磷酸化过程对水稻 OsAMT1;1 铵吸收功能的调控机制研究

Ammonium nitrogen is the main nitrogen form in paddy soil, and the absorption and utilization of it is taken by the AMT protein in plants. The AMT mediated ammonium uptake is of significant importance for rice growth and high yield. However, due to significant feedback suppression regulation in the process of AMT-mediated ammonium uptake in rice roots, raise of the ammonium uptake through the large amount of nitrogen fertilizer input and the functional AMT transgenic methods in the high-yielding cultivation mode were largely limited. Protein phosphorylation / dephosphorylation is an intrinsic control strategy for plants to respond to environmental changes. In view of the transcriptome data, we found that a large number of phosphorylation related genes responded to the change of NH4 +. The present application mainly focus on the changes of the genes related to the phosphorylation process upon ammonium fluctuation, and extracts the relevant differentially expressed genes from the completed transcriptome data, to study its role in the functional characterization of rice OsAMT1;1, and phosphorylation traget sites in OsAMT1; 1 were verified by site-directed mutagenesis and electrophysiological techniques. Used OsAMT1;1 as an example, we anticipated to reveal the phosphorylation process and its mechanism of promoting ammonium uptake capacity, and to provide the basis for break the feedback regulation, to match the high ammonium uptake demand of high yield rice under high ammonium fertilizer.

铵态氮是稻田土壤中主要的氮素形态，而植物对该形态氮素的吸收和利用是由AMT来承担的。AMT负责的铵吸收对于水稻生长以及高产极为重要。然而，由于水稻根系由AMT介导的铵吸收过程存在显著的反馈抑制调节现象，高产种植模式下通过氮肥的大量投入以及转AMT功能基因等方法来提高水稻铵吸收能力受到限制。蛋白磷酸化/去磷酸化是植物应对环境变化而采取的一种内在调控策略。鉴于前期转录组测序发现大量磷酸化过程相关基因响应了供NH4+变化，本申请主要研究不同供铵状况下磷酸化过程相关基因的变化，从已完成的转录组测序数据中抽取相关差异表达基因；研究其对水稻OsAMT1;1吸铵功能的调控特征和作用位点；同时通过定点突变和电生理技术验证OsAMT1;1蛋白序列中潜在磷酸化位点的作用效果，以OsAMT1;1为例揭示可促进其铵吸收能力的磷酸化过程及其作用机制，打破其反馈调节，匹配高产水稻所需的“高铵下持续高效吸铵”功能。

水稻根系由AMT介导的铵吸收过程存在显著的反馈抑制调节现象。寻求打破其反馈调节的措施，匹配高产水稻所需的“高铵下持续高效吸铵”是本项目的研究重点。本项目采用的主要研究技术手段是电生理。鉴于目前AMT电生理研究中底物吸收信号小，不易获取可靠结果；同时两种传统铵转运蛋白AMT电生理记录方法存在各自优缺点的客观事实，本研究首先建立了一种整合了两者优点，又避免了其各自缺点的更稳定可靠的电生理记录方法。AMT介导铵吸收的反馈抑制主要是通过磷酸化位于其碳末端的高度保守的一个苏氨酸-T460来实现的。借助上述改进的电生理方法及其它研究手段，本项目筛选得到一个特殊的基因ZmAMT1.1a，与目前已经报道的AMT基因不同，该基因具有功能不受保守性磷酸化位点T460调控的特征，ZmAMT1.1a未来可用作打破作物反馈抑制的候选基因。