关键词: 基因敲除, λ-Red, rpoH, σ32 , 热激应答

Abstract:

The objective of this study was to construct a rpoH gene deficiency variant strain of Salmonella enteritidis IFO3313 called IFO3313-Δ rpoH and characterize the heat shock response of wild type and mutant strains under different temperatures. The kanamycin-resistant DNA cassette with 39 bp short homologous sequences on both ends generated by PCR was electroporated into Salmonella enteritidis IFO3313. Recombination between linear DNA cassettes and Salmonella enteritidis chromosomes took place with the help of λ-Red recombinase system and was confirmed by PCR method. Based on this, the heat shock response and the repair of sub-lethal heat injured wild type and mutant strains were compared using different media at different temperature. The wild type strain presented stronger heat-resistance and repair capacity in tryptic soy yeast agar (TSYA), compared to the mutant. False-negative results from the detection of the sub-lethal heat injured Salmonella enteritidis could occur in the desoxycholate–hydrogen sulfite–lactose agar (DHL). The rpoH gene knockout in Salmonella enteritidis using λ-Red recombination system provides us better understanding to the mechanism of heat shock and repair of Salmonella enteritidis.

Key words: gene knockout, λ-Red recombinase system, rpoH gene, σ32, heat shock response