拟南芥Rab5鸟苷酸交换因子VPS9a响应盐胁迫和JA信号机制研究

Rab5 GTP酶为真核生物保守的GTP酶，调控囊泡内吞途径。Rab5 GTP酶可在活性GTP结合状态和非活性GDP结合状态之间循环，这个过程由特异性的鸟苷酸交换因子（guanine nucleotide exchange factors, GEFs）催化。在模式植物拟南芥中，VPS9a作为营养生长时期唯一的GEF激活所有的Rab5 GTP酶。VPS9a蛋白N端为真核生物保守的GEF 结构域，而C端为大片段内在无序区域（intrinsically disordered region, IDR），仅在陆生植物广泛存在，但功能尚不清楚。本项目旨在研究VPS9a依赖于C端无序序列的植物特有功能。首先，生理学实验证实VPS9a能快速响应盐和渗透胁迫富集到细胞内凝聚物中，且该效应依赖于C端IDR结构域。进一步细胞学实验表明VPS9a在盐胁迫下形成的凝聚物与下游Rab5 GTP酶 ARA7明显分离，但与FM4-64染料标记的囊泡及RABA2a标记的反式高尔基体高度共定位，暗示VPS9a在盐胁迫下改变内膜分选途径。

接下来，我们通过酵母双杂交实验筛选VPS9a互作蛋白。我们筛选到翻译延伸因子EF1A为VPS9a互作因子，并通过生化实验证实VPS9a通过N端GEF结构域与EF1A互作。进一步体外翻译实验表明，VPS9a蛋白强烈抑制翻译效率，最高可达90 %。然而，VPS9a与EF1A在盐胁迫处理前后均没有显著共定位。因此，VPS9a抑制翻译的作用可能与盐胁迫响应没有直接关系。

我们也研究了VPS9a在激素信号通路中的作用。研究结果表明， VPS9a转录水平在茉莉酸（Jasmonate, JA）处理后明显下调。生信分析发现VPS9a启动子上存在JA信号途径关键转录因子MYC结合的G -box位点，并通过体外凝胶迁移阻滞实验得以证实。生理学实验表明VPS9a功能减弱会导致突变体幼苗对JA处理超敏感，主根明显缩短。因此，VPS9a也参与JA激素信号途径调控植物主根发育。

综上所述，本课题研究揭示了进化中保守的Rab5 GEF VPS9a通过C端IDR结构域感知盐胁迫信号，可能以独立于Rab5途径的作用方式调控植物盐胁迫响应。同时，我们也意外发现VPS9a可通过N端GEF结构域抑制翻译，虽然该结果的生理学意义尚不清楚。我们也初步探究了VPS9a在JA激素信号通路中的重要作用。

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