Screening of Retroauricular Gland Immune Repair Genes by scRNA-seq in Bufo bufo gargarizans Cantor
Bufo bufo gargarizans Cantor is a kind of traditional medicinal animal and one of the original species of BUFONIS VENENUM.In order to explore the genes related to immune repair of its retroauricualr gland,single-cell RNA-sequencing(scRNA-seq)was carried out with adult B.bufo gargarizan retroauricular gland tissue samples using the Illumina sequencing technology.Cell subgroups were classified after construction of the scRNA-seq library,the up-regulated genes and those related to immune repair in the cell subgroups were analyzed,and real-time fluorescence quantitative PCR(qRT-PCR)was used to screen the candidate genes initially.The results showed that the retroauricular gland of a toad had 8 414 cells used for library con-struction and paired-end sequencing.The average reads and median gene number per cell were 46 326 and 734,respectively,and the number of high-quality cell available for additional analysis was 7 687.Twelve cell subgroup were obtained through cluster analysis,and it was hypothesized that Cluster 10 was plasma cell origi-nated from connective tissue.The 217 up-regulated genes in Cluster 10 were analyzed by GO and KEGG analy-ses,and a number of biological processes and pathway related to body immunity were identified,including bi-ological processes such as lymphocyte activation,lymphocyte differentiation and leukocyte activation,and im-mune related pathways such as cell receptor signaling pathway,Fc epsilon RI signaling pathway and chemo-kine signaling pathway.The MYO1F and CYBB genes were select from the differential genes of Cluster 10 as the immune repair genes of the retroauricular gland in B.bufo gargarizans,which were verified by qRT-PCR and speculated to have positive regulatory relationships with immune repair.The findings of this study could provide theoretical references for the subsequent excavation of immune repair genes and the repair mechanism research,and provide reference information for single cell genomics study in toad retroauricular gland.
万方数据
维普
