凡纳滨对虾致病菌Shewanella putrefaciens WS13中crp基因无痕敲除体系构建及功能验证
Journal of Northeast Agricultural University（2023）
江苏师范大学
Abstract
以crp为靶标基因,建立凡纳滨对虾腐败菌株腐败希瓦氏菌(Shewanella putrefaciens)WS13无痕敲除方法,初步研究crp基因对S.putrefaciens WS13生物被膜形成的影响.利用Pir蛋白依赖的质粒pMMB1构建缺失载体,利用质粒pBBR1MCS-2-PaacC1构建回补载体,通过同源重组方法,实现对crp基因的无痕敲除,琼脂糖凝胶电泳和测序比对结果表明成功获得crp缺失株(Δcrp)和回补株(Ccrp).通过对S.putrefaciens WS13野生型(WT)、Δcrp和回补株Ccrp形成的生物被膜作定量分析,发现突变株Δcrp生物被膜量明显下降,回补菌株Ccrp的生物被膜量相对Δcrp明显上升,揭示crp影响S.putrefaciens WS13生物被膜形成.研究结果表明,质粒pMMB1和pBBR1MCS-2-PaacC1可用于S.putrefaciens WS13基因无痕敲除与回补,成功实现对靶标基因crp无痕敲除,并证实crp是影响WS13生物被膜形成的关键基因.
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Key words
Litopenaeus vannamei,Shewanella putrefaciens WS13,traceless knockout method
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