Biosynthetic inhibitor of locusta migratoria aggregation pheromone 4-vinylanisole, and use thereof

Abstract

The present application provides a biosynthetic inhibitor of Locusta migratoria aggregation pheromone 4-vinylanisole, and a use thereof. The biosynthetic inhibitor of Locusta migratoria aggregation pheromone 4-vinylanisole reduces the activity of 4-vinylphenol methyltransferase, wherein the 4-vinylphenol methyltransferase contains an amino acid sequence selected from the following: (a) a sequence as shown in either of SEQ ID NO: 1 and SEQ ID NO: 3; and (b) a sequence having the activity of catalyzing the generation of Locusta migratoria aggregation pheromone 4-vinylanisole and derived from the sequence shown in either of SEQ ID NO: 1 or SEQ ID NO: 3 after deletion, insertion and / or substitution of one or more amino acid residues. The inhibitor can be used for biological control of Locusta migratoria, i.e., keeping Locusta migratoria in a harmless solitary state instead of killing Locusta migratoria, thereby providing a new method for biological control of Locusta migratoria, and achieving a wide prospect for application.

Description

A biosynthesis inhibitor of locust aggregation pheromone 4-vinylanisole and its use Technical Field

[0001] The present application belongs to the technical field of pest control, and specifically relates to a biosynthesis inhibitor of the locust aggregation pheromone 4-vinylanisole and its use. Background Art

[0002] Frequent locust outbreaks pose a serious threat to global agriculture, economy and environmental security. Aggregation pheromones play a vital role in attracting and aggregating individuals and maintaining locust populations. After more than 50 years of exploration, 4-vinylanisole (4VA) was recently discovered to be the aggregation pheromone of the migratory locust (Locusta migratoria) through chemical analysis, behavioral verification, electrophysiological recording, olfactory receptor identification, gene knockout and other methods. 4VA is mainly released by the hind legs of gregarious locusts and has no gender difference. Although 4VA is produced by gregarious locusts, the aggregation of 4-5 solitary locusts can induce 4VA production.

[0003] Therefore, the production and release of 4VA is the result of increased population density. In addition, 4VA also plays an important role in promoting the consistent maturation of female locusts and the interaction of individuals of the same species. OR35 was identified as the specific odor receptor for 4VA, and OR35 mutant locusts created by CRISPR-Cas9 showed a loss of attraction behavior. OR35 gene knockout resulted in the loss of locust perception of 4VA, hindering the acquisition and maintenance of group behavior. Therefore, 4VA is essential for the survival and reproduction of locusts. Exploring the biosynthesis pathway of 4VA and developing corresponding inhibitors have broad application prospects for the biological control of migratory locusts. Summary of the Invention

[0004] The present application provides a biosynthesis inhibitor of the locust aggregation pheromone 4-vinylanisole and its use. This application identifies for the first time a method for the biosynthesis pathway of the locust aggregation pheromone 4-vinylanisole, as well as the complete biosynthesis pathway of 4VA. It also screens and identifies two rate-limiting methyltransferases in the synthesis process. Based on these rate-limiting methyltransferases, an inhibitor for inhibiting the biosynthesis of 4VA is developed. The inhibitor can be used in the biological control of locusts. The inhibitor can maintain the locusts in a harmless, dispersive state rather than causing their death, providing a new method for biological control of locusts and having broad application prospects.

[0005] In a first aspect, the present application provides a 4-vinylphenol methyltransferase that catalyzes the production of the locust aggregation pheromone 4-vinylanisole, wherein the 4-vinylphenol methyltransferase comprises an amino acid sequence selected from the group consisting of:

[0006] (a) the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3;

[0007] (b) A sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole obtained by deleting, inserting and / or substituting one or more amino acid residues from the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3.

[0008] In a second aspect, the present application provides a method for synthesizing the locust aggregation pheromone 4-vinylanisole, which comprises: using 4-vinylphenol as a substrate and obtaining 4-vinylanisole under the catalysis of 4-vinylphenol methyltransferase.

[0009] Preferably, the synthesis step of 4-vinylphenol comprises: using phenylalanine as a substrate, obtaining cinnamic acid under the catalysis of phenylalanine ammonia lyase, obtaining p-hydroxycinnamic acid under the catalysis of cinnamate 4-hydroxylase, and obtaining 4-vinylphenol under the catalysis of p-hydroxycinnamic acid decarboxylase.

[0010] In this application, the identification process of the method for the biosynthetic pathway of the locust aggregation pheromone 4VA includes:

[0011] 1) Fifth-instar gregarious locust nymphs were starved and volatiles were collected using solid-phase microextraction (SPME) to determine whether 4VA was biosynthesized from the plants they consumed.

[0012] 2) Fifth-instar gregarious locust nymphs were fed artificial diet, and volatiles were collected using SPME.

[0013] 3) Fifth-instar gregarious locust nymphs were fed and injected with deuterated compounds, and volatiles were collected from the fifth-instar gregarious locust nymphs using the SPME method to identify the biosynthetic precursors of 4VA.

[0014] 4) UHPLC-MS / MS was used to perform metabolome analysis of locust intestinal and hemolymph metabolites to identify possible 4VA biosynthesis pathways.

[0015] 5) Fifth-instar gregarious locust nymphs were fed and injected with deuterated intermediates, and volatiles were collected by SPME to identify the intermediates in 4VA biosynthesis.

[0016] 6) Derivatization of biosynthetic intermediates in fifth-instar gregarious locusts was performed and analyzed using GC-MS / MS to determine whether plants can provide these intermediates to locusts.

[0017] This application also found the key methyltransferase 4VPMTs in the 4VA biosynthesis pathway, which includes the following steps:

[0018] 1) The biosynthetic intermediates in the fifth-instar gregarious and solitary locust nymphs were derivatized and analyzed using GC-MS / MS.

[0019] 2) Fifth-instar solitary locust nymphs were fed and injected with deuterated compounds, and volatiles were collected using SPME to determine the differences in 4VA production between gregarious and solitary locusts.

[0020] 3) Identify the genes that control locust 4VA production through transcriptome analysis, qPCR experimental verification and RNAi interference experiments.

[0021] 4) Verify the function of the gene that controls locust 4VA production through in vitro expression and kinetic parameter characterization.

[0022] In this application, the biosynthetic pathway of the locust aggregation pheromone 4-vinylanisole was identified for the first time by starvation treatment, exogenous feeding, or injection of stable isotope-labeled intermediates. The final determination of the 4VA synthesis pathway in locusts is: phenylalanine (Phe)-cinnamic acid (CA)-p-hydroxycinnamic acid (p-HCA)-4-vinylphenol (4VP)-4VA.

[0023] The present application further verified that two rate-limiting methyltransferases, 4VPMT1 and 4VPMT2, control the synthesis of 4VP to 4VA in locusts through transcriptome analysis, qPCR verification, in vivo interference, and in vitro enzyme activity detection. Further inhibitor screening, biochemical analysis, in vivo injection, and in vitro enzyme activity detection confirmed that 4-nitrophenol can effectively inhibit the biosynthesis of 4VA both in vitro and in vivo, and can be applied to the biological control of migratory locusts. The present application can keep locusts in a harmless scattered state instead of killing them, providing a new method for biological control of migratory locusts and has broad application prospects.

[0024] In a third aspect, the present application provides a nucleic acid molecule encoding the 4-vinylphenol methyltransferase that catalyzes the production of the locust aggregation pheromone 4-vinylanisole as described in the first aspect.

[0025] Preferably, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:

[0026] (A) the sequence shown in any one of SEQ ID NO: 2 and SEQ ID NO: 4;

[0027] (B) A sequence that is at least 90% identical to the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3, and encodes a sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole.

[0028] In a fourth aspect, the present application provides an expression vector comprising the nucleic acid molecule described in the third aspect.

[0029] In a fifth aspect, the present application provides a recombinant cell, which contains the expression vector described in the fourth aspect, or the nucleic acid molecule described in the third aspect is integrated into the genome of the recombinant cell.

[0030] In a sixth aspect, the present application provides a use of a 4-vinylphenol methyltransferase gene or a 4-vinylphenol methyltransferase in screening locust aggregation inhibitors, wherein the 4-vinylphenol methyltransferase comprises an amino acid sequence selected from the group consisting of:

[0031] (a) the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3;

[0032] (b) A sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole obtained by deleting, inserting and / or substituting one or more amino acid residues from the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3.

[0033] Preferably, the 4-vinylphenol methyltransferase gene contains a nucleotide sequence selected from the group consisting of:

[0034] (A) the sequence shown in any one of SEQ ID NO: 2 and SEQ ID NO: 4;

[0035] (B) A sequence that is at least 90% identical to the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3, and encodes a sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole.

[0036] In the present application, a substance capable of inhibiting the expression of the 4-vinylphenol methyltransferase gene is screened from a variety of chemical substances as a locust aggregation inhibitor; or a substance capable of reducing the activity of 4-vinylphenol methyltransferase is screened from a variety of chemical substances as a locust aggregation inhibitor; wherein the screening is carried out using the expression level of the 4-vinylphenol methyltransferase gene or the activity of the 4-vinylphenol methyltransferase as a detection indicator.

