Antiviral peptide and application thereof in prevention and treatment of rice virus diseases

Abstract

The invention discloses an antiviral peptide and application thereof in preventing and treating rice virus diseases. According to the invention, the traditional thought of finding antiviral peptides from soil microorganisms is broken through, and possible antiviral peptides are innovatively found from animal organisms; eight antiviral peptide sequences are finally determined through a series of earlier-stage researches, and verification shows that the antiviral peptide with the natural source provided by the invention has a remarkable prevention and treatment effect on the rice virus diseases, provides a new pesticide for prevention and treatment of the rice virus diseases, is harmless to the environment and has a very obvious effect.

Description

Technical Field

[0001] The present invention relates to the field of plant disease prevention and control, and in particular to an antiviral peptide and an application thereof in preventing and controlling rice viral diseases. Background Art

[0002] With the development of global agriculture, rice, as one of the world's major food crops, is crucial to human food security. However, rice production faces numerous threats from pests and diseases, among which viral diseases are a significant factor affecting rice yield and quality. The occurrence of viral diseases not only reduces rice production but also compromises rice quality and food safety. There are numerous types of rice viruses, including the southern rice black-streaked dwarf virus (SRBSDV), rice gall dwarf virus (RGDV), and rice stripe mosaic virus (RSMV). Rice viral diseases are primarily transmitted by vectors, such as the white-backed planthopper (Sogatella furcifera) for SRBSDV and the electric leafhopper (Lepidoptera: RGDV) and RSMV. Once infected, the viruses rapidly multiply within the rice plant, causing symptoms such as yellowing, curling, and necrosis of leaves, and in severe cases, even the death of the entire plant. Currently, the main methods for controlling rice viral diseases include chemical pesticides and the breeding of virus-resistant varieties, but these methods often have limitations.

[0003] While chemical pesticides can control the spread of viral diseases in the short term, long-term use can lead to increased resistance among pests and diseases, potentially polluting the environment and impacting human health. Breeding viral-resistant varieties, while a long-term and effective strategy, requires significant time and resources, and the promotion and application of new varieties presents challenges.

[0004] In recent years, with the advancement of biotechnology, antiviral peptides (AVPs), a new type of bioactive substance, have become a research hotspot due to their broad-spectrum antiviral activity, low toxicity, and resistance to drug resistance. Antiviral peptides are small molecule polypeptides naturally produced in animals with antiviral activity. Currently, there are few reports on antiviral peptides for the prevention and treatment of rice viral diseases. Summary of the Invention

[0005] The purpose of the present invention is to address the above problems and provide an antiviral peptide and its application in preventing and controlling rice viral diseases.

[0006] In order to achieve its purpose, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides an antiviral peptide, which is any of the following polypeptides:

[0008] The antiviral peptide RdAVP1 whose amino acid sequence is shown in SEQ ID NO.25;

[0009] The antiviral peptide RdAVP2 whose amino acid sequence is shown in SEQ ID NO.26;

[0010] The antiviral peptide RdAVP3 whose amino acid sequence is shown in SEQ ID NO.27;

[0011] The antiviral peptide RdAVP4 whose amino acid sequence is shown in SEQ ID NO.28;

[0012] The antiviral peptide RdAVP5 whose amino acid sequence is shown in SEQ ID NO.29;

[0013] The antiviral peptide RdAVP6 whose amino acid sequence is shown in SEQ ID NO.30;

[0014] The antiviral peptide RdAVP7 whose amino acid sequence is shown in SEQ ID NO.31;

[0015] The amino acid sequence of the antiviral peptide RdAVP8 is shown in SEQ ID NO.32.

[0016] The second aspect of the present invention provides a biomaterial related to the above-mentioned antiviral peptide, wherein the biomaterial is any one of the following:

[0017] (1) a nucleic acid molecule encoding the above-mentioned antiviral peptide;

[0018] (2) an expression cassette containing the nucleic acid molecule described in (1);

[0019] (3) a recombinant vector containing the nucleic acid molecule described in (1);

[0020] (4) a recombinant vector containing the expression cassette described in (2);

[0021] (5) A recombinant microorganism containing the nucleic acid molecule described in (1);

[0022] (6) a recombinant microorganism containing the expression cassette described in (2);

[0023] (7) a recombinant microorganism containing the recombinant vector described in (3);

[0024] (8) A recombinant microorganism containing the recombinant vector described in (4).

[0025] The biomaterial, wherein (1) the nucleic acid molecule encoding the above-mentioned antiviral peptide, the nucleotide sequences of the nucleic acid molecules encoding the antiviral peptides shown in SEQ ID NOs. 25 to 32 are shown in SEQ ID NOs. 17 to 24, respectively.

[0026] The third aspect of the present invention provides a product for preventing and controlling rice virus diseases or resisting rice virus disease pathogens, wherein the active ingredient is the above-mentioned antiviral peptide or the above-mentioned biological material.

