盐藻胞质型3-磷酸甘油脱氢酶基因的克隆及性质鉴定

Cloning and Characterization of a Cytoplasmic Glycerol 3-phosphate Dehydrogenase Gene from Dunaliella salina

3-磷酸甘油脱氢酶(GPDH)是盐藻(Dunaliella salina)甘油合成途径上的关键酶,本研究首次报道了一条胞质型GPDH基因的克隆(命名为Ds GPDH1),经大肠杆菌表达和融合蛋白的纯化及性质鉴定,也研究了在盐胁迫期间DsGPDH1和DsGPDH2的表达变化。研究发现DsGPDH1和DsGPDH2相比,少了一段信号肽序列,但包含完整的SerB结构域和GPDH结构域。通过大肠杆菌制备的融合DsGPDH1蛋白高度可溶,但却没有NADH依赖的脱氢酶活性,其SerB结构域是否具有磷酸化酶的活性还没有研究清楚。在高盐胁迫时DsGPDH1和DsGPDH2都表现出诱导性高表达,在低盐胁迫时,DsGPDH1的表达被高度抑制,但DsGPDH2的表达变化却不太明显。GPDH同工酶在盐藻甘油合成中的具体分工仍然不清楚,但本研究迈出了坚实的一步。

Glycerol 3-phosphate dehydrogenase(GPDH)is the key enzyme on the glycerol synthesis pathway of Dunaliella salina.This paper reports for the first time the cloning,E.coli expression,recombinant protein purification and characterization of a cytoplasmic GPDH isoform(named DsGPDH1),the expression profile of DsGPDH1 is also investigated under salt stress and compared to that of Ds GPDH2.The results show that Ds GPDH1 lacks a signal peptide compare to DsGPDH2,but contains the entire SerB domain and the entire GPD domain.The fusion DsGPDH1 protein prepared by Escherichia coli is highly soluble but does not have NADH-dependent dehydrogenase activity,and whether the phosphoenzyme activity in the SerB structure domain has not been studied.Under high salt stress,DsGPDH1 and DsGPDH2 show high induced expression;under low salt stress,Ds GPDH1 expression is highly inhibited,but the change of DsGPDH2 is not obvious.The specific division of labor of GPDH isozymes in glycerol synthesis of salina is still unclear,but this study is a solid step forward.