Screening and Analysis of Immune Genes Related to Disease Resistance in Tibetan Plateau Sheep

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INTRODUCTION
T ibetan sheep are one of the three original sheep breeds in China.They represent an excellent variety living in the Qinghai-Tibet Plateau, with Qinghai as the main production area (Yan et al., 2014).Because of the characteristics of the ecological environment, Tibetan sheep of type plateau account for the highest proportion.Tibetan sheep have a long history in China and can better adapt to cold conditions in the region after continuous breeding (Xie, 2016).Qinghai is an important animal husbandry production base in China.Due to the influence of the Qinghai Tibet Plateau, the large amount of grassland and national customs, a pattern of cultivation of herbivorous livestock has evolved.Among such livestock, sheep have become an important pillar of the animal husbandry economy of Qinghai.Qinghai Tibetan sheep are largest in number and of the best quality and have the most important cultural attributes, which allows them to have irreplaceable biological and economic characteristics.Through long-term natural selection and artificial selection, Tibetan sheep have adapted to the harsh environment of the Qinghai-Tibet Plateau and formed strong disease resistance.With the development of molecular biology and genetic e-ngineering technology, the search for disease resistance genes and genetic breeding to improve the resistance of livestock to diseases via genetic changes has been performed, and research to discover Tibetan sheep disease resi-stance genes has played an important role in the development of germplasm resources.
The immune system is generally divided into two categories, innate immunity and acquired immunity, according to the types of participating cells, the types and

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methods of identification, the forms of acquisition, and the effector mechanism (Liu et al., 2020).Innate immunity refers to an immune response that is mediated by innate genetic immunity.The innate immune system acts as a protective mechanism for the body to resist infection by foreign pathogens, which was gradually established during long-term germline development and evolution within the animal body in the struggle against invasive microorga-nisms (Janeway and Medzhitov, 2002).Innate immunity recognizes invading patho-gens through special pattern-recognition receptors (PRRs) and senses pathogenassociated molecular patterns (PAMPs) (McCartney and Colonna, 2009).Early genetic analysis found that higher vertebrates possess acquired immunity characterized by genes encoding RAG, MHC, TCR and BCR (Holmes and Bryceson, 2016).Eukaryotes recognize and eliminate invading pathogenic microorganisms through innate immunity and adaptive immunity.For example, dendritic cells can express the Toll-like receptors TLR7 or TLR9; among them, TLR7 can bind single-stranded RNA, and TLR9 can bind double-stranded DNA (Colonna et al., 2004).When stimulated by virus infection, the body can produce IFN-α, thus exerting an antiviral effect (Marshak-Rothstein and Rifikin, 2007).
Resistance is the ability of the host to tolerate pathogen infection, shorten the life cycle of the pathogen in the body, and resist the effects of the disease.Such disease resistance has long been found to be inherited stably by offspring (Hutt, 1958).The genetic variation in disease resistance exists between different species and individuals, thus forming the genetic basis for disease resistance selection (Liu, 2007).When stimulated by external factors, it can lead to resistance to disease and can produce antibodies in animals.Disease resistance can be classified as general disease resistance or special disease resistance, which have a different genetic basis.General resistance is affected by the combination of multiple genes and the environment and is not limited to a specific pathogen.Specific resistance is resistance to a specific disease or pathogen.It is mainly controlled by one gene or affected by other gene loci and environmental factors to varying degrees (Yan and Yin, 2007).Because general resistance to disease reflects the overall defence ability of the body against disease, improving the overall immune function of the body can improve general resistance to disease.However, innate immunity is particularly important in improving general resistance (Zhao, 2013).Finding genes that help improve immunity is of great significance for studying the molecular mechanisms of disease resistance and breeding for disease resistance.Candidate genes related to immune resistance in sheep are mainly Nramp1, MHC, TLR, and MBL (Yao et al., 2014).
RNA-Seq has been widely used in livestock and poultry research (Wang et al., 2015).Meng et al. (2015) discovered 263 cashmere goat meat quality-related genes by RNA-Seq.Analysis showed that the candidate genes affecting the quality of cashmere goat meat were mainly ADIPOQ, PDK, and CD36.Niu (2016) used the Illumina platform to perform high-throughput sequencing of mRNA in the skin tissue of white and beaver-coloured rex rabbits, and 12,408 differentially expressed genes were found, 8 of which were involved in the cytochrome metabolism pathway.Canovas (2010) Liu (2015) analysed the liver tissues of tailed and untailed Lanzhou big tail sheep by RNA-Seq technology and identified genes related to fat deposition, such as EAAT2.Geng et al. (2013) used RNA-seq to examine the changes in skin in the development cycle of cashmere sacs and identified 1332 differentially expressed genes.The development and growth of hair follicles may be related to the Wnt, Shh, TGF-β and Notch signalling pathways, and differentially expressed genes play an important role in these signalling pathways.Chen (2014) used RNA-Seq technology to sequence the transcriptome of the livers of normal-fed Lande goose and stuffed Lande goose, and a total of 802 differentially expressed genes were detected in the fatty liver of the stuffed geese group.Yang (2016) found six highly expressed genes related to the wolf innate immune system through RNAseq.Therefore, this study was conducted to perform high-throughput transcriptome sequenci-ng of disease resistance-related immune genes in Tibetan sheep and the functional verification of disease resistance-related differentially expres-sed genes using real-time quantitative PCR.This study was untaken to investigate the differential expression of disease resistance-related immune genes between Tibetan sheep of type plateau and small-tailed Han sheep, so as to explore disease resistance-related immune genes.The findings described here should be highly useful for breeding highly disease-resistant Tibetan sheep.

