拟南芥铵钾平衡调控基因AMOS2的克隆及功能分析

Ammonium (NH4+) is necessary for normal life in most organisms, but is toxic to the cells when excess. NH4+/K+ homeostasis is thought to be key to protect the plants from ammonium stress, but molecular mechanisms behind the interaction of K+ and NH4+ toxicity is not known. In this study, we use Arabidopsis ammonium overly sensitive 2 (amos2) mutant identified previously in our lab, which defecting NH4+/K+ homeostasis compared with wild type in response to NH4+ stress, to clone the AMOS2 gene and explore the gene function and the AMOS1-mediated NH4+/K+ homeostasis molecular pathway by combination of modern plant molecular biological and plant nutrition approaches. Firstly, to research the characteristic and function of AMOS2 by analyzing the expression orientation and important promoter binding elements for response to the vary NH4+/K+ radio, combination of electrophysiological techniques and gene chip technology. Secondly, to confirm the molecular pathway by analyzing the difference between WT and mutant in NH4+/K+ uptake, transport and cytosolic homeostasis. Collectively, this program is designed to reveal AMOS2 gene function in control NH4+/K+ homeostasis and the role of AMOS2 in protecting plant development from ammonium stress. It is very helpful for the improvement of ammonium use efficiency in higher plants under high ammonium stress.

铵是植物生长必需的养分离子，但其过量时易造成毒害。在铵胁迫下维持植物体内有效的铵钾平衡是提高植物忍耐铵毒害的关键。但目前对铵钾平衡调控过程的分子生物学机制研究尚未完全展开。本研究以本实验室先前筛选验证的铵胁迫下铵钾调控失衡的拟南芥突变体amos2为核心材料，应用现代植物生物学和营养学等先进技术，克隆铵钾平衡调控基因AMOS2，并以此为突破口研究AMOS2基因功能及其调控铵钾平衡的分子生理途径。通过分析AMOS2表达定位受不同铵钾条件调控的特征及其启动子区应答铵钾信号的关键元件，并拟结合电生理、基因芯片技术，初步研究AMOS2基因的特征与功能；分析铵胁迫下WT和amos2体内铵钾吸收、转运及胞质内平衡的差异，选定AMOS2的作用途径。研究结果将有助于更深入地研究高铵下拟南芥通过调控内部铵钾平衡来提高铵抗性的生物学途径及机理，丰富对植物感知适应高铵环境的认识，为提高铵态氮利用率提供新的理论知识

铵是植物生长发育必需的养分离子，但其过量时易造成毒害。侧根是植物养分吸收的重要组织也是对高铵响应的一个非常灵敏的指标。本研究是以本实验室先前从T-DNA插入库筛选验证的铵胁迫下侧根铵敏感的拟南芥突变体 amos2 为核心材料，克隆铵钾平衡调控侧根发育基因 AMOS2，阐明该基因与突变体表型之间的关系，明确 AMOS2 基因调控植物侧根铵敏感的途径，了解植物铵毒害的分子机理。结合图位克隆技术和二代测序等手段，成功克隆获得AMOS2基因，定位于第一染色体上端（由于文章尚未发表，具体位置暂时保密），并利用其等位基因突变体进行了杂交验证。研究结果表明：AMOS2基因在高铵调控钾离子由根向叶运输中发挥作用，维持地上部铵/钾的相对平衡，AMOS2基因突变会导致地上部铵钾失衡，造成铵积累，从而影响地上部乙烯水平及后续的侧根及地上部发育过程。此外，AMOS2基因在正常情况下也可能参与了生长素的合成或分配过程，从而影响无铵条件下的侧根发育。研究结果表明AMOS2基因可能是重要的调控地上部铵/钾平衡来影响高铵下生长素-乙烯互作影响侧根发育的调控者。

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