New uses of plant-derived glycosides

Abstract

The present invention discloses a new use of a plant-derived glycoside compound, relating to the fields of pesticides and plant protection. The plant-derived glycoside compound is galangin or isomandibolic acid. At the same time, the present invention discloses an agent for resisting tobacco mosaic virus or preventing and controlling crop diseases, comprising the plant-derived glycoside compound as described above. During treatment, an effective amount of the plant-derived glycoside compound can be applied, sprayed, or injected onto tobacco leaves or crops. The present invention first discovered that galangin and isomandibolic acid have good anti-TMV activity, wherein the anti-TMV activity of galangin reaches 82.22%, and the anti-TMV activity of isomandibolic acid reaches 75.00%, providing a material basis for anti-TMV research; molecular docking technology is used to simulate the binding mode of galangin and isomandibolic acid with target proteins, and three potential targets of galangin and isomandibolic acid are screened out based on the minimum binding energy and the number of hydrogen bonds, providing a theoretical basis for anti-TMV research.

Description

Technical Field

[0001] The present invention relates to the fields of pesticides and plant protection, and in particular to new uses of plant-derived glycoside compounds, and in particular to uses of plant-derived glycoside compounds in resisting tobacco mosaic virus or preventing crop diseases. Background Art

[0002] Drug Affinity Response Target Stability (DARTS) is a label-free probe technology in chemoproteomics. Due to its high simplicity, efficiency, versatility, and accuracy, it has been widely used to identify target proteins in animals, fungi, bacteria, and plants.

[0003] Tobacco mosaic virus (TMV) is one of the most well-known viruses, and many studies have used it as a model. TMV also poses significant risks to agricultural production. Reports indicate that tobacco yield losses in infected tobacco fields can reach 30% to 70%. TMV accounts for 50% to 70% of all economic losses in tobacco production caused by tobacco diseases. Globally, TMV causes over $100 million in losses annually. However, chemical pesticides lack specificity for plant viruses, making them ineffective in controlling them. Furthermore, long-term use of chemical pesticides can easily lead to the development of resistance in viruses and cause significant environmental pollution. Therefore, the development of natural pesticides that are less polluting and less susceptible to developing resistance is crucial.

[0004] my country is rich in natural resources, especially plant resources. Many natural products from plants have good anti-TMV activity. Therefore, the screening of natural products from tobacco leaves and flowers provides an important way to develop anti-TMV inhibitors. However, there are currently no reports on the anti-TMV activity of relevant plant-derived glycoside compounds. Summary of the Invention

[0005] The present invention provides a new use of a plant-derived glycoside compound, aiming to solve the problems existing in the above-mentioned background technology.

[0006] In order to achieve the above technical objectives, the present invention mainly adopts the following technical solutions:

[0007] In a first aspect, the present invention discloses the use of a plant-derived glycoside compound in resisting tobacco mosaic virus or preventing and controlling crop diseases, wherein the plant-derived glycoside compound is cephaloside represented by formula (I) or isomangiferin represented by formula (II);

[0008]

[0009] Preferably, the cerebroside or isomandibularin is derived from natural plants, produced by biotechnology, or artificially synthesized.

[0010] More preferably, the isomandibolic acid comes from an extract of the Anemarrhena asphodeloides plant. The preparation method of the Anemarrhena asphodeloides plant extract is: adding ethanol to the Anemarrhena asphodeloides plant powder, separating the residue after ultrasound and collecting the filtrate, then adding ethanol to the medicinal material, continuing ultrasound, vacuum filtering and combining the two filtrates, and concentrating to half the mass of the input plant material; the effective concentration of the isomandibolic acid is ≥0.1 mg / mL.

[0011] In a preferred embodiment of the present invention, the crop disease pathogen is selected from any one of tobacco mosaic virus, Rhizoctonia solani, and cucumber root rot fungus.

[0012] In a second aspect, the present invention discloses an agent for resisting tobacco mosaic virus or preventing and controlling crop diseases, comprising the plant-derived glycoside compound as described in the first aspect.

[0013] In a third aspect, the present invention discloses a method for preventing and controlling tobacco mosaic virus, comprising applying, spraying or injecting the agent described in the second aspect onto tobacco leaves or crops.

[0014] In a preferred embodiment of the present invention, the targets of the anti-tobacco mosaic virus effect of the cephaloside or isomandibolic acid are glycolate oxidase, chloroplast triphosphoglycerate kinase or mitochondrial ATP synthase F1 subunit.

[0015] Furthermore, the scutellarin or isomandibolic acid is embedded in the active pocket of glycolate oxidase, and the hydroxyl hydrogen atoms at positions 2 and 3 on the scutellarin sugar ring form hydrogen bonds with the carbonyl oxygen of Gly163, the hydroxyl oxygen atom at position 3 on the sugar ring forms a hydrogen bond with the imine hydrogen atom of Arg165, the hydroxyl hydrogen atom at position 4 on the sugar ring forms a hydrogen bond with the carbonyl atom of Arg165, and the hydroxyl hydrogen atom of the hydroxymethyl group at position 5 on the sugar ring forms a hydrogen bond with the carbonyl atom of Asp168; the hydroxyl hydrogen atoms at positions 6 and 7 on isomandibolic acid form hydrogen bonds with the carbonyl oxygen of Gly163, the hydroxyl oxygen atom at position 6 forms a hydrogen bond with the imine hydrogen atom of Arg165, and the hydroxyl oxygen atom at position 7 forms a hydrogen bond with the hydrogen atom on Arg165 C=NH.