RNA extraction and quality inspection
Tibetan sheep of type plateau were fed in breeding field (Xining, Qinghai, China), and small-tailed Han sheep were fed in breeding field (Ledu, Qinghai, China).The spleen tissue of Tibetan sheep of type plateau and smalltailed Han sheep were collected in this experiment and stored in the spleen -80 °C until use.All the experimental

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animals were approved by the Animal Ethics Committee of Agriculture and Animal Husbandry, Qinghai University.Total RNA from spleen tissue from Tibetan sheep of type plateau and small-tailed Han sheep was extracted using an EASY Spin Plus Tissue/Cell RNA Extraction Kit (Beijing Aidlab Biotechnologies Co., Beijing, China) as described by the manufacturer.A NanoPhotometer spectrophotometer (IMPLEN, CA, USA) and an RNA Nano 6000 Assay Kit with the Bioanalyser 2100 system (Agilent Technologies, CA, USA) were used to detect RNA integrity and purity.

RNA sequencing and data processing
Using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® to construct cDNA libraries, sequenced using an Illumina HiSeq-PE150 plat-form by Novogene Bioinformatics Technology Co. (Beijing, China).The raw reads were cleaned by removing reads with adapters, reads in which unknown bases accounted for > 10% of the sequence length, and low-quality reads.The Q20 and Q30 values were then calculated.The levels of gene expression were calculated using the reads per kb transcriptome per million mapped reads method for each sample.HT Seq software was used to analyse the differential expression in spleen tissue from different varieties and to perform the significance analysis.

Screening of DEGs
Differentially expressed genes between two samples were calculated using DEGseq software, and the P values were adjusted using the q value.The smaller the q value, the more significant the difference in gene expression was, and a q value < 0.005 was set as the threshold for significant differential expression.If the log2 fold change value was > 0, the expression of the DEG was determined to be upregulated, whereas if the log2 fold change value was < 0, the DEG was downregulated.

GO and KEGG pathway enrichment analysis of DEGs
GO enrichment analysis of the DEGs was implemented with the GOseq package, and the GO terms for which the corrected P value was < 0.05 were defined as significantly enriched.Then, KEGG pathway analysis of the DEGs was implemented by using KOBAS, and pathways with a corrected P value of ≤ 0.05 were considered to be significantly enriched among the DEGs.

