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

• Published in: Plant science (Limerick) Vol. 163; no. 2; pp. 241 - 251
• Main Authors: Polashock, James J, Griesbach, Robert J, Sullivan, Raymond F, Vorsa, Nicholi
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 01-08-2002; Elsevier Science
• Subjects: Agronomy. Soil science and plant productions; Anthocyanin; Biological and medical sciences; Classical and quantitative genetics. Population genetics. Molecular genetics; Flavonoid; Fundamental and applied biological sciences. Psychology; Generalities. Genetics. Plant material; Genes. Genome; Genetics and breeding of economic plants; Metabolism; Molecular and cellular biology; Molecular genetics; Plant physiology and development; Storage and secretion, pigments, phytochrome; Substrate specificity; Vaccinium macrocarpon; Anthocyanin; Flavonoid; Substrate specificity; Vaccinium macrocarpon; COROLLA; Allelism; Small fruits; Gene overexpression; Stimulant plant; Dihydrokaempferol 4-reductase; Nicotiana tabacum; Organic pigment; Gene; Dicotyledones; Angiospermae; Transgenic plant; Solanaceae; Molecular cloning; Nucleotide sequence; Enzyme; Interspecific comparison; Color; Gene expression; Spermatophyta; Ericaceae; Oxidoreductases
• ISSN: 0168-9452; 1873-2259
• DOI: 10.1016/S0168-9452(02)00087-0

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.