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| Cloning and Analysis of a Novel Gene Codifying for Violaxanthin De-epoxidase from Lycium chinense |
| ZHANG Xu-qiang1, JI Jing1, WANG Gang1, ZHONG Ying1, DIAO Jin-jin1, GUAN Chun-feng1, WU Jiang2,3, JIN Chao1 |
1. School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China;
2. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China;
3. Department of Agronomy, Tianjin Agricultural University, Tianjin 300384, China |
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Abstract Objective: Violaxanthinde-epoxidase play an important role in the violaxanthin cycle.The aim of the study is toclone the full-length cDNA ofLcVDE from Lycium chinense,to predict the architectural feature, functional sites and the secondary structures of LcVDE through bioinformatics analysis and to confirm its functions. Method:The full-length cDNA of LcVDE was cloned from Lycium chinense using rapid-amplification of cDNA ends essay and RT-PCR. Bioinformatics software were used to analyse the amino acid sequences of LcVDE protein, and constructed the prokaryotic expression vectorp ET-VDE and transformed into E.coli BL21. Overexpression of LcVDE in the E.coli BL21 was induced by IPTG, and in the vitro system the LcVDE function was confirmed. Results: Bioinformatics analysis showed that the open reading frame of LcVDE gene is 1 413bp,encodes a putative polypeptide of 470aa with molecular mass of 53.61 kDa and isoelectric point of 5.77.According to the analysis of SDS-PAGE, the LcVDE over-expressed in E.coli.The protein was added to thevitro reaction system, absorption spectrometry and HPLC indicated that the expressed protein catalyzed violaxanthin de-epoxidationreaction. Conclusion: In this study, the protein encoded by the LcVDE gene has the same function toviolaxanthin de-epoxidase.
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Received: 15 November 2013
Published: 25 January 2014
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