Effects Of DEV Infection On Related Genes Of JAT-STAT Signaling Pathway In Duck

Duck enteritis virus is a threat to the healthy development of waterfowl breeding one of the important pathogens can cause ducks and geese and other water birds died,But the mechanism of infection is not yet fully understood.The DEV infected ducks were used to take high-throughput sequencing of their spleens.the data were analyzed and the JAK-STAT signaling pathway was significantly different.JAK-STAT signaling pathway is an important pathway for cytokine signaling and is activated by various cytokines,growth factors,and receptors.which is widely involved in cell proliferation,differentiation,apoptosis,immune regulation,angiogenesis,oxidative stress,inflammatory response and tumor formation and other processes.Based on the previous study,by the fluorescence quantitative PCR and ELISA were used to verify the expression of genes and proteins associated with JAK-STAT signaling pathway after DEV infection.In order to provide important theoretical data for the elucidation of the pathogenesis of DEV.1.Cloning and Bioinformatics Analysis of Genes Associated with JAK-STAT Signaling PathwayIn order to study and analyze the JAK2、STAT3 and SOCS1 gene of Sansui duck,the JAK2、STAT3 and SOCS1 gene was amplified,cloned and sequenced using bioinformatic softwares and methods,The secondary structure,conserved domain analysis,transmembrane domain,signal peptide and tertiary structure were predicted.The results showed that the length of JAK2 gene was 3393 bp,encoding1130 amino acids.The homology of this nucleotide sequences reached 94.5% with gallus,and the homology of amino acid was 97.6%.The results of phylogenetic tree analysis indicated that there was a close relationship between Sansui duck and gallus.The secondary structure of the encoding protein is based with random curl,a1 phahelix and extended strand region,with a N-terminal FERM domain and SH2 domain,It wasn’t found the transmembrane domains and the signal peptide,and with the curved spiral tertiary structure.The results showed that the length of STAT3 gene was 2112 bp,encoding 703 amino acids.The homology of this nucleotide sequences reached 91.6% with gallus,and the homology of amino acid was 98.8%.The results of phylogenetic tree analysis indicated that there was a close relationship between Sansui duck and gallus.The secondary structure of the encoding protein is based with random curl,a1 phahelix and extended strand region,with a alpha domain and SH2 domain,It wasn’t found the transmembrane domains and the signal peptide,and with the curved spiral tertiary structure.The results showed that the length of SOCS1 gene of Sansui duck was 624 bp,which encode 207 amino acids.The homology of this nucleotide sequences reached 97.6% with that of anser,and the homology of amino acid was 99.5%.The secondary structure of the encoded protein is based with random curl,a1 phahelix and extended strand region,with a central SH2 domain and a SOCS box,and not found the transmembrane domains and the signal peptide,and with the curved spiral tertiary structure.These results laid the foundation for the study of the effect of DEV infection on the JAK-STAT signaling pathway in Sansui ducks.2.Changes of JAK-STAT Pathway Related Genes in Duck Tissues of infected DEVThe specific primers were designed based on the sequences of JAK2,STAT3 and SOCS1 genes obtain above.After establishment a real-time fluorescent quantitative PCR method,the healthy Sansui ducks were selected and infected DEV.Duck tissue samples collected at different time of infection,tissue total RNA extraction.Detection of JAK2,STAT3 and SOCS1 gene transcription levels in duck tissues by real-time fluorescence quantitative PCR.The results showed that: using JAK2,STAT3 and SOCS1 positive recombinant plasmids as templates,the real-time fluorescence quantitative PCR amplification curve has good parallelism,the melting curve decreased slowly with the dilution gradient,with a single melting peak,no non-specific amplification curve and primer dimer,the amplification efficiency of PCR method E,linear regression R2 and slope K are all close to the ideal value.The results show that the q-PCR method of JAK2,STAT3 and SOCS1 genes has been successfully established in this experiment.Based on the established q-PCR method,DEV infected Ducks from 12 h to 96 h,The transcription level of JAK2 gene in heart,spleen,brain and muscle decreased first and then increased.In the liver,lung,kidney,thymus,duodenum and bursa generally increased first and then decreased.The transcriptional level of STAT3 gene in heart,liver,bursa and muscle decreased first and then increased.In the spleen,lung,kidney,brain,thymus and duodenum increased first and then decreased.the transcriptional level of SOCS1 gene in heart,lung,kidney,brain,duodenum,bursa and muscle showed a general upward trend.In the liver and brain generally increased first and then decreased.In the spleen decreased first and then increased.3.Expression of JAK-STAT Pathway Related Protein in Duck Tissues of infected DEVCentrifugation supernatant of duck tissue samples collected at different time after infected of DEV,The expression levels of JAK2,STAT3,and SOCS1 proteins were detected using commercially available ELISA kits.The results showed that:DEV infected Ducks from12 h to 96 h,the expression of JAK2 and STAT3 protein in heart,liver,spleen,kidney,lung,brain,thymus,duodenum,bursa and muscle were lower than those in normal control group.the expression of SOCS1 protein in heart,liver,spleen,kidney,lung,brain,thymus,duodenum,bursa and muscle was higher than that in normal control group.Conclusion1.The study successfully cloned JAK2 、STAT3 and SOCS1 gene of Sansui duck.The full-length JAK2 gene,STAT3 gene and SOCS1 gene were 3393 bp,2112bp and 624 bp,respectively,encoding 1130,703 and 207 amino acids,respectively2.DEV infection has different effects on the transcription levels of JAK-STAT signaling pathway related genes JAK2,STAT3 and SOCS1 in duck tissues.3.DEV infection could down-regulate the expression of JAK2 protein and STAT3 protein in JAK-STAT signaling pathway and up-regulate the expression of SOCS1 protein in duck tissues.