Bioactive peptide in tobacco stem, screening preparation of bioactive peptide and application of bioactive peptide in resisting tobacco mosaic virus

Abstract

The invention discloses a bioactive peptide in a tobacco stem, screening preparation of the bioactive peptide and application of the bioactive peptide in resisting tobacco mosaic virus, and belongs to the technical field of tobacco mosaic virus control. The bioactive peptide in the tobacco stem is selected from a bioactive peptide IPF with an amino acid sequence of Ile-Pro-Phe, a bioactive peptide FGW with an amino acid sequence of Phe-Gly-Trp, a bioactive peptide PYF with an amino acid sequence of Pro-Tyr-Phe or a bioactive peptide FFAP with an amino acid sequence of Phe-Phe-Ala-Pro; the preparation method comprises the following steps: crushing tobacco stems, degreasing and removing impurities, extracting crude protein from the tobacco stems, preparing protein hydrolysate from the tobacco stems, separating active components from the hydrolysate, identifying amino acid sequences of the active components, screening active peptide fragments and synthesizing the active peptide fragments. Related tobacco mosaic virus resisting activity experiments prove that the bioactive peptide in the tobacco stem has an antiviral effect, a new raw material is provided for developing a novel tobacco mosaic virus resisting preparation, meanwhile, the treatment cost of agricultural by-products can be reduced, and sustainable agricultural development is promoted.

Description

Technical Field

[0001] This invention relates to the field of tobacco mosaic virus control technology, specifically to a bioactive peptide from tobacco stems, its screening and preparation, and its application against tobacco mosaic virus. Background Technology

[0002] Tobacco mosaic virus (TMV) is considered one of the representative plant viruses affecting crop health and one of the most serious viruses infecting tobacco plants. TMV has a wide host range and causes different symptoms in infected hosts. It is estimated that TMV infects at least 350 species, including tobacco, tomato, pepper, cucumber, and some ornamental flowers. TMV can invade host cells, causing chlorosis of plant leaves, and in severe cases, plant death, resulting in significant economic damage to crops, and in severe cases, reducing tobacco yield by 50% to 70%.

[0003] Driven by the green concept of plant protection, environmentally friendly, low-cost, and non-resistant biological control technologies are increasingly becoming a research focus. Bioactive peptides, as an important class of substances, exhibit significant application potential in crop disease control due to their advantages such as small molecular weight, strong targeting, and high environmental compatibility.

[0004] Tobacco stalks, as one of the main solid wastes from tobacco processing, are not only costly to dispose of using traditional methods (such as incineration and landfill), but also pose environmental risks. In-depth exploration of the high-value-added functional components in tobacco stalks can effectively reduce the cost of by-product disposal and decrease agricultural reliance on chemically synthesized pesticides. In particular, the development and research of antiviral active peptides in tobacco stalks can not only alleviate the environmental pressure of this waste, but also provide a new approach for creating highly efficient, low-toxicity, and low-residue green plant protection agents. Research in this direction is of significant importance for promoting the low-cost and high-efficiency development of agricultural antiviral agents and for facilitating the sustainable transformation of agriculture. Summary of the Invention

[0005] This invention addresses the current lack of research reports on small-molecule active substances from tobacco stems, particularly the lack of information on the preparation, screening, and activity evaluation of bioactive peptides from tobacco stems. It provides a method for the screening, preparation, and application of bioactive peptides from tobacco stems against tobacco mosaic virus.

[0006] Therefore, in a first aspect, the present invention provides a bioactive peptide for use in a cigarette holder, which is selected from bioactive peptide IPF, bioactive peptide FGW, bioactive peptide PYF, or bioactive peptide FFAP; wherein... The amino acid sequence of the bioactive peptide IPF is Ile-Pro-Phe; The amino acid sequence of the bioactive peptide FGW is Phe-Gly-Trp; The amino acid sequence of the bioactive peptide PYF is Pro-Tyr-Phe; The amino acid sequence of the bioactive peptide FFAP is Phe-Phe-Ala-Pro.

