硫氧还蛋白(Trx)和谷氧还蛋白(Grx) S-亚硝基化调控冷藏桃果实线粒体氧化应激的信号途径

Oxidative damage caused by reactive oxygen species (ROS) is a major factor to destroy the integrity and functions of mitochondrial membrane, and reduces significantly the qualities of postharvest fruits. This project investigates the S-nitrosylations of thioredox (Trx) and glutaredox (Grx) and the regulation mechanism of nitric oxide (NO) on redox signaling and oxidative stress in mitochondria of peaches during cold storage with modern analytical instruments and methods of chemical kinetics and molecular biology. (1) By using surface plasmon resonance (SPR), two-dimensional electrophoresis and other technologies, the translation modifications of Trx、Grx、nuclear factor NF-κB and other proteins in mitochondria by NO are detected, and chemical kinetics of the interactions between NO and special Cys residues are studied. (2) The genes encoding Trx, Grx and NF-κB are isolated, identified and the functions are analyzed. The effects of S-nitrosylation on the expressions of Trx, Grx and NF-κB are studied. (3) The effects of S-nitrosylation on the Trx and Grx system to modulate mitochondrial redox balance are studied. (4) The regulations by Trx and Grx S-nitrosylation on the following redox signaling pathway mediated by NF-κB are also studied. The results of this project will provide evidences for improving the qualities of postharvest peach fruits.

活性氧引起的氧化应激损伤是破坏采后果实线粒体膜完整和功能的重要因素，严重降低了果实贮藏品质。本项目将现代仪器分析方法和化学动力学、分子生物学技术相结合，研究一氧化氮（NO）对桃果实线粒体硫氧还蛋白（Trx）和谷氧还蛋白（Grx）S-亚硝基化作用进而调控线粒体氧化还原应激的信号机理。（1）利用表面等离子体共振、双向电泳等研究NO对Trx、Grx、核因子NF-κB等的化学修饰及与特定Cys残基作用的化学动力学；（2）分离鉴定线粒体编码Trx、Grx、NF-κB的基因并分析其功能，探讨S-亚硝基化对Trx、Grx、NF-κB等基因表达的影响；（3）研究S-亚硝基化对Trx、Grx系统调控线粒体氧化应激途径的影响；（4）Trx、Grx亚硝基化对后续NF-κB介导的信号通路的调控作用。这将为改善采后桃果实品质提供理论依据。

线粒体氧化还原信号在线粒体控制的程序性死亡过程中起着至关重要的作用。本研究以不同浓度的NO溶液和NO清除剂等处理的肥城桃果实为材料，检测其线粒体氧化还原应激相关指标，旨在研究NO调控采后果实线粒体氧化应激的分子机理，对于维持良好的果实品质，延长果实采后贮藏期具有重要的意义。.采用表面等离子体共振(SPR)法研究线粒体S-亚硝基化蛋白。对比Trx蛋白经亚硝基谷胱甘肽、适量还原剂DTT和对照的波数，结果表明NO可以将Trx上的巯基基团封闭，即发生亚硝基化作用，而还原性试剂如DTT可以还原亚硝基化产物。.采用分子生物学的方法，分析Trx和Grx这两种构成氧化还原调控系统的蛋白的全长cDNA，并进行蛋白表达、蛋白纯化与复性，获得基因全长和重组蛋白。研究不同浓度梯度的NO溶液对重组蛋白的荧光猝灭作用。并运用荧光定量PCR研究表明NO处理能够提高Trx和常温条件下Grx蛋白的基因表达量，但降低了冷藏条件下Grx蛋白的基因表达量，其中15 μmol L-1 NO处理的效果最为明显。.采用紫外分光光度法、荧光分光光度法研究线粒体的内源NO释放、抗氧化酶活性、还原性物质、丙二醛（MDA）含量和线粒体氧化还原电势。研究表明，NO能够提高线粒体氧化还原电势，维持了氧化应激下线粒体氧化还原平衡状态，提高了线粒体抗氧化酶活性和还原性物质含量，提高了线粒体的内源NO含量。.以上研究表明，NO处理能有效维持线粒体完整性和功能，降低贮藏期间果蔬线粒体损伤，保持良好的果实贮藏品质。