Slr0351的表达及其在Synechocystis sp.PCC 6803中功能的初步研究  

Expression and preliminary function analysis of Slr0351 in Synechocystis sp. PCC 6803

作　　者：马琼[1,2] 郑小江[1,2] 赵开弘[3] 周明[3] 王胜鹏[4] 

机构地区：[1]湖北民族学院生物资源保护与利用湖北省重点实验室,湖北恩施445000 [2]湖北民族学院生物科学与技术学院,湖北恩施445000 [3]华中农业大学农业微生物学国家重点实验室,湖北武汉430070 [4]湖北省农业科学院果树茶叶研究所,湖北武汉430071

出　　处：《水产学报》2015年第1期16-23,共8页Journal of Fisheries of China

基　　金：湖北省自然科学基金面上项目(2014CFB400);国家自然科学基金(31070743;31400586);湖北民族学院科技创新团队项目(MY2014T005);湖北民族学院博士科研启动基金项目(MY2013B013);湖北民族学院林学省级重点学科

摘　　要：Slr0351是Synechocystis sp.PCC 6803中的未知功能蛋白,其同源蛋白广泛存在于各种蓝藻和铁硫杆菌中。为了确定Slr0351的性质,构建了表达质粒p ET-slr0351,并在E.coli中表达Slr0351。在无氧条件下采用亲和层析纯化方法获得了Slr0351,无氧条件下Slr0351呈棕红色,在460 nm处有[2Fe-2S]铁硫簇的特征吸收峰,棕红色Slr0351对氧气敏感,能被连二亚硫酸钠还原,由此表明Slr0351为铁硫蛋白。为了获知slr0351的功能,基于基因同源重组交换的原理,利用Kana抗性片段替换slr0351,将Synechocystis sp.PCC 6803中的slr0351基因缺失,构建了Δslr0351突变体。利用紫外-可见吸收光谱仪扫描了Δslr0351与野生型Synechocystis sp.PCC 6803(WT)的吸收光谱,发现正常光照条件下Δslr0351的叶绿素a仅为WT的68.8%,slr0351的缺失使蓝藻中叶绿素a含量降低。比较了藻细胞在缺乏不同营养元素和不同光照条件下的生长速率差异,与WT相比,Δslr0351具有如下特征:(1)对缺Fe和缺S胁迫条件更敏感;(2)在弱光照条件下Δslr0351的光能利用效率和生长速率更低,该现象与Δslr0351中叶绿素a含量的降低有关。Slr0351 is an unknown protein and its homologs exist widely in many cyanobacteria and iron- sulfur bacteria. We constructed pET-s/r0351 and expressed Slr0351 successfully in E. coli BL21. Using affinity chromatography purification, we obtained purified Slr0351. Under anaerobic conditions, Slr0351 was brown and had absorption peak at 460 nm,which was the characteristic absorption peak of 2Fe -2S cluster. The brown Slr0351 is sensitive to oxygen and could be reduced with sodium dithionite,showing that Slr0351 was iron-sulfur protein. By homologous double-crossover, s/r0351 was replaced by Kana, resulting in the knockout mutant of Synechocystis sp. PCC 6803 ,△s/r0351. Via scanning the absorption spectra of △s/r0351 and WT with ultraviolet-visible spectrometer, we found that chlorophyll a content of △slr0351 is only 68.8 % that of WT under the medium light condition. The depletion of s/r0351 reducing the content of chlorophyll a in Synechocystis cells. Compared the growth rate of cyanobacteria under different culture conditions, △slr0351 showed the following changes compared to the wild type： （1）more sensitive to sulfur and iron starvation;（2） lower efficiency of light utilization and lower growth rate under LL conditions, which is related to the lower content of chlorophyll a in △slr0351. This paper is beneficial to gene function research of Cyanobacteria, and provides a certain reference value for the treatment of Cyanobacteria bloom and pollution in freshwater.

关 键 词：蓝藻 SYNECHOCYSTIS SP.PCC 6803 Slr0351 铁硫蛋白 

分 类 号：Q753[生物学—分子生物学] S917.3[农业科学—水产科学]