Isolation and characterization of a gene encoding cinnamoyl-CoA reductase from Isatis indigotica Fort.

A novel cinnamoyl-CoA reductase gene, designated as Iiccr (GenBank Accession No. GQ872418) was cloned from Isatis indigotica Fort. The full-length cDNA of Iiccr was 1368 bp with an ORF of 1026 bp that putatively encoded a polypeptide of 341 amino acids, with a predicted molecular mass of 37.50 kDa. The deduced amino acid sequence of IiCCR shared high homology with other known CCRs. No intron was detected in the genomic sequence of Iiccr. Southern-blot analysis revealed that Iiccr was a high-copy gene and real-time quantitative PCR analysis indicated that Iiccr was constitutively expressed in roots, stems and leaves of I. indigotica, with the highest expression level in roots. The results from treatment experiments using different signaling components for plant defense responses including methyl jasmonate (MeJA), gibberellins (GA(3)), abscisic acid (ABA) and ultraviolet-B revealed that expression of IiCCR had a prominent diversity. The full-length of ORF was sub-cloned into prokaryotic expression vector pET32a(+), which was then transferred into E. coli BL21(DE3). The recombinant protein had high expression level in E. coli BL21(DE3) with IPTG induction. A 2.6 kb long promoter sequence was isolated and its putative regulatory elements and potential specific transcription factor binding sites were analyzed. This study will enable us to further understand the role of IiCCR in the synthesis of phenylpropanoid compounds in I. indigotica Fort. at the molecular level.