Cloning and Characterization of a Flavanone 3-Hydroxylase Gene from Ginkgo biloba

Abstract

Flavanone 3-hydroxylase (F3H) activity is necessary for the biosynthesis of flavonoids, the main ingredients of Gingko biloba extract. The full-length cDNA and genomic DNA sequences of F3H gene were isolated from G. biloba for the first time. The full-length cDNA of G. biloba F3H gene (designated as GbF3H) contained a 1071 bp open reading frame (ORF) encoding a 357-amino-acid protein with a calculated molecular weight of about 40 kDa and isoelectric point (pI) of 5.57. The genomic DNA analysis showed that GbF3H gene had three exons and two introns. The deduced GbF3H protein showed high identities to other plant F3Hs. The conserved amino acids ligating ferrous iron and residues participating in 2-oxoglutarate binding (R-X-S) were found in GbF3H at the similar positions like other F3Hs. Three-dimensional structure modeling showed that GbF3H had a jerry roll in the enzyme core consisted of beta-sheet, a typical structure shared by all 2-oxoglutarate-dependent dioxygenases including F3Hs. Phylogenetic tree analysis revealed that GbF3H shared the same ancestor in evolution with other F3Hs and had a further relationship with other angiosperms species. Southern blot analysis indicated that GbF3H belonged to a multi-gene family. Transcription analysis revealed that GbF3H expressed in stem and leaf with the highest transcription level in leaf. The isolation and characterization of GbF3H gene will be helpful to further study the role of GbF3H gene in the biosynthesis of flavonoids in G. biloba.