【作者】 杨明峰；

【导师】 王宝山；

【作者基本信息】 山东师范大学 ， 植物学， 2002， 硕士

【摘要】 在盐渍条件下，碱蓬的根和地上部分在生长、离子积累、质子泵活性、V-H~+-ATPase的表达等几个方面存在明显差异，表明盐胁迫下地上部分和根在耐盐方面的作用和耐盐机理不同，二者共同影响着植物的耐盐性。NaCl和KCl对碱蓬上述各方面的效应也有显著差别。本实验目的是研究NaCl和KCl对碱蓬生长及液泡膜质子泵活性的效应，以盐生植物碱蓬为材料分别用NaCl和KCl处理，测定根和地上部分的有关生理指标，通过蔗糖密度梯度离心提取了碱蓬根和地上部分的液泡膜微囊，测定了V-H~+-ATPase、V-H~+-PPase活性，并进行Western blot分析以确定V-H~+-ATPase各亚基表达情况，主要结果如下： 1 NaCl处理对碱蓬根和地上部分生长和液泡膜质子泵的效应 1．1生长和离子积累 从碱蓬地上部分鲜重和干重变化趋势看，低盐胁迫(200mmol／L)明显促进碱蓬地上部分的生长，高盐胁迫(400mmol／L NaCl)与低盐胁迫相比抑制碱蓬地上部分的生长；在各种浓度的盐胁迫条件下，根的生长受盐胁迫影响小，说明了盐胁迫下地上部分生长变化大，而根在盐胁迫下保持稳定。根的肉质化程度远低于地上部分，但是根在盐处理下肉质化程度也增加。 碱蓬用正常Hoagland培养液培养时有聚Na~+作用，根吸收的大部分Na~+运往地上部分积累起来，可以作为渗透调节剂，增强吸水能力。盐胁迫下，Cl~+-、Na~+主要积累在地上部分，大约有97％的Na~+积累在地上部分，根只占3％，因而地上部分细胞汁液渗透势大大低于根。由于碱蓬的根鲜重占整株鲜重的1╱10左右，而且盐胁迫下无机盐相对含量很低，所以碱蓬的地上部分是积累盐的主要部位，根在抗盐中的作用不在于积累无机盐。在非盐胁迫条件下碱蓬地上部分除了选择K~+作为渗透调节剂以外，Na~+也作为一种重要渗透调节剂，根倾向于选择K~+作为渗透调节剂。 1．2液泡膜质子泵活性与亚基表达 碱蓬根和地上部分V-H~+-ATPase的亚基有差异。从地上部分的V-H~+-ATPase中检测到66kDa、55kDa、36kDa和18kDa等4种亚基；根只显示1种亚基(18kDa)，其余3个亚基与本实验所用抗体无明显反应。可见，根和地上部分V-H~+-ATPase的组成性构成不同，存在异构现象(isoform)，也有可能是有其他三种亚基虽然相同但是蛋白量很少。200mmol／L NaCl胁迫后，地上部分V-H~+-ATPase各亚基的数目及种类没有变化，表达量明显增加，各亚基增加幅度相似。根内V-H~+-ATPase的18 kDa亚基也明显受盐刺激而表达增加，与地上部分不同的是，根的印迹图上出现一个新肽段(16 kDa)，可能是18kDa亚基的降解产物。NaCl胁迫后，地上部分V-H~+-ATPase各亚基表达量明显增加，其水解活性也相应增加，由于没有新亚基的出现而且盐胁迫后亚基表达形式相同，所以V-H~+-ATPase水解活性增加的原因是盐胁迫刺激丁V-H~+-ATPase表达而不是结构改变。根内V-H~+-ATPase 18kDa亚基也受盐刺激而明显增加，和地上部分相比，根V-H~+-ATPase的18kDa亚基更容易受盐胁迫影响而发生降解，因而根内V-H~+-ATPase活性受 NaCI血KCI斟松对仪8住犬及般尬殿瘩子鱼捞注肘级应 到影响，其V－H”－ATPase活性没有象地上部分那样大幅度增加。盐胁迫后，地上部分 V－H”－PPase水解活性略有增加，根中的变化不显著。根中质子泵表达量虽有增加，但其 活性增加幅度不大，而且根中Na”等积累很少，表明根中液泡膜质子泵在根系水平上的 离子区域化中不起主要作用（对于根的皮层来说可能有重要作用），而地上部分质子泵 （主要是V－H”－ATPase）在地上部分离于区域化中可能担当重要角色。 2 KCI处理对碱蓬生长和液泡膜质子泵的效应 二．二生长和离子积累 碱蓬能很好的适应NaCI胁迫，NaCI胁迫不但没有造成伤害，还促进了碱蓬生长。 低浓度（100 nunol／L）KCI既没有抑制生长也没有促进生长，而高浓度（200，400 mmol几） KCI对碱蓬不但无利，反而有害，严重抑制碱蓬的生长，甚至导致碱蓬死亡。KCI胁迫 下，碱蓬幼苗含水量比对照下降，KCI浓度越大下降越显著，KCI对碱蓬伤害大的一个 原因是水分吸收速率小于蒸腾速率，使碱蓬水分代谢严重失衡。碱蓬在NaCI胁迫后， 吸水速率大于蒸腾速率，不但没有失水，含水量反而高于对照。KCI胁迫下，碱篷不发 生肉质化，而同浓度的NaCI促进碱蓬肉质化，Na”可能是促进碱蓬肉质化的主要因子， 而 K”和 CI不能促进碱蓬肉质化。KCI胁迫下碱蓬细胞汁液0 浓度增加幅度远大于 NaCI胁迫下细胞汁液CI“浓度增加幅度，KCI胁迫下在地上部分积累过多的CI”可能是 KCI胁迫伤害程度大的原因之一。KCI胁迫比NaCI胁迫时碱蓬的相对电导率升幅大， 表明 KCI对细胞膜伤害程度大。 2．二质子泵活性与亚基表达 相同浓度的 NaCI和 KCI（200。of几）胁迫后，地上部分 V－H”－ATPase各亚基数 目和表达形式与对照相同。＊＊口胁迫后，地上部分?更多还原

