Cloning, Analysis, Expression And Functional Identification Of COMT Gene Sequence

Ligusticum chuanxiong Hort(LC) has been used as one of traditional medicines for promoting the circulation of the blood and Qi、expelling wind and relieving pain. Ferulicacid is the majoractive ingredient and index component of LC. The formation of ferulic acid in LC occurs through thebiosynthetic pathway of skikimate pathay starting with alomatic amino acids.The caffeic acid gets converted into ferulic acid by methylation reaction in which caffeic acid methyltransferase(COMT) plays a very important role.In this study,Full length c4-comt sequence gene(GenBank accession no. KR106206) was isolated from LC according to the partial sequence in the transcriptome.c4-comt gene Cdna is 1171bp in length and contains an 1089bp open reading frame(ORF) encoding 362 amino acids residues.The calculated isoelectric point and molecular weight of the mature protein were predicted to be 5.95 and 39.932kda respectively.The amino acids sewuence showed a high match with other COMT,and fell into a cluster with the COMT in Ammi majus.The deduced amino acids sequences have seven conserved motifs,four are putative SAM binding domains and other are putative caffeic acids binding domains.Meanwhile,three-dimensionalmodel of the protein was build based on homology modeling method.COMT-pet28 are combinant plasmidwas constructed and expressed in E.coli BL21.The molecular mass of expression products,checked by SDS-PAGE,was 43kDa.The expression conditions were optimized,37℃,2mM IPTG and 4 hours were the best conditions.The recombinant COMT proteinwas purified by Ni2+ affinity column chromatograph.The enzymatic reaction occur under conditions pH7,37℃,and detected by HPLC extent. Meanwhile, the reaction products were purified by reverse phase column chromatography, and identified as ferulic acid by 1H NMR.The optimum reaction temperature and pH of recombinant COMT enzyme are 37℃, pH7 respectively.Site-directed mutagenes’s method was used to introduce mutations in the site His268 to proof it was the key amino acids residues in COMT.