[0037] In a seventh aspect, the present application provides a 4-vinylphenol methyltransferase gene inhibitor or a use of a 4-vinylphenol methyltransferase inhibitor in the preparation of a locust control drug, wherein the 4-vinylphenol methyltransferase comprises an amino acid sequence selected from the group consisting of:

[0038] (a) the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3;

[0039] (b) A sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole obtained by deleting, inserting and / or substituting one or more amino acid residues from the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3.

[0040] Preferably, the 4-vinylphenol methyltransferase gene contains a nucleotide sequence selected from the group consisting of:

[0041] (A) the sequence shown in any one of SEQ ID NO: 2 and SEQ ID NO: 4;

[0042] (B) A sequence that is at least 90% identical to the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3, and encodes a sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole.

[0043] Preferably, the drug has a 4-vinylphenol methyltransferase gene inhibitor or a 4-vinylphenol methyltransferase inhibitor as an active ingredient.

[0044] In an eighth aspect, the present application provides a use of a multi-substituted benzene ring structure and its analogs in the preparation of a 4-vinylphenol methyltransferase inhibitor, wherein the inhibitor is a competitive substance for 4-vinylphenol, a substrate of 4-vinylphenol methyltransferase;

[0045] The general structural formula of the polysubstituted benzene ring structure and its analogs is shown in Formula I:

[0046] wherein R1 is selected from the group consisting of hydrogen, hydroxy, thiol, methoxy, methylthio, methyl, amino, or halogen;

[0047] R2, R3, R4 or R5 are each independently selected from: hydrogen, hydroxy, methyl, methoxy, nitro;

[0048] R6 is selected from the group consisting of hydrogen, alkyl, alkenyl, cyano, hydroxy, carboxyl, methoxy, halogen, nitro, ester, oxime, amide, and carbonyl;

[0049] The X atom is selected from: C or N.

[0050] Preferably, the inhibitor inhibits the biosynthesis of 4-vinylanisole in vivo or in vitro.

[0051] In the ninth aspect, the present application provides the use of the multi-substituted benzene ring structure and its analogs described in the eighth aspect in the preparation of locust control drugs.

[0052] Preferably, the drug also includes pharmaceutically acceptable excipients.

[0053] Compared with the prior art, this application has the following beneficial effects:

[0054] This application identifies the complete biosynthetic pathway of 4VA and identifies the key enzymes 4VPMT1 and 4VPMT2 that control 4VA production. 4VPMT1 and 4VPMT2 serve as biosynthetic switches for 4VA synthesis, paving the way for the development of 4-nitrophenol inhibitors to inhibit the enzymatic activity of 4VPMTs and thus the biosynthesis of 4VA, providing an effective and sustainable strategy for locust control. This application can keep locusts in a harmless, dispersed state rather than causing their death, providing a new method for biological control of migratory locusts and has broad application prospects.

[0055] Compared with chemical pesticide control methods for locust control, the use of inhibitors screened in this application, such as 4-nitrophenol, for control has the following advantages:

[0056] 1) Compared with chemical pesticides, 4-nitrophenol can keep locusts in a harmless scattered state instead of killing them;

[0057] 2) It has a significant inhibitory effect on the biosynthesis of the locust aggregation pheromone 4VA, and can be artificially released to interfere with their aggregation, thereby controlling their large-scale swarming and thus forming locust plagues;

[0058] 3) The required dosage of 4-nitrophenol is low, which can greatly reduce the cost of prevention and control, and the prevention and control effect is very efficient.

[0059] 4) The method for identifying the biosynthesis of the locust aggregation pheromone 4VA provided in this application is the first to identify the biosynthesis of 4VA in animals, and has the advantages of being simple, easy to perform, and accurate in analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 is a graph showing the release of 4VA by gregarious locusts after starvation treatment.

[0061] Figure 2 is a schematic diagram of the main component structure of wheat seedlings, a plant eaten by locusts.

[0062] FIG3 is a graph showing the release of 4VA after feeding artificial feed containing lignin.

[0063] FIG4 is a diagram showing the verification results of the precursors for 4VA biosynthesis.

[0064] FIG5 is a graph showing the results of species detection of compounds in the locust intestine and hemolymph.

[0065] Figure 6 shows two chemically plausible biosynthetic pathways from phenylalanine to 4VA.

[0066] FIG. 7 is a diagram showing the verification results of the intermediates in the biosynthesis of 4VA.

[0067] FIG8 is the GC-MS / MS combined technology detection and analysis results of wheat seedlings.

[0068] It was verified that locusts can obtain phenylalanine, cinnamic acid and para-hydroxycinnamic acid directly from host plants.

[0069] FIG9 shows the detection results of four intermediates in 4VA biosynthesis in solitary locusts.

[0070] FIG10 shows the detection results of the difference in 4VA production between gregarious and solitary locusts.

[0071] Figure 11 shows the expression level detection results of genes annotated as methyltransferases in the hind legs of gregarious and solitary locusts.

[0072] FIG12 is a diagram showing the results of in vitro enzyme activity detection of 4VPMT1 and 4VPMT2.

[0073] FIG13 is a diagram showing the inhibitory effects of 4-nitrophenol and 4-trifluoromethylphenol on 4VA production in the screening of analogs designed based on 4VP.

[0074] FIG14 is a diagram showing the results of enzyme activity detection of 4-nitrophenol and 4-trifluoromethylphenol.

[0075] FIG15 is a graph showing the inhibitory effect of 4-nitrocatechol on 4VA production among analogs designed based on 4-nitrophenol.

[0076] FIG16 is an in vitro enzyme activity assay to identify the IC of 4-nitrocatechol to 4VPMT1 50 value.

[0077] Figure 17 is the conversion rate of 4-nitrophenol by 4VPMTs

[0078] FIG18 is a graph showing the inhibitory effect of 4-nitroanisole, the methylation product of 4-nitrophenol, on 4VA production.

[0079] FIG19 is a kinetic parameter characterization of 4VPMT1 to 4-nitrophenol.

[0080] FIG20 is a graph showing the inhibitory effect of 4-nitrophenol on 4VA production and social behavior in an in vitro feeding experiment.

[0081] FIG21 is a graph showing the inhibitory effect of 4-nitrophenol on 4VA production in an in vivo injection experiment.

[0082] FIG22 is a graph showing the inhibitory effect of tolcapone on 4VA production based on computer-assisted inhibitor screening.

[0083] FIG23 is a graph showing the inhibitory effect of tolcapone analogs on 4VA production identified in an in vitro enzyme activity assay.

[0084] FIG24 is an in vitro enzyme activity assay to identify the IC of tolcapone against 4VPMT1 50 value.

[0085] FIG25 is a graph showing the conversion rate of tolcapone by 4VPMT1.

[0086] FIG26 is a graph showing the inhibitory effect of tolcapone on 4VA production in an in vitro feeding experiment.

[0087] FIG27 is a graph showing the inhibitory effect of tolcapone on 4VA production in an in vivo injection experiment.

[0088] FIG28 is a schematic diagram of the complete biosynthetic pathway of 4VA. DETAILED DESCRIPTION

[0089] The technical solution of the present application is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0090] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0091] Example 1: Identification of the synthetic pathway of the locust aggregation pheromone 4VA

[0092] 1. Starve the fifth-instar gregarious locust nymphs and collect volatiles using SPME

[0093] 1.1 Implementation Method

[0094] The experiment used 5th-instar gregarious locust nymphs. 100 intact gregarious locusts of equal size were divided into a control group and an experimental group. Both groups were housed in plexiglass cages (15 cm × 15 cm × 15 cm). Control locusts were fed wheat seedlings, while experimental locusts were starved for 1, 2, 4, 6, and 8 hours. Volatile compounds were collected using SPME.

[0095] First, the extraction tip was preactivated at 250°C for 5 minutes. The experiment included 6 biological replicates, each containing 4 locusts. The 4 locusts were placed in a 20 mL sample vial (Agilent, PL, 5182-0837) and sealed with a cap. The handle rod (SPME Holder, 57330-U) was kept horizontal to the sample vial, and then the needle was penetrated through the sample vial septum 3-4 cm from the bottle mouth and slowly pushed into the sample vial. The fiber tip (SPME Fiber, PDMS / DVB-65μm, 57310-U) was pushed out to expose it to the headspace volatiles produced by the reaction and adsorbed at 30°C for 30 minutes. After adsorption, the fiber tip was retracted and the needle was withdrawn from the sample vial. The release level of 4VA was quantitatively determined by GC-MS analysis.

