| China has abundant local chicken resources,which provide a lot of materials for the breeding of disease-resistant strains.However,due to the difficulty in identifying the disease-resistant traits,the resources of local chicken used in disease-resistant strains in China are still few.Avian leucosis(AL)is a tumor disease caused by avian leukosis virus(ALV).Because of the high variability of viruses and complex pathogenic mechanism,AL is widespread in our country and leads to a huge economic loss.There is still no effective vaccine or drug which can be for prevention and control of AL.Breeding ALV-J resistant strains is one of the strategies for prevention and treatment of AL.The screening of significant immune markers will greatly promote the development of disease-resistant strains.It has been proved that different breeds have different resistance to AL,which indicates that the genetic background is closely related to the immune resistance of poultry.We selected the Pengxian yellow chicken and the Tibetan chicken as two representative breeds of local chicken to carry out the analysis of genomic differential mutation.We selected two local chicken species of Pengxian yellow chicken and Tibetan chicken to infect ALV-J.Considering that the ALV-J strain NX0101 we used was from Avian broilers,we added Avian broilers as susceptible species to compare with the Pengxian yellow chicken and Tibetan chicken.According to the pathogenic results,the spleen tissues of the three infected groups were detected by DIA at 15 days of age.We analyzed the differentially expressed proteins(DEPs)between different groups and performed functional enrichment analysis and pathway enrichment analysis.According to the omics results,TLR3,TRIF,TRAF3 and TBK1 genes in TRIF dependengt pathway were selected to explore their functions in virus recognition and immune response in cells.The main results are as follows:(1)There are a large number of differential mutations in the genome of Pengxian yellow chicken and Tibetan chicken,which are mainly located in the intergene region and the intron region of the gene.After gene annotation of these differential mutations,it was found that these genes enriched the functions of metabolism,immunity and other aspects.Differences mutation immune related genes are mainly involved in the Notch signaling pathway,the RIG-I like receptor signaling pathways,NOD receptors signaling pathways,Wnt signaling pathways,MAPK signal pathways,toll-like receptors signaling pathways and produce Ig A intestinal immune network which related to immune response.(2)The infectivity to ALV-J of Pengxian yellow chicken,Tibetan chicken and Avian broilers was different.In our results,Tibetan chicken was the most tolerant,while Avian broilers were the most sensitive.A large number of DEPs can be obtained from the three breeds through comparison between different groups.These differential proteins can be used as potential markers of spleen development,potential markers of immune response after infection of ALV-J,and potential markers of infection of ALV-J between different breeds,respectively.(3)After poly I:C induction,the expression levels of TLR3,TRAF3,TBK1,IRF7 and IFN? were significantly increased,which was consistent with previous studies.There was a downregulation of IFN? expression at the beginning of induction,and then the expression level returned to normal.(4)After ALV-J infection,the expression of TLR3,TRAF3,TRIF,TBK1,IRF7,IFN?and IFN? in CEF cells was significantly down-regulated.(5)After the abnormal expression of TLR3,TRIF,TRAF3,TBK1,poly I:C induced the cells.The expression of IFN?,IFN? and IRF7 in the cells were upregulated.The results suggest the IFN? may be reserve cell response to stimulation factor,start to activate when abnormal cells.(6)The cells with abnormal expression of TLR3,TRIF,TRAF3 and TBK1 were stimulated with ALV-J,and the expression of genes related to the signaling pathway in the cells were complicated.In this study,through genomic differential mutation analysis and spleen proteome differential protein analysis after ALV-J infection,the results showed that TLR3 and other differential proteins could be used as immune marker factors in poultry disease-resistant breeding,providing a theoretical basis for the screening of markers.In vitro experiments were conducted to study the expression of TLR3,TRIF,TRAF3 and TBK1 genes in the TRIF-dependent pathway in poly I:C stimulation and ALV-J infection,which enriched the function of the signaling pathway in poly I:C and provided an effective reference for the study of the complex mechanism of cell recognition of ALV-J and initiation of immune response. |
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