耐盐菊芋两个钠(钾)氢逆向转运蛋白调控钾钠平衡和耐盐力差异的作用机制

The compartmentation of sodium (Na+) from cytosol to vacuole mediated by tonoplast Na+(K+)/H+ antiporter (NHX) is one of the major mechanisms for salinity tolerance of the plants to adapt saline soil. Very recently, we have cloned two NHX genes named as HtNHX1 and HtNHX2 from the salt tolerant plant, Helianthus tuberosus. The two genes are highly homology to known tonoplast NHX genes in plants. Their sequences of open reading frames are identical except 342 base pairs missing in the middle part of HtNHX2. Both HtNHX1 and HtNHX2 were able to complement a tonoplast Na+/H+ antiporter mutated yeast strain in salt tolerance. Individual over-expression of HtNHX1 and HtNHX2 in rice could increase both Na+ and K+ uptake and translocation, and enhance salt tolerance, however, the HtNHX2 expression lines of rice showed much stronger salt tolerance and Na+ compartmentation in the shoot. In this project, we will explore the molecular mechanism of the salt tolerance improvemetn by the protein truncation due to missing the 114 amino acids in HtNHX2. We will detect whether the truncation results the following changes in HtNHX2 in comparison to HtNHX1: 1) subcellular localization; 2) C-terminal orientation; 3) activity and/or selectivity to Na+ over K+ in tonoplast, 4) partial structure which leads to the binding of HtNHX2 with specific regulatory proteins. We will also try to identify the key domains in HeNHX1 and HtNHX2 which control their activity and selectivity for Na+/H+ or K+/H+ exchanger and generate the chimeric NHX gene(s) through modification of HtNHX1 and HtNHX2. The physiological functions of the modified NHX genes and isolated specific regulatory proteins will be examed by detecting the growth performance,uptake and compartmentation of Na+ and K+ in their transgenic rice under different salinity treatments. Accomplishing this study, we will stand a good chance to elucidate the phsiological and molecular mechanisms underlying how the tonoplast located NHX functions in improving plant salt tolerance, and obtain the powerful NHX genes to be used for molecular breeding salt tolerant crops in future.

植物在高盐环境下生存的一个重要机制是通过液泡膜Na+/H+逆向转运蛋白NHX将细胞质Na+转运到液泡以维持细胞质高K+低Na+比例。本课题组从强耐盐菊芋中克隆到2个与液泡膜NHX高度同源、盐胁迫表达的基因HtNHX1和HtNHX2，后者较前者缺失114个氨基酸连续片段。在转基因水稻中发现两者均能促进K+、Na+吸收和转运，但HtNHX2能极显著提高地上部Na+含量和抗盐能力。本项目将通过分析HtNHX1与HtNHX2蛋白结构中的差异（缺失）片段对其亚细胞定位、Na+/H+和K+/H+交换的选择性和亲和力、互作调控蛋白、以及转基因水稻中抗盐相关基因表达、液泡和细胞质K+/Na+浓度的影响，明确 HtNHX2及其人工修饰改造和互作调控蛋白可否通过提高液泡膜转运Na+的选择性和活力以提高水稻的抗盐能力，为未来农作物抗盐提供有价值的基因和遗传材料。

植物液泡膜定位的Na+、H+逆向转运蛋白（NHX）被认为对植物抗盐有重要的作用。我们从耐盐耐贫瘠的菊芋中克隆了两个NHX同源基因，分别定名为HtNHX1 和HtNHX2。HtNHX2基因的编码区所有序列几乎与HtNHX1一致，但比HtNHX1 少114个氨基酸。我们对这两个基因在提高水稻抗盐和耐贫瘠方面的功能做了详细和深入的分析，所获得的主要结果和结论如下：1、在正常养分供应条件下，HtNHX2和HtNHX1均具有相似的提高水稻抗盐的功效，但在缺钾条件下，只有HtNHX2 具有提高耐钾缺乏和抗盐效果。无论是表达HtNHX1还是HtNHX2均极其显著提高水稻体内钾、钠的含量。我们发现，这两个基因提高水稻抗盐程度取决于栽培介质中钾的供应水平。无论是营养液水培还是在不同供钾水平下的土壤栽培均证实了这个新发现。2、发现HtNHX2还可以大幅提高水稻的耐贫瘠性，而HtNHX1则没有这个效果。针对这一新的发现，我们增加了一系列的水培、土壤盆钵和大田实验，进一步验证了HtNHX2提高水稻的耐低钾和耐氮磷低养分供应的特征。与此相反，表达HtNHX1反而促使水稻在缺钾和贫瘠条件下生长更差，产量更低。尽管国际上对植物的NHX基因的功能已经做了较多的研究，但是就我们所知，目前还没有任何关于NHX可以提高植物耐贫瘠的报道。3、深入研究了HtNHX1、HtNHX2在提高水稻耐盐和贫瘠差异的分子机制。我们通过在酵母、BY2 细胞和水稻的原生质中表达分析，均发现HtNHX1定位在液泡膜上，而HtNHX2则定位在小囊泡膜上。通过同源序列比对，相对于液泡膜型NHX蛋白，发现囊泡型NHX蛋白以及HtNHX2均缺损8个连续的氨基酸。裁截这8个氨基酸的HtNHX1de不能被定位到液泡膜。发现HtNHX1和HtNHX2均可以回补拟南芥囊泡型NHX双突体对盐的敏感性，但是只有HtNHX1提高了酵母和拟南芥对潮霉素的抗性。因此，HtNHX1和HtNHX2的功能差异主要取决于其表达定位。以上部分结果显示了这两个基因的应用潜力，获得了中国发明专利。