Screening Of Differentially Expressed Genes Against Mycoplasma Ovipneumoniae Based On RNA-Seq

Objective: To establish a MO disease model,Bashbay sheep and crossbred sheep were infected with Mycoplasma ovipneumoniae(MO)in this study.Then their lung were collected and performed transcriptomics analysis by RNA-Seq,which included Differently Expressed Genes(DEGs)analysis,Gene Ontology(GO)enrichment analysis and Kyoto encyclopoedia of genes and genomes(KEGG)enrichment analysis.Some relevant DEGs were screened,which paved the road for the further analysis of the molecular mechanism between host and pathogen.Methods:(1)The comparison of two breeds sheep infected with MO.6 Bashbay sheep and 6 Argali crossbred sheep were artificially infected with MO to observe their clinical symptoms and pathological anatomical changes.The concentration of IL-1?,IL-6,IL-9 and IFN-? in serum were measured by ELISA Kit.Lung tissue sections were made to observe histopathological changes and histopathological scores were made.(2)The establishment of a case model of mycoplasma ovipneumonia: The experimental sheep were seronegative by the MO ELISA diagnostic Kit.Then,the Bashbay sheep and Argali crossed sheep were artificially infected with MO to establish a case model of mycoplasma ovipneumonia,and their clinical symptoms were recorded daily and body temperature was measured every day.The serum was checked by MO ELISA diagnostic Kit.(3)Lung tissue transcriptomics study: The experimental sheep(9 Bashbay sheep,9 Argali crossed sheep)were slaughtered at 0d,4 dpi,14 dpi respectively.RNA was extracted from lung tissues,and RNA-Seq technology was used to detected DEGs with p-values <0.05 and Foldchange values> 2 or <0.667.GO and KEGG enrichment analysis were performed with GOseq package and KOBAS software.Finally,the quantitative PCR was applied to verify the sequencing results.(1)Transcriptome analysis of Bashbay sheep infected with MO compared to uninfected sheep through the above method;(2)Transcriptome analysis of Argali crossed sheep infected with MO compared to uninfected sheep through the above method;(3)Transcriptome analysis of Bashbayi sheep compared to Argali crossed infected with MO through the above method.Results:(1)The results manifested that post-infectious hybrid sheep showed more severe and typical clinical symptoms and pathological anatomical changes than Bashbay sheep.Furthermore,histopathology scores of hybrid sheep were significantly higher than Bashbay sheep.The results of related cytokine tests showed that the concentrations of IL-1?,IL-6,IL-9,and IFN-? in serum of hybrid sheep were significantly higher than those of Bashbay sheep.This showed that Bashbay sheep were anti-MO infection to some extent,and hybrid sheep were more susceptible to MO.(2)The results indicated that the body temperature of Bashbay sheep was elevated transiently,while the body temperature of the crossed sheep was higher for a long time.Argali hybrid sheep showed more typical and more severe clinical symptoms and pathological changes of mycoplasma pneumonia.After the MO infection,the serum of all experimental sheep were serppositive by the MO ELISA diagnostic kit.Those results demonstated that the cases model of mycoplasma ovipneumonia were successfully established.(3)The results of transcriptomics analysis are as following.(1)The transcriptomics results of Bashbay sheep showed 1048 DEGs were screened at 4dpi compared to 0d,575 of which were up-regulated and 473 were down-regulated,and 2823 DEGs were screened at 14 dpi vs 0d,1362 of which were up-regulated and 1461 were down-regulated.KEGG analysis found that the DEGs at 4dpi and 14 dpi compared to 0d were enriched in 245 and 287 pathways respectively,the most closely releted to MO infection of which were TLR signaling pathway(p=2.39 E-17).Two pathways(First: LAMP-TLR2/TLR6-MyD88-MKK6-AP1-IL1B;Second: LAMP-TLR8-MyD88-IRF5-RANTES)were obtained from the figure of TLR signaling pathway that DEGs related with MO infection were mapped to.GO analysis found that the DEGs of Bashbay sheep in different group were enriched to 1580 and 4561 terms,the most closely releted to MO infection of which were described as positive regulation of inflammatory response(1.27E-07)screened via p value.(2)The transcriptomics results of Argali crossed sheep showed that 156 DEGs(up-regulated: 44;down-regulated: 112)were screened at 4dpi compared to 0d,and 367 DEGs(up-regulated: 35;down-regulated: 332)were screened at 14 dpi compared to 0d.KEGG analysis found that the DEGs at 4dpi and 14 dpi compared to 0d were enriched in 109 and 150 pathways respectively,the most closely releted to MO infection of which were Primary immunodefenciency(p=2.98E-09).GO analysis found that the DEGs of Bashbay sheep in different group were enriched to 497 and 928 terms,the most closely releted to MO infection of which were described as impaired ciliary movement(p=1.72E-19)screened via p value,and the DEGs enriched to this term included: DNAH11,DNAI2,CFAP221,HYDIN,and FOXJ1.(3)The transcriptomics results of two breeds sheep showed that 212 DEGs(up-regulated: 146;down-regulated: 66)were screened from Bashbay sheep compared to crossed sheep at 4dpi,and 311 DEGs(up-regulated: 158;down-regulated: 153)were screened from Bashbay sheep compared to crossed sheep at 14 dpi.Combined with GO and KEGG analysis,some interesting DEGs associated with MO infection were obtained,such as SPLUC1(BPIFA1),SP-A(SFTPA-1),P2X7 R,DQA,SOSC1,HO-1(HSP32),NF-?B,NF-E2.Conclusion: In this study,some DEGs and pathways relevant with MO infection were screened out,which paved the road for the further analysis of the molecular mechanism between host and pathogen.