交替氧化酶调控线粒体活性氧在壳聚糖诱导马铃薯抗干腐病反应中的作用机理研究

Mitochondria are the main source of reactive oxygen species in postharvest fruits and vegetables, and alternative oxidase has important roles to maintain metabolic and signaling homeostasis in mitochondria. Our previous results showed that preharvest treatment with chitosan could induce resistance of potato tubers against dry rot, and reactive oxygen species involved in this process. However, the regulation mechanisms of reactive oxygen species in induced resistance remained unknown. In this proposal, potato tubers will be used as materials, and multidisciplinary approaches involving the postharvest pathology, cell biology, molecular biology, physiology and biochemistry will be used to reveal the types of alternative oxidase gene and proteins implicated in reactive oxygen species homeostasis in potato tubers treated with chitosan and salicylhydroxamic acid (SHAM, an inhibitor of alternative oxidase). With the utilization of modern analyzed methods including organelle positioning observation, transmission electron microscope (TEM), laser confocal microscope (LSCM), fluorescence spectrophotometer, iTRAQ and LC-MS/MS technology, quatitative real-time Polymerase Chain Reaction (qRT-PCR) to explain the mechanism of reactive oxygen species production mediated by alternative oxidase in potato tubers at the cellular, molecular and biochemical level. Expected results of this proposal can elucidate complex relationships of alternative oxidase and reactive oxygen species production homeostasis in potato tubers induced by chitosan, and also provide theoretical basis for further understanding mechanism of induced resistance in fruits and vegetables.

线粒体是采后果蔬细胞活性氧的主要来源，交替氧化酶是线粒体中调控代谢和信号动态平衡的关键。我们前期发现，采前壳聚糖处理可诱导马铃薯块茎对干腐病的抗性，活性氧参与了诱导抗性过程，但详细机制不明确。本项目拟选用马铃薯为试材，综合采后病理学、细胞生物学、分子生物学和生理生化等多学科方法，通过壳聚糖结合AOX抑制剂SHAM处理，利用细胞器定位观察、透射电镜（TEM）、激光共聚焦显微镜（LSCM）、荧光分光光度计、iTRAQ、qRT-PCR等技术手段，在细胞、分子和生化水平上解析参与壳聚糖诱导马铃薯块茎防御F. sulphureum中，AOX基因的表达及蛋白的种类、对线粒体ROS动态平衡的调控作用，为揭示壳聚糖诱导果实抗病性中的AOX与ROS的积累关系，丰富采后病害的诱抗机理提供理论基础。

本研究通过采前及采后多聚氨基葡萄糖（壳聚糖和壳寡糖）的处理，研究其对马铃薯采前生长及采后块茎抗干腐病的抗病力和贮藏性能的影响。研究结果表明：.1）采前壳聚糖处理提高了马铃薯植株田间的株高、茎粗、单株结薯数和单株产量，并有效降低了植株田间的晚疫病病情指数。.2）采前壳聚糖处理加速了块茎愈伤期间伤口部位SPP、SPA及木质素的积累速度明显加快；有效抑制了损伤块茎失重率和发病率，缩短了愈伤时间。壳聚糖处理可加速块茎愈伤，增强块茎抵御接骨木镰刀菌侵染的能力。.3） 块茎发生干腐病的早期，胞内呼吸强度增加，交替呼吸途径被激活。壳聚糖可快速诱导块茎与病原菌互作早期AOX的表达和交替呼吸途径的容量快速提高，AOX在响应病害早期进程中具有重要作用。.4）采前壳聚糖处理降低了马铃薯块茎采后干腐病的发生。发病早期胞内产生大量ROS，且定位在细胞膜上，使胞内氧化还原电势增高，细胞膜透性和氧化损伤增强，壳聚糖处理可降低ROS的水平，降低氧化还原电势， AOX可能参与了这一过程。 .5）采前壳聚糖处理通过调节NOX、SOD、CAT、POD以及AG循环，调节块茎采后ROS产生及清除体系。通过转录组和蛋白组的数据分析发现，谷胱甘肽介导的AG循环在调节胞内ROS的动态平衡调节过程中具有重要作用。通过SHAM抑制处理，AOX可能作为调节线粒体内能量的产生，调控氧化磷酸化过程的靶点参与壳聚糖对ROS代谢系统的调节。.6） 壳聚糖处理诱导块茎AOX表达量增多，ROS水平下降，调节干腐病的发生。转录组和蛋白组数据壳聚糖处理组块茎发生干腐病早期，显示氧化磷酸化代谢通路、与抗病有关的苯丙烷代谢通路的上游丙酮酸代谢通路相关基因蛋白均上调表达，但壳聚糖对AOX的诱导作用以及对ROS的调控，仍需对组学的数据深度挖掘进一步分析。.7） 寡聚氨基葡萄糖（壳寡糖）处理可促进块茎芽的萌发，但以50mg/L的处理浓度较佳。壳寡糖促进芽萌发过程不会出现如赤霉素处理后的芽疯长现象。.