【作者】 张阿英；

【导师】 蒋明义；

【作者基本信息】 南京农业大学 ， 植物学， 2006， 博士

【摘要】 植物激素脱落酸（abscisic acid，ABA）不仅在植物的生长发育过程中而且还在植物对各种环境胁迫的反应中起重要作用。ABA可以引起一氧化氮（nitric oxide，NO）和活性氧（reactive oxygen species，ROS）的产生，诱导抗氧化基因的表达，提高植物抗氧化防护能力。ABA、NO、ROS都可以活化促分裂原活化蛋白激酶（mitogen-activated protein kinases，MAPK），活化的MAPK调节植物对多种胁迫反应的生理过程。ROS在ABA诱导的抗氧化防护中起重要作用。然而，有关ABA诱导抗氧化防护的详细机制仍有待阐明。 本研究以玉米叶片为材料，研究了在ABA诱导的抗氧化防护过程中MAPK的作用以及H₂O₂、MAPK与NO之间的关系。主要的研究结果如下： 利用髓鞘碱性蛋白（myelin basic protein，MBP）作为底物，采用凝胶激酶分析方法研究了ABA处理后玉米叶片MAPK的活化情况。100μM ABA处理明显活化一个46 kDa的MBP激酶，以ABA缺失突变体vp5为材料，用10％PEG处理2h，结果发现在野生型中MBP激酶活性明显增加，而突变体中却增加很少，表明水分胁迫诱导的内源ABA积累能够活化MBP激酶。用酪氨酸磷酸化单克隆抗体4G10进行免疫沉淀凝胶激酶分析结果显示，ABA处理的玉米叶片蛋白质与对照的相比具有很高的酪氨酸磷酸化的MBP激酶活性。用两种专一性的MAPKK抑制剂PD98059和U0126预处理都抑制了ABA诱导的MBP激酶活性。这些结果表明ABA活化的46 kDa的MBP激酶是一种MAPK。而两种专一性的蛋白质酪氨酸磷酸酶（protein tyrosine phosphatase，PTP）抑制剂，PAO（phenylarsine oxide）和3，4DP（Methyl-3,4-dephostatin）预处理并不影响ABA诱导的MBP激酶活性。 ABA处理不仅诱导几种抗氧化防护基因转录水平增加，如CAT1（编码CAT1同工酶）、cAPX（编码细胞质APX同工酶）与GR1（编码质体GR同工酶），还诱导过氧化氢酶（catalase，CAT）、抗坏血酸过氧化物酶（ascorbate peroxidase，APX）、谷胱甘肽还原酶（glutathione reductase，GR）、超氧化物歧化酶（superoxide dismutase，SOD）活性的增加。两种专一性的MAPKK抑制剂PD98059和U0126预处理明显抑制了ABA诱导的抗氧化防护基因表达和酶活性增加，但是两种专一性的PTP抑制剂PAO和3，4DP预处理并不影响ABA诱导的抗氧化防护基因表达与酶活性。表明是MAPK而不是PTP参与ABA诱导的抗氧化防护。 H₂O₂抑制剂和清除剂DPI（diphenyleneiodonium）、咪唑、DMTU（dimethythiourea）和Tiron预处理结果显示ROS的抑制剂或清除剂几乎完全抑制了ABA诱导的MAPK更多还原

【Abstract】 Although the phytohormone abscisic acid （ABA） regulates many important aspects of plant growth and development, its main function is to regulate plant adaptive responses to various adverse environmental conditions. ABA can cause the generation of nitric oxide （NO） and reactive oxygen species （ROS） in various plant cells or tissues, induce the expression of antioxidant genes, and enhance the capacity of antioxidant defense systems, including enzymatic and non-enzymatic constituents. ABA, NO and ROS can activate mitogen-activated protein kinases （MAPKs）, and the activation of MAPKs play important roles in the regulation of physiological status in plant response to various environmental stresses. ROS are an important intermediate component in the ABA-induced antioxidant defense. However, the detailed mechanism about how ABA induces antioxidant defense in plant cells remains to be determined.In the present study, the role of MAPK and the relationship between H₂O₂, MAPK, and NO in the ABA-induced antioxidant defense were investigated in leaves of maize （Zea mays L.） plants. The results as follows:To investigate the effects of ABA on the activation of MAPKs in leaves of maize plants, in-gel kinase assays were performed on protein extracts from the leaves of maize plants treated with ABA, using myelin basic protein （MBP） as a substrate. Treatment with 100 μM ABA led to a significant increase in the activity of a 46 kDa kinase. To investigate whether the MBP kinase can be activated by endogenous ABA, the ABA-deficient maize vp5 mutant was used. Treatment with 10% PEG for 2 h resulted in a significant increase in the activity of the MBP kinase in the wild-type, but only a slight increase in the mutant, indicating that water stress-induced ABA accumulation can activate the MBP kinase. To demonstrate the exogenous and endogenous ABA-activated MBP kinase is a MAPK-like enzyme, protein extracts from control- or ABA-treated leaves were immunoprecipitated with anti-phosphotyrosine monoclonal antibody 4G10, and then subjected to the in-gel kinase assay. Treatment with 100 μM ABA resulted in an increase in immunoprecipitated tyrosine phosphorylated-MBP kinase activity, when compared to the control. Moreover, pretreatment with the widely-used specific MAPKK inhibitors PD98059 and U0126 inhibited the increase in the activity of the MBP kinase induced by ABA. These results obtained from the above clearly suggest that the ABA-activated 46 kDa MBP kinase is a MAPK-like enzyme. However, pretreatment with PAO and 3,4 DP, two specific inhibitors更多还原