J Integr Plant Biol. ›› 2009, Vol. 51 ›› Issue (11): 993-1001.DOI: 10.1111/j.1744-7909.2009.00848.x

• Metabolism and Biochemistry • Previous Articles     Next Articles

Molecular Characterization of a Dehydroascorbate Reductase from Pinus bungeana

Hai-Ling Yang1, Ying-Ru Zhao1, Cai Ling Wang1, Zhi-Ling Yang2, Qing-Yin Zeng2 and Hai Lu1*   

  1. 1 College of Life Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China
    2 State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China
  • Received:2008-10-27 Accepted:2009-05-10 Published:2009-11-10
  • About author: *Author for correspondence Tel(Fax): +86 10 6233 8063; E-mail: luhai1974@yahoo.com


Dehydroascorbate reductase (DHAR) plays a critical role in the ascorbate-glutathione recycling reaction for most higher plants. To date, studies on DHAR in higher plants have focused largely on Arabidopsis and agricultural plants, and there is virtually no information on the molecular characteristics of DHAR in gymnosperms. The present study reports the cloning and characteristics of a DHAR (PbDHAR) from a pine, Pinus bungeana Zucc. ex Endl. The PbDHAR gene encodes a protein of 215 amino acid residues with a calculated molecular mass of 24.26 kDa. The predicted 3-D structure of PbDHAR showed a typical glutathione S-transferase fold. Reverse transcription-polymerase chain reaction revealed that the PbDHAR was a constitutive expression gene in P. bungeana. The expression level of PbDHAR mRNA in P. bungeana seedlings did not show significant change under high temperature stress. The recombinant PbDHAR was overexpressed in Escherichia coli following purification with affinity chromatography. The recombinant PbDHAR exhibited enzymatic activity (19.84 μmol/min per mg) and high affinity (a Km of 0.08 mM) towards the substrates dehydroascorbate (DHA). Moreover, the recombinant PbDHAR was a thermostable enzyme, and retained 77% of its initial activity at 55 ◦C. The present study is the first to provide a detailed molecular characterization of the DHAR in P. bungeana.

Yang HL, Zhao YR, Wang CL, Yang ZL, Zeng QY, Lu H (2009). Molecular characterization of a dehydroascorbate reductase from Pinus bungeana. J. Integr. Plant Biol. 51(11), 993-1001.

Key words: cloning, dehydroascorbate reductase, enzyme activity, gymnosperm.

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