能量代谢和线粒体活性氧在硅诱导甜瓜果实抗病反应中的作用机理研究

During the defense process of muskmelon fruits induced by sodium silicate and challenged with Trichothecium roseum, burst of reactive oxygen species (ROS) is the earliest response, activations of phenylpropane pathway and pathogenesis-related proteins accumulation are final responses. Energy metabolism has been verified to play an important role in resistance responces of fruit. However, after the induced resistance treatment and challenge inoculation, the changes of energy metabolism in muskmelon fruits remain unknown. The relationships among fruits disease resistance, energy level, and mitochondrial ROS have also no systematic and in-depth researches. In this project, muskmelon fruits are chosen as materials, and multidisciplinary approaches involving the postharvest pathology, cell biology, molecular biology, physiological and biochemistry would be used to analysize the process of energy metabolism in mitochondria of fruits induced by sodium silicate and challenged with T. roseum. With the aid of modern analyzed methods such as organelle positioning observation, proteomics analysis, qRT-PCR, and Western blot, the energy metabolism, generation of mitochondrial ROS and disease resistance would be analysized at the subcellular, biochemistry, and molecular level of fruits. Expected results of the project will elucidate complex relationships of ROS and energy metabolism during disease resistance, and also provide theoretical basis for further understanding mechanism of induced resistance in fruits treated by sodium silicate.

活性氧（ROS）爆发是硅酸钠诱导厚皮甜瓜果实防御Trichothecium roseum侵染的早期事件，苯丙烷代谢活化以及病程相关蛋白积累是抗病的最后反应。已证明能量代谢在抗性反应中发挥着重要的作用。但是，诱抗处理以及挑战接种后果实体内能量代谢如何变化，能量水平与果实抗病性和线粒体ROS之间存在何种关系尚缺乏系统而又深入的研究。本项目拟选用西北地区特色水果厚皮甜瓜为试材，综合采后病理学、细胞生物学、生理生化和分子生物学等多学科方法，利用细胞器定位观察、生物化学分析、蛋白组学分析、实时荧光定量PCR(qRT-PCR)及Western blot等技术手段，在亚细胞、生化和分子水平上解析硅酸钠处理及T. roseum挑战接种所诱导的抗病反应中果实线粒体内能量代谢的变化，揭示能量代谢与线粒体ROS和果实抗病性的关系，为阐明硅酸钠诱导甜瓜果实的抗病机理提供理论依据。

1.本研究以“玉金香”厚皮甜瓜为试材，用100 mmol/L硅酸钠常温浸泡10 min，处理后12 h接种粉红单端孢（Trichothecium roseum），接种后第5天对照组已经明显发病，硅处理组病斑面积仅为对照的44.80%，第7天对照组病斑面积扩展严重，硅处理组仅为对照的57.96%，结果证明硅处理能有效控制甜瓜果实损伤接种Trichothecium roseum的病斑扩展。.2.研究了甜瓜线粒体中与能量代谢关系最密切的几种酶活，包括H+ - ATP酶、Ca2+ATP酶、琥珀酸脱氢酶和细胞色素氧化酶，硅处理使得挑战接种的甜瓜酶活性有显著性增加。同时，硅处理使接种后的甜瓜果实细胞内的ATP含量上升并且保持在较高水平。.3.病原菌接种挑战厚皮甜瓜采后果实组织与应激反应有关的代谢途径PPP加强，PPP途径中关键限速酶G6PDH和6PGDH在硅处理挑战接种后活力显著增加。同时，由G6PDH 催化产生提供还原力的 NADPH 含量也显著增加，而与EMP-TCA有关的NADH含量显著降低。.4.结果显示硅酸钠处理可诱导H2O2的积累并且延迟了O2-.产生速率峰值的出现，挑战接种后处理组H2O2含量和O2-.产生速率快速升高而积累了更多的活性氧，这说明硅酸钠可提高甜瓜对采后病害的抵抗与其调节果实活性氧的产生相关。同时，我们通过激光共聚焦扫描显微镜观察用两种不同的荧光染料分别定位的活性氧和线粒体信号，发现在硅处理挑战接种后的果实可以诱导氧爆的发生，共定位结果证实活性氧来源与线粒体有关。.5.首次通过Percoll密度梯度离心法获得了甜瓜果实线粒体提取、纯化的可靠方法。采用iTRAQ技术分析了硅酸钠以及T. roseum挑战接种引起甜瓜果实线粒体蛋白质组的51种蛋白差异表达。生物信息学分析，通过细胞组分定位我们发现，几乎所有的这些差异蛋白均定位于线粒体。分子功能分析显示，这些蛋白主要作用于金属离子绑定、钙离子结合以及氧化还原活性。而生物过程主要涉及初级代谢，涉及到8大类分别是：呼吸电子传递链, 三羧酸循环, 糖酵解途径, 氧化还原过程, 防御和胁迫响应 , 线粒体载体, 蛋白和氨基酸代谢。