| Antimicrobial peptides(AMPs)are an important part of the innate immune system of plants,which have strong broad-spectrum antimicrobial activities.As an alternative of antibiotics,AMPs have good application prospects.As a major germplasm resource,Solanum demissum is abundant in resistance genes.A new method developed by our research group is the screening of resistance genes from the c DNA libraries at higher flux using built-in indicator bacteria,meaning that the c DNA expression library was transferred into the pathogen indicator bacteria so that these expressed foreign proteins can play an antibacterial function against them from inside.In this study,S.demissum c DNA library was established by Bacillus subtilis expression system.Using autolysis as the screening standard,27 strains with stable autolysis were obtained from 3650 transformants in the c DNA library.The extracellular proteins of these strains were extracted by ammonium sulfate precipitation method,and their antibacterial activities were detected.Ten monoclonal strains with strong antibacterial activities were screened.After sequence alignment,six new antimicrobial peptides were obtained.Sd R2332 and Sd R2770 were selected for further study.The thermal stability,enzymatic stability and UV stability of Sd R2332 and Sd R2770 were determined.After treatment at 50℃ and 80℃,the antibacterial activities of Sd R2332 and Sd R2770 did not change,but after treatment at 100℃,the antibacterial activities of Sd R2332 and Sd R2770 decreased to a certain extent.In the enzyme stability test,the antibacterial activity of antimicrobial peptides treated with trypsin and lipase had no significant change,but the antibacterial activities of Sd R2332 and Sd R2770 were decreased in varying degrees after treated with the other five kinds of protease.After UV treatment for 2 h and 4 h,the antibacterial activities of antimicrobial peptides had no significant change.These results show that the antibacterial peptides Sd R2332 and Sd R2770 have excellent thermal stability,UV stability and certain enzyme stability.The antimicrobial peptides Sd R2332 and Sd R2770 were purified by using His-tag and Tobacco Etch Virus-tag,and then SDS-PAGE electrophoresis was performed.The results showed that the bands of Sd R2332 and Sd R2770 were about 9.1 k Da and 3.6k Da,respectively,which were consistent with the prediction,indicating that the target protein was successfully expressed.Then the minimum inhibitory concentration(MIC)of antimicrobial peptides Sd R2332 and Sd R2770 were determined,and polymyxin B(MIC:5.00-25.0 μm)was selected as the positive control.The results showed that the antimicrobial peptides Sd R2332(MIC:14.70-27.0 μm)and Sd R2770(MIC:8.20-20.0μm)showed good broad-spectrum antibacterial activities against both Gram-positive and Gram-negative bacteria,and could kill pathogens at low concentration.The results of hemolytic test showed that the hemolytic activity of antimicrobial peptides Sd R2332 and Sd R2770 on mice red blood cells was still lower than 5% even at the concentration of 5×MIC,indicating that the antimicrobial peptides Sd R2332 and Sd R2770 had no toxic effect on mammals.After treatment with antimicrobial peptides,R.solanacearum appeared obvious ROS burst phenomenon and the cells had a certain degree of shrinkage and damage under the electron microscope,which indicated that the antimicrobial peptides might act on the cell membrane of the pathogen to kill the pathogen.In q RT-PCR analysis,the expression of Sd R2770 in plants inoculated with R.solancearum was significantly up-regulated,which may be related to plant disease resistance.However,the expression of Sd R2332 did not change significantly.In conclusion,two new antimicrobial peptides Sd R2332 and Sd R2770 with broadspectrum antimicrobial activities were screened by B.subtilis expression system.They have stable physicochemical properties and are safe for human and livestock,which is helpful for further study on the function of resistance genes of S.demissum. |
