燕麦劣变种子线粒体AsA-GSH循环抗氧化机制与差异蛋白表达

Seed deterioration is the key factor that causes the loss of seed vigor, and also is the direct reason for the economic loss of agricultural production due to declining of seed quality. There are very important theoretical value and practical meaning to study and reveal the mechanisms of seed deterioration for maintaining seed vigor and exerting genetic potential. Oat (Avena sativa) seeds with 10% and 16% moisture content were adopted in this study and aged at 45 °C to obtain samples with different vigor levels. Changes of mitochondrial ultrastructure including membrane system were observed by transmission electron microscope; for ascorbic acid-glutathione (AsA-GSH) cycle, exogenous antioxidants were used to analyze and compare the changing rules of antioxidant enzymes and metabolic products. Furthermore, the types and expression levels of mitochondrial proteins were determined by two-dimensional electrophoresis and iTRAQ; functional genes associated with expression of differential proteins were identified by qRT-PCR. The objectives were to determine the changes in ultrastructure and function of embryonic mitochondria and antioxidation mechanisms of AsA-GSH cycle during seed deterioration at safe or high moisture content, and to provide theoretical basis for exploration the scavenging mechanism of active oxygen free radicals in mitochondria.

种子劣变是引起种子活力丧失的关键因素，也是导致种子质量下降和经济损失的直接原因。研究种子劣变的生理机制对保持种子活力和发挥其遗传潜能具有非常重要的理论价值和现实意义。试验以10%和16%含水量的燕麦种子为材料，采用45℃控制劣变处理获得不同活力水平的种子样品。通过透射电镜观测不同活力水平种子的线粒体超微结构变化；针对抗坏血酸—谷胱甘肽（AsA-GSH）循环，采用外源抗氧化剂处理分析比较抗氧化酶和代谢产物的变化规律；利用双向电泳和质谱分析以及iTRAQ技术确定线粒体差异蛋白的种类及表达水平，并结合qRT-PCR鉴定与差异蛋白表达相关的功能基因。试验目的在于研究安全含水量和高含水量种子贮藏劣变过程中，种胚线粒体的结构和功能变化规律以及抗坏血酸—谷胱甘肽循环的抗氧化机制，为探索燕麦劣变种子线粒体活性氧自由基清除的作用机理提供理论依据。

种子贮藏过程中的老化导致其活力下降，发芽延迟，并影响后续幼苗的生长发育。农业生产中，提高老化种子的活力、发芽率及田间出苗率，增强幼苗对逆境的抵抗能力，对保持生物产量和避免经济损失具有重要意义。以燕麦老化种子和种胚为材料，通过外源抗氧化剂处理对老化种子发芽及幼苗生长特征、线粒体抗氧化酶防御系统变化、胚细胞超微结构变化以及种胚中参与能量代谢和其他相关代谢通路差异蛋白的表达等方面开展研究，主要结果如下：老化处理导致线粒体细胞超微结构受损，降低AsA-GSH抗氧化循环中GR、MDHAR和DHAR活性，提高H2O2含量。添加外源NO、MT等抗氧化剂能提高劣变种子活力。NO处理可以显著增加线粒体CAT、GR、DHAR和MDHAR活性，显著降低H2O2含量，且三羧酸循环中succinate-CoA ligase和fumarate hydratase等含量显著增加，说明NO可通过促进AsA-GSH抗氧化循环和三羧酸循环改善劣变种子的发芽速率和幼苗生长。200 μM MT引发24h更利于胚细胞结构的修复，线粒体结构恢复正常，嵴清晰，内外膜双层结构完整。200 μM MT引发促进劣变种子发芽及幼苗生长，降低MDA、H2O2和O2•-的含量，提高抗氧化酶的活性，修复细胞核和线粒体等细胞结构。MT引发36 h促进劣变种子发芽及幼苗生长，降低MDA、H2O2和OH•的含量，提高SOD、MDHAR和DHAR的活性。种子老化造成燕麦胚细胞参与能量代谢、氨基酸代谢和蛋白质合成等相关通路差异蛋白表达下调，MT引发处理均能改变参与蔗糖代谢、糖酵解、丙酮酸代谢、TCA循环、脯氨酸代谢、蛋白质合成（HSPs、核糖体蛋白、翻译起始因子）通路差异蛋白的表达趋势。通过调控蔗糖代谢SuSy、糖酵解PEPCase2、TCA循环MDH、脂肪酸β-氧化KATLP和蛋白质合成通路RPS27和eIF-3E/H的表达，不同浓度MT处理在提高劣变种子发芽中发挥重要作用。而苯丙素生物合成通路可能在MT时间处理中对于提高劣变种子发芽有着新作用。综合研究结果表明，线粒体AsA-GSH抗氧化循环的生理代谢异常与种子劣变密切相关，外源添加抗氧化剂对于缓解劣变种子细胞结构损伤、保持细胞代谢功能和种子正常萌发具有关键作用。但劣变种子细胞生理调控路径和代谢响应的机制不清楚，需要进一步深入研究。

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