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Cloning and Function Characterization of Two Dehydroascorbate Reductases from Kiwifruit (Actinidia chinensis L.)

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Abstract

Dehydroascorbate reductase (DHAR, EC 1.8.5.1) plays a critical role in the regeneration of l-ascorbic acid (AsA). To date, there is virtually no information on the molecular characteristics of DHAR in kiwifruit, an economically and nutritionally important horticultural crop with remarkably high AsA concentration. Here, we isolated two cDNAs encoding putative DHARs (designated as AcDHAR1 and AcDHAR2) from Actinidia chinensis cv. Hongyang. Both in silico and subcellular localization analyses demonstrated that AcDHAR1 and AcDHAR2 were targeted to cytosol and chloroplast, respectively. The recombinant AcDHAR1 and AcDHAR2 were expressed in Escherichia coli and purified using Ni-affinity chromatography. Enzymatic study shows both of them are thermostable and possess a relatively high affinity to dehydroascorbate with an optimal pH ranging from 6 to 8. In addition, transgenic Arabidopsis thaliana plants separately expressing either AcDHAR1 or AcDHAR2 were shown to have significantly increased AsA concentration and enhanced tolerance to salinity. The present study suggested that AcDHAR1 and AcDHAR2 may play a protective role in response to environmental stimuli in kiwifruit.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 31471157 and 31461143008), the National Basic Research Program of China (973 Program) (No. 2011CB100401), a Key Project from the Government of Sichuan Province (No. 2013NZ0014), a Project from the Government of Anhui Province (No. 2012AKKG0739), and a National Science Fund for Distinguished Young Scholars (No. 30825030).

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    Authors and Affiliations

    1. Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu, 610064, China

      Fangfang Liu, Xiuhong Guo, Yangchun Yao & Yongsheng Liu

    2. School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, China

      Wei Tang, Wei Zhang, Shuqing Cao, Yi Han & Yongsheng Liu

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    1. Fangfang Liu
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    Correspondence to Yi Han or Yongsheng Liu.

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    Fangfang Liu and Xiuhong Guo contributed equally to this work.

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    Liu, F., Guo, X., Yao, Y. et al. Cloning and Function Characterization of Two Dehydroascorbate Reductases from Kiwifruit (Actinidia chinensis L.). Plant Mol Biol Rep 34, 815–826 (2016). https://doi.org/10.1007/s11105-015-0965-8

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