基于xkdB假基因座位的瓦雷兹芽孢杆菌FZB42超折叠GFP标记研究

Labeling of Bacillus velezensis FZB42 by expressing superfolder GFP at the locus of the pseudogene xkdB

【目的】利用一个GFP的超折叠变体SfGFP(superfolder GFP)对瓦雷兹芽孢杆菌(Bacillus velezensis)FZB42菌株进行标记,以期确立一种瓦雷兹芽孢杆菌及近缘芽孢杆菌通用的高亮GFP标记手段,同时,为了方便后续生物膜和分子互作的相关研究,测试SfGFP基因插入位点,假基因xkdB,作为外源基因表达座位的可行性。【方法】利用基因工程技术构建了一系列质粒,然后通过同源重组的方式,分别获得xkdB敲除菌株FBS373和SfGFP标记菌株FBS374,分别测试这些菌株在生长速度、碳源利用、荧光亮度、生物膜形成、swarming运动性等方面的差异。【结果】本研究成功构建了SfGFP标记的瓦雷兹芽孢杆菌FZB42,其荧光亮度是gfp+变体标记菌株的5倍以上;xkdB基因敲除对瓦雷兹芽孢杆菌FZB42生长速度、不同碳源利用、生物膜形成和运动性等方面无明显影响。【结论】通过本研究我们确认了xkdB基因位点作为瓦雷兹芽孢杆菌FZB42基因组上外源基因表达的中性位点的可行性,同时,通过在xkdB基因座位表达了SfGFP基因,成功对FZB42进行了高亮标记,对同类菌株的标记具有较好的借鉴价值。

[Objective]We used a super-bright variant of GFP,superfolder GFP(SfGFP),to label the Bacillus velezensis FZB42 strain,so as to establish a universal GFP labeling method for B.velezensis.This helps to develop a viable technique to study the colonization mechanism of beneficial rhizobacteria.[Methods]A mutant strain was generated by integrating SfGFP into a neutral locus called xkdB in the genome of FZB42,and then the fluorescence intensities of the mutant strains carrying different GFPs were detected to evaluate the influence of xkdB knockout.[Results]In this study,a marker strain labeled with SfGFP for FZB42 was constructed,whose fluorescence intensity was more than 5 times that of the gfp+variant marker strain.The knockout of xkdB did not affect the growth rate,carbon source utilization,motility or biofilm formation,which confirmed the feasibility of using xkdB as a neutral locus for exogenous gene expression.[Conclusion]In this study,we confirmed a new locus suitable for the expression of foreign genes in the FZB42 genome,and exogenously expressed SfGFP at this locus,thus labeling FZB42 with fluorescence.This study provides a reference for the labeling of similar strains.