【作者】 尹龙飞；

【导师】 华跃进；

【作者基本信息】 浙江大学 ， 生物物理学， 2010， 博士

【摘要】 耐辐射球菌不仅对电离辐射具有惊人的抗性,而且对过氧化氢、紫外线和干燥以及其它物理化学诱变因子都具有极强的抗性。迄今为止,我们对它的极端抗性的分子机制了解的还很有限。研究证明电离辐射对DNA直接损伤为20%,80%DNA的损伤是由氧化压力间接产生的,说明了耐辐射球菌的抗氧化能力是其电离辐射抗性一个重要因素。活性氧应激子OxyR是一种广泛存在于细菌中的LysR型转录调节因子家族的成员,大肠杆菌OxyR能够感受过氧化氢胁迫并启动细胞的抗氧化系统。本文对耐辐射球菌中氧化相关的转录因子OxyR2(DRA0336)在抗氧化过程中的作用进行了一定的研究,有助于从另一个角度深入理解这极端抗性。本论文还利用基因芯片技术对紫外辐射后耐辐射球菌的全基因组表达进行了分析。1、首先生物信息学分析发现除了OxyR (DR0615)耐辐射球菌中可能存在E. coli OxyR的另一个同源物OxyR2 (DRA0336)。多序列比对表明涉及DNA结合结构域、蛋白四聚化、激活区域和形成疏水中心的多个关键氨基酸位点和一个重要的半光氨酸位点C208都是十分保守,这是第一次发现细菌中可能存在两个OxyR同源物。利用完全缺失突变的方法构建了oxyR2基因的单突变菌株、oxyR2和oxyR基因的双突变菌株DM,同时构建完整补偿株MOxyR2-MC和N端截短48个核苷酸的补偿株MOxyR2-CΔN,再利用定点突变法构建半胱氨酸定点突变的补偿株包括MOxyR2-MC228,MOxyR2-MC272和MOxyR2-MC290。2、对各个突变株和补偿株的特性进行分析,过氧化氢胁迫下的生存率分析表明oxyR2基因的敲除导致突变株对过氧化氢氧化胁迫很敏感,进一步分析表明突变株内的过氧化氢酶总活性降低以及ROS积累更多。双突变DM比单突变对过氧化氢处理更敏感,过氧化氢酶更低以及ROS积累更多。补偿实验表明耐辐射球菌的全基因以和N端截短48个核苷酸的oxyR2都能完全补偿其氧化抗性,但耐辐射球菌OxyR2不能补偿E coli的OxyR突变株GS09。半胱氨酸定点突变的补偿株分析表明OxyR2的C228是耐辐射球菌在氧化胁迫过程中存活的一个关键位点。QRT-PCR结果表明耐辐射球菌的oxyR和oxyR2是持续表达的而且彼此的突变株并不存在由于氧化胁迫而导致的转录补偿。这些实验结果说明oxyR2是耐辐射球菌抗氧化过程所必须的。3、利用耐辐射球菌全基因组的olig芯片分析了DRA0336突变株全基因组的转录水平,比较了突变株和野生株在20mM过氧化氢处理后转录水平差异。发现MOxyR2中共有103个基因改变了其表达模式,表现出ROS的应急反应：抗氧化相关基因,铁代谢相关基因以及容易产生ROS的相关基因受到明显抑制。4、用基因芯片技术从转录水平研究耐辐射球菌紫外辐射后全基因组的表达谱,耐辐射球菌在800J/m2紫外辐射后的总共有167个基因发生表达模式改变,其中87个基因表达上调和81个基因表达下调,DNA修复相关的基因uvsE, uvrA2, uvrB, uvrC, uvrD, recA和pprA都受到紫外辐射的诱导。同时芯片数据表明耐辐射在紫外辐射后没有表现出一个经典的SOS反应机制,而且DNA复制的起始因子dnaA和分裂有关因子minE的下调说明紫外辐射后可能存在一个停滞的修复期。更多还原

【Abstract】 Deinococcus radiodurans is characterized by its extraordinary resistance to all kinds of DNA damaging agents, such as ionizing radiation, hydrogen peroxide, UV radiation, desiccation and other physical and chemical DNA damaging agents However, to date the molecular mechanism responsible for the astonishing resistance is not well understand. It has been demonstrated that damaging effects of ionizing radiation are due to the indirect oxidative stress to DNA. This indicates that the antioxidation ability is importantly contributed to the resistance to ionizing radiation. OxyR which belongs to the LysR family of transcriptional regulator widely exit in bacteria. E. coli OxyR regulates expression of the majority of genes response to hydrogen peroxide. In this study, the function of D. radiodurans OxyR2 (DRA0336) were investigated. In addition, the genome transcriptional profiling of D. radiodurans following 800 J/m2 UV radiation was studied. The results as below:1. Analysis of the D. radiodurans R1 genome using the BLAST program with the E. coli OxyR amino acid sequence as a query sequence revealed the presence of another putative homolog DRA0336 (OxyR2). Multiple amino acid sequence alignment indicated amino acid residues of OxyR2 involved in DNA binding domain, protein tetramerization, possible activating region and key cysteine residue C208 are conserved. It is first found that there exit two homologues in one bacterium. To investigate the function, we constructed the deletion mutant of oxyR2 and the double mutant of oxyR oxyR2/oxyR2. MOxyR2-C strains and MOxyR2-CΔN strains, complemented by full OxyR2 or OxyR2 with N terminal absence of 16 amino acids respectively, were also constructed. PCR site-direct mutagenesis was performed to construct MOxyR2-MC228, MOxyR2-MC272和MOxyR2-MC290 strains.2. All mutant strains and complementary strains were characterized. Survival rate assay with H2O2 treatment indicated that MOxyR2 was significantly more sensitive to H2O2 compared to the wild type. The complementation experiment showed that tans-expression of the OxyR2 or OxyR2 with N terminal absence of 16 amino acids in MOxyR2 could fully restore the H2O2 resistance. C228 is a crucial site for survival in H2O2 stress. The double mutant DM was significantly more sensitive to H2O2 than any of the single mutants. MOxyR2 accumulated much more ROS than the wild type R1 and DM accumulated the highest level of ROS. Deletion of oxyR2 attenuates the enzymatic activity of catalase and DM had almost no increase after the treatment with H2O2. These results demonstrate that the oxyR2 gene is responsible for the sensitivity to hydrogen peroxide.3. To test the role of OxyR2 in D. radiodurans under H2O2 treatment, the whole genome expression profile of MOxyR2 was performed in comparison with R1 after treatment with 20 mM H2O2 during the exponential growth phase. The result showed that total 103 genes changed the expression pattern and exhibited response to ROS emergency. One obvious character was that many genes related with production of ROS, antioxidation and iron metabolism significantly down-regulated.4. To better understand the response mechanism how D. radiodurans coped with the UV irradiation, the investigation of the global gene expression profile in response to UV irradiation was performed. Overall,167 of the total predicted D. radiodurans genes represented on the chip were pronouncedly differentially expressed following UV irradiation of 800 J/m2 after 30 min of post-irradiation incubation. DNA repair gene including uvsE, uvrA2, uvrB, uvrC, uvrD, pprA and recA were induced. The microarray results indicate that there may be no SOS response after UV radiation in D. radiodurans. DNA replication initiation factor dnaA and minE related with mitosis were obviously down-regulated. These indicated that there may exit a period of stasis characterized by repair phase.更多还原