基于激光镊子拉曼系统和单细胞分析技术的杆菌孢子萌发机理的研究

项目名称： 基于激光镊子拉曼系统和单细胞分析技术的杆菌孢子萌发机理的研究

项目编号： No.30860010

项目类型： 地区科学基金项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 黄庶识

作者单位： 广西科学院

项目金额： 24万元

中文摘要： 项目通过建立灵敏、特异和可靠的单细胞拉曼光谱系统，在单细胞水平上，实时记录芽孢个体在不同条件下萌发过程中的生理生化变化，尤其2，6-吡啶二羧酸钙（Ca2+-DPA）浓度变化过程以及其释放的行为特征。项目取得的主要结果是①#26500;建了可编程温度控制实验平台，实现实验过程温度灵活控制；②#24314;立单个细菌芽孢吡啶二羧酸浓度测定的方法；③#21333;个孢子萌发过程中，阐述芽孢在Ca2+-DPA释放完全1-15分钟内，部分蛋白质的二级结构变化，以及DPA释放完全后20-60分钟生理状态；④#25289;曼光谱的方法结合PCA方法可以方便地对Bt与Bc加以鉴别；⑤#36890;过研究不同萌发剂诱导孢子Ca2+-DPA释放的行为特征，验证孢子萌发的多条途径性；混合两种营养性萌发剂可以加快孢子Ca2+-DPA的释放，促进孢子萌发，证明芽孢存在多种受体响应不同类型的萌发剂；⑥#27695;气胁迫孢子，引起孢子核内蛋白质发生了变性导致其构象变化，在光谱上表现为1655 cm-1发生位移，漂移时间大约1分钟左右完成；⑦#32467;合多元统计方法，分析氯气、强碱、甲醛与孢子内生物大分子相互作用的光谱数据，可以得到包括蛋白质空间结构变化的信息以及膜脂和膜蛋白的构象变化信息。

中文关键词： 息激光镊子；拉曼光谱；芽孢；单细胞分析；萌发机理

英文摘要： The physiological and biochemical changes of germination process of individual bacterial dormant spores, especially the variation process of Ca2+-DPA concentration and the behavior of Ca2+-DPA release，were recorded real-time by Laser tweezers Raman spectroscopy system in sensitivity, specificity and reliability at the single cell level. The main conclusions from this work were as follows. 1) The programmable temperature controller was constructed as a experimental platform successfully for flexible process temperature control; 2) The determination method of Ca2+-DPA concentration in a single bacterial spore has been established; 3) The changes of the secondary structure of partial protein and the physiological state of spores were expatiate while a single spore germinating for 15 minutes after Ca2+-DPA release completely, as well as the changes from 20minute to 60 minutes; 4) Bacillus thuringiensis (Bt) and Bacillus cereus (Bc) can be identified by the Raman spectroscopy method combined with principal component analysis (PCA); 5) The germination pathway even more than one way was validated by investigating the behavior of Ca2+-DPA release after induced with different germination agent; A mixture of two kinds of nutritional germinant could accelerate Ca2+-DPA release from spores and promote spore germination, indicated that there are multiple receptors in spores membrane in response to different types of germinant; 6) The protein within spore core denatured and led to its conformational changes after stressed with chlorine stress, in which the spectrum peak drifted from 1655 cm-1 to 1669 cm-1 , the shift time about 1 minutes to complete; 7) The change information of spatial structure of proteins, even the membrane lipid and membrane protein conformation, could be obtained after analyzing spectral data of biological macromolecules in spores which stressed with chlorine, alkali and formaldehyde combined with multivariate statistical analysis method; the result had great value for illustrating the resistance mechanism of spores.

英文关键词： Laser tweezers; Raman spectrum; single-cell analysis; endospore; germination mechanism