Biosynthesis inhibitor of migratory locust aggregation pheromone 4-vinyl anisole and application thereof

Abstract

The invention provides a biosynthesis inhibitor of migratory locust aggregation pheromone 4-vinyl anisole and application of the biosynthesis inhibitor. The biosynthesis inhibitor of migratory locust aggregation pheromone 4-vinylanisole reduces the activity of 4-vinylphenol methyltransferase, and the 4-vinylphenol methyltransferase contains an amino acid sequence selected from the following sequences: (a) a sequence as shown in any one of SEQ ID No.1 and SEQ ID No.3; and (b) a sequence which is obtained by deleting, inserting and / or substituting one or more amino acid residues in the sequence shown in any one of SEQ ID No.1 and SEQ ID No.3 and has the activity of catalyzing generation of migratory locust aggregation pheromone 4-vinyl anisole. The inhibitor can be applied to biological prevention and control of migratory locusts, can keep locusts in a harmless scattered state instead of killing the locusts, provides a new method for biological prevention and control of migratory locusts, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of pest control, and particularly relates to a biosynthesis inhibitor of the locust aggregation pheromone 4-vinylanisole and a use thereof. 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 has broad application prospects for the biological control of migratory locusts. Summary of the Invention

[0004] In response to the shortcomings of the prior art, the present invention aims to provide a biosynthesis inhibitor of the locust aggregation pheromone 4-vinylanisole and its use. The present invention identifies for the first time the biosynthetic pathway of the locust aggregation pheromone 4-vinylanisole, screens and identifies two rate-limiting methyltransferases involved in the synthesis process, and develops an inhibitor of 4VA biosynthesis based on these rate-limiting methyltransferases. The inhibitor can be used in the biological control of locusts. The inhibitor can maintain the locusts in a harmless, dispersive state rather than killing them, providing a new method for biological control of locusts with broad application prospects.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention 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:

[0007] (a) the sequence shown in any one of SEQ ID No. 1 and SEQ ID No. 3;

[0008] (b) A sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole obtained by deleting, inserting and / or replacing one or more amino acid residues from the sequence shown in any one of SEQ ID No. 1 and SEQ ID No. 3.

[0009] In a second aspect, the present invention 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.

[0010] 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.

[0011] In the present invention, the identification process of the method for the biosynthetic pathway of the locust aggregation pheromone 4VA includes:

[0012] 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.

[0013] 2. The fifth-instar gregarious locust nymphs were fed artificial diet, and the volatiles were collected using the SPME method.

[0014] 3. Fifth-instar gregarious locust nymphs were fed and injected with deuterated compounds, and volatiles from fifth-instar gregarious locust nymphs were collected using the SPME method to identify the biosynthetic precursors of 4VA.

[0015] 4. UHPLC-MS / MS was used to perform metabolome analysis of locust intestinal and hemolymph metabolites to search for possible 4VA biosynthesis pathways.

[0016] 5. Fifth-instar gregarious locust nymphs were fed and injected with deuterated intermediates, and volatiles were collected using SPME to identify the 4VA biosynthetic intermediates.

[0017] 6. The biosynthetic intermediates in the fifth-instar gregarious locust nymphs were derivatized and analyzed using GC-MS / MS to determine whether plants can provide these intermediates to locusts.

[0018] The present invention also found the key methyltransferase 4VPMTs in the 4VA biosynthesis pathway, which includes the following steps:

[0019] 1. The biosynthetic intermediates in the fifth-instar gregarious and solitary locust nymphs were derivatized and analyzed using GC-MS / MS.

[0020] 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.

[0021] 3. Identify the genes that control locust 4VA production through transcriptome analysis, qPCR experimental verification and RNAi interference experiments.

[0022] 4. Verify the function of the gene that controls locust 4VA production through in vitro expression and kinetic parameter characterization.

[0023] In this study, the biosynthetic pathway of the locust aggregation pheromone 4-vinylanisole was first identified by starvation, exogenous feeding, or injection of stable isotope-labeled intermediates. The 4VA synthesis pathway in locusts was ultimately determined to be: phenylalanine (Phe)-cinnamic acid (CA)-p-hydroxycinnamic acid (p-HCA)-4-vinylphenol (4VP)-4VA.

[0024] The present invention further verified, through transcriptome analysis, qPCR validation, in vivo interference, and in vitro enzyme activity assays, that two rate-limiting methyltransferases, 4VPMT1 and 4VPMT2, control the synthesis of 4VP to 4VA in locusts. Further inhibitor screening, biochemical analysis, in vivo injection, and in vitro enzyme activity assays confirmed that 4-nitrophenol effectively inhibits 4VA biosynthesis both in vitro and in vivo, suggesting potential application in the biological control of migratory locusts. This invention can maintain the harmless dispersal of locusts rather than killing them, providing a new method for biological control of migratory locusts with broad application prospects.

