耐辐射异常球菌osmC与ohr基因突变株的构建及功能研究

Construction and Functional Analysis of osmC Gene Disruptant and ohr Gene Disruptant in Deinococcus radiodurans R1

摘要: 渗透胁迫诱导蛋白OsmC与有机氢过氧化物抗性蛋白Ohr都属于OsmC超家族抗氧化物酶，在细菌抵御有机或无机过氧化反应的过程中起重要作用。耐辐射异常球菌(D. radiodurans R1)中osmC基因(DR_1538)与ohr基因(DR_1857)分别编码了OsmC超家族中的这两种抗氧化物酶。运用体外三段融合PCR方法和线性转化方法，首次构建了壮观霉素抗性完全缺失突变株ΔosmC和卡那霉素抗性完全缺失突变株Δohr。对ΔosmC、Δohr突变株和野生型菌株分别都进行CHP(异丙苯基过氧化氢)、H₂O₂ 和NaCl胁迫处理，结果显示，与野生型相比，ΔosmC突变株对H₂O₂异常敏感，而Δohr突变株对CHP更敏感，两个突变株对NaCl胁迫都没有大的变化。QRT-PCR结果显示在H₂O₂和NaCl胁迫下野生型菌株中osmC基因都上调，其中H₂O₂胁迫下基因上调水平显著；在CHP和NaCl胁迫下野生型菌株中ohr基因基因上调大约2倍左右。根据结果推测同属于OsmC超家族的两个过氧化物酶选择底物的偏好性与作用方式可能并不一样，OsmC蛋白可能主要是以无机过氧化物作为底物，而Ohr蛋白可能主要是以有机过氧化物为底物。 The osmotic stress induced protein (OsmC) and organic hydroperoxide resistance protein (Ohr) were two antioxidant enzymes which play an important role in bacteria defensing organic or inorganic peroxidation. The osmC gene (DR_1538) and ohr gene (DR_1857) of Deinococcus radiodurans (D. radiodurans R1) encoded the two antioxidant enzymes in the OsmC superfamily. Fusion PCR of three sections in vitro and linear transformation method were used to firstly construct the spectinomycin-resistant mutant stain (ΔosmC) and the kanamycin-resistant mutant strain (Δohr). The ΔosmC, Δohr mutants strain and wild-type strain were treated with CHP (cumene hydroperoxide), H₂O₂ and NaCl stress treatments. The result showed that ΔosmC mutant is extremely sensitive to H₂O₂ compared with the wild type, while Δohr mutant is more sensitive to CHP and the two mutant strains had no changes after NaCl stress. QRT-PCR results displayed that the osmC gene of the wild-type strain were up-regulated in both H₂O₂ and NaCl stress treatments, and the osmC gene in H₂O₂ stress levels was significantly upregulated. The ohr genes in wild-type strain after CHP and NaCl stress treatments upregulated approximately 2-folds. From above, it was presumed that there may be different at the preference of two peroxidases in OsmC superfamily selecting substrates and the mode of those interacting with their substrate; OsmC proteins may mainly use inorganic peroxide as its substrate and Ohr protein may be primarily based on the organic peroxide as its substrate.