达坂喜盐芽孢杆菌D-8^T在低渗冲击下的双向凝胶电泳分析  

Two-dimensional gel electrophoresis analysis of moderately halophilic bacterium Halobacillus dabanensis D-8^T under hypoosmotic shock condition

作　　者：冯德芹[1] 解利石[1] 李小红[1] 杨苏声[1] 

机构地区：[1]中国农业大学生物学院农业部农业微生物资源及其应用重点开放实验室,北京100094

出　　处：《微生物学报》2006年第5期740-744,共5页Acta Microbiologica Sinica

基　　金：国家"863计划"(2003AA241150)~~

摘　　要：为了解革兰氏阳性中度嗜盐菌适应低渗冲击的机制，采用双向凝胶电泳技术，研究达坂喜盐芽孢杆菌（Halobacillus dabanensis）D-8^T在低渗冲击下的差异蛋白表达谱。利用Imagemaster^TM 2D Platinum软件分析到大约650个蛋白点，大多数蛋白分子量分布在17．5～66kDa，等电点为4．0～5．9，偏酸性。在20％盐浓度中生长的D-8菌株受到0％盐浓度的低渗冲击5min及50min后34个蛋白点的表达发生改变，包括20个表达上调的点和14个点表达下调。用MALDI—TOF／MS及MASCOT软件鉴定了4个与低渗胁迫有关的蛋白，分别为热激蛋白DanK、柱状决定蛋白、青霉素结合蛋白和5-莽草酸烯醇式丙酮酸-3-磷酸合成酶。其中，热激蛋白适应低渗胁迫时表达上调为首次报道。Halobacillus dabanensis D-8^T was isolated from the saline deposits of Daban lake in Xinjiang of China, and is able to grow in complex medium containing 0.5 % to 25 % salt. To figure out the survival mechanisms of Gram-positive moderately halophilic bacteria under hypoosmotic shock conditions, two-dimensional gel electrophoresis （2-DE） was carried out to investigate differential protein expression profiles of H. dabanensis D-8^T in response to low osmotic challenge. The 2-D gels were stored as dry gels and their images were taken by ImageScanner and analyzed by ImagemasterTM 2D Platinum software. About 650 protein spots were detected in 2-D gel. Most of proteins were distributed in molecular mass of 17.5 - 66kDa and the range of isoelectric point 4.0 - 5.9. A total of 34 protein spots were found to alter their expression after strain D-8T was subjected to hypoosmotic shock from 20% to 0% salinity for 5 min and 50 min. Among them, the expression of 20 protein spots is up-regulated including 6 new protein spots, while that of 14 protein spots is down-regulated in answer to sudden osmotic down-shift. Protein spots of interest were excised from the gels and digested by trypsin. By means of matrix-assisted laser desorption ionization-time of flight mass spectrometry （MALDI-TOF/MS） and MASCOT search engine, 4 up-regulated protein spots were identified with peptide mass fingerprint, and are similar to heat shock protein DanK, rod shape-determining protein, penicillin-binding protein （ PBP- 1A） and 5-enolpyruvoylshikimate-3-phosphate synthase, respectively. Noticeably, PBP-1A firstly was up-regulated after shock of 5 min but disappeared after shock of 50 min. This indicated that the strain activate a minor mechanism of peptidoglycan synthesis to compensate the major synthesis mechanism for ceils survival through a down-shift challenge. In addition, this paper was the first report that heat shock proteins were up-regulated in response to sudden osmotic downshift.

关 键 词：达坂喜盐芽孢杆菌 低渗冲击 双向凝胶电泳 蛋白表达 

分 类 号：Q93-3[生物学—微生物学]