[0096] Volatile compounds in SPME samples were quantified using a Bruker gas chromatography system (456-GC) and a triple quadrupole (TQ) mass spectrometer (Scion TQ MS / MS, Bruker Daltonics, Bremen, Germany) equipped with a DB-1 mass spectrometer column (30 m × 0.25 mm × 0.25 μm film thickness, Agilent Technologies). Data were analyzed and processed using a Bruker Chemical Analysis Mass Spectrometry Workstation (MS Data Review, Data Process, version 8.0). Standard curves for volatile compounds were constructed using different concentrations of 4VA (0.1 ng / μL, 1 ng / μL, 10 ng / μL, and 100 ng / μL) as a standard (purity ≥95%, Sigma-Aldrich) using the same thermal program and MRM method.

[0097] 1.2 Results

[0098] To investigate whether 4VA is biosynthesized from the plants they consume, this example first measured 4VA release in gregarious locusts after starvation. The results showed that after two hours of starvation, 4VA release in gregarious locusts decreased significantly. After eight hours of starvation, 4VA release was close to zero. These results suggest that locusts synthesize 4VA using building blocks from their host plants (Figure 1).

[0099] 2. The fifth-instar gregarious locust nymphs were fed artificial diet and volatiles were collected using SPME

[0100] 2.1 Implementation Method

[0101] Artificial diets for locusts were prepared according to the ingredient list (Tables 1 and 2). 400 mL of ultrapure water was thoroughly mixed with all compounds free of vitamin C and vitamin B1, then sterilized in an autoclave for 20 minutes to completely dissolve the agar powder. The sterilized solution was cooled to 30-40°C at room temperature, and vitamin C and vitamin B1 were added and stirred thoroughly. Before cooling and solidification, 1 g of lignin powder (Sigma-Aldrich, 370959) was added to 50 mL of the artificial diet, stirred thoroughly, and stored at 4°C until ready for use. Fifth-instar gregarious locust nymphs were selected for the experiment and starved for 6-8 hours. The experimental group of locusts was fed the artificial diet supplemented with lignin, while the control group was fed the artificial diet without lignin, with fresh artificial diet added every 12 hours. After 24 hours of feeding, volatiles were collected using SPME, and 4VA release levels were quantified using GC-MS analysis.

[0102] Table 1

[0103] Table 2

[0104] 2.2 Results

[0105] The main components of wheat seedlings, a plant eaten by locusts, include lignin, amino acids, cellulose, and hemicellulose (Figure 2). Since 4VA contains a benzene ring group, lignin, tyrosine, and phenylalanine are considered to be potential precursors for the biosynthesis of 4VA. In order to determine whether lignin (a polymer structure) participates in the biosynthesis of 4VA, this example detected the release of 4VA after gregarious locusts were fed an artificial diet supplemented with lignin. This example found that the release of 4VA did not increase in the group fed with lignin compared to the control group (Figure 3). Therefore, lignin does not participate in the biosynthesis of 4VA.

[0106] 3. Feeding and injecting deuterated compounds to fifth-instar gregarious locust nymphs, and collecting volatiles using SPME

[0107] 3.1 Implementation Method

[0108] Phenylalanine-d5 and tyrosine-d4 were dissolved in sterile water to a concentration of 10 μg / μL. 2 μL of the deuterated compounds were injected into the peritoneal cavity of fifth-instar gregarious locusts using a manual syringe (Agilent, Australia, 5190-1483). A control group was injected with 2 μL of sterile water. Volatiles were collected from the locusts 12, 24, and 48 hours after injection using SPME, and 4VA release levels were quantified by GC-MS analysis.

[0109] Phenylalanine-d5 and tyrosine-d4 were dissolved in sterile water to a concentration of 10 μg / μL. The solutions were evenly sprayed on the stems and leaves of wheat seedlings and added to artificial diets. Experimental groups fed 5th-instar gregarious locusts with wheat seedlings supplemented with deuterated compounds and artificial diets, respectively. A control group fed wheat seedlings sprayed with sterile water and artificial diets without deuterated compounds. Volatile compounds were collected 12, 24, and 48 hours after treatment, and 4VA release levels were quantified using GC-MS.

[0110] 3.2 Results

[0111] To investigate whether tyrosine and phenylalanine participate in the biosynthesis of 4VA, deuterated tyrosine and phenylalanine were directly injected or fed to gregarious locusts. Figure 4 shows the validation results of precursors for 4VA biosynthesis, where plant-derived phenylalanine is a precursor for 4VA biosynthesis.

[0112] The experiment found that injecting tyrosine and phenylalanine into gregarious locusts did not produce deuterated 4VA (Figures 4A and 4B). The same results were observed when feeding artificial diets containing deuterated tyrosine and phenylalanine (Figures 4C and 4D). When feeding plants containing deuterated phenylalanine, the release of deuterated 4VA (Figure 4F) in gregarious locusts was significantly induced, while feeding plants containing deuterated tyrosine did not produce deuterated 4VA (Figure 4E). Therefore, 4VA is biosynthesized from phenylalanine with the participation of plants.

[0113] 4. Metabolome analysis of locust intestinal and hemolymph metabolites using UHPLC-MS / MS

[0114] 4.1 Implementation Method

[0115] 100 mg of tissue was ground in liquid nitrogen and resuspended in pre-chilled 80% methanol and 0.1% formic acid. After mixing, the sample was incubated on ice for 5 minutes and centrifuged at 15,000 g, 4°C for 20 minutes. The supernatant was diluted with LC-MS-grade water to a final methanol concentration of 53%. The sample was transferred to a fresh centrifuge tube and centrifuged at 15,000 g, 4°C for 20 minutes. The supernatant was then aspirated and analyzed by LC-MS / MS.

[0116] UHPLC-MS / MS analysis was performed using a Vanquish UHPLC system (ThermoFisher, Germany) and an Orbitrap Q Exactive™ HF mass spectrometer (ThermoFisher, Germany) from Novogene Co., Ltd. (Beijing, China). Samples were injected into a Hypesil Gold column (100×2.1 mm, 1.9 μm) using a 17 min linear gradient at a flow rate of 0.2 mL / min. The eluents for the positive polarity mode were eluent A (0.1% FA in water) and eluent B (methanol). The eluents for the negative polarity mode were eluent A (5 mM ammonium acetate, pH 9.0) and eluent B (methanol). The solvent gradient was set as follows:

[0117] The Q ExactiveTM HF mass spectrometer was operated in positive / negative polarity mode with a spray voltage of 3.2 kV, a capillary temperature of 320 °C, a sheath gas flow rate of 40 arb, and an auxiliary gas flow rate of 10 arb.

[0118] Raw data files generated by UHPLC-MS / MS were processed using Compound Discoverer 3.1 (CD3.1, ThermoFisher) to perform peak alignment, peak picking, and quantification for each metabolite. Key parameters were: retention time tolerance, 0.2 min; actual mass tolerance, 5 ppm; signal intensity tolerance, 30%; signal-to-noise ratio, 3; and minimum intensity, 100,000. Peak intensities were then normalized to total spectral intensity. Normalized data were used to predict molecular formulas based on additive ions, molecular ion peaks, and fragment ions. Peaks were then matched against the mzCloud (https: / / www.mzcloud.org / ), mzVault, and MassList databases to obtain accurate qualitative and relative quantitative results.

[0119] 4.2 Results

[0120] In order to explore other biosynthetic intermediates from phenylalanine to 4VA, this example detected the intestinal and hemolymph metabolites of locusts. There were 628 and 448 compounds detected in the locust intestine and hemolymph, which were divided into 11 and 10 categories, respectively (Figure 5). Based on the phenylalanine metabolism annotated in KEGG, the metabolites in the intestine and hemolymph were located, and two chemically reasonable biosynthetic pathways from phenylalanine to 4VA were obtained. The first pathway is phenylalanine-phenylacetaldehyde-phenylethanol-p-hydroxyphenylethanol-4-vinylphenol-4VA. The second pathway is phenylalanine-cinnamic acid (CA)-p-hydroxycinnamic acid (p-HCA)-4-vinylphenol (4VP)-4VA (Figure 6).

[0121] 5. Feeding and injecting deuterated intermediates to fifth-instar gregarious locust nymphs, and collecting volatiles using SPME

[0122] 5.1 Implementation Method

[0123] Phenylacetaldehyde-d6, cinnamic acid-d6, p-hydroxycinnamic acid-d4, and 4-vinylphenol-d4 were dissolved in sterile water to a concentration of 10 μg / μL. 2 μL of the deuterated compounds were injected into the peritoneal cavity of fifth-instar gregarious locusts using a manual syringe (Agilent, Australia, 5190-1483). A control group was injected with 2 μL of sterile water. Volatiles were collected 12 hours after injection using SPME, and 4VA release levels were quantified by GC-MS analysis.

[0124] Phenylacetaldehyde-d6 and cinnamic acid-d6 were dissolved in sterile water to a concentration of 10 μg / μL. The solutions were evenly sprayed on the stems and leaves of wheat seedlings and added to artificial diets. Five-instar gregarious locusts were fed the wheat seedlings supplemented with the deuterated compounds and the artificial diet, respectively. A control group was fed wheat seedlings sprayed with sterile water and the artificial diet without the deuterated compounds. Volatiles were collected from the locusts 12 hours after feeding using SPME, and 4VA release levels were quantified using GC-MS.

