南极表层海水低温微生物筛选及其低温适应机制探讨

Polar microorganism has a special cold-adapted mechanism during evolution. Cold shock proteins (Csps) are one of the most important factors in the antifreeze mechanism of psychrophile. However, few studies focused on the function and mechanism of psychrophile. In this study, surface seawaters of Drake Passage, Antarctic were used as materials. First, culture-dependent method was employed to isolate and investigate the diversity of psychrophile from surface seawaters located in the different longitude and latitude. Second, psychrophile which responded to cold shock was picked up and Csp genes were cloned. Function of the Csp genes were predicted by bioinformatics and EMSA technology. Dynamic expression of the Csp genes during the cold shock response were also characterized by Real Time PCR and Western blot. Third, Csp genes were transformed into E.coli to investigate the ability of cold-resistance in E.coli and further revealed the biological function of Csps. Furthermore, 2D- gel electrophoresis, co-Immunoprecipitation and mass spectrometry were employed to study the cold-adapted mechanism in Antarctic psychrophile. The results not only increase the knowledge of psychrophile resources from Antarctic, but also have a profound influence on the discovery of novel genes in relation to cold shock response, and the development of cold-adapted mechanism.

极地微生物在自然选择的作用下形成了一套独特的与低温环境相适应的分子机制。冷激蛋白（Csps）是低温微生物抗冻保护机制研究中最重要的内容之一。然而，目前对极地低温微生物Csps功能与作用机制的研究还十分有限。在本项目中，以南极表层海水为研究对象，首先利用培养手段筛选不同经纬度海水样品中的嗜冷菌/适冷菌，并阐明其多样性。在此基础上，选择具有冷激反应的低温菌，获得Csps全长序列，通过生物信息学与EMSA技术预测其功能。并利用Real Time PCR和Western blot检测其在冷激反应中的动态表达。进而通过检测转入Csps基因的大肠杆菌抗低温/冷冻的能力，揭示Csps的生物学功能。并利用双向凝胶电泳、免疫共沉淀与质谱技术探讨Csps在南极嗜冷菌/适冷菌低温适应性中的作用机制。研究结果不仅拓展对南极低温微生物资源的了解，而且对抗低温功能基因的发现和适冷机制的发展具有重要的理论和实践意义。

极地微生物在自然选择的作用下形成了一套独特的与低温环境相适应的分子机制。冷激蛋白（Csps）是低温微生物抗冻保护机制研究中最重要的内容之一。然而，目前对极地低温微生物Csps功能与作用机制的研究还十分有限。在本项目中，以南极表层海水为研究对象，首先利用培养手段筛选得到海水样品中128株低温微生物，并阐明其多样性。在此基础上，选择具有冷激反应的适冷菌Psychrobacter pulmonis Ant24，获得CspA和CspE全长序列，通过生物信息学与EMSA技术证明其具有分子伴侣功能。Real Time PCR检测结果显示CspA和CspE基因分别在适冷菌不同生长时期表达并在冷激反应24h表达量明显升高。之后又进一步证明转入Csps基因的大肠杆菌抗低温/冷冻的能力。利用双向凝胶电泳分析参与冷激反应的其他蛋白并探讨Csps在适冷菌低温适应性中的作用机制。研究结果不仅拓展对南极低温微生物资源的了解，而且对抗低温功能基因的发现和适冷机制的发展具有重要的理论和实践意义。