Cloning and sequence analysis of MCS gene in Artemisia annua and its prokaryotic expression / 中草药

ABSTRACT

Objective: To obtain the indispensable key enzyme involved in the MEP pathway, the 2C-methyl-D-erythritol-2, 4-cyclodi-phosphate synthase gene (MCS) was cloned from Artemisia annua, and bioinformatic analysis, prokaryotic expression, and tissue-specific expression were conducted. Methods: According to AaMCS EST sequence, the full length of cDNA and genomic sequences were obtained by the design of specific primers, rapid-amplification of cDNA ends (RACE) and genome amplification. The coding region of MCS gene was cloned into the expression vector pET-21a (+) by gene recombination technology, the recombinant plasmid pET-21a (+)-MCS was transformed into E. coli BL21 (DE3), and the expression of recombinant protein was induced by IPTG. Semi-quantitative RT-PCR was used to detect the expression of AaMCS transcripts. Results: The AaMCS cDNA was found to be 994 bp containing an open reading frame (ORF) of 681 bp that translated into a putative peptide of 226 amino acid, The full length of MCS was 2540 bp consisting of three exons and two introns. A recombinant pET-21a (+)-MCS was constructed by genetically fusing the MCS to pET-21a (+) vector system, and was successfully expressed in E. coli BL21. The tissue expression patterns indicated that the expression level of AaMCS transcripts in flowers was higher than that in the roots and stems. Conclusion: The MCS gene is cloned from A. annua, and the stable prokaryotic expression system of pET-21a (+)-MCS is constructed. This work is helpful for investigating the activities and other physiological functions of MCS protein.