【作者】 狄廷均；

【导师】 沈其荣； 朱毅勇；

【作者基本信息】 南京农业大学 ， 植物营养学， 2008， 硕士

【摘要】 水稻是典型的以吸收铵态氮（NH₄⁺-N）作为氮源的水生作物。目前,水稻根系吸收铵态氮所引起的根际强烈酸化的适应机制尚不清楚,因此研究比较了pH 6.5和pH3.0条件下水稻根系细胞膜H⁺-ATPase对铵态氮营养和硝态氮（NO₃^--N）营养的适应差异。为此,把全硝和全铵营养液pH分别调到pH 3.0和pH 6.5。恒定pH培养4 d后,用两相法分离水稻（Oryza sativa L.“japonica”）根系细胞膜。研究结果表明:1.离体条件下,不论是铵态氮还是硝态氮营养,pH 3.0条件下培养的水稻根系细胞膜H⁺-ATPase的水解活性、最大初速度V_max、米氏常数K_m、H⁺泵活性、细胞膜对H⁺的通透性以及跨膜pH梯度都显著高于pH 6.5条件下培养的水稻根系。2.不同氮素营养和不同pH条件下,水稻细胞H⁺-ATPase对钒酸盐的敏感性不变。3.H⁺-ATPase多克隆抗体免疫分析发现,低pH条件下培养的水稻根系细胞膜H⁺-ATPase的酶浓度显著高于高pH,而不受氮素形态影响。4.在转录水平,低pH条件下培养的水稻根系细胞膜H⁺-ATPase基因OSA1、OSA3、OSA7、OSA8和OSA9的表达明显强于高pH。但pH改变对OSA2的表达没有影响。试验中,没有检测到OSA4、OSA5、OSA6和OSA10的PCR扩增产物。5.试验证明,水稻根系细胞膜H⁺-ATPase是通过增加同工酶基因表达,提高细胞H⁺-ATPase的水解活性和泵活性来适应根际低酸环境,这种适应性不依赖于氮素形态。小白菜一种典型的高硝酸盐累积的蔬菜。硝酸盐在小白菜体内不同组织中的积累存在显著差异,为了细胞膜H⁺-ATPase对这种差异的适应,用两相法分离了水培小白菜品种上海青（Brassica chinensis L.）叶柄和叶片细胞膜,并测定了与硝酸盐运输相关的质子泵的水解活性。研究结果如下:1.小白菜细胞膜纯度在90%以上。2.离体条件下,叶柄细胞膜H⁺-ATPase的水解活性显著高于叶片。3.叶柄H⁺-ATPase最佳pH值在pH 6.6,而叶片在pH值在6.4至6.6之间。并且,叶柄细胞膜上H⁺-ATPase的K_m值显著高于叶片。4.将H⁺-ATPase在20℃及30℃时测定所得的V_max代入Arrhenius方程计算后发现,叶柄H⁺-ATPase的活化能也高于叶片。5.Western Blot结果说明,叶柄细胞膜H⁺-ATPase酶浓度要显著高于叶片H⁺-ATPase。试验表明,硝酸盐在不同组织中的积累差异与其细胞膜H⁺-ATPase活性大小、K_m值、活化能以及H⁺-ATPase的蛋白浓度都密切相关。因此,叶柄细胞膜H⁺-ATPase活性高很可能是增强硝酸盐吸收的一个重要原因。更多还原

【Abstract】 Rice plants prefer ammonium to nitrate in paddy soils.The mechanisms responsible for adaptation of rice roots to the NH₄⁺ nutrition-associated strong rhizosphere acidification were not fully elucidated.In the present study the adaptation of plasma membrane H⁺-ATPase activity in rice roots to ammonium versus nitrate nutrition was investigated at pH 6.5 and 3.0.Nutrient solution was gradually acidified to pH 3.0 to allow adaptation of rice roots to low pH.After 4 d cultivation at final pH levels plasma membrane was isolated from rice（Oryza sativa L."japonica"） roots by two-phase method system.The results indicated that:1.In vitro,hydrolytic H⁺-ATPase activity,maximum initial velocity(V_max),Mechaelis constant（Kin）,H⁺-pumping activity,H⁺ permeability of plasma membrane and pH gradient across the plasma membrane of membrane vesicles,were significantly higher for rice roots grown at pH 3.0 than pH 6.5,irrespective of nitrogen forms supplied to rice plants.2.Vanadate sensitivity of the plasma membrane H⁺-ATPase remained unchanged.3.Immunoassays using polyclonal antibodies against H⁺-ATPase showed significant higher enzyme concentration of the plasma membrane H⁺-ATPase in the membrane fraction obtained from rice roots grown at low pH than at high pH,irrespective of nitrogen forms supplied.4.On the transcriptional level,the expression of OSA1,OSA3,OSA7,OSAS,and OSA9 from roots at low pH was much higher than those at high pH.The transcription rate of OSA2 remained unchanged at low pH.However,for OSA4,OSA5,OSA6 and OSA10 no PCR products could be found.5.In conclusion,various isoforms of plasma membrane H⁺-ATPase of rice roots were highly regulated after adaptation to low nutrient solution pH,resulting higher hydrolytic activity and H⁺-pumping activity of plasma membrane H⁺-ATPase.This adaptation was independent of nitrogen forms supplied.Pakchoi is a typical vegetable with high nitrate conten.In order to investigate the adaptation of the plasma membrane H⁺-ATPase to the significant difference of the accumulation of nitrate in different tissues of Pakchoi（Brassica chinensis L.cv. ’Shanghaiqing’）,the plasma membrane of petiole and leaf was isolated by two-phase method system.The hydrolytic activity of plasma membrane H⁺-ATPase in relation to the NO₃^transport was studied.The results were below:1.The purity of plasma membrane was above 90%.2.The H⁺-ATPase hydrolytic activity of petiole was significantly higher than that of leaf,in vitro.3.The pH optimum of the H⁺-ATPase activity for petiole was pH 6.6,but for leaf was between 6.4 and 6.6.In addition,the K_m for the petiole plasma membrane H⁺-ATPase was higher than that for the leaf.4.V_max at two temperatures（20℃and 30℃） was used to calculate the active energy by Arrhenius equation and results showed that the active energy for the H⁺-ATPase of petiole was higher than that of leaf.5.The westem blot showed that the enzyme concentration of plasma membrane H⁺-ATPase from petiole was higher than that from leaf.The results indicated that nitrate content was high in petiole and this was related to the high hydrolytic activity of its plasma membrane H⁺-ATPase,K_m value,active energy and the high enzyme concentration of H⁺-ATPase.It is may be true that the higher activity of plasma membrane H⁺-ATPase in petiole caused more uptake of NO₃^than in leaf.更多还原