樟树4-二磷酸胞苷-2-C-甲基-D-赤藓醇激酶基因的克隆及表达分析

发布时间：2019-06-03 02:09

【摘要】：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。
[Abstract]:Cytidine 4-diphosphate 2 / C-methyl-D-monositol kinase (4-diphosphocytidyl-2-C-methyl-D-erythritol kinase,CMK) is a biosynthesis pathway of plant terpenoids, methyl gibberellin-4-phosphate pathway (methylerythritol-4-phosphate pathway,). The fourth key enzyme on DXP/MEP. Based on the data of camphor transcriptional group, the 4-cytidine diphosphate 2 / C-methyl-D-erythritol kinase gene was cloned from Cinnamomum camphora by RT-PCR (reverse transcription-PCR). The sequence of Cc CMK1 (Gene Bank accession number Ku376098 was analyzed by bioinformatics analysis. The results showed that the open reading frame (ORF) of the gene was 1 212 bp, encoding 403amino acids, and its relative molecular weight was 44.46k Da,. The isoelectric point (theoretical p I) is 4.99. Transmembrane structure analysis showed that CMK protein did not have transmembrane structure. Signal peptide analysis showed that it was a non-secretory protein and there was no signal peptide. Subcellular localization showed that it was located in chloroplasts. The expression of Cc CMK1 gene in Cinnamomum camphora, Cinnamomum camphora and Cinnamomum camphora was detected by fluorescence real-time PCR. The results showed that the expression of Cc CMK1 gene was the highest in Cinnamomum camphora and the lowest in Cinnamomum camphora. It provides a research basis for the mining of key enzyme gene elements in the biosynthesis pathway of terpenoids in camphor trees.
【作者单位】：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安徽中医药大学
【基金】：国家自然科学基金项目(30901963)
【分类号】：S567.19;Q943.2

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