| Paenibacillus polymyxa can produce a variety of antimicrobial substances. It has an effective activity against bacteria, fungi and virus and also can prevent kinds of plant diseases. LI-F type antibiotics are natural broad-spectrum antimicrobial substances which are safe and stable that make them have a great potential application in the field of medicine, agriculture and food. However, the low yield of the wild strains makes the production costs increase. It causes that the LI-F type antibiotics can not be mass-produced, thus limiting their industrial applications.The research using Paenibacillus polymyxa JSa-9 as the original strain through different breeding ways to improve the yield of LI-F in order to obtain a stable high-yield strain, lays the foundation for industrial production and applications in the future. The results are as follows:1. Established a standard curve equation by HPLC to calculate the yield of LI-F type antibiotics by using preparative liquid chromatograph. LI-F type antibiotics were isolated and purified by preparative liquid chromatography, the purity was above 93.31%. The standard curve equation was:y=3401x-280.9, R2= 0.9991, calculated the yield of LI-F type antibiotics in the fermentation broth of Paenibacillus polymyxa JSa-9 was 88.19 mg/L.2. Studied on breeding high-yielding auxotrophic strains by different physical and chemical mutagens. Strain JSa-9 cells were treated with 1-methyl-3-nitro-l-nitroso-guanidin, Co-y ray irradiation and the low energy N+ ion implantation energy. Counting the positive mutation rate and the death rate, determined the following conditions of mutations:the optimal mutagenesis concentration of NTG and dose of 60Co-γ ray were respectively identified as 50μg·mL-1 and 200Gy.The optimal low energy N+ ion implantation energy was 20 keV and the optimal injection time were 30s. A phenylalanine auxotrophic strain N1-37 and a histidine auxotrophic strain N2-27 were ultimately screened. The results of the inhibition experiments on Staphylococcus aureus, Fusarium oxysporum and Fusarium oxysporum showed that the inhibition activity of strain N1-37 and strain N2-27 respectively increased 45.54% and 32.35%,23.12% and 21.85%,18.08% and 15.59% compared with original strain JSa-9. The yields of LI-F type antibiotics produced by strain N1-37 and strain N2-27 were separately 1.71-fold and 1.4-fold of strain JSa-9.3. The protoplast fusion technology of two auxotrophic strains N1-37 and N2-27 were studied.The optimal conditions for the preparation of protoplast were as follows: cultured the seeds solution for 8h to collect cells; the lysozyme concentration was 0.1 mg/mL; fusion temperature was 37℃; fusion time was 7 minutes. The protoplasm formation rate reached more than 97%, and the renewable rate was better than 30%. The optimized conditions for PEG6000 induced fusion of the protoplasts were:the concentration of PEG6000 was 40%, pH ratio was 9.0 the fusion time was 15 minutes and the temperature was 37℃. A high-yielding fusion strain F5-15 was obtained. The results of the inhibition experiment on Staphylococcus aureus, Fusarium oxysporum and Fusarium oxysporum showed that the inhibition activity of strain F5-15 respectively increased 84.70%,49.32% and 38.96%. The yield of LI-F type antibiotics produced by strain F5-15 were 3.1-fold of strain JSa-9.4. The original strain JSa-9 was as a control, using real-time quantitative PCR method to analyze the gene expressions of the synthase genes fus A1、fus A2、fus C1 and fus C2 of LI-F type antibiotics in strain F5-15. First, extracted RNA through Trizol method, measured R(OD260/OD280) value was 1.93, indicating that the RNA was high purity. Then, reverse transcription synthesis of cDNA as a template,16S rDNA as a reference gene, by real-time quantitative PCR, the results showed that the expressions of the target genes was separately 2.1-fold,10.48-fold,2.48-fold and 11.8-fold of strain JSa-9. |
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