Os-miRNA1320与PHD/ERF转录因子互作调控水稻耐冷性的分子机制研究

Low temperature/cold stress, as a large obstacle to the production of rice in cold region, severely affects rice yield and quality. In our previous study, we suggested the involvement of miRNAs in rice responses to cold stress. However, until now, the precise mechanism of miRNAs in regulating rice cold tolerance is largely unknown. In this project, we select one cold stress responsive miRNA in rice, miR1320, and will analyze its expression profiles and biological function in cold stress responses by combining gain-of-function and loss-of-function technologies. Then, we will carry out 5’-RACE analysis to identify whether PHD14/ERF5 are miR1320 targets. In an attempt to further reveal the regulatory mechanism between miR1320 and PHD14/ERF5, we will analyze the negative correlation of their expression profiles under cold stress and in different tissues, and the effect of miR1320 on the cleavage of PHD14/ERF5 mRNA and/or inhibition of PHD14/ERF5 translation. Furthermore, we will characterize the biological function of PHD14/ERF5 in cold stress responses by combining overexpression and CRISPR technologies. In addition, in order to uncover the molecular basis of PHD14/ERF5 in regulating plant cold stress responses, we will investigate the subcellular localization, DNA binding affinity, transcriptional activation or inhibition, and dimerization of PHD14/ERF5 transcription factors. Taken together, results in this project will reveal the molecular mechanism of miR1320 in regulating rice cold tolerance. These findings will be of great benefit to elucidate the signal transduction and regulatory network in rice responses to cold stress, and provide theoretical basis and candidate genes for designer breeding by molecular modules of cold tolerant rice.

低温冷害是寒地稻作生产的主要障碍，严重影响水稻产量和质量。我们前期研究发现miRNAs参与水稻冷胁迫应答，但目前尚未明确miRNAs调控水稻耐冷性的具体作用机理。因此本项目以前期筛选的水稻冷胁迫应答miR1320为切入点，分析其冷胁迫表达模式，从功能获得和功能缺失2个角度解析其耐冷功能。然后通过5’-RACE验证、冷胁迫和组织表达模式负相关性分析、mRNA降解及蛋白翻译抑制调控作用研究，揭示miR1320与PHD14/ERF5靶向关系及调控机理。最后利用超量表达和CRISPR技术鉴定PHD14/ERF5在耐冷应答中的功能；通过亚细胞定位、DNA元件结合、转录激活/抑制及二聚化分析，解析PHD14/ERF5调控耐冷性的分子机理。本研究将揭示miR1320调控耐冷性的分子机制，为阐明水稻耐冷分子调控网络提供重要参考，并为通过多基因聚合分子设计育种，培育耐冷水稻新品种提供理论依据和基因资源。

低温冷害严重影响东北寒地水稻生产。我们前期发现miRNAs参与水稻冷胁迫应答，但尚未明确miRNAs调控水稻耐冷性的具体作用机理。本项目以前期筛选的水稻物种特异的、冷胁迫应答miR1320为切入点，分析了其在冷胁迫下的表达模式，发现miR1320表达受冷胁迫抑制。构建了miR1320-OX超量表达和miR1320-STTM表达敲低的转基因水稻株系，发现miR1320正调控水稻耐冷性。通过5’-RACE在体外验证了miR1320与PHD17和ERF096转录因子的靶向关系。通过qRT-PCR分析发现PHD17和ERF096表达受冷胁迫诱导；组织表达分析发现PHD17表达无组织特异性，而ERF096与miR1320具有相反的组织表达模式。此外，PHD17和ERF096基因表达在miR1320-OX株系中显著上调，而在miR1320-STTM株系中则显著下调。miR1320对PHD17在蛋白翻译水平上存在一定的抑制作用。转录因子特性分析发现PHD17是一个细胞核定位的转录激活因子，可以与GTGGAG基序结合。ERF096由于C端具有一个跨膜结构域，定位于细胞膜，TMD缺失后可定位于细胞核；ERF096全长不具有转录激活活性，TMD缺失突变ERF096-∆TM具有转录激活活性，且其转录激活域位于N端。利用超量表达、基因沉默及RNAi技术进行功能分析，表明PHD17和ERF096基因负调控水稻耐冷性。此外，miR1320-PHD17/ERF096调控冷胁迫下体内ROS稳态，调节CBF冷胁迫信号通路及冷应答marker基因的表达。综上，本研究表明miR1320通过靶向PHD17/ERF096转录因子调控水稻冷胁迫应答过程，为阐明水稻耐冷分子调控网络提供了重要参考，为通过水稻耐冷分子设计育种提供了理论依据和基因资源。