一氧化氮和低温对采后桃果实线粒体DNA氧化损伤的调控作用

Oxidative damage is typical for mitochondrial DNA (mtDNA), can change mtDNA integrity, induce mitochondrial dysfunction and promote the senescence of fruit. Nitric oxide (NO) can improve the antioxidative ability of fruit, maintain mitochondrial function and delay the senescence of fruit. However, few works are done to study the regulation mechanism by NO on mtDNA of fruit during storage. This project investigates the regulation by NO and low temperature on the oxidative damage of mtDNA in peaches during storage via experiments in vivo and in intro. By using surface plasmon resonance, proteomics and other technologies, the roles of NO and low temperature on the balance of mitochondrial reactive oxygen species through enzymatic and non-enzymatic pathways and the damage of mtDNA of peaches are studied. The regulations by NO and low temperature on mtDNA oxidative damage and copy number are studied through mtDNA replication and damage repair pathway. By using modern analytical instruments and methods of chemical biology, physical chemistry, physiology and biochemistry, the roles of NO and low temperature on gene expression of the key factors in the pathways of mtDNA replication and damage repair are studied, and the reactions among NO and the factors in the pathways of mtDNA replication and damage repair are calculated and the dynamic analyses are discussed. With methods of chemical biology and physical chemistry, this project studies the roles of NO and low temperature in regulating the oxidative damage of mtDNA of peach fruit during storage. The results of this project provide evidences to shed light on the regulation mechanism of NO on improving the quality of peach fruit during cold storage.

氧化损伤是一种典型的线粒体DNA（mtDNA）损伤，会引起线粒体功能障碍和果实衰老。一氧化氮（NO）能提高果实抗氧化能力，维持线粒体功能并延缓衰老，但NO对果实贮藏期间mtDNA的调控机理仍少有研究。本项目拟通过活体和离体实验来研究NO和低温对桃果实贮藏期间mtDNA氧化损伤的调控作用。利用表面等离子体共振和蛋白质组学等方法从酶促和非酶促代谢途径研究NO和低温对线粒体活性氧平衡的调控作用和对mtDNA的损伤情况；从mtDNA复制途径和损伤修复途径研究NO和低温对桃果实mtDNA氧化损伤和拷贝数的调控作用；采用化学生物学、物理化学和现代仪器分析手段研究NO和低温对mtDNA复制和损伤修复相关关键因子基因表达的影响，并对NO与mtDNA相关关键因子间的反应进行模拟计算和动力学分析。本项目从化学生物学和物理化学等方面研究NO和低温对mtDNA氧化损伤的调控作用，为提高桃果实贮藏品质提供理论依据。

线粒体DNA（mtDNA）是线粒体维持正常生理功能的必要条件，但是由于缺乏有效的抗氧化机制和有效修复系统，容易受到氧化损伤。冷藏是广泛使用的果品贮藏方式，但极易导致果实冷害，产生大量活性氧。一氧化氮（NO）既能参与线粒体活性氧防御，又可以缓解果实贮藏冷害。越来越多的证据提示NO、线粒体活性氧和mtDNA之间存在重要关系，但是NO调控果实mtDNA的损伤修复，维持mtDNA完整性的机制仍旧未知。本项目将桃果实分别用双蒸水、15 μmol L-1 NO和20 μmol L-1 c-PTIO溶液浸泡处理后于0 °C贮藏。结果发现，NO处理能够有效减轻桃果实的冷害损伤程度，维持桃果实贮藏品质，而c-PTIO处理桃果实冷害情况加重，贮藏品质降低。外源NO处理能够通过增强桃果实线粒体内酶促抗氧化系统、非酶促抗氧化系统，降低线粒体内活性氧含量。NO处理的桃果实线粒体在贮藏过程中完整性更高，耗氧量小，呼吸控制率高，细胞色素C含量少，肿胀度小，膜电势高，膜流动性大，细胞色素途径被促进，线粒体电子传递链被维持，能量代谢过程被促进，ATP合酶活性被提高。NO处理维持了桃果实mtDNA拷贝数和完整性，减少了损伤标志物8-OHdG的水平。DNA聚合酶、线粒体单链结合蛋白、引物聚合酶等mtDNA复制关键因子的表达以及碱基切除修复过程关键因子的表达在NO处理中被上调以维持mtDNA拷贝数。理论计算证明mtDNA复制关键因子与NO反应存在作用位点，能进行谷胱甘肽化或亚硝基化反应。冷藏桃果实中NO可以调控活性氧代谢，调控mtDNA复制和修复过程减轻其损伤，进而影响维持线粒体功能并缓解贮藏冷害。