【作者】 文彬；

【导师】 潘瑞炽； 王小菁；

【作者基本信息】 华南师范大学 ， 植物学， 2002， 博士

【摘要】 植物质膜H⁺-ATPase（EC.3.6.1.35）是植物生命活动的“主宰酶”，调控着植物细胞许多重要的生理功能。它通过外泌H⁺产生跨膜的电化学梯度，驱动离子和营养物质的吸收，调节细胞的pH值，维持细胞正常的膨压，参与细胞分裂和生长、器官和气孔的运动，以及植物的耐盐性等抗性反应。因此，质膜H⁺-ATPase活力受激素、光、植物毒素、环境胁迫等外部和内部因子的影响。有人认为H⁺-ATPase可能是激素直接作用的靶分子。JA及其衍生物（总称为JAs）是近些年来倍受植物学家青睐的植物内源有机化合物，被认为同IAA、ABA等激素一样具有广谱的生理效应。但目前人们对JAs信号转导途径知之尚少。本文首次研究了MeJA对质膜H⁺-ATPase水解活力的影响，Ca²⁺对MeJA刺激作用的影响，并对这一过程中发生的可逆磷酸化作用进行了研究，通过与FC作用对比的方法初步探讨了MeJA作用的可能方式。1．绿豆下胚轴质膜H⁺-ATPase基本特性的研究 黑暗条件下生长三天龄的绿豆黄化幼苗，取弯钩下1-2cm长的下胚轴，机械法破碎组织，两相分配法分离、提取和纯化质膜。利用不同细胞器膜上ATPase或其它酶的标志性抑制剂来检测质膜纯度。植物材料与两相系统重量之比为32：8g时，用一次两相系统提取、分离，就可获得纯度较高的质膜微囊。提取缓冲液中BSA的浓度对微囊的致密性有很大的影响，0.5％BSA下获得的质膜微囊，有较好的的致密性。 绿豆下胚轴质膜H⁺-ATPase水解活力受温度的影响，37℃时，体外反应的水解活力最高。水解活力还与反应体系酸碱度有关，pH7.0时，水解活力最高。反复冻融可使酶水解活力下降。 质膜H⁺-ATPase水解活力依赖于Mg²⁺，而K⁺对酶活力有一定的刺激 茉莉酸甲酯对绿豆下胚轴质膜H＋ATh，W－－水解活力影响的研究4作用。x”和 ugh之间有一定的协同作用，二者同时存在时，酶的活力最高。二．MeJA和FC对质膜扛－ATPase水解活力的影响 MeJA对质膜汁tTPase有刺激作用，且刺激程度与pH有关，pH7刀j．5范围内 MeJA对质膜 H＋－AThase刺激作用较强。 活体实验表明：50 n moMi的 MeJA处理绿豆幼苗 shr，对质膜 H”－AThase刺激作用最强，可达 30％左右。离体实验表明：10 n mol／L的 MeJA处理 Zhr对质膜H“－ATPase的刺激作用最强，酶的水解活力增加约30％。 活体实验中，FC对质膜H”－Ahase的刺激程度可达72％，离体条件下，FC和MeJA刺激程度相同，均为30％左右，且两者之间没有叠加效应。3．MeJA和FC在刺激质膜H”．tllrase过程中可能发生的磷酸化和去磷酸化 作用 1)磷酸酶抑制剂斑螫素、岗田酸可以增强MeJA对酶的刺激作用，岗田酸的效应较强，而斑鳌素的作用略弱，增幅分别为60％和50％左右。蛋白激酶抑制剂星形抱菌素和白屈菜红碱作用下，MeJA对酶刺激作用完全受到抑制，酶活力表现为对照水平，白屈菜红碱作用略强于星形抱菌素。 二)岗田酸和斑鳌素对 FC刺激酶活力的作用有增强，增幅分别为 60％和50％；蛋白激酶抑制剂星形抱菌素和白屈菜红碱则可完全抑制FC对酶的刺激作用。 3)用产p叭h标记质膜H”eJP ase的实验表明，同位素标记结果与蛋白激酶和蛋白磷酸酶抑制剂的实验结果趋势基本一致。MeJA和FC处理后，酶活力增加，同位素的标记量也增加，这说明酶的活力增加，其磷酸化程度也增高；斑螫素和岗田酸进一步促进MeJA和FC的作用，同位素标记量进一步增强，岗田酸的作用比斑螫素作用强；蛋白激酶抑制剂白屈菜红碱对MeJA的刺激作用有抑制作用，同位素标记比MeJA单独处理的低。4．Cah对质膜H＼ATPase水解活力以及MeJA和FC作用的影响 华南师范大学博士学位论文5 l)Cah对质膜 r栅ase水解活力有显著的刺激作用，离体实验中，711 moljL的 Ca卜使酶活力增加一倍。但 Cay对 MeJA和 FC的刺激作用没有进一步的促进作用。 2)Cd”存在时，斑螫素对 MeJA的刺激作用没有进一步的增强，而岗田酸略有增强作用；蛋白激酶抑制剂星形抱菌素和白屈菜红碱抑制MeJA的刺激作用，加入叨”后，酶水解活力与对照水平相似，对MeJA的刺激作用并没有明显影响。 3)Cah存在时，磷酸酶抑制剂斑螫素和岗田酸对 FC刺激质膜 H＼AThase活力作用有进一步增强效果，此时酶活力增加 70％左右；蛋白激酶抑制剂星形抱菌素和白屈菜红碱对FC作用的抑制程度也进一步增强，酶活力表现为接近于无Ca》条件下FC的作用水平。5．MeJA刺激质膜 H＋人TPase分子机理的初步探讨 以FC为对照，研究MeJA的刺激作用。在MeJA刺激质膜H”－ATPase过程中，用胰蛋白酶酶切处理，发现酶活力的变化与FC处理后的相同。初步推测，MeJA刺激质膜 H＋－AThase的作用方式可能与 FC相类似，位点都在酶蛋白的C－端。更多还原

