盐生植物白刺Na+/H+逆向转运蛋白提高植物耐盐性的分子调控机理

The mechanism of that halophytes adapt to salt-stress environment, involves in complex factors, such as cell structure, physiological processes and metabolism regulation. Among these complicated processes, the Na+/H+ transporters play important roles in plant salt-tolerance. The cytomembrane Na+/H+ antiporter proteins (SOS) can transport cytoplasmic Na+ to the exocellular, and the vacuolar Na+/H+ antiporter proteins (NHX) can deliver Na+ into the vacuoles against the electrochemical gradient. The compartmentation of Na+ provides an effective mechanism to avert the deleterious effects of Na+ on cytosol, and maintains an osmotic potential by Na+ in vacuoles. In previous studies, we isolated NsNHX1 and NsSOS1 genes from the halophyte Nitraria sibirica, and confirmed that these genes play critical roles in adapting to salt-stress environment of plants, and will likely be useful for improving salt-tolerance of crops. In the project, we plan to analyze the molecular regulation mechanism of the two genes in improving salt-tolerance of plants using transgenic Arabidopsis thaliana and Populus tomentosa over-expressing the two genes with molecular biological techniques, such as mRNA sequencing, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) etc. The expectant results will provide theoretical and experimental basis for salt-tolerance improvement of agricultural crops and forest trees using the two genes, and for popularization and application of the transgenic crops.

盐生植物适应高盐环境涉及复杂的细胞结构、生理过程和代谢调控机制，其中Na+/H+逆向转运蛋白扮演至关重要的角色。质膜上的Na+/H+逆向转运蛋白（SOS）将细胞质中的Na+逆向运送到胞外，液泡膜上的Na+/H+逆向转运蛋白（NHX）将细胞质中的Na+逆浓度梯度运送到液泡中区隔化集中，以减少Na+对细胞器的毒害并维持细胞的渗透平衡。前期研究中我们分离了盐生植物西伯利亚白刺的NsNHX1和NsSOS1基因，并通过遗传转化拟南芥和毛白杨的方法证实它们在植物适应高盐环境中发挥关键作用，可作为重要的候选基因应用于作物的耐盐性遗传改良。本申请项目拟以前期获得的转基因拟南芥和毛白杨为材料，利用转录组分析、双分子荧光互补、免疫共沉淀等分子生物学技术，分析NsNHX1和NsSOS1提高植物耐盐性的分子调控机理，进而为应用它们进行农作物和林木的遗传改良及转基因作物的推广应用提供理论和实验依据。

前期研究证实盐生植物西伯利亚白刺的NsNHX1和NsSOS1基因在植物适应高盐环境中发挥关键作用，可作为重要的候选基因应用于作物的耐盐性遗传改良。本项目以前期获得的转基因拟南芥和毛白杨为材料，利用转录组和蛋白互作分析等分子生物学技术，分析了NsNHX1和NsSOS1提高转基因植物耐盐性的分子调控机理。. 在盐胁迫条件下，NsNHX1和NsSOS1的表达水平显著上调，显示其在白刺耐盐机制中发挥关键作用。启动子驱动GUS报告基因在杨树中异源表达结果表明，NsSOS1的表达受茉莉酸甲酯、赤霉素、NaCl、低温和甘露醇的诱导；NsNHX1的表达受茉莉酸甲酯、赤霉素、乙烯、NaCl和甘露醇的诱导。超表达NsNHX1和NsSOS1转基因杨树耐盐性检测发现，转基因杨树的株高增长量、叶绿素含量、叶片相对含水量、根生物量、抗氧化酶类活性和脯氨酸含量均高于非转基因植株。过表达NsSOS1减少了转基因植株Na+的积累，过表达NsNHX1增加了转基因植株Na+的含量，表明NsSOS1介导的Na+外排，NsNHX1介导的Na+区隔化，对提高转基因杨树耐盐性具有重要作用。转录组分析发现，NsSOS1和NsNHX1在转基因杨树中的过表达引起了植物激素信号转导、植物-病原体互作、植物MAPK信号途径和苯丙烷生物合成等途径中相关基因表达水平的显著改变，这些途径协同作用增强了转基因杨树对盐胁迫的耐受性。蛋白互作分析显示，NsNHX1与杨树的PagCML11，NsSOS1与杨树的PagSOS2均不存在物理互作，推测它们在杨树中可能有不同的调控方式。以上研究结果为应用NsNHX1和NsSOS1进行农作物和林木的遗传改良及转基因作物的推广应用提供了理论和实验依据。