Real-Time PCR analysis
A total of 14 DEGs were randomly chosen for validation by Real-Time PCR analysis.β-actin was selected as an internal control.Primer sequences are listed in (Table I).RNA was converted to first-strand cDNA by using the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China).The final concentration of cDNA was 35 ng.Four replicates were prepared for each gene in each Real-Time PCR assay.The reaction mixtures (25 µl) consisted of 12.5 µL SYBR Premix DimerEraser (2x), 0.75 µl of each of the forward and reverse primers, 2 µl of cDNA, and 9 µl of ddH 2 O.The thermal cycler parameters consisted of an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s, followed by melting curve analysis from 65-95 °C using a CFX96 Real Time System.The levels of relative gene expression were evaluated acc-ording to the 2 −△△Ct method.

Differential expression analysis
The quality assessment results for the sequencing data are shown in Table II, which show a low error rate and the high-quality of the sequences.RNA sequencing analysis identified 280 genes that were significantly differentially expressed in the two types of Tibetan sheep (Fig. 1).Among them, the Tibetan sheep of type plateau showed the

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upregulation of 104 genes and the downregulation of 176 genes.Partic-ularly, in the Tibetan sheep of type plateau, genes encoding protein phosphatase and tumour necrosis factor were upregulated, while genes encoding ABC (ATPbinding cassette) were downregulated.

GO enrichment analysis
To better understand the functional differences in gene expression, the biological functions of the DEGs were characterized by GO enrichment analysis.The upregulated DEGs were classified according to 30 significantly enriched GO terms, including 9 involved in biological process, 19 involved in molecular function, and two involved in cellular components (Figs. 2, 3 and Supplementary Fig. 1).In the biological process category, the significantly enriched GO entries included cardiovascular system development, antigen processing and presentation and response to acid chemicals.The cellular component categories included extrac-ellular region (partial), extracellular region, extracellular space and plasma membrane (partial).No GO terms were significantly enriched for the upregulated DEGs in the molecular function category (Fig. 2).The upregulated DEGs were generally related to the "immune response" and "immune system process" categories (Fig. 3), and the downregulated DEGs were involved with the extracellular region, extrac-ellular region (partial), extracellular matrix, response to organic substance, and other categories (Supplementary Fig. 1).

KEGG pathway enrichment analysis
We also conducted KEGG pathway enrichment analysis of the DEGs to further characterize their biological functions, which were measured in terms of the enrichment, q value, and the number of genes enriched for a particular pathway.The 20 most significant pathways are displayed in the scatter diagram (Fig. 4).Among them, the pathways showing upregulated gene enrichment were involved in antigen processing and presentation (Supplementary Fig. 2), and allograft rejection (Supplementary Fig. 3), and T cell receptor signalling pathway (Supplementary Fig. 4), and B cell receptor signalling pathway (Supplementary Fig. 5), and autoimmune diseases.Ten differrentially expressed genes involved in the antigen processing and presentation pathway were upregulated.

Real-Time PCR validation of RNA sequencing
To verify the RNA sequencing results, Real-Time PCR analysis was performed using 14 randomly selected DEGs.After standardizing the exp-ression level of each gene relative to that of β-actin, the gene expression ratios was calculated.The results showed that nine genes were upregulated and five genes were downregulated (Fig. 5), and the expression differences were consistent with the transcriptome sequencing results.

DISCUSSION
In this study, GO functional analysis of differentially expressed genes in Tibetan sheep of type plateau spleen and small-tailed Han sheep spleen showed that most differentially expressed genes were annotated in the cellular component category for 19 components, including the extracellular matrix, extracellular region, extracellular space, plasma membrane (partial), MHC protein complex, and T cell receptor complex.This illustrates that the spleen interacts with ligands/receptors during functioning.Macrophages perform cell migration during the functioning of the spleen and can engulf bacteria, selfaging and apoptotic substances in circulating blood.They can also respond to blood-borne antigens and synthesize and secrete bioactive substances outside of cells, such as complement, IFN, and erythropoietin, and perform phagocytosis (Zhao, 2013).KEGG enrichment analysis of differentially expressed genes in Tibetan sheep of type plateau spleen and smalltailed Han sheep spleen showed that the pathways with more differentially expressed genes were involved in Graft-versus-host disease (GVHD), antigen processing and presentation, allograft rejection, viral myocarditis, TCR, and protein digestion and absorption.Most of the differentially expressed genes that were enriched in these pathways were upregulated genes, which can explain why Tibetan sheep of type plateau have higher disease resistance than small-tailed sheep.

