铝胁迫下14-3-3蛋白调控铝敏感型大豆硝态氮吸收的分子机理研究

Aluminum (Al) toxicity is an important obstruction factor for nitrate uptake by plants from acidic soils. Nitrate uptake by plants is achieved through nitrate transporters accompanied with the co-transport of H+, which is extruded by plasma membrane (PM) H+-ATPase. 14-3-3 proteins can interact with PM H+-ATPase to regulate its activity and H+ extrution，which might be thereby involved in regulation of the nitrate uptake by plants. On the other hand, 14-3-3 proteins can impact on antioxidant enzyme activity and H2O2 content by regulation of antioxidant enzyme expression. The H2O2 can decrease the phosphorylatin of PM H+-ATPase and its interaction with 14-3-3 proteins. The preliminary studies of this project showed that Al stress reduced the expression of 14-3-3 proteins, increased the content of H2O2 and decreaed PM H+-ATPase activity and nitrate uptake in an Al-sensitive soybean (SB) roots. Using physiologyical and molecular methods, this project will investigate the correlation among the expression level of nitrate transporter, 14-3-3 protein expression and antioxidant enzyme activity, H2O2content and its interaction with PM H+-ATPase as well as nitrate uptake by SB roots. The results will be clarified the molecular mechanism of 14-3-3 protein-regulated nitrate uptake in SB roots under Al stress. The mechanism will be verified in transgenic soybeans with alteracted-expression of 14-3-3 proteins. The evidence obtained by this project can provide a novel strategy and generesources for genetic engineering to improve plant NO3- uptake from acidic soils.

铝毒是影响植物从酸性土壤吸收养分的一个主要障碍因子，植物吸收硝态氮通过硝态氮转运蛋白的作用伴随质膜H+-ATP酶泵出胞外的H+同向运输完成。植物14-3-3蛋白可通过与磷酸化质膜H+-ATP酶结合调控其活性和H+分泌,因此可能参与植物硝态氮吸收的调控；还可能通过调控抗氧化酶表达影响抗氧化酶活性和H2O2含量，H2O2可降低质膜H+-ATP酶磷酸化及其与14-3-3蛋白结合。我们前期研究发现铝胁迫降低铝敏感型大豆SB根中14-3-3蛋白表达，增加H2O2含量并降低根质膜 H+-ATP酶活性和硝态氮吸收量。本研究用生理生化和分子生物学方法解析铝胁迫SB根中硝态氮转运蛋白表达；14-3-3蛋白表达和抗氧化酶活性、H2O2含量及其与质膜H+-ATP酶的互作和硝态氮吸收的相关性，研究铝胁迫下SB根中14-3-3蛋白调控硝态氮吸收分子机理，为增强植物吸收酸性土壤氮素基因工程提供新的操作策略和基因资源。

铝毒是影响植物从酸性土壤吸收养分的一个主要障碍因子，但影响植物吸收硝太氮的分子机理仍不清楚。本研究通过对大豆实施铝胁迫，使用质膜H+-ATPase的激活剂和抑制剂，添加H2O2和H2O2的清除剂抗坏血酸ASA以及对14-3-3蛋白基因过表达和抑制表达烟草植株进行生理生化指标分析，阐明14-3-3蛋白调控铝胁迫下铝敏感型小黑豆SB硝态氮吸收分子机理。铝胁迫下铝抗性丹波黑大豆RB能降低自身H2O2含量，提高质膜H+-ATPase活性及表达量，促进质膜H+-ATPase与14-3-3蛋白的结合，促进硝态氮吸收；而SB则表现相反结果。铝胁迫下RB拥有比SB更强的抗性和更好的硝态氮营养吸收。铝胁迫下激活剂能提高质膜H+-ATPase活性和对硝态氮吸收，而抑制剂抑制质膜H+-ATPase活性和对硝态氮的吸收，质膜H+-ATPase活性与硝态氮吸收密切相关。通过外源抑制剂抑制RB的质膜H+-ATPase表达时，RB的硝态氮吸收量也随之降低；通过外源激活剂激活SB质膜H+-ATPase表达时，SB的硝态氮吸收量随之得到了大幅提升，这表明质膜H+-ATPase在大豆硝态氮吸收中扮演着重要角色。铝胁迫下RB的H2O2含量下降、质膜H+-ATPase表达活性升高、促进硝态氮吸收，而SB呈相反的结果。外源添加H2O2会导致大豆内源H2O2的增加，质膜H+-ATPase磷酸化及与14-3-3互作也同时降低，导致硝态氮吸收量下降，但添加H2O2清除剂ASA后，出现相反的结果。铝胁迫可能通过影响大豆根H2O2含量来影响大豆质膜H+-ATPase活性，从而影响硝态氮吸收。通过过表达和抑制表达14-3-3基因烟草，获得了蛋白高表达的5个株系和抑制表达的3个株系烟草。研究表明铝胁迫下 WT 和转基因烟草中 14-3-3 蛋白对质膜 H+-ATPase活性和NO3-吸收具有调控作用。铝胁迫降低大豆根14-3-3蛋白的表达量及其与质膜 H+-ATPase的结合，减少质膜H+-ATPase的活性和 H+泵出，从而减少大豆根对 NO3- 吸收；同时减少表达的14-3-3 蛋白调控抗氧化酶的表达，从而降低氧化酶活性，导致根细胞内H2O2含量上升，促进质膜H+-ATPase的磷酸化，从而降低它与14-3-3蛋白的结合，降低H+-ATPase活性及H+泵出和NO3-吸收。铝胁迫下这两种途径同时发挥作用来降低SB对