绿色荧光蛋白基因标记野生型生防枯草芽孢杆菌的研究

Genetically Marking of Natural Biocontrol Bacterium Bacillus subtilis Strains with Green Fluorescent Protein Gene

根据绿色荧光蛋白基因和枯草芽孢杆菌木糖诱导型启动子PxylR序列 ,分别设计两对特异引物primersPxyF R和primersgfpF R ,扩增获得了完整的启动子PxylR和gfp基因序列。进一步以上述产物混合物为模板 ,以primerPxyF primergfpR做引物进行重迭PCR ,获得了PxylR gfp重组翻译融合表达盒。经SphⅠ和KpnⅠ完全酶切后 ,将PxylR gfp表达盒分别插入大肠杆菌_苏云金芽孢杆菌穿梭载体pHT315和大肠杆菌 枯草芽孢杆菌穿梭载体pRP2 2。相应的重组表达质粒pGFP315和pGFP2 2转化枯草芽孢杆菌感受态细胞。前者在标准菌株 16 8中得到良好发光表型 ,后者则在标准菌株 16 8和野生目标菌株B916中均得到良好的发光表型。室内平板抑菌实验结果显示B916生防效果与出发菌株没有明显差异 ,遗传稳定性研究表明连续稀释培养约 175代后 ,工程菌株稳定性为 94 % ,质粒丢失频率低于 3 5× 10 - 4 代。

The full length sequence of the promoter and gfp gene were obtained respectively by PCR with two pairs unique primers Pxy F/R and primers gfp F/R, which were designed according to the gfp gene and promoter sequence of xylase operon from Bacillus subtilis 168, and the DNA template plasmids pHT315_xyIR and pGFPuv. Furthermore, the fused translational expression cassette PxylR gfp was constructed using overlapping PCR technique with the primers pair Pxy F/ gfp R and the mixture of above PCR production. After being digested by Kpn Ⅰ and Sph Ⅰ, PxylR gfp expression cassette was inserted into E.coli_B.thuringiensis shuttle vecter pHT315 and E.coli_B.subtilis shuttle vecter pRP22, and the resulted recombinant plasmids were named as pGFP315 and pGFP22 respectively. Both recombinant plasmids were transferred into B. subtilis lab strain 168 and the resulted transformants are bright green performance under 365 nm UV light. However, only pGFP22 can be introduced into the natural strain B916 The transformants containing pGFP22 have bright green performance under 365 nm UV light and was named B916 gfp . Antifungal activities testing results proved that there is no obvious difference between B916 and the engineered strains B916 gfp . Research results also showed that the stability of B916 gfp was 94% after growth about 175 generations at 37℃, and the losing rate of plasmid was less than 3 5×10 -4 per generation.