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The spleen, as an absolutely essential peripheral immune organ, is the base for the body to respond to antigens and produce immune effectors.Lymphocytes are the basic components of the immune system and are widely distributed throughout the body.T-lymphocytes and B-lymphocytes are stimulated and divide, proliferate, and undergo specific immune responses by antigen stimulation.Among them, the number of mature B lymphocytes is the largest; thus, the spleen mainly mediates a specific B lymphocyte immune response.When the pathogen enters the bloodstream and flows through the spleen, memory B cells are activated by antigen stimulation and return to the bone marrow through lymph fluid or blood to differentiate into mature plasma cells.A large number of antibodies (mainly IgG) produced in the peripheral immune organs are released into the blood circulation.Disease resistancerelated differentially expressed immune genes in Tibetan sheep of type plateau and small-tailed Han sheep may be involved in B cell receptor (BCR) and T cell receptor (TCR) activity.BCR plays a key role in the development and maintenance of B cells and regulates the expression of immune genes.BCR is composed of membrane-bound imm-unoglobulin (mIg) and disulfide-linked mIgα/ mIgβ (CD79a/CD79B) heterodimers and is one of the key indicators used for B cell characterization (Brezski and Monroe, 2008), The mIg of BCR can recognize antigens and bind antigens.In addition, both mIg α and mIg β intracellular regions have an immunoreceptor tyrosinebased activation motif (ITAM), which can transmit the received antigen signals downstream of the cell, so they play an important role in the immune response of B cells (Zeng, 2015).CD19 is a B cell surface marker molecule.Generally, CD19 is believed to form Cr2 with CD21, CD81 and CD225, and it is one of the positive regulators of BCR signalling (Wang, 2007).Its extracellu-lar region can bind to C3d attached to an antigen or antigenantibody complex, thereby cross-linking BCR with the aforementioned coreceptors and transmitting signals into the cell via CD19, activating PI3K, and thus initiating the PI3K/Akt signalling pathway cascade reaction (Zhang et al., 2012).The KEGG analysis results indicate that TCR signalling pathways are significantly enriched.T cells are the main functional cells in the acquired immune system and are responsible for recognizing antigens and assisting or inhibiting the production of antibodies by B cells.They can respond to specific antigens and mitogens and produce cytokines.TCR plays a key role in antigen recognition.The main function of TCR is to activate T cells after recognizing specific antigens (Yuan et al., 2006).The signalling of T cells upon activation is mediated by T cell surface antigen recognition antibodies, and foreign signals are transmitted to protein tyrosine kinases (PTKs) through receptors and related proteins.PTKs are key signal transduction molecules involved in intracellular signal transduction.The results show that the phosphorylation and dephosphorylation of proteins by CD45 are important for the process of transmembrane signalling and the activation of T cells (Mo et al., 2020).When the complex specifically recognizes and binds to the receptor, the receptor inputs a signal to the T cell to change the conformation of the surface receptor.This can enhance adhesion to macrophages and prolong contact between macrophages and T cells, thereby inducing antigenspecific T cell activation and proliferation.To increase the sensitivity of T cells to antigenic peptides, CD4/CD8 molecules can greatly enhance the affinity of T cells and macrophages (Yan et al., 2015).However, T cells can only recognize antigenic peptides processed by APC and MHC molecules (Tang, 2004).After the presentation of MHC molecules, the exogenous antigen interacts with TCR and triggers a T cell-mediated immune response, thereby eliminating invading pathogens (2016).In addition, TCR, under the action of serine/threonine kinases and their products, regulates the metabolism of T cells, thereby addressing the metabolic needs of T cells participating in the immune response (Navarro and Cantrell, 2012).
Innate immunity is the basis of the immune response, which is present in living organisms and can be passed on to offspring.Antigen presentation is a core link in the immune response process (Zhou, 2013).In the immune response, macrophages are important antigen-presenting cells that present antigens to immune-active cells and provide systemic stimulation, neutro-philic granulocytes and macrophages allow the pattern recognition receptors on the cell surface to recognize the molecules specific to pathogenic microorganisms, initiate the phagocytosis process, and mediate phagocytosis.The proteasome subunit induced by IFN-γ stimulates antigen presentation, can induce LMP2, LMP7 and MECL-1 expression, and stimulates the pres-entation of class I antigens (Fruh and Yang, 1999).LMP2, LMP7 and TAP are encoded in the MHC gene region.IFN-γ can induce the expression of various genes related to the processing and presentation of MHC-I molecules, so IFN-γ upregulates the expression of MHC-I molecules.According to the surface molecules of mature T cells, T cells can be divided into CD4 + T cells and CD8 + T cells (Fu et al., 2013).CD8 + T cells have MHC-I class restriction, and CD4 + T cells have MHC-II class restriction.MHC-II molecules are synth-esized in the endoplasmic reticulum and only exist in cells such as APC (Baldwin et al., 2004).Exogenous antigens internalized by phagocytosis are mainly presented to CD4 + T cells through MHC-II molecules, while a small proportion of cytosolic antigens derived from autophagy also bind to MHC-II (Crotzer and Blum, 2009).Most of the antigens involved in the presentation of MHC-I molecules are endogenously synthesized protein molecules that exist in the cytoplasm (and nucleus).Most endogenous antigens and some exogenous antigens taken up by cells are presented by MHC-I molecules through proteasome and TAP-dependent mechanisms (Wang et al., 2006).
The KEGG analysis showed the upregulation of immune rejectionrelated genes in Tibetan sheep of type plateau.Billingham (Billingham, 1966) proposed that transplant immune rejection is an immune response between donor T cells and recipient tissues.The key to its occurrence is the reaction of donor T cells, and donor immunoreactive cells can recognize different histoco-mpatible antigens (Wang et al., 2018).Mature T cells are the main immunolo-gically active

