A short peptide and its application in preparing antiviral drugs

Abstract

The present invention discloses a short peptide CLE7 and its use in the preparation of antiviral drugs. The amino acid sequence of the short peptide CLE7 is shown in SEQ ID NO: 1. Experimental studies have shown that injecting the short peptide CLE7 into Nicotiana benthamiana leaves can inhibit infection by TMV, ToMV, PVX, and TuMV. This indicates that the short peptide CLE7 can be used to prepare antiviral drugs, thereby inhibiting viral infection in plants. The present invention has important application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a short peptide and application thereof in the preparation of antiviral drugs. The amino acid sequence of the short peptide is shown in SEQ ID NO: 1. Background Art

[0002] Plant RNA viruses are the most diverse group of viruses found in plants and pose the most serious threat to plants. Their transmission vectors include soil, insects, and water, creating significant obstacles to the prevention and control of RNA viral diseases. Currently, chemical pesticides are still the most widely used method for the prevention and control of plant viral diseases. However, this method not only causes significant harm to the environment but also has little effect on non-insect-borne viruses. In agricultural operations, crop rotation and adjusting tillage times can also help prevent and control plant viral diseases, but these cannot completely address the harm caused by viral diseases. Therefore, enhancing the plant's own immune response through certain means is a future direction for the prevention and control of plant viral diseases.

[0003] Studies have shown that plant endogenous small peptides play an important role in activating plant immune responses. The CLE (CLAVATA3 / Embryo surrounding region-related) family is the largest family of plant peptide molecules reported to date and is also the most popular plant peptide hormone studied in the past decade. Studies have shown that this family plays an important role in different developmental processes and specific tissues of plants, including the development of stem apical meristem and root apical meristem, seed development, vascular bundle formation, nodule formation, embryo and endosperm development, regulation of root system structure, and regulation of stem cell homeostasis in the cambium. However, the function of CLE short peptides in activating plant immunity is still poorly understood. Summary of the Invention

[0004] The object of the present invention is to inhibit viruses from infecting plants.

[0005] The present invention first protects the short peptide CLE7, whose amino acid sequence is shown in SEQ ID NO: 1.

[0006] The present invention also protects the nucleic acid molecule encoding the short peptide CLE7.

[0007] The present invention also protects the use of the short peptide CLE7 or the nucleic acid molecule encoding the short peptide CLE7 in the preparation of antiviral drugs.

[0008] The present invention also protects the use of the short peptide CLE7 or the nucleic acid molecule encoding the short peptide CLE7 in inhibiting viral infection.

[0009] In any of the above applications, the virus may be at least one of TMV, ToMV, PVX and TuMV.

[0010] The present invention also provides a method for inhibiting plant virus infection, which may include the following steps: inoculating a plant with a short peptide CLE7, wherein the amino acid sequence of the short peptide CLE7 is shown in SEQ ID NO: 1.

[0011] In the above method, the inoculating the plant with the short peptide CLE7 can be performed by injecting the short peptide CLE7 into the plant or spraying the short peptide CLE7 onto the plant.

[0012] In the above method, the injection or spraying site may be leaves.

[0013] In the above method, the virus may be at least one of TMV, ToMV, PVX and TuMV.

[0014] Any of the plants mentioned above may be tobacco, and the tobacco may specifically be Nicotiana benthamiana.

[0015] After extensive experiments, the inventors discovered the short peptide CLE7 from Nicotiana benthamiana. Furthermore, they injected CLE7 into N. benthamiana leaves and then infected them with viruses (such as TMV, ToMV, PVX, or TuMV). The results showed that injection of the short peptide CLE7 into N. benthamiana leaves was less conducive to infection by TMV, ToMV, PVX, and TuMV, indicating that the short peptide CLE7 can inhibit infection by viruses (such as TMV, ToMV, PVX, and TuMV) to a certain extent. This suggests that the short peptide CLE7 can be used to prepare antiviral drugs, thereby inhibiting viral infection in plants. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The infection status of Nicotiana benthamiana injected with the short peptide CLE7 after inoculation with TMV, ToMV, PVX or TuMV.

[0017] Figure 2 Virus accumulation in Nicotiana benthamiana injected with the short peptide CLE7 after inoculation with TMV, ToMV, PVX, or TuMV. DETAILED DESCRIPTION

[0018] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0019] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0020] The ToMV-GFP infectious clone is described in the following literature: Liu Ni, Na Chengxiao, Dong Luran, Liao Qiansheng. Construction and expression of the ToMV tomato necrotic strain Agrobacterium-infectious clone. Journal of Agricultural Biotechnology, 2014, 22(8): 1027-1034.

