The Study On Antimicrobial Activity Against Plant Pathogenic Fungi And Prefractionation Of Antimicrobial Peptides From Insects

In this paper, the antimicrobial active crude extracts of antimicrobial peptides (AMP)were obtained from the larvae of Galleria mellonella, the larvae of Tenebrio molitor and the adult of Eupolyphaga sinensis after induction, the extracts were frozen, heated processing. The antimicrobial activity of crude extracts were detected against the 17 kinds of plant pathogenic fungi, to measure the antimicrobial spectrum of three kinds insects. The prefractionation conditions was groped by the separation with Sephadex G-50 Medium gel filtration, and the antimicrobial activity of various components after the separation was texted. By Tricine-glycerol-SDS-PAGE, the molecular weight range of the main active ingredient was determined. The results are as follow:1. After the induction by pricking with Escherichia coli germy needle, the antimicrobial extracts of AMP from the three kinds of insects was obtained by getting hemolymph directly from G.mellonella, by grinding extraction from T.molitor and E.sinensis. And the crude extracts were undertaken three treatment to divid into fresh, frozen, heated as three treatment.The crude extracts of AMP from each insect was undertaken antimicrobial activity detection against 17 kinds of plant pathogenic fungi: the extract of AMP from G.mellonella has visible inhibition against 10 kinds fungi among the all, it has the best inhibitory effect against Cytospora.coccodes (Wallr.)Hughes with fungistasis rate above 65%. The extract of AMP from T.molitor has visible inhibition against 7 kinds fungi among the all, it has the best inhibitory effect against Pythium aphanidermatum (Eds.)Fitzp with fungistasis rate at 55%. The extract of AMP from E.sinensis has visible inhibition against 11 kinds fungi among the all, it has the best inhibitory effect against Fusarium oxysporium Schl.f.sp. with fungistasis rate at 60%.After refrigeration and heating, the extract of AMP from the three kinds of insect retained their inhibitory activity against the plant pathogenic fungus. There is no difference between refrigeration or heating treatment and fresh extracts at inhibitory effect against the pathogenic fungus, it indicated that the antimicrobial extracts from the three kinds of insects were good characteristic with subzero-bearing and thermostability, so the antimicribial agent were inferred for the peptides material.2. The 50mM ammonium acetate was selected as suitability elution buffer. There are two separete components,â… andâ…¡, after the elution. The results of antimicrobial activity detection showed that each component retained a bit of antimicrobial activity which the inhibitory effect of componentâ…¡is better than componentâ… .3. The results of Tricine-glycerol SDS-PAGE showed that there are no visible cross between componentâ… andâ…¡which obtained after gel filtration of every extracts of AMP from every kind of inscet. It manifested that the extracts of AMP from three kinds of insects were completely separated by gel filtration separation. That indicated that it was fitting to prefractionation of extract of AMP from the three kinds of insect by Sephadex G-50 gel separation with 50mM ammonium4. By the analysis of electrophoretic software, the componentâ…¡with better antimicrobial activity which obtained in the gel filtration separation of extracts of AMP from three kinds of insect, are a series of porteins from 6kDa to 30kDa.