[Genetically marking of natural biocontrol bacterium Bacillus subtilis strains with green fluorescent protein gene]

The full length sequence of the promoter and gfp gene were obtained respectively by PCR with two pairs unique primers PxyF/R and primers gfpF/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 PxyF/gfpR 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 degrees C, and the losing rate of plasmid was less than 3.5 x 10(-4) per generation.