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

Abstract

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.

Cloning and Sequence Analysis of a Flavonol Synthase (FLS2) Gene from Ginkgo biloba

“…However, only AtFLS1 was shown to play a primary role in flavonol biosynthesis in A. thaliana (Owens et al, 2008). In G. biloba, GbFLS1 is a bifunctional enzyme within the flavonol biosynthetic pathway, but function of other FLS isoforms is still unknown (Xu et al, 2012). In this study, one FLS gene, GbFLS2, was isolated from G. biloba.…”

“…Flavonol Synthase, as a key enzyme involved in the flavonol biosynthetic pathway, has been studied in many plants (Li et al, 2013;Toh et al, 2013;Takahashi et al, 2007;Falcone Ferreyra et al, 2010;Xu et al, 2012;Mahajan et al, 2011). In plants, flavonol synthase could catalyse flavonoids synthesis.…”

“…To figure out biosynthetic pathway of flavonoids in G. biloba, each gene must be identified and characterized. In a previous study, we also reported the isolation and characterization of a flavonol synthase gene from G. biloba, function analysis indicated that GbFLS is a bifunctional enzyme within the flavonol biosynthetic pathway, recombinant GbFLS1 protein could catalyze the formation of dihydrokaempferol to kaempferol and the conversion of kaempferol from naringenin (Xu et al, 2012). In the current study, another flavonol synthase gene was isolated from G. biloba, the cDNA and protein structure was analyzed via bioinformatics methods.…”

“…Flavonol synthase (FLS), a member of the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily, plays a major role in the flavonols biosynthesis. So far, many FLS cDNAs have been isolated from different plant species, such as Tartary buckwheat (Li et al, 2013), Camellia sinensis (Lin et al, 2007), Acacia confusa (Toh et al, 2013), Citrus unshiu (Wellmann et al, 2002), Harosoy (Takahashi et al, 2007), Zea mays (Falcone Ferreyra et al, 2010) and G. biloba (Xu et al, 2012).…”

Phytogeographic and genetic variation inSorbus, a traditional antidiabetic medicine—adaptation in action in both a plant and a discipline

“…Flavonol synthase is a key enzyme in the antioxidant flavonoid pathway (Hukkanen et al, 2006; Fischer et al, 2007) and the gene expression of FLS has been demonstrated to control enzyme activity in many plants species (Xu et al, 2012). For example, in Vitis vinifera , gene expression of FLS increased during ripening of the fruit, coincident with the increase in flavonols per berry (Downey, Harvey & Robinson, 2003).…”