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ISHS Acta Horticulturae 1157: IX International Symposium on Grapevine Physiology and Biotechnology

Molecular cloning and sequence analysis of dihydroflavonol 4-reductase gene from North American native grapes (Vitis rotundifolia)

Authors:   A. Ananga, K. Acheampong, C. Zheng, E. Or, V. Georgiev, V. Tsolova
Keywords:   Vitis rotundifolia, dihydroflavonol 4-reductase, gene cloning, flavonoids, anthocyanins
DOI:   10.17660/ActaHortic.2017.1157.42
Abstract:
Muscadine grapes are known for their high antioxidant capacity, and there is a need to enhance the production of nutraceutical compounds in many cultivars. Dihydroflavonol 4-reductase (DFR) is a key enzyme controlling flavonoid synthesis in anthocyanin biosynthetic pathway. DFR catalyzes the reduction of dihydroflavonols to leucoanthocyanidins, a key 'late' step in the biosynthesis of anthocyanins. Here we report the cloning of full-length cDNA of DFR obtained from the berry skin of muscadine grapes NDASH 'Noble' cultivar using reverse transcription - polymerase chain reaction (RT-PCR). A typical translation initiation codon (ATG) and translation termination codon (TGA) were identified, indicating a full-length coding sequence of the DFR. Our data revealed that the cDNA sequences from muscadine DFR contained 1014 bp open reading frame which encodes a protein of 337 amino acid residues, corresponding to a molecular mass of 37.5 kDa, with a theoretical pI of 5.96. Phylogenetic analysis of the amino acid sequences indicates high identity to DFRs from V. bellula (98%), V. amurensis (97%), V. vinifera (97%), and V. lambrusca (96%) NDASH members of the Vitis family. Realtime-PCR analysis indicated that DFR gene is highly expressed in the berry skins of red cultivars and the mRNA levels of DFR reached the highest towards the end of veraison. The data revealed that even though there is close homology between V. vinifera and muscadines, differences in the nucleotide sequence of DFR exist. This work provides the fundamental framework for further studies on biological regulation of DFR activity in muscadines.

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