| 【Objective】Salmonella serotype paratyphi A is a kind of gram negative bacilli,and could cause acute infectious disease of digestive tract and enteric fever.σ factor,a regulatory gene,is common in gram negative bacteria and plays an important role in regulating the response of bacteria to various environmental stresses.In order to understand the thermal inactivation characterization of rpoE on Salmonella paratyphi A and to explore the effect of fat on the expression of rpoE gene of Salmonella paratyphi A in meat processing.At last,further the effect of fat on the transcriptome of the strain is going to explore using transcriptomics.【Methods】（1）A rpoE gene mutant strain of Salmonella serotype paratyphi A was prepared by using Red homologous recombination system.the first,the growth of rpoE gene mutant strains and wild strains at the optimum temperature were investigated,and then the vaccinated 108CFU/g Salmonella paratyphi A mutant strains and wild strains in different medium,then subjected to thermal treatment.The surviving cell number was counted on selective media,next the inactivation of mutant strains and wild strains were compared in meat products processing.（2）The first,the vaccinated 108CFU/g Salmonella paratyphi A mutant strains and wild strains in different medium（the sausage of 100% lean,the sausage of 50% lean）,then subjected to thermal treatment.The surviving cell number was counted on selective media,the inactivation of mutant strains and wild strains were compared and analyzed in meat products processing.The next real-time PCR method was used to detect the expression level of rpoE gene of wild strain under approximative conditions（55℃,30min）of meat products pasteurization in different medium.The effect of the fat on the expression level of rpoE was compared.（3）The vaccinated 108CFU/g Salmonella paratyphi A wild strains in different medium（the sausage of 100% lean,the sausage of 50% lean）,then subjected to thermal treatment 55℃,30 min.collect the bacteria and RNA extraction,S.paratyphi A libraries were sequenced by RNA-seq technology.Through the analysis of the differentially expressed gene results,to find the candidate genes affected by fat.【Results】（1）The mutant rpoE of Salmonella paratyphi A was constructed sucessdully,It provides a reliable data base for the Salmonella knockout technology.The results showed that the growth ability was no significant different between mutant and wild strains,The D values of the wild and the mutant strains were 14.0980 min and 10.8933 min at 55 ℃,at 63 ℃ were 1.3078 min and 0.3147 min,at 72 ℃ were 0.9885 min and 0.9135 min.and there was a significant difference（t<0.05）in the D value of between Salmonella paratyphi A mutant strains and wild strains,so the heat resistance of mutant strains were significantly weaker than the wild strain.（2）The results showed the D values of the mutant strains and wild-type strain has a significant difference（p<0.05）between the medium of sausages lean percentage 50% and 100%,bacterial strain in medium fat percentage 50% more heat resistant.The up-regulated expression level of rpoE gene in the medium of sausages lean percentage 50% and 100% were 3.53 and 3.29 times respectively,and there was no significant difference（p> 0.05）.（3）Further confirming that the expression of the rpoE gene is not affected by fat.The results showed that the response to stress（dp S、osm C、psp）,intracellular signal transduction（SPA4365）,negative regulation of cell division（sul A）,regulation of catalytic activity（wz C）,oxidation-reduction process（psp B 、 ahp C 、 Novel305）,regulation of transcription（mar R）gene were adapted to the stress environment in the medium which fat was equal to lean compared to the medium of pure lean meat.【Conclusion】The mutant rpoE of Salmonella paratyphi A was constructed sucessdully.In the process of meat product sterilization,the expression of rpoE gene was up-regulated in response to heat stress,which played an important role in the survival of strains.Meat fat has a protective effect on strains,but it is not regulated by the rpoE gene.The response to stress（dp S 、 osm C 、 psp）,intracellular signal transduction（SPA4365）,negative regulation of cell division（sul A）,regulation of catalytic activity（wz C）,oxidation-reduction process（psp B 、 ahp C 、 Novel305）,regulation of transcription（mar R）gene were adapted to the stress environment in the medium which fat was equal to lean compared to the medium of pure lean meat,which provide the basis for the expression of Salmonella paratyphi A in different media... |
