Arabidopsis Circadian Clock RVE8 LNK1 LNK2 Interactome LC-MS

The timing of many molecular and physiological processes in plants occurs at a specific time of day. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT INDUCIBLE AND CLOCK REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover new connections within the clock and between other pathways, here we use affinity purification coupled with mass spectrometry to discover time-of-day-specific protein interactors of the RVE8-LNK1/2 complex. Our clock proteins are tagged with a 6X-His-3X-FLAG C-terminal (HFC) affinity tag and purified through anti-FLAG immunoprecipitation and His-tag isolation affinity purification. We also performed affinity purification-mass spectrometry of 35S::YFP-COR27 and 35S::GFP-COR28, which were immunoprecipitated with anti-GFP TRAP beads. Experiment was performed on 10-day-old seedlings grown under 12 hours light: 12 hours dark 22 degrees Celsius.

植物体内诸多分子与生理过程的发生具有每日特定的时序节律。昼夜节律相关MYB类转录因子REVEILLE 8（RVE8）可与其转录共激活因子夜间光诱导且时钟调控蛋白1（LNK1）及LNK2相互作用，以促进晚间相时钟基因与耐寒因子的表达。过往相关研究多借助遗传手段挖掘生物钟通路内部及与其他通路间的新型关联，本研究则采用亲和纯化结合质谱分析法，鉴定RVE8-LNK1/2复合物的昼夜特异性蛋白互作因子。本研究中的时钟蛋白带有C端6X-His-3X-FLAG（HFC）亲和标签，通过抗FLAG免疫沉淀与His标签分离亲和纯化完成蛋白富集。此外，我们还对35S启动子驱动的YFP-COR27与GFP-COR28融合蛋白开展了亲和纯化质谱分析，实验中采用抗GFP Trap磁珠进行免疫沉淀。本次实验的材料为在12小时光照/12小时黑暗、22℃培养条件下生长的10日龄幼苗。