PvHSFA4a蛋白乙酰化修饰调控海滨雀稗耐镉的作用机制

Seashore paspalum is an excellent perennial turfgrass species with strong cadmium (Cd) tolerance. PvHSFA4a, a heat shock transcription factor from seashore paspalum performed a positive role in Cd-tolerance, while its molecular mechanism in protein modification level remained unclear. Acetylation is served as an important role in the regulation of protein activity, and the acetylation mechanism of plant HSF protein has rarely been reported. Our previous research exhibited that Cd stress promoted the acetylation modification of PvHSFA4a protein, and K76 lysine modification site of PvHSFA4a was preliminarily screened for controlling Cd-tolerance. Based on the above results, the following study will be carried out in this project: ① transgenic seashore paspalum plants over-expressed PvHSFA4a and its two artificial mutant alleles (K76R and K76Q) will be achieved respectively and the correlation of K76 site to Cd-tolerance will be identified; ② specific acetylation and deacetylation enzyme interacting with PvHSFA4a and regulating K76 site modification will be excavated, and their function will be further determined through transgenic strategy; ③ how the PvHSFA4a and its interaction enzymes coordinately controlled Cd-tolerance in seashore paspalum will be analyzed, through the following aspects including target gene expression, protein interaction with nucleic acid, protein localization and protein stability. This research will not only elucidate the molecular mechanism of acetylation modification regulating HSF in plants, but also provide crucial genes for Cd-tolerance breeding of perennial turfgrass and gramineous grass.

海滨雀稗是优异的多年生耐镉草坪草，其热激转录因子HSF家族蛋白PvHSFA4a正调控植物耐镉性，但该蛋白的翻译后修饰机制尚不明晰。研究表明乙酰化修饰在蛋白活性调控中发挥重要作用，而植物HSF蛋白的乙酰化机制未见报道。我们前期发现镉胁迫上调海滨雀稗PvHSFA4a蛋白的乙酰化修饰水平，并初步鉴定到调节PvHSFA4a功能的第76位赖氨酸修饰位点。在此基础上，本项目拟①获得PvHSFA4a及其突变基因（K76R和K76Q）的海滨雀稗转化植株，明确K76位点的耐镉功能；②找出与PvHSFA4a互作并调节K76位点修饰的乙酰化酶和去乙酰化酶，转基因鉴定其耐镉功能；③从靶基因表达、蛋白核酸互作、蛋白定位及蛋白稳定性等层面，解析PvHSFA4a及其修饰酶协同调控海滨雀稗耐镉的作用机制。本研究的实施，将不仅阐明乙酰化修饰调控植物HSF功能的分子基础，同时为多年生草坪草及禾草的耐镉育种提供优异基因资源。

海雀稗是优异的多年生耐镉草坪草，其热激转录因子PvHSFA4a正调控植物耐镉性，但该蛋白的翻译后修饰机制尚不明晰。研究表明乙酰化修饰在蛋白活性调控中发挥重要作用，而植物HSF蛋白的乙酰化机制未见报道。在此基础上，本研究围绕PvHSFA4a的乙酰化机制及其互作修饰酶展开探索，其研究结果如下：（1）从海雀稗转录组库中鉴定到18个去乙酰化酶家族成员，分为3类，呈现不同的定位和表达模式；（2）采用酵母双杂交点对点检测18个去乙酰化酶与PvHSFA4a互作，鉴定了3个互作成员PvHDAC6、PvHDAC9和PvHDAC15；亚细胞定位发现，PvHDAC15仅在细胞核中表达（与PvHSFA4a定位完全一致），而PvHDAC6、PvHDAC9在核质中均有表达；（3）通过荧光素酶互补、原核表达和pull-down实验证明了PvHDAC15与PvHSFA4a的互作信号最强；（4）将PvHDAC15同源转化海雀稗愈伤组织，经分子鉴定获得了转基因株系；镉胁迫下，过量表达的转基因海雀稗植株生长显著优于野生型，表现出更高的相对含水量和光化学效率、更低的活性氧水平，离子检测发现，转基因植株的地上部镉离子含量显著低于野生型，而根系中无明显差异；（5）乙酰化组学分析发现，镉胁迫提高了海雀稗根系总蛋白的乙酰化水平，经蛋白质免疫沉淀获得体内PvHSFA4a蛋白，发现其乙酰化水平受镉诱导上调，并通过LC-MS/MS鉴定到其中1个乙酰化赖氨酸位点K76，K76R和K76Q点突变蛋白在酵母和水稻中的耐镉功能丧失；（6）通过转录组测序、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；而两个突变蛋白（K76R和K76Q）均不能结合HSE元件。本项目实施，初步阐明乙酰化修饰调控植物HSF功能的分子基础，同时为多年生草坪草及禾草的耐镉育种提供优异基因资源。