Genetically Marking Of Natural Biocontrol Bacterium Bacillus Subtilis Strains With Green Fluorescent Protein Gene

With the development of biological control technology, Bacillus subtilis play more and more important role in biological control. To find the rule of Bacillus subtilis in microorganism populations and discover the mechanism of biocontrol, a steady, effective marking system is needed.The full length sequence of the promoter and gfp gene were obtained respectively by PCR with two pairs unique primers PxyF/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 plasmids pHT315xyIR and pGFPuv. Further more, the translational fusing expression cassette xylR-gfp was constructed using overlapping PCR techniques with the primers pair PxyF/gfpR and the above PCR productions mixture.After digested by Kpn and SphI, the xylR-gfp expression cassette was inserted into E.coli-B.subtilis shuttle vectors pHT315 and pRP22, and the resulted plasmids were named as pGFP315 and pGFP22 respectively. Both recombinant plasmids were transferred into B. subtilis standard strain 168, the transformants obtained showed bright green performance under UV. However, only pGFP22 can be introduced into B916. The transformants containing pGFP22 have bright green performance under UV and was named B916-gfp.Plasmid stability research indicated that the stability of B916-gfp was 94%, and the losing rate of the plasmid was less than about 3.5 X10-4 per generation.The diameters of B916 and B916-gfp inhibition zones against Rhizoctonia solani Kiihn are 28.5mm, 26.5mm, respectively. The result showed antagonistic ability of B916 and B916-gfp have no significant difference. The growth curve of B916-gfp certified the B916-gfp had the same growth ability as the B916.B916-gfp was recollected from leaves of rice, the result showed density on leaves was about 105cfu/cm2 on the 15th day after inoculation, the density on stem was about 105cfu/cm on the 13 th day after inoculation, the density decreased during the period ofstudy.The result observed by fluorescent microscope showed B916-gfp could live on mycelium of Rhizoctonia solani Kiihn. To discover the antagonistic mechanism, more experiments are needed.