[0007] A second aspect of the present invention provides a method for screening and preparing bioactive peptides from the above-mentioned tobacco stem, comprising: S1 Cigarette rod crushing, degreasing and impurity removal: The air-dried cigarette rod is crushed and sieved. Then, petroleum ether is added to the sieved cigarette rod powder, magnetically stirred, filtered, and air-dried to obtain pre-treated cigarette rod powder. S2 Tobacco Stem Crude Protein Extraction: The pretreated tobacco stem powder is mixed with distilled water, the pH is adjusted to alkaline, and protein extraction is carried out under heating and shaking conditions. After centrifugation, the supernatant is collected, and the pH is adjusted to the isoelectric point of the protein with acid. The precipitate is collected by centrifugation and freeze-dried to obtain tobacco stem crude protein. Preparation of S3 protein hydrolysate: Add the crude protein from the tobacco stalks to distilled water, adjust the pH with acid, add pepsin for hydrolysis, then adjust the pH to neutral, add neutral protease for secondary hydrolysis, then inactivate the enzyme, centrifuge to collect the supernatant, and freeze-dry to obtain the tobacco stalk protein hydrolysate. Preparation of active components of S4 hydrolysate: After dissolving the tobacco stalk protein hydrolysate, centrifuge and filter it using an ultrafiltration membrane with a molecular weight of 3 kDa. Collect the filtrate components and freeze-dry them to obtain the active components. S5 active component amino acid sequence identification: The peptide sequence of the active component was analyzed by nano LC-ESI-Q-Orbitrap-MS / MS; S6 Bioactive Peptide Screening: Through score screening, corresponding disease target screening, and molecular docking screening, candidate peptides with anti-tobacco mosaic virus activity were obtained. S7 Synthesis of active peptides: Based on the screening results of step S6, the four peptides with the highest binding energy are selected for peptide synthesis to obtain the bioactive peptide IPF, the bioactive peptide FGW, the bioactive peptide PYF and the bioactive peptide FFAP.

[0008] Further, in step S1, the sieving is done through a 50-mesh sieve, and the ratio of the sieved tobacco powder to the petroleum ether is 1g:20mL.

[0009] Further, in step S2, the pretreated tobacco powder is mixed with distilled water at a ratio of 1g:30mL, the pH is adjusted to 12 with sodium hydroxide, and the mixture is shaken at 50℃ and 150r / min for 2 hours. After centrifugation, the supernatant is collected, the pH is adjusted to 2.8 with hydrochloric acid, the precipitate is collected by centrifugation and freeze-dried to obtain the crude protein from the tobacco.

[0010] Further, in step S3, the crude protein from the tobacco stalks is added to distilled water, and the pH is adjusted to 2.5 with hydrochloric acid. Then, 3500 U / g of pepsin is added and the mixture is shaken at 37°C and 150 r / min for 2 hours. Subsequently, the pH is adjusted to neutral with sodium hydroxide, and then 4000 U / g of neutral protease is added. The mixture is shaken at 50°C and 150 r / min for 2 hours. After that, the enzyme is inactivated, the supernatant is collected by centrifugation, and the mixture is freeze-dried to obtain the hydrolysate of the tobacco stalk protein.

[0011] Further, in step S5, the preparation method of the injection solution for nano LC-ESI-Q-Orbitrap-MS / MS coupling includes: dissolving the active component powder in distilled water, desalting and purifying it using a reverse-phase C18 column, collecting the purified component and redissolving it in a 0.1% formic acid aqueous solution to form an injection solution with a final concentration of 1 μg / μL.

[0012] Furthermore, step S6 specifically includes: The PeptideRanker platform was used to predict peptide activity, and peptides with a score greater than 0.9 were screened. Based on the peak area of ​​the mass spectrometry, the top 200 peptides with the highest relative abundance were screened. Peptide sequences were converted into SMILES format and analyzed in multiple dimensions using the Swiss ADME system to examine solubility, glycoprotein substrate, number of hydrogen bond donors / receptors, skin permeability, bioavailability and synthetic feasibility, and the top 50 peptide sequences were screened. The three-dimensional structure of the screened peptide was constructed and semi-flexible docking was performed with the target protein of tobacco mosaic virus in Discovery Studio 2020 software. The peptide with the highest -CDOCKER interaction energy was selected as the candidate peptide for antiviral activity.

