lsrB基因对维氏气单胞菌TH0426菌株运动性影响的研究

Study on the effect of lsrB gene on the motility of Aeromonas veronii TH0426 strain

为了研究lsrB基因对维氏气单胞菌(A.veronii)TH0426菌株运动性的影响,试验以A.veronii TH0426菌株基因组为模板,扩增lsrB基因的上下游同源臂片段L1与L2,利用重叠PCR连接L1与L2,以得到的连接片段构建重组自杀性质粒pRE-112-lsrB,然后转化至E.coli WM3064感受态细胞内,得到接合转移菌株,通过同源重组的方法构建A.veronii的lsrB基因缺失株ΔlsrB,随后比较A.veronii TH0426菌株与ΔlsrB菌株的生长性能,并利用含3%琼脂的LB固体培养基检测ΔlsrB菌株的运动能力,最后利用实时荧光定量PCR方法检测ΔlsrB菌株中15个鞭毛主要调控基因的表达情况。结果表明:经PCR及测序鉴定后,成功扩增出lsrB基因的上下游同源臂片段L1与L2,通过重叠PCR得到基因片段L1L2,并成功构建出重组自杀性质粒pRE-112-lsrB和得到接合转移供体菌株WM3064(pRE-112-lsrB),通过一次接合转移与二次同源重组成功得到lsrB基因缺失菌株ΔlsrB;与A.veronii TH0426菌株相比,ΔlsrB菌株的生长性能保持不变,但在含3%琼脂的LB固体培养基中运动距离减小,同时ΔlsrB菌株中15个鞭毛主要调控基因的表达量显著高于A.veronii TH0426菌株(P<0.05)。说明lsrB基因可影响A.veronii TH0426菌株的运动性。

In order to study the effect of lsrB gene on the motility of Aeromonas veronii(A. veronii) strain TH0426, in the experiment, the genome of A. veronii TH0426 strain was used as the template to amplify the upstream and downstream homology arm fragments L1 and L2 of the lsrB gene, and to use overlapping PCR to connect L1 and L2 and to construct the recombinant suicide plasmid pRE-112-lsrB, which was transformed into competent cells of E. coli WM3064 strain to obtain a conjugative transfer strain, and the A. veronii lsrB gene deletion strain ΔlsrB was constructed by homologous recombination. Then, the growth performance of A. veronii TH0426 strain and ΔlsrB strain was compared, and the motility of ΔlsrB strain was detected by LB solid medium containing 3% agar. Finally, the expression of 15 major flagella-regulation genes in ΔlsrB strain was detected by real-time quantitative PCR method. The results showed that after PCR and sequencing identification, the upstream and downstream homology arm fragments L1 and L2 of the lsrB gene were successfully amplified, and the gene fragment L1 L2 was obtained by overlapping PCR;the recombinant suicide plasmid pRE-112-lsrB was successfully constructed and the conjugative transfer donor strain WM3064(pRE-112-lsrB) was obtained;the lsrB gene deletion strain ΔlsrB was successfully obtained after one conjugative transfer and two homologous recombination screening;compared with the A. veronii TH0426 strain, the growth performance of the ΔlsrB strain remained unchanged, but the movement distance was reduced in LB solid medium containing 3% agar, and the expression levels of 15 major flagella-regulation genes were significantly higher in the ΔlsrB strain than those in A. veronii TH0426 strain(P<0.05). The results indicated that lsrB gene played a role in the motility of A. veronii TH0426 strain.