[0016] Furthermore, galangin or isomandibolic acid is embedded in the active pocket of chloroplast triphosphoglycerate kinase, the oxygen atom of the glycosidic bond of galangin and the oxygen atom at position 6 of the sugar ring form a hydrogen bond with the imine hydrogen atom of His228, the hydroxyl oxygen atom of the hydroxymethyl group at position 5 of the sugar ring forms a hydrogen bond with the imine hydrogen atom of Gly455, the hydroxyl oxygen atom at position 2 of the sugar ring forms a hydrogen bond with the amino hydrogen atom of Arg196, the hydroxyl oxygen atom at position 3 of the sugar ring forms a hydrogen bond with the imine hydrogen atom of His138 and the amino hydrogen atom of Asn101, the hydroxyl oxygen atom at position 4 of the sugar ring forms a hydrogen bond with the amino hydrogen of Arg115, the five-membered ring ester carbonyl forms a hydrogen bond with the imine hydrogen atom of Arg229, and the carbonyl forms a hydrogen bond with the imine hydrogen of His138; the hydroxyl hydrogen atom at position 3 of isomandibolic acid forms a hydrogen bond with the Thr452 The oxygen atom of C=O forms a hydrogen bond, the hydroxyl oxygen atom at position 3 forms a hydrogen bond with the hydrogen atom of Arg115 amino group, the carbonyl oxygen forms a hydrogen bond with the hydrogen atom of Arg115 amino group, the hydroxyl hydrogen atom and oxygen atom at position 3 of the sugar ring form hydrogen bonds with the oxygen atom of Asp433 C=O and the hydrogen atom on the imine, respectively, the hydroxyl hydrogen atoms at positions 4 and 5 of the sugar ring form hydrogen bonds with the oxygen atom on Asp433 OH, and the hydroxyl hydrogen atoms at positions 6 and 7 of isomandibolic acid form hydrogen bonds with the oxygen atom of Glu439 OH.

[0017] Furthermore, galangin or isomandibolic acid is embedded in the active pocket of the F1 subunit of mitochondrial ATP synthase. The hydroxyl oxygen atom at position 2 of the sugar ring of galangin forms a hydrogen bond with the imine hydrogen atom of Val142, the hydroxyl oxygen atom of the hydroxymethyl group at position 5 of the sugar ring forms a hydrogen bond with the carbonyl oxygens of Are373 and Val374, and the oxygen atom on the five-membered ring ester group forms hydrogen bonds with the amino hydrogen of Arg139 and the imine hydrogen of Lys311, respectively; the hydroxyl oxygens at positions 2 and 3 of the sugar ring of isomandibolic acid form hydrogen bonds with the imine hydrogen atom of Ile136, the hydroxyl oxygen atom at position 2 of the sugar ring forms a hydrogen bond with the hydrogen on the imine of Ile137, the hydroxyl hydrogen atom at position 6 forms a hydrogen bond with the carbonyl oxygen of Ala133, and the hydroxyl hydrogen atom at position 7 forms a hydrogen bond with the carbonyl oxygen of Lys132.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention first discovered that cephaloside and isomangiferin have good anti-TMV activity, among which the anti-TMV activity of cephaloside reaches 82.22%, and the anti-TMV activity of isomangiferin reaches 75.00%, providing a material basis for anti-TMV research;

[0020] The present invention uses molecular docking technology to simulate the binding mode of cephaloside and isomandibolic acid with target proteins, and screens out three potential targets of cephaloside and isomandibolic acid based on the minimum binding energy and the number of hydrogen bonds, providing a theoretical basis for anti-TMV research.

[0021] The present invention discovers for the first time that the extract of Anemarrhena asphodeloides has good anti-TMV activity. When the content of isomandibulariae in the extract of Anemarrhena asphodeloides plant is 0.1 mg / mL, it has good protective activity (60.00%) and certain therapeutic activity (36.11%) against TMV, and the passivation activity (23.53%) is average, providing a basis for its use as a protective agent in the prevention and treatment of tobacco mosaic virus disease in plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Fluorescence area of ​​tobacco leaves after injection of TMV-GFP resuspension containing different compounds and agents;

[0023] Figure 2 This is the result of HPLC determination of isomandibolic acid;

[0024] Figure 3 This is the result of HPLC determination of Anemarrhena asphodeloides extract;

[0025] Figure 4 This is the effect diagram of the protective activity of Anemarrhena Rhizoma extract against TMV;

[0026] Figure 5 This is the therapeutic effect diagram of Anemarrhena Rhizoma extract against TMV;

[0027] Figure 6 This is the effect diagram of the anti-TMV passivation activity of Anemarrhena asphodeloides extract;

[0028] Figure 7 This is the silver staining result after SDS-PAGE separation after the DARTS experiment of cephaloside;

[0029] Figure 8 This is the silver staining result after SDS-PAGE separation after isomandibularin DARTS experiment;

[0030] Figure 9 This is the docking result diagram of cerebroside and GOX;

[0031] Figure 10 This is the docking result diagram of isomandibolic acid and GOX;

[0032] Figure 11 This is the docking result diagram of cephalothin and PGK;

[0033] Figure 12 This is the docking result diagram of isomandibolic acid and PGK;

[0034] Figure 13 This is the docking result diagram of cephalothin and ATPF1;

[0035] Figure 14 This is the docking result diagram of isomandibolic acid and ATPF1;

[0036] Figure 15 This is the result graph of the effect of protein dosage on absorbance;

[0037] Figure 16 This is the result of measuring GOX activity in the in vivo experiment using the smear method;

[0038] Figure 17 This is the result of GOX activity determination in the in vivo injection experiment;

[0039] Figure 18 This is the result of GOX activity assay after in vitro treatment with cephalosporin;

[0040] Figure 19 This is the result of GOX activity determination after in vitro isomangiferin treatment. DETAILED DESCRIPTION

[0041] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0042] The purpose of the present invention is to provide two plant-derived glycoside compounds that are resistant to tobacco mosaic virus and identify three potential targets of cephalosporin and isomangiferin.

