中国畜牧兽医2024，Vol.51Issue(7)：3077-3085,9.DOI:10.16431/j.cnki.1671-7236.2024.07.032
gltC基因缺失对单增李斯特菌10403S抗酸应激能力的影响
Effect of gltC Gene Deletion on Acid Stress Resistance of Listeria monocytogenes 10403S
摘要
Abstract
[Objective]The experiment was aimed to construct a strain of Listeria monocytogenes with gltC gene deletion and elucidate the effect of gltC gene on the acid stress resistance of Listeria monocytogenes.[Method]The effect of glutamic acid on the survival ability of the 10403S was determined by acid stress test with or without the addition of exogenous glutamic acid under lethal acidic conditions.The homologous recombination method was used to construct a strain with a deletion of gltC gene.The effect of gltC gene deletion on the growth capacity of the strain was determined by growth curves.The effect of gltC gene deletion on the acid stress capacity of Listeria monocytogenes was determined by the acid stress survival assay.The effect of the gltC gene deletion on the expression level of GadD2 protein was determined by Western blotting.By constructing the gfp reporter plasmid carrying the gltBD promoter region,the effect of gltC gene deletion on the transcription level of the gltBD promoter region was measured under acidic and neutral conditions.[Result]Acid stress results showed that adding exogenous glutamic acid could extremely significantly improve the survival ability of strain 10403S under acidic conditions at pH 2.5(P＜0.01).PCR results showed that the deletion strain 10403S△gltC was successfully constructed.The growth curve results showed that gltC gene deletion did not affect the growth ability of Listeria monocytogenes.The results of acid stress showed that the growth ability of the deletion strain 10403S△gltC was weaker than that of the parental strain 10403S in a lethal acidic environment at pH 2.5(P＜0.01).The results of the Western blotting showed that the deletion of gltC gene did not affect the expression level of GadD2 protein(P＞0.05).PCR results showed that gfp with gltBD promoter region reported plasmid was successfully constructed.GFP fluorescence analysis showed that the deletion of gltC gene extremely significantly decreased the transcription level of the gltBD promoter region under both acidic and neutral conditions(P＜0.01).[Conclusion]Deletion of gltC gene did not affect the normal growth of the Listeria monocytogenes 10403S,but the deletion strain 10403S△gltC reduced resistance to acid stress and decreased the level of transcription in the gltBD promoter region,without affecting the level of GadD2 protein expression.关键词
单增李斯特菌/谷氨酸合酶调控因子/gltC基因/抗酸应激/报告基因Key words
Listeria monocytogenes/glutamate synthase regulatory factor/gltC gene/acid stress resistance/reporter gene分类
农业科技引用本文复制引用
杨雨婷,韩旭,肖金华,方小伟,梁雄燕,刘晶,方春..gltC基因缺失对单增李斯特菌10403S抗酸应激能力的影响[J].中国畜牧兽医,2024,51(7):3077-3085,9.基金项目
湖北省教育厅科学技术研究项目(Q20221302) （Q20221302）
湖北省自然科学基金项目(2021CFB173) （2021CFB173）
