Use of a compound as a chitinase inhibitor in soybean cyst nematode

Abstract

The present application relates to "application of a compound in soybean cyst nematode chitinase inhibitor", belong to the technical field of leguminous plant symbiotic protection, the application of the compound and its pharmaceutically acceptable salt in soybean cyst nematode chitinase inhibitor, the present application is evaluated and researched through the inhibitory activity of compound by screening work, the inhibitor is screened finally, the inhibitor is likely to have good application prospect in controlling soybean cyst nematode disease aspect.The present application also provides a kind of chitinase inhibitor with the function of inhibiting the establishment of symbiosis of soybean cyst nematode to destroy, the number of nodule obtained by processing is flat with the number when only inoculating rhizobium, the inhibitory effect of Hg-CHI-1 on rhizobium nodule is completely restored, and it has good application prospect in the aspect of leguminous plant symbiotic protection.

Description

[0001] The present application is a divisional application of the invention patent application with the patent application number "2023101435527" and the invention name "Application of a compound in soybean cyst nematode chitinase inhibitor". TECHNICAL FIELD

[0002] The present application belongs to the technical field of legume symbiotic protection, and specifically relates to application of a compound as a soybean cyst nematode chitinase inhibitor in inhibiting soybean cyst nematode from destroying symbiotic establishment. BACKGROUND

[0003] Soybean cyst nematode (Soybean Cyst Nematode, Heterodera glycines Ichinohe, SCN) is a soil-borne endoparasitic nematode. Soybean cyst nematode can cause damage throughout the growth period of soybean, mainly damaging the roots of soybean. Generally, it will cause 5-10% yield loss, and the yield reduction of serious disease area can reach more than 30%, even no harvest, resulting in serious yield reduction and causing great economic loss.

[0004] The life cycle of soybean cyst nematode refers to the process from the egg of the nematode to the production of the egg again. Soybean cyst nematode is a specific parasitic nematode, which reproduces sexually to produce offspring in the form of cysts and overwinters in the soil. The egg of soybean cyst nematode is an embryonic egg formed in the cyst or egg sac. The first instar larva develops in the egg, molts once from the egg, and forms the second instar larva with infective activity. The second instar larva enters the soil, is attracted to the root tip by root exudates, and invades the plant root tip. Then it colonizes in the pericycle and secretes substances from the food canal gland to stimulate the feeding site and surrounding cells, forming a syncytium to provide nutrition for the development of the nematode.

[0005] In addition to the damage to soybean, soybean cyst nematode also affects the establishment of rhizobial nodule symbiosis, leading to reduced nitrogen fixation ability of root nodules, further leading to reduced yield of soybean. The beginning of the nodule formation process is that the legume host releases flavonoids to the rhizosphere through the root system. After the rhizobium successfully recognizes the flavonoid signal, it is attracted to the vicinity of the root system and initiates the expression of its own nodulation genes, producing and secreting a series of chitins with lipid modification, i.e. nodulation factors. After the legume host recognizes the nodulation factors, it will start to express symbiotic genes, and ultimately guide the rhizobium to form root nodules with nitrogen fixation ability.

[0006] Soybean cyst nematode excretes many effector proteins to help it complete the process of infection and colonization from being attracted to the root tip to the formation of syncytium. Among them, soybean cyst nematode chitinase Hg-CHI-1 (Gao, B. et al. Characterisation 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)) as an effector protein highly expressed in the early stage of infection, has nodule factor hydrolysis activity, and plays an important role in inhibiting the symbiotic process of rhizobium. Therefore, soybean cyst nematode chitinase is an ideal target for legume symbiotic protection agent.

[0007] Studies have shown that the eggs and pharynx of nematodes contain chitin, and chitin remodeling is a very 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 the remodeling of the pharynx. Therefore, soybean cyst nematode chitinase is also an ideal potential target for controlling soybean cyst nematode disease.

[0008] However, there are few reports on the development of inhibitors targeting nematode chitinase, and there are no reports on efficient inhibitors targeting soybean cyst nematode chitinase with legume symbiotic protection function. SUMMARY

[0009] In order to solve the technical problems in the above-mentioned field and find a high-efficiency inhibitor of nematode chitinase, the present application evaluates and studies the inhibitory activity of the compound by screening work, and finally screens the inhibitor.

[0010] The present application also provides an inhibitor of chitinase with the function of inhibiting the destruction of symbiotic establishment by soybean cyst nematode, and the number of nodules obtained by treatment is flat with the number when only inoculated with rhizobium, completely restoring the inhibition of Hg-CHI-1 on rhizobium nodule formation, and having good application prospect in legume symbiotic protection.

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

[0012] Use of a compound of formula (I), (II) or (III) and pharmaceutically acceptable salts thereof as a chitinase inhibitor of Heterodera glycines or use of a compound of formula (I), (II) or (III) and pharmaceutically acceptable salts thereof for controlling Heterodera glycines;

[0013]

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

[0015] R3 is mono- or di-substituted,

[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, and n is an integer from 0 to 3,

[0018]

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

[0020] R3 is mono- or di-substituted,

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

[0022] wherein n = 0 or 1.