[0043] The applicant analyzed and sorted the structural properties and biological activities of reported plant-derived natural products with antiviral activity. Two candidate compounds were selected from 2,590 antiviral natural products based on factors such as compound structure and mechanism of action. Anti-TMV activity was then determined using the half-leaf spot assay and Agrobacterium-mediated GFP fluorescence assay. Initial screening revealed that compounds such as schizoside (82.22%) and isomangiferin (75.00%) exhibited good anti-TMV activity.

[0044] The targets of scutellarin and isomangiferin were initially explored using the Drug Affinity Response Target Stability Technology (DARTS) technique. LC-MS identified glycolic oxidase (GOX), chloroplast triphosphoglycerate kinase (3-PGK), and mitochondrial ATP synthase F1 subunit 1 in N. benthamiana as potential targets of scutellarin and isomangiferin. Molecular docking was used to simulate the binding of scutellarin and isomangiferin to target proteins. Based on the minimum binding energy and the number of hydrogen bonds, GOX was further selected as a candidate target for scutellarin and isomangiferin. In vivo experiments revealed that GOX activity increased in N. benthamiana leaves treated with scutellarin injection or smearing. Both smearing and injection treatments demonstrated that scutellarin promoted GOX activity. In vitro experiments showed that 40 mg / L cephaloside and 20 mg / L isomandibolic acid promoted GOX activity, while concentrations above this level gradually inhibited GOX enzyme activity. Activity assays showed that cephaloside and isomandibolic acid increased GOX enzyme activity in tobacco leaves at lower concentrations.

[0045] The details are explained below.

[0046] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0047] Example 1

[0048] Screening and activity determination of plant-derived anti-TMV compounds

[0049] (1) Screening of plant-derived antiviral compounds. The natural product molecular information database of Taosu Company was searched. The structural properties and biological activities of plant-derived natural products with reported anti-TMV activity were analyzed and sorted out. From 2,590 antiviral natural products, some test compounds were selected based on factors such as compound structural similarity and biological receptors. The specific results are shown in Table 1. A total of two plant-derived glycoside anti-TMV compounds were screened.

[0050] Table 1 Plant-derived glycoside anti-TMV compounds

[0051]

[0052] (2) Half-leaf spot method. The anti-TMV activity of the selected plant-derived compounds was determined by the in vivo half-leaf spot method. Using dimethyl sulfoxide (DMSO) as the solvent, different plant-derived compounds at concentrations of 100 μg / mL and 500 μg / mL and the positive control Ningnanmycin (NNM) solution were prepared, and then mixed with the diluted virus solution to obtain a mixed agent. Select relatively thick and uniformly growing Sanxi tobacco plants at the 4-6 leaf stage, sprinkle quartz sand powder on the entire leaf, and take a small amount of mixed agent to wet the gauze with the main vein as the limit. Drop 100 μL of the mixed agent on the right half of the Sanxi tobacco leaf and gently rub it and inoculate it. The left half of the leaf was rubbed with DMSO solvent as a control. The gauze was changed every time a mixed agent was used. At least 3 pieces of suitable Sanxi tobacco leaves were treated for each plant as a repeated experiment. After treatment, the tobacco seedlings were cultured in an insect-free greenhouse. After 3-4 days, when the spot symptoms were obvious, the test results were statistically analyzed. The specific results are shown in Table 2. Both cephaloside and isomangiferin showed good anti-TMV activities, which were 82.22% and 75.00%, respectively.

[0053] Table 2 Determination of anti-TMV activity of plant-derived compounds

[0054]

[0055]

[0056] To determine the inactivation effect, a combination of different compounds and virus was applied for 30 minutes before inoculation into the right half of a Sansi tobacco leaf. The left half was smeared with a virus solvent at the same concentration as a control. At least three leaves of each strain were treated as replicates. After 3-4 days, the number of necrotic spots was recorded to determine the inactivation effect of the compounds on TMV in vitro.

[0057] Protective effect. First, different compounds were inoculated onto the right half of a Sansi tobacco leaf by friction inoculation. The left half was treated with solvent as a control. At least three suitable leaves per plant were treated as replicates. After treatment, the leaves were placed in an observation room. 24 hours later, the entire leaf was inoculated with TMV at a concentration of 50-60 μg / mL. After 3-4 days, the number of necrotic spots on each leaf was recorded on both the left and right halves to determine the protective effect of each compound on the tobacco plant after TMV infection.

[0058] Therapeutic effect. After application, TMV was inoculated onto whole-leaf Sansi tobacco leaves. Two hours later, the right half of the leaf was inoculated with the agent, while the left half was coated with the solvent as a control. At least three suitable leaves per plant were treated as replicates. After 3-4 days, the number of necrotic spots was recorded to determine the in vivo therapeutic effects of the different compounds on tobacco. The results are shown in Table 3. Both cephalosporin and isomangiferin exhibited good protective and therapeutic activity.

[0059] Table 3 Inhibition rate of compounds with different modes of action

[0060]

[0061] (3) Agrobacterium-mediated GFP fluorescence method. The specific experimental steps are as follows:

[0062] ① Agrobacterium transformation. Construct the TMV-GFP plasmid using the method described in the literature. Thaw the competent Agrobacterium culture stored at -80°C on ice. Then, add 1 μg of TMV-GFP plasmid to 50 μL of competent Agrobacterium culture. Gently flick to mix, let it rest on ice for 5 minutes, then quickly freeze it in liquid nitrogen for 1 minute. Transfer it to a 37°C water bath for 5 minutes for heat shock. Place it on ice until the temperature drops to 0°C. Add 500 μL of non-resistant LB liquid medium and incubate it at 28°C at 200 rpm for 3 hours. Spread the transformation product onto LB medium containing antibiotics. After 48 hours, check for single colonies, perform PCR on the culture, and select positive single colonies for future use.