【Abstract】 The proton-pump ATPase(FTH-ATPase) of the plant plasma membrane (PM), known as a "master enzyme" in plant, acts as a primary transporter by pumping protons out of the cell, thereby creating pH and electrical potential differences across the plasma membrane. This provides a driving force for solute transport at the plasma membrane. The plasma membrane H+-ATPase is involved in many important physiological functions, including cell elongation, stomata movement, stress responses and cellular responses to a number of factors such as plant regulators, light and fungal toxins. Even it is probably a direct target molecule of plant hormones. Jasmonic acid and its derivatives (JAs), a kind of plant endogenous compounds, have ubiquitous physiological effect on plant growth and development. But our present knowledge of JAs signaling pathway is very limited. In the present study we firstly investigate the effect of MeJA on plasma membrane H+-ATPase hydrolysis activity and the phosphorylation and dephosphorylation of the enzyme after MeJA treatment comparing to fusicosin (FC) function. Moreover, the role of the calcium ion in MeJA- and FC-induced increases of the plasma membrane H-ATPase was studied.1. Characteristic properties of the plasma membrane H+-ATPase3-d-old seedlings of etiolated mung bean (Vigna radiata L) were harvested and the hypocotyls (1-2 cm in length) under hook were used to prepare the plasma membrane vesicles by means of aqueous two-phase partition. The possible contamination of other organelle membranes was determined by using markers of enzyme inhibitors in different organelle membranes such as vanadate, azide, molybdate and NOs". The aqueous two-phase partition method is based on weight, when the proportion of the weight of hypocotyls and two-phase partition reached 32 ." 8g, the plasma membrane vesicles of mung bean were purified well. The concentration of BSA in homogenization medium influenced the latency activity ofthe PM tT-ATPase. 0.5% BSA was suitable in our experiment.Characteristic properties of the plasma membrane H+-ATPase were investigated firstly. An optimum pH for PM H+-ATPase hydrolysis activity was around 7.0, and the optimum temperature was 37 in vitro. Thaw/freezing decreased the enzyme hydrolysis activity in our experiment.Mg 2+ ion was necessary for PM hT-ATPase full hydrolysis activity and K+ stimulated enzyme activity slightly. ATPase activity reached to the highest when both Mg2+and K+were present in the reaction medium.2. The influence of MeJA and FC on the PM IT- ATPase hydrolysis activityPM IT^-ATPase hydrolysis activities were determined in responding to the treatment of MeJA. The enzyme activities reached to maximum when pH was 7.0 to 7.5 hi assay medium.Application of MeJA (50 u mol/L) to 3-d-old seedlings resulted in an increase in PM tT-ATPase activity in vivo. PM H+-ATPase hydrolysis activity increased 30% comparing to the control. The levels of stimulation by 10 u mol/L MeJA in vitro also showed about 30% increase of the enzyme activity 2hr after incubation of membrane vesicles with. MeJA.FC stimulated the PM H+-ATPase activity and the maximal increase reached to 72% in vitro, while it had the same stimulation (about 30%) of enzyme activity as MeJA had in vivo. The combination of MeJA and FC did not show significant additive effect on enzyme activity.3. Phosphorylation and dephosphorlation of PM H^-ATPase after treatments of MeJA and FC1) In vitro, phosphatase inhibitors, okadaic acid and cantharidin, enhanced MeJA-induced increase of the enzyme activity to 60% and 50%, respectively. Staurosporine and cheleythrine, two inhibitors of protein kinase, inhibited the stimulation of MeJA on PM H+-ATPase activity completely.2) Both protein kinase inhibitors and phosphatase inhibitors showed the same effect on FC-induced increases of enzyme activities.3) The results from y-32p tracing experiments showed that the level of isotop labeling on PM H+-ATPase increased after treatment with MeJA and FC, respectively. Okadaic更多还原