| The pesticide resistance in plant pathogens has seriously affected the effective control for crop diseases,especially the generation of multi-resistant bacteria has threatened the animal,plant and human health constantly.Antimicrobial peptides?AMPs?have received a considerable amount of attention due to their significant role against fungi,bacteria,parasites,viruses,inflammation and tumor cells,thus been excavated as new protein pesticides and biocontrol agents.Isatis indigotica Fortune,a Chinese traditional medicinal plant,belongs to the family Brassicaceae,and famous for a variety of medicinal properties including anti-microbe,anti-tumor,anti-leukemia,improve immunity,inhibit platelet aggregation,detoxification,anti-inflammatory and anti-allergic.Bacillus subtilis as an effective tool to study the expression of foreign proteins,does not produce endotoxins and has the capacity to secrete proteins directly into the extracellular media.It is widely used as an ideal host for the production and expression of heterologous proteins.In this study,B.subtilis expression system was used for the isolation and screening of AMPs from I.indigotica.The basic objective of this study was to explore new AMPs and provide the possible solution for the development of effective agents for drug-resistant pathogens.The main findings are shown as below:1. c DNA library was constructed with the titer of 5.6x106 CFU/m L,and screened for AMPs.Fourteen candidate genes were identified based on their encoded peptides with strong antimicrobial activities against gram-positive and gram-negative bacteria.From them,Ii R515 and Ii R915 were selected for further study.It was observed that Ii R515 and Ii R915 showed strong inhibition effect against Botrytis cinerea B05.10.Vectors of Ii R515 and Ii R915 and His-tag gene were constructed and the fusion peptides were expressed and purified.MICs of Ii R515 and Ii R915 against indicator bacteria ?Clavibacter fangii 1.1999,C.michiganensis YCKYBI,Xanthomonas oryzae pv oryzae XG-25,Ralstonia solanacearum R21-5?ranged from 10-20?M and 15-25?M, respectively.These two peptides were considered to be novel because no homologues were dected in NCBI BLAST?National Center for Biotechnology Information and Basic Local Alignment Search Tool?and the antimicrobial peptide database?APD?.2. Ii R515 and Ii R915 has stability properties with wide application potential.Ii R515 and Ii R915 exhibited strong antimicrobial reaction after treated at 100°C for 15 min and declared as thermally stable AMPs.Theses AMPs were found highly active at p H 3-9 and under UV light treatment?365 nm UV light for 150 min?.The activity of Ii R515 and Ii R915 was analyzed against seven different enzymes?Protease E,protease K, pepsin,trypsin,papain,a-amylase,lipase?and found that Ii R515 and Ii R915 could be degraded by proteinase K and E.Ii R515 and Ii R915 also exhibited the strong antimicrobial reaction after treatment with different chemical reagents?UREA,EDTA, SDS and TWEEN-20?.3. Ii R515 and Ii R915 can control the occurrence of crop diseases,obviously.They exhibited the significant inhibition against soil-borne pathogens,C.fangii 1.1999 and C.michiganensis YCKYBI.Detached leaf assay revealed that Ii R515 and Ii R915 had the potential to inhibit the growth of X.oryzae pv.oryzicola RH3 on rice leaves.It was also observed that these two AMPs can inhibit the growth of Phytophthora capsici LT263 on Nicotiana benthamiana leaves.Products from p TRV2 constructs of Ii R515 and Ii R915 in Agrobacterium tumefaciens EHA105 also inhibited the growth of B.cinerea B05.10 on N.benthamiana leaves.4. Ii R515 and Ii R915 were relatively safe for animal cells and didn't exhibit hemolytic activity against mammalian cells.Ii R515 and Ii R915 exhibited the notable impact on Caenorhabditis elegans N2 tropism with non-significant nematicidal activity. Non-notable impact of these peptides on C.elegans fecundity and body size was also observed after feeding with Ii R515-and Ii R915-transformed Bacillus strains, respectively.Hemolytic activity assay revealed Ii R515 and Ii R915 peptides as safe product for mammals and humans and,in future these AMPs may act as potential source for clinical medicine development.5. Antimicrobial peptides have a variety of modes of action including enhancement of pathogens cell wall and cytomembrane permeability as killing mechanism.Scanning electron microscopic?SEM?and transmission electron microscopic?TEM?images revealed the shrinkage,distortion,deformation and perforation on the cytomembrane of Ii R515?and Ii R915-transformed Bacillus cells as compared with WB800-e control.The Propidium iodide?PI?staining of Ii R515?and Ii R915-transformed Bacillus cells exhibited the red fluorescence under the Confocal laser scanning microscope?CLSM?as compared to WB800-e.Cytometric analysis also revealed significantly higher fluorescence intensity and intracellular particle complexity of the Ii R515?and Ii R915-transformed Bacillus cells as compared to control strains.The cell membrane fluidity analysis of the Ii R515?and Ii R915-transformed Bacillus cells also revealed the significant differences with WB800-e control.The electrical potential????of the cytoplasmic membrane was also significantly increased with increasing incubation time as compared to control.Based on these findings,the possible mode of action of Ii R515 and Ii R915 was to interrupt or rupture the cell membrane. |
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