黑暗限气条件下铜绿微囊藻细胞死亡的形态结构和生理生化变化

Morphological and biochemical changes of Microcystis aeruginosa PCC7806 subjected to dark and oxygen limitation

【目的】研究铜绿微囊藻细胞死亡过程中形态和生理生化变化,探讨蓝藻细胞死亡机制。【方法】通过黑暗限气处理模拟水华爆发后期水体环境,在处理后不同时间取样,对藻液的OD值,溶氧含量和pH值进行监测,使用透射电镜对细胞形态结构变化进行观察,通过胱天蛋白酶(Cysteine-dependent aspartate specificprotease,Caspase)活性检测、活性氧含量测定、末端脱氧核糖核酸转移酶介导的dUTP缺口末端标记(Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling,TUNEL)染色和琼脂糖凝胶电泳对处理后藻细胞的死亡生理进行研究。【结果】黑暗限气处理后,藻培养液pH值和溶解氧含量下降,处理12 h后藻液开始变黄,48 h后藻细胞全部死亡。电镜观察结果表明,藻细胞在黑暗限气处理所导致的死亡过程中出现空泡和类囊体、核糖体等内部结构解体但细胞壁仍保持完整等现象。活性氧含量和caspase活性检测表明,在藻细胞死亡过程中活性氧含量和caspase活性上升。TUNEL染色和琼脂糖凝胶电泳分析发现,藻细胞在死亡过程中DNA发生断裂和降解。【结论】铜绿微囊藻细胞在黑暗和限气处理中表现出和真核生物细胞程序性死亡相类似的死亡特征,这说明细胞死亡机制是保守的,原核细胞和真核细胞一样具有程序性死亡机制。

[ Objective ] To explore the mechanism of cell death in Microcystis aeruginosa PCC 7806. [Method] According to the water environment of the later period of algal bloom, M. aeruginosa PCC 7806 was treat with dark and 02 limitation. We observed the morphological changes using transmission electron microscope （TEM） and detected the reactive oxygen species （ROS） activity and Caspase3 activity in M. aeruginosa PCC7806 subjected to dark and 02 limitation. DNA status was also examined with the methods of Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） and agarose gel electrophoresis. [Result] Massive algae cell died after 48 h treatment under dark and 02 limitation. During cell death process, we observed some changes of cell organelles including ribosomes and thylakoids disorganization, cytoplasmic vacuolation, nucleoplasm diffusion and plasmolysis in M. aeruginosa PCC7806 subjected to darkness and O2 limitation. Meanwhile, we found that increased ROS reactivity and capase 3 activity were related to the cell death process of M. aeruginosa. DNA breakage and fragmentation were proved by TUNEL staining and agarose gel electrophoresis during cell death process. [ Conclusion] All results showed that cell death with characteristics similar to eukaryotic programmed cell death could be induced in M. aeruginosa PCC 7806 after treatment with darkness and 02 limitation. Therefore, we suggested that the mechanism of cell death are conserved during evolution according to the characteristics of cell death shared between eukaryotes and Microcystis .