【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.更多还原

【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.更多还原