| Rice bacterial blight is a common bacterial disease on rice caused by Xanthomonas oryzae pv.oryzae(Xoo).At present,the varieties of fungicides used to prevent and control rice blight are less,and the drug resistance is increasing year by year.It is an important strategy for the creation of new green fungicides to screen and discover small molecular compounds with strong specificity,using the key functional proteins in the pathogen as the molecular target.In recent years,with the development of molecular biology technology,the key enzymes involved in the bacterial fatty acid biosynthesis pathway(FASII)have attracted more and more attention.They are different from the multi-functional fatty acid synthase in eukaryotes,which are composed of several single functional enzymes and therefore have the characteristics of selective inhibition.?-ketoacyl-acyl carrier protein(ACP)synthase III(FabH)controls the initial step of bacterial fatty acid biosynthesis,catalyzes the condensation of malonic acid monoyl-ACP and acetyl-CoA to beta ketoyl-ACP,which is the key enzyme in the biosynthesis of bacterial fatty acids.Inhibition of FabH activity can interfere with bacterial fatty acid biosynthesis and inhibit bacterial growth and reproduction.In this study,an affinity chromatography column was made by immobilized technique to isolate FabH from Xoo.In conjunction with HPLC,the natural products with obvious inhibitory activity to FabH and Xoo were successfully separated,and the effect of the compound on rice white leaf blight was verified by pot experiments in greenhouse.The specific research contents include:First,this paper optimizes the heterologous expression and purification conditions of FabH,FabD and ACP protein of Xoo.The best purification conditions for FabHXoo and FabDXoo of Xoo are the first elution of 55mM imidazole,the first elution of 20mM imidazole and high concentration of 250mM imidazole,ACP is obtained by strong anion exchange.Second,the carrier was prepared by the ion crosslinking method,and the chitosan carrier was characterized by scanning electron microscope,transmission electron microscope,infrared spectrometer,specific surface area and particle size analyzer.By studying the temperature tolerance,the range of pH and the duration of the immobilized enzyme,we found that the immobilized enzyme had a significant increase in temperature tolerance,pH range and the duration of its holding effect compared with the free enzyme.Third,from 120 endophytic fungi preserved in our laboratory,the strains with good activity to Xoo and FabH were screened.The chemical screening method was used to determine the Alternaria alternata ZHJG5 of plant endophytic fungi as a research target.Three natural products,alteractone(1),dehydroaltenusin(2),and compound dihydroalterperylenol(4),were screened from the secondary metabolites of Alternaria alternata ZHJG5 by using the immobilized FabH-HPLC affinity chromatography screening system,and the concentration of FabH inhibition(IC50)was 30.59±3.07?M,76.36±2.64?M,27.84±4.16?M.The activity test results showed that the minimum inhibitory concentration(MIC)of compound 1,2 and 4 of Xoo were 16,8,and 4?g/mL,respectively.In vivo,compound 1 and 2 had a 46%and 42%control effect on rice white leaf blight,which was better than the positive control streptomycin sulfate(200?g/mL)Fourth,for compound 1,the interaction between compound 1 and FabH protein was studied by means of bio-layer interferometry(BLI),isothermal titration calorimetry(ITC),molecular simulation and so on,and the mode of action between the protein and the protein was clarified.The above results showed that we set up a rapid and efficient screening system of active compounds by using the immobilized enzyme HPLC combined with FabH as the target of Xoo,and successfully found a small molecular compound that had inhibitory effect on rice leaf blight pathogen,which was a new type of high efficiency and low value for the creation of independent intellectual property.Toxic and selective green pesticides provide lead compounds. |
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