【作者】 李欣；

【导师】 李红玉；

【作者基本信息】 兰州大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 本实验以植物病原细菌中黄单胞菌属的多个菌株为研究对象，以辣根过氧化物酶—酚红法和组织化学法为过氧化氢检测的基本方法，检测了多株植物病原细菌内源过氧化氢产生和积累情况，结果显示几乎所有黄单胞菌菌株细胞中都有内源过氧化氢的产生和积累，并且不同菌株中过氧化氢的水平有很大的差异。在透射电子显微镜下，组织化学染色结果显示黄单胞菌细胞中内源的过氧化氢主要定位在细胞壁上，在细胞质和质膜等其他位点没有检测到过氧化氢的积累。进一步的实验结果显示，上述病原菌内源产生的过氧化氢在细胞中具有重要的生理和病理学功能。实验发现整个细胞分裂周期中有稳定水平的过氧化氢积累于细胞壁上，其积累水平与细胞的生长速度有密切的联系。实验还发现，在细胞的分裂周期中，随着细胞分裂的进行，细胞内源的过氧化氢积累有显著的空间定位变化，在分裂的细胞中类间体结构和核区是两个新的过氧化氢大量积累的位点，这些过氧化氢积累与细胞分裂的进程密切相关。实验也显示黄单胞菌细胞中的内源过氧化氢通过在核区DNA上的附着在黄单胞菌的细胞增殖中起到积极的重要作用。另一方面，黄单胞菌细胞在受到损伤的状态下，细胞中的内源过氧化氢也出现过量的产生和积累，并在细胞壁之外出现间体中的过氧化氢的大量积累。间体出现的频率和大小以及其中过氧化氢的积累量均随着细胞损伤程度的增加而显著上升。实验结果暗示细胞损伤后间体中的过氧化氢积累对受损细胞是一种主动调控机制。在与植物的互作体系中，病原菌的avrXa7基因突变引起病原菌与水稻互作体系中的过氧化氢积累水平发生显著降低，暗示病原细菌很可能是水稻与白叶枯病菌互作体系中过氧化氢积累的一个主要来源。油菜黄单胞菌菜豆致病变种菌株的ahpC突变造成的病原菌内源过氧化氢水平的变化引起与水稻的互作体系中过氧化氢的平均积累水平显著的降低。暗示非寄主互作体系中细菌病原很可能也是互作体系中过氧化氢的一个潜在来源。细菌产生的过氧化氢很可能在非寄主互作中造成细菌周围植物细胞的损伤，对植物细胞具有潜在的毒性。无毒基因avrXa7基因突变后的突变体菌株与水稻互作时，不能引起植物的过敏反应。而ahpC基因突变后的突变体菌株与水稻互作时，虽然导致互作体系中的过氧化氢积累的水平显著的降低，但是仍然能够引起植物的过敏反应。由此推测在黄单胞病菌的致病机制中，过氧化氢处在avrXa7基因的下游，受到avrXa7或者更多毒性相关基因的调控，在病原菌与植物识别时起到一定的作用。更多还原

【Abstract】 Production and localization of endogenous hydrogen peroxide （H₂O₂） were investigated in strains of Xanthomonas using HRP-red phenol assay or by histochemical analysis under electron microscopy. Experiments identified the presence of endogenous H₂O₂ production in nearly all Xanthomonas strains tested. Furthermore, the levels of endogenous H₂O₂ production were different among Xanthomonas strains tested. Results showed that the accumulated H₂O₂ was mainly located in the cell wall; the H₂O₂ accumulation was undetectable in plasma membrane or cytoplasm of Xanthomonas cells. Further results showed that the endogenous H₂O₂ plays crucial roles in physiological or pathological process of bacterial cells. Steady-state level of H₂O₂ was localized in cell wall throughout the cell division cycle of bacteria. There is a close correlation between the endogenous H₂O₂ accumulation and the growth of bacteria. It was first reported by us that the H₂O₂ production and the localizations of the accumulated H₂O₂ changed during the process of cell division cycle. In addition to the cell wall localization of the accumulated H₂O₂, two other locations of H₂O₂ accumulation, nucleoids and the mesosome-like structures, were observed during the cell division cycle of Xanthomonas. It indicated that the abundant H₂O₂ accumulation was closely associated with the process of cellular division. The overall results intensely suggested that endogenous H₂O₂ production should play positive roles by integrating with chromosomes in cell division cycle of Xanthomonas. On the other hand, excess endogenous H₂O₂ accumulation was observed in cells under cellular injury. Intriguingly, mesosomes presented as an additional location of H₂O₂ accumulation within the injured cells. There was an association between the frequency and size of mesosomes and the quantity of excess H₂O₂ accumulation and the degree of cellular injury caused by cellular injury. Results indicated that it should be voluntary regulatory mechanisms of H₂O₂ accumulation in mesosomes for cells under cellular injury. The loss of the function of avirulence gene avrXa7 led to decrease in the virulence of strain and resulted in reduction of endogenous H₂O₂ accumulation in avrXa7 mutant Xanthomonas oryzae pv. oryzae strain. During the interaction with plant, the change of endogenous H₂O₂ accumulation caused by avrXa7 mutation in Xanthomonas oryzae pv. oryzae impacted on the responses of rice plants and also influence on the mean levels of H₂O₂ accumulation in the interaction systems. Our experiments suggested that bacterial pathogen is a potential source of the H₂O₂ accumulated in the interaction between rice and Xanthomonas oryzae pv. oryzae. Moreover, the change of endogenous H₂O₂ level caused by ahpC mutation of Xanthomonas campestris pv. phaseoli has also impact on the mean level of H₂O₂ accumulation during the interaction with plant. Our overall results suggested that bacterial pathogen is also a potential source of the H₂O₂ accumulated in the interaction between rice and Xanthomonas campestris pv. phaseoli. The H₂O₂ produced by bacteria may be a potent cause of membrane damage in plant cells undergoing the HR during non-host resistance. Either avrXa7 or ahpC mutation induced the decrease of H₂O₂ accumulation during the interaction between plant and bacterial pathogen. The ahpC mutant induced the hypersensitive response of plant, while the avrXa7 mutant did not. Author inferred that H₂O₂ is a downstream mediator of AvrXa7-dependent interaction between plant and bacterial pathogen. It was hypothesized that the H₂O₂ production is down-regulated by avrXa7 or other genes to act in the recognition with plant.更多还原