| Salmonella Enteritidis(SE),a highly transmissible food-borne zoonosis bacteria,can cause acute inflammation of the digestive tract by invading the digestive system of poultry and even lead to death,which has caused significant economic losses to the poultry industry.At the same time,the pathogen can affect human health by contaminating animal products.In this study,Salmonella enteritidis-negative Shouguang chickens were divided into two groups: 40 treated chickens and 30 control chickens.On the 3rd day after oral administration of Salmonella Enteritidis,caecum was collected,RNA was extracted and mixed into 3 RNA pools in each group(6 pools in the treated group and the control group)for mRNA and miRNA sequencing,and correlation analysis was performed to explore the regulation mechanism at two different levels.The results showed that :(1)The sequencing results were 26.54 G of raw data,4.42 G of sample data per RNA library,and 29 million reads were detected.A total of 19359 mRNAs were identified,1760 mRNAs were significantly expressed(|log2foldchange| ≥ 1,P<0.05),1046 mRNAs were up-regulated and 714 mRNAs were down-regulated.A total of 821 miRNAs were identified,174 miRNAs were significantly expressed(|log2foldchange| ≥ 1,P<0.05),of which,100up-regulated miRNAs and down-regulated 74 miRNAs.(2)Functional enrichment of differentially expressed mRNAs and miRNAs were consistent,and mainly enriched in immune,development and metabolism.The up-regulated mRNA was mainly enriched in immune response,intestinal immune network producing immunoglobulin A,Toll-like receptor signaling pathway and other immune related processes(P<0.05).Down-regulated mRNAs were mainly enriched to tissue development,WNT signaling pathway and cytochrome P450 metabolism(P<0.05).Up-regulated miRNA were mainly enriched to cell apoptosis,protein metabolism regulation,PPAR signaling pathway and other growth and metabolism pathways(P<0.05)Down-regulated miRNAs were enriched into TLR signaling pathway(P<0.05).(3)We analyzed mRNA and miRNA with opposite trends of differential expression and found that 169 up-regulated mRNA were overlapped with target genes of down-regulated miRNAs,and enriched in immune-related Biological Processes(P<0.05),such as immune response,inflammatory response,bacterial defense response,Toll-like receptor signaling pathway.221 down-regulated mRNAs were overlapped with target genes of up-regulated and mainly enriched in the growth,development and metabolic process of cell morphology regulation,Wnt signal pathway,drug metabolism-cytochrome P450,retinol metabolism(P<0.05).(4)The expression of CENPM and TLR1 A in cecum was not significantly different between treated and control groups.The expression of PGM1,TCF7L2 and NEK7 were0.65,0.66 and 0.18,respectively.The difference of SCNN1 A expression was 16.29,which was consistent with the result of sequencing.miR-15 a,miR-20b-5p and miR-16-1-3p were up-regulated by 2.06,1.65 and 1.46 times,respectively,and miR-1662 was down-regulated by0.41 times,which was consistent with the sequencing results.(5)DF1 cells were co-transfected with the constructed SCNN1 A dual Luciferase reporter vector and miR-20b-5p mimics/NC.The results showed that the values of wild-type vector and miR-20b-5p mimics were significantly lower than other four groups(P < 0.01).The binding site of wild-type vector and miR-20b-5p was the seed sequence of SCNN1 A 3’UTR region on the vector,there is a direct targeting relationship between gga-miR-20b-5p and SCNN1 A.The results showed that Salmonella enteritidis infection caused significant changes in mRNA and miRNA expression levels in Shouguang chickens.Up-regulated miRNA and down-regulated mRNA co-regulate growth and metabolism,down-regulated miRNA and up-regulated mRNA co-regulate immune process.Toll-like receptor signal pathway,WNT signal pathway,PPAR signal pathway,Fatty acid metabolism pathway,glucose metabolism pathway,PGM1,TCF7L2,SCNN1 A,NEK7,miR-15 a,miR-20b-5p,miR-1662 miRNA play an important role in SE infection. |
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