Study Of The Diversity And The Functional Gene Of The Fecal Microbial Community In Pygmy Loris(nycticebus Pygmaeus)by Bioinformatics Analysis

The pygmy loris(Nycticebus pygmaeus) is a first-class state-protected animal in China, of which the gastrointestinal microbial community plays important roles in the host diet, nutrition, immunity and so on. In order to deepen the knowledge of the diversity of its gastrointestinal microbes, to elucidate the community distribution of microbes in this environment, their population functions, their evolution and roles on the global materials cycle, and to promote the development of microorganism resources, this study is carried out combining traditional ecological approach with metagenomic technology and bioinformatics analysis. Firstly, it studies comprehensively the structure and potential function of the fecal microbial community of pygmy loris, and lays a foundation for promoting the preservation of this rare wild animal and the exploitation of its gastrointestinal microbes. It is also significant for further understanding of human being the evolution progress concerning its gastrointestinal microbes. Secondly, it studiesthe aromatic compounds metabolic genes of the pygmy loris. This research could provide a theoretical base for further studies on the metabolizing of aromatic compounds in natural environment and lay the groundwork for development and utilization of the intestinal microbe resources. The following results are obtained:1. By cloning and sequencing the 16 S rDNA molecules isolatedfrom the feces of the pygmy loris, we have shownthat the gastrointestinal microbial community of the pygmy loris appears to be dominated by FirmicutesandProteobacteria, particularly those related to the broad genus Pseudomonas. The application of 16 S rDNA sequence analysis to thestudy of bacterial populations in the pygmy lorisfeces has also uncovered large populations ofpreviously unidentified bacterial species.2. We have comprehensively studied the structure of the gastrointestinal microbes ofpygmy loris and analyzed its differences from humans and other animals by metagenomics shotgun sequencing and comparative metagenomics. In the pygmy loris fecal metagenome, Bacteroidetes was the most predominant phylum, followedby Proteobacteria, Actinobacteria and Firmicutes.Archaea, fungi, and viruses are minor populations of the pygmy loris fecal microbiome. All archaea are members of Crenarchaeota and Euryarchaeota, with methanogens being the most abundant and diverse. Three fungi phylotypes were present in the pygmy loris fecal microbiome,namely, Ascomycota, Basidiomycota, and Microsporidia. Only about 0.1% of sequences were of viral origin, and all sequences were classified as bacteriophages.The clustering-based comparisons at the phylogenetic level showed similarities among the gut microbiomes of the pygmy loris, humans, and other animals. Four phyla dominated the microbiomes, namely, Bacteroidetes,Proteobacteria, Actinobacteria, and Firmicutes. However, the relative proportion of the phyla was different. The pygmy loris metagenome was most distinguished by the greater prevalence of Verrucomicrobia compared with humans and other animals. In the phylogenetic comparison, the pygmy loris samples clustered with the mouse metagenomes and separated from those of the other animals and humans.3. We have studied the potential function of the gastrointestinal microbes ofpygmy loris and analyzed its differences from humans and other animals by metagenomics shotgun sequencing and comparative metagenomics. Carbohydrate metabolism is the most abundant functional category in the pygmy loris fecal metagenomes based on SEED subsystems annotation. Metabolism is the most abundant functional category, of which amino acid transport and metabolism(E)present the highest abundance in the pygmy loris fecal metagenomes based on COG annotation. Based on CAZymes annotation,the most frequently occurring CAZymes families in the pygmy loris metagenome were glycoside hydrolase(GH), followed by glycosyl transferases(GT), carbohydrate esterases(CE), polysaccharide lyases(PL)and carbohydrate binding modules(CBM). Candidate sequences that belong to GT2 are the most abundant, followed by members of GH3 and GH2. The enzymes involved in benzoate degradation were identified in the pygmy loris fecal metagenomesbased on KEGG annotation. Relatively abundant and diverse metabolic subsystems of aromatic compounds were found in the pygmy loris metagenome compared with all the other gut metagenomes. Metabolism-based hierarchical clustering demonstrates that, the pygmy loris, human, chicken, and dog samples clustered together, the miceand cow samples formed a separate cluster.4. Metabolic genes of aromatic compounds in the feces of the pygmy loris are studied by constructing metagenomic fosmid library and using functional screening to obtain 43 positive clones capable of metabolizing aromatic compounds, and the fosmids of which has been studied by high-throughput sequencing. The results show that the metabolic genes of aromatic compounds obtained from the library haveγ-Proteobacteria and Actinobacteria as their resources, covering the aerobic and hybrid metabolic pathway of aromatic compounds. In addition, a full metabolic gene cluster of phenylacetic acid(PAA) from Brevibacterium sp. was obtained. Compared with other PAA metabolic gene clusters of Actinobacteria and Gram-positive bacterium, there are obvious differences in the structure and distribution of genes in this gene cluster. Bioinformatics analysis shows that ORF of the paaK is 1,296 bp long, which consisting of 431 amino acids. The highest identity is under 65%between paaK and the PA-CoA ligase of bacteria from other genera. Besides, paaK have AMP binding motif and the typical conservative regions(DIYGLSE and YRTRD) of PA-CoA ligase.