穗发育关键基因APO1和APO2参与氮肥调控水稻每穗颖花数的分子机理

Nitrogen fertilizer is well known to increase rice yield. Application of nitrogen fertilizer is known to affect inflorescence development and increase flower numbers per panicle, but how nitrogen fertilizer regulates inflorescence development remains unclear. The effects of nitrogen fertilizer on panicle branching may be mediated by cytokinin, which accumulation in the inflorescence meristem can regulate panicle development, resulting in increased numbers of flowers and branches. In our published papers, nitrogen fertilizer enhances local cytokinin synthesis to increase flower numbers in the panicles of rice. Localized cytokinin biosynthesis is an important response to nitrogen. ABERRANT PANICLE ORGANIZATION1 (APO1) and APO2 are key regulators of panicle development in rice. Molecular basis for APO1 and APO2 interaction will be revealed by Co-immunoprecipitation and LC-MS/MS. The proteins related with nitrogen signaling and metabolism will be find from the study. I will also do global analysis of gene expression in shoot meristems between wild type and mutation plants by RNA-Seq. The experiment will provide some information about the downstream genes of APO2. Identifying targets of APO2 is clearly an essential step to understand APO1/APO2 function. I will identify binding sites of APO2 using ChIP-Seq. The results of Selex could help improve the accuracy of predicting transcription factor binding sites in ChIP-Seq data analysis. After finish these studies, we could find the relationship between nitrogen and APO1/APO2 pathway. After that, the metabolomics can be analysis according to the functions of new genes. The response to nitrogen fertilizer will also be analysis in wild type and mutation plants. In our study, the results will extensively improve our understanding of mechanisms of APO1/APO2 controlling meristem phase changes accompanying rice panicle formation. The findings expected to be obtained in this work will have a big impact to understand the roles of nitrogen fertilizer in rice panicle branching too. It can also help to invent new technologies to increase flower number per panicle in rice in the future.

增加每穗颖花数是施用氮肥提高水稻单产的主要途径之一，但氮肥调控水稻幼穗分化的分子机理尚不清楚。我们的研究表明氮肥能通过提高幼穗组织中的细胞分裂素含量促进枝梗和颖花分化。APO1和APO2是控制水稻幼穗分化的关键基因，并与细胞分裂素密切相关。本项目主要研究APO1和APO2在氮素信号和代谢方面的调控作用，明确这两个基因在氮肥调控水稻每穗颖花数中的分子机理。通过免疫共沉淀结合串联质谱技术，分离并鉴定与APO1和APO2结合的蛋白质，从中找到与氮相关的互作蛋白。同时使用转录组测序和ChIP-Seq技术，研究APO2下游调控基因，构建APO2基因调控网络，明确该调控网络与氮信号和代谢的关系。并根据以上的研究结果，有针对性的分析不同材料间代谢上的差异。通过本研究，可以从分子水平上明确APO1、APO2的功能，阐明氮肥调控水稻穗发育的分子机理。为进一步创新栽培措施，调控水稻每穗颖花数提供理论依据。

水稻是重要的粮食作物，水稻穗型与产量紧密相关。研究控制水稻穗型的基因功能以及发掘新的功能基因对提高水稻产量有重要意义。APO1与APO2蛋白可以直接相互作用并共同调控水稻幼穗分化。APO1是水稻的F-box蛋白，介导底物蛋白质的泛素化降解。本研究分析了APO1、APO2/RFL的功能，试图明确APO1和APO2/RFL的互作蛋白，以及构建与APO1和APO2/RFL互作基因的突变体。利用已有的apo1和apo2/rfl突变体，将gAPO1:mcherry、gAPO2:RFL分别转入apo1和apo2/rfl突变体，观察表型恢复状况。与野生型水稻相比，apo1和apo2/rfl突变体的穗形明显变小，二次枝梗数也明显减小。apo2/rfl突变体转入gRFL:GFP以后，穗型出现一定程度的恢复。apo1突变体转入gAPO1：mchery后，转基因植物表型为穗型恢复，并较野生型明显变大，可能是APO1表达量增加导致的。后进行酵母双杂筛选，发现SYD等10个蛋白质与APO2存在互作。利用CRISPR/Cas9技术构建互作基因突变体，syd表现为纯合致死，其他基因也出现多种不同表型。另外，通过比较apo2与野生型幼穗发育过程中转录组的表达变化，筛选了差异表达基因，同时创建了部分差异表达基因的突变体。其中PA21突变体表现出发育延迟、多分蘖的表型。通过我们的研究明确了APO1和APO2/RFL基因在蛋白层面对水稻穗分化的影响，为下一步了解它们的互作机制奠定基础。进一步探索并验证染色体重塑在APO2调控水稻穗发育中的功能，对于水稻产量的调控具有重要意义。