[0125] 5.2 Results

[0126] To determine the biosynthetic pathway for 4VA, this example conducted stable isotope labeling studies on gregarious locusts to detect the production of deuterated 4VA. Figure 7 shows the validation results for intermediates in 4VA biosynthesis, where cinnamic acid, p-hydroxycinnamic acid, and 4-vinylphenol are intermediates in 4VA biosynthesis.

[0127] The results showed that deuterated 4VA could not be produced after injection, feeding with artificial diet containing deuterated phenylacetaldehyde, or feeding with plants sprayed with deuterated phenylacetaldehyde (Figure 7, AC). Injection with deuterated cinnamic acid also did not produce deuterated 4VA (Figure 7D), while feeding with artificial diet and plants containing deuterated CA induced the production of deuterated 4VA (Figure 7, E and F). These results indicate that 4VA is biosynthesized through a second pathway. To further verify this biosynthetic pathway, deuterated p-HCA and 4VP were injected into gregarious locusts. Both compounds significantly induced the production of deuterated 4VA, indicating that p-HCA and 4VP are indeed intermediates in 4VA biosynthesis (Figure 7, G and H). In summary, locusts can independently convert cinnamic acid, p-hydroxycinnamic acid, and 4-vinylphenol into 4VA.

[0128] 6. Derivatization of biosynthetic intermediates in 5th instar gregarious nymphs and analysis using GC-MS / MS

[0129] 6.1 Implementation Method

[0130] Phenylalanine-d5 was dissolved in sterile water to a concentration of 10 μg / μL and then evenly sprayed on the stems and leaves of wheat seedlings. Deuterated compounds in wheat seedlings were derivatized and quantitatively analyzed using GC-MS / MS. Approximately 100 mg of tissue sample was weighed, transferred to a centrifuge tube containing 500 μL of extraction reagent (ethanol:acetonitrile, 9:1), and ground in a grinder (JXFSTRP-32L, Shanghai Jingxin Industrial Development, 60 Hz, 2 min). After standing at room temperature for 15 min, the sample was centrifuged at 4°C, 12,000 rpm for 10 min, and the supernatant was transferred to a new centrifuge tube and vacuum dried. Methoxyamine hydrochloride (20 mg / mL, dissolved in pyridine, 50 μL per sample) was added to the dried sample, mixed well, and incubated at 37°C for 90 min. Then, add N-methyl-N-trimethylsilyl trifluoroacetamide (70 μL per sample) to the sample, mix thoroughly, and incubate at 37°C for 90 minutes. Centrifuge at 4°C, 12,000 rpm for 10 minutes. Aspirate the supernatant and store at -20°C until assayed.

[0131] 6.2 Results

[0132] The conversion of phenylalanine to cinnamic acid to p-hydroxycinnamic acid is a conserved biosynthetic pathway for lignin in plants. To determine whether plants can provide these intermediates to locusts, the levels of these four intermediate compounds were measured in wheat seedlings sprayed with deuterated phenylalanine. The results showed that deuterated Phe, CA, and p-HCA were all detectable, while deuterated 4VP was not detected (Figure 8). Figure 8 confirms that locusts can directly obtain phenylalanine, cinnamic acid, and p-hydroxycinnamic acid from their host plants, but that host plants cannot directly produce 4VP or 4-VA.

[0133] Example 2: Identification of 4VPMTs as the key enzyme for 4VA production in locusts

[0134] 1. Derivatization of biosynthetic intermediates in the fifth-instar gregarious locust nymphs and analysis using GC-MS / MS

[0135] 1.1 Implementation Method

[0136] Phe, CA, p-HCA, and 4VP were derivatized and quantified in the gut, hemolymph, and legs of gregarious and solitary locusts using GC-MS / MS. Approximately 100 mg of tissue sample was weighed, transferred to a centrifuge tube containing 500 μL of extraction reagent (ethanol:acetonitrile, 9:1), and ground in a grinder (JXFSTRP-32L, Shanghai Jingxin Industrial Development, 60 Hz, 2 min). After standing at room temperature for 15 min, the sample was centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was transferred to a new centrifuge tube and vacuum-dried. Methoxyamine hydrochloride (20 mg / mL, dissolved in pyridine, 50 μL per sample) was added to the dried sample, mixed, and incubated at 37°C for 90 min. N-methyl-N-trimethylsilyl trifluoroacetamide (70 μL per sample) was then added to the sample, mixed, and incubated at 37°C for 90 min. After incubation, the cells were centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was aspirated and stored at -20°C for testing.

[0137] 1.2 Results

[0138] Since solitary locusts do not produce 4VA, this example further determined which of these four precursors contributes to the difference in 4VA production between gregarious and solitary locusts. By detecting the four intermediates in three tissues of gregarious and solitary locusts, it was found that Phe, CA, p-HCA, and 4VP were all detectable in different tissues of both gregarious and solitary locusts (Figure 9). This result indicates that the absence of 4VA in solitary locusts is not due to a deficiency in these four precursors.

[0139] 2. Fifth-instar solitary locust nymphs were fed and injected with deuterated compounds, and volatiles were collected using SPME

[0140] 2.1 Implementation Method

[0141] Phe-d5 and CA-d6 were dissolved in sterile water at a concentration of 10 μg / μL and sprayed evenly on the stems and leaves of wheat seedlings. Fifth-instar solitary locust nymphs were fed the deuterated wheat seedlings in the experimental group, while the control group was fed wheat seedlings sprayed with sterile water. Volatiles from the locusts were collected 12 hours after treatment using SPME and GC-MS analysis to quantify 4VA release.

[0142] p-HCA-d4 and 4VP-d4 were dissolved in sterile water to a concentration of 10 μg / μL. 2 μL of the deuterated compounds were injected into the peritoneal cavity of fifth-instar solitary locust larvae using a manual syringe (Agilent, Australia, 5190-1483). A control group was injected with 2 μL of sterile water. After 12 hours of treatment, volatiles were collected from the locusts and 4VP-d4 levels were quantitatively determined using SPME and GC-MS analysis.

[0143] 2.2 Results

[0144] To further verify this, this example applied deuterated compounds to solitary locusts and measured the release of deuterated 4VA. Figure 10 shows the difference in 4VA production between gregarious and solitary locusts. The difference in 4VA production between gregarious and solitary locusts may be due to the conversion of 4-vinylphenol to 4VA.

[0145] Testing revealed that feeding plants containing deuterated Phe and CA did not induce deuterated 4VA production in solitary locusts (Figure 10, A and B), and injection of deuterated p-HCA and 4VP did not induce deuterated 4VA production in solitary locusts (Figure 10, C and D). These results suggest that the chemical conversion of Phe to 4VP is not the rate-limiting step in 4VA biosynthesis in solitary locusts. Therefore, the conversion of 4VP to 4VA may contribute to the differences in 4VA production between gregarious and solitary locusts.

[0146] 3. Transcriptome analysis, qPCR experimental verification and RNAi interference experiment

[0147] 3.1 Implementation Method

[0148] The conversion of 4VP to 4VA is chemically mediated by a methylase. Therefore, we first analyzed the differential expression of annotated methyltransferase genes in the hind legs of gregarious and solitary locusts using transcriptome data. Total RNA was extracted from the hind legs (three samples, 5-6 insects each) using Trizol reagent. The quantity and purity of total RNA were determined using an Agilent 2100 Bioanalyzer (Agilent) to verify RNA integrity. After QC, poly-A RNA was enriched from eukaryotic total RNA using the TIANSeq mRNA Capture Kit (TIANGEN). Transcriptome sequencing libraries were then constructed using the TIANSeq Fast RNA Library Kit (Illumina) using the captured RNA as the starting material. The library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies) and diluted to 1 ng / μL. Insert size was then checked on the Agilent 2100 and quantified for higher accuracy by quantitative PCR (qPCR) (library activity >2 nM). Index-coded samples were clustered using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) on the cBot Cluster Generation System. After cluster generation, the samples were sequenced on the Illumina platform, generating 150-bp paired-end reads to analyze differential expression of annotated methyltransferase genes in the hind legs of gregarious and solitary locusts.

[0149] In order to verify the above-mentioned gene annotated as methyltransferase, this example was verified by qPCR experiment. 8 locust hind leg samples (2-3 locusts / sample) were collected and total RNA was extracted using Trizol reagent (Life Technology) and according to the manufacturer's instructions. DNase was used to eliminate DNA contamination in the RNA sample. 2 μg of total RNA was drawn and reverse transcribed using the MMLV reverse transcriptase kit (Promega, Madison, USA, Madison, USA) to analyze the expression levels of mRNAs. PCR amplification was performed using the miRcute miRNA qPCR Detection Kit (Tiangen) and the Real Master-Mix (SYBR Green) kit (Tiangen, Beijing, China) respectively. RP49 was used as an endogenous control for mRNAs. Melting curves were detected during the amplification process to determine the amplification specificity of the target gene, and the PCR amplification results were sequenced to verify the specificity of the primers. The primer sequences for qPCR detection are shown in Table 3.

