| The main contents of this thesis include two parts:The antibacterial mechanism of melittin against Xanthomonas oryzae pv.oryzae and The study on hemolysis of antibacterial peptidePart one:The antibacterial mechanism of melittin against Xanthomonas oryzae pv.oryzae.Xanthomonas oryzae pv.oryzae is one of the most destructive bacterial disease of rice,it is a Gram-negative bacteria which cause serious disease are responsible for severely reduced the quality and safely agricultural crop production and strongly influence human food supply.Most plant pathogenic bacteria based on synthetic chemicals are extensively used in agriculture.However,they are acutely toxic,causing long-term environmental pollution and even carcinogenic toward humans and other animals.Current day demands for food and environmental safely require a novel pesticide that shows high antimicrobial activity,with lower intrinsic toxicity to replace the tradition synthetic chemical pesticide in plant protection.Recently,antimicrobial peptides(AMPs)have attracted considerable attention by researchers which have been reported as candidates for plant protection products mainly against bacterial and fungal pathogens.melittin is a cationic peptide composed of 26 amino acid residues the major component of European honeybee venom from Apis mellifera,which is known to have strongly potential antibacterial activities,but no report on plant pathogenic bacteria.To gain the information on the mode of action of melittin,this study is focused on three aspects including the bacterial cell membrane,intracellular targets and protection of plants against diseases.The main results are as follows:1.An inhibition zone assay and the time-kill data of melittin was conducted to detect whether melittin showed antimicrobial activity to the Xanthomonas oryzae pv.oryzae.Rusults shows that at the concentration of 10?M,the antimicrobial activity of melittin has confirmed against the bacteria compared with the control and inhibited the growth of Xanthomonas oryzae pv.oryzae completely;Fluorescence microscopy showed melittin combine with the bacterial;the bacterial cell membrane integrity was examined by determination of the release of cytoplasm absorbing at 260nm and Scanning electron microscopy(SEM)together with transmission electron microscopy(TEM)allowed us the cytoplasm and membrane of bacteria were damaged by melittin,the intracellular components were tended to leach out;The T-ATPase level in lysed water was determined by using a commercial assay kit showed that the melittin did not significantly suppress the activity of T-ATPase.2.Confocal laser scanning microscopy showed that melittin penetrate the bacterial cell membrane accumulate in the cytoplasm;The fluorescence spectrophotometry measurements demonstrated that DNA and RNA synthesis was significantly reduced compared with the control group indicating that melittin had an adverse effect on nucleic acid synthesis;Gel retardation assay combine with four restriction enzymes all showed melittin can bind to the DNA or RNA;SDS-PAGE indicated that protein synthesis was significantly reduced following treatment of Xanthomonas oryzae pv.oryzae with melittin for 10h,some even lost.3.The result from the lesion measurement experiment demonstrates that the effect of melittin on the Xanthomonas oryzae pv.oryzae will lead to alleviate or inhibit and show the protection of plants against diseases.In summary,we first report the the antimicrobial mechanism of melittin to the Xanthomonas oryzae pv.oryzae,the antibacterial effects were attributed to its ability to interact with the bacterial cell wall and disrupt the cytoplasmic membrane by making holes and channels with leakage of cytoplasmic content.Meanwhile,melittin are able to penetrate bacterial membranes and reach in the cytoplasm showing that there is more than one mechanism of antimicrobial action.DNA?RNA and soluble protein reduced,DNA/RNA binding with melittin suggest that melittin may inhibite macromolecular biosynthesis and the expression of associated genes.These two modes eventually lead to bacterial cell death.melittin can inhibit the Xanthomonas oryzae pv.oryzae spreading,alleviating the illness showed that melittin may have potential applications as an antibacterial agent for phytopathogens.Part two:The study on hemolysis of antibacterial peptide.Antimicrobial peptides are the potential anticancer resources limited its directly application as an anticancer drugs because of causing the serious high hemolytic activity easily.Hemolysis is an important indicator of drug side effects,reducing the hemolytic activity of antimicrobial peptides for the development of antibacterial anticancer drugs is an urgent problem need to solve.However,the research on hemolytic mechanism of antimicrobial peptides still rarely and even unclear.This study selected three representative amphipathic a-helical antimicrobial peptides aureinl.2,buforinllb and melittin as materials,those peptides with different chain length,amino acid sequence and the number of positively charged,secondary structure,hemolytic and antibacterial antitumor activity are also different.This study further describes the hemolytic mechanism of antimicrobial peptides in order to promote the application of antimicrobial peptides in the field of anti-bacterial and anti-cancer.The main results are as follows:1.CD spectrum of the peptides was measured in distilled water and a membrane mimic condition in the 50%TFE solution.the peptides showed random coil structure in an aqueous solution,while displaying a typical a-helical spectrum with two peaks in 50%TFE solution.2.The determination of hemolytic activity showed that the three peptides could cause erythrocyte hemolysis in different degrees,the hemolytic activity of melittin was the strongest;Scanning electron microscopy showed that these peptides induced the red cells lacked integrity and membrane debris was visible;ATP level was closely associated with cellular metabolism and fate,melittin induced hemolysis is largely dependent ATP decline,the two others were not;SDS-PAGE analysis of membrane proteins revealed that these peptides all induced the appearance of several new protein bands.3.The effect of sialic acid,fucose,exogenous cholesterol and sugar molecules on the hemolysis showed that antibacterial peptides could interact with the sialic acid and fucose,exogenous cholesterol and sugar molecules could inhibit hemolysis in various degrees.In conclusion,this paper discusses the effect of different cationic antimicrobial peptides on erythrocyte membrane and erythrocyte membrane composition on hemolysis of antimicrobial peptides illustrated that red blood cell membrane components is closely related to hemolysis of antibacterial peptide.This difference may be due to insert the rat cell membrane in different ways for the peptide lead to different membrane changes,but eventually the cell membrane lysis.The difference of cationic antimicrobial peptides hemolysis is common results of antimicrobial peptide structure and red cell membrane components.The results of this study will provide the important theoretical basis for reducing hemolytic activity of antibacterial peptide in the use process and promoting the development of antimicrobial peptides for antimicrobial anticancer drugs. |
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