The Construction Of RpoE Mutant Strains And Primary Investigation On Function Of RpoE In Salmonella Enteritidis

Salmonella enteritidis is one of the common food-borne entericpathogens which is extremely easy to cause food poisoning in humans andlivestock. During the process of entering bodies from environment andinfecting the host cells, it is often suffered a lot of stimulations ofenvironmental stresses. σE(σ24) factor which encoded by rpoE gene haseffects on regulating heat shock genes at transcription level, which can helpto encode heat shock proteins to resist environmental stresses. In thisresearch, a rpoE gene mutant strain of Salmonella enteritidis wasconstructed by λ-Red homologous recombination system, then the growthability of mutant strain and the the expression levels of the heat shock geneswere inspected during the process of responding to environmental stresses toinvestigate the function of rpoE. The results are as follows:1. A rpoE gene mutant strain of Salmonella enteritidis called ΔrpoE wasgotten.By using Red homologous recombination system, a linear target fragmentwith the homologous arm of rpoE was transferred into Salmonella enteritidisby electroporation. With the help of recombinase, rpoE gene was replaced bythe target fragment. After appraisalling by electrophoresis and sequencing,finally we got the mutant strains ΔrpoE.2.The r p o E g e n e had a significant role in the regulation of cell growthability under environmental stresses.By designing four different simulative environment stresses—high andlow temperatures, acidity, oxidation, and high osmotic pressure, the growthcurves of wild and mutant strains under different conditions were drawncontrastively. The growth curves showed that the growth ability of mutantstrains was markedly weaker than wild strains. 3.Under the condition of thermal stress, the σEfactor had a remarkableregulation function in the expression of the key genes which can encode heatshock protein70(HSP70) of Salmonella enteritidis.At42℃, the expression level of key HSP70-coding genes—ftsA, hscA,eutJ, dnaK, grpE both in wild and mutant strains were inspected by RT-qPCR.The results showed that, gene expression quantities of ftsA, hscA, eutJ, dnaKin wild strains were significantly higher than ΔrpoE, and they wererespectively2.1times,1.63times,2.38times,2.61times as many as ΔrpoE,only grpE had the same expression level in wild and mutant strains.