【作者】 郁松林；

【导师】 黄卫东；

【作者基本信息】 中国农业大学 ， 果树学， 2004， 博士

【摘要】 本研究以葡萄‘京秀’（Vitis Vinifera L.cv.Jingxiu）一年生扦插苗萌芽后70天的苗和该品种坐果后30天的果实为试材，分别用40℃高温和150 μmol／L SA不同时间处理后，探讨了高温胁迫和水杨酸（SA）对葡萄叶片和果肉细胞内蛋白激酶的诱导和活性的影响，从分子水平上进一步研究了SA作为信号分子在高温胁迫中的作用。主要结果如下： 高温胁迫和SA处理均激活了叶片中能将底物MBP磷酸化的蛋白激酶，当MBP浓度为0.5mg/mL时该激酶活性达到最高值，其后，随底物浓度的增大，活性出现下降趋势。该蛋白激酶对底物组蛋白—Ⅲ（histone-Ⅲ）没有作用。反应体系中ATP浓度以50μmol／L为宜，浓度过高时激酶活性下降。Mg²⁺浓度为5 mmol／L时激酶活性达到最大值。Ca²⁺对激酶活性的影响较小。Mn²⁺对激酶活性的激活没有作用。凝胶中底物磷酸化放射自显影结果表明，被高温胁迫10～60min和SA处理30～180min所激活的蛋白激酶的分子量约为52kD，该蛋白激酶能将凝胶中所嵌入的髓鞘碱性蛋白（MBP）磷酸化，在放射自显影中表现出很高的放射活性，而对凝胶中的histone-Ⅲ则无作用。在溶液反应体系中该蛋白激酶对MBP也表现出很高的磷酸化活性，而对histone-Ⅲ却无作用。Ca²⁺对其活性变化无显著影响。在以MBP为底物的反应体系中，pH为7.5时，激酶的活性最高。酪氨酸特异性蛋白磷酸酶（YOP）对该激酶的活性有显著的钝化作用。结果表明该52kD蛋白激酶是MAPK家族中的一种。 采用果实圆片温育法，对果肉细胞进行高温和SA处理的结果表明：高温胁迫5～20 min和SA处理5～60min均分别激活了果肉细胞中的蛋白激酶，放射自显影显示出在52kD处凝胶中的MBP被磷酸化，以histone-Ⅲ为底物时，则在42kD处histone-Ⅲ被磷酸化。在液体反应体系中，以MBP为底物有Mg²⁺存在时，52kD蛋白激酶有很强的磷酸化活性，此时Ca²⁺对其活性没有激活作用。与叶片一样，YOP对52 kD蛋白激酶有钝化作用。以histone-Ⅲ为底物有Ca²⁺存在时，42 kD蛋白激酶有很强的磷酸化活性，对Ca²⁺有明显的依赖性。该激酶对CaM没有依赖性，Calmidazolium和W7对该激酶的活性有强烈的抑制作用。 高温胁迫诱导了葡萄叶片和果肉细胞中SA含量的迅速上升。对叶片而言，胁迫30min时，游离态和结合态SA含量均达到高峰，而果肉细胞中SA含量则在20min时达到高峰，随后其含量迅速下降。无论在叶片中还是果肉细胞中均以游离态SA的变化更为活跃。分析表明，SA含量的变化与蛋白激酶活性变化有高度的一致性。结果证明：高温胁迫和外源SA处理在叶片和果肉细胞中被激活的52kD MAPK类和42kD CDPK类的蛋白激酶，与SA有密切的关系，证明了SA的确是高温胁迫应激响应中的信号分子。更多还原

【Abstract】 One-year-old plants of 70 days after budding and berries of 30days after fruit setting of grape (Vtis vinifera L. cv. Jingxiu) were used as experiment materials. After they were treated by heat stress (40℃) and salicylic acid (SA) with concentration of 150 umol/L in different times, respectively, the effects of heat stress and SA on the induction and activities of protein kinases in grape leaves and pulp cells were studied. The signaling transduction of SA in cells stressed by heat was demonstrated from molecular level. The following results were obtained:Protein kinase was activated by heat stress and exogenous SA in cell tissues of grape leaves, which could phosphorylated MBP as artificial substrate in vitro. The activity of this protein kinase reached a peak value as the concentration of MBP was 0.5 mg/mL, and then, its activity declined as the concentration of MBP increased continuously. However, this protein kinase could not phosphorylate histone-III as substrate in vitro. The best concentration of ATP in reaction system was 50 umol/L. When the concentration of ATP in reaction solution exceeded 50 umol/L, the activity of protein represented a decreasing trend. As concentration of Mg2+ in solution was 5 mmol/L, the activity of protein kinase ascended a maximum value. The stimulation of Ca2+ to this protein kinase was extremely weak. Mn2+ had no effect of stimulation to this protein kinase, and it could not replace the role of Mg2+ in reaction system. The result of autoradiography of substrate phosphorylation in gel showed that the molecular weight of this protein kinase activated by heat stress 10-60 min and the treatment of SA 30-180 min was about 52 kD. This protein kinase could phosphorylate MBP as substrate embedded in gel, and the phosphorylating products labeled by 32P revealed a very high radiation activity in gel. But histone-III embedded in gel was not phosphorylated by this protein kinase and did not display any radiation activity in gel. The high phosphorylation activity of this protein kinase was found with MBP substrate in solution reaction system. After 60 min heat stress, the activity of this protein kinase reached its maximum value, and then declined rapidly. Compared with control, the activity of protein kinase had no difference when histone-III was used as substrate in reaction system. Ca2+ could not enhance the activity of this protein kinase, showing that this protein kinase was Ca2+-independent. The activity of protein kinase reached a peak value as pH was 7.5 in the reaction system with MBP substrate. The tyrosine-specific protein phosphatase (Yersinia enterocolitica protein, YOP) could significantly inactivate the phosphorylation activity of protein kinase. All results demonstrate that the 52 kD protein kinase activated by heat stress belong to the MAPK family.After the discs of grape berries were treated in the incubation solution with heat stress and SA, the types and activities of protein kinases in crud extracts of pulp cells of the berry discs were mensurated through the methods of in-gel protein activity assay and protein acitivity assay in solution. The results showed that a 52kD and a 42kD protein kinase were stimulated by heat stress 5-20 min and SA treatment 5-60 min. The phosphorylated MBP and histone-III were displayed by autoradiography at theposition of 52kD and 42kD in gels, respectively. The phosphorylation activity of 52kD protein kinase was very strong as artificial substrate MBP was added in reaction solution with Mg +, but Ca + in solution had no stimulation to 52kD protein kinase. Tyrosine-specific protein phosphatase Yersinia enterocolitica protein (YOP) could clearly inactivate 52kD protein kinase. The phosphorylation activity of 42kD protein kinase was strongly represented when histone-III was used as substrate in reaction solution with Ca2+, and 42kD protein kinase characterized Ca2+ dependent, and CaM independent. CaM antagonists Calmidazolium and W7 could significantly inhibit the phosphorylation activity of 42kD protein kinase to histone-I更多还原