盐生杜氏藻CPD光裂合酶FAD结合结构域Gln336在逆境胁迫下的修复活性  

Repair activity of Gln336 on the FAD binding domain of CPD photolyase in Dunaliella salina under stress

作　　者：姜腾飞[1] 王雨昊[1] 唐滋一 黄国印 徐辉[1] 乔代蓉[1] 曹毅[1] 

机构地区：[1]四川大学生命科学学院,微生物与代谢工程四川省重点实验室,成都610065

出　　处：《应用与环境生物学报》2017年第2期238-243,共6页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(31670078);四川省科技厅项目(2014GZX0005;2017TJ PT0001);国家微生物资源平台(NIMR-2016-8-1)资助~~

摘　　要：为了解盐生杜氏藻环丁烷嘧啶二聚体(CPD)光裂合酶的作用机制,通过定点突变的方法对其黄素腺嘌呤二核苷酸(FAD)结合结构域α13中保守氨基酸残基Gln336进行突变,并比较野生型菌株PGEX-4T-1-Ds PHR2(WT)和突变菌株PGEX-4T-1-Ds PHR2-Q336H(Q336H)表达的光裂合酶在体内外的光修复活性及其在不同盐浓度下修复光损伤的效果.结果显示:采用Dpn I法定点突变,成功获得盐生杜氏藻CPD光裂合酶突变体Q336H的基因,构建表达载体并导入到大肠杆菌BL21(DE3)中,构建了突变菌株Q336H.体内外活性研究发现,野生型菌株的CPD光裂合酶的活性显著大于突变菌株Q336H(P<0.05).在不同盐浓度条件下,野生型菌株存活率基本没有变化,而突变菌株Q336H随着盐浓度增加存活率迅速下降.在体外修复实验中,甘油浓度对突变酶Q336H活性影响显著大于对CPD光裂合酶活性影响(P<0.05).甘油浓度增加导致突变酶Q336H修复活性逐渐下降,而CPD光裂合酶活性变化趋势是先增加后下降.因此,Gln336对盐生杜氏藻CPD光裂合酶活性具有重要影响,而且可能是该酶在盐胁迫下发挥功能的关键氨基酸残基.In order to explore the repair mechanism of cyclobutane pyrimidine dimers （CPD） photolyase in Dunaliella salina, we mutated the α13 conserved amino acid residue Gln336 in the FAD binding domain by site-directed mutagenesis. Thereafter, we compared the repair activity of the photolyase in the wild-type strain PGEX-4T-1-DsPHR2 （WT） and the mutant strain PGEX-4T-1-DsPHR2-Q336H （Q336H） in vitro and in vitro and under different salt concentrations. The results indicated that through site-directed mutagenesis with Dpn I, we successfully obtained the Q336H gene, which was subsequently transformed into Escherichia coli BL21 （DE3） to construct a mutant Q336H. The results of in vivo and in vitro experiments demonstrated that the repair activity of the photolyase in the wild-type strain was significantly greater than that in the Q336H mutant （P 〈 0.05）. Under different salt concentrations, the survival rate declined rapidly as salinity increased in the mutant Q336H, while in the wild-type strain, there was no change in the survival rate. In vitro repair experiments showed that the influence of glycerol concentration on the enzymatic activity in the Q336H mutant was significantly greater than that in wild type CPD photolyase （P 〈 0.05）. The repair activity of the mutant enzyme Q336H decreased gradually as glycerol concentration increased, while for CPD photolyase, the trend for repair activity under different glycerol concentrations first increased and then decreased. Therefore, these results indicate that Gln336 is important for the repair activity of CPD photolyase in D. salina and may represent key amino acid residues under salt stress.

关 键 词：CPD光裂合酶 作用机制 FAD结合结构域 保守氨基酸 盐生杜氏藻 

分 类 号：Q943.2[生物学—植物学]