[0021] TuMV-GFP infectious clones are described in the following literature: Chen Shuxia, Wang Xiaowu, Cheng Zhihui. Construction of green fluorescent protein gene TuMv viral expression vector. Northwest Botanical Journal, 2005, 8:1395-1398.

[0022] The PVX-GFP infectious clone is described in the following literature: Sean, Chapman, Tony, Kavanagh, David, Baulcombe. Potato virus X as a vector for gene expression in plants. The Plant Journal. 1992.

[0023] The TMV-GFP infectious clone is described in the following references: An Mengnan, Xia Zihao, Zhang Chong, Wu Yuanhua, Zhao Xiuxiang, Xia Bo, and Wang Zhiping. A TMV infectious cloning vector and its construction method. 2021.

[0024] Example 1. Discovery of the short peptide CLE7

[0025] After extensive experiments, the inventors discovered a short peptide, CLE7, from Nicotiana benthamiana. The specific steps are as follows:

[0026] 1. Extraction of intercellular fluid protein of Nicotiana benthamiana

[0027] (1) Take TMV-infected Nicotiana benthamiana leaves, place them in ddH2O to rinse the surface, and then absorb the surface water with absorbent paper.

[0028] (2) After completing step (1), the cleaned leaves were immersed in a solution (specifically, Cockail containing 0.1% protease inhibitors), and the solution was completely penetrated into the leaves using a vacuum pump;

[0029] (3) After completing step (2), wipe the liquid on the surface of the leaf with absorbent paper, place it in a syringe (size of 20 mL), and then place it in a centrifuge tube (size of 50 mL), centrifuge at 4°C and 1000g for 1 min, and collect the liquid phase.

[0030] (4) The liquid phase collected in step (3) was added to a Merck Millipore ultrafiltration tube (specification: 0.5 ml, 3 kDa) and centrifuged at 4°C and 6000 g for 1 min; the effluent was collected in a new centrifuge tube (specification: 2 mL), and then the liquid was concentrated to a dry powder using a freeze-concentrator dryer; finally, it was dissolved in 200 μL of Cockail containing 0.1% protease inhibitor to obtain the Nicotiana benthamiana intercellular fluid protein.

[0031] 2. Desalination

[0032] A centrifuge tube (2 mL in size) was taken, and 100 μL of Nicotiana benthamiana intercellular fluid protein and 200 μL of an aqueous solution containing 0.5% TFA and 0.5% acetonitrile were added to obtain a sample.

[0033] First, use 200 μL of an aqueous solution containing 0.1% TFA and 80% acetonitrile to activate the C18 desalting column, and then use 400-600 μL of an aqueous solution containing 0.1% TFA and 1% acetonitrile to equilibrate the desalting column; then add the sample to the desalting column and allow the sample to slowly flow through the desalting column. The polypeptide is captured by the desalting column, and other non-hydrophobic small molecules such as salts flow out and are discarded; then add 200 μL of an aqueous solution containing 0.1% TFA and 0.5% acetonitrile to clean the desalting column and wash away residual salts; finally, add 300 μL of an aqueous solution containing 0.1% TFA and 80% acetonitrile and allow the liquid to slowly flow through the desalting column to elute the polypeptide. Use a new EP tube to collect the elution solution, and freeze-dry the eluate to obtain a dry powder.

[0034] 3. LCMS / MS analysis of peptides

[0035] The detection system used was a Thermo Scientific Easy-nLC 1200 (Thermo Scientific, P / N LC140) coupled to an Orbitrap Exploris 480 (Thermo Scientific, P / N BRE725533). The dry powder obtained in step 2 was dissolved in 10 μl of mobile phase A (0.1% formic acid in water). After that, 5 μl of the sample was loaded. The peptides were trapped on a trapping column (PepMap C18, 100 μm × 2 cm) at a flow rate of 10 μl / min for 3 minutes. The peptides were then separated by gradient elution chromatography on a nanoliter analytical column (PepMap C18, 75 μm × 25 cm). The separation gradient was from 5% to 30% mobile phase B (0.1% formic acid in acetonitrile) over 60 minutes. 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 heated 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 an orbitrap primary scan over the m / z 100-1600 range, with a resolution of 70,000 (at m / z 200). The maximum ion introduction time was 50 ms, and automatic gain control (AGC) was set to 5 × 10⁵. Subsequently, high-energy C-trap dissociation (HCD) was used to fragment the top 15 parent ions that met the cascade (MS / MS) fragmentation criteria and scanned with the orbitrap at a resolution of 17,500. The scan range was automatically controlled based on the parent ion mass-to-charge ratio, with a fixed minimum scan range of m / z = 100 and a maximum scan range of 2000. The minimum ion intensity for MS / MS was set to 13,000. For MS / MS, the maximum ion introduction time was 100 ms, the AGC control was set to 2.0 × 10⁵, and the precursor ion selection window was set to 1.6 Da. MS / MS acquisition was performed for ions of charge 1, 2, 3, and 4. Dynamic exclusion was set to perform one MS / MS run per precursor ion within 10 s, followed by a 40 s exclusion, and 30% collision energy.

