【作者】 冯兰东；

【导师】 王宝山；

【作者基本信息】 山东师范大学 ， 植物学， 2006， 硕士

【摘要】 V-ATPase是一种重要的膜结合初级质子转运体,能够利用细胞质中ATP水解产生的能量把细胞质中的H~+泵入液泡中,建立跨液泡膜的质子驱动力(protonmotive force,PMF),从而驱动溶质在细胞质和液泡间跨液泡膜转运,维持细胞质溶质等相对恒定,保证生命活动的正常进行。盐渍环境中,稀盐盐生植物通过将Na+等有害离子区域化到液泡中以缓解盐害。Na~+的区域化主要依赖于液泡膜Na+/H+逆向转运蛋白的运转,而液泡膜质子泵(V-H~+-ATPase、V-H~+-PPase)为其提供质子驱动力,V型H~+-ATPase不仅是一种重要的持家酶,更是一种重要的胁迫响应酶。本实验以典型的稀盐盐生植物盐地碱蓬为材料,200 mmol/L NaCl处理后测定有关生理指标及叶片中V-H~+-ATPase的活性,通过液泡膜微囊制备、免疫共沉淀、SDS-PAGE、Western-blot等实验方法,旨在探讨盐地碱蓬在正常生长和NaCl处理条件下V-H~+-ATPase亚基组成的种类,及盐胁迫条件下活性和某些亚基表达的变化。主要结果如下:1. NaCl处理对盐地碱蓬生长的影响200 mmol/L NaCl显著促进盐地碱蓬的生长,整株及叶片鲜重较对照均显著增加。地上部分植株大、分枝增加、叶片长、叶片肉质化程度高。低盐度生长条件下,盐地碱蓬有很强的吸收Na+能力。200 mmol/L NaCl处理后,盐地碱蓬叶细胞汁液的Na+浓度较对照有极显著增加(P<0.01)。Na~+在液泡中的积累,能够显著降低细胞水势,增强盐地碱蓬从盐渍环境中吸水的能力;胞质中Na~+浓度的降低,能够防止过多的Na~+对胞质酶的影响,保证了盐地碱蓬正常生理生化反应的进行。盐处理导致盐地碱蓬叶细胞汁液的K+浓度较对照显著降低,但植株的生长却明显好于对照,说明细胞内K+浓度在一定范围内的降低对盐地碱蓬的生长没有影响。2. NaCl处理对盐地碱蓬叶V-H~+-ATPase活性的影响200 mmol/L NaCl处理后,盐地碱蓬叶中V-H~+-ATPase水解活性较对照极显著增加(P<0.01)。叶片中的液泡膜质子泵活性高,必然会为与离子区域化有关的次级主动运输提供更多的能量,为液泡膜Na~+/H~+逆向转运蛋白提供更多的质子驱动力。因而更利于Na~+的液泡区域化,也必然会导致盐地碱蓬叶片的液泡维持较低的渗透势,更有利于植物的水分吸收,从而能更好的维持植物的正常生长。更多还原

