Establishment And Evaluation Of Transposon Insertion Mutant Library For Endophytic Biocontrol Strain Bacillus Cereus 0-9

Biological control plays an important role in agriculture as an environment-friendly method of comprehensive control of plant disease. Nowadays, the study of biological control is no longer confined to passively separate biocontrol bacteria from nature, but to find the mechanism of biocontrol bacteria by establish mutant libraries.Bacillus cereus 0-9, isolated from the health wheat root by our laboratory, showed the ability of forming biofilm on non-biological materials and the effective function on controlling the wheat sharp eyespot in the greenhouse; and could colonize the wheat rhizosphere well.In this study, we measured the growth curve of 0-9, and according to the curve, we choose the cells of 0-9, which is in the mid-logarithmic phase, as the material of making protoplasts. After comparing the effective of making protoplasts under different conditions, we ultimately chose the following condition: breaking the cells'wall at 42℃45min by using lysozyme (0.25 mg/mL), and we got the protoplasts of 0-9 with high regeneration rate.Then, plasmid pMarB carrying transponson TnYLB-1 was transformed into 0-9 protoplasts by electroporation. According to the transposon TnYLB-1 mediated insertional mutagenesis technique, the transposon TnYLB-1 successfully inserted the genome of 0-9 and 1125 mutants were screened that are resistant to kanamycin but erythromycin.In order to verify integration of TnYLB-1 into 0-9 chromosome and test whether the insertions are likely to be random, PCR and southern blotting analyses were performed on 5 kanamycin resistant clones that had been named 539,573,609,629,638 and showed obvious difference with the wild type. And the PCR amplification results proved the fact that the five kanamycin resistant clones have homologous sequence with transposon TnYLB-1, while the wild strain without; moreover, the result of southern blotting analyses told us three of the 5 kanamycin resistant clones, that is 539,609,629, have single unique insertions; however, the insertions on the chromosome DNA of 573and 638 were not detected.In addition, we compared the phenotypic characters between the 5 kanamycin resistant clones and the wild strain 0-9, such as the ability of forming biofilm, the characteristics of colony morphology and swimming, the effective of bio-control and the root-colonization ability; as a result, we found that there is obvious discrepancy between them. In sum, we think that the transposon TnYLB-1 was successfully inserted on the genome of Bacillus cereus 0-9 with randomness of insertion sites; therefore, we could isolate insertion mutations from the mutant library of 0-9, and research the relative function genes which play important role in preventing plant diseases.