[0013] In a third aspect, the invention provides the application of the bioactive peptides in the above-mentioned tobacco stems for the preparation of a formulation that inhibits tobacco mosaic virus.

[0014] Furthermore, the applications include: inactivation of tobacco mosaic virus, protection of tobacco plants infected with tobacco mosaic virus, and / or therapeutic effects on tobacco plants infected with tobacco mosaic virus.

[0015] Furthermore, the application includes disrupting the morphology of tobacco mosaic virus particles.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: The bioactive peptides derived from tobacco stalks provided by this invention have been experimentally proven to possess strong anti-Tobacco Mosaic Virus (TMV) activity. Specifically, they exhibit a significant inactivation effect on TMV, providing good protection and treatment for infected plants, and effectively disrupting the morphology of TMV virus particles. This invention offers a new alternative for developing high-efficiency agricultural antiviral agents, contributing to the promotion of sustainable agricultural development. This invention utilizes tobacco stalk waste to prepare antiviral bioactive peptides, addressing the current lack of research on the high-value utilization of tobacco waste, particularly the blank stage in the preparation of bioactive peptides from tobacco stalks. It also alleviates the pressure of waste disposal and demonstrates great potential for developing high-efficiency, low-risk green plant protection products. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0018] Figure 1 This is a flowchart illustrating the screening and preparation process of active peptides from tobacco stems, as provided in an embodiment of the present invention.

[0019] Figure 2 The total ion chromatogram of the active component provided in the embodiments of the present invention.

[0020] Figure 3 This is a schematic diagram of molecular docking of the synthesized active peptide in an embodiment of the present invention.

[0021] Figure 4 This is a secondary mass spectrum of the active peptide synthesized in the embodiments of the present invention.

[0022] Figure 5 Transmission electron microscopy (TEM) image showing the effect of active peptides on the morphology of TMV particles, as provided in an embodiment of the present invention. Detailed Implementation

[0023] To better understand the above technical solutions, the technical solutions of the embodiments of this application will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of this application and the specific features in the embodiments are detailed descriptions of the technical solutions of the embodiments of this application, rather than limitations on the technical solutions of this application. In the absence of conflict, the embodiments of this application and the technical features in the embodiments can be combined with each other.

[0024] Example 1: Method for screening and preparing bioactive peptides from tobacco stems used to inhibit tobacco mosaic virus A method for screening and preparing bioactive peptides from tobacco stems for inhibiting tobacco mosaic virus, such as... Figure 1As shown, the specific steps include the following: S1: Crushing, degreasing and impurity removal of tobacco rods: The naturally air-dried tobacco rods are crushed and passed through a 50-mesh sieve. Then, petroleum ether is added to the sieved tobacco rod powder at a material-to-liquid ratio of 1:20. The mixture is magnetically stirred twice, 2 hours each time, filtered, and air-dried to obtain powder. The material-to-liquid ratio is the ratio of the mass of the crushed and sieved tobacco rod powder to the volume of the petroleum ether solution.

[0025] S2: Extraction of crude protein from tobacco stalks: The powder treated in S1 was added to distilled water at a material-to-liquid ratio of 1:30. The pH was adjusted to 12 with sodium hydroxide. The mixture was shaken for 2 hours at 50°C and 150 rpm using a constant temperature water bath shaker. After cooling to room temperature, the mixture was centrifuged (4500 rpm) for 15 minutes. The supernatant was collected, and the pH was adjusted to 2.8 (isoelectric point of protein) with hydrochloric acid (6M). After standing at room temperature, the mixture was centrifuged (4500 rpm) for 15 minutes. The precipitate was collected and freeze-dried. The dried powder was the crude protein from tobacco stalks. The material-to-liquid ratio is the ratio of the mass of the air-dried powder to the volume of distilled water.

