The Construction Of GFP-tagged Bacillus Strains And The Primary Study Of The Colonization On The Wheat

Bacillus cereus (83-6, A-47, and M-22, et al.) which were isolated in the laboratory, are a kind of biocontrol agents which can enhance the disease resistance of plant, increase the yield, improve the quality of crops, and advance the stress resistance of plant successfully. The previous study implies that they can colonize in the inner body of some plant, but the approach which the bacillus to enter the plant is unclear. We utilize the green fluorescent protein (GFP) to tag the strains, so that we can study the behaviors of the strains.We had inserted the gfp gene which was under the control of 4412 promoter into an E.coli-Bacillus shuttle vector. Then the new vector (pGFP4412) was transferred into bacillus by electroporation. Bacteria harboring the vector pGFP4412 produced high level of GFP. Therefore, the transformants appeared bright green under blue light. The experiments of stability of the gfp-labeled bacillus strain (A47-gfp) indicate that its stability is 92%. This implied that the strain of A-47-gfp could satisfy the need of study in the special period or the whole life of the annul plant. Then we used the gfp-labled bacillus to study its colonization on the surface of wheat.The gfp-labeled bacillus can emit brightly green light under the blue light and the native bacteria can not. The grains of soil can produce yellow or brown fluorescence, sometimes emit red light. There is an obvious difference between the outline of bacillus and soil grains'. So it is easy to distinguish them. The leave and the stem of wheat can emit red fluorescence and then turn into lightly yellow light gradually. The color of rootstock is lighter than the old root's, but it is easy to distinguish them from the gfp-labeled bacillus. The results indicate that A-47-gfp can colonize on the surface of wheat and the bacteria were more strongly stuck on the roots than leaves; In the direction from root base to tip, the bacterial distribution showed a clear decreasing tendency in wheat root. We had found A-47-gfp in the albumen of wheat.