拟南芥受体激酶GSO1和GSO2调控芽再生的分子机制

Exploring molecular mechanism of plant regeneration is valuable and significant for in vitro plant propagation and transgenic breeding. By using the model plant Arabidopsis as research material and the shoot regeneration system, we found that the receptor kinase encoding gene GSO2 was expressed in the regenerative shoot meristem. Mutation of both GSO1 and GSO2 or overexpression of GSO2 resulted in the significantly decreased capacity of shoot regeneration, suggesting GSO acts in the process of shoot regeneration. However, the functional mechanism of GSO during shoot regeneration remains elusive. Twelve candidate proteins were characterized to be interacted with GSO2 by yeast two-hybrid screening. Next, we will detect the interaction between GSO and its candidate proteins by using the pull-down and immunoprecipitation assays. The regulatory signaling pathway would be revealed by RNA-seq analysis and quantitive real-time PCR assay. The phosphorylation levels and sites of GSO-interacting proteins can be detected by kinase substrate phosphorylation assays and mass spectrometry analysis, respectively. We aim to elucidate the regulatory mechanism of GSO-mediated shoot regeneration, and reveal the molecular mechanism of shoot regeneration. The findings would provide new cues for resolving the problem of low efficiency of plant genetic transformation.

深入研究植物芽再生的分子机制，对组培快繁和转基因育种具有重要的价值和理论意义。我们以模式植物拟南芥为研究材料，利用芽再生体系，发现拟南芥受体激酶编码基因GSO2在再生形成的茎端分生组织中表达，GSO1和GSO2的双突变或GSO2过表达均会导致芽的再生能力显著降低，说明GSO参与调控芽的再生。然而，GSO在芽再生过程中的作用机制并不清楚。通过酵母双杂筛库技术，目前鉴定到12个与GSO2激酶区互作的蛋白。在此基础上，我们拟通过pull-down和免疫共沉淀技术验证GSO与候选蛋白的互作，并结合RNA-seq技术和实时荧光定量PCR技术分析GSO介导的信号途径。通过激酶底物磷酸化实验检测GSO底物蛋白的磷酸化水平，并利用质谱分析确定GSO底物蛋白发生磷酸化的位点。本项目旨在阐明GSO在芽再生过程中的作用机制，揭示植物芽再生的分子基础，为解决植物转基因育种中基因遗传转化效率低的问题提供新的线索。

植物芽再生是实现基因遗传转化的重要手段，深入研究植物芽再生的分子机制对转基因育种具有重要的价值和理论意义。受体激酶GSO1和GSO2在植物的胚表皮形成、根凯氏带的形成、根的生长等过程中起着重要的调控作用，然而，GSOs是否参与调控芽的再生并不清楚。我们通过芽再生实验，发现gso1和gso2单突变体没有表型，而gso1gso2双突变体芽再生能力降低，表明GSO1和GSO2在芽再生过程中具有功能冗余性。进一步分析发现，过表达GSO1或GSO2均抑制芽的再生，说明GSO1和GSO2在调节芽再生过程中具有剂量效应。通过GUS染色，发现GSO1和GSO2均在愈伤组织和再生形成的分生组织中表达。通过RNA-seq分析，发现GSOs通过调节生长素、细胞分裂素、糖信号等多个途径影响芽的再生。GSOs突变导致生长素代谢调控基因IMAT1和分生组织调控基因WOX11表达水平降低，而GSO2过表达导致IMAT1和WOX11表达水平升高，并且IMAT1或WOX11突变均导致芽再生能力降低，说明GSOs可能部分通过影响IMAT1和WOX11的表达调控芽的再生。通过酵母双杂筛库，发现12个与GSO2互作的蛋白，通过酵母双杂和Pull-down实验进一步证明糖信号调节蛋白RHIP1与GSO2互作。通过磷酸化组学实验，发现GSOs突变导致575蛋白磷酸化水平发生改变，其中248个上调，327个下调。通过GO分析发现，差异表达的蛋白主要富集在逆境胁迫响应、RNA代谢、激素响应、细胞周期等过程。其中，gso1gso2突变体中生长素运输载体蛋白PIN3磷酸化水平降低，说明GSOs很可能通过影响PIN3的磷酸化水平调控芽的再生。综上，通过本项目研究，发现GSOs通过影响生长素、糖信号等途径调控芽的再生，丰富了植物芽再生的调控途径，为提高芽的再生能力提供新的线索。