磷脂酸: 基于FRET的分析及其在植物响应逆境中的作用机理研究

磷脂酸PA是结构最简单的磷脂，在所有生物体中起代谢调控和细胞信号作用。植物细胞内PA时空变化的测定方法尚未建立。本项目拟建立基于荧光能量共振转移（FRET）的质膜PA分析系统。选择、设计对PA能特异、高亲和结合的结构域，根据PRET原理构建PA探针 （Plant PA Biosensor，PPB）；获得编码探针的遗传材料。以野生型和PA降低的突变体为材料，结合药理学实验，在逆境和ABA处理时，用confocal检验PPB对质膜PA的特异性、灵敏性和可靠性。将PPB转基因到Ca2+通道突变体（osca1等），研究Ca2+信号对PA影响。反之，用Ca2+ 探针cameleon转PA降低的突变体，观察PA对胞内Ca2+水平的作用；电生理实验研究PA对OSCA1等Ca2+通道调控。本项目有望建立PA信号分析系统；对揭示信号分子PA和Ca2+之间的关系及其转导逆境信号的机理有重要意义。

The lipid phosphatidic acid (PA), simplest phospholipids, has important roles in cell signaling and metabolic regulation in all organisms. In plant cells, the method to monitor PA dynamics with high spatial and temporal resolution is therefore critically important, but yet to be established. We planned to establish fluorescence resonance energy transfer (FRET)-based systems to monitor the spatio-temporal dynamics of PA at plasma membrane. Initially, the proteins or fragments binding to PA with high specificity and affinity will be selected and designed for PA sensor (Plant PA Biosensor, PPB). PPB expression vector will be designed and transformed to wild- type and PLD-disrupted Arabidopsis. The constitutive and stable expression of PPB in transgenic plants will be observed using confocal laser scanning. We will determine the specificity, sensitivity, and reliability of the genetically encoded PA –sensors when WT and PA-lower mutants are treated with stresses and ABA, or pharmacologic approaches are used to reduce PA level in cells. To test whether Ca2+ messenger regulates PA level at PM, PPB vector will be transformed to Ca2+ channel mutants (osca1 etc.). On the contrary, to test if PA messenger affects Ca2+ signals, Ca2+ sensor cameleon will be transformed to lower-PA mutants. The electrophysiological experiments are to be done to test PA effects on Ca2+ channels (OSCA1 etc.). We hope set up PA analysis systems for the colleagues to dissect deeply how PA functions in plant cells. The project is also valuable for understanding the interaction between two important signal molecules PA and Ca2+, and the mechanisms of their functions in stress signaling.