| The problem of swine diseases has always been a key problem that plagues pig enterprises.Genetically improving the anti-disease ability of pig herds has become a tasks of contemporary breeding.We known,the anti-disease ability is often closely related to autoimmunity.As an important component of the immune system,T lymphocytes play a key role in fighting disease.Therefore,genetically studying the production mechanism of porcine T lymphocytes was of great significance for indirectly improving the disease resistance of pigs.However,at present,there were few studies on porcine T lymphocytes,especially the research on the differentiation and development of porcine T lymphocytes from the whole genome level.Therefore,in this study,we collected piglets thymus and mesenteric lymph node and made into leukocyte suspension.Then,we sorted four T cell subtypes of CD4~-CD8~-,CD4~+CD8~+,CD4~+and CD8~+in the thymus and three T cell subtypes of CD4~-CD8~-,CD4~+and CD8~+in mesenteric lymph nodes by flow cytometry with CD3,CD4 and CD8 antibodies.Then,these seven T cell subtypes were performed transcriptome sequencing.Finally,By combining the comparative transcriptome method and the WGCNA method to analyze and mine the key hub genes that affect the mutually exclusive differentiation of porcine T lymphocytes and their expression changes.The main results are as follows:(1)Flow cytometry and transcriptome sequencing obtained twenty-one sample’s transcriptome data for seven T cell subtypes.(2)We obtained 5403 differential expression genes via performing pairwise differential expression analysis for twenty-one samples based on the T cell mutually exclusive differentiation routes.We constructed co-expression networks of differential expression genes by WGCNA,which obtained seven main modules for various T cell subtypes.The GO analysis result of genes in each main module found that most genes were mainly related to immune regulation and T cell differentiation process.The PPI network analysis result showed that most genes had the function of being translated into proteins,and there was an obvious interaction relationship between proteins.(3)We have mined the hub genes for various T cell subtypes through the internal gene correlation analysis of each main module.We constructed regulatory network of hub genes for various T cell subtypes,and the result showed that the hub genes were highly connected.(4)We have further screened the key hub genes for various T cell subtypes by the seven network topology analysis algorithms of cytoHubba software.Network reconstruction of key hub genes was carried out,and it was found that key hub genes had similar regulation patterns.That was,there was regulation between key hub genes,at the same time,they also regulated other hub genes.In addition,key hub genes overlapped with important transcription factors related to the mutually exclusive differentiation of human and mouse T cells,and found significant differences,which indicated that there was a gene regulation pattern different from human and mouse in the mutually exclusive differentiation process of porcine T cell.(5)The analysis of key hub gene expression patterns and ROC curve analysis found that CDK6,BIRC5,BBS12,ST6GAL1,PNPLA7,KCNAB3 and ZFP82 genes were only highly expressed in their corresponding specific T cell subtypes,which indicated that key hub genes might determine the differentiation of various T cell subtypes,and could be used as potential candidate marker genes for corresponding T cell subtypes.We preliminarily inferred the pathway of mutually exclusive differentiation of porcine T cell and its gene expression change rule based on the above research results.That was,when a certain period of T cell subtypes began to differentiate,the key hub genes that determine the new state of T cell subtypes would be turned on or highly expressed,at the same time,the key hub genes that determine the original state would be turned off or lowly expressed.This study not only deepened the molecular understanding of the T cell mutually exclusive differentiation mechanism in porcine,but also provided certain candidate gene information for pig anti-disease breeding. |
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