[0025] In a third aspect, the present invention 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.

[0026] Preferably, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:

[0027] (A) the sequence shown in any one of SEQ ID No. 2 and SEQ ID No. 4;

[0028] (B) has at least 90% identity to the sequence shown in any one of SEQ ID No. 2 and SEQ ID No. 4, and encodes a sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole.

[0029] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the third aspect.

[0030] In a fifth aspect, the present invention provides a recombinant cell, wherein the recombinant cell 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.

[0031] In a sixth aspect, the present invention 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:

[0032] (a) the sequence shown in any one of SEQ ID No. 1 and SEQ ID No. 3;

[0033] (b) A sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole obtained by deleting, inserting and / or replacing one or more amino acid residues from the sequence shown in any one of SEQ ID No. 1 and SEQ ID No. 3.

[0034] Preferably, the 4-vinylphenol methyltransferase gene contains a nucleotide sequence selected from the group consisting of:

[0035] (A) the sequence shown in any one of SEQ ID No. 2 and SEQ ID No. 4;

[0036] (B) has at least 90% identity 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.

[0037] In the present invention, a substance capable of inhibiting the expression of a 4-vinylphenol methyltransferase gene is screened from a plurality of chemical substances as a locust aggregation inhibitor; or a substance capable of reducing the activity of 4-vinylphenol methyltransferase is screened from a plurality of chemical substances as a locust aggregation inhibitor; wherein the screening is performed using the expression level of the 4-vinylphenol methyltransferase gene or the activity of the 4-vinylphenol methyltransferase as a detection index.

[0038] In a seventh aspect, the present invention 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:

[0039] (a) the sequence shown in any one of SEQ ID No. 1 and SEQ ID No. 3;

[0040] (b) A sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole obtained by deleting, inserting and / or replacing one or more amino acid residues from the sequence shown in any one of SEQ ID No. 1 and SEQ ID No. 3.

[0041] Preferably, the 4-vinylphenol methyltransferase gene contains a nucleotide sequence selected from the group consisting of:

[0042] (A) the sequence shown in any one of SEQ ID No. 2 and SEQ ID No. 4;

[0043] (B) has at least 90% identity to the sequence shown in any one of SEQ ID No. 2 and SEQ ID No. 4, and encodes a sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole.

[0044] Preferably, the drug has a 4-vinylphenol methyltransferase gene inhibitor or a 4-vinylphenol methyltransferase inhibitor as an active ingredient.

[0045] In an eighth aspect, the present invention 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;

[0046] The general structural formula of the multi-substituted benzene ring structure and its analogs is shown in Formula I:

[0047]

[0048] wherein R1 is selected from the group consisting of hydrogen, hydroxy, thiol, methoxy, methylthio, methyl, amino, or halogen;

[0049] R2, R3, R4 or R5 are each independently selected from: hydrogen, hydroxy, methyl or methoxy;

[0050] R6 is selected from the group consisting of hydrogen, alkyl, alkenyl, cyano, hydroxy, carboxyl, methoxy, halogen, nitro, ester, oxime or amide;

[0051] The X atom is selected from: C or N.

[0052] Preferably, the inhibitor inhibits the biosynthesis of 4-vinylanisole in vivo or in vitro.

[0053] In a ninth aspect, the present invention 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.

[0054] Preferably, the drug also includes pharmaceutically acceptable excipients.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] The present invention 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. The inhibitor 4-nitrophenol was developed to inhibit the enzymatic activity of 4VPMTs, thereby inhibiting the biosynthesis of 4VA, providing an effective and sustainable strategy for locust control. The present invention can keep locusts in a harmless, dispersed state, rather than killing them, providing a new method for biological control of migratory locusts with broad application prospects. Compared to chemical pesticide control methods for migratory locusts, the use of inhibitors screened by the present invention, such as 4-nitrophenol, for control has the following advantages:

[0057] 1. Compared with chemical pesticides, 4-nitrophenol keeps locusts in a harmless scattered state instead of killing them;

[0058] 2. It has a significant inhibitory effect on the biosynthesis of the locust aggregation pheromone 4VA. It can be artificially released to interfere with their aggregation, thereby controlling their large-scale swarming and thus forming a locust plague;

[0059] 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.

[0060] 4. The method for identifying the biosynthesis of the locust aggregation pheromone 4VA provided by the present invention 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

[0061] Figure 1 This is a graph showing the release of 4VA by gregarious locusts after starvation treatment.

[0062] Figure 2 This is a schematic diagram of the main component structure of wheat seedlings, a plant eaten by locusts.

[0063] Figure 3 This is the result graph of 4VA release after feeding artificial feed containing lignin.

