| Humans and mammals are a complex system composed of various lives.Gastrointestinal tract is a dynamic,diverse and balanced ecosystem composed of bacteria,fungi,viruses and archaea.Just like fingerprints,gut microbiome is unique to each individual.Gut microbiota is easily influenced by diets,environment and host genetics and linked to host health(e.g.obesity,IBD,type 2 diabetes and non-alcoholic fatty liver disease).In addition,the diversity,structure and functional genes of gut microbiome are changed following the vertical axis of ages and the horizontal axis of gut location.Because of the same feeding conditions and controllable environment and diet,experimental pigs become an ideal animal model for studying the composition of gut microbiome along the ages and gut locations,and the effects of host genetics gut microbial composition.Following the development of 16 S r RNA gene and metagenomic sequencing technology,we can understand the composition and potential functional capacities of intestinal uncultured microorganisms in a specific environment.Many studies have also found gut microbiome is affected by host genotype by using GWAS analysis,but there is a controversial about what extent that host genetics affecting gut microbioal composition.In this thesis,two subjects were investigated.Firstly,we described the composition of the gut microbiome at different ages and gut locations in F6 and F7 generations from a mosaic population,and analyzed the influence of host genetics on the gut microbial composition by analyzing the correlation of host genetic kinship with the dissimilarity of gut microbiome.Secondly,we investigated of the molecular mechanism of the AO genotype influencing the abundance of Erysipelotrichaceae in the gut by combining isolation and culture of Erysipelotrichaceae strains,targeted metabolome and RNA-seq analysis.The main results of this study are listed as follows:(1)16S rRNA gene sequencing was performed in 5110 samples collected at three different ages(D25,D120,D240)and five gut locations(ileum content,ileum mucous,cecal content,cecal mucosa and feces)from(F6 and F7)pigs of the mosaic population.All these data were composed of 12 different datasets.We found that the alpha-diversity(Shannon index)was increased following the ages although gut microbiota was instable at early age.The microbial composition of pigs varied greatly among samples(Bray-Curtis distance).Similarly,the alpha-diversity was increased from the ileum to the feces,and the Bray-Curtis distance was gradually decreased.Compared to mice,the fecal microbial composition of pigs at 240 days of age was more similar to that of healthy humans.In addition,regardless of age and location,porcine gut microbiota is mainly composed of Firmicutes and Bacteroides.However,as the intermediate part between small intestine and large intestine,ileum also has a high abundance of Proteobacteria.Escherichia coli was dominated and Leuconostocaceae from Firmicutes was unique in the ileum contents.The gut core microbiome of Lachnospiraceae and Ruminococcaceae which can digest dietary fiber or complex polysaccharides in food were stabled in D120,D240 and CC.Those results have been verified in the two generations of pigs.This is confirmed the limitation of using a single sample type that only provide some information about the composition and structure of gut microbiome,and more complex and important results obtained need larger samples and more sample types.In addition,because of the controllable conditions,multiple ages and gut locations,we analyzed the correlation between the host kinship and microbiome dissimilarity,and found the closer host relationship had more similar microbiome that means the host kinship was significant negative with the microbiome dissimilarity.These results supported the hypothesis that host genetics can influence gut microbiome.(2)Members of the Erysipelotrichaceae have a high abundance in the intestinal tract of mammals,and have been reported to be associated with host metabolic disorders and inflammatory diseases.In our previous study,we found that the abundance of Erysipelotrichaceae strains in the cecum was associated with host AO genotypes.However,only a few members of Erysipelotrichaceae have been isolated and cultured,and their main characteristics,genomic information and the functional capacity of carbohydrate metabolism remain unknown.The mechanism of AO genotypes affecting the abundance of Erysipelotrichaceae strains in the gut remains unknown.In this study,we used eleven different kinds of commercially available media and successfully isolated five Erysipelotrichaceae strains from feces of healthy pigs,which contained two strains related to OTUs affected by ABO gene.The five isolates were Gram-positive,and their colonies on Gifu anaerobic medium(GAM)or modified GAM were approximately0.25–1.0 mm in diameter.The main characteristics of five isolates were circular,white,convex,moist,translucent,and contained colony margins.These isolates were performed whole-genome sequencing by Oxford Nanopore and Illumina platforms.Based on whole-genome sequences,we found that the five strains belong to Erysipelotrichaceae bacterium OH741＿COT-311,Eubacterium sp.AM28-29 and Faecalitalea cylindroides.The GC content of the five strains ranged from 34.1% to 37.37%.Functional annotation based on the KEGG pathways revealed tens to hundreds of strain-specific proteins among different strains.Prediction analysis of carbohydrate metabolism revealed different functional capacities for metabolizing carbohydrate substrates among Erysipelotrichaceae strains.The Gal NAc metabolism pathway and polysaccharide utilization loci(PUL)analysis revealed that the strains of Erysipelotrichaceae may have the ability to utilize Gal NAc and plant polysaccharides.For investigating of the molecular mechanism of AO genotypes influencing the abundance of Erysipelotrichaceae strains in the gut,we first determined the concentrations of Gal NAc in the cecum lumen using a LC-MS/MS,and found that the concentrations of free Gal NAc in the cecum lumen were significantly different between AA and OO pigs.The abundance of Erysipelotrichaceae strains was positively correlated with the concentration of free Gal NAc.The feeding experiments of Gal NAc in vitro and RNA-seq analysis found that Gal NAc could increase the abundance of Erysipelotrichaceae strains and changed the expression level of genes involving in the metabolism of Gal NAc.However,the different performance of two Erysipelotrichaceae strains in response to Gal NAc(the gene expression in the strain 4-15-1 is inducible,but both inducible and constitutive in strain4-8-110)suggested a new mechanism about the regulation of carbohydrate metabolism that is different from the typical repressor-based regulatory system. |