[0150] Table 3

[0151] To further validate the differential expression of the methyltransferase gene in the hind legs of gregarious and solitary locusts, an RNAi (RNAi) experiment was performed in this example. First, specific double-stranded RNAs (dsRNAs) for RNAi were synthesized based on the gene sequence using the T7 RiboMAX Express RNAi System (Promega, Madison, USA). The synthesized dsRNAs were injected into the second abdominal cavity of fifth-instar gregarious locust nymphs using a manual syringe (Agilent, Australia, 5190-1483). Relative mRNA expression and 4VA release were measured 72 hours after injection using qPCR and GC-MS / MS, respectively. RNAi primers are shown in Table 6.

[0152] Table 6

[0153] 3.2 Results

[0154] Figure 11 shows the expression level detection results of genes annotated as methyltransferases in the hind legs of gregarious and solitary locusts. Transcriptome data were used to analyze the expression differences of genes annotated as methyltransferases in the hind legs of gregarious and solitary locusts. It was found that the expression levels of nine genes annotated as methyltransferases were much higher in gregarious locusts than in solitary locusts (Figure 11A). qPCR experiments confirmed that the expression levels of six of these genes were significantly higher in gregarious locusts than in solitary locusts, namely LOCMI02868, LOCMI16699, LOCMI17143, LOCMI17606, LOCMI16705, and LOCMI03758 (Figure 11B). Further RNAi interference experiments confirmed that knocking out LOCMI16699 and LOCMI02868 significantly reduced the release of 4VA in gregarious locusts compared to the control group (Figures 11, C and D). Knocking out other genes did not change the release of 4VA (Figures 11, EH). These results suggest that LOCMI16699 and LOCMI02868 may control the production of 4VA in locusts.

[0155] 4. In vitro expression and kinetic parameter characterization

[0156] 4.1 Implementation Method

[0157] To verify the functions of LOCMI16699 and LOCMI02868, the two proteins were exogenously expressed. The CDS fragments encoding LOCMI16699 and LOCMI02868 were amplified by PCR, cloned into a pET-28a-derived vector with an N-terminal His tag, and transformed into Escherichia coli BL21 (DE3) strain. The cells were cultured at 37°C and the OD value was 0. 600When the p-value reached 0.5, IPTG was added to a final concentration of 0.4 mM and induced at 16°C for 14 h. The cells were harvested by centrifugation at 4500 g for 15 min and resuspended in 30 mM imidazole buffer (30 mM imidazole, 30 mM Tris-HCl, pH 8.0, 50 mM NaCl). After sonication, the cells were centrifuged at 15000 g for 15 min at 4°C, and the supernatant was transferred to a fresh centrifuge tube for subsequent column flow. First, a Ni-NTA nickel column was equilibrated with 30mM imidazole buffer (30mM imidazole, 30mM Tris-HCl, pH 8.0, 50mM NaCl). The supernatant after centrifugation was added, and the target protein was washed with 50mM imidazole buffer (50mM imidazole, 30mM Tris-HCl, pH 8.0, 50mM NaCl). Finally, the protein was eluted with 250mM imidazole buffer (250mM imidazole, 30mM Tris-HCl, pH 8.0, 50mM NaCl), and the eluate was collected. The expression level of the eluted protein was determined by SDS-PAGE (polyacrylamide gel electrophoresis). Protein samples were stored at -80°C.

[0158] Enzyme activity was determined by solid-phase microextraction (SPME) headspace quantification of product. First, 399 μL of the reaction mixture containing buffer (20 mM Na₂HPO₄-NaH₂PO₄, 50 mM NaCl, pH 7.5), 20 μg of LOCMI16699 protein, and 4 μL of the prosthetic group SAM (100 mM) were pipetted into a 5 mL vial (Cat: RY-10100). The reaction was initiated by adding 1 μL of the substrate 4VP at varying concentrations (0.04–100 mM). The vial was then sealed with a cap (Cat: RY-10100). Keep the handle rod (SPME Holder, 57330-U) and the sample bottle in a horizontal position, then penetrate the sample bottle septum at a distance of 3-4 cm from the bottle mouth, insert the needle into the bottle and slowly push the sample bottle into the bottle, push out the fiber head (SPME Fiber, PDMS / DVB-65μm, 57310-U) to expose it to the headspace volatiles produced by the reaction, and adsorb at 30°C for 10 minutes. After adsorption, retract the fiber head and then withdraw the needle from the sample bottle. The release level of 4VA was quantitatively determined by GC-MS analysis. The LOCMI02868 enzyme activity assay was similar to the above method. The added protein was 180μg, the substrate 4VP concentration was 4-320mM, and the adsorption was 20min. The amount of product in the reaction was calculated based on the standard curve of 4VA. GraphPad Prism 8 was used for data fitting to obtain K M 、k cat 、k cat / K M value.

[0159] 4.2 Results

[0160] To further verify the functions of LOCMI16699 and LOCMI02868, these two proteins were exogenously expressed in this example and their enzymatic activities were tested in vitro. Figure 12 shows the results of in vitro enzymatic activity testing of 4VPMT1 and 4VPMT2.

[0161] The results showed that LOCMI16699 and LOCMI02868 could catalyze the methylation of 4VP to 4VA in the presence of the prosthetic group SAM, and were therefore named 4-vinylphenol methyltransferase 1 (4VPMT1) and 4-vinylphenol methyltransferase 2 (4VPMT2), respectively (Figure 12, A and B). The kinetic parameters of 4VPMT1 and 4VPMT2 were also determined by in vitro enzyme activity assays. The kinetic parameters of 4VPMT1 showed that its K for the substrate 4VP was M and k cat The values ​​were 23.03 μM and 4.75 × 10 -4 s -1 In addition, as the concentration of 4VP gradually increased, the enzyme activity of 4VPMT1 decreased, and the Ki value was 94.88 μM (Figure 12C). M and k cat The values ​​were 309.7 μM and 1.45×10 -6 s -1 (Figure 12D). cat / K M =0.00468±0.00113M -1 s -1 ) compared to 4VPMT1(k cat / K M =20.6±11.8M -1 s -1 ) exhibited higher catalytic efficiency, indicating that 4VPMT1 is the primary factor in 4VP methylation in locusts. Therefore, 4VPMT1 and its homologous protein, 4VPMT2, can catalyze the production of 4VA both in vivo and in vitro. The amino acid sequence of 4VPMT1 is shown in SEQ ID NO:1, and the gene sequence encoding it is shown in SEQ ID NO:2; the amino acid sequence of 4VPMT2 is shown in SEQ ID NO:3, and the gene sequence encoding it is shown in SEQ ID NO:4.

[0162] Example 3: Development of 4VPMTs inhibitors

[0163] 1. Screening of designed substrate analogs based on the structural group of 4VP

[0164] 1.1 Implementation Method

[0165] In order to develop an effective 4VPMTs inhibitor to control locusts, this example systematically evaluates the ability of 4VP analogs to inhibit 4VP enzyme methylation as substrate competitors. First, using the styrene of the substrate 4VP as the basic skeleton, attempts were made to replace the phenolic hydroxyl group with other groups of different properties (such as amino, nitro, sulfhydryl, halogen, cyano, etc.) to design analogs. Then, attempts were made to add groups of different properties (such as hydroxyl, methoxy, etc.) or heterocyclic rings to the benzene ring to design analogs. Finally, using the phenol of the substrate 4VP as the basic skeleton, attempts were made to replace the vinyl group with groups of different properties (amino, alkyl, carboxyl, ester, trifluoromethyl, nitro, cyano, etc.) to design analogs. Analogs were added to the enzyme activity detection system at a concentration ten times higher than that of the substrate. 4VPMT1 enzyme activity was determined by quantitative headspace product amount using solid phase microextraction (SPME). First, pipette 399 μL of the reaction mixture into a 5 mL vial (Cat: RY-10100). This mixture contains buffer (20 mM Na₂HPO₄-NaH₂PO₄, 50 mM NaCl, pH 7.5), 20 μg of 4VPMT1 protein, 4 μL of the prosthetic group SAM (100 mM), 1 μL of each analog (4 mM), and finally 1 μL of the substrate 4VP (0.4 mM). The vial is then sealed with a cap (Cat: RY-10100). The SPME holder (57330-U) is held horizontally above the vial. The needle is inserted through the vial septum at a distance of 3-4 cm from the vial opening, slowly pushed into the vial, and the fiber (57310-U, PDMS / DVB-65 μm) is then introduced to expose it to the headspace volatiles generated by the reaction. Adsorption is allowed to proceed at 30°C for 10 min. After adsorption, the fiber tip was retracted and the needle was withdrawn from the sample vial. GC-MS and a 4VA standard curve were used to calculate the amount of product in reactions with and without the test compound. Data were analyzed using a two-tailed unpaired t-test in GraphPad Prism 8 software.

