Polypeptide and application thereof

Abstract

The invention belongs to the field of biological agriculture, and particularly relates to a polypeptide and application thereof. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 1, and the polypeptide is derived from nicotiana benthamiana. The polypeptide is screened and identified from nicotiana benthamiana through a mass spectrum technology, and experiments prove that virus diseases caused by turnip mosaic virus, potato X virus, tomato mosaic virus and tobacco mosaic virus on tobacco are remarkably inhibited by applying the polypeptide. The polypeptide is derived from plants, has the advantages of being green, safe and not prone to generating drug resistance, and provides new bioactive molecules for green prevention and treatment of plant virus diseases.

Description

Technical Field

[0001] This invention belongs to the field of bio-agriculture, specifically relating to a polypeptide and its applications. Background Technology

[0002] Plant viral diseases are among the major plant diseases, and their pathogens are viruses. Because viruses rely entirely on host cells to replicate and multiply, infection often leads to a significant decline in crop yield and quality.

[0003] Currently, the control of plant viral diseases mainly relies on chemical pesticides. However, there is a lack of highly effective and specialized antiviral chemical pesticides on the market, and long-term, large-scale use of chemical pesticides can easily lead to problems such as virus resistance, pesticide residues, and environmental pollution, which contradicts the concept of green and sustainable development in modern agriculture. Another strategy for controlling plant viral diseases is to use disease-resistant varieties, but traditional breeding cycles are long, and frequent viral genome mutations often lead to a rapid loss of crop resistance.

[0004] Against this backdrop, green control strategies based on plant immune inducers have attracted widespread attention. These formulations achieve broad-spectrum and long-lasting disease resistance by activating the plant's own immune system. Among them, plant-derived small peptides (typically composed of 5-60 amino acids) play a crucial role as important endogenous signaling or effector molecules in plant growth, development, and stress responses. Originating from the plant itself, they possess potential advantages such as good biocompatibility, easy degradation, strong target specificity, and low likelihood of inducing pathogen resistance, aligning with the needs of green agriculture and sustainable development. However, reports on the systematic extraction of endogenous peptides with clearly defined functions and direct or indirect antiviral activity from plants remain scarce. Therefore, discovering novel and highly effective antiviral peptides from the plant's own defense system has significant research value. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention utilizes mass spectrometry to screen and identify a plant-derived polypeptide from *Nicotiana benthamiana* that can effectively inhibit the infection of TuMV, PVX, ToMV, and TMV viruses, providing a new bioactive molecule for the green control of plant viral diseases.

[0006] Specifically, the present invention provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] Furthermore, the polypeptide is derived from Nicotiana benthamiana.

[0008] Furthermore, the polypeptide is derived from the intercellular fluid protein of Nicotiana benthamiana.

[0009] On the other hand, a pesticide composition is also provided, the pesticide composition comprising the polypeptide described in this invention, and an agriculturally acceptable carrier or adjuvant.

[0010] Furthermore, this invention also provides the application of the polypeptide described in this invention in the control of plant viral diseases, wherein the viral disease is caused by turnip mosaic virus. 芜菁花叶病毒 Potato virus X 马铃薯X病毒 Tomato mosaic virus 番茄花叶病毒 and tobacco mosaic virus 烟草花叶病毒 One or more of the following can trigger this.

[0011] Furthermore, in the aforementioned application, the plant is tobacco.

[0012] Furthermore, in the aforementioned application, the polypeptide is applied to the plant via spraying.

[0013] Finally, a method for preventing and controlling plant viral diseases is also provided, which involves applying the polypeptide or pesticide composition described in this invention to the plant or its growing environment.

[0014] Furthermore, in the method, the plant is tobacco.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention uses mass spectrometry to screen and identify a substance from *Nicotiana benthamiana* that can simultaneously inhibit turnip mosaic virus. 芜菁花叶病毒 Potato virus X 马铃薯X病毒 Tomato mosaic virus 番茄花叶病毒 and tobacco mosaic virus 烟草花叶病毒 A polypeptide that induces various plant viral diseases, the amino acid sequence of which is shown in SEQ ID NO:1. This polypeptide is derived from plants, has good environmental compatibility, low residual risk, and meets the needs of green agriculture and sustainable development. Attached Figure Description

[0016] Figure 1 The images show the virus infection results under different treatments under UV light. VIP1 represents the plant-derived polypeptide VIP1 treatment group, and H2O represents the water treatment control group.

