,等电点(pI)值为6.78;含有71 bp的5′非转译区(5′UTR)和232 bp的3′非转译区(3′UTR)。末尾具有完整的AATAA加尾信号和PolyA结构,说明所克隆基因的序列较为完整。经序列比对分析表明,SmCMK与其他植物CMK激酶家族具有较高的同源性。实时荧光定量PCR结果显示该基因受茉莉酸甲酯(MJ)诱导后表达水平显著升高,与丹参酮类成分含量受MJ诱导后的增加趋势一致,初步证明了SmCMK基因表达量与丹参酮类成分的积累之间的关系,为进一步研究丹参酮类成分的次生代谢调控机制奠定了基础。This paper firstly introduced the acquired full length cDNA of 4-（ cytidine 5＇-diphospho）- 2-C-methyl-D-erythritol kinase from hairy roots of Salvia miltiorrhiza （Abbr： SmCMK, GenBank number： EF534309）. Results of KEGG analysis showed that SmCMK was belong to the upstream of nonemevalonate pathway,, the only one kinase of the pathway. The full-length eDNA was deduced as encoding 4-（ cytidine 5＇-diphospho）-2-C-methylerythritol kinase （designated as SmCMK）, and the sequence had a 1 493 bp including 5＇ UTR 71 bp and 3＇ UTR 232 bp, an open reading frame （ORF） encoding a protein of 396 amino acid residues. The deduced protein had isoelectric point （pI） of 6. 78 and a calculated molecular weight about 43 kDa, similar to cloned diterpene of CMK from other species of plants such as Mentha piperita and Lycopersicon esculentum reported previously. Real time PCR results indicated that elicitors of MJ stimulated the increase of mRNA expression of SmCMK. At the same time, results of high performance liquid chromatography （HPLC）, used to examine the accumulation of diterpenoid tanshinones in hairy roots, showed that the contents of diterpenoid tanshinones in hairy root of Salvia miltiorrhiza were increased dramatically after treated with methyl jasmonate （MJ）. This result showed a positive correlation between the levels of mRNA expression and tanshinones accumulation in Salvia miltiorrhiza stimulated by MJ. It proved primarily that the increased expression level of mRNA of SmCMK helps to enhance tanshinones＇ accumulation, which will be the basis for further study on the mechanism of gene regulation of secondary metabolism of tanshinones.

关 键 词：丹参 SmCMK RACE 克隆 茉莉酸甲酯 

分 类 号：R915.2[医药卫生—微生物与生化药学]