CCCH型锌指蛋白C3H12参与水稻抗病反应的机理研究

Disease is one of the main reason for the great loss in rice production, so researching and exploiting rice defense genes provides new sources for breeding rice varieties with good resistance.CCCH-type zinc finger proteins are widely present in eukaryotes and play important roles in growth and stress response of plants. Previous studies have already proved that C3H12 which belongs to rice CCCH type zinc finger protein family was first found involving in disease resistance. However, it is still unclear about the substract type, active domain and binding model of C3H12. In this project, the substract type of C3H12 will be determined by comparing distinct binding activities of C3H12 with different substracts in stopped-flow assay. Then the active binding domain will be confirmed through comparing different binding activities of C3H12 mutants and its substract. Finally, the recognition mechanism of C3H12 with its substract will be illustrated based on the interaction 3D-model which is estabished by biological macromolecule crystal structure analysis and homology modeling tool. The project will aid to unravel the molecular mechanism of C3H12 in rice disease resistance and provide a new approach for advanced function research of other CCCH type zinc finger proteins in plants.

病害是造成水稻减产的主要原因之一，研究和发掘水稻自身抗病相关基因可为培育优良的抗性品种提供新的途径。CCCH型锌指蛋白广泛的存在于真核生物中，在植物的生长发育和对逆境的应答过程中扮演重要角色。前期研究已证实C3H12是水稻中首次发现的参与抗病反应的CCCH型锌指蛋白，但缺乏对C3H12的功能研究，对其结合底物类型、活性部位和与底物的结合模式并不了解。本项目将利用stopped-flow方法测定C3H12与不同底物的结合活性，明确C3H12蛋白结合的底物类型。再通过比较不同C3H12突变体与底物结合活性的差异，确定其与底物结合的活性部位。最后运用生物大分子晶体结构解析和同源建模技术，建立C3H12与底物互作三维模型，阐明C3H12蛋白与RNA底物的结合模式。本项目的开展有助于揭示C3H12参与水稻抗病反应的分子机理，并为植物中其他CCCH蛋白的功能深入研究提供了新的思路和实验依据。

病害是造成水稻减产的主要原因之一，研究和发掘水稻自身抗病相关基因可为培育优良的抗性品种提供新的途径。CCCH型锌指蛋白广泛的存在于真核生物中，在植物的生长发育和对逆境的应答过程中扮演重要角色。前期研究已证实C3H12是首次发现在水稻中参与抗病反应的CCCH型锌指蛋白，但缺乏对C3H12的功能研究，对其结合底物类型、活性部位和与底物的结合模式并不了解。于是我们对C3H12（WT）进行了一系列缺失突变，合成了互作的ARE19底物，3'端生物素标记RNA底物，采用RNA-蛋白体外互作的凝胶电泳迁移实验（Electrophoretic mobility shift assay，EMSA）来寻找蛋白质和RNA之间相互作用的位点。结果发现C3H12可与富含AU碱基的RNA底物结合，而C3H12 N端部分不直接与RNA结合，说明C3H12底物结合中心很可能包含在C端部分，更有趣的是串联两端锌指间特殊长序列连接片段似乎对RNA结合没有起到关键作用，这与之前的一些文献报道的类似CCCH蛋白不尽相同。随后通过核磁共振的方法解析了C3H12蛋白C端活性中心在复合体状态下的结构，建立了蛋白与RNA互作三维模型，从原子水平阐明了其互作模式，揭示了C3H12蛋白参与水稻抗病反应的机理，为植物中其他CCCH 蛋白的功能深入研究提供了新的思路和实验依据。