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cells involved in the occurrence of GVHD.Transplant immune rejection is thought to be related to the imbalance of cytokines secreted by the Th1 and Th2 subpopulations (Nikolic et al., 2000), and GVHD is closely related to Th1 cytokines, such as IL-12, IFN-γ, and Th2 cytokines, such as IL-4 and IL-10 (Levine, 2011).When pathogenic microorganisms or other foreign antigens enter the organism, they are first phagocytized and cleared by macropha-ges; macrophages can synthesize and secrete a variety of active substances (Zheng et al., 2016), including IL-1, IL-6, many enzymes (lysozyme, collagenase, etc.), GM-CSF, G-CSF, IFN-α, IFN-β, and TNF-α.Therefore, the expression and activity of AGVH, IL-1, IL-2, IL-6 and TNF-α serve an important function.
In conclusion, this study revealed that the disease resistance-related immune genes of plateau Tibetan sheep were significantly enriched in the immune response pathway, which can explain the higher disease resistance of Tibetan sheep of type plateau.Antigen processing and presentation is a significantly enriched pathway.In addition, cytokines secreted by mac-rophages play an important role in anti-implantation immune rejection.

Fig. 1 .
Fig. 1.Volcano plot of the differentially expressed genes.The significantly upregulated genes are marked with red dots in the figure, the significantly downregulated genes are marked with green dots in the figure, and the genes without significant differences are marked with blue dots in the figure.

Fig. 2 .Fig. 3 .
Fig. 2. Enriched GO terms of upregulated and downregulated DEGs.A represents biological processes, B represents cellular components, and C represents molecular functions.

Fig. 4 .Fig. 5 .
Fig. 4. Scatter diagram of upregulated (A) and downregulated (B) genes involvement pathway.The dots in the figure represent the number of differentially expressed genes in the pathway, and the colours of the dots represent various Q value boundaries.When the Q value < 0.05, the gene is significantly enriched.The enrichment factor refers to the ratio of the number of differentially expressed genes enriched in the pathway to the number of all genes.The smaller the enrichment factor, the smaller the degree of enrichment.