盐穗木金属硫蛋白的原核表达及功能活性分析

Analysis of Prokaryotic Expression and Functional Activity of Halostachys Caspica Metallothionein

为了探究盐穗木金属硫蛋白(HcMT)对金属离子的结合能力和转基因工程菌对非生物协迫的耐受性,将HcMT基因亚克隆至原核表达载体pET-32a上.诱导表达后,用5-Br-PAPS方法测定融合蛋白TrxAHcMT与Zn^(2+)离子的结合能力,并用原子吸收分光光度法测定其与多种金属离子的结合能力,用含有非生物协迫因素的培养基培养转基因工程菌,探究其对非生物协迫的耐受性.结果显示在诱导过程中添加Zn^(2+),融合蛋白TrxA-HcMT反应体系中吸光值与对照TrxA相比显著增高.TrxA-HcMT能够结合多种金属离子,其中对Cu2+的结合能力最强,是对照的20.3倍,对Cd^(2+)的结合能力次之,是对照的5.6倍.在H_2O_2、NaHCO_3、Na_2CO_3非生物协迫的条件下,转HcMT基因工程菌比对照组菌的耐受性更好.

To investigate the metal-binding ability and tolerance of transformed bacteria to abiotic stress of Halostachys caspica metallothionein,the HcMT gene was subcloned into prokaryotic expression vector pET-32 a.After inducing expression,the binding capacity of the fusion protein TrxA-HcMT and Zn^（2＋） were determined by 5-Br-PAPS method,and the binding capacity of the fusion protein with various metal ions was determined by atomic absorption spectrophotometry.The transformed bacteria were cultured with culture medium containing abiotic stress factors to study its tolerance to abiotic stress.The results showed that the absorbance value of the fusion protein TrxA-HcMT reaction system was significantly higher than that of the control TrxA with the addition of Zn^（2＋） in the induction process.TrxA-HcMT can combine a variety of metal ions,which is the strongest binding capacity of Cu^（2＋）,is 20.3 times of the control,the combination of the ability of Cd^（2＋） is 5.6 times of the control.Under the abiotic stress of H_2O_2,NaHCO_3 and Na_2CO_3,the HcMT gene transformed bacteria was better tolerated than the control bacteria.