独行菜 LaMCT 基因的克隆、序列分析与原核表达.

The active components of Lepidium apetalum, cardiac glycosides have been well studied both chemically and pharmacologically, but little is known about the cardiac glycosides biosynthesis pathway. Using cultivated seedlings as material, the transcriptome data of Lepidium apetalum, designing specific primers and using PCR method, an open reading frame (ORF) of key enzyme 2-C-methylerythritol-4-phosphate cytidylyltransferase (MCT) gene which involved in MEP pathway was isolated and named as LaMCT (GenBank accession no. KT832554). The sequence analysis and prokaryotic expression were also performed. Sequence analysis showed that LaMCT has an ORF of 912 bp, which encoded a protein of 304 amino acid residues. Subcelluar localization and conserved domain analysis indicated that LaMCT protein located in chloroplast had no signal peptide and transmembrane domain, but had a isoprenoid synthase conserved domain. Phylogenetic analysis revealed that LaMCT protein showed the highest homology, 94% similarity, with MCT protein from Arabidopsis thaliana. Through the construction of pET-32a-LaMCT vector, the recombinant LaMCT protein was successfully expressed in Escherichia coli BL21 (DE3) cells. Finally, the recombinant LaMCT protein was purified through Ni2+ affinity chromatography. The LaMCT gene was cloned from L. apetalum, and the stable prokaryotic expression system of pET-32a-LaMCT was constructed. This study will provide some basic information for the further antibody preparation of LaMCT protein and would be helpful in functional research of LaMCT gene involved in cardiac glycosides biosynthesis pathway. [ABSTRACT FROM AUTHOR]