Cloning, Bioinformatic Analysis, and Prokaryotic Expression of LaMK Gene in Lepidium apetalum / 中国药学杂志

ABSTRACT

OBJECTIVE: To clone the mevalonate kinase (MK) gene involved in cardiac glycosides biosynthesis pathway of Lepidium apetalum, and carry out bioinformatic analysis, prokaryotic expression, purification, and tissue-specific expression analysis. METHODS: By analyzing the transcriptome data of L. apetalum and designing specific primers, the cDNA of LaMK gene was isolated from L. apetalum (GenBank accession No. KX290928).Escherichia coli BL21 (DE3) cells were transformed with the prokaryotic expression vector pET-32a-LaMK and used for prokaryotic expression of recombinant LaMK protein. RESULTS: The open reading frame (ORF) of LaMK was 1 137 bp, which encoded a protein of 379 amino acid residues, with a predicted molecular weight of 40.43×103. Bioinformatic analysis showed that LaMK protein may locate in cytoplasm, had no transmembrane domain and signal peptide, and exhibited specific N-terminal domain and C-terminal domain of GHMP kinase super family, as well as the binding site of ATP. Phylogenetic analysis indicated that LaMK protein had the highest homology with MK protein from cruciferous plants (such as AtMK from Arabidopsis thaliana). Through construction of the prokaryotic expression vector, the recombinant LaMK protein was successfully expressed in Escherichia coli BL21 (DE3) cells. Furthermore, the recombinant LaMK protein was purified by Ni2+ affinity chromatography. Real-time PCR analysis indicated that LaMK was expressed at a high level in flowers, low levels in leaves and roots, lower expression level in stems, and the lowest level in seedlings. CONCLUSION: In this study, the LaMK gene is cloned from L. apetalum, the prokaryotic expression system is established, and the purified recombinant LaMK protein is obtained. This study lays the foundation for preparation of the antibody of LaMK protein and research of the function of LaMK gene involved in cardiac glycosides biosynthesis pathway.