Isolation and Characterization of a Cinnamoyl-CoA Reductase cDNA from Camellia oleifera

Cinnamoyl-CoA reductase (CCR: EC 1.2.1.44), the entry-point enzyme of the lignin specific biosynthetic pathway, catalyzes the conversion of cinnamoyl-CoA esters to their corresponding cinnamaldehydes. In order to reveal the biosynthesis and regulation rhythm of lignin in Camellia oleifera, a full-length cDNA of cinnamoyl-CoA reductase was obtained and named as Co-CCR by using 5 RACE and 3 RACE technology based on the partial sequence of CCR gene acquired from the Camellia oleifera cDNA and EST libraries in the research. The full-length cDNA of Co-CCR gene derived from the conjunction of 5'RACE and 3'RACE products was 1364 bp, and contained a 990 bp ORF encoding 329 amino acid residues, 76 bp and 298 bp non-coding region at 5'end and 3'end respectively. Multiple sequence alignment showed that the deduced polypeptide shared 81% similarity at the amino acid level with defined CCR genes from other plant species and they all contain the common signature sequences thought to be the catalytic site as well as the putative NADP binding domain. Evolutionary analysis showed that CCRs in higher angiosperms were classified into four monophyletic groups, and Co-CCR was classified with Arabidopsis thaliana and Glycine max. The characters of Co-CCR operated by Antheprot 5.0 suggested that the theoretical molecular weight was about 36.16 kD, pi was 5.485, Co-CCR could be anchored to membrane by four transmembrane structures and Co-CCR was a soluble protein in water. 14 alpha-helices and 11 beta-sheets were found in the second structure of Co-CCR and its theoretical 3D structure implied a binding location for NADPH and an active site.