利用水稻内源PEPCK提高水稻光合效率的机制研究

PEPCK是C4光合途径关键酶。申请者前期发现水稻也存在PEPCK，能脱羧参与光合碳固定并在水稻中建立部分C4通路，PEPCK蛋白序列N端有保守的磷酸化位点；网站预测并用酵母双杂交筛水稻蛋白质文库发现，水稻PEPCK可能与钙调蛋白家族成员存在相互作用；高表达水稻PEPCK能提高光合效率，但光合响应机制尚不清楚。申请者假设：PEPCK酶活可能受到磷酸化调控，并与钙调蛋白相互作用。本申请拟用水稻PEPCK过表达和缺失突变体进行研究，用免疫印迹和放射自显影，分析PEPCK的磷酸化状态和活性调控因子；用非标记定量蛋白质组学和基于质谱的蛋白磷酸化分析，结合点突变模拟磷酸化和去磷酸化，解析水稻PEPCK的磷酸化调控原理；用酵母双杂交和免疫共沉淀鉴定水稻PEPCK的互作蛋白，最终阐明利用水稻PEPCK提高光合效率的机制，为探索针对性技术调控水稻内源C4基因促进光合效率、提升光合潜能乃至水稻增产提供依据。

Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme in the C4 pathway of photosynthesis. Interestingly, our previous studies showed that PEPCK also exited in rice, besides, PEPCK had role in the carbon fixation by decarboxylation and established part of the C4 pathway in rice. Protein-sequence-analysis showed that there was a putative Ser phosphorylated site in the N terminal of PEPCK. We used a yeast two-hybrid system approach and predicted its interaction partner protein, a member of Cam complexes. Overexpressing PEPCK in rice could improve its photosynthetic efficiency, however, the mechanism of PEPCK in photosynthetic physiology is still unknown. Based above, we proposed a hypothesis that the activity of PEPCK was regulated by phosphorylation, and might interacted with a Cam complexes protein. To confirm the key role of PEPCK in the photosynthetic and metabolic pathway, in the present proposal, we plan to use the mutants which were with knockout and over-expression of PEPCK to investigate the molecular mechanism of PEPCK by western-blot and auto-radiography, and analyze its phosphorylation status and activity regulatory factors of PEPCK. Then, we plan to use Label-free quantitative proteomics, mass spectroscopy-based protein phosphorylation analysis, and combining with the point mutation to mimic phosphorylation and dephosphorylaion, we determine the protein phosphorylated sites qualitatively and quantitatively, and analyze the phosphorylation regulatory mechanism of PEPCK in rice. Furthermore, we plan to use a yeast two-hybrid screening system and identify its interaction partner protein of PEPCK in rice, and use the co-immunoprecipitation to further confirm their interaction relationship. Finally, we aim to verify the hypothesis of PEPCK in this proposal. Through the research, the role and its regulatory mechanism of PEPCK in rice would be understood, and it would help to explore more effective techniques for rice C4 enzymes usage in rice breeding, and provide theoretical and practical foundation for improving rice photosynthetic efficiency even production.