黏液型铜绿假单胞菌PA2815基因的敲除与功能鉴定

Gene knockout and functional identification of PA2815 gene from mucoid Pseudomonas aeruginosa

目的构建黏液型铜绿假单胞菌64(PA-64)株2815基因敲除株PA-64/ΔPA2815,建立有效的PA2815基因敲除平台。方法利用自杀质粒p CVD442及基因同源重组技术敲除PA-64株PA2815基因,并采用PCR及测序检测方法筛选获得PA2815基因被Gm抗性基因取代的克隆,命名为PA-64/ΔPA2815。比较敲除株与野生株之间的生长特性、抗生素的敏感性、藻酸盐以及绿脓菌素分泌的差异。结果 PA-64/ΔPA2815菌株与野生株的PA2815基因两侧同源臂片段PCR产物相比缺少1 480 bp,成功构建了黏液型铜绿假单胞菌PA2815基因敲除株。体外实验证实敲除株相比野生株生长速度略慢,藻酸盐分泌显著降低,差异有统计学意义(P<0.05),而绿脓菌素分泌显著升高(P<0.05),但在抗生素的敏感性方面差异无统计学意义(P>0.05)。结论利用同源重组原理成功构建黏液型铜绿假单胞菌PA2815基因敲除平台;PA2815基因不影响黏液型铜绿假单胞菌对抗生素的敏感性,但可以正向调控其藻酸盐的分泌和菌落生长状态,负向调控绿脓菌素的分泌。

Objective To construct mucoid Pseudomonas aeruginosa 64（ PA-64） PA2815 gene knockout strain, and establish an efficient PA2815 gene knockout platform. Methods The PA2815 gene was knocked out from the genome of PA-64 by using suicide vector p CVD442 and homologous recombination, and then the PA2815 gene was replaced by GM resistance gene; confirmed by PCR and sequencing; the recombinant strain was named PA-64/ΔPA2815. The survival abilities, drugs sensitivity, alginate and pyocyanin of PA-64 and PA-64/ΔPA2815 were separately examined. Results PA2815 gene homologous arm fragments of PA-64/ΔPA2815 was lack of 1 480 bp compared with the wild strain. The mucoid PA2815 gene knockout strain was successfully constructed. The PA-64/ΔPA2815 knockout strain had a little weaker survival abilities, significantly lower alginate secretion and higher pyocyanin secretion than wild strain in vitro（P〈0.05）, but there was no difference in the drug sensitivity（P〉0.05）. Conclusion PA2815 gene knockout platform was successfully constructed by homologous recombination. The PA2815 gene does not affect the susceptibility of mucoid Pseudomonas aeruginosa to drugs, but it could positively regulate the secretion of alginate and colony growth state, simultaneously,negatively regulates pyocyanin secretion.