Expression Profiles Of The SPOP And MyD88 Genes In Chicken Tissue And Their Interaction Proteins Screening And Functional Analysis

Speckletype POZ protein（SPOP）is an E3 ubiquitin ligase adaptor protein,which affect the biological functions of substrate proteins to directly or indirectly regulate a variety of biological processes,such as the development of skeletal,pancreatic and nervous system,X chromosome inactivation,regulation of gene transcription and expression,and DNA repair.Myeloid Differentiation Factor 88（MyD88）not only plays central role in signaling pathways,but also participates in the occurrence and development of various diseases,including inflammatory and autoimmune diseases,neurological and metabolic diseases.It has been shown that SPOP can modify the expression of MyD88 through the ubiquitination non-degradation pathway to inhibit the NF-κB signaling pathway,which in turn leads to pro-inflammatory cytokine production.Currently,the researches on SPOP and MyD88 are mainly focused on human and mice,while th ere is little information about the research of poultry,especially in the aspects of the tissue expression characteristics,subcellular localization and cellular interaction proteins of chicken SPOP and MyD88.Therefore,in this study,the CDS regions of chicken SPOP and MyD88 genes were firstly amplified by RT-PCR and then analyzed by bioinformatics.In addition,the expression characteristics of SPOP and MyD88 genes in different tissues and immune organs of chickens were detected and analyzed by q RT-PCR.Furthermore,co-immunoprecipitation（Co-IP）combined with protein fingerprinting technology were performed to screen and identify the cellular proteins interacting with chicken SPOP and MyD88 proteins,which were then used to perform functional analysis of these cellular interaction proteins.Meanwhile,chicken embryo fibroblasts（DF-1）cells were infected with virulent Newcastle disease virus（NDV）to study the effect of NDV infection on the expression of chicken SPOP and MyD88 genes and the production of inflammatory cytokines.These results will lay foundation for further investigating the growth and development of chicken tissues and the replication of NDV regulated by SPOP and MyD88 interaction.The main results are as follows:1.The CDS regions of chicken SPOP and MyD88 genes were successfully amplified from the c DNA of DF-1 cells,and their CDS sequences were 1125 bp and 900 bp,which encoded 374 and299 amino acids,respectively.The molecular formula of chicken SPOP protein was C₁₈₆₆H₂₉₂₆N₄₉₆O₅₅₉S₂₈,the relative molecular weight and theoretical isoelectric point（p I）of chicken was 42 k Da and 5.58,respectively.The molecular formula of chicken MyD88 protein was C₁₅₀₂H₂₃₉₄N₄₁₀O₄₃₈S₁₈,while the relative molecular weight and p I were 34 k Da and 5.93,respectively.The secondary structures of chicken SPOP and MyD88 proteins were mainly composed ofα-helix and irregular coiling.The three functional structural domains（MATH,BTB-POZ and BACK）of the chicken SPOP protein were more conserved compared to that of human and mammals,whereas the two functional structural domains（Death and TIR）of chicken MyD88protein had multiple amino acid site variants.The results of q RT-PCR showed that SPOP and MyD88 genes were expressed in all chicken tissues at different developmental stages of chicken embryos,but the relative expression was highest in the lung（P<0.01）.In addition,SPOP and MyD88 genes were expressed in the chicken immune organs of different months,and their expressions in spleen showed the tendency of decline at first and then rise,but there was a significant difference in thymus（P<0.05）.2.The recombinant eukaryotic expression vector p CMV-HA-SPOP of chicken SPOP gene was transfected into DF-1 cells,and the cell proteins interacting with chicken SPOP protein were screened and identified by Co-IP and protein spectrum identification technology,and the function analysis of these interaction proteins was carried out.The results showed that the chicken SPOP recombinant protein HA-SPOP was correctly expressed in DF-1 cells and was mainly localized in the nucleus and cytoplasm.A total of 158 cellular proteins interacting with chicken SPOP protein were screened,and they were mainly distributed in the nucleus,cytoskeleton and cytoplasm,which were involved in biological processes such as enzyme activity,nucleic acid binding,and protein processes,as well as signaling pathways such as metabolism,ribosome composition,and biological regulation.The results of protein-protein interaction networks revealed a complex network of interactions among the cellular proteins,of which PSME3,PSMD11 and H2AFZ had obvious interactions with chicken SPOP protein.It was interesting that chicken SPOP protein was found to interact with PSMD11 protein by fluorescence co-localization and Co-IP assays,which changed the subcellular localization of PMSD11 protein in cells.3.The recombinant eukaryotic expression vector p CMV-Myc-MyD88 of chicken MyD88 gene was transfected into DF-1 cells,and the cellular proteins interacting with chicken MyD88 protein were screened and identified by Co-IP and protein spectrum identification technology,and the function analysis of these interaction proteins was performed.The results showed that the recombinant protein Myc-MyD88 was correctly expressed in DF-1 cells and mainly distributed in the nucleus and cytoplasm.A total of 29 cellular proteins interacting with chicken MyD88 were screened.They mainly located at the cytoplasm and cytoskeleton,which played key roles in actin binding,RNA binding and transcription regulation,and participated in signaling pathways of metabolic,biosynthesis,ribosome composition and transcription regulation.According to the analysis of protein interaction network diagram,the correlation coefficient between chicken MyD88protein and SPOP and RPS27A protein was the largest.4.Co-IP experiment demonstrated that chicken SPOP could interact with MyD88 protein.The results of virulent NDV infection experiment showed that the expression of SPOP gene exhibited a downward trend first and then an upward trend,while the expression of MyD88 gene had a gradual upward trend,of which the increase was most obvious at 24 h after NDV infection.q RT-PCR analysis found that the m RNA level of chicken SPOP decreased by 3.6 times at the early stage of NDV infection,and then increased by 12.7 times late in NDV infection.The m RNA level of chicken MyD88 increased 10 times at the early stage of NDV infection,and then increased 2.6 times late in NDV infection.Meanwhile,the results of cells infected with NDV at different time points revealed that the contents of TNF-α,IFN-γand IL-1βgradually increased,while the contents of IL-12,NF-κB,INF-βand IL-6 increased at first and then decreased.The above results provided a basis for further studying the mechanism of SPOP and MyD88 interaction regulating the replication of NDV.