[0166] 1.2 Results

[0167] First, analogs were designed based on the styrene backbone of the substrate 4VP. When these analogs were added to the enzyme activity assay at concentrations tenfold higher than the substrate, the most potent inhibitory analogs reduced 4VPMT1 activity by less than 50% (Figure 13A). This result suggests that the phenolic hydroxyl group on 4VP plays a crucial role in substrate binding and recognition by 4VPMTs.

[0168] Then, among the analogues designed by adding groups of different properties to the benzene ring or replacing the benzene ring with heterocycles, 4-vinylbenzene-1,2-diol had the best inhibitory effect, which reduced the enzyme activity of 4VPMT1 by 6 times (Figure 13B).

[0169] Finally, analogues were designed using the phenol of the substrate 4VP as the basic skeleton. Detection revealed that among the 17 screened analogues with different functional groups, 4-nitrophenol and 4-trifluoromethylphenol reduced the enzyme activity of 4VPMT1 by 339-fold and 348-fold, respectively ( Figure 13C ).

[0170] 2. Identification of the IC values ​​of 4-nitrophenol and 4-trifluoromethylphenol for 4VPMTs 50 value

[0171] 2.1 Implementation Method

[0172] In order to further compare the inhibitory effects of 4-nitrophenol and 4-trifluoromethylphenol, this example performed IC 50 Detection. 4VPMT1 IC 50 The values ​​were obtained by in vitro enzyme activity assay. The reaction system included: buffer (20mM Na2HPO4-NaH2PO4, pH 7.5, 50mM NaCl), 20μg 4VPMT1 protein, 4μL SAM (100mM), 1μL of 4-nitrophenol and 4-trifluoromethylphenol at different concentrations (0.00004-40mM), and finally 1μL 4VP (0.4mM). Adsorption was carried out at 30°C for 10 minutes. The 4VPMT2 reaction system consists of: buffer (20mM Na2HPO4-NaH2PO4, pH 7.5, 50mM NaCl), 180μg 4VPMT1 protein, 4μL SAM (100mM), 1μL of 4-nitrophenol and 4-trifluoromethylphenol at varying concentrations (0.00004-20mM), and finally 1μL 4VP (30mM). Adsorption was allowed to proceed at 30°C for 20min. The product amount in the reaction was calculated using GC-MS and a 4VPMT1 standard curve. IC 50 The values ​​were obtained by fitting the relative activities of the test compounds at different concentrations using the dose-response-inhibition function in GraphPad Prism 8 software.

[0173] 2.2 Results

[0174] Figure 14 shows the results of enzyme activity assays for 4-nitrophenol and 4-trifluoromethylphenol. By performing enzyme activity assays for 4-nitrophenol and 4-trifluoromethylphenol under the same in vitro conditions, it was found that the IC values ​​of 4VPMT1 and 4VPMT2 for 4-nitrophenol were 50The IC values ​​of 4-trifluoromethylphenol for 4VPMT1 and 4VPMT2 were 184.2 nM and 177.7 nM, respectively (Figure 14, A and C). 50 The values ​​were 411.6 nM and 550.1 nM, respectively ( FIG14 , B and D). These results indicate that 4-nitrophenol can inhibit 4VPMT1 and 4VPMT2 at lower concentrations.

[0175] 3. Design of analogs based on the structural groups of 4-nitrophenol

[0176] 3.1 Implementation Method

[0177] To further identify analogs with enhanced inhibitory effects, this example attempted to replace the structural groups of 4-nitrophenol with groups of similar properties and systematically evaluated the ability of 4-nitrophenol analogs to inhibit 4VP methylation as substrate competitors. 4VPMT1 enzyme activity was determined by headspace quantification using solid-phase microextraction (SPME). First, 399 μL of the reaction mixture containing buffer (20 mM Na₂HPO₄-NaH₂PO₄, 50 mM NaCl, pH 7.5), 20 μg of 4VPMT1 protein, 4 μL of the prosthetic group SAM (100 mM), and 1 μL of each analog (0.4 mM) was pipetted into a 5 mL vial (Cat: RY-10100). The mixture contained 1 μL of the substrate 4VP (0.4 mM) and was adsorbed at 30°C for 10 min. An additional 1 μL of 4-nitrophenol (0.4 mM) was added as a positive control. The amount of product in the reactions with and without test compound was calculated using GC-MS and a 4VA standard curve. Data were analyzed using a two-tailed unpaired t-test in GraphPad Prism 8 software.

[0178] 3.2 Results

[0179] Figure 15 shows the inhibitory effect of 4-nitrocatechol on 4VA production among analogs designed based on 4-nitrophenol. The test found that among the analogs designed by replacing the structural groups of 4-nitrophenol with groups having the same properties, 4-nitrocatechol with the best inhibitory effect reduced the enzyme activity of 4VPMT1 by 40 times, while 4-nitrophenol reduced the enzyme activity of 4VPMT1 by 18 times ( Figure 15 ). 4-Nitrocatechol has the best inhibitory effect on 4VA production.

[0180] 4. Identification of the IC of 4-nitrocatechol for 4VPMT1 50 value

[0181] 4.1 Implementation Method

[0182] In order to further compare the inhibitory effect of 4-nitrocatechol, this example conducted IC50 Detection. 4VPMT1 IC 50 IC values ​​were obtained through in vitro enzyme activity assays. The reaction system consisted of: buffer (20mM Na2HPO4-NaH2PO4, pH 7.5, 50mM NaCl), 20μg 4VPMT1 protein, 4μL SAM (100mM), 1μL of 4-nitrocatechol at varying concentrations (0.0004-4mM), and finally 1μL 4VP (0.4mM). Adsorption was allowed to proceed at 30°C for 10 minutes. GC-MS and a 4VA standard curve were used to calculate the amount of product in the reaction. IC values ​​were calculated. 50 The values ​​were obtained by fitting the relative activities of the test compounds at different concentrations using the dose-response-inhibition function in GraphPad Prism 8 software.

[0183] 4.2 Results

[0184] By detecting the enzyme activity of 4-nitrocatechol under the same conditions in vitro, this example found that the IC 50 The value was 311.5nM (Figure 16). 50 The value was 184.2 nM, which was higher than the concentration of 4-nitrocatechol required to inhibit the enzyme activity by half. These results indicate that 4-nitrophenol can inhibit 4VPMT1 and 4VPMT2 at lower concentrations.

[0185] 5. In vitro enzyme activity assay to identify the conversion rate of 4VPMTs to the inhibitor 4-nitrophenol and the natural product 4-vinylphenol

[0186] 5.1 Implementation Method

[0187] In chemical conversion, 4-nitrophenol can be enzymatically methylated to produce 4-nitroanisole. To further investigate the properties of 4-nitrophenol, this example performed an in vitro enzymatic activity assay. The reaction system consisted of a buffer solution (20mM Na2HPO4-NaH2PO4, pH 7.5, 50mM NaCl), 20μg 4VPMT1 protein, 4μL SAM (100mM), followed by the addition of 1μL 4-nitrophenol (0.4mM) and HO, respectively, and adsorption at 30°C for 10min. The reaction system for 4VPMT2 consisted of a buffer solution (20mM Na2HPO4-NaH2PO4, pH 7.5), 50mM NaCl, 180μg 4VPMT2 protein, 4μL SAM (100mM), followed by the addition of 1μL 4-nitrophenol (30mM) and HO, respectively, and adsorption at 30°C for 20min. The amount of product in reactions with and without test compound was calculated using GC-MS and a standard curve of 4-nitroanisole. Data were analyzed using a two-tailed unpaired t-test in GraphPad Prism 8 software.

[0188] To further verify the conversion efficiency of 4VPMTs for the natural product 4VP and the inhibitor 4-nitrophenol, in vitro enzymatic activity assays were performed. The reaction system consisted of: buffer (20 mM Na2HPO4-NaH2PO4, pH 7.5, 50 mM NaCl), 20 μg 4VPMT1 protein, 4 μL SAM (100 mM), followed by the addition of 1 μL of 4VP (0.4 mM) and 4-nitrophenol (0.4 mM), respectively, and adsorption at 30°C for 10 min. The reaction system for 4VPMT2 consisted of: buffer (20 mM Na2HPO4-NaH2PO4, pH 7.5, 50 mM NaCl), 180 μg 4VPMT2 protein, 4 μL SAM (100 mM), followed by the addition of 1 μL of 4VP (0.4 mM) and 4-nitrophenol (0.4 mM), respectively, and adsorption at 30°C for 20 min. The amount of product in the reaction was calculated using GC-MS, a standard curve of 4VA and 4-nitroanisole. Data were analyzed using a two-tailed unpaired t-test in GraphPad Prism 8 software.