[0017] Figure 2 This image shows the quantitative distribution of fluorescent lesion area under different treatments. VIP1 represents the plant-derived polypeptide VIP1 treatment group, and H2O represents the water treatment control group. Detailed Implementation

[0018] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0019] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0020] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0021] Example 1 This embodiment describes the screening of plant-derived polypeptide VIP1 from Nicotiana benthamiana.

[0022] 1. Extraction of intercellular fluid protein from Nicotiana benthamiana. (1) Take turnip mosaic virus ( 芜菁花叶病毒 The TuMV-infected tobacco leaves were placed in ddH2O to rinse the surface clean, and then the surface water was dried with absorbent paper.

[0023] (2) After completing step (1), soak the cleaned leaves in a solution (a Cocktail containing 0.1% protease inhibitor) and use a vacuum pump to completely immerse the leaves in the solution; (3) After completing step (2), wipe the liquid off the surface of the leaf with absorbent paper, place it in a 20mL syringe, then put it into a 50mL centrifuge tube, centrifuge at 1000g for 1min at 4℃, and collect the liquid phase.

[0024] (4) Add the liquid phase collected in step (3) into a Merck Millipore ultrafiltration tube (specification: 0.5 mL, 3 kDa), centrifuge at 4 °C and 6000 g for 1 min; collect the effluent into a new 2 mL centrifuge tube, and then concentrate the liquid to dry powder using a freeze-drying concentrator; then dissolve it in 200 μL of Cocktail containing 0.1% protease inhibitor to obtain Benjamin intercellular fluid protein.

[0025] 2. Desalination Take a 2 mL centrifuge tube, add 100 µL of Benjamin Benjamin intercellular fluid protein and 200 µL of an aqueous solution containing 0.5% TFA and 0.5% acetonitrile to obtain the sample.

[0026] First, the C18 desalting column was activated with 200 µL of an aqueous solution containing 0.1% TFA and 80% acetonitrile. Then, the column was equilibrated with 400–600 µL of an aqueous solution containing 0.1% TFA and 1% acetonitrile. Next, the sample was added to the desalting column and allowed to flow slowly through it. The peptides were captured by the column, while non-hydrophobic small molecules such as salts were eluted and discarded. Then, 200 µL of an aqueous solution containing 0.1% TFA and 0.5% acetonitrile was added to wash the column to remove residual salts. Finally, 300 µL of an aqueous solution containing 0.1% TFA and 80% acetonitrile was added and allowed to flow slowly through the column to elute the peptides. The eluent was collected using a new EP tube and freeze-dried to obtain a dry powder.

[0027] 3. LCMS / MS analysis of peptides The detection system consisted of a Thermo Scientific Easy-nLC 1200 (P / N LC140) coupled with an Orbitrap Exploris 480 (P / N BRE725533). The dried powder obtained from desalting step 2 was dissolved in 10 µL of mobile phase A (0.1% formic acid aqueous solution), followed by a 5 µL sample loading. The peptides were captured on a PepMap C18 (100 μm × 2 cm) column at a flow rate of 10 µL / min for 3 min. Subsequently, the peptides were separated by gradient elution chromatography on a nano-scale analytical column (PepMap C18 (75 μm × 25 cm)). The separation gradient was achieved by increasing mobile phase B (0.1% formic acid in acetonitrile) from 5% to 30% over 60 min. The chromatographic flow rate was 200 nL / min, and the column temperature was 55 °C. The ion source spray voltage was 2.0 kV, and the mass spectrometer heating capillary was set to 320 °C. Data-dependent mode was used for automatic switching between MS and MS / MS acquisition. Full-scan MS was performed using Orbitrap for the first-stage scan, with a scan range of m / z 100–1600 and a resolution of 70,000 (at m / z 200). The maximum ion introduction time was 50 ms, and the automatic gain control (AGC) was set to 5 × 10⁻⁶. 5 Subsequently, high-energy C-trap dissociation (HCD) was used to fragment the top 15 precursor ions that met the MS / MS fragmentation criteria, and the fragments were scanned using orbitrap at a resolution of 17,500. The scan range was automatically controlled based on the precursor ion mass-to-charge ratio, with a minimum scan range fixed at m / z = 100 and a maximum of 2000. The minimum ion intensity value for MS / MS was set to 13,000. The maximum ion introduction time for MS / MS was 100 ms, and the AGC control was set to 2.0 × 10⁻⁶. 5 The precursor ion selection window was set to 1.6 Daltons. MS / MS was used to acquire ions with charges of 1, 2, 3, and 4. Dynamic exclusion was set to perform one MS / MS scan for each precursor ion within 10 seconds, followed by exclusion for 40 seconds at 30% collision energy.

