肥城桃果实<em>2-Cys Prx</em>基因克隆、生物信息学分析及原核表达

doi:10.7668/hbnxb.2018.03.009

摘要/Abstract

摘要： 为了深入研究2-Cys Prx基因调控桃果实氧化还原信号分子作用机制，结合了PCR技术和RT-PCR技术通过体外克隆的方法得到桃果实2-Cys Prx基因，通过对序列分析表明，2-Cys Prx基因全长1 424 bp，其中，编码区813 bp，共编码270个氨基酸。对其分子结构进行分析，2-Cys Prx蛋白分子量为29.7 ku，为疏水型蛋白，且是膜外蛋白。在线软件预测2-Cys Prx蛋白分子式为C₁₃₄₂H₂₁₁₀N₃₄₈O₄₀₃S₄，分子量29.696 ku，由4 207个原子组成，理论等电点为6.23。使用ProtScale分析该蛋白疏水性平均值为-0.126，说明2-Cys Prx为亲水性蛋白。TMHMM对跨膜结构进行预测，发现，2-Cys Prx氨基酸序列中不存在跨膜结构域，为膜外蛋白。此外，对该蛋白二级结构进行预测，发现2-Cys Prx有4个α螺旋结构、9个β折叠结构及无规则卷曲构成。系统进化树分析表明，肥城桃果实2-Cys Prx与其他植物氨基酸序列有较高同源性，其中与樱桃的亲缘关系最近，相似度达到了98.15%，表明植物体内2-Cys Prx具有较高的保守性。体外构建重组蛋白pET-30a-2-Cys Prx成功在大肠杆菌BL21中表达，为下一步研究其功能与结构，从而探讨2-Cys Prx基因调控桃果实氧化还原信号作用机理奠定了基础。

关键词: 桃果实, 2-Cys Prx, 基因克隆, 蛋白诱导表达

Abstract: In order to study the regulation mechanism of 2-Cys Prx gene to redox signal molecules. 2-Cys Prx of Feicheng peach was studied by gene cloning and prokaryotic expression including PCR technique and RT-PCR technique. The experimental results showed that the full length of 2-Cys Prx gene was 1 424 bp induced 813 bp of coding sequence encoding a polypeptide of 270 amino acids,with predicted molecular weight of 29.7 ku. The predicted protein molecular formula of 2-Cys Prx was C₁₃₄₂H₂₁₁₀N₃₄₈O₄₀₃S₄ with a molecular weight of 29.696 ku,which contained 4 207 atoms. For 2-Cys Prx,the theoretical isoelectric point was about 6.23,and the total average hydrophobicity index was -0.126,which indicated that 2-Cys Prx protein was a hydrophilic protein. The two level prediction of 2-Cys Prx protein composed of 9 segment of fold and 4 segment of alpha helix and the irregular. Phylogenetic tree analysis showed that the 2-Cys Prx was most closely related to Prunus avium,with 98.15% amino acid homology rate. The high homology of amino acids of 2-Cys Prx in peach fruit and other plants,indicating that 2-Cys Prx was more conservative. These results would lay a foundation for researching the structure and function of 2-Cys Prx protein to explore the mechanism of 2-Cys Prx regulation of redox signal molecules in the further study.

Key words: Peach fruit, 2-Cys Prx, Gene cloning, Protein induced expression

中图分类号:

Q78
S662.03

张永淋, 高彬, 周杰, 朱树华. 肥城桃果实2-Cys Prx基因克隆、生物信息学分析及原核表达[J]. 华北农学报, 2018, 33(3): 50-55. doi: 10.7668/hbnxb.2018.03.009.

ZHANG Yonglin, GAO Bin, ZHOU Jie, ZHU Shuhua. Cloning,Bioinformatics Analysis and Protein Induced Expression of 2-Cys Prx Gene in Feicheng Peach Fruits[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(3): 50-55. doi: 10.7668/hbnxb.2018.03.009.

