【作者】 马婷婷；

【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】 Deinococcus属的细菌对电离辐射都具有很强的抗性。与该类中其它成员不同,Deinococcus geothermalis可以在高达55℃下进行培养,因此D.geothermalis作为遗传工程菌株,被应用于由于放射性物质(如137Cs和90Sr)长期衰变而导致环境温度升高的放射性废物的生物治理工程中。目前,虽然通过比较基因组分析还不能清楚解释Deinococcus. radiodurans或者D.geothermalis强大的DNA修复原理,但是高效的DNA修复蛋白和细胞清除系统都对它们的抗性表型具有重大贡献。研究D. geothermalis的自发突变模式不仅有助于了解嗜温生长条件与其野生突变发生之间的关系,对该菌的抗性机制的研究也有一定的助益。同时,突变率也是研究细菌的适应性和进化机制的重要参数。2003年,Garibyan等利用rpoB/Rifr系统法分析了Escherichia.coli的突变谱。细菌RNA聚合酶的β亚基是一段可以结合在利福平上的保守序列,E. coli经利福平诱导后会导致该序列的氨基酸发生改变。目前,该rpoB/Rifr突变分析系统已被广泛应用于E. coili, Bacillus subtilis, Helicobacter pylori, D. radiodurans, Mycobacterium tuberculosis, Pseudomonas stutzeri, Neisseria species等。该系统能被用于分析D. radiodurans的突变谱主要基于rpoB基因的保守性。2008年,华孝挺等人就利用rpoB/Rifr系统法分析了耐辐射球菌recQ突变株和四突变株uvsE. uvrAl. uvrA2. recQ的突变情况,结果证明recQ参与了耐辐射球菌的紫外损伤修复途径。此外,细菌在有氧代谢过程中产生的ROS (reactive oxygen species)是诱发自发突变的原因之一。耐辐射球菌中的抗氧化酶包括SOD (superoxide dismutase)和CAT(catalase),它们对该菌清除ROS和抵抗氧化压力具有重要作用。因此,抗氧化酶对D. geothermalis的突变率和突变谱可能也存在着一定的影响。在本文中,我们利用rpoB/Rifr系统法分析了D. geothermalis的突变率和突变谱,这是第一次对该菌的突变情况进行研究。我们发现,30℃培养的细菌发生了更多的碱基对替换,且30℃培养时该菌的SOD和CAT酶的活性和基因表达水平也比50℃要低。这表明,当D. geothermalis在30℃培养时其突变率升高的同时会伴随着抗氧化酶的活性的下降。更多还原

【Abstract】 The genus Deinococcus is well known for its extreme resistance to ionizing radiation. Deinococcus geothermalis is distinguished from other Deinococcus species by its ability to grow at temperatures as high as 55℃. The radioresistant and thermophilic bacterium D. geothermalis can potentially be used as a genetic-engineering bacterium for the bioremediation of radioactive wastes in situ, in environments where temperatures are elevated by the decay of long-lived radionuclides, e.g.,137Cs and 90Sr. However, the mechanism underlying its growth in detrimental environments is poorly understood. Comparative genome analysis failed to identify a unique DNA repair systems in D. radiodurans or D. geothermalis, but highly efficient DNA repair proteins and cell-cleaning systems have been suggested to contribute to their resistant phenotypes.The investigation of spontaneous mutations patterns is not only helpful in understanding the relationships between the nature of mutagenesis and thermophilic growth behaviour, but may also reveal the cellular resistance mechanism used by this bacterium The mutation rate is also an important parameter in studying bacterial adaptation and evolutionary mechanisms. An rpoB/Rif system was developed to analyze mutations in Escherichia coli. The resistance of E. coli to rifampin is attributed almost exclusively to amino acid exchanges in a limited region of the P subunit of the RNA polymerase which is involved in rifampin binding, and the related sequence is highly conserved among prokaryotes.The rpoB/Rif mutation analysis system has been widely used, including E. coili,Bacillus subtilis, Helicobacter pylori and D. radiodurans, Mycobacterium tuberculosis, Pseudomonas stutzeri, Neisseria species.The rpoB/Rifr mutational analysis system was extended to analyze mutations in mesophilic D. radiodurans, based on the conservation of the rpoB gene. We have previously used this system to determine the mutation rates of the mutant recQ in D.radiodurans and the four-mutation strain uvsE uvrAl uvrA2 recQ. The results indicated that recQ is involved in the ultraviolet damage repair pathway of D.radiodurans. Moreover, reactive oxygen species (ROS), generated as by-products of aerobic metabolism, are thought to be the inducers of spontaneous mutations of bacteria. The antioxidant enzymes including superoxide dismutase (SOD) and catalase (CAT) plays an important role in the resistance of D. radiodurans to oxidative stress by their ROS scavenging activity. Hence, antioxidant enzyme may have effects on the mutation rates and spectra of D. geothermalis.In this study, we determined the mutation rates and spectra of D. geothermalis using the rpoB/Rifr mutational analysis system. The results provided the first detailed description of mutagenesis in D. geothermalis. More base-pair substitutions (BPSs) were detected from D. geothermalis grown at 30℃than that at the optimal growth temperature (50℃). The activities and gene expression levels of SOD and CAT of D. geothermalis grown at 50℃are significantly higher than those grown at 30℃. These results indicated that the elevation of mutation rate when D. geothermalis grown at 30℃was associated with a decline of antioxidant enzymes in this extreme bacterium.更多还原