nrdF基因介导禽致病性大肠杆菌DE205B的抗氧化应激

Gene nrdF meditates tolerance to oxidative stress in avian pathogenic Escherichia coli DE205B

[目的]本文旨在研究前噬菌体phiv205-3片段对禽致病性大肠杆菌DE205B氧化应激耐受力的影响。[方法]利用Red同源重组技术敲除前噬菌体phiv205-3片段,构建前噬菌体片段缺失株DE205BΔphiv205-3,检测野生株和前噬菌体片段缺失株对氧化应激的耐受力;进一步敲除前噬菌体片段中氧化应激耐受相关基因nrdE、nrdF、nrdH、nrdI,构建前噬菌体基因缺失株DE205BΔnrdE、DE205BΔnrdF、DE205BΔnrdH和DE205BΔnrdI,并通过基因序列分析和荧光定量PCR(qPCR)检测这些缺失菌株和野生菌株的氧化应激耐受力。[结果]与野生株相比,前噬菌体片段缺失株DE205BΔphiv205-3氧化应激耐受力下降33%(P<0.05);qPCR检测结果显示,前噬菌体基因nrdF在氧化应激情况下转录水平显著提高(P<0.05);对构建前噬菌体单基因缺失株检测结果显示:与野生株相比,缺失株DE205BΔnrdF在氧化应激环境下的存活率下降21%(P<0.05),而缺失株DE205BΔnrdE、DE205BΔnrdH和DE205BΔnrdI的存活率无显著变化。[结论]前噬菌体片段编码的nrdF基因有助于增强宿主细菌DE205B氧化应激耐受力。

[Objectives]This study was performed to determine the effect of prophage phiv205-3 on oxidative tolerance of avian pathogenic Escherichia coli(APEC).[Methods]Prophage deletion mutant strain was constructed by lambda red homologous recombination method and the oxidative tolerance of the deletion and the wild type strain was studied.For further analysis,the deletion mutants of prophage genes nrdE,nrdF,nrdH and nrdI were constructed,designated as DE205BΔnrdE,DE205BΔnrdF,DE205BΔnrdH and DE205BΔnrdI.Then the oxidative tolerance of these deletion mutants and the wild strain was investigated by gene sequence analysis and flourescent quantitative PCR(qPCR).[Results]Compared to the wild type strain,the oxidative tolerance of the phage-deficient strain significantly decreased by 33%(P<0.05).The results of qPCR showed that the transcription level of nrdF significantly increased(P<0.05)under oxidative stress in the wild-type strain.So the nrdF deletion mutant was constructed,and the results showed that the survival rate of the deletion strain DE205BΔnrdF was reduced by 21%(P<0.05)compared with that of wild type strain in oxidative stress,while the survival rate of DE205BΔnrdE,DE205BΔnrdH and DE205BΔnrdI did not show obvious change.[Conclusions]The nrdF in prophage phiv205-3 enhanced the resistance to oxidative stresses of avian pathogenic E.coli DE205B.