Cloning Of Aegilops Tauschii Cosson Antimicrobial Genes Using Bacillus Subtilis Expression System

Antimicrobial peptides(AMPs)are an important part of the innate immune system,and they are widely present in many organisms in nature.In recent years,due to the emergence of pathogen resistance,AMPs have received considerable attention.However,there are still have some difficulties in screening for new AMPs.In this study,a method for efficient screening of AMPs has been established in our laboratory through the Busillus subtilis expression system.B.subtilis expression system was used to construct Aegilops tauschii Cosson c DNA library,and screened after the library quality evaluation.The screening was based on the antibacterial protein causing damage to the host cells and thus cause autolysis.Under the Scanning Electron Microscope(SEM),the candidate protein strains can be observed as shrink and perforate.Moreover,37 AMPs were obtained through preliminary screening.In addition,the candidate AMPs were extracted by the ammonium sulfate method,the inhibitory effects on bacteria were observed,and 6 candidate AMPs that inhibited multiple bacteria were screened again.In this study,2 candidate AMPs with broad spectrum resistance were selected for further research and the secondary structure prediction tool was used to predict the biological information of the 6 candidate AMPs.The 2 AMPs used in this study have both?-helix and?-sheet,so we speculate that peptides containing both?-helix and?-sheet have more significant antibacterial activity.Thermal stability experiments were performed on 2 candidate AMPs,and the results showed that the 2 antibacterial peptides still had antibacterial activity even under high temperature treatment at 100?.The results of hemolytic experiments showed that the hemolysis rate of the 2 candidate AMPs was less than 3%under the treatment of the highest concentration of 1000?g/m L.After incubating the purified peptide At R100 with bacterial cells,it was found that bacterial cell membranes treated with the candidate AMPs were damaged.Propidium iodide(PI)can enter cells through damaged cell membranes and show red under fluorescence,indicating that the screened antibacterial peptide has the activity of destroying bacterial cell membranes.Liquid fermentation and spray drying were performed on 2 candidate AMPs and empty vector strains to obtain B.subtilis powder.When the powder was diluted to 1×10~6 CFU/m L,2 candidate AMPs were found to have the effect of inhibiting tomato Botrytis cinerea.Overall experiments have shown that the 2 candidate AMPs have potential as biological agents.In summary,this study used the B.subtilis expression system to screen for AMPs,and obtained two new AMPs with antibacterial and antifungal activity.