[0189] 5.2 Results

[0190] Testing revealed that both 4VPMT1 and 4VPMT2 methylated 4-nitrophenol to 4-nitroanisole ( Figure 17 , A and B). Furthermore, under identical assay conditions, the conversion rates of 4-nitrophenol by 4VPMT1 and 4VPMT2 were significantly lower than that of the natural product 4VP ( Figure 17 , C and D). These results indicate that 4-nitrophenol is an effective and stable inhibitor of 4VA production in locusts.

[0191] 6. In vitro enzyme activity assay to verify the effect of 4-nitroanisole on 4VA production

[0192] 6.1 Implementation Method

[0193] The above experiments confirmed that 4-nitrophenol can be enzymatically methylated to 4-nitroanisole during the inhibition process. To further investigate the properties of 4-nitroanisole, an in vitro enzymatic activity assay was performed. The reaction system consisted of: buffer (20 mM Na₂HPO₄-NaH₂PO₄, pH 7.5, 50 mM NaCl), 20 μg 4VPMT1 protein, and 4 μL SAM (100 mM). The experimental group received 1 μL of 4-nitroanisole (0.4 mM) and the substrate 4VP (0.4 mM), while the control group received only 1 μL of the substrate 4VP (0.4 mM). Adsorption was allowed to proceed at 30°C for 10 min. The amount of product in reactions with and without the test compound was calculated using GC-MS and a 4VPMT1 standard curve. Data were analyzed using a two-tailed unpaired t-test in GraphPad Prism 8 software.

[0194] 6.2 Results

[0195] The results showed that the enzyme activity of 4VPMT1 decreased by 1.5 times after the addition of 4-nitroanisole compared with the control group (Figure 18). These results indicate that 4-nitroanisole, the methylation product of the inhibitor 4-nitrophenol, has an inhibitory effect on the production of 4VA.

[0196] 7. In vitro enzyme activity assay to identify the kinetic parameters of 4VPMT1 for 4-nitrophenol

[0197] 7.1 Implementation Methods

[0198] First, 399 μL of the reaction mixture was pipetted into a 5 mL sample vial (Cat: RY-10100). The mixture contained buffer (20 mM Na2HPO4-NaH2PO4, pH 7.5, 50 mM NaCl), 200 μg of 4VPMT1 protein, and 4 μL of the prosthetic group SAM (100 mM). Finally, 1 μL of 4-nitrophenol at different concentrations (0.04-40 mM) was added and adsorbed at 30°C for 30 min. The amount of product in the reaction was calculated using GC-MS analysis and a standard curve of 4-nitroanisole. Data fitting was performed using GraphPad Prism 8 to obtain K values. M , k cat , k cat / K M value.

[0199] 7.2 Results

[0200] The kinetic parameters of 4VPMT1 showed that its K for substrate 4-nitrophenol was M and k cat The values ​​were 15.99 μM and 1.55 × 10 -5 s -1 At the same time, 4VPMT1 has a k cat / K M The value is 0.97±0.3M -1 s -1 )(Figure 19). These results indicate that 4-nitrophenol, as a substrate competitor, can inhibit the methylation of 4VP enzyme, thereby inhibiting the production of 4VA in locusts.

[0201] 8. In vitro feeding of 4-nitrophenol

[0202] 8.1 Implementation Methods

[0203] In order to further explore the effects of 4-nitrophenol on the morphological changes of gregarious locusts and the aggregation process of solitary locusts, gregarious locusts and solitary locusts treated with swarming were fed with wheat seedlings sprayed with 4-nitrophenol, and their behavioral states were detected using the Arena behavioral analysis method.

[0204] To further investigate the effects of 4-nitrophenol on phenotypic variation in gregarious locusts, fifth-instar gregarious locusts were fed wheat seedlings sprayed with 2 mL of a 2 mM 4-nitrophenol solution. A control group was fed wheat seedlings sprayed with sterile water. Volatiles were collected 24, 48, and 72 hours after treatment, and 4-VA release levels were quantitatively determined using GC-MS analysis. A field behavioral device was also used to monitor behavioral changes in gregarious locusts.

[0205] To further investigate the effects of 4-nitrophenol on the aggregation of solitary locusts, this example first subjected solitary locusts to a swarming treatment. Ten solitary nymphs in the early fifth instar were placed in a 10 cm × 10 cm × 10 cm cage, and 20 gregarious nymphs in the early fifth instar were added as a stimulus group. During this period, the experimental group was fed wheat seedlings sprayed with 4-nitrophenol, while the control group was fed wheat seedlings sprayed with sterile water. Volatiles were collected 24, 48, and 72 hours after treatment, and 4-VA release levels were quantitatively determined using GC-MS analysis. Simultaneously, behavioral changes in the solitary locusts were monitored using a mine behavioral device.

[0206] 8.2 Results

[0207] To investigate the effect of 4-nitrophenol on phenotypic variation in gregarious locusts, we fed wheat seedlings sprayed with 4-nitrophenol, and measured their behavioral state 72 hours after treatment using the Arena behavioral assay (Figure 20A). The results showed that 4VA release decreased significantly after 48 hours of 4-nitrophenol feeding compared to the control group (Figure 20B). Furthermore, the behavior of gregarious locusts shifted significantly toward solitary behavior (Figure 20C).

[0208] In addition, the solitary locusts that had been swarmed were fed wheat seedlings sprayed with 4-nitrophenol, and their behavioral states were monitored (Figure 20D). The test found that the 4VA release of the solitary locusts in the group fed 4-nitrophenol was significantly reduced 24 hours after swarming compared to the control group fed water (Figure 20E). The solitary locusts in the swarming treatment that fed 4-nitrophenol still exhibited solitary behavior, while the control group exhibited significant gregarious behavior (Figure 20F). These results indicate that 4-nitrophenol can effectively inhibit the gregarious behavior of locusts.

[0209] 9. In vivo injection of 4-nitrophenol

[0210] 9.1 Implementation Methods

[0211] To further investigate the effects of 4-nitrophenol on 4VPMTs, this example involved the injection of 4-nitrophenol into locusts. 2 μL of aqueous 4-nitrophenol solutions at varying concentrations (0.005 mM, 0.05 mM, 0.5 mM, 5 mM, and 50 mM) were injected into the abdominal cavity of fifth-instar gregarious locust nymphs. 4VA release was measured 2 hours after injection using GC-MS / MS. 4VA release was also measured using GC-MS 2, 4, 8, and 12 hours after injection of 2 μL of 0.5 mM 4-nitrophenol. All control groups received 2 μL of water.

[0212] 9.2 Results

[0213] The experiment found that 4-nitrophenol significantly inhibited 4VA production within an injection concentration range of 0.1-100 nmol (Figure 21A). Furthermore, 1 nmol of 4-nitrophenol significantly inhibited 4VA production 2 and 4 hours after injection, while 4VA release remained unchanged 8 and 12 hours after injection (Figure 21B). This suggests that 4-nitrophenol can inhibit 4VA production in locusts.

[0214] 10. Computer-assisted screening of 4VPMTs inhibitors

[0215] 10.1 Implementation Methods

[0216] To more rapidly and comprehensively develop new 4VPMT inhibitors for locust control, this study used GNINA software23 to perform a virtual screening of a library of 100,000 active small molecule compounds based on the structure of the target protein 4VPMTs to identify potential target compounds. During the screening process, all small molecules were ranked from low to high based on their scores. Ultimately, 100 candidate molecules were selected for enzyme activity testing, and the small molecule concentration was tenfold higher than the substrate concentration in the assay system. First, 399 μL of the reaction mixture containing buffer (20 mM Na₂HPO₄-NaH₂PO₄, 50 mM NaCl, pH 7.5), 20 μg of 4VPMT1 protein, 4 μL of a 100 mM prosthetic group SAM, 0.4 μL of various analogs (10 mM), and finally, 1 μL of the substrate 4VPMT (0.4 mM) was added. Adsorption was allowed to proceed at 30°C for 10 min. The amount of product in the reactions with and without the test compound was calculated using GC-MS. The data were analyzed using a two-tailed unpaired t-test in GraphPad Prism 8 software.

[0217] 10.2 Results

[0218] Testing revealed that tolcapone, the most potent inhibitor among the 100 candidate small molecules, reduced 4VPMT1 enzymatic activity by 7-fold (Figure 22). Furthermore, structural analogs of tolcapone were tested for their inhibitory activity against 4VPMT1, and it was found that niticapolone, entacapone, and 5-nitrovanillin all exhibited inhibitory activity against 4VPMT1 (Figure 23).

[0219] 11. Identification of the IC of Tolcapone for 4VPMT1 50 value

[0220] 11.1 Implementation Methods

[0221] To further explore the inhibitory effect of tolcapone, the IC of tolcapone on 4VPMT1 was detected in vitro. 50 The reaction system consisted of: buffer (20mM Na2HPO4-NaH2PO4, pH 7.5, 50mM NaCl), 20μg 4VPMT1 protein, 4μL SAM (100mM), 1μL tolcapone at varying concentrations (0.004-400mM), and finally 1μL 4VP (0.4mM). Adsorption was performed at 30°C for 10 minutes. The amount of product in the reaction was calculated using GC-MS and a 4VA standard curve. IC 50 The values ​​were obtained by fitting the relative activities of the test compounds at different concentrations using the dose-response-inhibition function in GraphPad Prism 8 software.

