Transcriptome analysis of chick spleens stimulated by nonreplicating Toxoplasma uracil auxotrophs

【Objective】The purpose of the study was to explore the transcriptomic changes of chick spleens infected by nonreplicating Toxoplasma uracil auxotrophs(NRTUAs),to clarify the regulatory effect of NRTUAs on the innate immunity in chick spleens,so as to provide theoretical basis for the development of anti-Toxoplasma gondii vaccine in poultry.【Method】 14-day-old chickens were infected with NRTUAs and phosphate buffer saline(PBS)respectively. 3 d after infection,spleen tissue samples were collected for transcriptome sequencing,and differentially expressed genes(DEGs) between NRTUAs and PBS groups were screened. Then GO functional annotation,KEGG signaling pathway enrichment and protein regulatory network analysis were performed. The accuracy of transcriptome sequencing was verified by realtime fluorescence quantitative PCR.【Result】Compared with PBS group,there were 499 DEGs(286 were up-regulated DEGs and 213 were down-regulated DEGs)screened from the chick spleens infected with NRTUAs,and the NRTUAs group had more up-regulated DEGs than down-regulated DEGs. GO functional annotation analysis found that DEGs was annotated on 58 functional items in three major categories of biological process(BP),cellular component(CC)and molecular function(MF). They mainly involved functional entries of positive regulation of establishment of protein localization to telomere,protein stabilization,chaperonin-containing T-complex,endoplasmic reticulum lumen,endoplasmic reticulum, integral component of endoplasmic reticulum membrane, spindle midzone and unfolded protein binding. KEGG signaling pathway enrichment analysis showed that DEGs were mainly enriched in endoplasmic reticulum protein processing pathway in endoplasmic reticulum,RNA transport,cell cycle,calcium signaling pathway,focal adhesion,cellular senescence,neuroactive ligand-receptor interaction,cytokine-cytokine receptor interaction pathway and amino acid biosynthesis pathways. The results of protein interaction network analysis showed that the expression of DEGs coding proteins PLK1,CCNB1,CDK1,CDC20,CCNA2 and NDC1 were up-regulated in the cell cycle pathway. Real-time fluorescence quantitative PCR verification results showed that 2 DEGs(CXCL12 and VPREB3)were up-regulated,and the other 8 DEGs were down-regulated,which basically conformed with the transcriptome sequencing results.【Conclusion】 The expression of immune-related genes is up-regulated in chick spleens infected with NRTUAs,and most of DEGs are enriched in the related signaling pathways of endoplasmic reticulum protein synthesis and cell cycle. The activity of spleen cells is greatly enhanced,which is to accelerate the establishment of cell connections and initiate the innate immune response to actively fight against the invasion of T. gondii.