Molecular cloning and characterization of a flavonoid 3'-hydroxylase gene from purple-fleshed sweet potato (Ipomoea batatas)

Flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21) is an important enzyme which determines the hydroxylation pattern of anthocyanins. In this study, the full-length cDNA and genomic DNA of F3'H were isolated and characterized from the purple-fleshed sweet potato (Ipomoea batatas). IbF3''H was 1,789 bp containing a 1,554 bp open reading frame (ORF) encoding 518 amino acids. Comparative and bioinformatic analysis revealed that IbF3'H was highly homologous with F3'Hs from other plant species. Conserved domain search revealed that IbF3'H was a cytochrome P450 dependent enzyme. Three F3'H-specific motifs (V(75)VVAAS(80), G(427)GEK(430) and V(433)DVKG(437)) were conserved in IbF3'H. Phylogenetic analysis revealed that IbF3'H was clustered into the same subgroup with the homologues from I. purpurea, I. tricolor and I. nil. There were multiple copies of the IbF3'H gene in the genome of I. batatas. IbF3'H was constitutively expressed in all tested tissues including fibrous roots, thick roots, storage roots, stems and leaves. During storage root formation, IbF3'H was expressed most abundantly in the storage roots, suggesting that the anthocyanin biosynthesis is also active in the under-ground organs. IbF3'H expression was associated with anthocyanin accumulation in five different sweet potato cultivars tested. Complementative analysis implied that the full-length cDNA of IbF3'H could encode a functional protein and had a special catalytic activity of flavonoid 3'-hydroxylase.