[0028] 4. Database retrieval and peptide identification The raw data obtained from the LCMS / MS analysis of the above-mentioned peptides were processed and analyzed using PEAKS software. PEAKS can perform de novo sequencing and protein identification (PEAKS DB). The parameters set for de novo sequencing and database search were as follows: deep-sea actinomycete protein database, non-enzymatic digestion, mass spectrometry tolerance of 10 ppm, mass spectrometry tolerance of 0.02 Da, no fixed modifications, and variable modifications set to methionine oxidation and N-terminal acetylation, with charges set to +1, +2, +3, and +4. The false positive rate (FDR) for peptide identification was set to 1%. For the results of de novo sequencing, ALC (%) stands for average local confidence level. Generally, a confidence level greater than 80% is considered reliable, and a confidence level greater than 95% is considered very reliable. Finally, the plant-derived peptide VIP1 (hereinafter referred to as VIP1) was discovered. The amino acid sequence of the plant-derived peptide VIP1 is shown in SEQ ID NO:1. The specific amino acid sequence of the plant-derived peptide VIP1 is: KLGSTTFLVSDSD.

[0029] Example 2 This embodiment evaluates the antiviral activity of the plant-derived polypeptide VIP1.

[0030] (1) When using plant-derived polypeptide VIP1 as the active ingredient in plant virus inhibitors, first dissolve and dilute the plant-derived polypeptide VIP1 with sterile water to a working concentration of 50 micromoles (μM) for later use.

[0031] (2) Tobacco was inoculated with turnip mosaic virus (Agrobacterium) using the Agrobacterium tumefaciens infiltration method. 芜菁花叶病毒 TuMV), Potato Virus X ( 马铃薯病毒 X, PVX), Tomato Mosaic Virus (X, PVX), 番茄花叶病毒 Tobacco Mosaic Virus (ToMV) 烟草花叶病毒 The four viruses (ToMV, TMV) were analyzed using the following steps: Infectious clones of Agrobacterium, carrying viral fusion with green fluorescent protein (GFP), namely ToMV-GFP, TuMV-GFP, PVX-GFP, and TMV-GFP, were inoculated into 2 mL of YEP (yeast extract peptone medium) and cultured at 28°C with shaking for 12–16 hours. The cells were then collected by centrifugation at 5000 rpm for 2 min. The cells were resuspended in the infection solution, and the Agrobacterium concentration was adjusted to OD0.05. 600=0.0001, thus obtaining the Agrobacterium tumefaciens bacterial suspension. The preparation method of the yeast extract peptone medium (YEP) is as follows: weigh 10g yeast extract, 10g peptone, and 5g sodium chloride, dissolve in distilled water, then bring the volume to 1L with distilled water and adjust the pH to 7.0; the infection solution consists of 10mM 2-morpholinoethanesulfonic acid hydrate, 10mM... MgCl2 and 200 μM acetylsyl syringone; the construction method of the viral infectious clone ToMV-GFP was based on the literature (Chen Shuxia, Wang Xiaowu, Cheng Zhihui. Construction of green fluorescent protein gene TuMv viral expression vector [J]. Northwest Botanical Journal, 2005, 25(8):1395-1398.); the construction method of the viral infectious clone TuMV-GFP was based on the literature (Chen Shuxia, Wang Xiaowu, Cheng Zhihui. Construction of green fluorescent protein gene TuMv viral expression vector [J]. Northwest Botanical Journal, 2005, 25(8):1395-1398.); the construction method of the viral infectious clone PVX-GFP was based on the literature (CHAPMANS, KAVANAGH T, BAULCOMBE D. Potato virus X as a vector for gene expression in plants [J]. The Plant Journal, 1992, 2(4): 549-557.); The method for constructing the viral infectious clone TMV-GFP is based on the patent document (CN112143643A, A TMV infectious cloning vector and its construction method, Shenyang Agricultural University, January 15, 2021).

