Molecular Cloning and Function Analysis of an Anthocyanidin Synthase Gene from Ginkgo biloba, and Its Expression in Abiotic Stress Responses

  • Xu, Feng (College of Horticulture and Gardening, Yangtze University) ;
  • Cheng, Hua (College of Horticulture and Gardening, Yangtze University) ;
  • Cai, Rong (College of Horticulture and Gardening, Yangtze University) ;
  • Li, Lin Ling (College of Life Science and Engineering, Huanggang Normal University) ;
  • Chang, Jie (College of Horticulture and Gardening, Yangtze University) ;
  • Zhu, Jun (College of Horticulture and Gardening, Yangtze University) ;
  • Zhang, Feng Xia (College of Horticulture and Gardening, Yangtze University) ;
  • Chen, Liu Ji (College of Horticulture and Gardening, Yangtze University) ;
  • Wang, Yan (College of Horticulture and Gardening, Yangtze University) ;
  • Cheng, Shu Han (College of Horticulture Science and Engineering, Shandong Agricultural University) ;
  • Cheng, Shui Yuan (College of Life Science and Engineering, Huanggang Normal University)
  • Received : 2008.04.28
  • Accepted : 2008.09.09
  • Published : 2008.12.31

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 pET-28a 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.

Keywords

abiotic stresses;anthocyanidin synthase;enzyme activity;GbANS;gene expression;Ginkgo biloba;in vitro

Acknowledgement

Supported by : Natural Science Foundation of Hubei Province, Youth Talent Foundation of Hubei Province

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