Construction And Biological Characteristics Of ?rpoE Mutant Strain Of Pasteurella Multocida From Tibetan Yak

The yak is an "all-round" livestock on the Qinghai-Tibet Plateau.It is also a necessity for local life and production in Tibet,providing livelihood and economic resources for farmers and herdsmen.In recent years,Pasteurella multocida(Pm)has caused many hemorrhagic sepsis in Tibetan yak,which has a certain infectiousness and mortality rate,which has caused certain economic losses to the development of the yak breeding industry in the region.In order to clarify the genome,bioinformatics characteristics,genotype and the role of rpoE gene of the Pm strain Tibet-Pm1 from yak origin in the strain,this study used whole-genome extraction and the use of nanopore single-molecule real-time electrical signal sequencing technology(Oxford Nanopore Technologies,ONT)to perform whole-genome sequencing;Perform genome analysis,functional annotation analysis,proprietary database annotation analysis,protein subcellular location,and drawing genome circles on the sequencing results.Tibet-Pm1 was analyzed for capsule typing,lipopolysaccharide typing,multi-gene sequence typing,Pasteurella multocida Kmt-specific genes,rpoE genes,and bacterial universal 16 S r DNA determination by PCR amplification,on the basis of the whole gene prediction,the rpoE gene was cloned and sequenced,gene sequence analysis,bioinformatics analysis,and the ST type of Pasteurella multocida was identified online.Use homologous recombination technology to knock out the rpoE gene of Tibet-Pm1 strain,and conduct biochemical characteristics test,stress resistance test,cell adhesion test,apoptosis test,pathogenicity test,and bacterial loading of mutant strains andw ild virus strains.Quantitative test to further understand the role of rpoE gene in Tibet-Pm1.It will provide a theoretical basis for understanding the genomics of Tibetan yak-derived Pm,the pathogenic mechanism of rpoE gene and vaccine research.The findings of this study include the following.By sequencing the entire genome of Tibet-Pm1 strain,the total length of its gene sequence is 2 331 688 bp,2 138 coding genesar e predicted,with a total length of 2 068 839 bp.The predicted len gth of the repeat sequence is 3 266 bp,accounting for 0.14% ofthe total length.In the prediction of non-coding RNA,there are 19 r RNAs in 3 families and 59 t RNAs in 33 families.There are 43 oth er nc RNA families,27 families,5 gene islands,962 promoters,2 091 Pm in the non-redundant protein library,37 proteins in the GO database,and 48 in the kegg function annotation There are 25 types of proteins in the egg NOG database annotations.The glycosyltransferases in the CAZy database annotations account for 51.47%.There are 660 transporters in the TCDB database.Threeresis tance proteins are predicted in the CARD database.Protein subcellular localization analysis,it was found to contain 190 signal peptides,509 transmembrane proteins and 190 secreted proteins.The positional relationship between the genome components can be clearly analyzed through the genome circle diagram,rpoE gene analysis showed that it was a RNA polymerase sigma-70 factor,possibly a virulence factor of Tibet-Pm1 strains,and the gene seque nce accession number is CP072655.By PCR identification,sequencing,website comparison,TibetPm1 strain was B:L2:ST44 genotype,The genetic similarity between rpoE gene and Pasteurella multocida rpoE India was 100%,rpoE gene sequence is 576 bp,long Coding 191 amino acids,22.0 ku,predicted molecular weight Theoretical isoelectric point 4.86,Intracellular hydrophilic proteins with unstable,non-transmembrane structure,non-signaling peptide,conserved potential phosphorylation,A maximum of 67.02% ?-helix in secondary structure,The tertiaryst ructure is a rod microbending model,Multiple epitopes,it is predicted that it can interact with 10 proteins,and some of the results were consistent with rpoE analysis in the whole genome.The suicide vector p BC-SK-?rpoE was successfully constructe d through PCR identification,sequencing,restriction digestion and ligation of the target gene and vector,and the suicide vector was transformed into Tibet-Pm1 competent cells by electrotransform ation technology and passed forward screening The mutant strain with Kana gene replacing the rpoE gene was successfully screened.PCR identification and sequencing showed that the mutant strain can be inherited stably during passage.Through the research and analysis of the biological characteristics of the wild strains and the rpoE gene-deficient strains,there is no difference in the biochemical characteristics of the two strains.In the stress resistance test,the growth status of the mutant strains at 20 ? and 42 ? is greatly affected.After environmental mutations,the survival rate of mutant strains is lower,the biofilm of rpoE gene-deficient strains is affected,the number of adherent cells is reduced,the number of apoptosis is reduced,the pathogenic ity to rabbits is reduced,and the bacterial load of internal organs is significantly reduced.In summary,through the analysis of the Pm gene from Tibet yak and the study of the biological characteristics of the ?rpoE mutant strain,the complete genome sequence of the Tibet-Pm1 strain and the prediction of its gene function characteristics were clarified,and the genotype of the Tibet-Pm1 strain was identified,Predicted the functional characteristics of the protein encoded by the rpoE gene,successfully constructed the Pm suicide vector p BCSK-?rpoE,and screened the rpoE gene mutant strains,and explored the biological characteristics of the mutant strains and wild virus strains,indicating that rpoE The encoded protein is a vaccine candidate antigen protein with good immunogenicity.rpoE has a certain regulatory effect on the stress resistance,biofilm,adhesion,and pathogenicity of Tibet-Pm1 strain.It will lay a theoretical foundation for further in-depth study of Pm vaccines derived from Tibetan yak and exploration of the pathogenic mechanism of rpoE genes in Tibet-Pm1 strains.