[0026] S3: Preparation of protein hydrolysates from tobacco stalks: Crude tobacco stalk protein was added to distilled water, and the pH was adjusted to 2.5 with hydrochloric acid (6M). Pepsin (3500 U / g) was then added, and the mixture was placed in a constant temperature water bath shaker at 37°C and 150 rpm for 2 hours. After removal, the pH was adjusted to 7 with sodium hydroxide, and neutral protease (4000 U / g) was added. The mixture was then placed in a constant temperature water bath shaker at 50°C and 150 rpm for 2 hours, and the enzyme was inactivated in a boiling water bath for 10 minutes. The mixture was centrifuged (4500 rpm) for 15 minutes, and the supernatant was collected and freeze-dried to obtain the tobacco stalk protein hydrolysates.

[0027] S4: Separation of active components from hydrolysis products: The tobacco stalk protein hydrolysis products obtained in S3 were dissolved in distilled water, centrifuged (4000 r / min) using a 3 kDa ultrafiltration membrane for 30 min each time, for a total of 3 centrifugations, and the components after filtration were freeze-dried to obtain the active components.

[0028] S5: Amino Acid Sequence Identification of Active Components: Peptide sequence analysis of the active components was performed using nano LC-ESI-Q-Orbitrap-MS / MS. An appropriate amount of the active component powder was dissolved in distilled water. The sample was desalted and purified using a reverse-phase C18 desalting column (2.1 × 100 mm, 5 μm), and then redissolved in 0.1% formic acid aqueous solution (LC-MS grade) to form an injection solution with a final concentration of 1 μg / μL. Analysis was performed using an EASY-nano LC 1200 system coupled with an Orbitrap Fusion Lumos triple-mass spectrometer. The results are as follows: Figure 2 As shown. From Figure 2It can be seen that the active components are mainly concentrated in the first 30 minutes. The high peak intensity and symmetrical peak shape indicate that the target active peptide components have been effectively separated and enriched in the chromatographic system, and have high purity.

[0029] S6: Screening of Active Peptides: Peptide sequences were used for multi-parameter activity prediction on the PeptideRanker bioinformatics platform. A threshold of Score > 0.9 was set to screen for highly confident active peptides. Then, the top 200 peptides by relative abundance were selected based on peak area. The peptide sequences were then imported into the BIOPEP-UWM database SMILE module, outputting standard SMILES format. The Swiss ADME online prediction system was used for multi-dimensional analysis, screening for peptide sequences with high solubility, glycoprotein substrate properties, 2-8 hydrogen bond acceptors, 4-7 hydrogen bond donors, bioavailability score > 0.7, and synthetic feasibility score > 3, resulting in a top 50 peptide sequence selection. Initial three-dimensional conformations of the peptide sequences were constructed using Pymol 2.5.0 software, and energy minimization optimization was performed. High-resolution crystal structures of the target receptor protein (PDB ID: 1EI7) were obtained from a protein database and implemented using the CDOCKER module in Discovery Studio 2020 software. A semi-flexible docking approach was used, generating 50 independent docking conformations for each ligand system. The dominant conformation with the highest -CDOCKER interaction energy was selected as the final complex model. The results are as follows: Figure 3 As shown. From Figure 3 It can be seen that the four bioactive peptide molecules achieve highly complementary binding with specific amino acid residues (such as ASP and ARG) at the target active sites through key intermolecular forces such as hydrogen bonds, hydrophobic interactions, and π-π stacking. This binding mode reveals the potential interaction mechanism and provides crucial structural evidence for explaining their biological functions or guiding subsequent structural optimization.

[0030] S7: Synthesis of Active Peptides: Based on the molecular docking results obtained in S6, the four peptides with the highest binding energies were selected and commissioned to a biotechnology company for peptide synthesis. The secondary mass spectra of the synthesized peptides are shown below. Figure 4 As shown. From Figure 4 As can be seen, the fragment ion peaks in the mass spectrum have formed a continuous and complete series of b- and / or y-ions, and the mass difference between adjacent fragment ions perfectly matches the expected amino acid sequence of the target peptide. This indicates that the target peptide has been successfully synthesized and its amino acid sequence has been definitively verified. Furthermore, no obvious abnormal fragment ion peaks corresponding to missing sequences or other byproducts appeared in the spectrum, indicating high synthesis purity and effectively confirming the molecular structure of the target peptide.

