利用绿色荧光蛋白标记革兰氏阴性细菌的研究

Labeling gram-negative bacteria using green fluorescent protein

构建了具有不同抗性且能够组成型表达绿色荧光蛋白的一系列转座子质粒pTnMod-OCm-G、pTnMod-OTc-G、pTnMod-OKm3-G和pTnMod-OGm-G,并通过三亲本杂交的方法,成功地将荧光蛋白基因分别插入到多环芳烃降解菌株Sphingomonas sp.12A和Pseudomonas sp.12B的基因组内,获得了具有降解多环芳烃特性,同时在没有抗生素选择压力下连续传代多次仍能够稳定组成型表达荧光的转化子.结果表明,该系列转座子不仅适合其它革兰氏阴性菌的遗传标记,也为进一步研究降解菌在污染环境中的存活能力和生态安全奠定了基础.

In this study,four transposable plasmids were constructed,pTnMod-OCm-G,pTnMod-OTc-G,pTnMod-OKm3-G and pTnMod-OGm-G,which can constitutively express green fluorescent protein. Via triparental mating,the GFP gene was inserted into genomes of Sphingomonas sp. 12A and Pseudomonas sp. 12B,two polycyclic aromatic hydrocarbons-degrading bacterial strains. The transformants,which can degrade polycyclic aromatic hydrocarbons,can constitutively express GFP without the presence of antibiotics through many generations of propagation. The transposons can be transferred to other gram negative bacteria and expressed stably. Therefore,they have potential to be used in research on bacterial survival and ecological safety in contaminated environments.