Application of compound in chitinase inhibitor of soybean cyst nematode

Abstract

The invention relates to application of a compound in a chitinase inhibitor of soybean cyst nematode, belongs to the technical field of symbiotic protection of leguminous plants, and particularly relates to application of a compound shown in a formula (I), a formula (II) or a formula (III) and pharmaceutically acceptable salt thereof in the chitinase inhibitor of the soybean cyst nematode. The inhibition activity of the compound is evaluated and studied through the inhibition effect, the inhibitor is finally screened, and the inhibitor possibly has a good application prospect in the aspect of controlling soybean cyst nematode diseases. The invention further provides an inhibitor of chitinase capable of inhibiting soybean cyst nematode from destroying symbiosis establishment, the number of nodules obtained through treatment is equal to the number of nodules obtained when nodules are only inoculated, the inhibition effect of Hg-CHI-1 on nodules of the nodules of the nodules of the nodules of the nodules of the nodules of the nodules of the nodules of the nodules of the nodules of the nodules of the nodules of the nodules of the nodules of the nodules of the nodules of # imgabs0 #

Description

[0001] This application is a divisional application of the invention patent with patent application number "2023101435527" and invention name "Application of a compound as a chitinase inhibitor of soybean cyst nematode". Technical Field

[0002] The invention belongs to the technical field of symbiotic protection of leguminous plants, and particularly relates to application of a compound as a soybean cyst nematode chitinase inhibitor in inhibiting the soybean cyst nematode from destroying symbiotic establishment. Background Art

[0003] Soybean Cyst Nematode (Heterodera glycines Ichinohe, SCN) is a soil-borne endoparasitic nematode that can cause damage to soybeans throughout the entire growth period, mainly to the roots of soybeans. Generally, it causes a 5-10% yield loss. In severely infected plots, the yield reduction can reach more than 30%, or even no grains can be harvested, resulting in serious yield reduction and causing great economic losses.

[0004] The life cycle of soybean cyst nematode refers to the process from nematode egg to egg production. Soybean cyst nematode is an obligate parasitic nematode that produces offspring through sexual reproduction and overwinters in the soil in the form of cysts. The eggs of soybean cyst nematode are embryonic eggs, which are formed in cysts or egg capsules. The first-instar larvae develop in the eggs, molt once and hatch from the eggs to form second-instar larvae with infectious activity. The second-instar larvae enter the soil, are attracted by the root secretions, find the root tips of the plant and invade, then colonize in the pericycle, and secrete substances from the esophageal glands to stimulate the feeding site and surrounding cells to form a syncytium, which provides nutrition for the development of the nematodes.

[0005] In addition to harming soybeans, soybean cyst nematodes also affect the establishment of rhizobia nodulation symbiosis, resulting in a weakening of the nodule's nitrogen-fixing ability, which further leads to a reduction in soybean yield. The nodule formation process begins with the release of flavonoids by the leguminous host into the rhizosphere environment through the root system. After the rhizobia successfully recognize the flavonoid signal, they will be attracted to the vicinity of the root system and activate the expression of their own nodulation genes, producing and secreting a series of lipid-modified chitosans, namely nodulation factors. After the leguminous host recognizes the nodulation factor, it will begin to activate the expression of symbiotic genes, ultimately guiding the rhizobia to form nodules with nitrogen-fixing capabilities.

[0006] Soybean cyst nematodes secrete many effector proteins to help them complete the infection and colonization process from being attracted to the root tip to forming syncytium. Among them, soybean cyst nematode chitinase Hg-CHI-1 (Gao, B. et al. Characterization and developmental expression of a chitinase gene in Heterodera glycines. Int J Parasitol 32, 1293-1300, doi: 10.1016 / s0020-7519(02)00110-8(2002)) is an effector protein highly expressed in the early stage of infection, has nodulation factor hydrolysis activity, and plays an important role in the process of soybean cyst nematode inhibiting rhizobium symbiosis. Therefore, soybean cyst nematode chitinase is an ideal target for legume symbiotic protectants.

[0007] Studies have shown that nematode eggs and pharynx contain chitin, and chitin remodeling is a critical physiological process during the hatching and infection of soybean cyst nematodes. It can be inferred that chitinase plays a key role in the hatching of nematode eggs and pharynx remodeling. Therefore, soybean cyst nematode chitinase is also an ideal potential target for the prevention and control of soybean cyst nematode disease.