[0064] Figure 4This figure shows the verification results of the precursor of 4VA biosynthesis.

[0065] Figure 5 This is a graph showing the results of species detection of compounds in the locust intestine and hemolymph.

[0066] Figure 6 There are two chemically plausible biosynthetic pathways from phenylalanine to 4VA.

[0067] Figure 7 This figure shows the verification results of the intermediates in the biosynthesis of 4VA.

[0068] Figure 8 This is the result of GC-MS / MS combined technology detection and analysis of wheat seedlings.

[0069] Figure 9 These are the detection results of four intermediates in 4VA biosynthesis in solitary locusts.

[0070] Figure 10 This is the result of detecting the difference in 4VA production between gregarious and solitary locusts.

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

[0072] Figure 12 This is the result of in vitro enzyme activity detection of 4VPMT1 and 4VPMT2.

[0073] Figure 13 This is a diagram showing the inhibitory effect of 4-nitrophenol and 4-trifluoromethylphenol on 4VA production in the screening of analogs based on 4VP design.

[0074] Figure 14 This is the result of enzyme activity detection of 4-nitrophenol and 4-trifluoromethylphenol.

[0075] Figure 15 This is a diagram showing the inhibitory effect of 4-nitrocatechol on 4VA production among analogs designed based on 4-nitrophenol.

[0076] Figure 16 The IC50 value of 4-nitrocatechol against 4VPMT1 was determined in an in vitro enzyme activity assay.

[0077] Figure 17 This figure shows the inhibitory effect of 4-nitrophenol on the production of 4VA in an in vivo injection experiment.

[0078] Figure 18 This is the conversion rate of 4-nitrophenol by 4VPMTs.

[0079] Figure 19This is a diagram showing the inhibitory effect of 4-nitroanisole, the methylation product of 4-nitrophenol, on the production of 4VA.

[0080] Figure 20 It is the kinetic parameter characterization of 4VPMT1 on 4-nitrophenol.

[0081] Figure 21 Schematic diagram of the complete biosynthetic pathway of 4VA. DETAILED DESCRIPTION

[0082] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0083] 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.

[0084] Example 1 Identification of the Synthetic Pathway of Locust Aggregation Pheromone 4VA

[0085] 1. Starve the fifth-instar gregarious locust nymphs and collect volatiles using SPME

[0086] In order to investigate whether 4VA is biosynthesized from the consumed plants, this example first tested the release of 4VA by gregarious locusts after starvation.

[0087] 1.1 Implementation Method: The experiment used fifth-instar gregarious locust nymphs. 100 intact gregarious locusts of equal size were divided into a control group and an experimental group. Both groups of locusts were housed in plexiglass cages (15 cm × 15 cm × 15 cm). The control group of locusts was fed wheat seedlings, while the experimental group of locusts was starved for 1, 2, 4, 6, and 8 hours. Volatile compounds were collected from the locusts using SPME. 4VA release levels were quantitatively determined using GC-MS analysis. Volatile compounds in the 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).

[0088] 1.2 Results: After 2 hours of starvation, the release of 4VA by gregarious locusts decreased significantly. After 8 hours of starvation, the release of 4VA was close to zero. The results indicate that locusts synthesize 4VA using building blocks from host plants ( Figure 1 ).

[0089] 2. The fifth-instar gregarious locust nymphs were fed artificial diet and volatiles were collected using SPME

[0090] The main components of wheat seedlings, a plant eaten by locusts, include lignin, amino acids, cellulose, and hemicellulose ( Figure 2 Because 4VA contains a benzene ring, lignin, tyrosine, and phenylalanine are considered potential precursors for 4VA biosynthesis. To determine whether lignin (a polymer structure) participates in 4VA biosynthesis, this example measured the release of 4VA after gregarious locusts were fed an artificial diet supplemented with lignin.

[0091] 2.1 Implementation method: The artificial feed for feeding locusts was prepared according to the ingredient list (Table 1 and Table 2). 1 g of lignin powder (Sigma-Aldrich, 370959) was added to 50 mL of artificial feed to obtain artificial feed with added lignin, stirred evenly, and placed at 4°C for use. 5-year-old gregarious locust nymphs were selected for the experiment and starved for 6-8 hours. The locusts in the experimental group were fed with artificial feed with added lignin, and the locusts in the control group were fed with artificial feed without added lignin, and fresh artificial feed was added once every 12 hours. After feeding the locusts for 24 hours, the locust volatiles were collected by SPME, and the release level of 4VA was quantitatively determined by GC-MS analysis.

