灯盏花Exo70A1与ARC1互作调控自交不亲和信号转导途径的机制研究

Self-incompatibility is an important mechanism to avoid inbreeding for angiosperms in process of reproduction. Exo70A1 and ARC1 are key genes in the SI signal transduction pathway and are involved in the SI. However, the mechanism of SSI is still not elucidated outside the Brassica. Recently, we found that Erigeron breviscapus is show to has a classic characteristics of the SI. Exo70A1 gene and ARC1 gene were cloned successfully, the identification of protein interaction after yeast two hybrid of Exo70A1 and ARC1. Thus, it was suggested to be studied as the following some aspects: the expression analysis profile of Exo70A1 gene and ARC1 gene to determine the correlation between two genes and SI. To analysis their functions of two genes through transgenic experiments. Identification of the interacton models between Exo70A1 protein and ARC1 protein by yeast Two-Hybrid System. The study of ubiquitylation involve two proteins explore whether ubiquitination occurs during the interaction between ARC1 and Exo70A1. Our study contributes to explore the molecular of SI signaling in the Asteraceae.

自交不亲和性是被子植物在有性生殖过程中避免近亲繁殖的重要机制。Exo70A1和ARC1是自交不亲和信号转导途径中的关键基因，参与自交不亲和信号转导过程，但在芸薹属以外孢子体型自交不亲和性信号通路中的调控作用尚不清楚。本项目前期研究发现，灯盏花具典型的孢子体自交不亲和性，克隆到其Exo70A1基因及ARC1基因，并证实二者存在相互作用，为此，本项目拟开展以下内容：通过ARC1和Exo70A1基因表达谱分析，确认它们与自交不亲和性的相关性，转基因实验验证两个基因功能；酵母双杂交明确两个蛋白互作模式及其特异性；ARC1和Exo70A1泛素化研究探讨两者互作是否发生泛素化，及其对自交不亲和信号途径的调控。这些研究有助于进一步理解菊科植物自交不亲和信号传导机制。

自交不亲和性是被子植物在有性生殖过程中避免近亲繁殖的重要机制，Exo70A1和ARC1是自交不亲和信号转导途径中的关键基因，但在芸薹属以外孢子体型自交不亲和性信号通路中的调控作用尚不清楚。该研究以自交不亲和的菊科植物灯盏花（Erigeron breviscapus）为材料，基于基因组和转录组数据对ARC1、Exo70A1的基因家族进行生物信息学综合分析，挖掘到多个自交不亲和性相关的EbARC1、EbExo70A1基因，其中EbARC1、EbARC1-13和EbARC1-18在自交授粉后上调表达；EbExo70A1在自交授粉后下调表达，它们可能参与自交不亲和反应。从灯盏花中克隆到EbARC1基因全长序列，编码732个氨基酸残基，保守性较高；qRT-PCR显示EbARC1在根茎叶花各组织中均有表达，它随着花器官成熟表达量逐渐增加；亚细胞定位显示EbARC1在细胞核中表达；过表达的转基因拟南芥EbARC1-OE植株结实率明显降低，花丝变短，花药位置低于柱头；野生型和EbARC1-OE自交和杂交染色结果表明EbARC1具有调控自交不亲和的功能。为探索灯盏花EbARC1调控自交不亲和分子作用机制，构建了灯盏花柱头cDNA酵母文库，利用EbARC1蛋白为诱饵，从酵母文库中筛选到18个互作蛋白，主要包括Exo70A1蛋白、U-box蛋白、E3泛素化蛋白连接酶DRIP2-like等。从灯盏花中克隆到Exo70A1基因全长序列，编码638个氨基酸残基；EbExo70A1蛋白序列中只包含一个Exo70超家族结构域，EbExo70A1基因在根茎叶花各组织中均有表达，但在成熟花中基因表达量低于未成熟花；亚细胞定位表明EbExo70A1在细胞核中微弱表达；序列分析发现EbExo70A1序列上存在多个潜在的泛素化修饰位点，其中340位的氨基酸残基可能性最大，EbARC1可能通过该位点泛素化降解EbExo70A1介导灯盏花的自交不亲和反应。酵母双杂交和双分子荧光互补试验显示EbARC1和EbExo70A1存在互作，蛋白序列截短试验证明EbARC1-（1-368）和EbExo70A1-（1-284）是其互作的关键区域。本项目证实了EbARC1具有调控自交不亲和的功能，根据EbARC1和EbExo70A1相互作用关系，推测EbARC1通过泛素化EbExo70A1介导了灯盏花自交不亲和的分子调控机制。