[0008] However, there are few reports on the development of nematode chitinase inhibitors, and there are even fewer reports on highly effective inhibitors of soybean cyst nematode chitinase with symbiotic protection functions for legumes. Summary of the invention

[0009] In order to solve the technical problems in the above-mentioned field and find an inhibitor that effectively inhibits nematode chitinase, the present invention conducted a screening work, evaluated the inhibitory activity of the compound through the inhibitory effect, and finally screened out the inhibitor.

[0010] The present invention also provides a chitinase inhibitor capable of inhibiting the destructive function of soybean cyst nematode on symbiosis establishment. The number of nodules obtained by treatment is equal to the number obtained when only rhizobia are inoculated, and the inhibitory effect of Hg-CHI-1 on rhizobium nodulation is completely restored. The invention has good application prospects in the symbiotic protection of leguminous plants.

[0011] Based on the key role of chitin and chitinase in the growth and development of soybean cyst nematode, this inhibitor is likely to have good application prospects in controlling soybean cyst nematode disease.

[0012] Use of the compound of formula (I), formula (II) or formula (III) and pharmaceutically acceptable salts thereof in soybean cyst nematode chitinase inhibitors, or use of the compound of formula (I), formula (II) or formula (III) and pharmaceutically acceptable salts thereof in controlling soybean cyst nematodes;

[0013]

[0014] Wherein R1 is hydrogen or C1-C8 alkyl,

[0015] R3 represents a single or multiple substitution,

[0016] R2, R3, R4 are independently selected from halogen, hydroxy, cyano, C1-C8 alkyl, C1-C8 alkoxy;

[0017] Ar is represented by one of the following formulae, wherein n is an integer from 0 to 3,

[0018]

[0019] Wherein R1 is hydrogen or C1-C4 alkyl,

[0020] R3 represents mono- or di-substitution,

[0021] R2, R3, R4 are independently selected from halogen, hydroxyl, cyano, C1-C4 alkyl, C1-C4 alkoxy;

[0022] Where n=0 or 1.

[0023] Wherein R2 represents a C1-C4 alkyl group, R3 is independently selected from halogen, hydroxyl, C1-C4 alkyl group, C1-C4 alkoxy group; and R4 represents a cyano group.

[0024] The compound is one of the structures shown below,

[0025] .

[0027] The effective concentration of the inhibitor is not less than 10 μM.

[0028] The inhibitor was used effectively at a concentration of 100 μM.

[0029] The application of the following compounds as symbiotic protective agents for leguminous plants,

[0030]

[0031] The compounds were used effectively at a concentration of 100 μM.

[0032] The present invention provides data obtained by evaluating the inhibitory activity of compounds and the activity as symbiotic protectors of leguminous plants. The results show that compounds No. 14, 15, 16, 17, 25, 27, 30, 31 and 32 exhibit certain inhibitory activity against chitinase Hg-CHI-1. Among the screened compounds, compound No. 32, i.e., 1516b, has the activity of completely restoring the symbiotic damage of Hg-CHI-1 to rhizobia.

[0033] The method for using the compound of the present invention as a symbiotic protective agent for leguminous plants is as follows: using dimethyl sulfoxide as a solvent to dissolve the compound, first incubating it with Hg-CHI-1, and then inoculating soybean roots with rhizobia to study the effect of Hg-CHI-1 on rhizobium nodule formation after inhibitor treatment.

[0034] The data of the examples of the present invention prove that 1516b has good application prospects in the symbiotic protection of legumes. When the concentration of the screened inhibitor is 100 μM, the number of nodules obtained by treatment is equal to the number when only rhizobia are inoculated, and the inhibitory effect of Hg-CHI-1 on rhizobium nodulation is completely restored. This shows that the inhibitor can effectively inhibit Hg-CHI-1 and thus achieve a good symbiotic protection function. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 To analyze the effect of high inhibitory activity compounds on the inhibition of nodulation by exogenous addition of Hg-CHI-1.

[0036] Figure 2 is the K of compound 32 to Hg-CHI-1 i Determination. DETAILED DESCRIPTION

[0037] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The biological materials or reagents used in the following examples, unless otherwise specified, are conventional biological materials or reagents known to those skilled in the art, or reagents prepared by conventional experimental methods.

