| Pinellia ternata has high economic value as a medicinal plant,and has a large number of endophyte resources in the plant.Endophytes can promote plant growth and also contain abundant resistance genes.Therefore,it is of great significance to study endophytes of P.ternata to improve their yield.Antibiotics are widely used in agricultural production because of their fast control effect,but the problem of drug resistance cannot be ignored.Antibacterial peptides have become potential substitutes for antibiotics due to their advantages of being less able to be overcome by pathogens and possessing natural antibacterial activities.In this study,163 endophytic bacteria and 53 endophytic fungi were isolated from P.ternata,and 13 endophytic bacteria and 6 endophytic fungi were identified by confrontation test with different pathogens.The endophytic fungal antagonistic effect of F23 was the most significant one.It was identified as Humicola fuscoatra by morphological and molecularanalyses.In this study,through the built-in library resistance gene screening technology of indicator bacteria established by our group,the H.fuscoatra is used as raw material and established H.fuscoatra c DNA library using Bacillus subtilis efficient secretion expression system.Taking the autolysis of the strains as the screening criterion,4336 strains in the library were screened,and 61 monoclonal strains with stable autolysis were obtained.Then,their extracellular crude protein was extracted by ammonium sulfate precipitation,five Gram-positive bacteria and Gram-negative bacteria were used as target bacteria to analyze their antibacterial effects,and the antibacterial peptide Hf R3283 and Hf R3877 with obvious and stable antibacterial effect were screened out and for further antibacterial peptide activity study.The research results showed that the antibacterial effects of antimicrobial peptides Hf R3283 and Hf R3877 remained stable under different conditions of UV and temperature treatments,while in the experiments after different bioenzyme treatments,they still maintained a stable antibacterial effect under the influence of lipase.It can be seen that the antimicrobial peptides Hf R3283 and Hf R3877 have good physicochemical properties and can be further investigated.In this study,the antimicrobial peptides Hf R3283 and Hf R3877 were purified by Ni-NTA plus protein TEV enzyme tag,and the resulting products were subjected to SDS-PAGE electrophoresis.The electrophoresis results were consistent with the predicted sizes,13.8 k Da and 21.5 k Da,respectively.Using polymyxin B as a positive control,the purified antimicrobial peptides Hf R3283 and Hf R3877 proteins were used for Minimum Inhibitory Concentration(MIC)determination,which showed that the MIC value of antimicrobial peptides Hf R3283 was in the range of 10.0-18.2 μg/m L interval and the antimicrobial peptides Hf R3877 was in the 13.8-20.0 μg/m L interval,and these two antimicrobial peptides showed significant antibacterial activity against Gram-negative and Gram-positive bacteria at very low concentrations.Subsequently,antimicrobial peptides Hf R3283 and Hf R3877 were cultured against five pathogenic fungi,and it was found that antimicrobial peptides Hf R3283 and Hf R3877 exhibited significant inhibitory effects on the growth of five pathogenic fungi hyphae.We used antimicrobial peptides at 5×MIC concentration to measure hemolysis of the hemoglobin of pigs to be treated with Hf R3283 and Hf R3877,which showed that the hemolysis rate was less than 1%.It was proved that the antimicrobial peptides Hf R3283 and Hf R3877 not only have broad-spectrum resistance but also have a good safety profile.In order to study the antibacterial mechanism,we used antimicrobial peptides Hf R3283 and Hf R3877 to treat X.oryzae pv.oryzae and C.michiganensis cells.Under scanning electron microscopy after observation,it was found that the cell surface of the two pathogens treated with antimicrobial peptides Hf R3283 and Hf R3877 were deformed,shrunken,or even ruptured,while the cell surface of the two pathogens treated with PBS buffer were smooth without damage.Thus,it was proved that the two antimicrobial peptides achieve bacteriostatic activity by damaging bacterial cell membranes.In order to test the control effect of antimicrobial peptides Hf R3283 and Hf R3877 on living plants,we inoculated the pathogenic bacteria treated with polymyxin B on tomato plants as a positive control,and inoculated the pathogenic bacteria treated with PBS buffer on tomato plants as a negative control.In contrast,the results showed that the tomato plants inoculated with the pathogenic bacteria treated with antimicrobial peptides Hf R3283 and Hf R3877 grew normally without obvious symptoms.In conclusion,in this study,163 endophytic bacteria and 53 endophytic fungi were isolated from P.ternata plants.Then,the H.fuscoatra c DNA library was established using the B.subtilis secretion expression system,and two novel antimicrobial peptides Hf R3283 and Hf R3877 with broad-spectrum antibacterial activity were screened from them.They have stable physicochemical properties and are relative safe,and also have good antibacterial activity on live tomato plants. |
