title: 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.
authors.affiliation: College of Horticulture and Gardening, Yangtze University, Jingzhou 434025, China. xufeng198@126.com
authors.forename: Feng
authors.initials: F
authors.lastname: Xu
authors.forename: Linling
authors.initials: L
authors.lastname: Li
authors.forename: Weiwei
authors.initials: W
authors.lastname: Zhang
authors.forename: Hua
authors.initials: H
authors.lastname: Cheng
authors.forename: Nannan
authors.initials: N
authors.lastname: Sun
authors.forename: Shuiyuan
authors.initials: S
authors.lastname: Cheng
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000040:Abscisic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D000704:Analysis of Variance; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003080:Cold Temperature; D019295:Computational Biology; D018076:DNA, Complementary / Q000235:genetics; D019143:Evolution, Molecular; D005419:Flavonoids / Q000378:metabolism; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant / Q000187:drug effects* / Q000235:genetics / Q000528:radiation effects; D020441:Ginkgo biloba / Q000201:enzymology*; D044949:Kaempferols / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020156:Salicylic Acid / Q000494:pharmacology; D036226:Seedlings / Q000201:enzymology; D017422:Sequence Analysis, DNA; D013395:Sucrose / Q000494:pharmacology; D014466:Ultraviolet Rays
chemical_list: D018076:DNA, Complementary; D005419:Flavonoids; D044949:Kaempferols; D009943:Organophosphorus Compounds; D010940:Plant Proteins; D013395:Sucrose; D000040:Abscisic Acid; C006552:kaempferol; C080220:aromadedrin; D010088:Oxidoreductases; C085313:flavonol synthase; D020156:Salicylic Acid; C005073:ethephon