Abstract

The enzyme 4-coumarate: CoA ligase (4CL) activates cinnamic acid and its hydroxylated derivatives by forming the corresponding CoA thioesters. These serve as substrates for biosynthesis of phenylpropanoid-derived secondary metabolites for the medicinal herbal Isatis indigotica. To investigate the function of 4CL gene, we have characterized this gene namedIi4CL with GenBank Accession No. GQ872418. The full-length cDNA of Ii4CL was 1967 bp and contained a 1632 bp open reading frame (ORF) encoding a 543 amino acid protein. Phylogenetic analysis places the Ii4CL into a third group distinct from the common type I and type II 4CL. Real-time quantitative polymerase chain reaction (PCR) analysis indicated thatIi4CL was expressed in roots, stems, leaves and flowers of I. indigotica, with the highest expression level in roots and flowers. The elicitor treatment experiments using methyl jasmonate (MeJA), abscisic acid (ABA) and UV-B revealed that Ii4CL respond to these elicitors in different manners. The full-length of ORF was sub-cloned into bacterial expression vector pET32a(+) and transferred into Escherichia coli BL21(DE3). The recombinant protein had high expression level in E. coli BL21(DE3) with isopropyl-β-D-thiogalactoside (IPTG) induction.

Key words: Phenylpropanoid pathway, UV-B, lignan, Isatis indigotica Fort., 4-coumarate:CoA ligase (4CL).