| The entomopathogenic nematode-symbiotic complex,which has rapid lethal toxicity to host insects,good production potential for being cultured in vitro,a wide range of hosts and is safe for plants,vertebrates and various invertebrates,has become the internationally biological pesticides with broad prospects and been widely used for pest control in the fields of agriculture,forestry and health.In the whole life cycle of entomopathogenic nematodes,the symbiotic bacteria which is in the intestinal of nematodes made the key role.They produce kinds of secondary metabolites under the competitive niche conditions.These natural products have the activity of antifungal,antibacterial and killing insects.Isolation and culture of entomopathogenic nematode symbiotic bacteria is an important way to obtain the secondary metabolites.which will be significance for developing a new bio-insecticides.Molecular biology research techniques,gene sequencing techniques and related bioinformatics analysis techniques are used to study the function of the secondary metabolites from the pathogenic nematede symbiotic bacteria.The results of the study are as follows:1.Good ecological environments,such as tussah production bases,orchards and cultivated land,were selected for getting the soil samples in this study.A total of 108 soil samples were collected from 11 counties in Liaoning Province.A total of 40 stains entomopathogenic nematodes were obtained and isolated by the method of Gallerua mellonella trapping.After small-scale fermentation and chemical screening of 10 stains primary symbiotic bacteria,we determined SN44 and SN52 as research objects.2.The symbiotic bacteria of entomopathogenic nematodes were identified by the traditional bacterial taxonomy,together with the molecular method of 16 S r DNA.The SN44 strain was Photorhabdus luminescence subsp.Kayaii,and SN52 strain was Xenorhabdus bovienii BE.3.Three secondary metabolites,identified as IPS,Y15-2 and Y-11 respectively,were identified for the compounds productions of P.luminescence SN44.Among them,Y-11 was isolated from Photorhabdus for the first time.A parallel liquid fermentation of cultured primary strains was carried out for obtaining the crude extract.Through TLC and liquid phase analysis,two compounds,Y15-2 and IPS were obtained from the crude extract of SN44.IPS is one of stibene compounds,which has antifungal,antibacterial and insecticidal activities.The crude extract of SN44 was isolated and purified by column chromatography,TLC and liquid phase preparation after large fermentation.The pure sample,named Y-11,was first identified by 1H NMR spectrum,13 C NMR spectrum,mass spectrum and two-dimensional spectrum.Antheraea pernyi,one of the model insects for pesticide test,was used as the research object.The secondary metabolite IPS,secreted by SN44,was tested for the inhibition of phenoloxidase activity.The result showed that the IC50 was 50.8 ?g/m L.4.In order to get new secondary metabolites,the same technical procedures and means were used in isolating the secondary metabolite of SN52.After column chromatography and liquid phase preparation,4 compounds were isolated and purified from crude extracts of SN52.Their chemical structures were determined by mass spectrometric analysis and comprehensive nuclear magnetic resonance spectroscopy.The results showed that they were new compounds,anthracene derivative,and named Xenocyloin G?Xenocyloin H?Xenocyloin I and Xenocyloin J.Xenocyloins G-J had significant antiplatelet aggregation induced by collagenase.Xenocyloin H had the strongest activity,and the inhibition rate reached 96±0.1% at a concentration of 50 ?M.The IC50 of Xenocylion G and Xenocylion H were 31.7±4.4 ?M and 27.5±3.5 ?M respectively. |
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