目的 从独行菜Lepidium apetalum中克隆萜类合成途径关键酶磷酸甲羟戊酸激酶（phosphomevalonate kinase，PMK）基因，进行序列分析和原核表达。方法 根据独行菜转录组数据中LaPMK基因序列设计特异性引物，克隆得到LaPMK基因的开放阅读框（open reading frame，ORF），构建pET32a-LaPMK原核表达载体，在大肠杆菌BL21（DE3）中用IPTG诱导表达LaPMK融合蛋白。结果 LaPMK基因ORF全长为1 518 bp（Genbank注册号KT004541），编码505个氨基酸。生物信息学分析结果显示LaPMK蛋白没有跨膜区，不含信号肽，位于细胞质中，含有GHMP激酶家族特异的N末端和C末端的保守结构域。系统进化树结果显示LaPMK蛋白与芜菁的PMK蛋白具有92%的序列相似性，亲缘关系较近。构建pET32a-LaPMK原核表达载体，在大肠杆菌中成功表达LaPMK融合蛋白。结论 克隆了LaPMK基因，建立其稳定的原核表达体系，为LaPMK蛋白纯化、抗体的制备，进一步研究LaPMK基因在独行菜萜类合成途径中的作用奠定了基础。

Objective To obtain the key enzyme gene involved in terpenoid biosynthesis pathway, phosphomevalonate kinase (PMK) gene was cloned from Lepidium apetalum, and sequence analysis and prokaryotic expression were performed. Methods Based on the transcriptome data of L. apetalum, by designing specific primers of LaPMK gene, an open reading frame (ORF) of LaPMK gene was isolated from L. apetalum. Escherichia coli BL21 (DE3) cells were transformed with the prokaryotic expression vector pET32a-LaPMK and used for prokaryotic expression under IPTG induction. Results LaPMK gene has ORF of 1 518 bp (GenBank accession number KT004541), which encoded a protein of 505 amino acid residues. Bioinformatic analysis indicated that LaPMK protein which located in cytoplasm had no transmembrane domain and signal peptide, and exhibited the specific N-terminal domain and C-terminal domain of GHMP kinase super family. Phylogenetic analysis indicated that LaPMK protein showed the highest homology, 92% similarity, with PMK protein from Brassica rapa. The recombinant LaPMK protein was successfully expressed in E. coli BL21 (DE3) cells. Conclusion The LaPMK gene is cloned from L. apetalum, and the stable prokaryotic expression system of pET32a-LaPMK is constructed. This study will provide the fundamental information for the further purification and the antibody preparation of LaPMK protein be helpful for the functional researches of LaPMK gene in terpenoid biosynthesis pathway.

国家重点基础研究发展计划“973计划”项目（2013CB531802）；河南中医学院博士科研基金（BSJJ2011-07）