[0038] Example 1 Cloning and expression of Hg-CHI-1

[0039] Sequence analysis of Hg-CHI-1 (GenBank: AF468679.1) revealed that amino acids 1-23 were signal peptides and amino acids 25-350 were GH18 chitinase domains. The GH18 chitinase domain (25-350) was selected for gene synthesis, and the synthesized gene was cloned into the pPIC9 vector. The restriction endonuclease sites of Hg-CHI-1 were analyzed, and the plasmid was linearized using the restriction endonuclease PmeI. The linearized plasmid was recovered using a DNA fragment recovery kit (TaKaRa). The linearized plasmid was then transformed into Pichia pastoris GS115 using the electroporation method. The selected positive monoclones were activated with YPD liquid medium (1% yeast powder, 2% peptone, 2% glucose) until the OD value was 1.3-1.5, and then inoculated into BMGY liquid medium (1% yeast powder, 2% peptone, 1% glycerol, 0.2% biotin, 1.34% yeast nitrogen base, 100mM phosphate buffer, pH 6.0) at a ratio of 1:100, and cultured overnight at 30°C shaking at 200rpm until the OD value was 2.0. Then the cells were collected by centrifugation at room temperature, and the centrifugation condition was 3000g. Then the cells were transferred to BMMY liquid medium (1% yeast powder, 2% peptone, 1% methanol, 1.34% yeast nitrogen base, 0.2% biotin, 1% (v / v) methanol, 100mM phosphate buffer, pH 6.0), and cultured for 5 days under the same culture conditions, during which 1% (v / v) methanol was added every day. Then centrifuge at 8000g for 30 minutes at room temperature and collect the supernatant. Slowly add 500g / L ammonium sulfate to the fermentation broth supernatant at 4°C. To prevent the local salt concentration from being too high, the whole process needs to be stirred continuously. After the ammonium sulfate is completely dissolved, let it stand overnight. Collect the precipitate by centrifugation at 4°C, then dissolve it with buffer A at 4°C, and centrifuge it again at 4 degrees Celsius to remove undissolved impurities. The supernatant is filtered through a 0.22μm pore size filter to remove fine impurities again, and then a desalting pre-packed column (GE healthcare) is used to remove salt from the sample.The desalted sample was loaded onto a HisTrap HP affinity chromatography column (GE healthcare) that had been equilibrated with buffer A (20 mM sodium phosphate, 0.5 M NaCl, pH 7.4 3.12 g NaH2PO4, 29.22 g NaCl, dissolved in 900 mL distilled water, adjusted to pH 7.4 with hydrochloric acid or NaOH, and fixed to 1 L), and then equilibrated again with 20 column volumes of buffer A. Impure proteins were then eluted using 96% buffer A + 4% buffer B (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4 3.12 g NaH2PO4, 29.22 g NaCl, 34.04 g imidazole, dissolved in 900 mL distilled water, adjusted to pH 7.4 with hydrochloric acid or NaOH, and fixed to 1 L) and 90% buffer A + 10% buffer B, and 60% buffer A + 40% buffer B: Elute the target protein.

[0040] Example 2 Determination of the Inhibitory Efficiency of Compounds on Hg-CHI-1

[0041] Chitinase Hg-CHI-1 was used as the target to screen the inhibitors of the 32 compounds listed in Table I. The specific steps are as follows:

[0042] Positive control: 3 sets of parallel positive controls were set up. Under the conditions of 30°C reaction temperature and 100μL reaction system, 2nmol / L chitinase and 50μmol / L substrate (MU-(GlcNAc)2) were incubated in 20mmol / L pH 6.0 phosphate buffer for 30min, and then 100μL 0.5mol / L sodium carbonate solution was added to terminate the reaction. The reaction solution was excited with 360nm wavelength excitation light and the absorbance value at 450nm wavelength was measured.

[0043] Experimental group: 3 parallel experimental groups were set up. Under the conditions of 30°C reaction temperature and 100 μL reaction system, 2 nmol / L chitinase and 50 μmol / L substrate (MU-(GlcNAc)2) and the compound with a concentration of 100 μM in Table I were incubated in 20 mmol / L pH 6.0 phosphate buffer for 30 min, and then 100 μL 0.5 mol / L sodium carbonate solution was added to terminate the reaction. The reaction solution was excited with 360 nm wavelength excitation light and the absorbance value at 450 nm wavelength was measured.

