Construction Of SinI Mutant And The Influence On The Biofilm Formation And Antimicrobial Activity In Bacillus Amyloliquefaciens

In this research,we mainly analyzed the sinI gene of B. amyloliquefaciens PEBA20 which is isolated from poplar, sinl gene is not only regulate the biofilm formation, but also coordinate the bacteriostatic activity, spore formation and various cellular processes of the bacteria. Homologous recombination method is used to build the missing sinl gene mutation. And then verify the influence of B. amyloliquefaciens PEBA20 on biofilm formation, antibacterial activity and spore formation.The main research results are as follows:1. We designed practicable procedure for the construction of PEBA20 variant with impaired sinl gene. Two homologous recombinant arms, sinIF and sinIR, were amplified by PCR and connected successfully by overlap extension PCR. Then we have obtained recombinant suicide plasmid of sinIF-sinIR-pK18mobsacB and transformed it into PEBA20 competent cell by electroporation. Kanamycin and sucrose was used to select the PEBA20 sinl mutant strains. Using B. amyloliquefaciens PEB A20 DNA as a template for PCR to select positive clone, and further determined sinI gene deletion mutant strains by sequencing.2. To observe the biofilm formation dynamic characteristics of the B. amyloliquefaciens PEBA20 wild strains and sinl gene deletion mutant strains on solid-gas interface and liquid-gas interface. The results showed that the wild strain and the mutant strains both have the ability of biofilm formation, but compared with wild strains, complex degree of biofilm of sinl gene deletion mutant strains is degraded, we found that sinl gene deletion mutant strains can not form complex fold structure of biofilm on liquid-gas interface. Therefore, sinl gene may be lead to the ability of biofilm formation degraded even lossed.3. Keeping on observing the phenotypic of motility of B. amyloliquefaciens PEBA20 sinl gene deletion mutant strains. The results showed that compared with wild strains, flagellum-dependent swarming motility of the mutant strains is falling. So sinl gene deletion affect the development of swarming motility.4. By bacteriostatic activity determination in vitro, the B. amyloliquefaciens PEBA20 wild type strains showed great inhibitory effect to Valsa mali, Botryospuaeria berengeriana and Colletotrichum gloeosporioides on colony growth and hyphae form. The Microscopic observation showed that hyphae forms at the edge of the inhibition of pathogenic bacteria happened different degrees abnormal changes. B. amyloliquefaciens PEBA20 producing antibiotic substances caused pathogen mycelial morphology distortion, and then influenced the growth.5. Analyzed the antibiotic activity of B. amyloliquefaciens PEBA20 wild strains and sinl gene deletion mutant strains with four fungi and three bacterial. The results showed that compared with the wild strains, the antimicrobial effect of sinl mutant strains about fungi and bacterial are all decreased, and the significant difference level of these fungi and bacteria is more remarkable. This suggests that the sinl gene deletion affects the antibacterial activity of biocontrol bacteria about fungi and bacteria.6. Measuring and comparing bacteria growth curve, spore and body shape of B. amyloliquefaciens PEBA20 sinl mutant strains. Results showed that the sinl mutant population growth was significantly inhibited. Via detecting B. amyloliquefaciens PEBA20 sinl mutant strains, spores still did not be found until 120 h. sinI gene deletion influence the spore production of strains, and the thallus of the mutant strains is relatively short in comparison with wild strains.