[0092] Table 1

[0093]

[0094] Table 2

[0095] Component (Weigela salt) V / V% Component (Weigela salt) V / V% <![CDATA[CaCO3]]> 21.0 <![CDATA[KH2PO4]]> 31.0 <![CDATA[FeSO4.7H2O]]> 1.56 NaCl 10.5 <![CDATA[MgSO4]]> 9.0 <![CDATA[Ca3(PO4)2]]> 14.9 KCl 12.0 <![CDATA[CuSO4.5H2O]]> 0.039

[0096] 2.2 Results: This example found that the lignin-fed group did not increase the release of 4VA compared to the control group ( Figure 3 ). Therefore, lignin does not participate in the biosynthesis of 4VA.

[0097] 3. Fifth-instar gregarious locust nymphs were fed and injected with deuterated compounds, and volatiles were collected using SPME

[0098] In order to investigate whether tyrosine and phenylalanine participate in the biosynthesis of 4VA, in this example, deuterated tyrosine and phenylalanine were directly injected or fed to gregarious locusts.

[0099] 3.1 Implementation Method: 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.

[0100] 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.

[0101] 3.2 Results: Figure 4 This is a diagram showing the verification results of the precursors of 4VA biosynthesis, in which phenylalanine from plants is the precursor of 4VA biosynthesis. The experiment found that the injection of tyrosine and phenylalanine into the body of gregarious locusts did not produce deuterated 4VA ( Figure 4 A and 4B), and the same results were observed when the rats were fed an artificial diet containing deuterated tyrosine and phenylalanine ( Figure 4 C and 4D). When fed with plants containing deuterated phenylalanine, deuterated 4VA ( Figure 4 F) in gregarious locusts, while no deuterated 4VA ( Figure 4 E) Therefore, 4VA is biosynthesized from phenylalanine with the participation of plants.

[0102] 4. Metabolome analysis of locust intestinal and hemolymph metabolites using UHPLC-MS / MS

[0103] To explore other biosynthetic intermediates from phenylalanine to 4VA, this example examined the gut and hemolymph metabolites of locusts.

[0104] 4.1 Implementation Method: Grind 100 mg of tissue in liquid nitrogen and resuspend in pre-chilled 80% methanol and 0.1% formic acid. After mixing, incubate the sample on ice for 5 minutes and centrifuge at 15,000 g, 4°C for 20 minutes. Dilute the supernatant with LC-MS-grade water to a final methanol concentration of 53%. Transfer the sample to a fresh centrifuge tube and centrifuge at 15,000 g, 4°C for 20 minutes. Finally, aspirate the supernatant for analysis on an LC-MS / MS system.

[0105] 4.2 Results: 628 and 448 compounds were detected in the locust intestine and hemolymph, which were divided into 11 and 10 categories, respectively ( Figure 5 Based on the annotated phenylalanine metabolism in KEGG, the metabolites in the intestine and hemolymph were located, and two chemically plausible 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 ).

[0106] 5. Feeding and injecting deuterated intermediates to fifth-instar gregarious locust nymphs, and collecting volatiles using SPME

[0107] In order to determine which is the biosynthetic pathway of 4VA, this example conducted a stable isotope labeling study on gregarious locusts to detect whether deuterated 4VA is produced.

[0108] 5.1 Implementation Method: 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 compound was injected into the peritoneal cavity of 5th-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.

[0109] 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.

[0110] 5.2 Results: Figure 7 This is a diagram showing the verification results of the intermediates of 4VA biosynthesis, among which cinnamic acid, p-hydroxycinnamic acid and 4-vinylphenol are the intermediates of 4VA biosynthesis. The results showed that deuterated 4VA ( Figure 7 , AC). Injection of deuterated cinnamic acid also failed to produce deuterated 4VA ( Figure 7D), while feeding artificial feed and plants containing deuterated CA will induce the production of deuterated 4VA ( Figure 7 , E and F). These results indicate that 4VA is biosynthesized via a second pathway. To further validate 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.

[0111] 6. Derivatization of biosynthetic intermediates in 5th instar gregarious nymphs and analysis using GC-MS / MS

[0112] The conversion of phenylalanine to cinnamic acid to p-hydroxycinnamic acid is a conserved lignin biosynthetic pathway in plants. To determine whether plants could provide these intermediates to locusts, the researchers measured the levels of these four intermediate compounds in wheat seedlings sprayed with deuterated phenylalanine.

[0113] 6.1 Implementation method: 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. About 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 minutes, centrifuge at 4°C and 12,000 rpm for 10 minutes, and the supernatant was transferred to a new centrifuge tube for vacuum drying. 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 minutes. 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.

[0114] 6.2 Results: The test results showed that deuterated Phe, CA and p-HCA were all detected, but deuterated 4VP was not detected. Figure 8 ). Figure 8 It was verified that locusts can directly obtain phenylalanine, cinnamic acid and para-hydroxycinnamic acid from host plants, but host plants cannot directly produce 4VP or 4-VA.

