【作者】 石锦平；

【导师】 范礼斌； 张成岗；

【作者基本信息】 安徽医科大学 ， 遗传学， 2011， 硕士

【摘要】 秀丽线虫(Caenorhabditis elegans,C. elegans)是1974年Brenner首次公开报道的模式生物,其优点很多,是进行遗传、基因研究方面的首选模式生物。线虫生长周期短,实验室饲养简便,具备完整的发育和解剖学特征,已完成全基因组测序,基因操作便捷,如转基因、基因突变、RNA干涉等。本研究将线虫应用于Ngb对氧化损伤的作用研究及缺氧状态下细胞骨架的变化分析,旨在促进对低氧应答机制的理解及氧化应激损伤保护的防护基础研究。1、利用秀丽线虫研究Ngb抗氧化损伤的作用Ngb是新近发现的氧结合珠蛋白超家族成员,表达于神经系统,具有抗氧化损伤作用。Ngb含有两种重要的氨基酸:组氨酸和半胱氨酸,对Ngb的蛋白质结构及功能具有重要作用。为了探讨Ngb的抗氧化作用及作用机制,我们使用hNgb转基因线虫来进行百草枯(paraquat,PQ)处理,结果显示Ngb具有抗氧化作用。使用免疫共沉淀分析Ngb的相互作用蛋白,但未见明显结果,有可能Ngb是通过自身的重要结构来发挥抗氧化作用。我们还构建了Ngb的双突变表达载体,进行显微注射,以期获得Ngb的突变株系,进一步探讨Ngb抗氧化损伤的作用机制。秀丽线虫体内有33种globin,远多于脊椎动物,其中glb-13是人源Ngb(hNgb)的同源物,氨基酸序列具有20%的相似性,有相同的功能域。为了探讨glb-13的抗氧化作用及其发挥作用的信号通路,我们通过线虫的交配构建了线虫重要通路hif-1与daf-16和glb-13的双突变株系,并进行PQ处理来统计成虫率,结果显示glb-13(tm2825)突变株系不能恢复PQ引起的氧化损伤,hif-1的glb-13双突变株系的抗氧化作用低于glb-13(tm2825),说明hif-1在glb-13抗氧化作用的信号通路上可能发挥重要作用。在mRNA水平上,glb-13表达水平上调,与其抗氧化表型吻合,glb-13具有抗氧化应激作用,而glb-13的抗氧化应激作用不受HIF-1调控,可能与DAF-16和Nrf-2相关。同时,我们构建了以glb-13为启动子的表达载体,通过EGFP的可视化显示glb-13在线虫体内的分布与表达,以期获得其转基因株系,从而从蛋白质水平探讨glb-13的抗氧化作用。从Ngb的同源物角度进行Ngb的抗氧化作用研究,反映了Ngb具有抗氧化的作用,因此研究glb-13的功能对于理解Ngb的功能进化性具有重要意义。2、利用秀丽线虫研究细胞骨架蛋白在缺氧中的作用机制细胞骨架蛋白是支撑细胞的重要结构,不可或缺。细胞骨架蛋白的功能非常广泛且十分重要,参与细胞的基本生命活动,如细胞运动、物质运输和能量转换、细胞分化、信号转导等。其中,细胞骨架在抗缺氧方面的作用同样重要。为了探讨细胞骨架蛋白在缺氧中发挥的作用,选取原肌球蛋白(tropomyosin)突变株系lev-11(x12)研究细胞骨架在缺氧中的作用。结果显示lev-11突变后对缺氧敏感,蛋白水平显示LEV-11上调,说明细胞骨架蛋白能对抗缺氧引起的损伤。更多还原

【Abstract】 qBrenner first reported C. elegans(Caenorhabditis elegans) as a model organism in 1974. C. elegans has many advantages, and is the preferred model in genetic studies because of short life cycle and lifespan, simple laboratory rearing, characteristics of full development and anatomy. It has completely-sequenced genome and convenient genetic manipulation, such as transgene, gene mutation, RNA interference. Therefore, C. elegans was used in this study to probe the response of Ngb on oxidation injuries and cytoskeleton changes under hypoxia stress.1. Anti-oxidative Mechanism of Ngb in C. elegansNgb is a recently discovered as oxygen binding globin and specifically expressed in the nervous system. Ngb has two kinds of important amino acids: histidine and cysteine, which play important roles in the structure and anti-oxidative function of this protein. To investigate the antioxidant role of Ngb and the underlying mechanism, we used the human Ngb (hNgb) transgenic strain of C. elegans to treat with paraquat (PQ). Results showed that Ngb has antioxidant function. We used co-immunoprecipitation to pull down the potential interacting proteins of Ngb, but had no significant results. Ngb may play antioxidant role through its structure. We also constructed the histidine and cysteine double mutants of Ngb expression vector for microinjection, in order to obtain Ngb mutant transgenic strains and further exploring the oxidative mechanism of Ngb.C. elegans has 33 globins, which is far more than vertebrates. glb-13 is the homolog of hNgb, which has 20% similarity and the same functional domains. To investigate its antioxidant role and the signaling pathway, we constructed the mutant strain by mating the major pathways of hif-1 and glb-13 double mutant strains, then treated with PQ for adult rate and mRNA expression analysis. Results showed that the glb-13(tm2825) mutant strains can not be restored upon PQ induced oxidative damage. The antioxidative effects of the hif-1 and glb-13 double mutant strains were weaker than that of the strain glb-13(tm2825). Therefore, our result indicated that glb-13 is antioxidant and HIF-1 does not influence the antioxidative function of glb-13, which DAF-16 and Nrf-2 might plays important roles in the antioxidant signaling pathway of glb-13. We also constructed the glb-13 promoter of expression vector to observe the expression and distribution of glb-13 tagged EGFP in C. elegans in order to detect the protein level of glb-13. Taken together, the antioxidant of glb-13 also agreed with the antioxidant role of Ngb. It’s important for understanding the evolution of Ngb.2. Hypoxia Response Mechanism of Cytoskeleton in C. elegansCytoskeleton, which support the cell structure, is indispensable for cell functions. Cytoskeleton is involved in cell movement, material transport and energy conversion, cell differentiation, signal transduction. The anti-hypoxia function is equally important. To investigate the role in hypoxia, the tropomyosin mutant strain lev-11(x12) was selected. Our results showed that the lev-11(x12) mutant is sensitive to hypoxia, and the LEV-11 protein level increased under hypoxia stress. Therefore, these data indicated that cytoskeleton can recover the damage induced by hypoxia.更多还原