[0031] Example 2: Activity test of active peptides in tobacco stems against tobacco mosaic virus The active peptides prepared in Example 1 were used to test their anti-tobacco mosaic virus activity. A peptide solution with a concentration of 500 μg / mL was prepared: 1 mg of active peptide (IPF, FGW, PYF, FFAP) solid powder was weighed and dissolved in 2 mL of phosphate buffer solution (PBS).

[0032] Select robust K326 tobacco plants with uniform growth stages. From each plant, select three leaves of similar growth. Inoculate each leaf with 200 μL (32 μg / mL) of TMV suspension by rubbing. After standing for 30 minutes, rinse thoroughly with sterile water. Once obvious signs of TMV infection appear, use a perforator to evenly cut the leaves into small circles with a diameter of 1 cm. These circles are then randomly mixed and used for the following experiment: Treatment group: Take 4 mixed discs and immerse them in a centrifuge tube containing a peptide solution (500 μg / mL) dissolved in PBS; Blank group: Take 4 discs after mixing and immerse them in a centrifuge tube containing PBS buffer; Control group: Four mixed discs were immersed in centrifuge tubes containing ningnanmycin solution (500 μg / mL) dissolved in PBS.

[0033] Place all centrifuge tubes in a 28°C light incubator and incubate for 48 hours. After 48 hours, measure the TMV virus concentration according to the steps of the TMV virus concentration kit. Calculate the TMV inhibition rate using the formula below. Perform three replicates for each sample.

[0034]

[0035] Table 1. Inhibitory effect of bioactive peptides in tobacco pipes on TMV

[0036] Table 1 shows that the active peptides have strong inhibitory activity against TMV, and their activity is higher than that of the positive control ningnanmycin.

[0037] Example 3: Experiment on the effect of active peptides in tobacco stems in inactivating tobacco mosaic virus and protecting and treating tobacco plants. Passivation: An equal volume of active peptide solution (500 μg / mL) and 32 μg / mL TMV virus solution were mixed and incubated at 4℃ for 1 h to form a virus + sample complex. Physiologically homogeneous heart-leaf tobacco (…) was selected. Nicotiana glutinosa L. Plants were selected. Three leaves of similar developmental stages were chosen from each plant. The left half of the leaf was inoculated with 100 μL of virus + sample complex (treatment group), and the right half of the leaf was inoculated with an equal volume of 32 μg / mL TMV virus solution (positive control group). The treated plants were placed in a controlled greenhouse (25℃, 16h light / 8h dark). After 5 days, the number of local necrotic spots on each half of the leaf was counted, and the relative inhibition rate was calculated using the formula below.

[0038] Protective effect: Select heart-leaf tobacco with uniform physiological state ( Nicotiana glutinosa L. Three leaves of the same developmental stage were selected from each plant. The left half of the leaf was rubbed with 100 μL of active peptide solution (500 μg / mL) as the treatment group, and the right half of the leaf was inoculated with an equal volume of PBS buffer (pH=7.2) as the blank control group. Six hours later, both halves of the leaf were inoculated with 100 μL of 32 μg / mL TMV virus solution. After treatment, the plants were placed in a controlled greenhouse (25±1℃, 16h light / 8h dark). After 5 days, the number of local necrotic spots on each half of the leaf was counted, and the relative inhibition rate was calculated using the formula below.

[0039] Therapeutic effect: Plants with uniform physiological condition and heart-leaf leaves were selected, and three mature leaves at the same developmental stage were chosen from each plant. First, a basic infection model was established by rubbing 100 μL of TMV virus solution (32 μg / mL) onto both the left and right halves of the leaves. Six hours later, 100 μL of the test sample (500 μg / mL) was inoculated into the left half of the leaf as the treatment group, while the other half was injected with an equal volume of PBS (pH=7.2) as the control group. After treatment, the plants were transferred to a controlled greenhouse (25±1℃, 16h / 8h light / dark cycle). Five days later, the number of localized necrotic spots on the half-leaf of each treatment group was counted, and the relative inhibition rate was calculated using the formula below.

