| Vibrio mimicus is one of the most prevalent pathogeny in aquaculture?can infect shrimp,crab and fish?,which closely resembles vibrio cholerae.V.mimicus was initially considered an atypical v.cholerae and V.mimicus can cause gastroenteritis,diarrhea and food poisoning.Vibrio is not only a causative agent of aquatic animal disease but also has bad effects on food safety and human health.In recent years,v.mimicus is an emerging pathogen for Silurifomes,the epidemiological features of v.mimicus showed that short disease duration and high mortality rate,and lead to large economic loss in Silurifomes farmed.Immunoprophylaxis are becoming a powerful tool for preventing and controlling aquatic diseases,attenuated vaccines based on gene deletion were the important research direction for development of fish vaccine.It was to be clear the function of target deleted genes before construction of attenuated vaccine.At present,it is not clear for genomics and pathogenic genes in v.mimicus isolated from siluriformes.Therefore,virulent strain of v.mimicus which isolated from yellow catfish in 2014?SCCF01 strain?was studied as thesis subject,whole-genome sequence analysis and comparative genome analysis of v.mimicus strain SCCF01 were performed.Based on complete genome sequence of v.mimicus strain SCCF01,three of virulence genes deletion mutants will be constructed by natural transformation.Biological function and pathogenic role of these virulence genes will be illustrated by comparing influence on phenotypic characteristics?growth,movement,adhesion and invasion etc?after genes deletion,and virulence genes which virulence declined will further research their function.Specific experimental results are as follows:1.Complete genome sequencing and annotation of v.mimicus strain SCCF01 and component analysisThe genome DNA of SCCF01 strain was extracted by bacteria DNA kit and the integrality,purity and concentration were estimated.DNA samples which is qualified through the examination were sequencing by PacBio RS platform.Polymerase read bases were filtrated using quality shearing software and the assembled contigs were polished using Denovo to generate 2 circular chromosomes.The consists of two chromosomes of3,213,040 and 1,272,975 bp with G+C content of 46.61%and 45.88%,respectively.The sequences were submitted to GenBank under the accession no.CP016383?chromosome I?and CP016384?chromosome II?.Subsequently,genome annotation?functional annotation,resistance genes annotation and virulence genes annotation?was performed using COG,GO,KEGG,ARDB and VFDB database,and genomic image was created by Circos v0.64for.The information of gene function,classification and metabolic pathways were obtained by functional annotation,genomic image was designed for visualizing SCCF01 strain genomic data,20 resistance genes were discovered by resistance genes annotation,the information of virulence?adhesion,flagellum system,exotoxin and secretory system etc?were obtained by virulence genes annotation.Finally,component analysis of two chromosomes was performed by Prodigal,barrnap,tRNAscan-SE,RepeatMasker,IslandViewer and PHAST.The results of prediction showed that 31 rRNA and 106 tRNA were contained in the chromosome,the information of repeated sequence,genomic island and prophage were obtained.2.Comparative Genome Analysis of V.mimicus strain SCCF01Linear pairwise comparison,gene family analysis,single nucleotide polymorphisms?SNP?annotation and phylogenetic analysis were performed in order to resolve the origin of strain SCCF01 and genomic structural variation.Genomic linear relationship between strain SCCF01 and standard strain of v.mimicus by linear pairwise comparison,however it was unable to determine relationship.Gene family analysis predicted that evolutionary direction of strain SCCF01 was clinical strain?environmental strain?SCCF01 strain;SNP annotation and phylogenetic analysis showed that strain SCCF01 was related more closely to environmental strain.According to gene family analysis,SNP annotation and phylogenetic analysis,strain SCCF01 was more likely diverged from environmental strain.In addition,the difference of species and genus was compared with strain SCCF01 in order to obtain rough difference information;the relationship of core gene and specific gene across strain SCCF01 and other v.mimicus was obtained by Core-Pan gene analysis;genomic structural variation between strain SCCF01 and other v.mimicus was determined by SV and Indel detection.Finally,Phylogenetic tree of usual vibrio base on genome showed that strain SCCF01 appeared in the cluster for type of v.mimicus and further proved that strain SCCF01 was determined to be v.mimicus on genome level.3.Construction and identification for gene deleted mutants of v.mimicus strain SCCF01Three of virulence genes?hemolysin gene,type II secretion system gene cluster and tow of TonB system?were deleted by homologous recombination using natural transformation and obtained recombinants of 3 virulence genes/cluster