Application of a chitin hydrolase inhibitor in regulating insect growth activity
A chitin hydrolase and inhibitor technology, which can be used in applications, plant growth regulators, insecticides, etc., can solve problems such as reducing insecticidal effect
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Embodiment 1
[0039] Using the enzymes OfHex1, OfChi-h, OfChtI and OfChtII as targets, 1680 microbial secondary metabolites were screened for inhibitors. Specific steps are as follows:
[0040] Positive control: Set up 2 parallel positive controls. Under the condition of 30℃ reaction temperature and 100μL reaction system, glycosyl hydrolase, 10μmol / L substrate and 2μL DMSO were incubated in 20mmol / L pH 6.0 phosphate buffer for 30min, and then 100μL 0.5mol / L L sodium carbonate solution terminates the reaction, and the reaction solution is excited by excitation light with a wavelength of 360nm, and then the absorbance value at a wavelength of 450nm is measured. When the enzyme is OfHex1, the substrate is MU-GlcNAc (4-methylumbelliferyl-N-acetyl-β-D-glucosamine), and when the enzyme is OfChi-h, OfChtI and OfChtII, the substrate is MU-(GlcNAc ) 2 (4-Methylumbelliferyl-β-D-N,N'-diacetylchitobioside).
[0041] Experimental group: set up 3 parallel experimental groups. Under the conditions of...
Embodiment 2
[0046] Inhibition constant K i Determination:
[0047] When the enzyme is OfHex1, the substrate is MU-GlcNAc (4-methylumbelliferyl-N-acetyl-β-D-glucosamine), and when the enzyme is OfChi-h, OfChtI and OfChtII, the substrate is MU-(GlcNAc ) 2 (4-methylumbelliferyl-β-D-N,N'-diacetyl chitobioside), three sets of substrate concentration gradients are set up for the reaction, and multiple sets of suitable compound concentration gradients are taken under each set of substrate concentrations Inhibitory activity assays were performed. The reaction system was 100 μL, the buffer environment was 20 mM phosphate buffer, pH 6.0, the reaction temperature was 30 ° C, the reaction time was 30 min, and then 100 μL of 0.5 M sodium carbonate solution was added to terminate the reaction, and the released 4-methylumbelliferone passed through The absorbance value was measured at a wavelength of 450nm after excitation with 360nm excitation light. The data is plotted by the Dixon method, and the ...
Embodiment 3
[0049] The specific steps for evaluating the insecticidal activity of shikonin and its derivatives are as follows:
[0050] In the experiment, healthy Spodoptera frugiperda and armyworm larvae on the first day of the third instar were selected as experimental materials, and a control group and an experimental group were set up. The compound Shikonin (dextroshikonin) was dissolved in methanol and mixed in feed at a concentration of 10 mM (1 g feed = 1 mL feed). The larvae of the control group were injected with the feed added with methanol. The larvae were cultured at 26°C, with a relative humidity of 70%-90%, 16 hours of light per day and 8 hours of darkness until the end of the larval stage, during which the number and phenotype of normal larvae, dead larvae, normal pupae and abnormal pupae were tested every day Make statistics.
[0051] The statistical results are plotted as image 3 , image 3 It is the graph of the growth effect of 10mM compound Shikonin on Spodoptera ...
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