[0063] ② Agrobacterium suspension culture. Expand the positive clone bacterial solution to 5mL saturated bacterial solution and culture at 28℃, 220r / min for 48h. After the culture is completed, the suspension culture is carried out at 28℃, 200r / min for 16h at a ratio of saturated bacterial solution: suspension solution = 1:100. After that, the suspension bacterial solution is filtered with gauze to remove flocs. Then, centrifuge at 4500r / min for 5min to collect the bacterial precipitate. Use the suspension solution to adjust the OD 600 The required bacterial solution concentration is 0.5, and it is allowed to stand at room temperature for 2-3 hours.

[0064] ③ Preparation of compound solution: Prepare 10μM and 50μM compound solutions using Agrobacterium resuspended culture, incubate at room temperature for 0.5h and then inject.

[0065] ④ Injection observation. The co-injection method was used to inject the Agrobacterium resuspension containing the compound into the Nicotiana benthamiana leaves using a needleless syringe. The back of the leaf was injected, and 3 leaves were injected per tobacco plant, 100 μL was injected per leaf, and one point was injected on each side of the main vein of each leaf. The diameter of each injection point did not exceed 2 cm. After the injection, a digital camera (under ultraviolet light) was used to take pictures every 2 hours, and the changes in the fluorescence area on the leaf surface were recorded. The observation was continued for about 5-8 days. The changes in the fluorescence area were counted, and the control effect of the compound on TMV was calculated to obtain the plant-derived compound with the best anti-TMV activity. The specific results are as follows. Figure 1 As shown, the green fluorescence area of ​​Nicotiana benthamiana leaves after drug treatment was significantly less than that in the 1% DMSO group. The proliferation of TMV in Nicotiana benthamiana leaves was inhibited after drug treatment. The compounds showed TMV inhibitory effect, among which the inhibitory effect of cephalosporin on TMV was comparable to that of the positive control NNM.

[0066] Example 2

[0067] Extraction and bioactivity determination of isomangiferin from Anemarrhena asphodeloides

[0068] (1) Extraction of active ingredients from Anemarrhena Rhizoma. Grind dried Anemarrhena Rhizoma. Accurately weigh 20 g of the extract and place it in a conical flask containing 240 mL of 70% ethanol solution. Extract the extract at 51°C and ultrasonic power of 400 W for 30 min. Collect the filtrate. Add 120 mL of 70% ethanol to the extract and ultrasonically extract it for 30 min under the same conditions. Filter and combine the extracts. Vacuum filter the extract at 50°C until the concentrate weighs 10 g.

[0069] Weigh 1 g of the filtrate into a 50 mL volumetric flask, add 30 mL of anhydrous methanol, and sonicate at room temperature for 30 minutes. After cooling the solution to room temperature, dilute to 50 mL. Detection was by HPLC using acetonitrile and 0.1% phosphate buffer as the mobile phase at a wavelength of 316 nm.

[0070] (2) Tobacco mosaic virus (TMV) activity assay of Anemarrhena extract. The protective activity of Anemarrhena extract against TMV was determined using the whole leaf spot method. The final concentration of isomandibulariacein in Anemarrhena extract was diluted to 100 mg / L, and 100 mg / L Ningnanmycin (NNM) was used as a positive control. Sansi tobacco plants with thick and uniform growth at the 4-6 leaf stage were selected.

[0071] Protective effect: In the experimental group, 200 μL of the agent was dripped onto each leaf and gently rubbed onto the entire leaf. One Sansi tobacco plant was rubbed with water as a control. At least three suitable Sansi tobacco leaves were selected for each plant as replicates. After 24 hours, the entire leaf was sprinkled with quartz sand powder and rubbed with TMV. After treatment, the tobacco seedlings were cultured in an insect-free greenhouse. After 3-4 days, when the blight symptoms were obvious, such as Figure 4 As shown, the test results were statistically analyzed and the inhibition rate was calculated.

[0072] Table 4 Protective effect of Anemarrhena Rhizoma extract against TMV

[0073]

[0074] Treatment effect: TMV was inoculated on Sansi tobacco leaves by friction. After 2 hours, 200 μL of the drug was dripped onto each leaf of the experimental group and lightly rubbed on the whole leaf. One Sansi tobacco plant was inoculated with water by friction as a control. At least 3 suitable tobacco leaves were treated for each plant as a replicate experiment. After 3-4 days, the number of necrotic spots was recorded. Figure 5 As shown, the statistical test results were used to calculate the inhibition.

[0075] Table 5 Anti-TMV therapeutic effects of Anemarrhena Rhizoma extract

[0076]

[0077] Passivation effect: Mix different compounds with the virus for 30 minutes and then inoculate them into Sansi tobacco. Treat at least 3 leaves of each plant as replicates. After 3-4 days, record the number of dead spots. Figure 6 As shown, the test results were statistically analyzed and the inhibition rate was calculated.

[0078] Table 6 Anti-TMV passivation effect of Anemarrhena Rhizoma extract

[0079]

[0080] Example 3

[0081] Identification of target proteins of cephaloside and isomandibolic acid

[0082] (1) Total protein extraction from Nicotiana benthamiana leaves. Take tobacco mosaic virus-infected Nicotiana benthamiana leaves, grind them into powder under liquid nitrogen conditions, and transfer them to 1.5 mL centrifuge tubes. Add 500 μL of protein extraction buffer (1XPBS, 5‰ Triton X-100, 1mM DTT, 150mM NaCl, 10mM MgCl2, 1mM PMSF) per gram of sample, mix well with a pipette, and let stand on ice for 3 hours. Centrifuge at 12000 r / min and 4°C for 10 minutes, and then collect the supernatant. Add protein extraction buffer to the precipitate again, mix well, and centrifuge at 12000 r / min and 4°C for 10 minutes. Combine the supernatants and use Nanodrop to determine the protein sample concentration. Aliquot 500 μL of the protein solution into each 1.5 mL centrifuge tube, freeze in liquid nitrogen, and store at -80°C.