[0115] Example 2 Identification of 4VPMTs as the key enzyme for 4VA production in locusts

[0116] 1. Derivatization of biosynthetic intermediates in the fifth-instar gregarious locust nymphs and analysis using GC-MS / MS

[0117] Since solitary locusts do not produce 4VA, this example further determined which of these four precursors led to the difference in 4VA production between gregarious and solitary locusts.

[0118] 1.1 Implementation method: GC-MS / MS was used to derivatize and quantify Phe, CA, p-HCA, and 4VP in the intestine, hemolymph, and legs of gregarious and solitary locusts.

[0119] 1.2 Results: By detecting four intermediates in three tissues of gregarious and solitary locusts, it was found that Phe, CA, p-HCA and 4VP could be detected in different tissues of gregarious and solitary locusts ( Figure 9 ), the results indicate that the absence of 4VA in solitary locusts is not due to the lack of these four precursors.

[0120] 2. The fifth-instar solitary locust nymphs were fed and injected with deuterated compounds, and the volatiles were collected by SPME

[0121] For further verification, in this example, the deuterated compound was applied to solitary locusts, and the release level of deuterated 4VA was detected.

[0122] 2.1 Implementation Method: 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. The experimental group fed wheat seedlings supplemented with the deuterated compounds to 5th-instar solitary locust nymphs, while the control group fed wheat seedlings sprayed with sterile water. After 12 hours of treatment, locust volatiles were collected and 4VA release levels were quantified using SPME and GC-MS analysis.

[0123] 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.

[0124] 2.2 Results: Figure 10 The results of the study showed that the difference in 4VA production between gregarious and solitary locusts was due to the conversion of 4-vinylphenol to 4VA. The results showed that feeding plants containing deuterated Phe and CA did not induce the production of deuterated 4VA in solitary locusts. Figure 10, A and B), injection of deuterated p-HCA and 4VP did not induce solitary locusts to produce deuterated 4VA ( 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.

[0125] The sources of the deuterated compounds used in the examples are shown in Table 3.

[0126] Table 3

[0127] sample source Phenylalanine-d5 Purchased from Sigma Tyrosine-d4 Purchased from CIL Phenylacetaldehyde-d6 Synthesized from styrene-d8 according to the literature (Synthesis 2009, No.15, 2505–2508). Cinnamic acid-d6 Synthesis from styrene-d8 via olefin metathesis Parahydroxycinnamic acid-d4 Synthesized from phenol-d5 via para-iodination and Heck reaction 4-Vinylphenol-d4 Synthesized from phenol-d5 via para-iodination and Suzuki reaction

[0128] 3. Transcriptome analysis, qPCR experimental verification and RNAi interference experiment

[0129] 3.1 Implementation Method: The conversion of 4VP to 4VA is a methylation reaction mediated by methylases in chemical terms. Therefore, we first analyzed the expression differences of genes annotated as methyltransferases in the hind legs of gregarious and solitary locusts using transcriptome data.

[0130] To validate genes annotated as methyltransferases, this example used qPCR. RP49 was used as an endogenous control for mRNAs. Melting curves were monitored during amplification to confirm the amplification specificity of the target gene, and the PCR amplification results were sequenced to verify primer specificity. The primer sequences for qPCR are shown in Table 4.

[0131] Table 4

[0132]

[0133]

[0134] To further verify the differential expression of the methyltransferase gene in the hind legs of gregarious and solitary locusts, an RNAi interference experiment was performed in this example. The RNAi primers are shown in Table 5.

[0135] Table 5

[0136] Gene sequence Upstream primer (5'-3') sequence Downstream primer (5'-3') LOCMI02868 SEQ ID No. 23 ctactgctacgacactcat SEQ ID No.24 catggaggtcgtgatgtt LOCMI16699 SEQ ID No.25 catctcgctccaggttgtg SEQ ID No.26 gcagaagtccagcatctcc LOCMI03758 SEQ ID No.27 cgctcgctacatcaatca SEQ ID No. 28 tcgtatgccaactcttcac LOCMI17143 SEQ ID No. 29 tggtgaagcggtgtttac SEQ ID No.30 ctcgccacagctatcatag LOCMI16705 SEQ ID No.31 cctgaaggcacgaagatag SEQ ID No.32 ctgcatgtagggcttcca LOCMI17606 SEQ ID No.33 aggaggagaggctgtatt SEQ ID No.34 cgatgcttctaggttctga

[0137] 3.2 Results: Figure 11 The results of the expression level test of methyltransferase genes in the hind legs of gregarious and solitary locusts were obtained. The expression differences of methyltransferase genes in the hind legs of gregarious and solitary locusts were analyzed by transcriptome data. It was found that the expression levels of 9 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 genes were significantly higher in gregarious locusts than in solitary locusts, namely LOCMI02868, LOCMI16699, LOCMI17143, LOCMI17606, LOCMI16705, and LOCMI03758 ( Figure 11 B). Further RNAi interference experiments confirmed that knocking out LOCMI16699 and LOCMI02868 significantly reduced the release of 4VA in gregarious locusts compared with the control group ( Figure 11 , C and D), after knocking out other genes, the release of 4VA did not change ( Figure 11 , EH). These results suggest that LOCMI16699 and LOCMI02868 may control the production of 4VA in locusts.

