应用单细胞拉曼分析技术结合PCA方法, 以枯草芽孢杆菌芽孢为对象, 以强碱为诱导剂实时记录芽孢生理变化过程, 探究芽孢在强碱胁迫下的致死机制。 实验表明, 枯草芽孢杆菌芽孢和其萌发后芽孢在一定范围均有一定的耐受强碱能力, 但萌发后芽孢的耐受力明显下降, 主要原因是释放了维持其抗性及稳定性的特有Ca2+-DPA的保护; 芽胞受强碱胁迫也会有少量Ca2+-DPA释放过程的行为特征, 但通过分析所记录的光谱数据结果表明, 其释放行为不同于由L-丙氨酸引起的芽孢的正常Ca2+-DPA释放过程。 通过PCA比较分析强碱胁迫芽孢和萌发后芽孢Ca2+-DPA释放过程, 主要是强碱破坏芽孢膜结构, 膜损伤后强碱比较容易进到芽孢内部从而破坏蛋白质的主链结构链及核酸; 芽孢膜通道也可能受到强碱的伤害, 造成微量Ca2+-DPA的释放。

To research the lethal mechanism of spores stressed by alkali, laser tweezers Raman spectroscopy (LTRS) combined with principal components analysis (PCA) was used to study the physiological process of single spore with alkali stress. The results showed that both spores and germinated spores had tolerance with alkali in a certain range, but the ability of spores was obviously lower than that of spores due to the release of their Ca2+-DPA which plays a key role in spores resistance as well as spores resistance to many stresses; A small amount of Ca2+-DPA of spores was observed to release after alkali stress, however, the behavior of release was different with the normal Ca2+-DPA release behavior induced by L-alanine; The data before and after alkali stress of the spores and g.spores with PCA reflected that alkali mainly injured the membrane of spores, and alkali could be easily enter into the inner structure of spores to damage the structure of protein backbone and injure the nucleic acid of spores. We show that the alkali could result in the small amount of Ca2+-DPA released by destroying the member channel of spores.