[0036] 4. Database search and peptide identification

[0037] The raw data obtained in step 3 was processed and analyzed using PEAKS software. PEAKS can perform de novo sequencing and protein identification (PEAKS DB). The following parameters were set for de novo sequencing and database retrieval: deep-sea actinomycete protein database, non-enzymatic digestion, a primary mass spectrometry tolerance of 10 ppm, a secondary mass spectrometry tolerance of 0.02 Da, no fixed modifications, variable modifications set to methionine oxidation and N-terminal acetylation, and charges set to +1, +2, +3, and +4. The false positive rate (FDR) for peptide identification was set to 1%. For de novo sequencing results, ALC (%) stands for average local confidence. The confidence level of de novo sequencing data results is generally greater than 80% for reasonable confidence and greater than 95% for very confidence. The short peptide CLE7 was ultimately discovered, with the amino acid sequence of CLE7 being DRVAPGGPDPQHHS (SEQ ID NO: 1).

[0038] Example 2: Application of short peptide CLE7 in plant antiviral activities

[0039] The short peptide CLE7 in this example was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0040] The short peptide CLE7 diluent is obtained by diluting the short peptide CLE7 with sterile water. The concentration of the short peptide CLE7 in the short peptide CLE7 diluent is 300 μg / mL.

[0041] 1. Take 24 Nicotiana benthamiana plants that are 3 weeks old and randomly divide them into two groups, the experimental group and the control group, with 12 Nicotiana benthamiana plants in each group. The following treatments are carried out:

[0042] Experimental group: 0.5 ml of the short peptide CLE7 dilution was injected into the second round of leaves of Nicotiana benthamiana using a sterile syringe. The injection area was approximately 4-5 cm 2 Mark the injection area with a marker and let it sit for 6 hours.

[0043] Control group: Use a sterile syringe to inject 0.5 ml of sterile water into the second whorl of leaves of Nicotiana benthamiana, with an injection area of ​​approximately 4-5 cm 2 Mark the injection area with a marker and let it sit for 6 hours.

[0044] 2. After completing step 1, inoculate the virus (TMV, ToMV, PVX or TuMV) by Agrobacterium infiltration. The specific steps are as follows:

[0045] (1) The ToMV-GFP infectious clone was inoculated into 7 ml YEP liquid medium and cultured with shaking at 28°C for 12-16 h. The precipitate was then collected by centrifugation at 10,000 rpm for 2 min. The precipitate was then resuspended in tobacco infiltration solution (10 ml tobacco infiltration solution consists of 1 ml 100 mM MES solution, 0.1 ml 1 M MgCl2 solution, 20 μl 2 mM As solution and 8.88 ml ddH2O) to obtain the OD 600nm It is a 0.3 ToMV virus solution.

[0046] According to the above steps, the ToMV-GFP infectious clone was replaced with the TuMV-GFP infectious clone, PVX-GFP infectious clone and TMV-GFP infectious clone, and the OD values ​​were obtained in sequence. 600nm TuMV virus solution of 0.3, OD 600nm The PVX virus solution and OD of 0.3 600nm It is a 0.3 TMV virus solution.

[0047] (2) Take Nicotiana benthamiana that has completed step 1 and inject 0.2 ml of virus solution (ToMV virus solution, TuMV virus solution, PVX virus solution or TMV virus solution) into the leaf (the leaf injected with virus solution is the inoculated leaf) using a sterile syringe. The injection area is about 1 cm 2 The injection site was at the same location treated with the short peptide CLE7 dilution or sterile water.

[0048] 3. After completing step 2, culture the Nicotiana benthamiana at 25°C for 5-7 days.

[0049] 4. After completing step 3, take the inoculated leaves and systemic leaves of Nicotiana benthamiana (systemic leaves are the upper leaves of the inoculated leaves) respectively and observe the virus infection under UV light.

[0050] Observation results are shown in Figure 1 (CK was the control group and CLE7 was the experimental group.) The results showed that the amount of virus accumulated in the experimental group was significantly less than that in the control group.

