热激转录因子PvHSFA4a调控海滨雀稗耐镉的分子机制

Seashore paspalum is a widely used warm-season turfgrass species that exhibit superb tolerance to cadmium (Cd). Heat shock factors (HSF) are a family of genes previously known to be involved in regulating various abiotic stress tolerances. It was found that a members of HSF, HSFA4a, may be uniquely involved in the regulating Cd-tolerance, while the molecular mechanism of HSFA4a remained unclear. Our recent study showed that PvHSFA4a cloned from seashore paspalum specifically only regulated Cd-tolerance and performed an important role through its nitrosylation modification. Based on these results, we will perform researches on two aspects, (1) determine the downstream pathways directly regulated by PvHSFA4a, which will be revealed through several strategies including transcriptomics, proteomics, bioinformatics, promoter cloning, protein interactions with nucleic acid, function identification of target gene, in transgenic materials of PvHSFA4a overexpression and RNAi suppression expression; (2) determine the molecular mechanism of PvHSFA4a regulated Cd-tolerance through nitrosylation modification, which will be examined by several methods including protein nitrosylation detection in vivo and in vitro, the expression of target genes, protein interactions with nucleic acid, protein localization, protein interactions. Execution of this research can not only enrich the understanding of the molecular mechanism how HSFA4a regulates Cd-tolerance of plants, but also provide excellent genes for molecular breeding of Cd-tolerance turfgrass based on HSFA4a genes.

海滨雀稗是广泛应用的暖季型草坪草，具有很强的耐镉性。已有研究表明植物热激转录因子HSF介导了不同的非生物胁迫抗性，其中HSFA4a成员参与了耐镉调控，但分子机制尚不清楚。本课题组前期发现海滨雀稗PvHSFA4a特异调控耐镉性，同时该蛋白的亚硝基化修饰对正调控耐镉起重要作用。在此基础上，本项目拟从两个方面展开研究，（1）获得PvHSFA4a过量和RNAi抑制表达的转基因材料，结合转录组学、蛋白组学、生物信息学、启动子克隆、蛋白核酸互作、靶基因功能鉴定等手段解析PvHSFA4a特异调控耐镉的下游分子途径；（2）通过体内体外PvHSFA4a蛋白亚硝基化水平检测、靶基因表达、蛋白核酸互作、蛋白定位、蛋白互作等方法明确PvHSFA4a蛋白亚硝基化修饰调控耐镉的分子机制。本研究的实施，将深化对HSFA4a调控植物耐镉分子机制的理解，也将为基于HSFA4a基因的草坪草耐镉分子育种提供优异的基因资源。

海滨雀稗是广泛应用的暖季型草坪草，具有很强的耐镉性。已有研究表明植物热激转录因 子HSF介导了不同的非生物胁迫抗性，其中HSFA4a成员参与了耐镉调控，但分子机制尚不清楚。前期通过海滨雀稗表达文库筛选获得了耐镉转录因子PvHSFA4a，在此基础上，本研究围绕其直接调控的下游耐镉途径和亚硝基化机制展开探索，其主要研究结果如下：（1）PvHSFA4a受镉胁迫诱导上调表达，定位于细胞核并具有转录自激活活性；（2）过量表达PvHSFA4a显著提高酵母、海滨雀稗和水稻的耐镉性，并促进了镉离子在根和茎中的积累；（3）在通过转录组测序、蛋白组学分析、候选靶基因筛选、定量表达分析、酵母单杂交和凝胶阻滞实验，发现PvHSFA4a能直接与MT-1和CLT1启动子上的HSE元件结合，进而诱导其表达，提高耐镉性；（4）对野生型和转PvHSFA4a基因的海滨雀稗材料进行NO处理发现，NO能显著增强PvHSFA4a的耐镉功能；通过体外PvHSFA4a蛋白原核表达、亚硝基化反应和生物素抗体检测，发现PvHSFA4a存在亚硝基化修饰途径；（5）采用生物信息学软件分析结合点突变的转基因耐镉功能鉴定，发现PvHSFA4a蛋白的C49S位点突变导致其耐镉功能丧失，而C49W位点突变仍保持其耐镉功能，表明C49位点是PvHSFA4a蛋白的亚硝基化修饰靶位点；（6）通过体外凝胶阻滞实验证明，NO处理能显著增强PvHSFA4a与两个靶基因启动子上的HSE元件的结合能力，进而促进其表达，提高耐镉性；在前人的研究基础上，通过酵母双杂交实验，我们也发现PvHSFA4a能与植物体内的NO供体蛋白过氧化氢酶CAT互作。本项目的实施，一方面明晰了HSFA4a直接调控的下游耐镉靶基因，同时从蛋白亚硝基化修饰层面探究了HSFA4a的分子作用机制，将为基于HSFA4a基因的草坪草耐镉分子育种提供思路。