拟南芥RAF22和RAF28通过磷酸化IAA16调控生长素信号介导的胚胎发生机制研究

Auxin plays a key role in plant embryogenesis, whereas the molecular mechanisms underlying auxin response in embryogenesis are unclear. We found two RAF kinases, RAF22 and RAF28, are important components of auxin-mediated embryogenesis, and the kinase activity of RAF22 and RAF28 is required for embryonic development in Arabidopsis thaliana. Recently, we identified auxin signal response protein IAA16 as a partner of RAF22 and RAF28 by interaction screening. Combining previous research that IAA16 may be involved in embryonic development, in this project, Pull-down and Co-IP interaction analysis, in vivo and in vitro phosphorylation analysis and IAA16 protein stability analysis will be conducted to further verify that RAF22 and RAF28 directly phosphorylate IAA16 and regulate its stability. Moreover, genetic analysis will be carried on to prove that RAF22/RAF28-IAA16 cascade regulates auxin signaling-mediated embryogenesis. The study of RAF22/RAF28-IAA16 cascade will explore the molecular mechanism of embryogenesis, which provides theoretical basis for plant development mechanism and crop germplasm research.

生长素是植物胚胎发生过程的主要调控激素，但是，目前胚胎发生过程中的生长素响应机理仍不十分清楚。项目组前期研究证明拟南芥RAF22和RAF28通过其激酶活性调控生长素介导的胚胎发生过程。近期申请者利用相互作用筛选，鉴定发现RAF22和RAF28与生长素信号响应蛋白IAA16相互作用，而且已有研究证明IAA16可能参与调控胚胎发育。本项目拟在此基础上，进一步通过Pull-down和Co-IP等相互作用分析、体内和体外磷酸化分析以及IAA16的蛋白稳定性分析等手段，证明RAF22和RAF28直接磷酸化IAA16，并且调节其稳定性；通过突变体表型及遗传分析，证明RAF22/RAF28-IAA16级联调控生长素信号介导的胚胎发生过程。本研究将为胚胎发生的调控机理、植物发育机制和农作物种质研究提供理论依据。

生长素是植物生长发育各阶段的主要调控激素，调控根生长和胚胎发育等生命过程，但是目前生长素响应机理仍不十分清楚。本研究发现拟南芥MAPKKK家族成员RAF22调控生长素介导的根生长，低浓度生长素抑制了RAF22对根伸长的负调控；通过转录组数据的深入分析，筛选了若干个RAF22的可能底物；进一步通过酵母双杂交验证，首次鉴定到IAA16和ATDOT2与RAF22的相互作用，二者均为生长素途径中的直接调控因子。项目的研究对于进一步阐明RAF类MAPKKK家族的功能及体内作用方式提供了重要的理论依据，对于完善生长素途径的调控机制具有重要的科学意义。