番茄SlBRI1磷酸化位点的农艺学价值及调控机理的研究

Appropriately enhancing or weakening Brassinostroid (BR) signal strength to an optimum level by either exogenous application of BR or endogenous regulation of BR related gene expression can significantly increase crop yield and stress-tolerance. However, it is difficult to control BR signal strength precisely, which resulted in unstable effect with BR signal manipulation and failed to meet the possible potential. BRASSINOSTEROID INSENSITIVE1 (BRI1) is the key rate-limiting enzyme in the BR signal pathway which transmits BR signal through its kinase cyto-domain phosphorylation. In the last study of our group it is demonstrated that phosphorylation of phosphorylation sites of BRI1 play spectrum differentiation roles in both BR signal strength and botanic characters. This project will focus on phosphorylation of tomato SlBRI1 on BR signal transduction and identify the phosphorylation sites of SlBRI1 in vivo with immunoprecipitation (IP) followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Both dephosphorylated and phosphorryted-mimic site-directed mutants of individual SlBRI1 phosphorylation sites will be constructed and transformed into SlBRI1 weak mutant cu3-abs1. The biologic role and agronomic potential of each phosphorylation sites will be evaluated by its restoration of cu3-abs1 and botanic characters. Interacted protein screening and gene editing will be then used to reveal the regulatory mechanism of the important phosphorylation site. Finally, our study may provide molecular basis for establishing high-yielding crop varieties with biosafety via controlling BR signal strength precisely.

油菜素内酯BR外源施用和其路径基因表达调控使BR信号强度恰好地略有增强或减弱时，可显著提高作物产量和抗性。现有途径难以精准调控BR信号强度，致使效果不稳定而未能发挥其潜力。油菜素内酯受体BRI1是其信号的关键限速环节，借助磷酸化转导信号。本组近期研究发现，拟南芥不同BRI1单磷酸化位点突变的转基因系的BR信号强度存在差异，植物学性状表现系列变化。本项目拟以番茄为材料，采用蛋白免疫沉淀和质谱分析相结合鉴定番茄油菜素内酯受体SlBRI1的活体磷酸化位点，突变SlBRI1的磷酸化位点模拟磷酸化和非磷酸化构建植物表达载体，转化SlBRI1的弱突变材料cu3-abs1，依据各个位点突变转基因系的表型恢复程度和农艺性状表现，评价位点的生物学功能和农艺学价值。选择农艺学上有重要意义的位点，筛选互作组分，以体外互作和基因编辑等方法研究其调控机理，为精准调控BR信号，创建生物安全的优良番茄育种材料奠定基础。

油菜素内酯BR调控植物生长发育的许多方面，受体BRI1是BR路径的关键组分，并通过BRI1胞内域磷酸化位点的磷酸化而被激活，转导BR信号进而调控生长发育。本研究通过优化免疫沉淀方法，创建多种SlBRI1番茄转基因材料，纯化番茄SlBRI1活体蛋白并确认激酶的磷酸化，鉴定了SlBRI1的磷酸化位点。借助磷酸化位点的定点突变，对14个潜在的磷酸化位点的激酶活性和生物学效应进行了研究。通过SlBRI1基因位点突变的番茄转基因系，对T1050、S1040、T825等5个重要磷酸化位点的生物学功能进行了深入研究。在田间试验条件下，对4个影响番茄产量、抗热性、果实品质等重要农艺学性状的磷酸化位点进行了农艺学研究。采用多学科研究手段，对筛选到的4组BR路径新的相关组分中的SlPPL5、SlPHB3, SlBIR3和下游3个转录因子的生物学功能和作用机理进行了研究，增加了番茄BR信号转导路径的新知识。该研究揭示了番茄BR受体SlBRI1磷酸化位点对激酶活性，BR信号转导、生物学功能和重要农艺性状的影响，发现了BR路径的新组分及其作用机理。研究表明，番茄SlBRI1的单个磷酸化位点的突变可使BR信号强度恰好地略有增强或减弱时，可显著提高作物的产量和抗性，不仅为番茄SlBRI1的分子编辑育种奠定了基础，也可为其他作物的分子设计育种提供参考。该研究按照本项目计划任务书执行，完成了相关研究内容，达到了科学研究、学生培养和论文发表等预期研究目标。