[0051] 5. After completing step 3, take Nicotiana benthamiana cells for Western blotting and Ponceau red staining. The specific steps are as follows:

[0052] (1) Extraction of total protein

[0053] After completing step 3, the inoculated leaves or systemic leaves (systemic leaves are the upper leaves of the inoculated leaves) of Nicotiana benthamiana from the control group and the experimental group were respectively taken and ground into powder in liquid nitrogen; then 0.2 g of the powder was taken into a 2 ml sterile centrifuge tube, 0.2 ml of cell lysis buffer (pH 7.4, 20 mM / L Tris-HCl buffer containing 200 mM / L NaCl, 1.0 mM / L EDTA, 1.0 mM / L DTT) was added, vortexed and allowed to stand on ice for 20 min; then centrifuged at 4°C and 14000g for 10 min, and the supernatant was collected; 0.2 ml of the supernatant was mixed with 50 μl of loading buffer (GenScript, Beijing) in a 1.5 ml sterile centrifuge tube to obtain a mixture; the mixture was boiled in a water bath for 5 min, placed on ice for 5 min, and centrifuged at 4°C and 14000g for 10 min to obtain a processed protein mixed sample.

[0054] (2) Western-blotting

[0055] (2-1) The treated protein mixture obtained in step (1) was electrophoresed in an SDS-PAGE gel (GenScript, Beijing) until the target band (GFP molecular weight of approximately 35 kD) was separated by a marker (Takara, Shanghai), and then transferred to a membrane. Before transfer, the nitrocellulose membrane was soaked in transfer buffer. A sponge pad, protein gel, nitrocellulose membrane, and sponge pad were placed in a membrane transfer apparatus (GenScript, Beijing) from the negative electrode to the positive electrode, taking care to remove any bubbles in the gap. The membrane was transferred at a constant current of 0.3 A for 14 minutes.

[0056] (2-2) After transfer, place the nitrocellulose membrane in pre-prepared Ponceau stain solution (1 L of Ponceau stain solution is prepared as follows: mix 0.1 g Ponceau, 5 ml acetic acid, and 95 ml ddH2O, then dilute to 1 L with ddH2O). Shake on a horizontal shaker until a distinct red band appears. Discard the Ponceau stain and rinse the membrane with pure water until the red background disappears and the protein bands are clearly visible. Compare the thickness of each sample to ensure consistent sample loading and take a photo. Rinse the membrane again with pure water until the red bands are almost gone. Place the membrane in 5% (m / v) skim milk in 1× PBS and block on a horizontal shaker for 1 hour.

[0057] (2-3) After blocking, discard the blocking solution and wash the membrane three times with 1× PBS for 10 minutes each time; add 5% (m / V) skim milk containing GFP antibody at a ratio of 1:40,000 and incubate on a horizontal shaker for 1 hour; discard the primary antibody and wash the membrane three times with 1× PBS for 10 minutes each time; add 5% (m / V) skim milk containing the corresponding secondary antibody at a ratio of 1:40,000 and incubate on a horizontal shaker for 1 hour; discard the secondary antibody and wash the membrane three times with 1× PBS for 10 minutes each time.

[0058] (2-4) Discard PBS, place the membrane in a color development bag, add color development solution, develop in the dark for 2-5 minutes, and expose in an AI600 machine.

[0059] Test results are shown in Figure 2 (CK was the control group and CLE7 was the experimental group.) The results showed that the accumulation of virus in the experimental group was significantly reduced compared with the control group.

[0060] The above results indicate that injection of the short peptide CLE7 into Nicotiana benthamiana leaves is not conducive to the infection of TMV, ToMV, PVX and TuMV, that is, the short peptide CLE7 can inhibit the infection of viruses (such as TMV, ToMV, PVX and TuMV) to a certain extent.

[0061] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A short peptide CLE7, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the short peptide CLE7 according to claim 1.

3. Use of the short peptide CLE7 according to claim 1 or the nucleic acid molecule according to claim 2 in the preparation of antiviral drugs; The virus is at least one of TMV, ToMV, PVX and TuMV.

4. Use of the short peptide CLE7 according to claim 1 or the nucleic acid molecule according to claim 2 in inhibiting viral infection; The virus is at least one of TMV, ToMV, PVX and TuMV.

5. A method for inhibiting tobacco virus infection, comprising the following steps: injecting a short peptide CLE7 into tobacco; the amino acid sequence of the short peptide CLE7 is shown in SEQ ID NO: 1; The virus is at least one of TMV, ToMV, PVX and TuMV.

6. The method according to claim 5, characterized in that: The injection site is the leaf blade.

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