【作者】 陈娟；

【导师】 黄文超；

【作者基本信息】 武汉大学 ， 遗传学， 2020， 硕士

【摘要】 烯醇化酶是生物中普遍存在的一类多功能蛋白,在糖酵解通路和糖异生过程中参与催化2-磷酸甘油酸(2-PGA)向磷酸烯醇式丙酮酸(PEP)的可逆转化。此外,烯醇化酶也参与植物的抗逆性,基因调控等生物学过程。基于烯醇化酶在生物体内的功能多样性和对生物生长发育的重要性,我们对水稻烯醇化酶OsENO4进行了结构和功能两方面的探索研究。首先我们体外构建了OsENO4的原核表达载体,并在大肠杆菌中成功表达出OsENO4蛋白,通过Ni-IDA亲和层析和Mono Q离子交换层析纯化出了具有高纯度和均一构象的蛋白。在经过大量的晶体条件筛选和优化后,我们得到了质量较高的OsENO4晶体,通过X-射线晶体衍射收集到了3.0?的衍射数据。我们以人源烯醇化酶Hu ENOα的蛋白结构(PDB:2PSN)为置换模版,成功解析了OsENO4的晶体结构,并使用Pymol软件对其进行可视化分析。结构显示OsENO4由两个大的结构域组成,具有典型的α/β桶状结构,不同物种的烯醇化酶结构基本完全重叠,体现出了烯醇化酶在蛋白结构上的高度保守性。不同的是,OsENO4的结构呈现单体形式,可能与植物特有的插入序列有关,此外这些序列在弓形虫烯醇化酶中也存在,因而二者在该部分结构上表现出高同源性。随后分子对接呈现的OsENO4与底物2-PGA的结合模式与人源烯醇化酶Hu ENOγ(PDB:3UJE)相比,同样地也显示出了高度保守性。OsENO4是目前为止第一个被解析结构的植物烯醇化酶,可以为以后植物烯醇化酶的功能和进化研究提供一定的结构基础。接下来为了继续摸索OsENO4在水稻生长发育过程中的其他可能作用,我们通过CRISPR/Cas9技术对日本晴材料进行OsENO4基因敲除,转基因过程中愈伤死亡,水稻烯醇化酶OsENO4基因的功能仍需要进一步摸索探究。更多还原

【Abstract】 Enolase is a multifunctional protein throughout a broad spectrum of organisms,which is the enzyme responsible for the conversion of 2-phosphoglycerate(2-PGA)to phosphoenolpyruvate(PEP)during glycolysis and the reverse reaction during gluconeogenesis.It has been proposed that involved in biological processes such as plant stress resistance,gene regulation and so on.Based on the functional diversity of enolase in organisms and its importance to biological growth and development,we have carried out structure and function the two aspects exploration research to the rice enolase OsENO4.First,we constructed a prokaryotic expression vector and successfully expressed OsENO4 in Ecoli,which had high purity and homogeneous conformation purified by Ni-IDA affinity chromatography and Mono Q ion exchange chromatography.Then the high quality OsENO4 crystals were obtained after numerous crystal conditions screening and optimization,and the diffraction data of3.0 A were received in X-ray crystal diffraction.Then we used the structure of human enolase Hu ENOα(PDB: 2PSN)as a replacement template to analyze OsENO4s’ and used Pymol to visualize it.It shows that OsENO4 consists of two large domains with a typical α/β barrel.The structures of enolases from different species are almost completely overlapped,which indicates the highly conserved three-dimensional structure of enolase.Differently,OsENO4 presents as a monomeric form,which may be related to its plant-specific insertions.In addition,these insertions are also present in the enolase of toxoplasma gondii,the two enolase exhibit high homology in this part of the structure.The subsequent binding pattern of OsENO4 protein to substrate2-PGA presenting by molecular-docking also showes a high degree of conservation compared with counterpart in structure of Hu ENOγ(PDB: 3uje).The analysis of OsENO4’s crystal structure makes it the first plant enolase to be resolved protein structure so far,which provides a structural basis for further functional and evolutionary studies of plant enolase.To further explore the other possible roles of OsENO4 in rice growth and development,the gene-knockout vector of OsENO4 was performed by CRISPR/Cas9 and then transformed into Nipponbare,which resulted in callus death.The function of rice enolase OsENO4 gene needs further exploration.更多还原