[0138] 4. In vitro expression and kinetic parameter characterization

[0139] 4.1 Implementation: To verify the function of LOCMI16699 and LOCMI02868, we exogenously expressed these two proteins. Enzyme activity was assayed by headspace quantification of product 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 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 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.

[0140] 4.2 Results: Figure 12 The results of in vitro enzyme activity assays for 4VPMT1 and 4VPMT2 were shown. The assays revealed that LOCMI16699 and LOCMI02868 could catalyze the methylation of 4VP to 4VA in the presence of the prosthetic group SAM. Therefore, they were 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 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 94.88μM ( Figure 12 C) At the same time, 4VPMT2 has a K M and k cat The values were 309.7 μM and 1.45×10 -6 s -1 ( Figure 12 D). With 4VPMT2(k 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.

[0141] Example 3: Development of 4VPMTs inhibitors

[0142] 1. Screening of designed substrate analogs based on the structural group of 4VP

[0143] In order to develop an effective 4VPMTs inhibitor to control locusts, this example systematically evaluated the ability of 4VP analogs as substrate competitors to inhibit 4VP enzyme methylation.

[0144] 1.1 Implementation method: 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, thiol, 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 solid phase microextraction (SPME) headspace quantitative product amount. 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 a 100 mM prosthetic group (SAM), 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 performed 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.

[0145] 1.2 Results: First, analogs were designed based on the styrene of the substrate 4VP. When the analogs were added to the enzyme activity detection system at a concentration ten times higher than that of the substrate, the results showed that the best inhibitory effect of these analogs could reduce the enzyme activity of 4VPMT1 by less than 50% ( Figure 13 A). The results showed that the phenolic hydroxyl group on 4VP plays an important role in substrate binding and recognition of 4VPMTs. Then, among the analogs designed by adding groups of different properties to the benzene ring or replacing the benzene ring with heterocycles, 4-vinylbenzene-1,2-diol, which had the best inhibitory effect, reduced the enzyme activity of 4VPMT1 by 6 times ( Figure 13 B). Finally, analogs were designed using the phenol of the substrate 4VP as the basic skeleton. Detection revealed that among the 17 analogs with different functional groups screened, 4-nitrophenol and 4-trifluoromethylphenol reduced the enzyme activity of 4VPMT1 by 339-fold and 348-fold, respectively. Figure 13 C).

[0146] 2. Identify the IC50 values of 4-nitrophenol and 4-trifluoromethylphenol against 4VPMTs

[0147] In order to further compare the inhibitory effects of 4-nitrophenol and 4-trifluoromethylphenol, IC50 tests were performed on them under the same conditions in this example.

[0148] 2.1 Implementation Method: The IC50 value of 4VPMT1 was determined using an in vitro enzymatic activity assay. The reaction system consisted of: buffer (20 mM Na2HPO4-NaH2PO4, pH 7.5, 50 mM NaCl), 20 μg of 4VPMT1 protein, 4 μg of SAM (100 mM), 1 μL of 4-nitrophenol and 4-trifluoromethylphenol at varying concentrations (0.00004-40 mM), and finally, 1 μL of 4VP (0.4 mM). Adsorption was performed at 30°C for 10 min. The 4VPMT2 reaction system consisted of: buffer (20 mM Na₂HPO₄-NaH₂PO₄, pH 7.5, 50 mM NaCl), 180 μg 4VPMT1 protein, 4 μL SAM (100 mM), 1 μL of varying concentrations of 4-nitrophenol and 4-trifluoromethylphenol (0.00004–20 mM), and finally 1 μL 4VP (30 mM). Adsorption was allowed to proceed at 30°C for 20 min. Product levels were calculated using GC-MS and a 4VPMT standard curve. IC₅₀ values were calculated using the dose-response-inhibition function in GraphPad Prism 8 software by fitting the relative activity of the test compound at varying concentrations.

