The Open Plant Science Journal, 2008, 2, 31-36 31 1874-2947/08 2008 Bentham Open Open Access Molecular C loning and C haracterization of Cinnamate-4-Hydroxylase Gene from Rubus coreanus Seung Sik Lee 1 , Eun Mi Lee 1 , Byung Chull An 1 , Shyamkumar Barampuram 1 , Jae-Sung Kim 1 , Jae- Young Cho 2 and Byung Yeoup Chung 1,* 1 Advanced Radiation Technology Institute (ARTI), Korea Atomic Energy Research Institute (KAERI), Jeongeup 580- 185, Korea. 2 Department of Applied Life Sciences, Chonbuk National University, Jeonju 561-756, Korea 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. Keywords: Cinnamate-4-hydroxylase, phenylpropanoid pathway, RACE, Rubus coreanus, southern blot. INTRODUCTION Rubus coreanus Miquel (Rosaceae) is a Korean raspberry distributed in southern parts of the Korea, China and Japan. The unripe fruits of Korean raspberry have been used as a traditional medicine for the treatment of impotence, sperma- torrhea, enuresis, asthma, allergic diseases, and it also has been used as a stomachic and tonic in Korea [1]. Recently, it was reported that berry extracts inhibit the growth and in- duce apoptosis of different human cancer cell lines in vitro [2]. The biological activities of berries are partially deter- mined by their content of a diverse range of phytochemicals such as flavonoids (anthocyanins, flavonols, and flavanols), tannins (proanthocyanidins, ellagitannins, and gallotannins), stilbenoids (e.g., resveratrol), phenolic acids (hydroxyben- zoic and hydroxycinnamic acid derivatives), and lignins [3, 4]. The quality of ripe raspberry fruits is critically dependent on the accumulation of specific anthocyanin pigments and characteristic flavor components which are biosynthetically produced in the phenylpropanoid pathway [4-6]. Cinnamate 4-hydroxylase (C4H) is a member of the cy- tochrome P450 monooxygenase superfamily and it catalyzes the first oxygenation step during a phenylpropanoid metabo- lism, the hydroxylation of trans-cinnamate to p-coumarate (4-hydroxy trans-cinnamate), and the phenylpropanoid bio- synthesis branch pathways lead to the synthesis of a variety *Address correspondence to this author at the Advanced Radiation Tech- nology Institute (ARTI), Korea Atomic Energy Research Institute (KAERI), Jeongeup 580-185, Korea; Tel: +82-63-570-3331; Fax: +82-63-570-3339; E-mail: bychung@kaeri.re.kr of compounds including lignin monomers, flavonoids, hy- droxycinnamic esters and coumarins. These compounds are necessary for UV-protection, differentiation of tissues and defense giving reason for the induction of C4H upon irradia- tion, wounding, elicitors, and pathogen infection of plants [7]. In addition, C4H is regulated in a pattern of temporal and spatial gene expression correlated with its role in lignifi- cations [8, 9]. Many C4Hs have been isolated and characterized in herbal plants. However, there are scanty reports about the cloning of C4H genes from woody plant species. In woody plants, C4H is particularly important for the biosynthesis of lignin, the second most abundant biopolymer after cellulose, and C4H is likely to play a pivotal role in the ability of a phenylpropanoid metabolism to channel carbon from a pri- mary metabolism into a biosynthesis of lignin and other polymers in trees. The C4H gene from raspberry which has been used as a traditional Korean medicinal woody-plant was firstly isolated in this study. We report on the cloning and characterization of a full-length cDNA sequence of the C4H gene from R. coreanus. This report will be helpful to further research the role of the RcoC4H gene during a phen- ylpropanoid metabolism in R. coreanus. MATERIALS AND METHODS Plant Growth Conditions and Materials Korean native bramble (Rubus coreanus Miq.) was grown in experimental plots at the Korean Black Raspberry Research Center, Gochang, Republic of Korea, under ambi- ent conditions. The leaf, stem and flower were collected