Isolation, characterization, and function analysis of a flavonol synthase gene from Ginkgo biloba

• DOI: 10.1007/s11033-011-0978-9
• Publication Date: 2011-06-04T08:29:47Z
• Authors: Feng Xu; Linling Li; Weiwei Zhang; Hua Cheng; Nannan Sun; Shuiyuan Cheng; Yan Wang
• Keywords: Flavonoids; 0301 basic medicine; Analysis of Variance; DNA, Complementary; Base Sequence; Gene Expression Profiling; Molecular Sequence Data; Computational Biology; Ginkgo biloba; Cold Temperature; Evolution, Molecular; Open Reading Frames; 03 medical and health sciences; Organophosphorus Compounds; Gene Expression Regulation, Plant; Amino Acid Sequence; Kaempferols; Oxidoreductases; Promoter Regions, Genetic; Chromatography, High Pressure Liquid; Abscisic Acid; Plant Proteins

Flavonols are produced by the desaturation of dihydroflavanols, which is catalyzed by flavonol synthase (FLS). FLS belongs to the 2-oxoglutarate iron-dependent oxygenase family. The full-length cDNA and genomic DNA sequences of the FLS gene (designated as GbFLS) were isolated from Ginkgo biloba. The full-length cDNA of GbFLS contained a 1023-bp open reading frame encoding a 340-amino-acid protein. The GbFLS genomic DNA had three exons and two introns. The deduced GbFLS protein showed high identities with other plant FLSs. The conserved amino acids (H-X-D) ligating ferrous iron and residues (R-X-S) participating in 2-oxoglutarate binding were found in GbFLS at similar positions like other FLSs. GbFLS was found to be expressed in all tested tissues including roots, stems, leaves, and fruits. Expression profiling analyses revealed that GbFLS expression was induced by all of the six tested abiotic stresses, namely, UV-B, abscisic acid, cold, sucrose, salicylic acid, and ethephon, consistent with the in silico analysis results of the promoter region. The recombinant protein was successfully expressed in the E. coli strain BL21 (DE3) with a pET-28a vector. The in vitro enzyme activity assay by high performance liquid chromatography indicated that recombinant GbFLS protein could catalyze the formation of dihydrokaempferol to kaempferol and the conversion of kaempferol from naringenin, suggesting that GbFLS is a bifunctional enzyme within the flavonol biosynthetic pathway.