[0149] 2.2 Results: Figure 14 The results of enzyme activity assays on 4-nitrophenol and 4-trifluoromethylphenol were obtained. Under the same in vitro conditions, the enzyme activity assays on 4-nitrophenol and 4-trifluoromethylphenol revealed that the IC50 values of 4VPMT1 and 4VPMT2 for 4-nitrophenol were 184.2 nM and 177.7 nM, respectively. Figure 14 , A and C). The IC50 values of 4-trifluoromethylphenol against 4VPMT1 and 4VPMT2 were 411.6 nM and 550.1 nM, respectively ( Figure 14 , B and D). These results indicate that 4-nitrophenol can inhibit 4VPMT1 and 4VPMT2 at lower concentrations.

[0150] 3. Design of analogues based on the structural groups of 4-nitrophenol

[0151] In order to further search for analogues with better inhibitory effects, this example attempts to replace the structural groups of 4-nitrophenol with groups of the same nature, and systematically evaluates the ability of 4-nitrophenol analogues to inhibit 4VP enzyme methylation as substrate competitors.

[0152] 3.1 Implementation Method: 4VPMT1 enzymatic activity was determined by headspace quantification of product 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), 1 μL of each analog (0.4 mM), and finally 1 μL of the substrate 4VP (0.4 mM) were added to a 5 mL vial (Cat: RY-10100). Adsorption was allowed to proceed at 30°C for 10 min. 1 μL of 4-nitrophenol (0.4 mM) was also added as a positive control. GC-MS and a 4VPMT standard curve were used to calculate product amounts in reactions with and without test compounds. Data were analyzed using a two-tailed unpaired t-test in GraphPad Prism 8 software.

[0153] 3.2 Results: Figure 15 The inhibitory effect of 4-nitrocatechol on 4VA was investigated based on the analogs designed based on 4-nitrophenol. The results showed 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 had the best inhibitory effect on 4VA production.

[0154] 4. Identification of the IC50 value of 4-nitrocatechol against 4VPMT1

[0155] In order to further compare the inhibitory effect of 4-nitrocatechol, this example performed an IC50 test under the same conditions.

[0156] 4.1 Implementation Method: The IC50 value of 4VPMT1 was determined using an in vitro enzymatic activity assay. The reaction system consisted of: buffer (20 mM Na2HPO4-NaH2PO4, pH 7.5, 50 mM NaCl), 20 μg 4VPMT1 protein, 4 μg SAM (100 mM), 1 μL of 4-nitrocatechol at varying concentrations (0.0004-4 mM), and finally, 1 μL of 4VPMT1 (0.4 mM). Adsorption was performed at 30°C for 10 min. The amount of product in the reaction was calculated using GC-MS and a 4VA standard curve. IC50 values were calculated by fitting the relative activity of the test compound at varying concentrations using the dose-response-inhibition function in GraphPad Prism 8 software.

[0157] 4.2 Results: By detecting the enzyme activity of 4-nitrocatechol under the same conditions in vitro, this example found that the IC 50 The value is 311.5nM ( Figure 16 ). With 4-nitrophenol (IC 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.

[0158] 5. Intracorporeal injection of 4-nitrophenol

[0159] In order to further study the effect of 4-nitrophenol on 4VPMTs, 4-nitrophenol was injected into locusts in this example.

[0160] 5.1 Implementation Method: 2 μL of aqueous 4-nitrophenol solutions of varying concentrations (0.005 mM, 0.05 mM, 0.5 mM, 5 mM, and 50 mM) were injected into the peritoneal cavity of fifth-instar gregarious locust nymphs. 4VA release was measured 2 hours after injection using GC-MS / MS. Additionally, 2 μL of 4-nitrophenol (0.5 mM) was injected, and 4VA release was measured using GC-MS at 2, 4, 8, and 12 hours after injection. All control groups received 2 μL of water.

[0161] 5.2 Results: The experiment found that 4-nitrophenol could significantly inhibit the production of 4VA within the injection concentration range of 0.1-100 nmol ( Figure 17 A). In addition, the production of 4VA was significantly inhibited 2h and 4h after injection of 1 nmol of 4-nitrophenol, while the release of 4VA did not change 8h and 12h after injection ( Figure 17 B) This indicates that 4-nitrophenol can inhibit the production of 4VA in locusts.

[0162] 6. In vitro enzyme activity assay to identify the conversion rate of 4VPMTs to the inhibitor 4-nitrophenol and the natural product 4-vinylphenol

[0163] In chemical transformation, 4-nitrophenol can be enzymatically methylated to generate 4-nitroanisole. In order to further study the properties of 4-nitrophenol, this example performed an in vitro enzyme activity assay.

[0164] 6.1 Implementation Method: The reaction system for enzyme activity assays 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 4-nitrophenol (0.4 mM) and HO, 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 4-nitrophenol (30 mM) and HO, respectively, and adsorption at 30°C for 20 min. GC-MS and a 4-nitroanisole standard curve were used to calculate the amount of product in reactions with and without the test compound. Data were analyzed using two-tailed unpaired t-test in GraphPad Prism 8 software.

