耐辐射奇球菌铁结合蛋白DRA0258的抗氧化功能

Antioxidant function of the iron binding protein DRA0258 in Deinococcus radiodurans

摘要:

摘要：【目的】通过对极端环境耐受的耐辐射奇球菌Deinococcus radiodurans R1全基因组进行序列比对分析，获得具有铁储备蛋白Ferritin类似功能基序的未知功能蛋白DRA0258，采用分子生物学技术对该蛋白的功能和性质进行了验证和分析。【方法】首先对DRA0258进行克隆表达和纯化，并经络合物显色法测定蛋白上铁结合含量；通过三段连接敲除法构建dra0258突变株，检测突变株在双氧水协迫下的生存率、总抗氧化活性及过氧化氢酶活性；利用实时定量PCR检测突变株内抗氧化酶类及铁转运相关性调控蛋白的基因转录水平。【结果】经体内外蛋白铁含量检测证实DRA0258具有一定的铁结合能力；双氧水生存率实验表明dra0258的缺失导致细胞的抗氧化能力显著下降；过氧化氢酶活性、总抗氧化活性检测及抗氧化酶类的基因转录水平检测证实dra0258基因的缺失导致细胞内一些抗氧化基因转录水平下调，细胞的抗氧化应激系统受到损伤，并影响了一些铁调控网络蛋白的基因转录水平。【结论】本研究证实DRA0258是一种铁结合蛋白，该编码基因的缺失影响胞内铁转运系统并使细胞抗氧化能力下调。

关键词:关键词:耐辐射奇球菌，铁储备蛋白，转录水平，过氧化氢酶活性，总抗氧化活性

Abstract:

Abstract: [Objective] The complete genome of the extreme environmental resistant bacterium Deiococcus radiodurans R1 was analyzed by sequence comparative method and putative ferritin-like protein DRA0258 was screened. Molecular techniques were applied to validate and analyze its function. [Methods] We applied sequence alignment to analyze amino acid sequence of the hypothetical protein DRA0258 and detected its iron binding activity after purification. We used triple-fraction-ligation method to construct dra0258 null mutant and detected its survival rate under H2O2 treatment, catalase activity and total antioxidant capacity, using QRT-PCR to examine the relative transcriptional level change of the antioxidant relative enzymes and iron transport relative proteins. [Results] We confirmed DRA0258 obtained a certain iron binding activity. The survival rate assay with H2O2 treatment suggested that deletion of dra0258 reduced the cellular antioxidant activity of D. radiodurans. The attenuation of catalase activity, total antioxidant capacity as well as the reduction of relative transcriptional levels of antioxidant related genes verified that both the oxidative stress response systems and the iron regulation network were damaged. [Conclusion] This study verified DRA0258 is an iron-binding protein. Deletion of this gene would affect cellular iron transport system and reduce cellular antioxidant capability.

Key words:Keywords: Deinococcus radiodurans, ferritin, transcriptional level, catalase activity, total antioxidant capacity