[0044] The inhibitory activity was calculated according to the following formula

[0045] Inhibition percentage = (positive control - experimental group) / positive control * 100

[0046] Table I. Information on compounds screened as Hg-CHI-1 inhibitors

[0047]

[0048]

[0049] The results showed that among all the 32 compounds screened, compounds No. 14, 15, 16, 17, 25, 27, 30, 31 and 32 showed certain inhibitory activity against chitinase Hg-CHI-1 and could also be used to control soybean cyst nematodes.

[0050] Example 3 Effect of Compounds and Hg-CHI-1 on Rhizobium Nodulation

[0051] Soybeans (Williams 82) were germinated for 5-7 days, and rhizobia (Bradyrhizobium japonicumUSDA110) were cultured to OD600 of 0.8-1.2 for use. Hg-CHI-1 (1 mg / mL) was incubated with the selected compounds with inhibitory activity (100 μM) at room temperature for 1 hour, and the germinated soybeans were transplanted into sterile vermiculite, and then inoculated into the soybean roots at the same time as rhizobia (OD600 diluted to 0.01). After 14 days, the number of nodules on the soybean roots was counted. Figure 1 As shown, Hg-CHI-1 reduced the number of nodules of rhizobia, while the presence of compound No. 32, 1516b, restored the phenotype of the number of nodules of rhizobia, while other compounds with inhibitory activity did not show phenotype biological activity. This result fully demonstrates that compound 1516b has the function of protecting soybean nodulation.

[0052] Example 4 Determination of the inhibition constant of 1516b against Hg-CHI-1

[0053] MU-(GlcNAc)2 was used as the substrate, and three groups of substrate concentration gradients were set up for the reaction, with final concentrations of 5μM, 10μM and 15μM, respectively. At each group of substrate concentrations, multiple groups of appropriate compound concentration gradients were taken for inhibitory activity determination. The reaction system was 100μL, the buffer environment was 20mM phosphate buffer, pH6.0, the final enzyme concentration was 2nM, the reaction temperature was 30°C, the reaction time was 20min, and then 100μL of 0.5M sodium carbonate solution was added to terminate the reaction. The released MU was excited by 360nm excitation light and its absorbance was measured at a wavelength of 450nm. The data were plotted using the Dixon method, and the inhibition constant K of compound 1516b against Hg-CHI-1 i The value is 118.9μM. Figure 2 shown.

[0054] The general method for determining the inhibition constant is to use three substrate concentrations for determination, draw three linear relationships between the inverse of the reaction rate and the compound concentration, and the negative number of the intersection of the three lines is the inhibition constant. The inhibition constant Ki reflects the inhibitory strength of the inhibitor on the target. The smaller the value, the stronger the inhibition ability. The inhibition constant K of the compound 1516b of the present invention on Hg-CHI-1 i The value was 118.9 μM, indicating that the compound had a strong inhibitory ability against Hg-CHI-1.

Claims

1. Use of the compound of formula (I) and its pharmaceutically acceptable salt in the preparation of soybean cyst nematode chitinase inhibitors, or use of the compound of formula (I) and its pharmaceutically acceptable salt in the prevention and treatment of soybean cyst nematodes, Wherein R1 is hydrogen or C1-C8 alkyl, R3 represents a single or multiple substitution, R2 and R3 are independently selected from halogen, hydroxyl, cyano, C1-C8 alkyl, C1-C8 alkoxy; n is an integer from 0 to 3.

2. The use according to claim 1, wherein R1 is hydrogen or C1-C4 alkyl, R3 represents mono- or di-substitution, R2 and R3 are independently selected from halogen, hydroxyl, cyano, C1-C4 alkyl, C1-C4 alkoxy; wherein n=0 or 1.

3. The use according to claim 2, wherein R2 is a C1-C4 alkyl group, and R3 is independently selected from halogen, hydroxyl, C1-C4 alkyl group, and C1-C4 alkoxy group.

4. The use according to claim 3, wherein the compound is one of the following structures:

5. The use according to any one of claims 1 to 4, wherein the effective concentration of the inhibitor is not less than 10 μM.

6. The use according to claim 5, wherein the effective concentration of the inhibitor is 100 μM.

Images