茅苍术AlCMK基因的克隆及原核表达分析

Cloning and prokaryotic expression analysis of AlCMK from Atractylodes lancea

4-二磷酸胞苷-2-C-甲基-D-赤藓醇激酶[4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinase,CMK]是生成萜类化合物MEP途径的关键酶之一。本研究以茅苍术转录组数据为依据,克隆获得CMK基因序列,命名为AlCMK(GenBank登录号为OM283293)。研究结果表明,AlCMK包含一个全长为1230 bp的开放阅读框(ORF),编码409个氨基酸,推测其相对分子质量为44752.53,蛋白理论等电点为6.67;跨膜结构分析表明其无跨膜结构,二级结构显示其主要由无规则卷曲(48.17%)结构组成;同源氨基酸序列分析表明,茅苍术与除虫菊、桂花、杜仲、忍冬、丹参CMK基因编码的氨基酸序列具有较高同源性;系统进化树分析表明,茅苍术AlCMK与菊科植物CMK蛋白亲缘关系较近;构建pET-32a-AlCMK原核表达载体,并转化至大肠杆菌BL21(DE3)表达感受态,蛋白表达结果显示其分子质量约为65 kDa;利用qRT-PCR分析AlCMK基因在不同组织和茉莉酸甲酯(MeJA)处理后的表达模式,同时采用ELISA试剂盒法测定茅苍术CMK酶活性,结果表明,不同产地茅苍术AlCMK基因具有组织表达差异性,且受外源MeJA诱导表达,酶活结果显示不同产地茅苍术CMK酶活性均在叶中较高;亚细胞定位显示该基因位于叶绿体中。本研究为进一步阐明茅苍术中萜类化合物合成途径AlCMK基因的生物学功能提供参考。

4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase(CMK)was one of the key enzymes in the methylerythritol-4-phosphate(MEP)pathway to generate terpenoids.In this study,based on the transcriptome data of Atractylodes lancea,the sequence of the CMK gene was cloned,named AlCMK(GenBank accession number OM283293).The results showed that AlCMK contains a 1230 bp open reading frame(ORF)encoding 409 amino acids.The deduced protein had a theoretical molecular weight of 44752.53 and an isoelectric point of 6.67.Transmembrane structure analysis showed that there was no transmembrane structure,and the secondary structure of AlCMK was predicted to be mainly composed of random coil.Homologous alignment revealed that AlCMK shared high sequence identity with the CMK proteins of Tanacetum cinerariifolium,Osmanthus fragrans,Eucommia ulmoides,Lonicera japonica and Salvia miltiorrhiza.Phylogenetic analysis indicated that AlCMK protein had the higher homology with CMK protein of Compositae.The pET-32a-AlCMK prokaryotic expression vector was constructed and a fusion protein with molecular mass of about 65 kDa was expressed in the E.coli BL21(DE3).The qRT-PCR was used to analyze the expression pattern of AlCMK gene in different tissues and after MeJA treatment.Meanwhile,the enzyme activity was determined by ELISA kit.The results showed that AlCMK gene was tissue-expressed in different origins and its expression was induced by MeJA,and the results of the enzyme activity assay showed that the AlCMK enzyme activity in different regions was higher in the leaves.The subcellular localization showed that AlCMK was located in the chloroplast.This study provides a reference for further elucidating the biological function of AlCMK gene in terpenoid synthesis pathway in Atractylodes lancea.