谷氨酸脱氢酶gdhA基因缺失对单增李斯特菌抗氧化应激的影响

doi:10.16431/j.cnki.1671-7236.2024.09.007

摘要/Abstract

摘要： 【目的】旨在阐明谷氨酸脱氢酶gdhA基因对单增李斯特菌抗氧化应激能力的影响，并探明glnR基因对gdhA基因的调控作用。【方法】采用同源重组技术构建单增李斯特菌gdhA基因缺失株；通过生长曲线测定亲本株10403S、缺失株10403S-ΔgdhA和回补株10403S-CΔgdhA菌株的生长能力；利用抗氧化应激存活试验检测gdhA基因缺失对单增李斯特菌抗氧化应激能力的影响。诱导GdhA蛋白表达并纯化，制备兔多克隆抗体，并通过Western blotting检测抗体特异性。通过构建携带gdhA基因启动子区域的GFP报告质粒，测定氧化应激条件下glnR基因缺失对gdhA基因转录水平的影响。使用Western blotting检测氧化应激条件下glnR基因缺失对GdhA蛋白表达水平的影响，并检测glnR基因缺失对菌株存活能力的影响。【结果】生长曲线结果显示，gdhA基因缺失不影响单增李斯特菌在正常条件下的生长能力。在20 mmol/L H₂O₂条件下，缺失株10403S-ΔgdhA存活能力极显著高于亲本株10403S(P＜0.01)。Western blotting结果显示，GdhA兔多克隆抗体制备成功。GFP荧光分析结果显示，glnR基因缺失后，gdhA基因转录水平极显著上调(P＜0.01)；在氧化应激条件下glnR基因缺失后GdhA蛋白表达水平极显著升高(P＜0.01)；glnR基因缺失菌株存活能力显著下降(P＜0.05)。【结论】谷氨酸脱氢酶编码基因gdhA缺失不影响单增李斯特菌10403S正常生长，但显著提高其氧化应激条件下的存活能力，gdhA基因转录水平及GdhA蛋白表达水平受glnR基因负调控。

关键词: 单增李斯特菌; 谷氨酸脱氢酶; gdhA基因; 氧化应激; glnR基因

Abstract: 【Objective】 The aim of this study was to elucidate the effect of glutamate dehydrogenase gdhA gene on the antioxidant stress ability of Listeria monocytogenes, and explore the regulatory effect of glnR gene on gdhA gene. 【Method】 The gdhA gene deletion strain of Listeria monocytogenes was constructed by homologous recombination technique.The growth ability of parental strain 10403S, deletion strain 10403S-ΔgdhA and complementation strain 10403S-CΔgdhA were measured by growth curves.The antioxidant stress survival test was used to detect the effect of gdhA gene deletion on the antioxidant stress ability of Listeria monocytogenes.The rabbit polyclonal antibody was prepared by inducing the expression and purification of GdhA protein, and the specificity of the antibody was detected by Western blotting.GFP reporter plasmid carrying gdhA gene promoter region was constructed to determine the effect of glnR gene deletion on gdhA gene transcription level under oxidative stress.Western blotting was used to detect the effect of glnR gene deletion on the expression level of GdhA protein under oxidative stress, and to detect the effect of glnR gene deletion on the survival ability of strains. 【Result】 The results of growth curve showed that the deletion of gdhA gene did not affect the growth ability of Listeria monocytogenes under normal conditions.The survival ability of deletion strain 10403S-ΔgdhA was extremely significantly higher than that of the parental strain 10403S under the condition of oxidative stress with 20 mmol/L H₂O₂ (P＜0.01).Western blotting detection indicated that the rabbit polyclonal antibody to GdhA was successfully produced.GFP fluorescence analysis showed that the transcription level of the gdhA gene was extremely significantly increased after the deletion of glnR gene (P＜0.01).Under oxidative stress, the expression level of GdhA protein was extremely significantly increased after glnR gene deletion (P＜0.01).glnR gene deletion significantly decreased the viability of strains (P＜0.05). 【Conclusion】 The deletion of gdhA gene, which encoded for glutamate dehydrogenase, did not affect the normal growth of Listeria monocytogenes parental strain 10403S, but significantly increased the survival ability under the oxidative stress.The transcription level of gdhA gene and the expression level of GdhA protein were negatively regulated by glnR gene.

Key words: Listeria monocytogenes; glutamate dehydrogenase; gdhA gene; oxidative stress; glnR gene

中图分类号:

S852.61⁺5

田篮鑫, 杨雨婷, 秦祎, 张政, 田光明, 方春. 谷氨酸脱氢酶gdhA基因缺失对单增李斯特菌抗氧化应激的影响[J]. 中国畜牧兽医, 2024, 51(9): 3771-3779.

TIAN Lanxin, YANG Yuting, QIN Yi, ZHANG Zheng, TIAN Guangming, FANG Chun. Effects of Glutamate Dehydrogenase gdhA Gene Deficiency on Antioxidant Stress in Listeria monocytogenes[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 3771-3779.

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