【Abstract】 Significant differences were observed in the responses of growth, ion accumulation, tonoplast proton pump activities, expression of V-H+-ATPase of shoots and roots of Suaeda salsa to salt stress, suggesting the cooperation and different functions of shoots and roots in salt resistance. There are significant differences in the responses of Suaeda salsa to NaCI and KC1 in the above aspects. The aim of the present study is to investigate the effects of NaCI and KC1 on growth parameters and vacuolar proton pumps of the halophyte Suaeda salsa. The Suaeda salsa seedlings were cultured under different NaCI and KC1 concentrations, and the tonoplast-enriched vesicles were isolated by sucrose density gradient from shoots and roots of Suaeda salsa plants. In addition to the determination of growth, V-H+-ATPase and V-H+-PPase activities, the subunit composition of V-H+-ATPase was studied by western blot analysis in the tonoplast preparations from plants grown under different conditions in order to correlate changes in enzyme activity with structural modifications. The main results are shown as follows.1 Effects of NaCI on the growth and tonoplast proton pumps of shoots and roots of Suaeda salsa.1.1 Growth and ion accumulationThe fresh weight and dry weight of Suaeda salsa shoots were markedly stimulated by low salinity while reduced by high salinity. The growth of roots was not affected so much as shoots indicating that the growth of shoots were changed drastically under salt stress while roots remained steady in the same conditions. The succulent degree of roots enhanced by salt treatment is much lower than that of shoots.In normal environment, the roots of Suaeda salsa uptaked Na+ and transported it to shoots where NaT act as osmotica which lowered water potential and enhanced water-absorption ability of plants. Cl~ and Na+ mainly accumulated in shoots under salt stress. About 97% of NaTwas stored in shoots and only 3% Na+in roots. Therefore, osmolarity of the cell sap was much lower in the shoots than in the roots at a given salt concentration. The shoots of Suaeda salsa were the main parts of NaT accumulation and the roots did not play a major role in ion accumulation. The shoots of Suaeda salsa used both Na+ and K+ as osmotica while roots prefer to use K+ as osmotica.1.2 Activities and subunits of tonoplast proton pumpsThe subunit composition of V-H+-ATPase in shoots and roots were different for that the antiserum immunostained four polypeptides from the membrane fractions of shoots with apparent molecular masses of 66, 55, 36 and 18 kDa which most probably represent V-H+-ATPase subunits A, B, D and c respectively while the three high molecular mass polypeptides were absent in the roots. The three undetected V-H"-ATPase subunits might not cross-react with the antiserum used in this study. Therefore, the constitutive composition of V-H+-ATPase subunits was different in roots and shoots, and there may be V-H+-ATPase isoforms in Suaeda salsa or three subunits protein amount was too low to be detected in roots. The patterns of V-H+-ATPase subunits in the shoots were not affected by NaCI stress thoughV-H+-ATPase protein amount and activity were significantly increased. A 18 kDa subunit of V-H+-ATPase in root was enhanced by salt stress and an additional polypeptide (16 kDa)was detected in the fractions of roots of NaCl-treated plants. It might be suspected that the 16 kDa polypeptide found in roots under NaCl stress was a hydrolyzed fragment of 18 kDa subunit. Under NaCl stress, Protein amounts of V-H+-ATPase subunits increased significantly and the activities of V-I-T-ATPase also increased. The increase of V-H+-ATPase activity was not due to structural changes of the enzyme, but to a quantitative up-regulation of V-H+-ATPase protein for there were no new subunits and the subunits displayed in the same pattern. A 18 kDa subunit of V-H+-ATPase in the roots was enhanced by salt stress, but the subunit was possibly degraded by salt stress so that the activity of V-H+-ATPase in the roots did not increase d更多还原