【作者】 詹红丹；

【导师】 华跃进；

【作者基本信息】 浙江大学 ， 生物物理学， 2011， 硕士

【摘要】 耐辐射球菌(Deinococcus radiodurans)是世界上最耐辐射的物种之一,对各种DNA损伤介质具有超强的抗性,已成为研究基因组完整性和稳定性的一种理想模式生物。本实验室的研究结果显示,一个被注释为CRP/FNR家族的基因ddrl(基因编号为dr0997),在DNA受损时被显著诱导表达,可能贡献于耐辐射球菌的极端抗性。因此,我们重点围绕该基因进行了研究。1.分别构建了dr0997及其同源基因dr1646的基因缺失突变株,并发现突变株相对于野生株,生长情况出现了明显的延滞。2.分析了△DR0997突变株对DNA损伤因子的抗性。结果表明：dr0997和dr1646对UV和氧化损伤抗性的贡献都不大。而△DR0997突变株对Y-射线辐照处理很敏感,表明该基因可能与电离辐射造成的双链断裂修复有关。3.用DNA微阵列技术分析了突变株相对于野生株的转录谱变化。本研究覆盖了3033个基因,占整个基因组的95.26%。DR0997的缺失导致28个基因的转录水平明显上调,另外79个基因明显下调,这些基因很多涉及NADPH、乙酰辅酶A合成酶等的转录合成,表明dr0997可能参与调控了相关途径。4.表达并纯化了DR0997重组蛋白。通过测序,我们证实对该基因在Genebank的ORF是错误的,其实际长度为612 bp,我们以新序列为标准克隆了dr0997基因,并最终成功在体外表达并纯化了DR0997重组蛋白。综上所述,耐辐射球菌的DR0997基因对其极端抗性很重要。它是一个具有特异性DNA结合活性的代谢调节基因,主要通过调控与细菌抗氧化和DNA修复相关的途径起作用。它与重要蛋白PprI和RecA的相互关系正在探索之中。更多还原

【Abstract】 Deinococcus radiodurans R1 is one of the most radioresistant bacteria on the earth. This particular species can stand a wide range of DNA-damaging agents such as gamma-radiation, UV, H2O2 and desiccation. For many years, it has been an ideal model organism to investigate the integrity and stability of the genome. However, the underlying molecular mechanisms remain poorly understood. System singaling the stresses from eniverment are required in all three kindom of lifes. Phosphorylation on histidine and aspirate are the main signal-transduction pathways in prokaryote, named two-compenents system, which consists of a kinase and a response regulator. Previous studies of our laboratory showed that DR2418, a key participator of the radio-resistance of Deinococcus radiodurans, binds to the promoter region of dr0997, encoding of cAMP receptor protein (CRP). Proteins of CRP family are very important to Deinococcus radiodurans, and regulate more than 200 genes. Therefore, we focused on this gene in this study.1. The gene dr0997 and its homologue gene dr1646 were successfully knocked out with a kanamycin-resistance gene cassette. We found that the growth of both knockout strains were remarkablely delayed, indicating both genes are required for the metabolism.2. We investigated the survival of the mutant strainsΔDR0997 andΔDR1646 under stresses of DNA-damaging agents. We found that both mutants are sensitive to gamma-radiation, indicating that both genes may be involved in the recovery of D. radiodurans from radiation damage. However, absence of DR0997 only caused a very modest reduction of survival capability when cells were treated UV radiation or H2O2.3. Then, DNA microarray was used to analyze the alteration of transcription profiles when DR0997 is abscent.3033 genes were detected in the microarray, taking up of 95.26% in all predicted ORF. We found that 79 genes were significantly down-regulated, while 28 genes were significantly up-regulated in the mutant compared to these in the wildtype strain. Many of these genes are involved in the synthesis of NADPH and acetyl coenzyme A. These suggest that DR0997 may be involved in the regulation of these pathways.4. To further investigate the biological function of DR0997 protein, the recombinant DR0997 protein was induced and purified here. The gene dr0997 was cloned and resequenced. It has been comfirmed that the ORF of this gene consists of only 612 bp, which is smaller than the predicted one in the Genebank. We also successfully constructed the strain for recombinant DR0997 protein expression and purified the recombinant protein.Taken together, the gene dr0997 plays an important role in the radioresistance of Deinococcus radiodurans Rl, and regulates the carbohydrate metabolism. The work to reveal the underlying mechanisms is proceeding.更多还原