[0083] (2) The protein sample concentration was determined using Nanodrop. 15 mL of protein sample, 3 mL of 10× TNC buffer, 3 mL of cephalosin (0, 0.5, 1.0, 2.0 mg / mL) solution, and 6 mL of double-distilled water were added to a 200 μL PCR tube, vortexed to mix, and incubated on ice for 2 h. After incubation, equal volumes of Pronase (0, 0.005, 0.01, 0.025, 0.5, 1.0 mg / mL) were added in sequence and gently shaken to mix, and then allowed to stand at room temperature for 10 min. After enzymatic hydrolysis, 8 μL of Loading buffer was added, vortexed to mix, and heated in a 95°C metal bath for 10 min to terminate the reaction.

[0084] (3) SDS-PAGE separation and mass spectrometry detection. Take 10.00 μL of the prepared protein sample and run it on an SDS-PAGE gel (12% separation gel + stacking gel) at 80V for 3.5 hours. After running the gel, silver staining is used for color development. The steps for silver staining imaging are as follows:

[0085] ① Fixation. After electrophoresis, place the gel in 100 mL of fixative solution and shake on a shaker at room temperature overnight.

[0086] ②Wash with 30% ethanol. Discard the fixative solution, add 100 mL of 30% ethanol, and shake on a shaker at room temperature for 10 minutes.

[0087] ③ Wash with water. Discard the 30% ethanol, add 100 mL of Milli-Q grade pure water, and shake on a shaker at room temperature for 10 minutes.

[0088] ④ Sensitization. Discard the water and add 100 mL of silver staining enhancement solution (1X). Shake on a shaker at room temperature for 2 minutes.

[0089] ⑤ Wash with water (2 times in total). Discard the original solution and add 100 mL of Milli-Q grade pure water. Shake on a shaker at room temperature for 1 minute. Discard the water. Add another 100 mL of Milli-Q grade pure water and shake on a shaker at room temperature for 1 minute.

[0090] ⑥ Silver staining: Discard the water, add 100 mL of silver solution (1×), and shake on a shaker at room temperature for 10 minutes.

[0091] ⑦ Water wash. Discard the original solution and add 100 mL of Milli-Q grade pure water. Shake on a shaker at room temperature for 1-1.5 minutes. The water wash time should not exceed 1.5 minutes.

[0092] ⑧ Color development. Discard the water, add 100 mL of silver staining solution, and shake on a shaker at room temperature until the desired protein bands appear.

[0093] ⑨ Stop. Discard the silver staining solution, add 100 mL of silver staining stop solution (1×), and shake on a shaker at room temperature for 10 minutes.

[0094] ⑩ Wash with water. Discard the silver staining stop solution, add 100 mL of Milli-Q grade pure water, and shake on a shaker at room temperature for 2-5 minutes.

[0095] The stained gel is stored in Milli-Q grade pure water or double distilled water for analysis and detection. After the expected band is cut out, mass spectrometry detection is performed. Figure 7 and Figure 8 As shown, the low-concentration treatment group showed no distinct protein bands compared to the untreated group, while the high-concentration treatment group exhibited a distinct protein band at 75 kDa, distinct from the untreated group, suggesting that this protein band represents the target protein of the compound. Since the molecular weights of the four proteins encoded by TMV are not near 75 kDa, it is hypothesized that this protein band represents the 75 kDa protein of N. benthamiana. The red-boxed region was excised for mass spectrometry. The LC-MS identification results are shown in Tables 7 and 8. Common target proteins of scutellarin and isomandibolic acid were screened for subsequent experiments. The three proteins screened were: N. benthamiana glycolate oxidase, N. benthamiana chloroplast triphosphoglycerate kinase, and N. benthamiana mitochondrial ATP synthase F1 subunit.

[0096] Table 7 Mass spectrometry detection results of cephaloside treatment

[0097]

[0098]

[0099] Table 8 Mass spectrometry detection results of isomandibolic acid

[0100]

[0101]

[0102]

[0103] (4) Molecular docking of glycolate oxidase with cephaloside and isomandibolic acid. The crystal structure of Nicotiana benthamiana GOX has been solved, and the three-dimensional structure of Nicotiana benthamiana GOX protein with FMN ligand can be downloaded directly from the PDB official website (PDB ID: 5ZBM). The 3D structure of cephaloside and isomandibolic acid can be searched on the Chempub website. Download the 3D structure and save it as an sdf file. Then convert the format using the openbabele software and save it as a pdb file.

[0104] The software used for molecular docking was AutoDock4.

[0105] ① Acceptor treatment: In AutoDock Tools, GOX was treated with dehydration and polar hydrogenation, and saved as a pdbqt file (GOX.pdbqt).

[0106] ② Ligand processing: Set the rotatable bonds of the small molecule in AutoDock Tools and save it as a pdbqt file (A03.pdbqt).

[0107] ③AutoGrid. Set the center 3D coordinates and spatial size of the docking box to closely encompass the GOX active site (x = 60, y = 62, z = 58; x center = -19, y center = 7, z center = -12.250). Save this as a grid parameter file (.gpf) and run autogrid.exe to generate a grid file (.map) and a log file (.glg) for each atom type in the ligand.

