肠炎沙门氏菌rpoH基因缺陷株的构建及其热激响应特性

Knockout of rpoH Gene in Salmonella enteritidis and Characterization of Heat Shock Response

构建肠炎沙门氏菌Salmonella enteritidis IFO3313株的rpoH基因缺陷株IFO3313-ΔrpoH,比较不同温度下缺陷株与野生株的热激响应特性。利用λ-Red重组系统对Salmonella enteritidisIFO3313的rpoH基因进行缺失突变,并使用PCR方法对其进行验证,在此基础上使用不同培养基对缺陷株与野生株进行不同温度下的热激应答结果和亚致死热损伤修复能力的考察:野生株比缺陷株有更强的热激耐受能力,在DHL平板上对亚致死热损伤的肠炎沙门氏菌的分离检测可能具有假阴性,野生株比缺陷株在TSYA平板上有更强的修复能力。利用λ-Red重组系统敲除了肠炎沙门氏菌rpoH基因,有助于了解肠炎沙门氏菌的热激亚致死及其修复机制,对亚致死状态食源性病原微生物的检测做出初步研究。

The objective of this study was to construct a rpoH gene deficiency variant strain of Salmonella enteritidis IFO3313 called IFO3313- △ 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 enten＇tidis 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.