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植物细胞活性氧爆发在植物的抗病以及信号转导中起着非常重要的作用,植物内活性氧产生及代谢受到复杂而精确的机制调控,从而维持正常的活性氧水平以发挥其生理功能.然而,在单细胞水平开展活性氧爆发实时监测及其调控机制研究一直受到很大的挑战.本文以碳纤维微盘电极(CFMDE)为基底电极,利用Nafion的模板效应,采用电化学沉积法制得纳米铂颗粒修饰电极(NPt/Nafion/CFMDE);同时采用基于聚二甲基硅氧烷(PDMS)的软光刻技术,制备了一种高效固定植物悬浮细胞的琼脂糖阵列微孔芯片.使用NPt/Nafion/CFMDE实时监测了单个拟南芥原生质体活性氧爆发,并证明电化学监测活性氧的主要成分为过氧化氢.在此基础上,采用浅层培养法培养原生质体再生植物细胞壁.电化学监测结果表明,与单个原生质体相比,植物细胞在受到刺激时释放的过氧化氢量显著降低;然而当采用过氧化物酶抑制剂抑制植物细胞壁上过氧化物酶活性后,植物细胞释放过氧化氢量显著回升.研究结果表明,细胞壁在活性氧爆发过程具有很好的调控功能,可望促进植物细胞活性氧爆发及其调控机制的研究.
Plant cell reactive oxygen species (ROS) burst plays a very important role in disease resistance and signal transduction in plants. The production and metabolism of reactive oxygen species (ROS) in plants are regulated by complex and precise mechanisms to maintain their normal ROS levels and exert their physiological functions. However, the research on the real-time monitoring and regulation of reactive oxygen species (ROS) at the single-cell level has always been a challenge.In this paper, carbon fiber micro-disk electrode (CFMDE) was used as the substrate electrode and Nafion’s template effect was used to prepare nano- (NPt / Nafion / CFMDE), and an agarose array microwell chip with high efficient immobilization of plant suspension cells was prepared by using soft photolithography technique based on polydimethylsiloxane (PDMS) Nafion / CFMDE real-time monitoring of single Arabidopsis protoplasts reactive oxygen species outbreak, and the main component of the electrochemical monitoring of reactive oxygen species hydrogen peroxide, on this basis, the use of shallow culture protoplast regeneration of plant cell wall. The monitoring results show that the amount of hydrogen peroxide released by plant cells when stimulated is significantly lower than that of a single protoplast; however, when using peroxidation Inhibition of peroxidase by plant enzyme inhibitors inhibited the release of hydrogen peroxide from plant cells, which showed that the cell wall had a good regulatory function in the process of reactive oxygen species (ROS) burst and could promote the release of reactive oxygen species And its regulation mechanism.