【作者】 李双石；

【导师】 李颖章；

【作者基本信息】 中国农业大学 ， 植物学， 2004， 硕士

【摘要】 本实验研究了棉花黄萎病菌—大丽轮枝菌毒素（VD-toxin）与拟南芥幼苗互作反应中外源SA、NO供体SNP、NO合酶抑制剂NNA等不同处理对拟南芥幼苗H₂O₂含量、CAT和APX活性及CAT基因mRNA表达量的影响，并对NO、H₂O₂的积累部位进行染色检测。 外源SA、NO供体处理拟南芥幼苗均能诱导H₂O₂的积累，NO供体的诱导作用最强；NO合酶抑制剂处理则未表现出H₂O₂含量的增强：H₂O₂的积累部位主要在叶片的表皮毛和维管束组织。在大丽轮枝菌毒素与拟南芥互作反应中，外源SA在转录和蛋白水平上均表现出对CAT的抑制作用，对APX活性也存在抑制作用，SA对CAT的抑制作用比对APX的抑制作用更强，表明，在现有的实验体系中，CAT对H₂O₂的亲合性可能大于APX对H₂O₂的亲合性，CAT是降解H₂O₂的主要酶系。NO在毒素与拟南芥的互作过程中也表现出对CAT的抑制作用，结果表明，H₂O₂可能作为信号分子参与了SA和NO调控的拟南芥的防卫反应，SA和NO或许在转录和蛋白水平上通过对CAT的抑制作用而促进H₂O₂的积累。 本实验结果进一步验证了NO作用的双重性，即在通常情况下，NO表现出的是抗氧化性，而当毒素与拟南芥互作时，NO通过抑制CAT活性，促进H₂O₂的积累，传递抗病信号，表现其氧化性的一面。 用NO特异荧光染料DAF-2DA染色得知，毒素、SA、H₂O₂单独及组合处理拟南芥下表皮条均能诱导NO的积累，NO主要积累于在保卫细胞腹壁上，在保卫细胞的胞质和表皮细胞的胞壁及胞质中也有NO的积累。 综上所述，在大丽轮枝菌毒素与拟南芥幼苗的互作反应中，信号分子NO、SA和H₂O₂之间可能存在复杂的相互关系，这种相关性在诱导拟南芥幼苗的防卫反应中起重要的调控作用。更多还原

【Abstract】 In this experiment, seedlings of Arabidopsis thaliana (Col) were observed after being treated by Verlicillium dahliae (VD-toxin), exogenous salicylic acid (SA), nitric oxide donor (SNP) and nitric oxide synthase inhibitor (NNA), then we investigated the changes of endogenous H2O2 content, the activity of the antioxidant enzymes catalase (CAT,EC: 1.11.1.6) and ascorbate peroxidase (APX,EC:1.11.1.11) and mRNA levels of CAT3 in different stress conditions,we also identified the localizations of H2O2 and NO accumulated in the leaves of Arabidopsis.The accumulation of hydrogen peroxide (H2O2) in Arabidopsis was induced by the treatment of SA and SNP. The effect of SNP was the most intense. H2O2 level was not increased in Arabidopsis treated with NNA. It was identified with the results of DAB staining of H2O2 accumulates in the leaves of Arabidopsis. H2O2 was cytochemically detected in the cells of epidermal hair and the cell wall of vascular parenchyma cell. CAT and APX were the two major H2O2-scavenging enzymes in plants. When seedlings of Arabidopsis thaliana were treated by VD-toxin, the activity of CAT and CAT3 mRNA level were decreased after SA treatment. The activity of APX was also decresed after SA and VD-toxin treatment.The activity of CAT was decreasded more obviously. In the VD-toxin infected Arabidopsis, NO also restrained the activity of CAT and decreased the level of CAT3 mRNA . The results suggested that H2O2 acted as a signal to involve the defense reactions which is induced by SA and NO in Arabidopsis.The dual effects of NO were validated in this research. NO was either oxidative or antioxidative which depends on the circumstances. Generally NO took part in the antioxidant cellular system by elevating the activities of CAT and APX .While in the VD-toxin infected Arabidopsis, NO supressed the activity of CAT and may participate in redox signaling during the activation of defense responses which follows the pathognen attack.We use a specific NO probe diaminofluorescein diacetate (DAF-2DA) to visualize the changes of NO content with different treatments and its localization . The NO burst in Arabidopsis was induced by VD-toxin, SA and H2O2. NO accumulated in the guard cells and the epidermal cells of the lowerepidermal layer of Arabidopsis.These results revealed that there may be a complicated interaction among the three signal moleculars NO,SA and H2O2 in the Responses of Arabidopsis to Toxin of Verticillium dahliae. The interaction played important function in the defence reactions of Arabidopsis.更多还原