Abstract
Paratuberculosis is a major endemic disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection and leads to huge economic loss in the dairy sector worldwide. Alternative splicing (AS) events, playing indispensable regulatory roles in many protein functions and biological pathways, are shown to be associated with complex traits and diseases. In this study, by integrating the RNA sequencing (RNA-seq) data of 24 samples from three tissues (peripheral blood, jejunum and salivary gland) of Holstein cows, we obtained 2,706,541,696 uniquely mapped reads in total that represented 12,870 expressed genes, and detected 4285 differentially expressed genes (DEGs) between MAP-infected and healthy cows (p < 0.05). Of them, 92 differentially expressed splicing factors (DESFs) were included. Further, 119, 150 and 68 differential alternative splicing (DAS) events between MAP-infected and healthy cows were identified in peripheral blood, jejunum and salivary glands, respectively. Of note, six DAS events were highly and significantly correlated with the DESFs (R2 > 0.9; p < 0.01), and their corresponding genes (COPI coat complex subunit gamma 2 gene (COPG2), kinesin family member 2C gene (KIF2C), exocyst complex component 7 (EXOC7), Rab9 effector protein with kelch motifs gene (RABEPK), deoxyribonuclease 1 gene (DNASE1) and early endosome antigen 1 gene (EEA1)) were significantly enriched in immune response such as vesicle-mediated transport, regulation of acute inflammatory response and tuberculosis through gene ontology (GO) and KEGG analysis. KS test showed that the DAS events in the EXOC7 and KIF2C genes indeed displayed differences between MAP-infected cows and healthy cows. The DAS in EXOC7 might produce a new protein sequence with lack of 23 amino acids, and the DAS in KIF2C induced a stop codon of premature occurrence and resulted in a lack of functional domain. In summary, this study identified the DAS events and corresponding genes related to MAP-infection base on the RNA-seq data from multiple tissues of Holstein cows, providing novel insights into the regulatory mechanisms underpinning paratuberculosis in dairy cattle.
Highlights
Through gene ontology (GO) and KEGG analysis, we found the differentially expressed genes (DEGs) in jejunum were significantly enriched in biological processes and pathways related to adaptive immune response, including response to cytokine, cytokine production and Th1 and Th2 cell differentiation, whereas the DEGs in salivary gland were enriched in innate immune response and defense response
Paratuberculosis is an infectious disease that seriously endangers the health of dairy cows and causes huge economic losses to dairy farms
It is caused by Mycobacterium avium subspecies paratuberculosis (MAP) that invades macrophages and inhibits the immune response pathway
Summary
MAP infections have already caused serious economic losses in dairy farms due to decreased milk production and increased management costs [3,4]. Paratuberculosis is difficult to prevent and control because of its long incubation periods and convenient transmissible ways of pathogen such as feces and infected milk. In 2007, Gonda et al first detected a quantitative trait locus (QTL) associated with susceptibility to MAP infection on BTA20 in US Holsteins [5]. Several QTLs for MAP resistance or susceptibility were identified on chromosomes 7, 16 and 22 with genome-wide association studies (GWASs) in Holstein cattle [6,7]. Gao et al identified 30 immune-related genes associated with paratuberculosis by performing GWAS [8] and RNA sequencing (RNA-seq) in jejunum tissue in Chinese Holsteins, such as NOD2, SLC11A1, TLR, SPI10, IL10RA, BolFNG and PGLYRP1 [9]
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