【摘要】：目的 Eha是一個影響遲緩愛德華菌(Et)胞內(nèi)生存的轉(zhuǎn)錄調(diào)控因子,本研究有助于揭示其調(diào)控Et抵御酸的分子機(jī)制。方法用ATPase抑制劑洛霉素A1抑制巨噬細(xì)胞的酸化,菌落計數(shù)法比較酸化對野生株和eha基因缺失株胞內(nèi)存活數(shù)目的影響;比較兩種細(xì)菌在酸性應(yīng)激實驗中存活率的差異;構(gòu)建pMP220-P_(eha)LacZ質(zhì)粒,采用β-半乳糖苷酶實驗檢測eha基因的啟動子在不同酸性pH值下和不同培養(yǎng)時間的轉(zhuǎn)錄活性;選擇Eha轉(zhuǎn)錄水平最高的一個酸性pH值和培養(yǎng)時間,分別提取兩種細(xì)菌RNA,進(jìn)行RNA-Sequencing;并用qRT-PCR驗證其結(jié)果。結(jié)果野生株ET13在巨噬細(xì)胞內(nèi)和不同pH酸環(huán)境中的存活率明顯高于缺失株,阻止酸化胞內(nèi)菌數(shù)明顯高于未阻止酸化的胞內(nèi)菌數(shù)(P0.05)。對數(shù)期細(xì)菌pH6.3培養(yǎng)基生長2h,RNA-Sequencing結(jié)果表明:eha基因缺失株轉(zhuǎn)錄水平和野生株相比,147個差異顯著表達(dá)的基因(DEGs)(|log2Ratio|≥1),其中113個上調(diào),34個基因下調(diào),qRT-PCR隨機(jī)抽樣,和RNA-Sequencing表達(dá)趨勢呈強(qiáng)相關(guān)。147個基因采用GO數(shù)據(jù)庫進(jìn)行功能聚類,分成25類,主要涉及細(xì)菌加工、定位、代謝、結(jié)合、催化、運輸、細(xì)胞成份;基于KEGG通路的富集分析,有130個可以富集到55條通路中,包括與氨基酸、核苷酸、脂質(zhì)代謝及鐵的轉(zhuǎn)運等路徑,涉及基因較多的有雙組分系統(tǒng)、ABC轉(zhuǎn)運系統(tǒng)、不同環(huán)境中的微生物代謝和次級代謝產(chǎn)物等路徑。結(jié)論在酸性生存環(huán)境,Eha對Et的轉(zhuǎn)錄組呈多途徑、多基因的適應(yīng)性的全局性調(diào)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，

本文編號：2281831