基于RNA-seq分析Eha调控迟缓爱德华菌抵抗酸化的作用
发布时间：2018-10-19 17:27
【摘要】：目的 Eha是一个影响迟缓爱德华菌(Et)胞内生存的转录调控因子,本研究有助于揭示其调控Et抵御酸的分子机制。方法用ATPase抑制剂洛霉素A1抑制巨噬细胞的酸化,菌落计数法比较酸化对野生株和eha基因缺失株胞内存活数目的影响;比较两种细菌在酸性应激实验中存活率的差异;构建pMP220-P_(eha)LacZ质粒,采用β-半乳糖苷酶实验检测eha基因的启动子在不同酸性pH值下和不同培养时间的转录活性;选择Eha转录水平最高的一个酸性pH值和培养时间,分别提取两种细菌RNA,进行RNA-Sequencing;并用qRT-PCR验证其结果。结果野生株ET13在巨噬细胞内和不同pH酸环境中的存活率明显高于缺失株,阻止酸化胞内菌数明显高于未阻止酸化的胞内菌数(P0.05)。对数期细菌pH6.3培养基生长2h,RNA-Sequencing结果表明:eha基因缺失株转录水平和野生株相比,147个差异显著表达的基因(DEGs)(|log2Ratio|≥1),其中113个上调,34个基因下调,qRT-PCR随机抽样,和RNA-Sequencing表达趋势呈强相关。147个基因采用GO数据库进行功能聚类,分成25类,主要涉及细菌加工、定位、代谢、结合、催化、运输、细胞成份;基于KEGG通路的富集分析,有130个可以富集到55条通路中,包括与氨基酸、核苷酸、脂质代谢及铁的转运等路径,涉及基因较多的有双组分系统、ABC转运系统、不同环境中的微生物代谢和次级代谢产物等路径。结论在酸性生存环境,Eha对Et的转录组呈多途径、多基因的适应性的全局性调控。
[Abstract]:Objective Eha is a transcriptional regulator affecting the intracellular survival of (Et). This study is helpful to reveal the molecular mechanism of its regulation of acid resistance of Et. Methods ATPase inhibitor rotomycin A1 was used to inhibit macrophage acidification, and colony counting was used to compare the effects of acidification on the number of intracellular survival of wild and eha gene deficient strains, and to compare the difference of survival rate between the two bacteria in acid stress test. PMP220-P_ (eha) LacZ plasmid was constructed, and 尾 -galactosidase assay was used to detect the transcriptional activity of the promoter of eha gene under different acidic pH values and at different incubation times, and to select the highest Eha transcription level of acid pH value and culture time, and 尾 -galactosidase assay was used to detect the transcriptional activity of eha gene promoter. Two kinds of bacterial RNA, were extracted for RNA-Sequencing; and qRT-PCR was used to verify the results. Results the survival rate of wild strain ET13 in macrophages and in different pH acid environment was significantly higher than that of the missing strain, and the number of intracellular bacteria preventing acidification was significantly higher than that of non-acidification bacteria (P0.05). The results of RNA-Sequencing showed that 147 differentially expressed genes (DEGs) (log2Ratio 鈮，
本文编号：2281831
[Abstract]:Objective Eha is a transcriptional regulator affecting the intracellular survival of (Et). This study is helpful to reveal the molecular mechanism of its regulation of acid resistance of Et. Methods ATPase inhibitor rotomycin A1 was used to inhibit macrophage acidification, and colony counting was used to compare the effects of acidification on the number of intracellular survival of wild and eha gene deficient strains, and to compare the difference of survival rate between the two bacteria in acid stress test. PMP220-P_ (eha) LacZ plasmid was constructed, and 尾 -galactosidase assay was used to detect the transcriptional activity of the promoter of eha gene under different acidic pH values and at different incubation times, and to select the highest Eha transcription level of acid pH value and culture time, and 尾 -galactosidase assay was used to detect the transcriptional activity of eha gene promoter. Two kinds of bacterial RNA, were extracted for RNA-Sequencing; and qRT-PCR was used to verify the results. Results the survival rate of wild strain ET13 in macrophages and in different pH acid environment was significantly higher than that of the missing strain, and the number of intracellular bacteria preventing acidification was significantly higher than that of non-acidification bacteria (P0.05). The results of RNA-Sequencing showed that 147 differentially expressed genes (DEGs) (log2Ratio 鈮，
本文编号：2281831
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