Molecular characterization of a dehydroascorbate reductase from Pinus bungeana.
Molecular characterization of a dehydroascorbate reductase from Pinus bungeana.
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DOI：
10.1111/j.1744-7909.2009.00848.x
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发表时间：
2009-11
影响因子：
11.4
通讯作者：
Hai-Ling Yang;Yingdong Zhao;Cai-Ling Wang;Zhiling Yang;Q. Zeng;Hai Lu
中科院分区：
文献类型：
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作者：
Hai-Ling Yang;Yingdong Zhao;Cai-Ling Wang;Zhiling Yang;Q. Zeng;Hai Lu
摘要脱氢抗坏血酸还原酶（DHAR）在大多数高等植物的抗坏血酸-谷胱甘肽循环反应中起着至关重要的作用。迄今为止，高等植物中DHAR的研究主要集中在拟南芥和农业植物上，裸子植物中DHAR的分子特征研究几乎没有。本文报道了白皮松（Pinus bungeana Zucc） DHAR基因的克隆及其特征。Endl交货。PbDHAR基因编码一个由215个氨基酸残基组成的蛋白质，计算分子质量为24.26 kDa。预测PbDHAR的三维结构表现为典型的谷胱甘肽s -转移酶折叠。逆转录聚合酶链反应表明，PbDHAR基因是白杨的组成表达基因。高温胁迫下，白杨幼苗PbDHAR mRNA表达量无显著变化。经亲和层析纯化后，重组PbDHAR在大肠杆菌中过表达。重组PbDHAR具有19.84微mol/min / mg的酶活性和对底物脱氢抗坏血酸（DHA）的高亲和力（K(m)为0.08 mM）。此外，重组PbDHAR是一种耐热酶，在55℃下仍保持77%的初始活性。本研究首次提供了白木中DHAR的详细分子表征。
Abstract 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 micromol/min per mg) and high affinity (a K(m) 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 degrees C. The present study is the first to provide a detailed molecular characterization of the DHAR in P. bungeana.