耐辐射球菌基因DRB0099缺失突变株的构建及逆境分析

Construction of the gene drb0099 deleted mutant and adversity analysis in the extremely radioresistant bacterium Deinococcus radiodurans

摘要:

耐辐射球菌(Deinococcus radiodurans R1)有着极强的辐射抗性。研究其抗辐射的机理对于处理放射性废料有着潜在的应用价值。在耐辐射球菌的基因组中, 许多序列的功能未知。其中DRB0099尤为引人注意。将DRB0099缺失突变构建该基因的突变株。对野生型和突变体进行比较后发现, 在正常生长条件下的前期阶段(0~16 h), 突变体生长速度比野生型慢。16 h 以后, 野生型逐渐进入稳定生长期。这时, 突变株的生长速度高于野生型。但是, 野生型的浓度一直高于突变株。表明在DRB0099被删除后, 耐辐射球菌的生长可能受到了阻滞。在紫外线照射的条件下, 尽管野生型随着照射剂量的增加, 存活率越来越低, 但是要比突变体高许多。野生型具有比突变体更强的修复DNA双链断裂的能力。DRB0099可能直接参与了对DNA的修复。突变体对H2O2的敏感程度高于野生型, 表明野生型耐辐射球菌在对抗活性氧保护其蛋白质、DNA或者DNA修复方面具有比突变体更强的功能。在低浓度H2O2处理条件下, 尽管野生型和突变体的存活率都出现下降趋势, 但二者的差值并不大。随着H2O2剂量的增加, 二者的差值越来越大。表明随着活性氧浓度的增加, 蛋白质和DNA损伤的数量增加, 失去DRB0099基因功能的突变体比野生型更容易受到损伤。在紫外线照射处理或者H2O2处理条件下, DRB0099能够保护蛋白质和DNA。

Abstract:

Deinococcus radiodurans R1 has extraordinary resistance to radiation. DRB0099 might play an important role in protecting the bacterium against radiation. To verify the inference, we deleted drb0099 and constructed the mutant. Comparing with the wild type, the mutant grew more slowly in the beginning cultivation stage (0~16 h) under normal conditions. After being cultivated for 16 h, the mutant grew faster than the wild type. The biomass concentration of the wild type was always higher than that of the mutant. The mutant cell’s fission during the growing phase might be blocked. When treated with UV, although Deinococcus radiodurans R1 cultures’ survival fraction was lower with UV treatment time increased, the survival fraction was much higher than that of⊿drb0099. The wild type could repair DNA double strands breaks better than the mutant. The gene drb0099 might directly relate with the DNA repair system. The mutant was more sensitive to H2O2 than the wild type. The wild type could better protect protein and DNA against reactive oxygen species (ROS) or in DNA repair. When treated with low concentration of H2O2, although the survival fraction of both R1 and the mutant decreased, the difference was small. However, with the concentration of H2O2 increased, the difference value increased. The mutant without drb0099 was more easily injured than the wild type with ROS increased. Under UV or H2O2 treatment, DRB0099 could protect protein and DNA from oxidation.