玉米醇溶蛋白转录因子Opaque2的翻译后修饰调控研究

Maize is major crop and mainly used as animal feeding. The protein quality is the most important nutritional quality in maize. However, common maize has relative poor protein quality. The improvement of maize protein quality is a long-term goal for maize breeding. Zeins are the prominent storage proteins in maize seeds, account for more than 60% of the total seed proteins, and are the major determining factor for maize seed protein quality. Opaque2 (O2) is the major transcriptional factor for zein genes, and has been used as most important functional target gene in maize protein quality breeding for more than 50 years. The applicant’s lab had years’ research experience on the regulation of zein biosynthesis. In recent years, we have made important progress on the studies of O2 downstream targets and O2 interacting proteins. Our pilot sutdy clearly indicated that O2 has phosphorylation and ubiquitination modifications, and had obtained good preliminary results. In this project, we propose to carry out extensive analysis on the phosphorylation and ubiquitination modifications of Opaque2. We will determine the modification sites and modification types, and characterize their regulatory functions and underlying mechanisms. We will also seek for the corresponding upstream modifiers, and explore the potential upstream signal pathways. This study will expand our understanding about the regulatory mechanisms for zein gene expression, and provide guidelines for future breeding programs on high protein quality maize.

玉米作为主要作物，主要用于饲料，蛋白品质是玉米最重要的营养品质。但一般玉米的蛋白品质较低，改良玉米蛋白品质是长期的育种目标。醇溶蛋白是玉米籽粒最主要的储藏蛋白，约占籽粒总蛋白60%，是玉米蛋白品质的重要决定因素。Opaque2（O2）是已知的最主效的醇溶蛋白转录因子，也是近50年玉米蛋白品质育种中最倚重的育种功能基因。申请人实验室长期从事玉米醇溶蛋白合成调控研究，近两年在O2的下游靶标基因和互作蛋白研究上取得了重要的进展。我们的前期研究发现O2存在磷酸化和泛素化修饰，并取得了较好的前期结果。本研究计划对O2的磷酸化和泛素化修饰开展系统研究，确定修饰位点和修饰类型，分析这些修饰的调控功能和相应机制；并通过寻找上游的修饰因子，探索醇溶蛋白表达调控的可能上游信号通路。本研究将深化醇溶蛋白的表达调控机制，为创制优质蛋白玉米提供理论指导。

Opaque2（O2）是玉米籽粒醇溶蛋白的主效转录因子，也是协调籽粒淀粉（产量）和蛋白（品质）的关键调控基因。但其翻译后的活性调控机制以及上游的信号通路尚不清楚。本项目针对O2的翻译后修饰调控机制，开展了O2泛素化修饰和磷酸化修饰研究，取得的主要进展如下：.(1) 在O2的泛素化修饰方面，鉴定到泛素修饰酶ZmRFWD3。发现通过单泛素化O2，促进O2进入细胞核，提高转录活性。鉴定到ZmRFWD3的修饰激酶SnRK1，通过磷酸化ZmRFWD3促进其降解。发现SnRK1的活性受糖信号诱导，揭示了SnRK1-ZmRFWD3-O2信号通路，连通了上游糖信号和下游的储藏蛋白合成。籽粒从母体获得的糖分，呈现昼夜的高低节律。上述信号通路也使ZmRFWD3以及O2活性呈现昼夜节律，从而协调了籽粒中淀粉-蛋白的合成（Plant Cell, 2020）。(2) 在磷酸化修饰方面，分析了O2体内的磷酸化修饰位点。鉴定了两个磷酸化修饰的激酶K3和K3-2，通过磷酸化修饰O2的S208位点，负调控O2转录活性和醇溶蛋白积累。结果正在整理发表中。(3)蛋白互作调控是另一种翻译后调控机制。新分离到27kD醇溶蛋白基因的转录因子ZmbZIP22，开展了与O2，PBF1，OHP等转录因子的互作调控研究，揭示了醇溶蛋白基因的多因子互作协同的转录调控机制（Plant Cell，2018）。(4) 鉴于O2泛素修饰信号通路涉及了母体糖分转运，籽粒的淀粉和蛋白合成，对三个相关突变体基因开展了功能研究。其中ENB1调控了母体糖分向胚乳的转运（Plant Cell，2022）；Sh4揭示了玉米胚乳淀粉合成的机制（Genetics，2021）；NZP1揭示了玉米储藏蛋白转运到蛋白体的机制（New Phytologist，2022）。.总之，本项目鉴定了翻译后修饰O2的1个泛素酶和2个激酶；揭示了O2在泛素化修饰和磷酸化修饰调控上的新机制；发现了一条连通母体糖信号和籽粒蛋白合成的信号通路；揭示了O2的多因子互作调控机制。在O2的翻译后调控机制和信号通路研究上取得了突出进展。发表研究论文5篇，其中Plant Cell3篇；特邀综述3篇；申请专利2项。圆满完成了各项任务指。