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4-二磷酸胞苷-2-C-甲基-D-赤藓醇激酶(4-diphosphocytidyl-2-C-methyl-D-erythritol kinase,CMK)是植物萜类化合物生物合成途径甲基赤藓醇-4-磷酸途径(methylerythritol-4-phosphate pathway,DXP/MEP)上的第4个关键酶。该研究根据樟树转录组数据,利用RT-PCR(reverse transcription-PCR)方法从药用植物樟树Cinnamomum camphora中克隆得到4-二磷酸胞苷-2-C-甲基-D-赤藓醇激酶基因,命名为Cc CMK1(Gene Bank登录号Ku376098),对其序列进行生物信息学分析,结果表明该基因开放阅读框(ORF)为1 212 bp,编码403个氨基酸,推测其相对分子质量为44.46 k Da,等电点(theoretical p I)为4.99。跨膜结构分析表明CMK蛋白不存在跨膜结构。信号肽分析表明其为非分泌蛋白,不存在信号肽。亚细胞定位表明其定位于叶绿体中。利用荧光实时PCR检测了龙脑樟、油樟、芳樟、脑樟和异樟5种化学型樟树中Cc CMK1基因的表达量,结果表明Cc CMK1基因在油樟中的表达量最高,龙脑樟中表达量最低,为樟树萜类化合物生物合成途径关键酶基因元件挖掘提供研究基础。
4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK) is a plant terpenoid biosynthesis pathway The 4th key enzyme on the alcohol-4-phosphate pathway (DXP / MEP). In this study, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase gene was cloned from the camphor Cinnamomum camphora by RT-PCR , Named as Cc CMK1 (Gene Bank accession number Ku376098). The bioinformatics analysis of the sequence showed that the open reading frame (ORF) of this gene was 1 212 bp, encoding 403 amino acids. The deduced molecular weight of this gene was 44.46 k Da, the theoretical pI is 4.99. Transmembrane structure analysis shows that there is no transmembrane structure in CMK protein. Signal peptide analysis revealed that it was a non-secreted protein, absent the signal peptide. Subcellular localization indicates that it is located in the chloroplast. Fluorescence real-time PCR was used to detect the expression of Cc CMK1 gene in Campylobalsmilium, Campylobacter camphor, Campanulaceae camphor, Cinnamomum camphora and C. camphora. The results showed that the expression level of Cc CMK1 gene was the highest in camphor camphor, Cinnamomum camphora was the lowest, which provided the research foundation for the excavation of the key enzyme gene elements of the camphor terpenoid biosynthesis pathway.