一个水稻小热休克蛋白的异源表达及寡聚特性分析

doi:10.16819/j.1001-7216.2017.7010 483

中国水稻科学 ›› 2017, Vol. 31 ›› Issue (5): 483-488.DOI: 10.16819/j.1001-7216.2017.7010 483

一个水稻小热休克蛋白的异源表达及寡聚特性分析

蔡年俊^1,², 郭留明^1,², 李静², 项聪英^1,², 羊健², 陈剑平^2,^*, 张恒木^2,^*()

¹浙江师范大学化学与生命科学学院, 浙江金华321004
²浙江省农业科学院病毒学与生物技术研究所,杭州 310021

收稿日期:2017-01-18 修回日期:2017-02-21 出版日期:2017-10-10 发布日期:2017-09-10
通讯作者: 陈剑平,张恒木
基金资助:
国家自然科学基金资助项目(31601603);浙江省自然科学基金资助项目(LQ14C140003)

Heterologous Expression and Oligomeric Identification of a Small Heat Shock Protein (SHSP) from Oryza sativa

Nianjun CAI^1,², Liuming GUO^1,², Jing LI², Congying XIANG^1,², Jian YANG², Jianping CHEN^2,^*, Hengmu ZHANG^2,^*()

¹College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China
²State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Key Laboratory of Biotechnology in Plant Protection of MOA and Zhejiang Province, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2017-01-18 Revised:2017-02-21 Online:2017-10-10 Published:2017-09-10
Contact: Jianping CHEN, Hengmu ZHANG

摘要/Abstract

摘要：

目的在前期研究中,本实验室已克隆了一个水稻小热休克蛋白基因(OsSHSP17.6),并发现该基因的表达明显受到热激和病毒侵染调控,表明该蛋白可能在逆境胁迫过程中起重要作用。本研究的目的在于进一步明确OsSHSP17.6的特性。方法在本研究中,进一步将该基因亚克隆至原核表达载体pET-32a并导入大肠杆菌E. coli BL21(DE3)pLysS诱导表达,通过亲和层析的方法纯化了该重组蛋白,进一步用于非变性聚丙烯酰胺凝胶电泳和Western blotting分析。结果异源表达的重组OsSHSP17.6能减轻IPTG对宿主菌的毒害。非变性聚丙烯酰胺凝胶电泳和Western blotting分析显示纯化的重组OsSHSP17.6蛋白在体外能形成同源二聚体和寡聚体。结论这些结果支持OsSHSP17.6是一个有功能的分子伴侣蛋白并表明该蛋白可能通过形成同源寡聚体的方式参与逆境胁迫反应,这为进一步明确OsSHSP17.6的功能机制奠定基础。

关键词: 小热休克蛋白, 异源表达, 寡聚化作用

Abstract:

【Objective】In our previous study, a small heat shock protein gene OsSHSP17.6 was cloned from Oryza sativa and its expression was shown to be significantly up-regulated by heat shock or viral infection, suggesting that the OsSHSP17.6 could play an important role in both biotic and abiotic stress responses. In this study, our objective is to further identify the characteristics of OsSHSP17.6. 【Method】 The OsSHSP17.6 gene was sub-cloned into the plasmid pET-32a, a prokaryotic expression vector, and transformed into Escherichia coli BL21(DE3)pLysS for inducible expression. Then the recombinant protein was purified with affinity chromatography and used for native PAGE and Western-blotting assays. 【Result】Its heterologous expression appeared to alleviate the poisonous effect of IPTG on the host E. coli. Native PAGE and Western-blotting assays showed that the purified recombinant OsSHSP17.6 could form homological dimers and oligomer in vitro. 【Conclusion】 Taken together, these findings supported the hypothesis that the protein should be functional molecular chaperone in vivo and indicated that OsSHPS17.6 could be involved in the stress response by its homological oligomerization, which could contribute to the functional identification of OsSHSP17.6.

Key words: small heat shock protein (SHSP), heterologous expression, oligomerization

中图分类号:

蔡年俊, 郭留明, 李静, 项聪英, 羊健, 陈剑平, 张恒木. 一个水稻小热休克蛋白的异源表达及寡聚特性分析[J]. 中国水稻科学, 2017, 31(5): 483-488.

Nianjun CAI, Liuming GUO, Jing LI, Congying XIANG, Jian YANG, Jianping CHEN, Hengmu ZHANG. Heterologous Expression and Oligomeric Identification of a Small Heat Shock Protein (SHSP) from Oryza sativa[J]. Chinese Journal OF Rice Science, 2017, 31(5): 483-488.

图/表 4

图1 OsSHSP17.6二级及高级结构预测

Fig. 1. Secondary and tertiary structure predictions of OsSHSP17.6.

图2 重组质粒pET-OsSHSP17.6的鉴定(A)及其重组表达分析(B)

Fig. 2. Identification of recombinant plasmid pET-OsSHSP17.6(A) and detection of recombinant OsSHSP17.6 (B) in E. coli.

图3 重组OsSHSP17.6蛋白的活性分析

Fig. 3. Activity assays of recombinant OsSHSP17.6 protein in E. coli.

图4 PAGE和Western blot分析纯化的重组OsSHSP17.6蛋白

Fig. 4. Assays of purified recombinant OsSHSP17.6 proteins by PAGE and Western blot.

参考文献 20

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一个水稻小热休克蛋白的异源表达及寡聚特性分析

Heterologous Expression and Oligomeric Identification of a Small Heat Shock Protein (SHSP) from Oryza sativa

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