Application of aryl hydroxamic acid and its derivatives as chitin deacetylase inhibitors and plant antifungal agents
A technology of deacetylase and chitin, which is applied in the direction of chemicals, biocides, and plant growth regulators for biological control, and can solve the problem of no PDA inhibitors
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Embodiment 1
[0045] The chitin deacetylase VdPDA1 was used as the target for inhibitor screening. Specific steps are as follows:
[0046] Positive control: Set up 3 parallel positive controls. With 30°C as the reaction temperature, under the conditions of 100 μL reaction system, 90nmol / L chitin deacetylase VdPDA1 and 1mmol / L substrate (GlcNAc) 3 Incubate in pH 8.0 buffer solution of 20mmol / L Tris-HCl and 100mmol / LNaCl for 10min, then add 20μL of fluorescamine solution with a final concentration of 7mM, mix well and react for 5min, add 150μL of reaction termination solution (dimethylformamide and Water was mixed at a ratio of 1:1) to terminate the reaction, and the fluorescence intensity was detected with a microplate reader at an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
[0047] Experimental group: set up 3 parallel experimental groups. With 30°C as the reaction temperature, under the conditions of 100 μL reaction system, 90 nmol / L chitin deacetylase VdPDA1 ...
Embodiment 2
[0055] Inhibition constant K i Determination:
[0056] K i Represents the dissociation constant of the enzyme-inhibitor complex, reflecting the strength of the inhibitory effect. With (GlcNAc) 3 As substrates, three sets of substrate concentration gradients were set up for the reaction, and the final concentrations were 0.1 mM, 0.2 mM and 0.5 mM, respectively. In each group of substrate concentrations, six groups of compound concentration gradients with final concentrations of 10, 5, 1, 0.5, 0.2, and 0.1 μM were used to measure the inhibitory activity. The reaction system was 100 μL, the buffer environment was 20mmol / L Tris-HCl and 100mmol / L NaCl, pH 8.0, the final enzyme concentration was 90nM, the reaction temperature was 30°C, and the reaction time was 10min, then 20μL of fluorescamine solution with a final concentration of 7mM was added, mixed After homogeneously reacting for 5 minutes, add 150 μL of reaction termination solution (dimethylformamide and water mixed at 1...
Embodiment 3
[0058] The specific steps for evaluating the antifungal activity of compound 1 against Verticillium dahliae are as follows:
[0059] 1. Preparation of Verticillium dahliae Spores
[0060] Cultivate Verticillium dahlia strains preserved in glycerin in complete medium at 25°C for 4-6 days, filter the mycelium with gauze, centrifuge at 4500rpm for 5min, discard the supernatant, collect a large number of spores in the bacterial liquid, and the spores are passed through wxya 2 O shock suspension, count the number of spores with a hemocytometer, and adjust the concentration of the spore suspension to 5.0×10 by diluting 7 spores / mL, used to dip the roots of cotton seedlings.
[0061] 2. Preparation of Cotton Seedlings
[0062] Cotton seeds were placed in an incubator at 28°C for 48 hours. Vermiculite and nutrient soil were mixed at a ratio of 1:1 as culture soil, and placed in black bottom pots. Five cotton seeds were inoculated in each pot, nutrient solution and water were added,...
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