Abstract:

The aim of this study was to clone full-length cDNA of dihydroflavonol 4-reductase gene (DFR) from Paphiopedilum concolor Pfitz., to analyze the sequence characteristics and to investigate its expression in different tissues. The cDNA of DFR gene was cloned from flowers of P. concolor by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Its sequence characteristics were analyzed by bioinformatics tools and its expression levels in different tissues were detected by semi-quantitative RT-PCR. The obtained DFR gene was 1267 bp in full-length, containing a 5’-untranslated region (5’-UTR) of 10 bp, a 3’-UTR of 183 bp, and an opening reading frame (ORF) of 1074 bp encoding a 357 predicted amino acids residues. The DFR protein contained a conserved NADB_Rossmann superfamily domain, an NADP (H) binding domain and a substrate binding site domain. The DFR protein shared 75%-80% identities with DFR proteins of Oncidium Gower Ramsey, Dendrobium hybrid, and Cymbidium hybrid. Phylogenetic tree analysis clearly showed that the DFR protein was close to DFR proteins in orchidaceae plants. Expression analyses revealed that DFR showed different expression patterns in tissues examined. The transcripts of DFR were the highest levels in mature flowers and vegetative leaves, moderate levels in columns, bracts, sepals and petals, lower levels in ovaries than in lips, lowest levels in flower stems, and almost no detection in roots. Prokaryotic expression showed that recombinant plasmid was efficiently expressed in Escherichia coli Rosetta-gami B (DE3). DFR gene was firstly isolated and characterized from Paphiopedilum orchid, which may participate in regulation of anthocyanin biosynthesis.