杨树C2H2锌指蛋白基因重复和盐胁迫响应的功能分化

Functional divergence among paralogous genes provide an important genetic source for plant evolutionary innovation. C2H2-ZFPs family (ZFPs, Zinc-finger protein) involve in a variety of processes such as the regulation of floral organogenesis, leaf initiation, lateral shoot initiation and stress responses. ZATs genes, belong to C2H2-ZFPs, may have undergone independent evolutionary trajectories since the origin of the genus Populus. In this project, we aim to examine how C2H2-ZFPs of Populus evolved in sequences and conserved domain/motifs; how the ZATs diversified functions involved in the response to salt stress and which gene regulating networks are involved by ZATs in the process of salt stress. First, we will investigate the evolution of C2H2-ZFPs from Populus via phylogenic analysis, identify the models of functional divergency, the history of gene duplication and evolutionary dynamics. Then, we will compare the possible divergence in orthologous or paralogous of these ZATs genes through over- expression and knockout of PalZATs in transgenic poplars. Third, we will detect the up-stream regulators of PalZATs via yeast one hybrid screening. The interaction between ZATs and regulators will be confirmed by EMSA (electrophoretic mobility shift assay) and ChIP (Chromatin Immunoprecipitation) methods, and their functions will be identified in transgenic poplars (P. alba) in which the regulators genes will be overexpressed or knockout. Finally, we will sum up all candidate regulators and draw the outline of gene regulation networks built by the diversified ZATs involved during salt stress. All these results will clarify the key roles of the ZATs divergence and select the most salt-tolerant poplar breed.

重复基因的功能分化为生物发育与环境适应提供了广泛的遗传基础。C2H2锌指蛋白（C2H2-ZFP）参与植物多种逆境响应过程。杨树C2H2-ZFPs家族中，ZAT亚家族基因发生了独立的重复进化，而这些基因在盐胁迫下的表达模式发生了分化，推测可能和杨树起源后为适应环境导致的重复基因功能分化相关。项目拟：⑴通过整个杨属C2H2-ZFPs基因的分子进化分析，推测其基因重复方式、进化历史及保留模式，确定ZAT亚家族基因的起源和序列分化；⑵从表达模式、生化功能及蛋白定位等方面，比较代表杨树物种4个PalZATs功能分化模式；⑶构建4个PalZATs的超表达、Cas-9定点敲除等遗传转化体系，分析PalZATs的功能；⑷构建酵母单杂文库，筛选4个PalZATs在盐胁迫响应中的上游调控蛋白，并进行生物学功能分析。结果将揭示杨树C2H2-ZFPs基因重复和盐胁迫响应的功能分化，为完善杨树抗盐的调控途径提供证据

盐和干旱胁迫会损害植物渗透稳态，极大地影响植物的生长发育。在受到渗透胁迫时，植物通过减小气孔开张以减少水分流失并保持渗透稳态，从而提高抗逆性。项目通过4年的执行期，主要完成了杨树C2H2锌指蛋白基因家族重复历史分析，并对其功能分化和分子调控功能进行了深入研究。结果发现：⑴ 杨属C2H2基因进化模式多样，在经历杨属物种近期基因组加倍事件后，转录因子OSIC（OSMOTIC STRESS INDUCED C2H2 1）发生了杨属特异性的基因扩张，其表达模式发生了显著分化。杨属中C2H2家族的多样化进化模式可能和其抗逆能力有关；⑵ 新疆杨OSIC1受干旱、高盐、ABA等胁迫诱导表达，提高水分利用效率，引起叶片气孔中H2O2积累，降低气孔开张度，增强新疆杨对胁迫的耐受能力； ⑶ 通过酵母单杂（Y1H）、EMSA、LUC等实验手段，验证了OSIC1能直接结合在CuAOζ基因的启动子上，并上调CuAOζ基因的表达。⑷ 体外和体内磷酸化实验表明，MPK3能特异性磷酸化OSIC1，进而提高了OSIC1转录因子与CuAOζ启动子的结合能力，增强对CuAOζ的表达调控，从而加速H2O2在保卫细胞中的积累，促进气孔关闭；⑸ ABA、干旱、高盐等诱导MPK3对OSIC1的磷酸化修饰，增强了其对CuAOζ的转录活性。OSIC1在转录和蛋白质水平上的调节有利于杨树通过气孔闭合应对渗透胁迫。⑹筛选到了多个C2H2(OSIC1)转录因子调控的下游基因，同时也通过比较转录组学技术，筛选到了影响OSIC1表达的circRNA，并对其进行了克隆和新疆杨遗传转化，这些为深入研究OSIC转录因子参与调控的逆境响应途径提供了重要参考。总之，我们的结果揭示了由OSIC1-PalCuAOζ模块介导的保卫细胞中H2O2产生的新转录调控机制。这些发现加深了我们对多年生木本植物（如杨树）如何响应盐和干旱引起的渗透胁迫的理解，并为杨树抗逆育种提供了重要的理论参考。