[0222] 11.2 Results

[0223] By detecting the enzyme activity of tolcapone in vitro, it was found that the IC 50 The value was 865.1 nM (Figure 24). These results indicate that 4-nitrophenol (IC 50 =184.2 nM) was able to inhibit 4VPMT1 at lower concentrations.

[0224] 12. In vitro enzyme activity assay to identify the conversion rate of 4VPMT1 to tolcapone

[0225] 12.1 Implementation Methods

[0226] Tolcapone can be enzymatically methylated to produce three methylated products during chemical conversion (Figure 25A). To further investigate the properties of tolcapone, this example performed an in vitro enzymatic activity assay. The reaction system consisted of a buffer (20mM Na2HPO4-NaH2PO4, pH 7.5, 50mM NaCl), 20μg 4VPMT1 protein, 4μL SAM (100mM), and finally 1μL tolcapone (4mM). A control group was treated with 1μL HO. Adsorption was carried out at 30°C for 10 minutes. Product peaks were observed using GC-MS in reactions with and without the test compound.

[0227] 12.2 Results

[0228] The results showed that compared with the control group, no target component peak was detected after the addition of tolcapone, indicating that 4VPMT1 could not methylate tolcapone ( FIG25B ).

[0229] 13. In vitro feeding of tolcapone

[0230] 13.1 Implementation Methods

[0231] To further explore the effects of tolcapone on the morphological changes of gregarious locusts and the aggregation process of solitary locusts, gregarious locusts and solitary locusts treated with swarming were fed with wheat seedlings sprayed with tolcapone.

[0232] To further investigate the effects of tolcapone on phenotypic variation in gregarious locusts, fifth-instar gregarious locusts were fed wheat seedlings sprayed with 2 mL of tolcapone (200 mM) in ethanol. A control group was fed wheat seedlings sprayed with ethanol. Volatiles were collected 24, 48, and 72 hours after treatment, and 4-VA release levels were quantitatively determined using GC-MS analysis. A field behavioral device was also used to monitor behavioral changes in gregarious locusts.

[0233] To further investigate the effects of tolcapone on the aggregation process of solitary locusts, this example first subjected solitary locusts to a swarming treatment. Ten solitary nymphs in the early fifth instar were placed in a 10 cm × 10 cm × 10 cm cage, and 20 gregarious nymphs in the early fifth instar were added as a stimulus group. During this period, the experimental group was fed wheat seedlings sprayed with 4-nitrophenol, while the control group was fed wheat seedlings sprayed with sterile water. Volatile compounds were collected 24, 48, and 72 hours after treatment, and 4-VA release levels were quantitatively determined using GC-MS analysis.

[0234] 13.2 Results

[0235] To investigate the effects of tolcapone on gregarious locusts, both gregarious and solitary locusts were fed wheat seedlings sprayed with tolcapone. Results showed that 4VA release decreased in gregarious locusts 72 hours after feeding with tolcapone compared to the control group (Figure 26A). Furthermore, 4VA release in solitary locusts significantly decreased 48 hours after the swarming treatment (Figure 26B). These results suggest that tolcapone effectively inhibits the gregarious behavior of locusts.

[0236] 14. Intracorporeal injection of tolcapone

[0237] 14.1 Implementation Methods

[0238] To further investigate the effects of tolcapone on 4VPMTs, this example involved injection of tolcapone into locusts. Tolcapone was diluted with ethanol to different concentrations (0.5 mM, 5 mM, and 50 mM) and injected into the peritoneal cavity of fifth-instar gregarious locust nymphs. A control group received ethanol. 4VPMT release was measured using GC-MS / MS 2 hours after injection.

[0239] 14.2 Results

[0240] The experiment found that tolcapone could significantly inhibit the production of 4VA at an injection concentration of 100 nmol, and the release of 4VA did not change within the injection concentration range of 1-10 nmol ( Figure 27 ), indicating that tolcapone can inhibit the production of 4VA in locusts.

[0241] In summary, this application, through experimental chemical composition analysis of the precursors and intermediates in the biosynthesis of the locust aggregation pheromone 4VA, has for the first time identified the complete biosynthetic pathway of 4VA (as shown in Figure 28), including all precursors and key enzymes that control 4VA production. 4VPMT1 and 4VPMT2 act as biosynthetic switches for 4VA synthesis, providing an effective and sustainable strategy for locust control by developing inhibitors such as 4-nitrophenol to inhibit the enzymatic activity of 4VPMTs and thus inhibit 4VA biosynthesis.

[0242] The applicant declares that the above is only a specific implementation method of the present application, but the protection scope of the present application is not limited thereto. Technical personnel in the relevant technical field should understand that any changes or substitutions that can be easily thought of by technical personnel in the relevant technical field within the technical scope disclosed in this application fall within the protection scope and disclosure scope of this application.

Claims

1. A 4-vinylphenol methyltransferase that catalyzes the production of the locust aggregation pheromone 4-vinylanisole, comprising an amino acid sequence selected from the group consisting of: (a) the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3; (b) A sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole obtained by deleting, inserting and / or substituting one or more amino acid residues from the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO:

3.

2. A method for synthesizing the locust aggregation pheromone 4-vinylanisole, comprising: 4-Vinylphenol is used as a substrate to obtain 4-vinylanisole under the catalysis of 4-vinylphenol methyltransferase.

3. The synthesis method according to claim 2, wherein The synthesis steps of 4-vinylphenol include: using phenylalanine as a substrate, obtaining cinnamic acid under the catalysis of phenylalanine ammonia lyase, obtaining p-hydroxycinnamic acid under the catalysis of cinnamate 4-hydroxylase, and obtaining 4-vinylphenol under the catalysis of p-hydroxycinnamic acid decarboxylase.

4. A nucleic acid molecule encoding the 4-vinylphenol methyltransferase according to claim 1 that catalyzes the production of the locust aggregation pheromone 4-vinylanisole; Preferably, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: (A) the sequence shown in any one of SEQ ID NO: 2 and SEQ ID NO: 4; (B) A sequence that is at least 90% identical to the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3, and encodes a sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole. An expression vector comprising the nucleic acid molecule according to claim 4 . A recombinant cell comprising the expression vector according to claim 5, or the nucleic acid molecule according to claim 4 is integrated into the genome of the recombinant cell.

7. Use of a 4-vinylphenol methyltransferase gene or 4-vinylphenol methyltransferase in screening locust aggregation inhibitors, wherein: The 4-vinylphenol methyltransferase comprises an amino acid sequence selected from the group consisting of: (a) the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3; (b) a sequence having activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole obtained by deleting, inserting and / or substituting one or more amino acid residues from the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3; Preferably, the 4-vinylphenol methyltransferase gene contains a nucleotide sequence selected from the group consisting of: (A) the sequence shown in any one of SEQ ID NO: 2 and SEQ ID NO: 4; (B) A sequence that is at least 90% identical to the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3, and encodes a sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole.

8. Use of a 4-vinylphenol methyltransferase gene inhibitor or a 4-vinylphenol methyltransferase inhibitor in the preparation of a locust control drug, wherein: The 4-vinylphenol methyltransferase comprises an amino acid sequence selected from the group consisting of: (a) the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3; (b) a sequence having activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole obtained by deleting, inserting and / or substituting one or more amino acid residues from the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3; Preferably, the 4-vinylphenol methyltransferase gene contains a nucleotide sequence selected from the group consisting of: (A) the sequence shown in any one of SEQ ID NO: 2 and SEQ ID NO: 4; (B) A sequence that is at least 90% identical to the sequence shown in any one of SEQ ID NO: 1 and SEQ ID NO: 3, and encodes a sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole.

9. The use according to claim 8, wherein The medicine takes 4-vinylphenol methyltransferase gene inhibitor or 4-vinylphenol methyltransferase inhibitor as active ingredient.

10. Use of a polysubstituted benzene ring structure and its analogs in the preparation of 4-vinylphenol methyltransferase inhibitors, wherein: The inhibitor is a competitive substance of 4-vinylphenol, a substrate of 4-vinylphenol methyltransferase; The general structural formula of the multi-substituted benzene ring structure and its analogs is shown in Formula I: wherein R1 is selected from the group consisting of hydrogen, hydroxy, thiol, methoxy, methylthio, methyl, amino, or halogen; R2, R3, R4 or R5 are each independently selected from: hydrogen, hydroxy, methyl or methoxy, nitro; R6 is selected from the group consisting of hydrogen, alkyl, alkenyl, cyano, hydroxy, carboxyl, methoxy, halogen, nitro, ester, oxime, amide, and carbonyl; and The X atom is selected from: C or N.

11. The use according to claim 10, wherein: The inhibitor inhibits the biosynthesis of 4-vinylanisole in vivo or in vitro.

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