水稻磷素感应器SPX4蛋白稳定性调节因子的鉴定及分子生理机制研究

Chemical fertilizer has been over applied on rice production in China, which has been heavily threatening the environment and wasting the mineral resources. Enhancing Pi-acquistion and utilization efficiency in rice has great significance for reducing chemical fertilizer application. Therefore, it is very important to comprehensively understand how crops sense Pi status in plant or environment. It is well known that SPX domain containing protein can regulate and sense the Pi-status in plant. However, how the internal or external Pi status affect the stability of SPX domain containing proteins remain obscure. Basing on our previous research results, we identified a ubiqutin E3 ligase that could interact with SPX4 (designed as SDEL1) by the method of yeast-two-hybrids assay from the rice cDNA library. Besides, SDEL1 could ubiquitylate SPX4 in vitro. Enhancing the expression of SDEL1 could result the Pi accumulation in shoot and root. On the opposite, malfunction SDEL1 could retard the growth of plant, which is similar to the phenotypic of the SPX4 overexpressors. Preliminary data indicated that SDEL1 is one candidate regulator of the stability of SPX4. Therefore, in current project, protein interaction analysis, transcriptome analysis, and plant nutrition physiological analysis will be used to determine the molecular mechanism of SDEL1 in regulating the stability of SPX4 protein in rice. The potential application of SDEL1 for breeding low phosphate tolerance rice cultivars will also be determined.

我国水稻生产过渡依赖化肥投入，造成严重的环境污染与资源浪费。提高水稻对磷养分的吸收利用效率对化肥减施增效具有重要意义。因此，探知作物如何感知体内外磷素变化至关重要。在植物应对低磷胁迫过程中，SPX结构域的蛋白是植物体内磷素状态的感应器。但是，外界磷素调节SPX蛋白稳定性的分子生理机制仍然不清楚。本课题组前期利用酵母双杂交的方法在水稻cDNA文库中筛选到一个能与SPX4相互作用的泛素化E3连接酶SDEL1。SDEL1在体外能使SPX4发生泛素化修饰；增强表达SDEL1会导致水稻地上部无机磷升高；突变SDEL1则抑制植株生长，无机磷含量降低。初步结果暗示，SDEL1是SPX4潜在的降解因子。因此，本研究将围绕SDEL1参与的SPX4蛋白稳定性调控体系，利用蛋白互作分析、转录组分析、以及养分生理实验，探讨SDEL1通过调节SPX4蛋白稳定性进而调控磷素养分平衡的营养分子机制及其潜在的运用前景。

植物的生长发育受到土壤中无机磷的含量影响，然而土壤中的无机磷容易被土壤中的铁、钙、铝等重金属离子固定而不能被植物吸收利用。在农业生产实践中,主要通过大量施用磷肥来满足作物的正常生长，这不仅引起环境污染还导致资源浪费。因此，提高水稻对磷养分的吸收利用效率对化肥减施增效具有重要意义。真核生物中含SPX结构域的蛋白(SPX-domain-containing protein, SPXs)在感受无机磷酸盐(Pi)、磷信号传递和转运过程中发挥着重要作用。植物在应对低磷胁迫过程中，SPX结构域的蛋白是植物体内磷素状态的感应器。负调控Pi信号网络核心调控因子PHRs (PHOSPATE STARVATION RESPONSE)的活性。水稻中的SPX4在低磷条件下极易被降解，但外界磷素调节SPX蛋白稳定性的分子生理机制仍然不清楚。本课题组利用酵母双杂交的方法在水稻cDNA文库中筛选到一个能与SPX4相互作用的泛素化E3连接酶SDEL1。水稻原生质体转化实验发现SDEL1和SDEL2均定位在细胞核和细胞质中，通过体内外泛素化实验证明了SDEL1能直接泛素化SPX4；利用生理及养分分析实验证明：在磷充足条件下，增强表达SDEL1和SDEL2导致水稻体内无机磷的超积累并诱导磷饥饿信号；相反，SDEL1和SDEL2的功能缺失突变体表现出磷含量降低和磷饥饿信号受到抑制。综上所述，我们解析了SDEL1降解SPX4的分子生理机制，发现了SDEL1通过调节SPX4蛋白稳定性进而调控磷信号和磷平衡。相关研究将为培育磷素养分高效的水稻品种提供理论依据及重要基因资源。