Transcriptome Analysis Of B.pseudomallei Infect RAW264.7

Burkholderia pseudomallei is the causative agent of melioidosis,a kind of tropical disease,and which widely distributed in the tropical regions between the Northern and the Southern latitude 20 degrees.Hainan,Guangdong and Guangxi province in China is the source of melioidosis.Melioidosis was not only listed as class I bioterrorism by the United States CDC,but also listed entry animal quarantine epidemic list in the second category of animal disease in China.Melioidosis has a mortality up to 40%,there is currently no vaccine available.In view of its threat to human public health security,it is of great social significance to accelerate the study of melioidosis.B.pseudomallei(BPHN1)strain was isolated and identified by Gram staining and 16S rRNA gene detection.The linear regression equation between B.pseudomallei and was established by the combination of B.pseudoomallei OD600rm.and plate count.Y=4E+08x+5E+06(R2-0.9836)(0 ? OD600nm ? 1.2).RAW264.7 cells were infected under different multiplicity of infection.The cell model of B.pseudomallei infected RAW264.7 was constructed by analyzing invasion rate and the morphological changes of the host cells.On the basis of B.pseudomallei infected RAW264.7 cell model,the total RNA of RAW264.7 cells infected with B.pseudomallei at 4h,8h,11h was extracted and sequenced by RNA-seq.The expression of different genes at different time points was analyzed by bioinformatics methods.The results showed that obtained 3383,1174 and 2583 differentially expressed genes was obtained from RAW-vs-Infect4,RAW-vs-Infect8 and RAW-vs-Infect11 respectively.And the number of down-regulated genes was higher than that of the up-regulated genes.We also performed pattern clustering,Gene Ontology(GO)functional annotation and pathway enrichment analysis of the gene expression patterns.In the process of B.pseudomallei infecting RAW264.7 cells,26 differentially expressed genes were obtained,among which 7 were significantly enriched.21.3%(1597/7510)differential expression genes could be annotated to 288 metabolic or signaling pathways,among which 85 were significantly enriched(P<0.05).We found that most of the significantly change gene were correlated to immunoinflammatory strong enrichment of the pathway,including Chemokine signaling pathway,TNF signaling pathway,p53 signaling pathway and so on.17 types of gene related to immune were selected and verified by qRT-PCR.The results showed that it were consistent with the trend of RNA-seq analysis,revealing a strong correlation between the qRT-PCR and RNA-seq data(R2=0.9306).CCL9,Ifnbl,TANF-?,Ptgs2,TNF-aip3,Zbp1,CCL5,Ifi202b,NFfkbia,Nfkbie,NMIK1,IRF7 and P21 were up-regulated,while the expression of E2F2,TFDP1,PLK1 and RAD51 were down-regulated.In this study,RNA-seq was used to analyze the changes of mRNA expression levels,B.pseudomallei infected RAW264.7 cells at different time points.The metabolic pathway and molecular regulation network of B.pseudomallei infected RAW264.7 cells were revealed at the molecular level,which provided a new idea for the clinical treatment of melioidosis.