铜离子与SALI3-2蛋白的相互作用

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.

Interaction of Copper(II) Ion with SALI3-2

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.