磷脂酰胆碱转运蛋白OsPCTP调控水稻耐冷的分子机制研究

Low temperature is one of the important factors affecting rice yield. Further study of the molecular mechanism of rice cold tolerance and exploring related gene resources are the fundamental ways to improve rice low temperatural resistance and cultivate new cold tolerant varieties. Previous researches show that the fatty acid unsaturated degree of phospholipids in cellular biomembranes was closely related to plant low temperature resistance, however the effect of plants phospholipid transfer among cellular membranes in low temperature response remains unclear. The applicant firstly identified a phosphatidylcholine transporter protein OsPCTP which involved in low temperature response of rice seedlings and affected the content of hydrogen peroxide in seedlings. We also obtained its interaction protein OsLSD1 through yeast library screening. Based on the different OsPCTP transgenic materials, we will explore the role of OsPCTP in phospholipid transfer and membrane lipid balance; Analyze the relationship between OsPCTP and OsCATs; Elucidate the regulation mechanism of OsPCTP on OsCATs enzyme activity and ROS dynamic balance and also analyze the natural variation of OsPCTP gene in core germplasm. On the basis of previous studies, this project will explore the mechanism of OsPCTP in rice low temperature resistance by molecular genetic, biochemical and metabolic methods, which will enrich the of rice low temperature response signaling pathway and provide theory basis for genetic improvement of rice low temperature adaptability.

低温是影响水稻产量的重要因素之一。深入研究水稻耐低温分子机制，发掘耐冷基因资源是提高水稻耐冷水平、培育耐冷新品种的根本途径。研究表明，生物膜中磷脂的不饱和度与植物的低温耐受性密切相关，而负责生物膜间磷脂转运的蛋白在低温胁迫响应中的作用目前尚不明确。申请人首次确定磷脂酰胆碱转运蛋白OsPCTP参与水稻幼苗低温胁迫响应过程；影响幼苗体内过氧化氢的含量；并通过酵母筛库获得其互作蛋白OsLSD1。本项目拟利用OsPCTP转基因材料，探究该基因在脂质转运及膜脂平衡中的作用；解析OsPCTP与过氧化氢酶OsCATs蛋白之间关系；阐明OsPCTP对OsCATs酶活活性及ROS动态平衡的调控机制；分析该基因在核心种质中的自然变异。本项目将在前期研究基础上，采用分子遗传、生化、代谢等技术方法，探讨OsPCTP调控水稻耐冷的分子机制，将丰富水稻低温响应信号通路及对水稻低温适应性的遗传改良提供理论依据。

低温是影响水稻产量的重要因素之一。生物膜中磷脂的不饱和度与植物的低温耐受性密切相关。我们的研究发现水稻的磷脂酰胆碱转运蛋白OsPCTP负调控水稻苗期低温耐受性，影响幼苗体内过氧化氢酶CAT活性以及过氧化氢含量。进一步研究发现，OsPCTP是组成性表达，在幼苗中具有高表达水平，并且受低温诱导表达，亚细胞定位分析发现该蛋白定位于胞质中，生化特性分析发现其具有磷脂酰胆碱（PC）和磷脂酰乙醇胺（PE）的结合活性。通过酵母双杂交、荧光素酶分子互作试验表明OsPCTP通过OsLSD1与OsCATs互作，进而影响过氧化氢酶活性。此外，我们通过酵母双杂、荧光素酶分子互作以及免疫共沉淀实验验证了OsPCTP可以直接与抗坏血酸过氧化物酶OsAPX1进行互作。上述结果表明，水稻OsPCTP主要是通过与ROS清除酶蛋白互作，影响其酶活活性，从而维持水稻体内ROS动态平衡来参与耐冷。本项目的研究结果，将丰富水稻低温响应信号通路及对水稻低温适应性的遗传改良提供理论依据。