[0027] The rice virus diseases include rice gall dwarf disease, rice stripe mosaic disease and southern rice black streaked dwarf disease.

[0028] The rice virus disease pathogens include rice gall dwarf virus RGDV, rice stripe mosaic virus RSMV and southern rice black streaked dwarf virus SRBSDV.

[0029] The fourth aspect of the present invention provides the use of the above-mentioned antiviral peptide or the above-mentioned biomaterial or any of the above-mentioned products in any of the following:

[0030] (1) Resistance to rice virus disease pathogens;

[0031] (2) Preparation of products resistant to rice virus disease pathogens;

[0032] (3) Prevent and control rice virus diseases;

[0033] (4) Preparation of products for preventing and treating rice viral diseases.

[0034] In the above application, the rice viral diseases include rice gall dwarf disease, rice stripe mosaic disease and southern rice black streaked dwarf disease;

[0035] The rice virus disease pathogens include rice gall dwarf virus RGDV, rice stripe mosaic virus RSMV and southern rice black streaked dwarf virus SRBSDV.

[0036] The application is to apply the product to diseased rice plants by soaking the roots of rice seedlings with the antiviral peptide solution or soaking the seedlings before transplanting.

[0037] The product is an antiviral peptide solution with a concentration of 0.5 to 4 mg / ml, preferably 2 to 3 mg / ml.

[0038] The beneficial effects of the present invention are: breaking the traditional idea of studying antiviral peptides from rice plant sources or rice soil microbial sources, and innovatively searching for possible antiviral peptides from the electric leafhopper, an insect vector of rice virus disease; after a series of preliminary studies, 8 antiviral peptide sequences were finally determined. It has been verified that the natural antiviral peptides provided by the present invention have a significant prevention and control effect on rice virus disease, providing a new pesticide for the prevention and control of rice virus disease (especially rice gall dwarf disease and rice stripe mosaic disease, which are currently very difficult to effectively prevent and control), which is harmless to the environment and has very obvious effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1The results are the results of testing the antiviral effect of antiviral peptides on rice gall dwarf virus RGDV in rice plants.

[0040] Figure 2 This is the comparison result of plant height of rice plants carrying RGDV after being treated with antiviral peptides.

[0041] Figure 3 The results are the results of testing the antiviral effect of antiviral peptides against rice stripe mosaic virus RSMV in rice plants.

[0042] Figure 4 This is the comparison result of plant height of rice plants carrying RSMV after being treated with antiviral peptides.

[0043] Figure 5 These are the results of RGDV detection in the body of the electric leafhopper after injection of antiviral peptides.

[0044] Figure 6 These are the results of RSMV detection in the body of the electric leafhopper after injection of antiviral peptides.

[0045] Figure 7 These are the results of SRBSDV detection in white-backed planthoppers after they were injected with antiviral peptides. DETAILED DESCRIPTION

[0046] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0047] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0048] Example 1 Screening of antiviral peptides against rice gall dwarf virus RGDV and rice stripe mosaic virus RSMV

[0049] In the early stage, the research team screened a number of potential insect antiviral peptide gene sequences from rice disease vector insects through the antiviral peptide database and bioinformatics. Based on the analysis, 8 of them were selected for further verification research experiments.

[0050] The target species is the electric leafhopper (Recilia dorsalis). Adults and nymphs of the electric leafhopper suck sap from rice leaves and sheaths, inhibiting plant growth and causing yellowing or complete plant wilt. They also transmit diseases such as rice gall stunt virus and rice stripe mosaic virus. A preliminary screening of antiviral peptide genes from the electric leafhopper was performed to amplify the genes.

[0051] (1) PCR amplification

[0052] Design PCR primers to amplify the full length of the gene: Design amplification primers based on the screened antiviral peptide gene sequence.

[0053] Total RNA was extracted from the electric leafhopper, reverse transcribed into cDNA, and then amplified by PCR. The amplification system consisted of 2 μL of cDNA, 1 μL of a 10 μM upstream primer, 1 μL of a 10 μM downstream primer, 25 μL of 2* Primer STAR Mix, and 21 μL of ddH₂O, for a total volume of 50 μL. The amplification protocol was as follows: 98°C for 5 min, followed by 36 cycles of 98°C for 20 s, 60°C for 30 s, and 72°C for 24 s, and finally 72°C for 10 min. The primer sequences for the corresponding antiviral peptide genes are shown in Table 1.

[0054] Table 1

[0055]

[0056]

[0057] The amplified product was recovered using a gel recovery kit and then connected to the pET-28a vector: the amplified product and the pET-28a prokaryotic expression vector plasmid were double-digested with EcoRI and BamHI and then connected using T4 DNA ligase to obtain a recombinant vector, which was then sequenced for verification.

[0058] The full-length sequence of the antiviral peptide gene was obtained by sequencing, and the gene was compared with the target species database gene. The signal peptide fragment in the antiviral peptide gene was removed and used to design specific amplification primers.

