Screening Of Garlic Antimicrobial Genes Using Bacillus Subtilis Expression System

Antimicrobial peptides(AMPs)have attracted more and more researcher's attention because of their wide applications in agriculture,food biopreservatives and medicinal value.Traditional techniques for screening peptides are time consuming and labor intensive,the probability of screening for new antimicrobial peptide encoding genes are very low.In order to resolve the above mentioned problems,this study was designed to establish a strategy for the screening of highly efficient AMPs by using Bacillus subtilis expression system.Garlic c DNA expression library was constructed by using the B.subtilis expression system,and evaluated the library quality together with the Escherichia coli garlic c DNA expression library preserved in our laboratory.B.subtilis and E.coli were used as target indicator bacteria,candidate proteins played antimicrobial roles from the inside of the indicator bacteria(internal effect).A total of(53/1700 transformants)and(170/3000 transformants)candidates antimicrobial proteins were screened by the B.subtilis system and E.coli system from garlic c DNA library,respectively.Changes in the cell morphology of the candidate bacteria were observed by Scanning Electron Microscopy(SEM),the results demonstrated that the bacteria of the candidate antimicrobial proteins were broken,damaged or wrinkled.We extracted crude proteins of candidate antimicrobial proteins and tested their activities under in vitro conditions.We selected 11 stable AMPs from the garlic c DNA library,which showed broad spectrum antibacterial and antifungal activities.These genes were named as novel genes,because they were not reported before when BLAST searched on NCBI.Through E.coli expression system only one peptide was identified with antibacterial activity.Therefore,after comparison of the two screening systems,it was confirmed B.subtilis screening system was more efficient and stable than the E.coli system.Bioinformatics tool was used to predict the secondary structure,isoelectric point,molecular mass and other properties of 11 AMPs,and these peptides were divided into 4 categories according to the secondary structure.After combining with the antibacterial data,it was speculated that peptide with both ?-helix and ?-sheet usually has strong antibacterial activity.In order to explore the application potential of AMPs,hemolysis experiments and thermal stability experiments were carried out.Results confirmed that the hemolytic activity was less than 5% at a peptide concentration of up to 1000 ?g/m L,and some peptides have antimicrobial activities under 100?.Application of AMPs on exogenous tobacco leaves can inhibit the Phytophthora capsici.The purified antimicrobial peptide As R416 was tested to treat bacterial cells,and the experimental results showed that the AMPs severely damage and destroy the integrity of the bacterial cell membrane.In conclusion,this study evaluated a novel,sensitive,and high-throughput screening strategy for the isolation of antimicrobial genes.Through this strategy 11 novel genes were identified with broad-spectrum resistance.Based on our findings,we speculated that plant,animal and human pathogenic bacteria,fungi or even cancer cells might be taken as the indicator target cells for screening specific antimicrobial genes.