http://www.hbnxb.net/CN/Y2018/V33/I3/50

参考文献

[1] Pulido P C,Cejudo F J. An antioxidant redox system in the nucleus of wheat seed cells suffering oxidative stress[J]. Plant Journal,2009,57(1):132-145.
[2] Camejo D. Salinity-induced changes in S-nitrosylation of pea mitochondrial proteins[J]. Proteomics,2013,79:87-99.
[3] Calderon A. Glutathionylation of pea chloroplast 2-Cys Prx and mitochondrial Prx Ⅱ F affects their structure and peroxidase activity and sulfiredoxin deglutathionylates only the 2-Cys Prx[J]. Front Plant Sci,2017,8:118.
[4] Cheng F,Yin L L,Zhou J,et al. Interactions between 2-Cys peroxiredoxins and ascorbate in autophagosome formation during the heat stress response in Solanum lycopersicum[J]. Journal of Experimental Botany,2016,67(6):1919-1933.
[5] Bhardwaj P K,Kumar S. 2-Cys peroxiredoxin responds to low temperature and other cues in Caragana jubata,a plant species of cold desert of Himalaya[J]. Molecular Biology Reports,2014,41(5):2951-2961.
[6] Park J,Lee S,Kang S W,et al. 2-cys peroxiredoxins:emerging hubs determining redox dependency of Mammalian signaling networks[J]. International Journal of Cell Biology,2014:715867.http://dx.doi.org/10.1155/2014/715867.
[7] Bernal-Bayard B P. Molecular recognition in the interaction of chloroplast 2-Cys peroxiredoxin with NADPH-thioredoxin reductase C(NTRC)and thioredoxin x[J]. FEBS Letters,2014,588(23):4342-4357.
[8] Moon J C,Hah Y S,Kim W Y,et al. Oxidative stress-dependent structural and functional switching of a human 2-Cys peroxiredoxin Isotype Ⅱ that enhances HeLa cell resistance to H₂O_2-induced cell death[J]. Journal of Biological Chemistry,2005,280(31):28775-28784.
[9] Hong S H,Lee S S,Chung J M,et al. Site-specific mutagenesis of yeast 2-Cys peroxiredoxin improves heat or oxidative stress tolerance by enhancing its chaperone or peroxidase function[J]. Protoplasma,2017,254(1):327-334.
[10] Awad J. 2-cysteine peroxiredoxins and thylakoid ascorbate peroxidase create a water-water cycle that is essential to protect the photosynthetic apparatus under high light stress conditions[J]. Plant Physiology,2015,167(4):1592-1603.
[11] Kim M D,Kim Y H,Kwon S Y,et al. Overexpression of 2-cysteine peroxiredoxin enhances tolerance to methyl viologen-mediated oxidative stress and high temperature in potato plants[J]. Plant Physiology and Biochemistry,2011,49(8):891-897.
[12] Cerveau D. Characterization of the Arabidopsis thaliana 2-Cys peroxiredoxin interactome[J]. Plant Science,2016,252:30-41.
[13] Dangoor I,Peled-Zehavi H,Wittenberg G,et al. A chloroplast light-regulated oxidative sensor for moderate light intensity in Arabidopsis[J]. Plant Cell,2012,24(5):1894-1906.
[14] Dietz K J. Thiol-based peroxidases and ascorbate peroxidases:why plants rely on multiple peroxidase systems in the photosynthesizing chloroplast?[J].Molecules and Cells,2016,39(1):20-25.
[15] Jing G,Zhou J,Zhu S. Effects of nitric oxide on mitochondrial oxidative defence in postharvest peach fruits[J]. Journal of the Science of Food and Agriculture,2016,96(6):1997-2003.
[16] 陈长宝,朱树华,周杰. 改良CTAB法提取成熟肥城桃果实的总RNA[J]. 山东农业科学,2009(5):102-104.
[17] Barrancomedina S,Kakorin S,Lázaro J J,et al. Thermodynamics of the dimer-decamer transition of reduced human and plant 2-cys peroxiredoxin[J]Biochemistry,2008,47(27):7196-7204.
[18] Lee E M,Lee S S,Tripathi B N,et al. Site-directed mutagenesis substituting cysteine for serine in 2-Cys peroxiredoxin (2-Cys Prx A) of Arabidopsis thaliana effectively improves its peroxidase and chaperone functions[J]. Annals of Botany,2015,116(4):713-725.
[19] Cerveau D,Ouahrani D,Marok M A,et al. Physiological relevance of plant 2-Cys peroxiredoxin overoxidation level and oligomerization status[J]. Plant Cell and Environment,2016,39(1):103-119.
[20] Pace P E,Peskin A V,Han M H,et al. Hyperoxidized peroxiredoxin 2 interacts with the protein disulfide-isomerase ERp46[J].Biochemical Journal,2013,453(3):475-485.
[21] Peskin A V,Low F M,Paton L N,et al. The high reactivity of peroxiredoxin 2 with H₂O₂ is not reflected in its reaction with other oxidants and thiol reagents[J]. Journal of Biological Chemistry,2007,282(16):11885-11892.
[22] Dietz K J. Peroxiredoxins in plants and cyanobacteria[J]. Antioxidants & Redox Signaling,2011,15(4):1129-1159.
[23] Gao J. Enhanced fermentative performance under stresses of multiple lignocellulose-derived inhibitors by overexpression of a typical 2-Cys peroxiredoxin from Kluyveromyces marxianus[J].Biotechnol Biofuels,2017,10:79.

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肥城桃果实2-Cys Prx基因克隆、生物信息学分析及原核表达

Cloning,Bioinformatics Analysis and Protein Induced Expression of 2-Cys Prx Gene in Feicheng Peach Fruits

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肥城桃果实2-Cys Prx基因克隆、生物信息学分析及原核表达

Cloning,Bioinformatics Analysis and Protein Induced Expression of 2-Cys Prx Gene in Feicheng Peach Fruits

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