[0165] 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.

[0166] 6.2 Results: It was found that both 4VPMT1 and 4VPMT2 could methylate 4-nitrophenol to 4-nitroanisole ( Figure 18). And under the same detection conditions, the conversion rate of 4-nitrophenol by 4VPMT1 and 4VPMT2 was much lower than that of the natural product 4VP( Figure 18 These results indicate that 4-nitrophenol is an effective and stable inhibitor of 4VA production in locusts.

[0167] 7. In vitro enzyme activity assay to verify the effect of 4-nitroanisole on 4VA production

[0168] 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, we performed an in vitro enzymatic activity assay.

[0169] 7.1 Implementation Method: The reaction system comprises: 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 0.4 mM 4-nitroanisole and 0.4 mM 4VP substrate, respectively; the control group received only 1 μL of 0.4 mM 4VP substrate. Adsorption was incubated at 30°C for 10 min. GC-MS and a 4VP 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.

[0170] 7.2 Results: The results showed that the enzyme activity of 4VPMT1 decreased by 1.5 times after adding 4-nitroanisole compared with the control group ( Figure 19 These results indicate that 4-nitroanisole, the methylation product of the inhibitor 4-nitrophenol, has an inhibitory effect on the production of 4VA.

[0171] 8. In vitro enzyme activity assay to identify the kinetic parameters of 4VPMT1 for 4-nitrophenol

[0172] 8.1 Implementation Method: First, 399 μL of the reaction mixture containing 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) were pipetted into a 5 mL sample vial (Cat: RY-10100). 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. M , k cat , k cat / K M value.

[0173] 8.2 Results: The kinetic parameters of 4VPMT1 showed that its K for the 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 20 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.

[0174] In summary, the present invention has identified the complete biosynthetic pathway of 4VA for the first time by analyzing the precursors and intermediates of the biosynthesis of the locust aggregation pheromone 4VA. Figure 21 The 4VPMTs (shown in Figure 2) comprise all precursors and key enzymes controlling 4VA production. 4VPMT1 and 4VPMT2 act as biosynthetic switches for 4VA synthesis. Based on this, the inhibitor 4-nitrophenol was developed to inhibit the enzymatic activity of 4VPMTs, thereby inhibiting 4VA biosynthesis, providing an effective and sustainable strategy for locust control.

[0175] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A 4-vinylphenol methyltransferase that catalyzes the production of the locust aggregation pheromone 4-vinylanisole, characterized in that 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 the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole obtained by deleting, inserting and / or replacing 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, characterized in that: The synthesis method comprises: using 4-vinylphenol as a substrate and obtaining 4-vinylanisole under the catalysis of 4-vinylphenol methyltransferase; 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.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the 4-vinylphenol methyltransferase that catalyzes the production of the locust aggregation pheromone 4-vinylanisole according to claim 1; 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) has at least 90% identity to the sequence shown in any one of SEQ ID No. 2 and SEQ ID No. 4, and encodes a sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole.

4. An expression vector, characterized in that The expression vector contains the nucleic acid molecule according to claim 3.

5. A recombinant cell, characterized in that The recombinant cell contains the expression vector according to claim 4, or the nucleic acid molecule according to claim 3 is integrated into the genome of the recombinant cell.

6. Use of a 4-vinylphenol methyltransferase gene or 4-vinylphenol methyltransferase in screening locust aggregation inhibitors, characterized in that: 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 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; 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) has at least 90% identity to the sequence shown in any one of SEQ ID No. 2 and SEQ ID No. 4, and encodes a sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole.

7. Use of a 4-vinylphenol methyltransferase gene inhibitor or a 4-vinylphenol methyltransferase inhibitor in the preparation of a locust control drug, characterized in that: 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 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; 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) has at least 90% identity to the sequence shown in any one of SEQ ID No. 2 and SEQ ID No. 4, and encodes a sequence having the activity of catalyzing the production of the locust aggregation pheromone 4-vinylanisole.

8. The use according to claim 7, characterized in that The medicine takes 4-vinylphenol methyltransferase gene inhibitor or 4-vinylphenol methyltransferase inhibitor as active ingredient.

9. Use of a polysubstituted benzene ring structure and its analogs in the preparation of 4-vinylphenol methyltransferase inhibitors, characterized in that: 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; R6 is selected from the group consisting of hydrogen, alkyl, alkenyl, cyano, hydroxy, carboxyl, methoxy, halogen, nitro, ester, oxime or amide; The X atom is selected from: C or N.

10. The use according to claim 9, characterized in that The inhibitor inhibits the biosynthesis of 4-vinylanisole in vivo or in vitro.

Images