西藏野生大麦HvHKT1转运蛋白的耐盐机制及其应用研究

Tibetan wild barley has been increasingly addressed because of its wide genetic diversity and high tolerance to abiotic stresses. In recent years, some wild barley accessions have been identified with high tolerance to salinity, acid soil and drought. The biological functions of high-affinity K+ transporters (HKT) are crucial for plant salinity tolerance. Gene cloning, fine-mapping, subcellular localization and analysis of spatial-temporal expression pattern of HvHKT1 were all accomplished in our previous studies. Here, the functions of HvHKT1 and its application in genetic improvement of barley salt tolerance will be investigated. The main contents of this study are as follows: (1) heterologous expression of HvHKT1 in yeast to uncover its ion specificity and transport capacity; (2) using Immunostaining and Yeast Two-hybrid System to identify cell-type expression of HvHKT1 and its interacting protein, respectively; (3) expression of HvHKT1 in Arabidopsis mutants and in combination of Microelectrode ion flux estimation (MIFE) to reveal its role in ion transportation and plant salt tolerance; (4) taking advantage of barley HvHKT1 over-expressors to explore the feasibility of HvHKT1 in genetic improvement of salinity tolerance. This program aims at comprehensively and systematically understanding the physiological function of HvHKT1 from Tibetan wild barley as well as its application in plant salt tolerance.

西藏野生大麦因其丰富的遗传多样性而备受关注，近年来已挖掘到多个对盐、铝毒、干旱等非生物胁迫具有很强耐性的种质材料。高亲和钾转运蛋白（HKT）在植物耐盐性上具有重要作用，在前期鉴定到西藏野生大麦高耐盐种质并完成HvHKT1基因克隆、遗传定位、亚细胞定位以及时空表达特性分析的基础上，本项目将围绕HvHKT1转运蛋白的功能解析，深入研究其耐盐分子机理，并明确其在大麦耐盐育种上的应用潜力。主要研究内容有：（1）利用酵母异源表达，明确HvHKT1离子亲和性和转运能力；（2）利用酵母双杂交和免疫组化，明确HvHKT1的互作蛋白和表达组织；（3）利用模式植物拟南芥和离子流测定技术（MIFE），明确HvHKT1在盐胁迫下的离子转运调控及其增强耐盐性的机理；（4）利用大麦成熟胚遗传转化株系，明确该基因在大麦耐盐遗传改良上的应用潜力。研究结果可望阐明西藏野生大麦HvHKT1转运蛋白的耐盐机理及其应用前景。

诸多研究表明，植物高亲和性钾转运蛋白（HKT）的生理学功能与植物盐胁迫下的耐性表现密切相关。禾谷类作物各物种都含有多个HKT编码基因，HKT基因功能研究是解析不同禾谷类作物的盐胁迫耐性机制和开展耐盐育种的基础。大麦与水稻、小麦等其它禾谷类作物相比，耐盐性较强，我们前期发现其耐性与HKT转运蛋白家族控制的组织离子平衡有关。但是，大麦HvHKT1;1转运蛋白的耐盐分子机理和生理机制仍不甚清楚，本研究综合运用酵母和爪蟾卵母细胞表达、遗传转化、基因沉默、免疫荧光、电压钳以及微电极离子流测定等手段，系统地明确了该基因功能及其参与的耐盐机制。大麦HvHKT1;1特异响应盐胁迫上调表达，是一个在木质部表达的质膜蛋白并且高度Na亲和，在盐胁迫下通过木质部卸载和径向运输钠离子到表皮细胞，而显著降低大麦和拟南芥地上部Na积累并增强植株耐盐性。同时，HvHKT1;1间接参与了盐胁迫下根际K和Ca的离子平衡，增强了植株应对渗透和氧化胁迫的能力。此外，离子组学和代谢组学分析完阐明了两类离子通道HKT1和SOS1对植物耐盐性的调控途径及机理，发现耐性差异材料在糖酵解和三羧酸循坏的启动优先级上存在差异。本研究鉴定并明确的大麦优异耐盐候选基因HvHKT1;1功能，肯定了该基因在大麦耐盐育种上的应用潜力，从而为促进大麦特异种质的发掘与利用以及作物耐盐遗传改良提供理论与技术支撑。