亚精胺在集胞藻PCC 6803抵抗氧化损伤中的分子机理研究

Aerobic cyanobacteria cannot avoid oxidative stresses caused by enhanced generation of reactive oxygen species (ROS) under chill-light stress. It is inferred that application of polyamines treatment would result in the increase in the activity of antioxidant enzymes and the expression of related genes in higher plants. However, the effects of polyamines on the resistance to oxidative stress in cyanobacteria remains to be elucidated. It has been proved that the synthesis de novo of proteins could been inhibited in the spermidine-minus mutant under chill-light stress. This project takes Synechocystis sp. PCC 6803 as the research model, using gene knockout and proteomics, the antioxidant enzymes which expressed differentially under chill-light stress can be found, and analyze through two aspects of translation and transcription; further more, the effect of spermidine on the activity of antioxidant enzymes can be known via the analysis of enzymes activity; at the same time, add exogenous spermidine into the mutant, and observe the effects of exogenous spermidine against oxidative damage of the mutant. In addition, expression and purification of recombinant protein and western blot analysis can ensure the effect of spermidine on the redox state of EF-Tu and EF-G; site directed mutagenesis can help to detect the activity of antioxidant enzymes with EF-Tu and EF-G being oxidized or not in the spermidine-minus mutant. Through this project, we can deeply understand the role of spermidine in oxidative damage caused under chill-light stress, ensure the molecular mechanism to add a new evidence to further reveal the mechanism in overwintering of cyanobacteria.

蓝藻在光照和低温双胁迫下会导致其细胞内的ROS含量增加，引发氧化胁迫。一定浓度的多胺被证实可提高高等植物体内抗氧化酶的活性，调控相关基因的表达，但多胺在蓝藻抵抗氧化胁迫中的作用却报道甚少。业已证明缺失亚精胺会导致藻株不能继续在低温光照下合成蛋白。本项目以集胞藻PCC 6803为研究对象，利用突变和蛋白组学的方法，找到在寒冷光照条件下差异表达的抗氧化酶，并对其编码基因从转录和翻译两个方面来分析；进一步，通过酶活测定，分析亚精胺缺失与否对抗氧化酶活性的影响。另外，重组蛋白的表达纯化结合western blot的技术，分析亚精胺对翻译延伸因子EF-Tu和EF-G氧化还原态的影响；定点诱变技术的应用，检测EF-Tu和EF-G不被氧化时，亚精胺缺失突变株中的抗氧化酶的酶活性。通过本项目，能深入了解亚精胺在抵抗由寒冷光照引起的氧化损伤中的分子机理，为进一步揭示蓝藻抗寒越冬机理增添新的佐证。