TAD1与翻译起始因子TAS1协同调控水稻株型的分子机理研究

Plant architecture is an important agronomic trait that makes great contribution to rice yield.TAD1 encodes a multi-subunit E3 ligase and functions as a co-activator of the anaphase-promoting complex (APC/C). The tad1 mutant showed an increased tiller number, a reduced plant height, and twisted leaves and panicles. Through genetic screening for tad1 suppressors, we identified a dominant mutant allele of eukaryotic translation initiation factor TAS1 that substantially rescues the tad1 mutant phenotypes. In this proposal, we plan to identify the role of TAS1 in shoot architecture regulation through transgenic complementary experiment and crispr/cas9 techniques; analyze interaction between TAD1 and TAS1 through using yeast two-hybrid system, GST pull-down, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation assay; investigate whether the APC/C TAD1 complex target TAS1 for ubiquitination and degradation through Cell-free protein degradation assay, in vitro ubiquitination assay, Western blotting and in vivo experiment; and identify differentially translated proteins-mediated by TAS1 through comparing the Wild type, tad1 and tas1 translation using Ribo-seq (Ribosome profiling) and conducting function validation. This project will uncover the molecular mechanism of TAD1 and TAS1 regulated plant development, especially tillering and plant height, and make contribution to the theoretical foundation for rice architecture improvement and yield increase.

株型是决定水稻产量的核心要素。TAD1基因编码细胞分裂后期促进复合体（APC/C，多亚基的E3连接酶）的一个共激活子，在水稻株型调控中发挥重要作用。TAD1突变后，植株表现矮生多分蘖表型，同时植株叶片和穗部扭曲。研究植物株型调控基因tad1的表型恢复突变体，分离克隆到一个翻译起始因子TAS1。本项目利用转基因互补实验和构建基因编辑突变体实验验证TAS1基因功能；通过酵母双杂、GST沉淀实验、双分子荧光互补和免疫共沉淀等实验，分析和鉴定TAD1与TAS1在体外和体内的相互作用方式；开展无细胞蛋白降解实验、体外泛素化实验、蛋白质印迹和体内实验来探究TAD1蛋白是否通过泛素化途径来调控TAS1的稳定性和活性；比较野生型、tad1和tas1的翻译组学来鉴定由TAS1介导的特异性差异翻译蛋白，并进行功能验证。通过对TAS1与TAD1的功能研究，进一步揭示两者协同调控水稻株型的分子机制。

按照研究计划，该项目主要是揭示TAD1如何调控TAS1介导的翻译控制来影响水稻株型的机制。分蘖和株高是影响水稻株型和产量的核心要素。前期研究工作表明TAD1在水稻株型调控中发挥重要作用，其突变后，植株表现矮生多分蘖的表型，同时叶片和穗部扭曲。采用MutMap我们快速鉴定和克隆到了一个新的突变基因-翻译起始因子TAS1,它可以恢复tad1突变体的表型。.研究工作按照计划如期执行，总的可以分为以下部分：.1.通过基因互补实验和构建CRISPR/Cas9基因编辑材料验证了TAS1基因在调控水稻株型的生物学功能；.2.通过酵母双杂、GST沉淀实验、双分子荧光互补和免疫共沉淀等实验，进一步证实了蛋白TAD1和TAS1在体外和体内的直接相互作用；.3.开展了无细胞蛋白降解实验、体外泛素化实验、 蛋白质印迹和体内实验，证明了TAD1蛋白通过泛素化途径来调控TAS1的稳定性和活性；.4.基本完成了TAS1介导的翻译组学比较，分析得到了比较明确的信号通路。.总体来说，研究工作的顺利完成，为揭示水稻在翻译水平上如何精确调控株型夯实了基础。