嗜水气单胞菌ATCC7966 luxS基因缺失株构建及特性研究  

Construction of luxS gene deletion mutant strain in Aeromonas hydrophila ATCC 7966

作　　者：崔一[1] 赵晶[2] 张福蓉 郭婉萍 陈明[1] 权春善[2] 范圣第[2] 

机构地区：[1]大连工业大学生物工程学院,大连116034 [2]大连民族大学生命科学学院 大连民族大学国家民委-教育部生物化工重点实验室,大连116600

出　　处：《中国人兽共患病学报》2016年第5期462-468,共7页Chinese Journal of Zoonoses

基　　金：辽宁省教育厅项目(L2013508);校基本科研业务费项目(DC201502020407)联合资助~~

摘　　要：目的研究群体感应信号分子AI-2合成酶编码基因luxS对嗜水气单胞菌ATCC 7966生理特性及毒力因子的影响。方法利用同源重组原理,构建含有中间为卡那霉素抗性基因,两侧为luxS基因上、下游同源序列片段的基因敲除质粒,将构建好的质粒转化大肠埃希氏菌SM 10(λpir)感受态细胞,利用接合法将质粒转入嗜水气单胞菌ATCC 7966,通过抗性筛选、PCR和DNA测序确认嗜水气单胞菌luxS基因缺失突变株。比较野生株ATCC 7966和luxS基因缺失突变株生长速度、AI-2合成、胞外蛋白酶合成及生物膜形成的差异。结果嗜水气单胞菌luxS基因缺失突变株ΔluxS构建成功。与野生株相比,突变株生长周期延长,基本丧失合成AI-2和胞外蛋白酶的能力;突变株生物膜形成能力降低,生长24h时生物膜形成能力为野生株的14.5%,36h时生物膜形成能力为野生株的60.0%,突变株生物膜形成速度明显比野生株缓慢,结构更为疏松。结论 luxS基因参与调控嗜水气单胞菌的生长、AI-2合成和毒力因子表达,本研究为进一步研究luxS基因在嗜水气单胞菌致病性中发挥的作用奠定基础。The influences of synthase encoding gene luxSof quorum sensing signal molecule AI-2on the physiological characteristics and virulence factors of the Aeromonas hydrophila ATCC 7966 was studied.Based on the principle of homologous recombination,recombinant gene knock-out vector was constructed consisting of Kanr cassette with the flanking homologous regions of gene luxS.The constructed recombinant plasmid was transformed into competent Escherichia coli SM 10（λpir）cells and was further transferred into Aeromonas hydrophila ATCC 7966 by conjugation.The luxSgene deletion mutant strain was confirmed by resistance screening,PCR and DNA sequencing.Growth rates,ability of AI-2synthesis,extracellular protease synthesis and biofilm formation were compared between the wild strain and luxSgene deletion mutant strain.Results suggested that luxSgene deletion mutant strain of Aeromonas hydrophila ATCC 7966 was successfully constructed.Compared with the wild strain,the growth cycle of the mutant strain was extended,the ability to synthesize AI-2and extracellular protease were lost.Biofilm forming ability of the mutant strain was decreased,which was 14.5% and 60.0% of that of the wild strain at 24 hours and 36 hours respectively.Compared with the wild strain,biofilm formation rate of the mutant strain was slower and biofilm structure was looser.It was indicated that luxSgene was involved in the regulation of the growth,AI-2synthesis and the expression of virulence factors of Aero monashydrophila.This study laid the foundation for further investigation of the role of luxS gene in the pathogenesis of Aeromonas hydrophila.

关 键 词：嗜水气单胞菌 LUXS AI-2 胞外蛋白酶 生物膜 

分 类 号：R378[医药卫生—病原生物学]