Molecular Cloning, Characterization, and Expression Analysis of Flavanone 3-Hydroxylase (F3H) Gene during Muscadine Grape Berry Development

Abstract

Flavonoids are natural antioxidants that include the groups of notable pigments such as anthocyanins and proanthocyanidins. Flavanone 3-Hydroxylase (F3H) is a key enzyme needed for the biosynthesis of flavonoids, the main ingredients of muscadine grape extracts. This study reports the first successful isolation, cloning and characterization of F3H gene from Vitis rotundifolia Michx. The full length cDNA of V. rotundifolia F3H gene (designated as VrF3H) had an open reading frame (ORF) of 1081 bp encoding 364 amino acids with a calculated molecular mass of 40.8kDa as well as an isoelectric point of 5.60. Comparative and in silico analyses revealed that the cloned VrF3H from muscadine grapes has high identity with F3H from other plant species. The deduced VrF3H protein showed similarities with other available plant F3H proteins, and the conserved amino acids ligating ferrous iron and residues participating in 2-oxo-glutarate binding were found in similar positions comparable to other F3Hs. Furthermore, three-dimensional structure modeling showed that F3H protein had the enzyme core consisting of β-sheet, a typical structure shared by all 2-oxoglutarate-dependent dioxygenases including F3Hs. Phylogenetic tree analysis indicated that VrF3H belongs to the Vitis F3H cluster. VrF3H transcripts were found to be abundantly expressed in the in-vitro red cells, véraison and physiologically mature red berries, but not expressed in the skins of the green berries. The isolation and characterization of VrF3H gene will enable further study in the role of VrF3H gene in the biosynthesis of flavonoids in V. rotundifolia.