【Abstract】 Vacuolar H~+-ATPase is a kind of membrane-bound proton transporter, whichuses the energy released from ATP and builds up a proton gradient across thetonoplast to drive translocation of ions and metabolites. Vacuolar H~+-ATPase in thetonoplast plays crucial roles in ion balance, metabolite translocation, plant growth andso on. In a saline environment, salt-dilution halophytes alleviate salt toxicity mainlythrough the capacity of sequestrating Na~+ into the vacuole. The transportation of Na~+against the concentration gradient from the cytoplasm into the vacuole via thetonoplast Na~+/H~+ antiporter is dependent on the activities of tonoplast proton pumps(V-H~+-ATPase and V-H~+-PPase), which establish an electrochemical H~+-gradientacross the tonoplast that energizes the transporters. Vacuolar H~+-ATPase not only isextraordinarily important as a house-keeping enzyme, but also functions as a stressresponse enzyme under environmental stresses. In the present study, salt-dilutionhalophyte S. salsa cultured under different salt concentrations was used to investigatethe activities, subunit composition and subunits expression of V-H~+-ATPase byWestern, Northern blot analysis and immunoprecipitation in addition to thedetermination of some physiological parameters. The main results were shown asfollows.1. Effects of NaCl treatment on the growth of S. salsaThe growth of S. salsa was enhanced by treatment of 200 mmol/L NaCl, and thefresh weight of leaves and whole plants was increased significantly compared withthe control. The number of shoot branching was increased. Furthermore, theNaCl-treated seedlings and leaves were bigger than the controls.S. salsa plants have high capacity of accumulating Na~+ in low Na~+ concentrationenvironment. The Na~+ concentrations of cell sap from leaves were greatly increasedunder 200 mmol/L NaCl treatment. As a typical salt-accumulating halophyte, Na~+ wasmainly accumulated in leaves, and then the osmotic potential of the cell sap was muchlower in leaves than that in the soil. So S. salsa plants can modulate the osmolarity tomaintain the normal growing state under salt stress. Furthermore, low Na~+concentration environment in cytoplast can protect many enzymes from Na~+ toxicityto maintain the normal growing state under salt stress. In saline condition, K+concentrations in the cell sap from leaves were decreased significantly compared withthe controls. Since the growth of S. salsa seedlings was promoted by salt stress, thedecrease of K+ concentration in the cell sap to a certain extent wouldn’t inhibit theplants growth.2. Effects of NaCl treatment on the activities of H+-ATPase in tonoplast vesiclesprepared from leaves of S. salsa.The V-H+-ATPase activities in leaves were significantly increased by 200mmol/L NaCl treatment. The increased proton transport activity of V-ATPase mayprovide stronger driving force for Na+/H+ antiporter and a wide range of secondaryactive transport processes, leading to osmotic adjustment and elimination of iontoxicity. With lower osmotic potential of the cell sap, S. salsa plants can maintain thenormal growing state under salt stress.3. Subunit composition of tonoplast H+-ATPase from Suaeda salsa L.The tonoplast vesicles were obtained from leaves of Suaeda salsa plants by thediscontinuous sucrose gradient centrifugation.By solubilizing tonoplast vesicles withTriton X-100 (2%, v/v) and purifying ATPase complex by immunoprecipitation, thepartial pure enzyme was obtained. Western blot analysis with the antisera ATP95 andATP88 directed against the V-ATPase holoenzyme leads to the similar results thatV-ATPase consists of about 12 subunits. The V1 subcomplex is probably formed bythe subunits A, B, C, D, E, F, G and H, and the V0 subcomplex by a、d、e and c(or c′).The subunit a, G and e were firstly identified in the present study in Suaeda salsa L.4. Effects of NaCl treatment on subunit expression of H+-ATPase in tonoplastvesicles prepared from leaves of S. salsa.Western blot analysis indicated that the expression of a、A、B and c subunits wassignificantly up-regulated by 200 mmol/L NaCl treatment. Moreover, the expressionof B subunit was coordinated with c subunit of V-H+-ATPase at transcript andtranslation levels under NaCl stress. Then the number of V-ATPase holoenzymewould be increased in membrane as well. There was a basically consistentrelationship between the western blot analysis and the hydrolysis activity.In conclusion, significant difference was observed in the responses of growth,ion accumulation, activities of tonoplast proton pumps and expression at the levels oftranscription and translation of tonoplast H+-ATPase of S. salsa to NaCl stress. Thegreat difference suggested that the salt tolerance of S. salsa was related with manyfactors. Investigation of expressions and activities of V-H+-ATPase of S. salsaindicated that the changes of hydrolytic activity of V-H+-ATPase were consistent withthe gene expressions and translations and Na+ accumulation in cell saps. TheV-H+-ATPase might play a very important role in Na+ sequestration into vacuoleunder salt environments. Tonoplast H+-ATPase from S. salsa consists of about 12subunits by immunoprecipitation, SDS-PAGE and Western analysis. The V1subcomplex is probably formed by the subunits A, B, C, D, E, F, G and H, and the V0subcomplex by a、d、e and c(or c′). The subunits a, G and e were tested firstly in S.salsa. The expression of some subunits of S. salsa, and the expression of somesubunits at transcript and translation levels were significantly up-regulated underNaCl stress.更多还原