高等学校化学学报

高等学校化学学报 ›› 2013, Vol. 34 ›› Issue (1): 128.doi: 10.7503/cjcu20120184

• 生物化学 • 上一篇    下一篇

铜离子与SALI3-2蛋白的相互作用

唐玉林1,3, 高占1,3, 徐宏2,3, 贺建芝2,3, 董一含2,3, 郑易之1,3   

  1. 1. 深圳市微生物基因工程重点实验室, 深圳 518060;
    2. 深圳市海洋生物资源与生态环境重点实验室, 深圳 518060;
    3. 深圳大学生命科学学院, 深圳 518060
  • 收稿日期:2012-03-05 发布日期:2012-12-31
  • 通讯作者: 唐玉林,女,博士,副教授,主要从事植物分子生物学研究.E-mail:yltang@szu.edu.cn;郑易之,女,博士,教授,主要从事植物抗逆分子生物学研究.E-mail:yzzheng@szu.edu.cn E-mail:yltang@szu.edu.cn;yzzheng@szu.edu.cn
  • 基金资助:

    国家自然科学基金(批准号: 30770184)和深圳市科技计划项目(批准号: 200714)资助.

Interaction of Copper(II) Ion with SALI3-2

TANG Yu-Lin1,3, GAO Zhan1,3, XU Hong2,3, HE Jian-Zhi2,3, DONG Yi-Han2,3, ZHENG Yi-Zhi1,3   

  1. 1. Shenzhen Key Laboratory of Microbial and Gene Engineering, Shenzhen 518060, China;
    2. The Key Laboratory for Marine Bioresource and Eco-environmental Science, Shenzhen 518060, China;
    3. College of Life Science, Shenzhen University, Shenzhen 518060, China
  • Received:2012-03-05 Published:2012-12-31

PDF (PC)

被引次数

8

摘要：

用1/2 MS固体培养基培养拟南芥幼苗, 研究了CuCl2对转Sali3-2基因植株生长的影响, 发现转基因植株Sali3-2的表达可提高其耐受Cu2+胁迫的能力. 进一步克隆Sali3-2基因, 表达并纯化了缺失信号肽的 SALI3-2 蛋白. 利用固定金属离子亲和色谱(IMAC)分析发现, Cu2+ 能够与 SALI3-2 蛋白结合. 通过荧光光谱及圆二色光谱(CD)进一步研究了Cu2+ 与 SALI3-2 蛋白间的键合机理. 结果表明, Cu2+ 可引起SALI3-2 蛋白内源性荧光猝灭, 其猝灭机制为静态猝灭; Cu2+ 与SALI3-2 蛋白的结合常数为8.89×106, 结合位点数为1.6. CD分析显示, Cu2+ 与SALI3-2 蛋白的结合未使SALI3-2 蛋白二级结构发生明显改变. 由此推测, 在高浓度铜离子胁迫下, SALI3-2蛋白通过结合一定数量的Cu2+使蛋白的构象发生改变, 这可能是SALI3-2蛋白的表达使植物耐受Cu2+胁迫能力提高的分子机制之一.

关键词: 铜离子, SALI3-2 蛋白, 金属离子亲和层析, 荧光光谱, 圆二色光谱

Abstract:

The effect of CuCl2 on the growth of the transgenic Arabidopsis carrying Sali3-2 gene was studied on 1/2 MS agar plates. The over-expression of Sali3-2 was elevated the tolerance of transgenic Arabidopsis seedling to Cu2+ stress. Sali3-2 gene was then cloned and the SALI3-2 protein deleted signal peptide was expressed and purified. SALI3-2 protein interaction with Cu2+ was further studied by immobilized metal ion affinity chromatography(IMAC). The results show that the protein can bind with Cu2+. The mechanism of Cu2+-SALI3-2 bonding was further analyzed by fuorescence spectrum and circular dichroism(CD) spectrum. Fluorescence analysis shows intrinsic fluorescence of SALI3-2 is quenched with the addition of Cu2+, which belongs to static fluorescence quenching. Relatively high affinity of SALI3-2 binding Cu2+is revealed with a constant of 8.89×106 and a number of binding sites of 1.6. The secondary structure of SALI3-2 is not changed obviously with the addition of Cu2+ as shown in CD spectra.These results suggest that SALI3-2 is a Cu2+-binding protein. It may function by altering its conformation through binding Cu2+ to trigger the regulation mechanism and elevate the tolerance of cells to Cu2+ stress. The research provides the direct insight into molecular mechanism of the role of SALI3-2 in plant tolerance to Cu2+stress.

Key words: Cu2+, SALI3-2 protein, Metal ion affinity chromatography, Fluorescence spectrum, Circular dichroism spectrum

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