title: Molecular cloning and function analysis of an anthocyanidin synthase gene from Ginkgo biloba, and its expression in abiotic stress responses.
abstract: Anthocyanidin synthase (ANS, leucoanthocyanidin oxygenase), a 2-oxoglutarate iron-dependent oxygenase, catalyzed the penultimate step in the biosynthesis of the anthocyanin class of flavonoids, from the colorless leucoanthocyanidins to the colored anthocyanidins. The full-length cDNA and genomic DNA sequences of ANS gene (designated as GbANS) were isolated from Ginkgo biloba for the first time. The full-length cDNA of GbANS contained a 1062-bp open reading frame (ORF) encoding a 354-amino-acid protein. The genomic DNA analysis showed that GbANS gene had three exons and two introns. The deduced GbANS protein showed high identities to other plant ANSs. The conserved amino acids (H-X-D) ligating ferrous iron and residues (R-X-S) participating in 2-oxoglutarate binding were found in GbANS at the similar positions like other ANSs. Southern blot analysis indicated that GbANS belonged to a multi-gene family. The expression analysis by real-time PCR showed that GbANS expressed in a tissue-specific manner in G. biloba. GbANS was also found to be up-regulated by all of the six tested abiotic stresses, UV-B, abscisic acid, sucrose, salicylic acid, cold and ethylene, consistent with the promoter region analysis of GbANS. The recombinant protein was successfully expressed in E. coli strain with pET28a vector. The in vitro enzyme activity assay by HPLC indicated that recombinant GbANS protein could catalyze the formation the cyanidin from leucocyanidin and conversion of dihydroquercetin to quercetin, suggesting GbANS is a bifunctional enzyme within the anthocyanidin and flavonol biosynthetic pathway.
authors.affiliation: College of Horticulture and Gardening, Yangtze University, Jingzhou 434025, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Xu
authors.forename: Hua
authors.initials: H
authors.lastname: Cheng
authors.forename: Rong
authors.initials: R
authors.lastname: Cai
authors.forename: Lin Ling
authors.initials: LL
authors.lastname: Li
authors.forename: Jie
authors.initials: J
authors.lastname: Chang
authors.forename: Jun
authors.initials: J
authors.lastname: Zhu
authors.forename: Feng Xia
authors.initials: FX
authors.lastname: Zhang
authors.forename: Liu Ji
authors.initials: LJ
authors.lastname: Chen
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Shu Han
authors.initials: SH
authors.lastname: Cheng
authors.forename: Shui Yuan
authors.initials: SY
authors.lastname: Cheng
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D019143:Evolution, Molecular; D020441:Ginkgo biloba / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D016415:Sequence Alignment; D013312:Stress, Physiological / Q000502:physiology
chemical_list: D018076:DNA, Complementary; D010105:Oxygenases; C118159:anthocyanidin synthase