[0108] ④AutoDock. When there is an existing grid parameter file for AutoGrid operation and ligand and receptor files, select the Lamarckian genetic algorithm to set the conformational search parameters, set the target conformations to 10, and select the default docking parameters. Save as a docking parameter file (.dpf), run autodock.exe, and obtain a record file (.dlg) containing the docking results. The docking results of galangin and GOX are shown in Table 9. The conformations generated by docking were divided into 10 clusters based on the RMSD values. Cluster 2 showed the lowest binding energy, and this conformation was further analyzed. Figure 9 The results show that galangin is embedded in the active pocket of GOX. Among these interactions, the hydroxyl hydrogen atoms at positions 2 and 3 of the sugar ring form hydrogen bonds with the carbonyl oxygen of Gly163, the hydroxyl oxygen atom at position 3 of the sugar ring forms a hydrogen bond with the imine hydrogen atom of Arg165, the hydroxyl hydrogen atom at position 4 of the sugar ring forms a hydrogen bond with the carbonyl atom of Arg165, and the hydroxyl hydrogen atom of the hydroxymethyl group at position 5 of the sugar ring forms a hydrogen bond with the carbonyl atom of Asp168. The docking results of isomandibolic acid and GOX are shown in Table 10. The conformations generated by docking were divided into 10 clusters based on the RMSD values. Cluster 4 showed the lowest binding energy and was further analyzed. Figure 10 The results showed that isomandibine is embedded in the active pocket of GOX. Among these interactions, the hydroxyl hydrogen atoms at positions 6 and 7 of isomandibine form hydrogen bonds with the carbonyl oxygen of Gly163, the hydroxyl oxygen atom at position 6 forms a hydrogen bond with the imine hydrogen atom of Arg165, and the hydroxyl oxygen atom at position 7 forms a hydrogen bond with the hydrogen atom on the C=NH of Arg165.

[0109] Table 9 Summary of docking results of cephalosporin and GOX

[0110]

[0111] Table 10 Summary of the docking results of isomandibolic acid and GOX

[0112]

[0113] (5) Molecular docking of triphosphoglycerate kinase 3-PGK with cephaloside and isomandibolic acid. The specific steps are as follows:

[0114] ① Homology modeling. The crystal structure of N. benthamiana 3-PGK has not yet been solved, so it is necessary to search for a template on the SWISS-MODEL website based on its protein sequence for homology modeling. Search NCBI (https: / / www.ncbi.nlm.nih.gov / ) to obtain the amino acid sequence of N. benthamiana 3-PGK. Enter the sequence into the SWISS-MODEL website and search for templates. Use the template with the highest similarity (1php.1.A) for homology modeling and save it as a pdb file (PGK.pdb).

[0115] ②Molecular docking. Set the central three-dimensional coordinates and spatial size of the docking box so that it can wrap the active site of PGK (Arg21, Arg38, His62, His167, Arg168, His170) as much as possible. (x=40,y=52,z=46;x center=-7,y center=1.250,z center=-7). The remaining steps are the same as those described in Example 3(4). The results of the docking of galangin with 3-PGK molecules are shown in Table 11. The conformations generated by docking were divided into 10 clusters based on the RMSD values. Cluster 4 showed the lowest binding energy, and its conformation was further analyzed. Figure 11 The results showed that galangin was embedded in the active pocket of 3-PGK. Among these interactions, the oxygen atom of the glycosidic bond of galangin and the oxygen atom at position 6 of the sugar ring formed hydrogen bonds with the imine hydrogen atom of His228, the hydroxyl oxygen atom of the hydroxymethyl group at position 5 of the sugar ring formed a hydrogen bond with the imine hydrogen atom of Gly455, the hydroxyl oxygen atom at position 2 of the sugar ring formed a hydrogen bond with the amino hydrogen atom of Arg196, the hydroxyl oxygen atom at position 3 of the sugar ring formed hydrogen bonds with the imine hydrogen atom of His138 and the amino hydrogen atom of Asn101, the hydroxyl oxygen atom at position 4 of the sugar ring formed a hydrogen bond with the amino hydrogen of Arg115, the five-membered ring ester carbonyl formed a hydrogen bond with the imine hydrogen atom of Arg229, and the carbonyl formed a hydrogen bond with the imine hydrogen of His138. The results of docking of isomandibolic acid with PGK molecules are shown in Table 12. The conformations generated by docking were divided into 10 clusters according to the RMSD values. Cluster 6 showed the lowest binding energy and the largest number of conformations, so this conformation was further analyzed. Figure 12The results showed that isomandibine is embedded in the active pocket of 3-PGK. Among these interactions, the hydrogen atom of the hydroxyl group at position 3 of isomandibine forms a hydrogen bond with the oxygen atom of Thr452 C=O, the oxygen atom of the hydroxyl group at position 3 forms a hydrogen bond with the hydrogen atom of Arg115 amino group, the carbonyl oxygen forms a hydrogen bond with the hydrogen atom of Arg115 amino group, the hydrogen atom and oxygen atom of the hydroxyl group at position 3 of the sugar ring form hydrogen bonds with the oxygen atom of Asp433 C=O and the hydrogen atom of the imine, respectively, the hydrogen atom of the hydroxyl group at positions 4 and 5 of the sugar ring form hydrogen bonds with the oxygen atom of Asp433 OH, and the hydrogen atom of the hydroxyl group at positions 6 and 7 of isomandibine forms a hydrogen bond with the oxygen atom of Glu439 OH.

[0116] Table 11 Summary of docking results of cephalosporin and 3-PGK

[0117]

[0118] Table 12 Summary of the docking results of isomandibolic acid and 3-PGK

[0119]

[0120]

[0121] (6) Molecular docking of ATP synthase F1 subunit ATPF1 with cephaloside and isomandibolic acid. The specific steps are as follows:

[0122] ① Homology modeling. The crystal structure of N. benthamiana ATPF1 has not yet been solved, so it was necessary to search for a template on the SWISS-MODEL website based on its protein sequence for homology modeling. The amino acid sequence of N. benthamiana ATPF1 was obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / ). The sequence was entered into the SWISS-MODEL website and a template search was performed. The template with the highest similarity (4asu.1.C) was used for homology modeling and saved as a pdb file (ATPF1.pdb).

