Construction And Preliminary Analysis Of Single Cell Transcriptional Map In The Lung Of Cattle

As one of the most important economical livestock in China,cattle have the highest feeding numbers in China,also have good environmental adaptability and disease resistance,including rich genetic resources and a wide range of gene banks.As an important functional organ of cattle,the lung not only plays an extremely important role in respiration,but also plays a role in regulating the transport,excretion,immunity and hematopoiesis of body water.However,due to the special physiological structure and function of the lung,it is more susceptible to external harmful factors during respiration and is the most vulnerable organ to get sick.In recent years,with the rapid development of single-cell sequencing,single-cell transcriptome sequencing has been widely used in different scientific research fields.It not only enables sequencing of all transcriptomes of a single cell,but also identifies cell types and cell states with high accuracy resolution based on the expression profile characteristics of individual cells and identifies new cell types,which is a powerful tool for analyzing the complex molecular cellular mechanisms behind the target phenotype.At present,lots of studies about the single cell level of organs such as retina,cortex,testis,skeletal muscle,tail intervertebral disc and gonad in cattle has been reported.However,as an important respiratory and hematopoietic organ in mammals,only human,mouse and pig lung cell maps has been reported,and the single cell maps of bovine lungs have not been reported.In this study,we delineated the complete single-cell transcriptional map of cattle lung for the first time,and screened the differentially expressed genes between cattle and plateau yaks using transcriptome sequencing on this basis,and mapped the expression of these genes to different cell categories of cattle.The main results are as follows:(1)In this study,we constructed a single-cell whole transcriptional map of the lungs of cattle for the first time,containing nine cell types and 39 cell subtypes.The nine cell types are as follows:B cells,Plasma cells,Endothelial cells,Epithelial cells,Fibroblasts,Macrophages,Monocytes,DCs and T cells.Among them,B cells contain two cell subtypes(Immature B cells and mature B cells),Endothelial cells contain four cell subtypes(a-Capillaries cell,Arterial cells,Vein cells and Lymphatic Endothelial cells),Epithelial cells contain six cell subtypes(Alveolar Epithelial TypeⅠCell,Alveolar Epithelial TypeⅡCell,Basal cells,Ciliated cells,Club cells and CAV1~+Epithelial cells),Fibroblasts contain six cell subtypes(Adventitial Fibroblasts,Col13a1~+Fib,Pericytes 2,Airway smooth muscle cells,Fib-1 and Fib-2),T cells contain 15 cell subtypes(Cycling T cells,Naive CD4~+T cells,Exhausted CD4~+T cells,Effector CD4~+T cells,Co-stimulatory cells,Th1 cells,CD4-C1,CD4-C2,CD4-C3,CD4-C4,Naive CD8~+T cells,Cytotoxic CD8~+T cells,CD8-C1 and CD8-C2).In addition,we found the presence of neutrophils and ILCs.(2)In this study,we defined one new cell type(CAV1~+Epithelial cells)in the Epithelial cell types,two new cell types(Fib-1 and Fib-2)in the Fibroblast types,and six new cell types(CD4-C1,CD4-C2,CD4-C3,CD4-C4,CD8-C1 and CD8-C2)in the T&NK cell types.(3)Based on transcriptome sequencing,we selected a total of 2318 differentially expressed genes in cattle vs yak,of which 1184 genes were up-regulated and 1134were down-regulated in cattle.Then by enrichment analysis,we found that differentially expressed genes were mainly concentrated in biological functions and signaling pathways related to immune response such as immune response,cell chemotaxis,natural killer cell activation and T cell-mediated cytotoxicity,some in signaling pathways related to hypoxic adaptability such as neuroactive ligand-receptor interaction,arachidonic acid metabolism,hematopoietic cell lineage and c AMP signaling pathway,as well as signaling pathways related to reproduction such as steroid hormone biosynthesis and ovarian steroid production.After mapping the differentially expressed genes to the nine cell types in the lungs of cattle,the results showed that a total of 347 differentially expressed genes were enriched in nine cell types,including 213 up-regulated genes and 134 down-regulated genes,and the differentially expressed genes were mainly enriched in immune cells.In addition,based on the mapping results,nine potential new marker genes were found,including PON3,CYP2B6,LY75,CXCL2,ACOD1,TREM2,CD22,CR2 and GIMAP4.This study enriches the single-cell data of the lungs,provides a broad reference for the study of the lungs of bovine animals,and also further deepens the understanding of the cell subtypes of lungs in bovine animals.