[0040]

[0041] Table 2. Inactivation effect of tobacco stalk active peptides on TMV, protection and therapeutic effects on plants.

[0042] Table 2 shows that all peptides exhibited superior inactivation of tobacco mosaic virus (TMV) and protection of tobacco plants compared to the commercially available drug ningnanmycin. The FGW peptide demonstrated the best overall anti-TMV activity, showing superior activity to ningnanmycin in all three aspects: TMV inactivation (81.13%), protection (83.16%), and therapeutic effect (65.63%).

[0043] Example 4: Experiment on the disruption of virus particle structure by active peptides in tobacco pipes The experiment was conducted to observe the destructive effect of the active peptide prepared in Example 1 on the structure of viral particles. The procedure is as follows: The sample solution (500 μg / mL) was prepared using phosphate buffer (pH=7.2) and mixed with the crude extract of tobacco mosaic virus (TMV) in a specific ratio. The mixture was incubated in a shaker at 20°C for 2 hours. After sufficient incubation, 10 μL of the mixture was spotted onto the surface of a copper mesh support membrane. After 1-2 minutes of sample adsorption, the copper mesh was held with tweezers, and residual liquid was gently absorbed by touching the edge of filter paper. Under semi-dry conditions, 2% phosphotungstic acid (PTA, pH=6.8) staining solution was added to cover the copper mesh. After staining for 2 minutes, the staining solution was removed, and the virus particles were gently dispersed by blowing with a syringe. After the copper mesh was completely dry, the morphology and structure of the virus particles were observed using a transmission electron microscope. The results are shown below. Figure 5 As shown.

[0044] like Figure 5 As shown, compared to the blank control group (K), all samples disrupted the morphology of TMV particles, exhibiting varying degrees of breakage, deformation, and structural depolymerization. The synthetic peptide FGW showed the most significant disruptive effect, with numerous fragmented viral particles observed. Transmission electron microscopy (TEM) images revealed that untreated TMV particles exhibited a typical rigid rod-like structure, while treated samples showed localized depressions, bending, and breakage on the surface of the viral particles, with some particles even completely disintegrating into short fragments. This weakened the supporting stability of the TMV coat protein and RNA, reducing the structural strength of the TMV particles and consequently decreasing TMV activity.

[0045] This invention is the first to obtain tobacco endogenous active peptides with good anti-tobacco mosaic virus activity from tobacco stems. The tobacco stem endogenous active peptides of this invention can be used for the research and development of anti-tobacco mosaic virus drugs.

[0046] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.

Claims

1. A bioactive peptide in a tobacco pipe, characterized in that, It is selected from bioactive peptides IPF, FGW, PYF, or FFAP; among them, The amino acid sequence of the bioactive peptide IPF is Ile-Pro-Phe; The amino acid sequence of the bioactive peptide FGW is Phe-Gly-Trp; The amino acid sequence of the bioactive peptide PYF is Pro-Tyr-Phe; The amino acid sequence of the bioactive peptide FFAP is Phe-Phe-Ala-Pro.

2. A method for screening and preparing bioactive peptides from tobacco stems, characterized in that, The method for screening and preparing bioactive peptides from tobacco stems according to claim 1 includes: S1 Cigarette rod crushing, degreasing and impurity removal: The air-dried cigarette rod is crushed and sieved. Then, petroleum ether is added to the sieved cigarette rod powder, magnetically stirred, filtered, and air-dried to obtain pre-treated cigarette rod powder. S2 Tobacco Stem Crude Protein Extraction: The pretreated tobacco stem powder is mixed with distilled water, the pH is adjusted to alkaline, and protein extraction is carried out under heating and shaking conditions. After centrifugation, the supernatant is collected, and the pH is adjusted to the isoelectric point of the protein with acid. The precipitate is collected by centrifugation and freeze-dried to obtain tobacco stem crude protein. Preparation of S3 protein hydrolysate: Add the crude protein from the tobacco stalks to distilled water, adjust the pH with acid, add pepsin for hydrolysis, then adjust the pH to neutral, add neutral protease for secondary hydrolysis, then inactivate the enzyme, centrifuge to collect the supernatant, and freeze-dry to obtain the tobacco stalk protein hydrolysate. Preparation of active components of S4 hydrolysate: After dissolving the tobacco stalk protein hydrolysate, centrifuge and filter it using an ultrafiltration membrane with a molecular weight of 3 kDa. Collect the filtrate components and freeze-dry them to obtain the active components. S5 active component amino acid sequence identification: peptide sequence analysis of the active component was performed using nano LC-ESI-Q-Orbitrap-MS / MS; S6 Bioactive Peptide Screening: Through score screening, corresponding disease target screening, and molecular docking screening, candidate peptides with anti-tobacco mosaic virus activity were obtained. S7 Synthesis of active peptides: Based on the screening results of step S6, the four peptides with the highest binding energy are selected for peptide synthesis to obtain the bioactive peptide IPF, the bioactive peptide FGW, the bioactive peptide PYF and the bioactive peptide FFAP.

