Method Research And Application Of Microbial Genome And Metagenome

Microbe are widely distributed in nature.There are many kinds and large quantities of microbe and they have complex interactions with each other,which have big effect on many biological processes.Therefore,researching on microbe is helpful for us to understand more about nature.Microbial genome and microbial metagenome have important research and application prospects in microbial research area.Microbial genome is a subject that studies the whole genetic material of microbe.The research on microbial genome can help to explain the association of many phenotypes and genetic materials,and lay the foundation for the analysis of more complex interactions,such as relationship between virulence and its genes.The study of microbial metagenome can analyze the species composition,quantity proportion and interaction model of microbe in natural microbial communities.Therefore,we attempt to research on the methodology and application of microbial genome and microbial metagenomeIn the parts of methodological study,we analyzed the existing analysis methods and research tools of microbial genome.And then we integrated a set of microbial genome automatic analysis process,including quality control after sequencing data produced,de novo assembling of genome,annotation of genome,screening of core genome and pan-genome,Screening of virulence genes and antibiotic resistance genes,extraction of gene variation sites,gene sequence alignment and evolutionary analysis,using the existing tools and methods as well as the programming tools written ourselves.At the same time,a set of general microbial metagenome analysis process was integrated,including quality control of sequencing data,16S sequence assembling,bacterial species identification and counting,and routine data analysis.In terms of application,we analyzed the genome of Listeria monocytogenes which is an important food-borne pathogen,gut metagenome of quarantine insects,and gut metagenomes of Bigu(a kind of in traditional Chinese fasting process)population.For food-borne Listeria monocytogenes,the most crucial phenotype isvirulence,so we analyzed the diversity nine important virulence genes of this bacteria,and they are PrfA,LLO,InlB,PlcA,PlcB,VirS,VirR,Hpt,ActA.First,to insure the research matter was the specie of listeria monocytogenes and to type this bacteria in a uniform method,we performed Multi-locus sequence typing(MLST).Using the seven standard housekeeping gene,fabcZ,bglA,cat,dapE,dat,ldh,lhkA,we identified sequence type,clonal complex and lineage of each bacteria sample.In variation sites analysis,it was found that the major virulence regulatory elements of Listeria monocytogenes,such as PrfA,VirR and VirS,had less variation and the variation pattern was simple.For other virulence factors,there are always three or four major variation pattern in each protein and in each pattern there are multiple sequence type(ST).Although the sequence type was not corresponding to the variation pattern strictly,the sequence type(ST)with close relationship was still clustered together.The Virulence Variation pattern of Listeria monocytogenes in different lineages have relatively huge difference,while the differences in the same lineage were partly related to the Multi-locus sequence typing(MLST).At the same time,we found some new variation sites in each virulence factors except conservative protein PrfA and VirR.Compared to the previous study,we found that the variation rarely appeared at major function region of each proteins.Next study,we analyzed the gut metagenome of quarantine insects in order to investigate the composition of the gut microbiota in these insects and trace the geographical source of quarantine insects through these microbe.We collected sufficient representative quarantine insects from many areas in China and obtained their gut microbial composition through experiments and preliminary analysis.Further analysis showed that there were three dominant phyla in gut microbiota of insects,and they are phylum Proteobacteria,phylum Firmicutes and phylum Bacteroidetes.In the whole,the trend of the proportion of these three groups was that phylum Bacteroidetes more than phylum Firmicutes more than phylum Proteobacteria.There was no significant difference among the other insects except the insect Eriosonra lanigerum with high alpha diversity of gut microbiota.The microbial environment of insects gut is complex.Species,feeding habits,sex and developmentalstatus of insects all affect on these microbe.Therefore,the geographical source of these insects could not be figured out by conventional unsupervised classification methods such as principal component analysis(PCA),but can be analyzed using supervised method like linear discriminant analysis(LDA).Linear discriminant method can trace geographical source of insect population at the class level or the order level of insects gut microbiota and trace insect individuals at the phylum level of insects gut microbiota.Comparing the composition of gut microbe of the insects which was same species but came from different geographical source,we found that there was no special bacteria phylum directly related to insects location.The whole pattern of the microbe was the related to insects geographical source.In the last study,we analyzed the gut metagenome of Bigu population in order to understand the impact of long term fasting on human gut microbiota.Fecal samples were collected at dayO,dayl-21,day22-31 and day3 1-51.DayO was the day before Bigu beginning,day1-21 refer to the days during Bigu process and people just take a few of extremely low-calorie foods,day22-31 were recover days when people could take foods incrementally,day3 1-51 was the days after whole Bigu process and people already take normal foods.It was found that the gut microbiota of these people was changed significantly during the whole study process.The proportion of Phylum Proteobacteria increased significantly in Bigu process(day1-21).With the end and recovery of Bigu(day22-31,day3 1-51),the proportion of Phylum Proteobacteria decreased gradually.But in normal diet stage after Bigu process(day3 1-51)The proportion of Phylum Proteobacteria was still higher than that before Bigu(dayO).The proportion of Phylum Bacteroidetes decreased significantly during the Bigu process and recover stages,but this proportion recovered significantly in the normal diet stage.The proportion of Phylum Firmicutes decreased significantly during the Bigu process,and then gradually recovered.Other phylum whose relative abundance was low had a transient increase,but then returned to their normal levels in a short time scale.