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

ABSTRACT

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.