Cloning and expression analysis of dihydroxyflavonol 4-reductase (DFR) in Ascocenda spp.

Abstract

Dihydroflavonol 4-reductase (DFR) gene is a key gene of anthocyanins biosynthesis pathway, which represent an importance pathway for orchid flower. In this study, cloning and expression analysis of DFR gene in Ascocenda spp. were carried out. Nucleotide analysis revealed that the Ascocenda DFR gene was 1,056 bp in length, and encoded a protein of 351 amino acid residues. A typical translation initiation codon (ATG) and translation termination codon (TGA), the most frequently found codon in plant were identified, indicating a full-length coding sequence of the DFR gene. The calculated molecular mass of the deduced polypeptide was 39.8 kDa and the predicted isoelectric point was 5.58. Homology analysis revealed that the amino acid sequence of the Ascocenda DFR gene product was 80 to 87% identity to amino acid sequences of DFR gene products of other orchids such as Bromheadia, Dendrobium, Cymbidium and Oncidium. It also showed a high degree of identity to the DFR gene products of other flowers such as Lilium, Tilipa, Allium, Gentiana and Chrysanthenum. Southern blot analysis indicate that DFR is presented as a single copy in the Ascocenda spp. genome. The AscoDFR gene was highly expressed in the flower stages 2 and 3 of development as well as in the sepal and petal of the orchid flower.

Keywords: Orchid, dihydroxyflavonol 4-reductase, anthocyanins, gene cloning