水稻miR1850靶向NPR3调控冷胁迫应答的分子机制研究

低温冷害是东北寒地水稻生产的主要障碍。我们前期通过构建水稻冷胁迫应答miRNA-mRNA调控网络，发现了水稻物种特异的miR1850及靶基因NPR3表达响应冷胁迫。但目前尚未有研究报道其是否调控、如何调控水稻耐冷性。本项目首先拟通过5’-RACE验证、表达负相关性分析及miR1850对NPR3基因表达的调控研究，验证miR1850与NPR3靶向关系；进一步综合利用超量表达、短串联靶标模拟STTM和CRISPR基因编辑技术，解析miR1850和NPR3基因在水稻耐冷应答中的功能；最后分析NPR3-HSFA1蛋白相互作用及其对HSFA1下游基因表达的调控，揭示miR1850是否通过NPR3调控HSFA1冷胁迫信号途径。通过上述研究将揭示miR1850靶向NPR3调控水稻冷胁迫应答的分子机制，为深入阐明miRNA参与的水稻耐冷分子调控网络提供重要参考，为耐冷水稻新品种培育提供理论依据和基因资源。

Low temperature/cold stress is a large obstacle to the rice production in Northeast China. In previous studies, we constructed a cold stress responsive miRNA-mRNA gene regulatory network, and found that expression of miR1850, which is a species-specific miRNA in rice, and its target gene NPR3 responded to cold stress. However, it remains obscure whether and how miR1850 and NPR3 regulate the cold tolerance in rice. Therefore, this project will verify the target relationship of miR1850-NPR3 through the 5’-RACE analysis. Meanwhile, we will investigate whether miR1850 and NPR3 show negative expression patterns under cold stress and in different tissues, and whether miR1850 overexpression and knockdown will alter the mRNA accumulation of NPR3. Then, we will characterize the biological function of miR1850 and NPR3 in regulating rice cold tolerance by coordinately using the overexpression, STTM (Short Tandem Target Mimic) and CRISPR technologies. Lastly, in order to investigate the potential role of miR1850-NPR3 in regulating HSFA1-mediated cold stress signal transduction pathway, we will identify the protein interaction between NPR3 and HSFA1 by using BiFc and CoIP, and investigate the regulation of miR1850 and NPR3 on the expression of HSFA1 downstream genes by using qRT-PCR. Taken together, studies in this project will finally reveal the molecular mechanism of miR1850 in regulating rice cold stress responses by targeting NPR3. These findings will be of great benefit to elucidate the miRNA-mediated signal transduction and regulatory network in rice responses to cold stress, and provide theoretical basis and candidate genes for breeding of cold tolerant rice cultivars.