西藏野生大麦硝酸盐转运蛋白HvNRT2.6的低氮耐性机理及应用研究

Nitrogen (N) is the most common limiting factor for crop productivity worldwide. An effective approach to reduce the application of N fertilizer is to develop low N (LN) tolerant crop cultivars. High-affinity nitrate transporter (NRT2) plays crucial roles in efficient nitrate uptake under LN stress in plants. Tibetan annual wild barley is a unique and precious plant germplasm in China, well-known for its wide genetic diversity. HvNRT2.6, a member of NRT2 family, was cloned from the elite accessions with LN tolerance identified from the Tibetan annual wild barley. In this study, the functions of HvNRT2.6 and its potential application in genetic improvement of LN tolerance in barley will be investigated. The main objectives of the current project are as follows: (1) to reveal the cell-type expression of HvNRT2.6 by using immunofluorescence analysis, based on the preliminary analysis of spatial-temporal expression pattern of the gene; (2) to determine nitrate affinity and transport capacity of this gene by employing heterologous expression of the gene in yeast and Xenopus oocytes, respectively; (3) to discover the roles of HvNRT2.6 in regulation of nitrate transport by using CRISPR/Cas9 system and Microelectrode ion flux estimation (MIFE) technologies; (4) to prove the feasibility of HvNRT2.6 in the application of genetic improvement for LN tolerance in barley by using transgenic methods. The results of the current project are expected to clarify the molecular mechanisms of LN tolerance of HvNRT2.6, providing elite genotypes or genes for the improvement of LN tolerance in barley as well as other crops.

氮素是作物产量最常见的限制因子之一。培育耐低氮新品种是减少氮肥施用量最有效的途径。高亲和性硝酸盐转运蛋白（NRT2）与植物低氮耐性密切相关。西藏野生大麦是我国独有的珍贵资源，遗传多样性丰富，前期研究显示蕴含优异的耐低氮种质，并从中鉴定与克隆到与耐低氮密切相关的HvNRT2.6。本项目拟围绕该基因的功能解析，深入研究其低氮耐性机理，并评估在大麦耐低氮遗传改良上的应用潜力。主要研究内容有：（1）在分析基因时空表达特性的基础上，利用免疫荧光技术，明确其组织定位；（2）通过基因的异源表达，明确其硝酸盐亲和性及转运能力；（3）利用CRISPR/Cas9基因编辑和微电级离子流测定技术，明确其硝酸盐吸收特性；（4）利用大麦遗传转化体系，明确其在大麦耐低氮遗传改良上的应用潜力。研究结果可望阐明西藏野生大麦HvNRT2.6的耐低氮机理，并为大麦耐低氮遗传改良提供优异种质与基因资源。

氮素是世界范围内作物产量最常见的限制因子之一。通过挖掘作物自身利用氮素的潜力，培育耐低氮或氮素高效利用的作物品种，是解决土壤缺氮及氮肥供应不足的最基本途径，对现代农业可持续发展至关重要。研究发现，植物高亲和硝酸盐转运蛋白（NRT2）家族基因在植物的耐低氮响应过程中具有重要作用。前期从大麦中鉴定到一个与低氮耐性密切相关的NRT2家族成员HvNRT2.6，本项目重点围绕HvNRT2.6的基因功能解析，深入研究其耐低氮机理，并评估在大麦耐低氮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，HvNRT2.6基因编码一个典型的膜蛋白；（2）HvNRT2.6基因在根部的表达量高于其他组织；在不同组织中的时间动态表达分析表明，该基因受低氮胁迫诱导上调表达，其对低氮胁迫的响应具有基因型、组织和时间特异性；（3）HvNRT2.6基因在拟南芥中的过表达增强了其在低氮环境中的适应性；其原因可能是HvNRT2.6基因的过表达减弱了低氮胁迫对拟南芥的NR和GS等同化酶活性的抑制作用，促进其氮素的吸收同化，从而增强其低氮耐性；（4）利用基因编辑技术和大麦遗传转化体系，成功获得HvNRT2.6基因过表达和敲除大麦遗传转化材料。结果显示，相比于野生型，HvNRT2.6基因敲除大麦的低氮耐性明显减弱，而HvNRT2.6基因过表达大麦的低氮耐性明显增强；通过离子组分析发现野生型与遗传转化大麦材料的离子含量迥异。可见，HvNRT2.6基因在大麦低氮应答过程中具有重要作用。研究结果可望深入阐明作物耐低氮的分子机理，丰富或创新作物低氮耐性理论，从而指导作物耐低氮遗传改良实践。