Cloning of a cDNA encoding the cranberry dihydroflavonol-4-reductase (DFR) and expression in transgenic tobacco

A clone representing a fragment of the dihydroflavonol-4-reductase (DFR) gene from cranberry was isolated from a genomic DNA library using the tomato DFR gene as a probe. Sequence analysis of the clone confirmed homology to published DFR gene sequences. 3′ and 5′ RACE (rapid amplification of cDNA ends) reactions from cranberry leaf total RNA were used to obtain the entire cDNA sequence. The sequence information was used to amplify a full-length clone by RT-PCR. Sequencing analysis to confirm the identity of the full-length DFR cDNA identified a putative second allele. Segregation analysis suggested that the two sequences are not allelic, but multi-locus. Nucleotide sequence homology of the full-length clones was highest to published DFR sequence from Camellia sinensis (about 80% identity) followed by Forsythia x intermedia, Antirrhinum majus, Rosa hybrida and Petunia hybrida. When expressed using the CaMV 35S promoter, the corolla of flowers of transgenic tobacco plants were much darker pink than the controls. Some flower parts not normally highly pigmented, such as the filaments, were also dark pink. These data confirm the identity and function of the cranberry clones and further suggest that overexpression of the cranberry DFR could be used to increase anthocyanin production in transgenic plants.