荒漠单细胞C4植物异子蓬C3和C4型PEPC同工酶的表达调控及其逆境响应机制研究

Phosphoenolpyruvate carboxylase (PEPC) is the key factor for CO2 concentrating in C4 plant. Plant PEPC exists different isozymes in one individual plant, its activity, function, expression and regulation pattern must be largely different. For single-cell C4 plant species, maintaining strictly spatial biochemical separation within one individual chlorenchyma cell is essential and crucial, its mechanism of CO2 concentrating and the expression and regulation characteristic of the major photosynthetic enzymes must be different with classical C4 species. So far, few has been reported on comparison of different isozymes (such as C3, C4 types) of PEPC in one C4 plant species. Therefore, in the present study, based on the current report on plant PEPC, combining our previous work on C3 and C4 types of PEPC in Suaeda aralocaspica, we plan to explore the function of C3 and C4 types of PEPC in single-cell C4 plant S. aralocaspica from the following aspects: ① Comparison of differences in protein property, enzymatic activity, phosphorylation pattern between C3 and C4 types of isozyme with in vitro protein expression system; ② comparison of changes of gene expression and enzymatic activity between C3 and C4 types of PEPC in different growth stages, tissues, and under various stress conditions; ③ comparison of differences in regulation at transcriptional (promoter function) and post-translational (phosphorylation and ubiquitination) levels between C3 and C4 types of PEPC isozyme. Thus, a better understanding can be achieved with the regulation mechanism of PEPC isozymes responding to growth and development and their contribution to environmental adaptability in single-cell C4 photosynthetic species S. aralocaspica, and it can also provide scientific and theoretical evidence for genetic improvement of crop with high photosynthetic efficiency.

磷酸烯醇丙酮酸羧化酶(PEPC)是C4植物CO2浓缩机制的关键因素。PEPC以多种同工酶存在于同种植物中，其活性、功能、表达调控均可能存在差异。单细胞C4植物需在同一细胞内维持严格生化区隔，其CO2浓缩机制及光合关键酶的调控特性必定与经典C4植物有显著区别。目前对同种C4植物中不同类型（C3和C4型）PEPC同工酶的比较研究鲜见报道。鉴于此，本项目基于目前对植物PEPC的报道，结合本小组对异子蓬C3、C4型PEPC的研究，拟对单细胞C4植物异子蓬PEPC及其同工酶（C3、C4型）开展深入探索，从两种同工酶体外表达、活性及磷酸化差异；不同发育阶段、组织及环境胁迫下同工酶基因的表达、酶活改变；同工酶在转录（启动子功能）和翻译后（磷酸化和泛素化）调控等方面进行比较研究，从而深入理解异子蓬单细胞C4途径PEPC同工酶响应生长发育的调控机制及对环境适应的贡献，并为农作物高光效遗传改良提供科学依据。

基于本研究设定的初始目标，我们首先搞清了异子蓬C3、C4型（后称为PEPC-1和PEPC-2）同工酶的体外酶活性、酶学特性及对抑制剂和激活剂的响应特性的差异（PEPC-2 > PEPC-1）；阐明了两种同工酶在大肠杆菌过表达后对各种非生物胁迫的耐受性（均显著耐受）。体外磷酸化后的酶学特性实验未获成功，有待将来深入探究。其次，本研究系统分析了两种PEPC基因在mRNA水平和蛋白质表达水平对生长发育及胁迫的响应模式，转录表达分析显示PEPC-1在各种处理下的响应显著高于PEPC-2；由于PEPC-1在抗体制备中出现问题（制备了两次），最后只得到了PEPC-2在蛋白水平上的结果以及其他光合关键酶的响应模式。PEPC-2在蛋白水平上的表达与总PEPC表达模式一致但与磷酸化PEPC以及总酶活性趋势存在显著差异。第三，我们克隆了两种PEPC基因的上游启动子序列，截段分析其功能显示两种PEPC启动子活性无显著区别；然而在异子蓬中两种基因转录表达模式显示PEPC-1对各种处理的响应显著高于PEPC-2。第四，解析了PEPC两种同工酶蛋白质磷酸化的情况，确定了PEPC-2磷酸化程度随光强度增强的变化模式，PEPC-1由于抗体制备问题未检测到结果；由于技术、经费及时间等问题导致蛋白质的泛素化研究未获得结果，还需今后深入研究。第五，我们获得了两种PEPC基因的全长序列（各包含10个外显子和9个内含子）；利用两种PEPC启动子和编码区序列开展了转基因研究，目前正在进行表型实验。第六，通过本项目与剑桥大学植物科学系建立了互访关系；与中国农业大学武维华院士建立了联合培养博士项目；第七，目前已发表论文9篇，其中SCI收录5篇；培养博士研究生3人，硕士研究生2人；获得新疆科协自然科学优秀学术论文三等奖1项。