deletion.Subsequently,selection markers of resistance gene in recombinants were removed by PCP20 plasmid and obtained corresponded genes deleted mutants.Identification of recombinants and deleted mutants for genomic level and transcriptomic level was performed by PCR technique,sanger sequencing and RT-PCR technique.The results of PCR showed that all of target genes in recombinants and deleted mutants has been deleted and target genes in recombinants has been removed by PCP20 plasmid,the target band was accordance with anticipated size based on FRT site etection,no residue of PCP20 plasmid in all of deleted mutants.The results of Sanger sequencing showed that upstream/downstream homologous arm of deleted mutants were accordance with wild type strain and proved that target genes have been deleted.The results of RT-PCR showed that target deleted genes could not be detected in RNA of recombinants and deleted mutants,and upstream/downstream open reading frame?ORF?of target deleted genes could be normal transcription.Lastly,continuous passage culture proved that deleted mutants could be steadily hereditary.In conclusion,these results based on genomic level and transcriptomic level proved that deleted mutants?hemolysin gene,type II secretion system gene cluster and TonB system?could be successfully constructed.4.Phenotypic characteristics of gene deleted mutants of v.mimicus strain SCCF01Compare wild type strain with deleted mutants?hemolysin genes,type II secretion system cluster genes and TonB system genes?on phenotypic characteristics.The results show that morphological and physiological-biochemical characteristics had no change after3 virulence genes/cluster were deleted,while colony size growing Luria-Bertani/TCBS medium and rate of biofilm formation was extremely significantly lower wild type strain?P<0.01?.After T2SS cluster genes were deleted,growth rate,ability of autoaggregation and adhesive ability were extremely significantly lower wild type strain?P<0.01?.After hemolysin genes or TonB system genes,deleted mutants displayed no difference with wild type strain on growth rate?P>0.05?,ability of autoaggregation and adhesive ability.The results of motility test showed that deletion of T2SS cluster genes or TonB system genes displayed extremely significant reduced mobility on 0.3%semisolid medium?P<0.01?.In order to explore which virulence genes deletion were most effective,mortality was analysed by water bath immersion with the different concentration of deleted mutants and wild type strain.The results showed that LD500 was no difference after deletion of hemolysin genes,LD500 was significant increase after deletion of T2SS cluster genes or TonB system genes.Fold attenuation were 307.26?deletion of T2SS cluster genes?,26.61?deletion of TonB1 system?,81.75?deletion of TonB2 system?and 141.25?deletion of TonB1 and TonB2 system?,respectively.Finally,Union-genes deleted matant?deletion of T2SS cluster genes and two of TonB system genes?was performed pathogenicity tests.Results showed that LD500 of union-genes deleted matant was 4.46E+07,fold attenuation were up to 668.34.5.Effects of T2SS on extracelluar protein and natural transformation,and the effects of TonB system on utilization of iron.This chapter further studied the effects of T2SS on extracelluar protein and natural transformation,and the effects of TonB system on utilization of iron,In order to explicate the function of T2SS and TonB system and and to lay the foundation for attenuated vaccine.The results of T2SS study showed that mutants of deleted T2SS displayed extracellular products located in 15-25KDa?4?,35-50kDa?1?and 50-70kDa?1?was lower than wild type strain,and enzymatic activity?Protease,lecithin and chitinase?was significantly lower wild type strain?P<0.05?;The results of natural transformation frequency showed that the stain which deleted T2SS cluster genes could not take up free DNA by natural competence for horizontal gene transfer.The results of TonB system study showed that ability of taking up Fe2+/Fe3+or lactoferrin was no difference after deletion of TonB system genes,mutants of deleted TonB1 system genes displayed ability of taking up heme and hemoglobin was significantly lower wild type strain;the results of dipyridyl sensitivity assays showed the dipyridyl sensitivity of the mutants?deletion of TonB1 or TonB2 system?was more sensitivity than wild type strain.In conclusion,the mutant which deleted T2SS cluster genes and TonB system genes was difficult to adapt to environment and could not take up free DNA for horizontal gene transfer.In conclusion,complete genome sequence of strain SCCF01 will provides genetic basis and sequence data.Successful application of the natural transformation method in this study will provide new methodological reference for bacterial gene editing.The results of biological characteristics of gene deleted attenuated mutants for v.mimicus will provides theoretical basis for the development of vaccine.The results of thesis are as follows:... |
PDF Full Text Request