【摘要】 目的:探讨非编码小RNA(sRNA_EsR240)调控迟缓爱德华菌(Edwardsiella tarda,Et)胞内生存和毒力的作用。方法:根据转录组测序,结合生物信息学方法分析ET13菌sRNA。采用RT-PCR检测在不同应激(低酸、营养缺乏、氧化和高盐)条件下sRNA的转录水平,筛选高表达的sRNA;Northern blotting和RACE试验检测EsR240的长度及转录起始和终止位点。采用BLAST软件对EsR240的保守性进行分析;IntaRNA软件预测EsR240靶基因;qRT-PCR检测这些靶基因的表达水平。采用自杀质粒同源重组的方法构建ET13菌的EsR240缺失株和互补株。比较野生株ET13、EsR240缺失株和互补株在胞外不同应激条件下的存活率,及其在巨噬细胞胞内存活能力和对小鼠毒力的差异。结果:根据ET13菌RNA测序的结果,与蛋白库注释基因相比对;比对不上的候选sRNA与sRNA库比对;用软件发现13个具有启动子和ρ-非依赖型终止子新的sRNA,RT-PCR显示其中8个sRNA在各种应激条件下的转录水平不一,EsR128、EsR139和EsR240转录水平均较高。Northern blotting验证了EsR240在Et生长期和稳定期均转录,大小是596 bp。RACE试验确定了EsR240的转录起始和终止位点。BLAST软件分析显示,EsR240在Et中普遍存在,符合sRNA的特征。IntaRNA软件预测EsR240靶基因及qRT-PCR结果显示,EsR240调控大部分预测靶基因的表达,这些靶基因注释可能为三磷酸腺苷结合盒(ATP binding cassette,ABC)-F转运体、FtsH蛋白酶的调控子YccA、Na~+/H~+反向转运体和糖苷水解酶等;在不同条件下,EsR240调控靶基因数目不同。EsR240的缺失明显影响Et在胞外不同应激条件下存活率和胞内生存能力,而且影响Et对小鼠的毒力。结论:EsR240通过调控靶基因影响Et的胞内生存,是一个正调控Et对小鼠毒力的sRNA。更多还原

【Abstract】 Objective: To investigate sRNA to regulate the intracellular survival and virulence of Edwardsiella tarda(Et). Methods: In this study,the analysis of ET13 sRNA was carried out according to the sequence of transcripts and bioinformatics methods. The transcription levels of sRNAs under different stress conditions(low acid,nutrition deficiency,oxidation and high salt) were detected by RT-PCR,and the high expression of sRNA was screened out. The length and transcription initiation and termination sites of EsR240 were found by Northern blotting and RACE assay. BLAST was used to analyze the conservatism of EsR240. IntaRNA software was used to predict EsR240 target genes,and qRT-PCR was used to detect the expression levels of these target genes. The EsR240 deletion strain and complementary strain of ET13 were constructed by suicide plasmid homologous recombination. The survival rate,intracellular viability and virulence of type ET13,its EsR240 deletion strain and complementary strain under different extracellular stress conditions were compared,as well as their ability to survive in macrophages and their virulence to mice. Results: According to the results of RNA-sequencing of ET13 strain,it was compared with the annotated gene of protein library,the candidate sRNA of non-alignment was compared with the sRNA library. 13 new sRNA,RT-PCR with promoters and ρ-independent terminators were found by software,showing that 8 of them had different transcription levels under various stress conditions,and EsR128,EsR139 and EsR240 transcription levels were relatively higher. Northern blotting confirmed that EsR240 was transcribed during the growth and stable stages of Et strain,with a size of 596 bp. The transcription initiation and termination sites of EsR240 were determined by RACE assay. BLAST analysis showed that EsR240 was common in Et strains,which was consistent with the characteristics of sRNA. IntaRNA was used to predict EsR240 target gene by software. qRT-PCR results showed that EsR240 regulated most of the predicted target gene expression. These target genes may be the adenosine triphosphate binding cassette(ATP binding cassette,ABC)-F transporter,the regulator of FtsH protease YccA,Na~+/H~+ reverse transporter and glycoside hydrolase,etc. Under different conditions,the number of target genes regulated by EsR240 was different. The deletion of EsR240 significantly affected the survival rate and intracellular viability of Et under different extracellular stress conditions,and affected the toxicity of Et to mice. Conclusion: EsR240 affects the intracellular survival of Et by regulating the target gene,which is a sRNA that regulates the virulence of Et to mice.更多还原