拟南芥乙烯受体ETR1的结构研究

  乙烯是一种气态植物激素，它是最简单的烯烃气体，由植物生物合成，通过乙烯信号通路调节植物的发育、生长和逆境反应。最早报道的乙烯反应之一是三重反应。研究人员通过遗传筛选试验，已识别出乙烯信号通路的许多成分。这些成分包括内质网膜上的乙烯受体家族；一种蛋白激酶，称为组成型三重反应蛋白1（CTR1）；一种内质网定位的跨膜蛋白，其生化活性未知，称为乙烯不敏感蛋白2（EIN2）；以及转录因子。根据研究结果建立了乙烯信号通路的经典模型，在没有乙烯的情况下，乙烯受体向CTR1发出信号，抑制EIN2并阻止下游信号传导。乙烯存在时，受体感知乙烯使其构象发生变化，抑制了CTR1的激活，从而解除对EIN2的抑制作用，EIN2改变转录和翻译状态，导致大多数乙烯反应。

  尽管对乙烯受体进行了数十年的研究，但其是如何感知乙烯并介导信号传导的机制仍然是不清楚的。若能通过结构生物学的手段获得乙烯受体蛋白高分辨的结构，无疑将极大地丰富我们对其信号传导机制的认知。这对于调节植物生长发育、开花及寿命具有重大的应用价值。本课题以乙烯受体ETR1蛋白为切入点，围绕ETR1、ETR1/CTR1复合物以及EIN2蛋白，利用结构生物学手段进行相关研究。本课题使用哺乳动物和昆虫表达系统，通过亲和层析的方法纯化目的蛋白，结合冷冻电镜技术解析蛋白结构。在本研究中，优化了ETR1（包括不同截短体）、CTR1（野生型和突变体）和EIN2蛋白的表达系统以及纯化方式，最终得到了性质较好，表达量较高的目的蛋白。通过pull down，验证了ETR1和CTR1具有相互作用。在ATP存在的情况下，CTR1磷酸化位点的突变体与ETR1的相互作用显著增强。EIN2的胞内区结构域可以与ETR1形成稳定复合物。制备了ETR1、ETR1/CTR1复合物和EIN2的冷冻电镜样品，对样品进行了相关优化，遗憾的是，最终未能获得这些蛋白样品的高分辨率冷冻电镜结构。但是，我们优化了一些蛋白纯化和复合物的组装方法，这将对后续相关复合物的研究提供一定的参考依据。

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