3. The method for screening and preparing bioactive peptides from tobacco stems according to claim 2, characterized in that, In step S1, the sieving is done through a 50-mesh sieve, and the ratio of the sieved tobacco powder to the petroleum ether is 1g:20mL.

4. The method for screening and preparing bioactive peptides from tobacco stems according to claim 2, characterized in that, In step S2, the pretreated tobacco powder is mixed with distilled water at a ratio of 1g:30mL, the pH is adjusted to 12 with sodium hydroxide, and the mixture is shaken at 50℃ and 150r / min for 2 hours. After centrifugation, the supernatant is collected, the pH is adjusted to 2.8 with hydrochloric acid, the precipitate is collected by centrifugation and freeze-dried to obtain the crude protein from the tobacco.

5. The method for screening and preparing bioactive peptides from tobacco stems according to claim 2, characterized in that, In step S3, the crude protein from the tobacco stalks is added to distilled water, and the pH is adjusted to 2.5 with hydrochloric acid. Then, 3500 U / g of pepsin is added and the mixture is shaken at 37°C and 150 r / min for 2 hours. Subsequently, the pH is adjusted to neutral with sodium hydroxide, and then 4000 U / g of neutral protease is added. The mixture is shaken at 50°C and 150 r / min for 2 hours. After enzyme inactivation, the supernatant is collected by centrifugation and freeze-dried to obtain the hydrolysate of the tobacco stalk protein.

6. The method for screening and preparing bioactive peptides from tobacco stems according to claim 2, characterized in that, In step S5, the preparation method of the injection solution for nano LC-ESI-Q-Orbitrap-MS / MS coupling includes: dissolving the active component powder in distilled water, desalting and purifying it using a reverse-phase C18 column, collecting the purified component and redissolving it in 0.1% formic acid aqueous solution to form an injection solution with a final concentration of 1 μg / μL.

7. The method for screening and preparing bioactive peptides from tobacco stems according to claim 2, characterized in that, Step S6 specifically includes: The PeptideRanker platform was used to predict peptide activity, and peptides with a score greater than 0.9 were screened. Based on the peak area of ​​the mass spectrometry, the top 200 peptides with the highest relative abundance were screened. Peptide sequences were converted into SMILES format and analyzed in multiple dimensions using the Swiss ADME system to examine solubility, glycoprotein substrate, number of hydrogen bond donors / receptors, skin permeability, bioavailability and synthetic feasibility, and the top 50 peptide sequences were screened. The three-dimensional structure of the screened peptide was constructed and semi-flexible docking was performed with the target protein of tobacco mosaic virus in Discovery Studio 2020 software. The peptide with the highest -CDOCKER interaction energy was selected as the candidate peptide for antiviral activity.

8. The application of a bioactive peptide in a tobacco pipe, characterized in that, The use of the bioactive peptide in the tobacco stem as described in claim 1 in the preparation of a formulation that inhibits tobacco mosaic virus.

9. The application according to claim 8, characterized in that, The applications include: inactivation of tobacco mosaic virus, protection of tobacco plants infected with tobacco mosaic virus, and / or treatment of tobacco plants infected with tobacco mosaic virus.

10. The application according to claim 8, characterized in that, The application includes disrupting the morphology of tobacco mosaic virus particles.

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