Molecular Cloning and Characterization of Cinnamate-4-Hydroxylase Gene from Rubus coreanus

Abstract

Cinnamate-4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid pathway, which synthesizes a variety of secondary metabolites to participate in differentiation and protection of plant tissues against environmental stresses. We isolated a full-length cDNA of the C4H gene from a Korean native bramble (Rubus coreanus Mique), using a reverse transcriptase-PCR and a rapid amplification of the cDNA ends (RACE)-PCR. The full-length cDNA of the RcoC4H gene contained a 1,515 bp open reading frame (ORF) encoding a 504 amino acid protein with a calculated molecular weight of about 57.9 kDa and an isoelectric point (pI) value of 9.1. The genomic DNA analysis revealed that the RcoC4H gene had three exons and two introns. The comparison of the deduced amino acid sequence of RcoC4H with other C4Hs was highly conserved among widely divergent plant species. Also, the P450-featured motifs such as the heme-binding domain, the T- containing binding pocket motif (AAIETT), the ERR triad and the tetrapeptide (PPGP) hinge motif necessary for an opti- mal orientation of the enzyme were highly conserved. Southern blot analysis indicated that RcoC4H exists as a single copy in R. coreanus. Reverse transcriptase PCR analysis showed that the gene is expressed at similar levels in the stem, leaf and flower.