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

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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.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (30971974), Natural Science Fund of Hubei Province (2008CDA061), and University-Industry Cooperation Fund of Hubei Educational Office (CXY2009B009).

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Authors and Affiliations

  1. College of Horticulture and Gardening, Yangtze University, Jingzhou, 434025, China

    Feng Xu, Weiwei Zhang & Nannan Sun

  2. College of Life Science and Engineering, Huanggang Normal University, Huanggang, 438000, China

    Linling Li, Hua Cheng & Shuiyuan Cheng

  3. Huanggang Department of Forestry of Hubei Province , Huanggang, 438000, China

    Yan Wang

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  1. Feng Xu
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  2. Linling Li
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  3. Weiwei Zhang
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  4. Hua Cheng
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Correspondence to Feng Xu or Shuiyuan Cheng.

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Xu, F., Li, L., Zhang, W. et al. Isolation, characterization, and function analysis of a flavonol synthase gene from Ginkgo biloba . Mol Biol Rep 39, 2285–2296 (2012). https://doi.org/10.1007/s11033-011-0978-9

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  • DOI: https://doi.org/10.1007/s11033-011-0978-9

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