杜仲2-甲基-D-赤藓糖醇-2,4-环焦磷酸合酶基因全长cDNA克隆与序列分析

Cloning and sequence of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase gene cDNA from Eucommia ulmoides

2-甲基-D-赤藓糖醇-2,4-环焦磷酸合酶（MDS）基因曾被认为是调控植物2-甲基-D-赤藓糖醇4-磷酸（MEP）途径的一个关键节点.为解析杜仲MDS基因序列信息和预测基因功能,以叶片cDNA为模板,采用反转录-聚合酶链式反应（RT-PCR）及cDNA末端快速扩增（RACE）技术分离出杜仲MDS基因的cDNA克隆,并通过一系列生物信息学方法进行序列分析.结果表明：EuMDS基因cDNA全长976 bp,5＇端非编码区长119 bp,3＇端非编码区长146bp,编码236个氨基酸.推导EuMDS氨基酸序列中包含转运肽序列（A1～A56）以及多个植物MDS蛋白保守的功能位点（A84,A87,A89,A121,A213,A217,A221,A223,A228）.推导EuMDS蛋白二级结构中α-螺旋占40.3％,β-折叠占13.6％,螺环结构占46.2％.推导EuMDS蛋白三级结构由3个亚单位组成,并相互围绕形成1个分子内腔.系统进化分析表明EuMDS蛋白与啤酒花MDS蛋白亲缘关系最为接近.预测所克隆的EuMDS基因在杜仲萜类生物合成中发挥重要功能.

2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase （MDS） gene had been regarded as a key regulating plot in plant 2-C-methyl-D-erythritol-4-phosphate （MEP） pathway.To dissect the MDS gene sequence information and predict the gene function of Eucommia ulmoides,the homologous MDS gene cDNA was isolated from leaves by the reverse transcription polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends （RACE） techniques,and the sequence analysis was also conducted by series of bioinformatic methods.Results showed that and the full-length cDNA of EuMDS was 976 bp,including a 5＇non-coding region of 119 bp and a 3＇non-coding region of 146 bp with 236 amino acids encoded.The transit peptide sequence （A1-A56） and multiple conserved functional sites （A84,A87,A89,A121,A213,A217,A221,A223,and A228） of plant MDS enzyme were found in the deduced coding sequence of EuMDS.The secondary structure of the EuMDS protein was predicted with proportions of α-helix to 40.3％,β-sheet to 13.6％,and loop/coil to 46.2％.The calculated protein tertiary structure of EuMDS was exhibited as a molecular cavity formed by three subunits.Phylogenetic analysis revealed that the evolutionary relationship of EuMDS protein was closest to the Humulus lupulus MDS protein.It was suggested that the cloned EuMDS exert an important function in Eucommia ulmoides terpene biosysthesis.