[0123] ②Molecular docking. Set the central three-dimensional coordinates and spatial size of the docking box so that it can wrap the active site (KTSIAIDTIIN) of ATPF1 as much as possible. (x=44, y=36, z=36; x center=4, y center=-5, z center=4). The remaining steps are the same as those described in Example 2(4). The results of the molecular docking of ketoconazole with ATPF1 are shown in Table 13. The conformations generated by docking were divided into 10 clusters based on the RMSD values. Cluster 5 showed the lowest binding energy. However, since cluster 10 had a large number of conformations and the binding energy was low and the difference was small, cluster 10 was selected for further analysis. Figure 13The results show that scutellarin is embedded in the active pocket of ATPF1. Among these interactions, the hydroxyl oxygen atom at position 2 of the scutellarin sugar ring forms a hydrogen bond with the imine hydrogen atom of Val142, the hydroxyl oxygen atom of the hydroxymethyl group at position 5 of the sugar ring forms hydrogen bonds with the carbonyl oxygens of Are373 and Val374, and the oxygen atom of the five-membered ester group forms hydrogen bonds with the amino hydrogen of Arg139 and the imine hydrogen of Lys311, respectively. The docking results of isomandibose with ATPF1 are shown in Table 14. The resulting conformations were divided into 10 clusters based on the RMSD values. Cluster 7 exhibited the lowest binding energy and a large number of conformations, so further analysis was performed on this conformation. Figure 14 It shows that the hydroxyl oxygen at positions 2 and 3 of the sugar ring of isomandibose forms a hydrogen bond with the imine hydrogen atom of Ile136, the hydroxyl oxygen at position 2 forms a hydrogen bond with the hydrogen on the imine of Ile137, the hydroxyl hydrogen atom at position 6 forms a hydrogen bond with the carbonyl oxygen of Ala133, and the hydroxyl hydrogen atom at position 7 forms a hydrogen bond with the carbonyl oxygen of Lys132

[0124] Table 13 Summary of docking results of scutellarin and ATPF1

[0125]

[0126]

[0127] Table 14 Summary of the docking results of isomandibolic acid and ATP F1

[0128]

[0129] Example 4

[0130] Target protein activity assay of cephaloside and isomangiferin

[0131] (1) In vivo treatment: Total protein extraction from Nicotiana benthamiana. Nicotiana benthamiana with similar growth characteristics and 6-9 leaves were selected for the experiment. 10 mg / L, 100 mg / L, and 500 mg / L cephaloside solutions were prepared in a 1% DMSO aqueous solution.

[0132] Apply 200 μL of 100 mg / L, 500 mg / L cephaloside solution, or 1% DMSO aqueous solution to each tobacco leaf. Apply 200 μL of the corresponding solution to each tobacco leaf. Apply 4 leaves per tobacco plant. Moisten the gauze with 200 μL of the corresponding solution before application.

[0133] Injection method: Use a needleless syringe to inject 10 mg / L or 100 mg / L cephaloside solutions or 1% DMSO solution into Nicotiana benthamiana leaves. Inject on the underside of the leaf. Inject 4 leaves per plant, 200 μL per leaf. Inject one spot on each side of the main vein of each leaf. Each injection point should be no more than 2 cm in diameter and should be marked.

[0134] The same untreated Nicotiana benthamiana strain was used as a control for both the smear and injection methods.

[0135] After 3 days, the treated Nicotiana benthamiana leaves were quickly frozen in liquid nitrogen, ground into powder under liquid nitrogen conditions, and aliquoted into pre-chilled 1.5 mL centrifuge tubes. 1 mL of protein extraction buffer (1X PBS, 5‰ Triton X-100, 1 mM DTT, 150 mM NaCl, 10 mM MgCl2, 1 mM PMSF, and 10 mL / tablet of protease inhibitors) was added to each tube and allowed to stand on ice for 3 hours. The treated samples were centrifuged at 12,000 r / min and 4°C for 10 minutes, and the supernatant was collected. This step was repeated twice, and the supernatants were combined. After the protein concentration was determined by Nanodrop, 500 μL of the supernatant was aliquoted into 1.5 mL centrifuge tubes, quickly frozen with liquid nitrogen, and stored at -80°C.

[0136] (2) GOX activity determination method. Since the error of ultraviolet absorption is the smallest in the range of 0.2-0.8, it is necessary to control the amount of protein so that the absorbance of the sample at 10 minutes is between 0.2-0.8. The experiment was carried out using protein samples extracted from untreated Nicotiana benthamiana. Take a 1.5mL centrifuge tube and add the corresponding volume of crude enzyme solution, Tris-HCl, sodium glycolate, and double-distilled water as shown in Table 16. Vortex and mix well, then incubate at 4°C for 1h. Then add the corresponding volume of phenylhydrazine hydrochloride, mix well, and add 100μL per well to the enzyme labeling plate. Measure the absorbance of the sample at 324nm at 10min. The specific results are as follows. Figure 17 1.72 mg of total protein was added for subsequent GOX activity determination.

[0137] Table 15 GOX activity assay system

[0138]

[0139] (3) In vivo GOX activity determination. Take a 1.5 mL centrifuge tube and add the corresponding volume of crude enzyme solution, Tris-HCl, sodium glycolate, and double-distilled water as shown in Table 16. Vortex and mix well, then add the corresponding volume of phenylhydrazine hydrochloride. After mixing, add 100 μL per well to the enzyme-labeled plate and measure the absorbance of the sample at 324 nm at 10 minutes. The specific results of the smear method are as follows: Figure 16 As shown in Figure 2, the GOX activity of Nicotiana benthamiana treated with cephaloside increased, and the increase in GOX activity was more obvious when treated with high concentrations. Figure 17 As shown in the figure, the GOX activity of Nicotiana benthamiana treated with low and high concentrations of cephaloside was greatly increased, but the difference was not large.

[0140] Table 16 In vivo GOX activity assay system

[0141]

[0142]

[0143] (4) In vitro GOX activity determination. Take a 1.5 mL centrifuge tube and add the corresponding volume of cephaloside or isomandibolic acid, crude enzyme solution, Tris-HCl, sodium glycolate, and double-distilled water as shown in Table 17. Vortex and mix well and incubate at 4°C for 1 hour. Then add the corresponding volume of phenylhydrazine hydrochloride, mix well and add 100 μL per well to the enzyme-labeled plate. Measure the absorbance of the sample at 324 nm at 10 minutes. The specific results are as follows. Figure 18 、 Figure 19 As shown in the results, both cephaloside and isomandibolic acid promoted GOX activity at low concentrations, and the promoting effect of cephaloside was more obvious; at high concentrations, both cephaloside and isomandibolic acid inhibited GOX activity.