[0023] wherein R2 is C1-C4 alkyl, and R3 is independently selected from halogen, hydroxy, C1-C4 alkyl, C1-C4 alkoxy; and R4 is cyano.

[0024] said compound is one of the following structures,

[0025]

[0026] the effective concentration of said inhibitor is not less than 10 μM.

[0027] the effective concentration of said inhibitor is 100 μM.

[0028] use of the following compound as a symbiotic protective agent for leguminous plants,

[0029]

[0030] the effective concentration of said compound is 100 μM.

[0031] The present application provides data obtained from evaluating the inhibitory activity of compounds and their activity as symbiotic protectants for legumes. The results show that compounds numbered 14, 15, 16, 17, 25, 27, 30, 31 and 32 exhibit some inhibitory activity against chitinase Hg-CHI-1. Among the compounds screened, compound numbered 32, i.e. 1516b, has full restorative activity of Hg-CHI-1 against the symbiotic disruption of rhizobia.

[0032] The method for using the compounds of the present application as symbiotic protectants for legumes is to dissolve the compounds using dimethyl sulfoxide as solvent, and to study the effect of Hg-CHI-1 on rhizobium nodule formation after inhibitor treatment by co-incubating with Hg-CHI-1 first and then inoculating soybean roots with rhizobium at the same time.

[0033] The data of the embodiments of the present application prove that 1516b has good application prospects in symbiotic protection of legumes. The number of nodules obtained by treating the selected inhibitor at a concentration of 100 μM is the same as that obtained by inoculating rhizobium only, which fully restores the inhibitory effect of Hg-CHI-1 on rhizobium nodulation. This shows that the inhibitor can effectively inhibit Hg-CHI-1 and thus achieve good symbiotic protection function. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 To analyze the effect of highly inhibitory active compounds on the inhibition of nodule formation by exogenous Hg-CHI-1,

[0035] Figure 2 To analyze the K i determination of compound 32 on Hg-CHI-1. DETAILED DESCRIPTION

[0036] The following non-limiting examples can enable those of ordinary skill in the art to more fully understand the present application, but in no way limit the present application. The biological materials or reagents used in the following examples, if not specifically stated, are conventional biological materials or reagents known to those skilled in the art, or reagents configured by conventional test methods.

[0037] Example 1 Cloning and expression of Hg-CHI-1

[0038] The sequence analysis of Hg-CHI-1 (GenBank: AF468679.1) showed that the amino acids 1-23 were signal peptide and the amino acids 25-350 were GH18 chitinase domain. The GH18 chitinase domain (25-350) was selected for gene synthesis and the synthesized gene was cloned into pPIC9 vector. The restriction enzyme sites of Hg-CHI-1 were analyzed and the linearized plasmid was obtained by restriction enzyme PmeI and DNA fragment recovery kit (TaKaRa). Then the linearized plasmid was transformed into Pichia pastoris GS115 by electroporation. The selected positive single clone was activated by YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) and inoculated into BMGY liquid medium (1% yeast extract, 2% peptone, 1% glycerol, 0.2% biotin, 1.34% yeast nitrogen base, 100 mM phosphate buffer, pH 6.0) at a ratio of 1:100 after the OD value reached 1.3-1.5. The culture was incubated at 30°C and 200 rpm overnight until the OD value reached 2.0. Then the bacteria were collected by centrifugation at room temperature and 3000g. The bacteria were then transferred to BMMY liquid medium (1% yeast extract, 2% peptone, 1% methanol, 1.34% yeast nitrogen base, 0.2% biotin, 1% (v / v) methanol, 100 mM phosphate buffer, pH 6.0) and incubated under the same conditions for 5 days, during which 1% (v / v) methanol was added every day. Then the supernatant was collected by centrifugation at 8000g for 30 minutes at room temperature. The fermentation supernatant was slowly added with 500g / L ammonium sulfate at 4°C to prevent local salt concentration from being too high. The whole process required continuous stirring. After the ammonium sulfate was completely dissolved, it was left overnight at 4°C. The precipitate was collected by centrifugation at 4°C, then dissolved with buffer A at 4°C, and again centrifuged at 4°C to remove undissolved impurities. The supernatant was filtered through a 0.22μm filter to remove fine impurities, and then a desalting pre-packed column (GE healthcare) was used to remove salt from the sample.The desalted sample was loaded into a HisTrap HP affinity chromatography column (GE healthcare) which 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, 900 mL of single distilled water was dissolved, and the pH value was adjusted to 7.4 using hydrochloric acid or NaOH, and the volume was made to 1 L), and then re-equilibrated with 20 column volumes of buffer A, and then the impurities were eluted with 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, 900 mL of single distilled water was dissolved, and the pH value was adjusted to 7.4 using hydrochloric acid or NaOH, and the volume was made to 1 L), and the target protein was eluted with 60% buffer A + 40% buffer B.

