BRI1各个磷酸化位点的生物学功能及分子调控机理的研究

Brassinosteroids play critical roles in multiple aspects of plant growth and development. BRI1 (Brassinosteroid Insensitive 1) is a key component in the pathway of BRs signal transduction. The phosphorylation of phosphorylation-sites on cytoplasm domain of Receptor-Like Kinases cause the conformational change of the receptor molecules, binding of downstream substrates and interacting components, endocytosis and degradation of the receptors and so on. BRI1 phosphorylation sites identification and preliminary functional analysis indicated that there was functional difference among the individual phosphorylation sites of BRI1. Such as T872 phosphorylation negatively regulated kinase activity, phosphorylation sites of the juxtamembrane domain closely related to BRI1 degradation, and site T1044 is involved in seeds set. This study will use the lines created by site-directed mutagenesis and flora-dip transformation technology. The biological function analysis of phosphoryaltion sites in vitro and in vivo have been studied to confirm the unique function of the phosphoryaltion sites. The interacting components to the phosphoryaltion sites have been screened among BRI1 interaction candidates published or screened by co-immunoprecipitatation. The important sites with unique function will be focused to recover the molecular mechanism regulating plant growth and development by means of multidisciplinary research methods. The result will not only enhance the understanding of the molecular mechanism of BRI1 signaling, but also establishes a paradigm for molecular mechanism research in all the 223 LRR-RLKs and the BRI1 in crops.

油菜素内酯调控植物生长发育的多个方面，油菜素内酯受体BRI1是油菜素内酯路径的关键组分，通过细胞内磷酸化位点的磷酸化而引起受体分子构象变化、受体激活、底物及调节组分的结合及受体分子的内吞和降解等。本人对BRI1磷酸化位点鉴定和初步的功能分析中发现，BRI1不同的磷酸化位点存在功能的分化，如T872磷酸化负向调控激酶活性，近膜区几个磷酸化位点涉及BRI1降解，T1044与结籽有关等。本研究拟用定点突变和遗传转化技术创建的材料，在体外和活体水平上，对BRI1的已知或推测的十多个磷酸化位点的功能进行研究，以确认有独特功能的重要磷酸化位点。在已知的BRI1互作组分或免疫沉淀获得的候选组分中筛选重要磷酸化位点的互作组分，借助多学科的研究手段揭示重要磷酸化位点的分子调控机理。该项研究不仅加深对BRI1分子调控机理的了解，也为223个LRR-RLKs类受体激酶及作物上BRI1的分子调控机理的研究提供参考

油菜素内酯BRs在植物生长发育的许多方面有重要功能，油菜素内酯受体BRI1作为油菜素内酯路径的关键组分，通过细胞内磷酸化位点的磷酸化而激活，影响BR信号转导和植物的生长发育。本研究以本人鉴定的BRI1磷酸化位点为基础，采用磷酸化位点定点突变技术创建了68个模拟磷酸化和非磷酸化的基因位点突变体。遗传转化了29个BRI1的全长基因突变体，获得600多份独立的转基因系。通过对29个BRI1突变系的T1代和23个T3代材料的开展度，株高和种子产量等性状的研究，将磷酸化位点与植物学性状相联系，发现了T1042等几个有重要农艺学价值的磷酸化位点。通过免疫沉淀、酵母杂交等方法选出了BRI1的3个互作组分，通过同源比对和酵母杂交发现4个互作组分。借助免疫沉淀和CRISPR-Cas9基因编辑等多学科研究手段对BRI1互作组分和位点特异互作组分进行了系统研究，揭示了BRI1磷酸化位点的磷酸化对激酶的活性,细胞内相关通路的信号转导和生物学功能的影响，为受体激酶及作物上BRI1的分子调控机理的研究，分子设计育种提供了参照方法和设计策略，并在番茄上进行了初步验证。该研究按照基金任务计划书执行，很好地完成了相关研究内容，达到了研究生培养和论文发表等预期的研究目标。