[0144] Table 17 In vitro GOX activity assay system

[0145]

[0146] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Use of a plant-derived glycoside compound in combating tobacco mosaic virus, wherein the plant-derived glycoside compound is cephaloside of formula (I) or isomangiferin of formula (II); 2. The use according to claim 1, characterized in that: The cerebroside or isomandibolic acid is derived from natural plants or is produced by biotechnology or is artificially synthesized.

3. The use according to claim 2, characterized in that: The isomandibolic acid comes from an extract of the Anemarrhena plant. The preparation method of the Anemarrhena plant extract is as follows: adding ethanol to the Anemarrhena plant powder, separating the residue after ultrasonication, collecting the filtrate, then adding ethanol to the medicinal material, continuing ultrasonication, vacuum filtering, combining the two filtrates, and concentrating to half the mass of the input plant material; the effective concentration of the isomandibolic acid is ≥0.1 mg / mL.

4. A method for preventing and treating tobacco mosaic virus, characterized in that: The plant-derived glycoside compound according to claim 1 is applied, sprayed or injected onto tobacco leaves.

5. The use according to claim 1, characterized in that: The targets of the anti-tobacco mosaic virus action of the scutellarin or isomandibolic acid are glycolate oxidase, chloroplast triphosphoglycerate kinase or mitochondrial ATP synthase F1 subunit.

6. The use according to claim 5, characterized in that: The scutellarin or isomandibolic acid is embedded in the active pocket of glycolate oxidase. The hydroxyl hydrogen atoms at positions 2 and 3 on the scutellarin sugar ring form hydrogen bonds with the carbonyl oxygen of Gly163, the hydroxyl oxygen atom at position 3 on the sugar ring forms a hydrogen bond with the imine hydrogen atom of Arg165, the hydroxyl hydrogen atom at position 4 on the sugar ring forms a hydrogen bond with the carbonyl atom of Arg165, and the hydroxyl hydrogen atom of the hydroxymethyl group at position 5 on the sugar ring forms a hydrogen bond with the carbonyl atom of Asp168; the hydroxyl hydrogen atoms at positions 6 and 7 on isomandibolic acid form hydrogen bonds with the carbonyl oxygen of Gly163, the hydroxyl oxygen atom at position 6 forms a hydrogen bond with the imine hydrogen atom of Arg165, and the hydroxyl oxygen atom at position 7 forms a hydrogen bond with the hydrogen atom on Arg165 C=NH.

7. The use according to claim 5, characterized in that: The glycosidic oxygen atom of galanthus or isomandibolic acid is embedded in the active pocket of chloroplast triphosphoglycerate kinase. The oxygen atom of the glycosidic bond of galanthus and the oxygen atom at position 6 of the sugar ring form a hydrogen bond with the imine hydrogen atom of His228, the hydroxyl oxygen atom of the hydroxymethyl group at position 5 of the sugar ring forms a hydrogen bond with the imine hydrogen atom of Gly455, the hydroxyl oxygen atom at position 2 of the sugar ring forms a hydrogen bond with the amino hydrogen atom of Arg196, the hydroxyl oxygen atom at position 3 of the sugar ring forms a hydrogen bond with the imine hydrogen atom of His138 and the amino hydrogen atom of Asn101, the hydroxyl oxygen atom at position 4 of the sugar ring forms a hydrogen bond with the amino hydrogen of Arg115, the five-membered ring ester carbonyl forms a hydrogen bond with the imine hydrogen atom of Arg229, and the carbonyl forms a hydrogen bond with the imine hydrogen of His138; the hydroxyl hydrogen atom at position 3 of isomandibolic acid forms a hydrogen bond with the Thr452 The oxygen atom of C=O forms a hydrogen bond, the hydroxyl oxygen atom at position 3 forms a hydrogen bond with the amino hydrogen atom of Arg115, the carbonyl oxygen forms a hydrogen bond with the amino hydrogen atom of Arg115, the hydroxyl hydrogen atom and oxygen atom at position 3 of the sugar ring form hydrogen bonds with the oxygen atom of Asp433 C=O and the hydrogen atom on the imine, respectively, the hydroxyl hydrogen atoms at positions 4 and 5 of the sugar ring form hydrogen bonds with the oxygen atom on Asp433O-H, and the hydroxyl hydrogen atoms at positions 6 and 7 of isomandibolic acid form hydrogen bonds with the oxygen atom of Glu439 OH.

8. The use according to claim 5, characterized in that: Echinopsin or isomandibolic acid is embedded in the active pocket of the F1 subunit of mitochondrial ATP synthase. The hydroxyl oxygen atom at position 2 of the sugar ring of echinopsin forms a hydrogen bond with the imine hydrogen atom of Val142, the hydroxyl oxygen atom of the hydroxymethyl group at position 5 of the sugar ring forms a hydrogen bond with the carbonyl oxygens of Are373 and Val374, and the oxygen atom on the five-membered ring ester group forms hydrogen bonds with the amino hydrogen of Arg139 and the imine hydrogen of Lys311, respectively; the hydroxyl oxygens at positions 2 and 3 of the sugar ring of isomandibolic acid form hydrogen bonds with the imine hydrogen atom of Ile136, the hydroxyl oxygen atom at position 2 of the sugar ring forms a hydrogen bond with the hydrogen on the imine of Ile137, the hydroxyl hydrogen atom at position 6 forms a hydrogen bond with the carbonyl oxygen of Ala133, and the hydroxyl hydrogen atom at position 7 forms a hydrogen bond with the carbonyl oxygen of Lys132.

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