[0039] Determination of the inhibitory efficiency of the compounds of Example 2 on Hg-CHI-1

[0040] Chitinase Hg-CHI-1 was taken as the target to screen 32 compounds listed in Table I as inhibitors. The specific steps are as follows:

[0041] Positive control: 3 groups of parallel positive controls were set. Under the conditions of 100 μL reaction system and 30°C reaction temperature, 2 nmol / L chitinase and 50 μmol / L substrate (MU-(GlcNAc)2) were incubated in 20 mmol / L phosphate buffer at pH 6.0 for 30 min, and then 100 μL of 0.5 mol / L sodium carbonate solution was added to terminate the reaction, and the reaction solution was excited with excitation light of 360 nm wavelength to measure the absorbance value at 450 nm wavelength.

[0042] Experimental group: 3 groups of parallel experimental groups were set. Under the conditions of 100 μL reaction system and 30°C reaction temperature, 2 nmol / L chitinase and 50 μmol / L substrate (MU-(GlcNAc)2) and the corresponding compound with a concentration of 100 μM in Table I were incubated in 20 mmol / L phosphate buffer at pH 6.0 for 30 min, and then 100 μL of 0.5 mol / L sodium carbonate solution was added to terminate the reaction, and the reaction solution was excited with excitation light of 360 nm wavelength to measure the absorbance value at 450 nm wavelength.

[0043] The inhibitory activity was calculated according to the following formula

[0044] Inhibition percentage = (positive control - experimental group) / positive control * 100

[0045] Table I. Information of compounds screened as Hg-CHI-1 inhibitors

[0046]

[0047]

[0048] The results showed that among all the 32 compounds screened, compounds No. 14, 15, 16, 17, 25, 27, 30, 31 and 32 exhibited certain inhibitory activity against chitinase Hg-CHI-1, and could be used for preventing and treating soybean cyst nematode.

[0049] Example 3 Test of the effect of compounds and Hg-CHI-1 on rhizobium nodulation

[0050] Soybean (Williams 82) was germinated for 5-7 days, and rhizobium (Bradyrhizobium japonicum USDA110) was cultured to OD600 of 0.8-1.2 for standby. Hg-CHI-1 (1 mg / mL) was incubated with the screened compounds (100 μM) having inhibitory activity at room temperature for 1 h, and the germinated soybean was transplanted into sterile vermiculite, and then inoculated with rhizobium (OD600 diluted to 0.01) at the same time. The number of nodules on the roots of soybean was counted after 14 days. As shown in Figure 1 , Hg-CHI-1 reduced the number of rhizobium nodules, and the presence of compound No. 32, i.e. 1516b, restored the phenotype of the number of rhizobium nodules, while the other compounds having inhibitory activity did not exhibit biological activity. This result fully demonstrated that compound 1516b had soybean nodulation protection function.

[0051] Example 4 Determination of the inhibition constant of 1516b against Hg-CHI-1

[0052] MU-(GlcNAc)2 was used as the substrate, and three groups of substrate concentration gradients were set, with final concentrations of 5 μM, 10 μM and 15 μM, respectively. Under each group of substrate concentration, multiple groups of appropriate compound concentration gradients were taken for inhibitory activity determination. The reaction system was 100 μL, the buffer environment was 20 mM phosphate buffer, pH 6.0, the enzyme final concentration was 2 nM, the reaction temperature was 30°C, the reaction time was 20 min, after which 100 μL of 0.5 M sodium carbonate solution was added to terminate the reaction, and the released MU was determined by measuring the absorbance value at 450 nm after excitation by 360 nm excitation light. The data was plotted by the Dixon method, and the inhibition constant K i value of compound 1516b against Hg-CHI-1 was 118.9 μM. The results are shown in Figure 2 .

[0053] The general method for determining the inhibition constant is to determine the concentration of three substrates, draw three linear relationships of the reciprocal of reaction rate and the concentration of the compound, and the negative number of the intersection point of the three lines is the inhibition constant. The inhibition constant (Ki) reflects the inhibition strength of the inhibitor to the target, and the smaller the value is, the stronger the inhibition ability is. The inhibition constant K i of the compound 1516b to Hg-CHI-1 is 118.9 μM, indicating that the compound has strong inhibition ability to Hg-CHI-1.

Claims

1. Use of a compound of formula (I) and pharmaceutically acceptable salts thereof in the manufacture of a soybean cyst nematode chitinase inhibitor, or use of a compound of formula (I) and pharmaceutically acceptable salts thereof in the control of soybean cyst nematode, wherein R1 is hydrogen or C1-C8 alkyl, R3 represents mono- or poly-substitution, R2, R3 are independently selected from halogen, hydroxy, cyano, C1-C8 alkyl, C1-C8 alkoxy; n is an integer from 0 to 3.

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

3. Use according to claim 2, wherein R2 represents C1-C4 alkyl, R3 are independently selected from halogen, hydroxy, C1-C4 alkyl, C1-C4 alkoxy.

4. Use of a compound and pharmaceutically acceptable salts thereof in the manufacture of a soybean cyst nematode chitinase inhibitor, or use of a compound and pharmaceutically acceptable salts thereof in the control of soybean cyst nematode, said compound being one of the following structures, 5. Use according to any one of claims 1 to 4, wherein the effective concentration of the inhibitor is not less than 10 μM.

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

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