The Dynamics Of Gut Microbiome And Immune Regulatory Mechanism Analysis Of Meishan Pigs At Different Growth Stages

The changes of intestinal microbiota and immune responses play crucial roles in the processes of growth and development of animal organisms.In order to systematically reveal changes in intestinal microbiota and the genetically molecular mechanisms underlying immune regulation during the periods from primary birth to adulthood,the representative pig breed was used in this study.The dynamics of porcine gut microbiota in six intestinal segments?duodenum,jejunum,ileum,cecum,colon and rectum?from pigs at eight postnatal intervals?days 1,7,14,21,28,35,120 and 180?were analyzed by 16S rDNA sequencing techniques in combination with OTU analysis,species classification and abundance analysis,Alpha diversity analysis,and Beta diversity analysis.In addition,the long noncoding RNA?IncRNA?sequencing was used to analyze the expression of IncRNA and mRNA in the spleen tissuesat different growth stages.The Weighted Gene Co-Expression Network Analysis?WGCNA?was performed to screen important IncRNAs,immune regulatory pathways,and functional genes.Firstly,the expression of immune-related genes in immune and intestinal tissues from different growth stages of Meishan pigs was validated by real-time quantitative PCR.Secondly,the target genes of lncRNA were predicted by combining the cis and trans analyses.The interaction between lncRNA and the target gene was verified by RNA immunoprecipitation?RIP?-PCRto confirm the target gene regulated by lncRNA.Finally we analyzed the regulatory effects of promoter methylation on gene expression in different developmental periods by pyrosequencing PCR technique.The main findings are as follows:1.The dynamic landscape of gut microbiota of Meishan pigs at differentpostnatal intervals.?1?Analyses of the 240 samples from six intestinal segments of pigs at eight growth stages identified a total of 4,465 OTUs.The six intestinal segments could be divided into three partsincluding the category of duodenum and jejunum,the category of ileum,and the the category of cecum,colon,and rectum.The Lactobacillus and Bacteroides were the dominant genera in duodenum-jejunum section,and Fusobacterium and Escherichia were dominant in the ileum.The Prevotella showed higher abundance in the cecum-rectum section.?2?The gut microbiota of newborn piglets showed high similarities in the six intestinal segments and evolved with time.The gut microbiota exhibited apparent temporal and spatial characteristics in different intestinal segments.Overall,the change regularity of microbiome in the large intestine was more stable than that in the small intestine.?3?Some bacteria associated with dietary changes or immunity modulation displayed tremendous changes in intestinal segments,illustrating the importance of the two host factors in shaping the segmental intestinal microbial communities of newborn mammals.In addition,the microbial compositions differ between different intestinal segments,which suggested the digestive function differences in different intestinal segments.2.The molecular mechanisms of immune regulation in Meishan porcine at eight postnatal intervals.?1?A total of 251.8 Gb clean reads were obtained by sequencing,and 3200 candidate lncRNAs were identified based on CNCI analysis,CPC analysis,PFAM analysis,and PLEK analysis.The differential expression analysis of IncRNAs and mRNA in spleen tissues of different ages showed that there were 123,53,146,35,129,310 and 276 differential lncRNAs and 2726,1067,2360,1015,2108,4860,4598 differential mRNAs in the 7-day-old,14-day-old,21-day-old,28-day-old,35-day-old,120-day-old,and 180-day-old group respectively,using the 1-day-old group as a control.WGCNA analysis showed that lncRNA and mRNA transcripts were divided into 13 modules,of which the purple and grey templates had significant correlation with time points.The KEGG pathway enrichment analysis of genes in the module showed that the purple module is highly enriched for immune pathways such as NF-kappa B signaling pathway,including immune regulatory genes such as TLR4,IL1?,and IFN-?,and two key IncRNAs,TCONS₀0112081 and TCONS₀0028679.The target gene prediction indicated that there was an immune-related target gene TNF-? within 10 kb downstream of TCONS₀0028679.RIP-PCR confirmed the interaction between TCONS₀0028679 and TNF-?.?2?Tissue expression tests at different growth stages showed that the TLR4,TNF-?,IL-1?and IFN-a genes were expressed in all immune tissue?spleen,thymus,lymph?and intestinal tissue?duodenum,jejunum and In the ileum?in different developmental growth stages of the Meishan pigs,and the expression of TLR4 gene was higher in spleen tissues and lower in other tissues.The expression levels of TLR4 in spleen and lymphoid tissues at 7 and 35 days were significantly lower than those at other different growth stages and the expression of TLR4 in the intestinal tissues was significantly lower at 21 and 35 days than at other days.The expression levels of IFN-?,IL-1?,and TNF-? genes vary greatly with the developmental stages.The expression levels of IFN-a at 21,120,and 180 days were significantly higher than those at other stages.The expression level of IL-1? in intestinal tissues at 120 and 180 days was significantly higher than other stages.The expression level of TNF-? at 21,180 days was significantly higher than at other stages.Cluster analysis revealed that the expression levels of TLR4,TNF-?,IL-1?,and IFN-? genes in the immune tissues?spleen,thymus,and lymph?were similar,and those four genes in intestinal tissues?duodenum,jejunum,and ileum?were similar.Correlation analysis of expression levels showed that there was a significant correlation between TLR4,TNF-?,IL-1?and IFN-? in the immune and intestinal tissues,suggesting that TLR4,TNF-?,IL-1? and IFN-?play a synergistic role in immune regulatory pathways.?3?There was one CpG islands?417 bp?in promoter region of porcine TNF-? gene,and methylation analysis revealed a trend for TNF-? promoter methylation level to increase with the age.The methylation level of spleen in Meishan pigs at 35 days was significantly higher than that at other days,followed by 21 days and 28 days.Correlation analysis showed significantly negative associations between methylation level and mRNA expression?Pearson'sr =-0.775,P =4.87E-07?.Combined with dual luciferase assays,the results indicated that the methylation level of promoter region showed reverse associations with TNF-? expression,of which four CpG sites?CpG-6,CpG-8,CpG-11 and CpG-15?displayed statistically significant negative associations with the expression level of TNF-?.The four CpG sites were predited to locate in the binding sites of three transcription factors ELK4,Spl and TFAP2B.In this study,we revealed the changes of intestinal microbiota and the molecular mechanisms of immune regulation in Meishan pigs at different growth stages by using 16S rDNA and IncRNA sequencing.We not only constructed a complete Meishan pig intestinal microbial database,but also screened some functional gene such as TLR4,IL-1?,IFN-a.We further unveiled the important immune regulation roles of lncRNA-TCONS₀0028679 and its target gene TNF-? in the growth and development of Meishan pigs.Our findings may provide foundations and theoretical basis for scientific and rational design of pig disease resistance breeding strategies from the microbiological and immunoregulatory level.