| Globally,chicken products are the most consumed food of animal origin.The chicken and egg products industry has seen tremendous success over the past few decades,with rapidly increasing consumption of chicken,which is considered a healthy alternative to red meat and other protein sources.However,stress-induced immunosuppression remains a hidden threat currently leading to immune failure and disease outbreaks in chicken flocks,causing huge economic losses to the modern poultry industry.How to scientifically and effectively improve the ability of poultry to cope with stress is a production problem that needs to be solved urgently in the poultry industry.In this study,based on the dexamethasone-induced chicken stress immunosuppression model from a systematic point of view,the RNA-seq technology was used to construct the spleen m RNA expression profile of chicken stress immunosuppression,and the relative quantification of the proteome using isobaric isotope multi-tags Using Tandem mass tags(TMT)to construct the differential proteomic profile of the spleen of chicken stress-induced immunosuppression,the two sets of biological data were combined to screen and identify the key candidate gene GBP4 L that stress affects immune function,and verified at the cellular and individual levels.GBP4 L gene,to study its immune regulation function,lay a foundation for the analysis of the molecular regulation mechanism of poultry stress affecting immune function,and provide a theoretical basis for scientific prevention and control of poultry stress response.The specific research results are as follows:Part I Analysis of the response mechanism of dexamethasone-induced splenic immunosuppression in chicks by transcriptome technology515 differentially expressed genes(DEGs)were identified by RNA-seq in the spleen of dexamethasone-induced chicken stress immunosuppression model.Cytokine-cytokine receptor interaction signaling pathway is strongly activated in stress-induced immunosuppression.Through GO,KEGG and PPI analysis,we found that IL8L2,IL8L1,CXCR5,CCR8 L,CCR9 and CCR10 may play an important role in stress-induced immunosuppression through cytokine-cytokine receptor interaction signaling pathway.GBP4 L may participate in the stress immunosuppressive response by interacting with ISG12-2 and MOV10 genes.TNFRSF13 B and TNFRSF13 C may play an important role in stressinduced immunosuppression through cytokine-cytokine receptor interaction signaling pathway and intestinal immune network signaling pathway producing Ig A.These results provide a valuable basis for further study of the molecular mechanism of stress-induced immunosuppression,and also provide new insights into better understanding the interaction between genes related to stress-induced immunosuppression.Part II Combined analysis of proteome and transcriptome to analyze the molecular mechanism of stress-induced immunosuppression in chicken spleenTMT-based analysis characterized the protein expression profile of chicken spleen tissue in a model of dexamethasone-induced stress-induced immunosuppression.A total of 590 differentially abundant proteins(DAPs)were identified that were significantly enriched in the following functional categories: ECM-receptor interaction,DNA replication,p53 signaling,PI3K-Akt signaling,and NF-kappa B signaling pathway.An integrated analysis of proteomic and transcriptomic data revealed that the protein levels of 62 DAPs correlated with their m RNA levels.Among these proteins,we focused on two significantly up-regulated DAP/DEGs,DPP4 and ALDH1A3,and two significantly down-regulated DAP/DEGs,GBP4 L and OASL.The above results again provide evidence for GBP4 L gene as a key gene involved in stress-induced immunosuppression.We speculate that the GBP4 L gene shows potential as an important regulatory target for poultry stress affecting immune function,which may be helpful for breeding chicken breeds with reduced stress levels.Part III Verification of immune function of key gene GBP4 L in vitroThe full-length CDS region of chicken GBP4 L gene was amplified and its bioinformatics function was analyzed.The results showed that the full-length CDS region of GBP4 L gene was 1878 bp,encoding 626 amino acids.The instability index was 39.85,which was a stable protein.The total average hydrophilicity was-0.456,which was a hydrophilic protein.GBP4 L protein is mainly composed of α-helix,β-sheet,extension chain and random coil.α-helix accounts for 62.30 % of the total secondary structure,β-sheet accounts for 3.67 %,extension chain accounts for 8.95 %,and random coil accounts for 25.08 %.Construction of pc DNA3.1-EGFP-GBP4 L recombinant plasmid and synthesis of si-GBP4 L gene interference fragment were transfected into HD11 cells,cell phenotype was detected.The results showed that GBP4 L gene could affect the expression of inflammatory factors in HD11 cells,down-regulate the expression of pro-inflammatory factors IFN-γ,IL-1β,IL-12 B,TNF-α,IL-6 and i NOS,and promote the transformation of HD11 cells from M1 type to M2 type.Chicken GBP4 L gene can effectively reduce the expression of IL-8 gene in HD11 cells induced by LPS,reduce the inflammatory response of HD11 cells,and promote the proliferation of HD11 cells and inhibit the apoptosis of HD11 cells.Part IV Verification of immune function of key gene GBP4 L in vivoForty-eight healthy Gushi cocks with similar body weight at 28 d were selected as the research objects.They were treated at 32 ± 1 °C for 4 weeks to construct the heat stress model of Gushi cocks.The p AAV-GBP4L-3FLAG virus purified by subcutaneous injection of neck was 2.43*1010 vg.The immunological indexes were detected.The results showed that the recombinant plasmid of p AAV-GBP4L-3FLAG adeno-associated virus was successfully constructed in this study,and the GBP4 L gene was significantly overexpressed in spleen tissue.Injection of recombinant adeno-associated virus GBP4 L plasmid can effectively alleviate the decrease of serum CD3+,CD4+,Ig G and Ig M levels in chickens caused by heat stress,and the increase of corticosterone and TNF-α,IL-1β,IL-6,TGF-β and NF-k B cytokines,alleviate the inflammatory response caused by heat stress and improve the immune state of the body.Injection of recombinant adeno-associated virus GBP4 L plasmid can effectively alleviate the increase of serum GLU,ALT,AST and TC levels and the decrease of CHE,ALP and TG levels in chickens caused by heat stress,thereby effectively alleviating the damage of liver and myocardium in chickens caused by heat stress.Injection of recombinant adeno-associated virus GBP4 L plasmid could increase the spleen and bursal immune organ indexes of heatstressed chickens and alleviate the spleen tissue damage caused by heat stress.In conclusion,this study integrated transcriptome and proteome sequencing data to screen out the key candidate gene GBP4 L for dexamethasone-induced stress-induced immunosuppression,and to verify its biological function in vivo and in vitro.It was found that GBP4 L gene can be negative Regulating the expression of pro-inflammatory cytokines in HD11 cells may be a brake on the body’s inflammatory response.It can prevent the occurrence of "cytokine storm" caused by excessive inflammatory response in the body by inhibiting the expression of cytokines.Injection of recombinant adeno-associated virus GBP4 L plasmid can effectively relieve heat stress in Gushi chickens.This study provides new insights into the mechanism of stress-induced immunosuppression,and provides a theoretical basis for GBP4 L gene as an important regulatory target of stress-induced immune function. |
