ncRNA伴侣分子hfq基因缺失对单核细胞增生李斯特菌环境适应能力的影响

Regulatory roles of ncRNA chaperone hfq gene deletion strain in adaptability of Listeria monocytogenes to environmental stress

Hfq作为一种保守的RNA分子伴侣蛋白,主要通过与nc RNA结合来参与调控单核细胞增生李斯特菌(LM)的生命活动.利用基因重叠延伸PCR(SOE-PCR)方法构建具有氯霉素抗性的p KSV7-Δhfq重组穿梭质粒,电转化至新疆野毒株LM-SB5感受态细胞后在42℃和氯霉素压力下进行同源重组,筛选LM-SB5 hfq基因缺失株,并分析hfq基因缺失株在不同温度、pH、高盐、乙醇、H_2O_2、Triton X-100胁迫环境下的适应能力.结果成功构建得到一株遗传性稳定的LM hfq基因缺失株LM-Δhfq;LM-Δhfq对5%NaCl、3.5%乙醇、0.1%H_2O_2的适应能力显著降低,对30℃、37℃、42℃、pH 4、pH 9及Triton X-100的适应能力没有显著变化,表明Hfq参与了LM对高盐、乙醇、H_2O_2胁迫环境的适应过程.本研究为进一步揭示Hfq蛋白在参与ncRNA调控细菌生命活动的机制奠定了基础.

The conserved RNA chaperone Hfq protein plays key roles in regulating biological processes of Listeria monocytogenes（LM） through binding to nc RNA. This research aimed to construct the hfq gene deletion strain of L. monocytogenes. The shuttle plasmid p KSV7- Δhfq with chloramphenicol resistance was gained by overlap extention PCR, then electroporated into the Xinjiang wild type LM-SB5 competent cell for homologous recombinant under the pressure of 42 ℃ and chloramphenicol. Recombinant LM strain was identified by PCR. LM hfq gene deletion strain LM-Δhfq was successfully screened by PCR, which had stable genetic characteristics. It was then compared with the LM–SB5 for adaptability to environmental stresses by measuring their growth at different temperatures, pH and under hypertonic, alcohol, aquae hydrogenii dioxidi and Triton X-100 conditions. The results showed that the growth of LM-Δhfq was reduced under environmental stresses of 5% NaCl, 3.5% alcohol and 0.1% aquae hydrogenii dioxidi conditions, but not significantly different from LM–SB5 at 30 ℃, 37 ℃, 42 ℃, pH4, pH9 and Triton X-100, indicating that Hfq is involved in regulating the adaptability of LM to environmental stresses of hypertonic, alcohol and aquae hydrogenii dioxidi. The results laid foundation for further study of the molecular mechanism of Hfq protein in LM as nc RNA chaperone.