Cloning and expression analysis of 5-phosphomevalonate kinase gene (CcPMK) in Cinnamomum camphora / 中国中药杂志

The 5-phosphomevalonate kinase(PMK) is a key enzyme in mevalonate(MVA) pathway which reversibly catalyzes the phosphorylation of mevalonate 5-phosphate(MVAP) to form mevalonate-5-diphosphate(MVAPP) in the presence of ATP and divalent metal ion such as Mg~(2+). In this research, on the basis of the transciptome database of Cinnamomum camphora, the PMK was cloned by cDNA from C. camphora, and was named CcPMK(GenBank number KU886266). The ORF of CcPMK was composed of 1 545 bp, encoding 514 amino acids. The bioinformatics analysis of CcPMK indicated that the molecular weight of the encoded protein was 56.14 kDa, with a theoretically isoelectric point of 7.64, and there was no signal peptide and transmembrane structure in putative protein. By multiple sequence alignment and phylogenetic tree analysis, we found that similarity between CcPMK and PMK amino acid sequence of other plants was as high as 75%. Among the similar sequences, 45% of them belonged to the alpha helix, while 16% belonged to the beta strand. CcPMK obtained 3 PMK protein family motifs and 1 ATP binding site Gly-Leu-Gly-Ser-Ser-Ala-Ala, and its 3 D structure contained a catalytic pocket structure, proving CcPMK as a member of PMK gene family. The result of phylogenetic tree showed that CcPMK was closely related to monocotyledon plants such as Phonenix dactylifera. The results of the Real-time PCR indicated that the expression level of CcPMK in borneol type was higher than that in linalool type, cineol type, iso-nerolidol type and camphor type. CcPMK expressed highest in roots and lowest in branches. Our results revealed that the expression level of CcPMK was different among five chemical types and different plant tissues, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.

Cloning, bioinformatic analysis, and prokaryotic expression of LaPMK gene in Lepidium apetalum / 中草药

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.