[0032] (3) Use a sterile syringe to inject 0.2 mL of Agrobacterium tumefaciens solution obtained in step (2) into tobacco leaves. After injection, divide the injected leaves into two groups, each containing 3 biological replicates. One group is sprayed with sterile water as a control, and the other group is sprayed with 50 μM of plant-derived polypeptide VIP1.

[0033] (4) After completing step (3), each treated leaf was placed in an artificial culture room at 22℃ for 3 days. On the 4th day, the accumulation of virus in the injected leaves was observed with ultraviolet light to evaluate the inhibitory activity of plant-derived polypeptide VIP1 on plant viruses. Observation results as follows Figure 1 As shown (mock is the control group, VIP1 is the experimental group treated with plant-derived peptide VIP1), the virus accumulation in the VIP1 group was significantly reduced compared to the control group. Virus infection was observed using a UV lamp 7–9 days after inoculation (the infected virus clone contains a GFP tag, which appears green under UV light after infection). The area of ​​green fluorescence produced by virus infection in leaves treated with plant-derived peptide VIP1 was significantly reduced, indicating that virus infection was inhibited.

[0034] (5) The area of ​​fluorescent lesions on leaves under different treatments was quantified using ImageJ software, and the virus accumulation was calculated. The results are as follows: Figure 2 As shown, the proportion of green fluorescence in the whole leaf was significantly reduced after treatment with the plant-derived peptide VIP1.

[0035] The above experimental results show that the plant-derived polypeptide VIP1 exhibits significant inhibitory effects against the tested viruses of the genera *Potatovirus Y*, *Potatovirus X*, and *Tobacco Mosaic Virus* (TuMV, PVX, ToMV, and TMV), indicating that the antiviral activity of the plant-derived polypeptide VIP1 is not limited to a single virus and has a broad spectrum. This suggests that the plant-derived polypeptide VIP1 can serve as a candidate molecule for developing novel, safe, and bioavailable plant virus inhibitors or resistance-inducing agents. This invention provides a new bioactive molecule for the green control of plant viral diseases.

[0036] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that, The polypeptide is derived from Nicotiana benthamiana.

3. The polypeptide according to claim 2, characterized in that, The polypeptide is derived from the intercellular fluid protein of Nicotiana benthamiana.

4. A pesticide composition, characterized in that, The pesticide composition comprises the polypeptide of claim 1, and an agriculturally acceptable carrier or adjuvant.

5. The application of the polypeptide according to claim 1 in the control of plant viral diseases, characterized in that, The viral disease is caused by turnip mosaic virus. Turnip mosaic virus Potato virus X Potato virus X Tomato mosaic virus Tomato mosaic virus and tobacco mosaic virus Tobacco mosaic virus One or more of the following can trigger this.

6. The application according to claim 5, characterized in that, The plant in question is tobacco.

7. The application according to claim 5, characterized in that, The polypeptide is applied to the plants by spraying.

8. A method for preventing and controlling plant viral diseases, characterized in that, Apply the polypeptide of claim 1 or the pesticide composition of claim 4 to the plant or its growing environment.

9. The method according to claim 8, characterized in that, The plant in question is tobacco.

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