Effect Of Bacillus Amyloliquefaciens TL On The Gene Expression Of The Broiler’s Ileum

Bacillus amyloliquefaciens is gram positive bacteria.It has high homology with bacillsus subtilis.It can produce amylase,protease and a variety of antibacterial substances.Studies have shown that Bacillus amyloliquefaciens TL had the same effect as aureomycin in promoting the growth of broilers.and it can promote the synthesis of protein and lipid metabolism.In the immune organ index,Bacillus amyloliquefaciens TL can stimulate the growth and development of broiler immune organs.In this study,we aim to explore the effect of Bacillus amyloliquefaciens TL on the gene expression of the broiler’s ileum in the early stage of broilers by transcriptomics techniques,and analysis of the relationship between gene expression and growth-promoting mechanisms in ileum tissue.Broilers in the probiotic group were fed with basal diet supplemented with Bacillus amyloliquefaciens TL,the antibiotic group were fed with basal diet with supplemented aureomycin,while the control group were fed with basal diet.The results of this research were as follow:1.Analysis of gene expression levels:FPKM greater than one as the screening criteria.The number of genes expressed in the control group were 14008.The number of genes expressed in the antibiotic group were 14211,and the number of genes expressed in the probiotic group were 14402.There were 13,600 genes that are expressed in all three groups.The number of uniquely expressed genes in the control group were 158,the number of uniquely expressed genes in the antibiotic group were 158,the number of uniquely expressed genes in the probiotic group were 158.FPKM greater than five hundred as the screening criteria.There were 172 highly abundant genes in the control group,there were 166 genes with high abundance in the probiotic group and 199 genes with high abundance in the antibiotic group.Pyruvate kinase（PKM）,prothymosin（PTMA）and heat stress protein（HSPs）were highly expressed in the probiotic and antibiotic groups,but not in the control group.The results showed that the energy metabolism rate of the probiotics group and antibiotic group was fast,the immune organs of probiotics and antibiotic groups developed early.the anti-oxidative stress ability of probiotics group and antibiotic group was strong.2.Screening differentially expressed genes:Log₂ fold change≥l or log₂ fold change≤-1,and FDR（False discovery rate）<0.05 for screening criteria to identify differentially expressed genes.The genes with log₂ fold change≥1 and FDR<0.05 were identified as up-regulated genes.The gene with log₂ fold change≤-1 and an FDR<0.05 was identified as a down-regulated gene.A total of 141 differentially expressed genes were detected in the ileum tissue in the antibiotic group compared with the control group,3genes were up-regulated,and 138 genes were down-regulated.A total of 510differentially expressed genes were detected in the ileum tissue of the probiotic group compared with the control group,up-regulated genes 163 and down-regulated genes 347.One down-regulated gene was detected in the ileum tissue in the antibiotic group compared to the probiotic group.Cluster analysis of differentially expressed genes,the results showed that the same differentially expressed genes in the probiotic group and antibiotic group had the same tendency in both groups and were not the same as the control group.Probiotics and antibiotics had similar mechanisms in promoting broilers growth.The Gene Ontology and KEGG analysis of differentially expressed genes:According to the GO analysis of differentially expressed genes,the gene function of differentially expressed genes were mainly related to inflammatory reaction,immune response,gene expression,ion transport,fat metabolism and so on.According to the KEGG analysis of differentially expressed genes,Butirosin and neomycin biosynthesis,Ubiquitin mediated proteolysis,Cytokine-cytokine receptor interaction,Insulin signaling pathway were highly aggregated in both groups.The analysis showed that after the addition of Bacillus amyloliquefaciens TL,pathogens were reduced,resulting in reduced immune stimulation,reduced immune activity and promoted fat deposition.3.RT-qPCR validate the RNA-seq deta:The results of RT-qPCR and in vitro cell experiments verified that the RT-qPCR results were basically consistent with the results of transcriptome data analysis,and its stimulation of small intestinal epithelial cells（IPEC-J2）can lead to the increase of LEPR gene expression in cells.The results showed that the sequencing data and the analysis result of the transcriptome were true and reliable.