Screening Of Key Genes Of Mastitis Induced By Staphylococcus Aureus By Using Expression Gene Chromatography And MicroRNA Sequencing

Bovine mastitis is an inflammatory reaction caused by the persistence of pathogens within the mammary glands. It has been considered to be one of the most economically important diseases in the dairy industry. Mastitis is a very complex disease for its multiple disease-causing factors. Usual treatments of mastitis are not very effective, and researchers turned to researches on resistance traits, hoping to find individual cows with mastitis resistance traits and good potential in breeding. Molecular genetic marking technology has provided a new way and reliable genetic markers for mastitis resistance molecular breeding in dairy cow.With the development of high-throughput sequencing technologies, the transcriptomic sequencing has become an important tool for system researches on gene expression and regulation. The transcriptomic technology could help us to research on individual transcriptomic information under different background, which could systemically expound the regulation of gene expressions.Staphylococcus aureus(S. aureus) is one of the main pathogenic bacteria causing the bovine mastitis, By studying the expression of the miRNAs and genes in mammary gland infected with staphylococcus aureus to find out the key genes fo resistance to mastitis, which was beneficial to develop a new set of bovine mastitis prevention and treatment.In this study, S. aureus was administered to the mammary gland of Chinese Holstein cows to construct mastitis model. Total RNA was isolated from bovine mammary gland tissue samples from the S. aureus group and control group. Affymetrix gene chip was used to analyze differentially expressed genes, Further, Q-PCR technique was used to verify the results of gene chip. GO and Pathway analysis were conducted on the differentially expressed genes. While miRNAs were analyzed by Solexa sequencing for the S. aureus group and control group; Q-PCR technology was also used to analyse the sequencing results validation. Using bioinformatics technology to predict the target genes of the micrornas, GO and KEGG analysis of target genes also were conducted.The results were addressed as follows:(1) In this study, bovine Mastitis model was successfully constructed by artificially infecting with S. aureus and the infected cows showed obvious clinical mastitis symptoms.(2) Amongst the total of 297 significant differentially expressed genes screened out by Microarray analysis,188 genes were up-regulated and 109 genes down-regulated (FC>2, P <0.05).8 significant differentially expressed genes(C3、TLR8、BoLA-DRB3、PTX3、CSF3、 SLC11A1、CD80、IL17A) were selected for Q-PCR anlysis, the results are consistent with the microarray. The differentially expressed genes were analyzed by GO analysis, these genes were involved in 151 GO Terms (P<0.05). Pathway analysis indicatede that a total of 15 significant Pathways were enriched with these differentially expressed genes, Hematopoietic cell lineage, Cytokine-cytokine receptor interaction, Chemokine signaling pathway, Natural killer cell mediated cytotoxicity, Primary immunodeficiency, MAPK signaling pathway, T cell receptor signaling pathway, Antigen processing and presentation, B cell receptor signaling pathway, Intestinal immune network for IgA production, Wnt signaling pathway, PPAR signaling pathway, Toll-like receptor signaling pathway, Jak-STAT signaling pathway, VEGF signaling pathway.(3) miRNAs were analyzed using Solexa sequencing for the S. aureus group and control group. A total of 21,293,853 and 19,142,094 reads were obtained from S.aureus and control group respectively, Through filtering reference sequences,20,847,000and 9,535,613 clean reads were remained finally. A total of 370 known bovine miRNAs and 341 novel miRNAs were detected for the S. aureus group and 358 known bovine miRNAs and 232 novel miRNAs for control group. A total of 77 miRNAs in the S. aureus group showed significant differences compared to the control group. GO analysis showed these target genes were involved in the regulation of cells, binding, etc, while KEGG analysis showed that these genes were enriched in endocytosis, olfactory transduction, pathways involved in cancer.(4) Correlation analysis between the miRNA and mRNA (Affymetrix gen chip) showed that there were a total of 233 pairs of miRNA/mRNA regulating contrarily,162 pairs of miRNA down-regulation, mRNA up-regulation; 71 pairs of miRNA up-regulation, mRNA down-regulation. The most target genes enriched were miR-382 and miR-424-5p, the number of the target genes enriched were 12. A total of 47 target genes associated with the 10 significant differently expessed miRNAs.8 significant differentially expressed genes(ST3GAL3、IL13RA2、ARID3A、ADAP1、SYNE2、SLCO4A1、GRAMD1C、SLC5A1) was selected for Q-PCR anlysis, the results are consistent with the microarray. These differentially expressed genes may be the key genes in the process of S. aureus-inAuced mastitis development.