全转录组揭示化合物M6对谷氨酸棒状杆菌基因表达的影响  

Whole Transcriptome Reveals the Effect of Compound M6 on Gene Expression of Corynebacterium Glutamicum

作　　者：杨丽[1] 唐静 李晓雨 梁彤 秦闫燕 林源[1] 杨延辉[1] YANG Li;TANG Jing;LI Xiaoyu;LIANG Tong;QIN Yanyan;LIN Yuan;YANG Yanhui(Ningxia Key Laboratory of Prevention and Control of Common Infectious Diseases,School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学基础医学院,宁夏回族自治区常见传染病防治重点实验室,银川750004

出　　处：《宁夏医科大学学报》2022年第7期702-711,共10页Journal of Ningxia Medical University

基　　金：国家自然科学基金项目(81560604);宁夏回族自治区重点研发计划项目(2021BEG03072)。

摘　　要：目的探究化合物M6所干预的谷氨酸棒状杆菌(Cg)基因表达网络,以期为深入研究M6的抗菌作用机制提供候选基因。方法利用Chemdraw 18.0软件展示M6的分子结构,二倍稀释法测定M6在多种微生物中的抗菌谱;模式菌选用与MTB细胞壁相似的Cg,刃天青法测定M6的最低抑菌浓度(MIC);用转录组测序分析M6对Cg基因表达的影响,实验组和对照组分别用1/2 MIC浓度的M6和相同浓度的DMSO处理Cg 24 h,每组设3次生物学重复;Trizol法提取总RNA并进行转录组测序;利用Bewtie 2软件分析差异表达基因(DEGs),用GO功能和KEGG通路分析DEGs富集及功能,STRING分析DEGs表达蛋白质之间的互作关系。结果大部分G^(-)菌和真菌对M6耐受,G^(+)菌对M6较敏感,特别是MTB最敏感(MIC为0.0625μg·mL-1);分子结构显示M6具备有限的分子构象。选用对M6敏感的Cg(MIC为0.5μg·mL^(-1))为模式菌进行后期的转录组测序,测序结果显示,M6干预使Cg差异表达的mRNA共452个(P≤0.05,|log2FC|≥1.0),其中346个高表达,106个低表达;差异倍数4倍以上的基因有40个,包括高表达的ctpA、ftn、dps、cmr、trxC和clpB等34个基因及6个低表达基因bioB、bioA、gip、hI_0095、DNA81和DNA345。GO功能富集分析显示,DEGs主要富集在核糖体结构组分、r RNA结合和核蛋白复合体等与核糖体相关的功能途径。KEGG通路功能分析显示,核糖体及阳离子抗菌肽耐药性通路中的DEGs富集程度最显著,以rplL、rplJ、rplQ、rpmD、ftn、dps、cmr、trxC、bioA和ugpE等差异性最大。在蛋白互作网络图中,位于中心节点的蛋白质所对应的基因分别为rplQ、rplJ、rspD、rpmD、rpoA、rplE和rplL等。结论M6具备显著的抗MTB活性。在调控Cg的基因表达方面,主要影响了Cg的核糖体及阳离子抗菌肽耐药性通路相关多个基因的表达。Objective To explore the gene expression network of corynebacterium glutamicum interfered by compound M6 to provide candidate genes for further study of M6 antibacterial mechanism.Methods The molecular structure of M6 was displayed by Chemdraw18.0 software,and the anti-microbial spectrum of M6 was measured by double dilution method.Cg similar to MTB cell wall was used for model bacteria,the minimum inhibitory concentration(MIC)of M6 was determined by using resazurin.Transcriptome sequencing was used to analyze the effect of M6 on Cg gene expression.The experimental group and the control group were treated with1/2 MIC of M6 and DMSO at the same concentration for 24 h,respectively.Results were generated from three biological replicates.Trizol was used to extract total RNA,and transcriptome sequencing was performed.Bewtie2 software was used to analyse different expression genes(DEGs).DEGs enrichment and function were annotated by GO function and KEGG pathway analysis.STRING analyzed the interaction between DEGs expressed proteins.Results The most of G-bacteria and fungi were resistant to M6,and G+bacteria were sensitive to M6,especially MTB(MIC was 0.062 5 μg·m L^(-1)).Molecular structure shown that M6 has limited molecular conformation.Cg was selected as model bacteria for later transcriptome sequencing according to sensitivity(MIC was 0.5 μg·m L^(-1)).As shown in sequencing results,a total of 452 m RNA were significantly differential expression in Cg(P≤0.05 and |log2FC|≥1.0),and there were 346 DEGs with high expression and106 DEGs with low expression.There were 40 genes with more than 4 times difference,including 34up-regulated genes such as ctp A,ftn,dps,cmr,trx C and clp B,and 6 down-regulated genes including bio B,bio A,gip,h I_0095,DNA81 and DNA345.GO functional enrichment analysis showed that DEGs were mainly involved in functional pathways associated with ribosomes such as ribosomal structural components,r RNA binding and riboprotein complexes.KEGG pathway analysis showed that DEGs enrichment was mos

关 键 词：转录组 结核分枝杆菌 谷氨酸棒状杆菌 化合物M6 核糖体 

分 类 号：R37[医药卫生—病原生物学] R91[医药卫生—基础医学]