野生大麦离子转运蛋白HvHKT7和HvHKT2;2的耐盐机理及应用研究

Some elite accessions with high salt tolerance have been identified from the Tibetan annual wild barley, a unique and precious plant germplasm in China. High-affinity potassium transporter (HKT) have been increasingly highlighted because of their crucial functions for salt tolerance in plants. In our previous studies, two genes HvHKT7 and HvHKT2;2 were cloned from the wild barley genotypes with high salt tolerance, and they belong to one of the two HKT subfamilies, respectively. Meanwhile, the preliminary analysis on the subcellular localization and spatial-temporal gene expression pattern of the two genes were conducted. Accordingly, the functions of HvHKT7 and HvHKT2;2 and their potential application in genetic improvement of salt tolerance in barley will be addressed in this study. The main objectives of the current project are as follows: (1) to understand ion affinity and transport capacity of the two genes by using heterologous expression of the genes in yeast and Xenopus oocytes, respectively; (2) to reveal the interacting proteins of HvHKT7 and HvHKT2;2 and their cell-type expression by using Yeast one-hybrid system and Immunofluorescence analysis; (3) to determine the functions of HvHKT7 and HvHKT2;2 in regulation of ion transportation by using Microelectrode ion flux estimation (MIFE) and Virus-induced gene silencing (VIGS) technologies; (4) to prove the feasibility of HvHKT7 and HvHKT2;2 in the application of genetic improvement for salt tolerance in barley by using transgenic methods. It may be expected that the results of the current project may be helpful for deeply understanding the molecular mechanisms of salt tolerance of HvHKT7 and HvHKT2;2, efficient identification of elite genotypes or genes for the improvement of salt tolerance in barley as well as other crops.

青藏高原一年生野生大麦是我国独有的珍贵资源，蕴含有优异的耐盐种质。高亲和性钾转运蛋白（HKT）因其与植物耐盐性相关而备受关注。前期研究鉴定到高耐盐的野生大麦种质，并从中克隆到两个HKT不同亚家族的成员HvHKT7和HvHKT2;2，在亚细胞定位与基因时空表达特征上进行初步分析。在此基础上，本项目拟围绕它们的功能解析，深入研究其耐盐机理，并评估在大麦耐盐遗传改良上的应用潜力。主要研究内容有：（1）利用酵母和爪蟾卵母细胞两个异源表达系统，明确其离子亲和性及转运能力；（2）利用酵母单杂交和免疫荧光技术，明确其调控蛋白和组织定位；（3）利用病毒诱导的基因沉默和微电级离子流测定技术，明确其离子吸收特性；（4）利用大麦遗传转化体系，明确它们在大麦耐盐遗传改良上的应用潜力。研究结果可望阐明野生大麦HvHKT7和HvHKT2;2耐盐机理，丰富和创新作物耐盐理论，并为大麦耐盐遗传改良提供优异种质及基因资源。

土壤盐害是影响全球作物生产的一种最主要的非生物逆境，严重威胁农业生产的可持续发展及粮食安全。因此，开展作物耐盐的生理与分子机理研究，选育作物耐盐新品种，具有重要的理论与实践价值。研究发现，植物高亲和性钾转运蛋白（HKT）家族基因在维持细胞内Na+/K+平衡中起着关键作用，与植物的耐盐性表现密切相关而备受关注。前期从大麦中分离到两个HKT不同亚家族的成员HvHKT7和HvHKT2;2，本项目重点围绕HvHKT7的功能解析，深入研究其耐盐机理，并评估在大麦耐盐遗传改良上的应用潜力。主要研究内容和结果如下：（1）亚细胞定位分析显示，HvHKT7基因编码蛋白靶向膜系统，为一个典型的膜蛋白；（2）HvHKT7基因在地上部的表达量高于根部表达量，且该基因受盐胁迫诱导上调表达，随着盐浓度的增加而增加，明确了其时空表达特征；（3）利用爪蟾卵母细胞异源表达系统，结合电压钳技术，明确HvHKT7为一个典型的Na离子转运体，对其它一价阳离子不亲和；（4）利用大麦遗传转化体系，成功获得HvHKT7基因的超表达和RNAi遗传转化材料。结果显示，相比于野生型，过表达HvHKT7基因株系的耐盐性明显增强，而RNAi的植株对盐胁迫十分敏感；通过离子组分析发现，超表达和RNAi遗传转化材料地上部Na离子含量迥异。可见，HvHKT7基因参与调控大麦的耐盐性与地上部Na离子的卸载相关。研究结果可望深入阐明作物耐盐的分子机理，丰富或创新作物耐盐理论，从而指导作物耐盐遗传改良实践。