[0059] The nucleotide sequences of the antiviral peptide genes are shown in Table 2:

[0060] Table 2

[0061]

[0062]

[0063] 2. Prokaryotic expression and purification of antiviral peptides

[0064] The nucleotide sequences listed in Table 2 were synthesized and constructed into the pET-28a vector by enzyme digestion (double digestion with EcoRI and BamHI) and ligation, and sequence verification was performed. The verified pET-28a-AVP vector was transformed into BL21 Escherichia coli. Single colonies were picked from the transformed plates and transferred to liquid LB medium with kanamycin at 220 rpm and 37°C, shaking until the OD600 value reached approximately 0.6-0.8. The cells were allowed to stand on ice for two minutes. IPTG was then added at a 1:200 dilution and shaken at 220 rpm and 37°C for 4-5 hours. The culture medium was removed by centrifugation, and the cells were resuspended in 1× PBS. The resuspended cells were supplemented with protease inhibitors (PMSF) and disrupted in a cell disruptor until clear. Protein induction was successful by Coomassie blue staining and Western blot analysis. Single and consistent Coomassie blue and Western blot bands were observed, indicating successful protein induction. After induction of target protein expression, the E. coli cells were harvested by centrifugation and resuspended in PBS in a 50 ml centrifuge tube. Dissolve the inclusion bodies with Binding wash buffer and incubate at room temperature for 30-60 minutes to fully dissolve the precipitate. Remove the antibody purification column, remove the bottom cap, and remove excess liquid. Equilibrate the column with a volume of Binding wash buffer twice the volume of the purification column filler, and flow through the column at a flow rate of 0.5-1 ml / min. Add the bacterial solution treated with Binding wash buffer from the top of the column. Wash the resin with Binding wash buffer twice the volume of the resin and collect the effluent. Repeat this step until the absorbance of the effluent liquid is at the baseline at 280 nm. Elute the His-tagged protein from the resin with Elution buffer twice the volume of the resin, and repeat this step twice. Purified antiviral peptides were obtained. The amino acid sequences of each antiviral peptide are shown in Table 3.

[0065] Table 3 Antiviral peptide sequences

[0066]

[0067]

[0068] 3. Detecting the antiviral ability of antiviral peptides using susceptible rice plants

[0069] Currently, in this technical field, rice viral diseases mainly include rice gall dwarf disease (pathogen: Rice gall dwarf virus (RGDV)) and rice stripe mosaic disease (pathogen: Rice stripemosaic virus (RSMV)). Because rice viruses cannot be artificially isolated and injected into plants, laboratory-reared electric leafhoppers carrying rice gall dwarf virus and rice stripe mosaic virus were used to conduct disease transmission experiments on rice seedlings.

[0070] Preparation of antiviral peptide solution: Take the purified antiviral peptide obtained above and add it to 1×PBS solution to prepare an antiviral peptide solution with a concentration of 2-3 mg / ml.

[0071] Prepare populations of electric leafhoppers carrying rice gall stunt virus and rice stripe mosaic virus, respectively. Then, use rice seedling trays to cultivate rice to a height of 3-4 cm, and place electric leafhoppers that have been identified as carrying rice gall stunt virus or rice stripe mosaic virus, respectively. Place 5 electric leafhoppers per rice plant, and feed the virus-carrying electric leafhoppers under a constant temperature of 28°C and light conditions (light:dark = 14:10). After feeding for 7 days, soak the rice in an antiviral peptide solution (2-3 mg / ml) for 48 hours (first pour out the water in the seedling tray, then add the antimicrobial peptide solution until the soil surface is immersed). At least 20 rice seedlings are treated with each antiviral peptide. After soaking the rice in the antiviral peptide solution for 48 hours, the antiviral peptide solution in the seedling tray was poured out of the seedling tray. The seedling tray was then incubated at a constant temperature of 28°C under a light intensity (light:dark = 14:10). After 20 days of incubation, the virus content in the rice leaves was detected: five leaves from different rice seedlings in the same seedling tray were randomly selected and combined into one sample. RNA from the main veins was extracted and the Ct value of the corresponding rice virus was detected by real-time fluorescence quantitative PCR using the HiScript II One Step RT-PCR Kit (Novozymes, China) (with rice actin as an internal reference). The primer sequences are shown in Table 4.

[0072] The relative virus content was calculated using a formula, and the changes in the Ct value of the virus titer in the rice of the treatment group and the control group soaked in antiviral peptides were detected at the nucleic acid level.

[0073] Excel software was used for data statistics, and GraphPad Prism 8.0 was used for data statistical analysis and drawing charts.

[0074] Table 4 Real-time fluorescence quantitative PCR primers

[0075]

[0076]

[0077] The results are as follows Figure 1-4 As shown in: Except for RdAVP7, all antiviral peptides have significant antiviral effects on rice gall dwarf virus RGDV ( Figure 1 ), the RdAVP7 treatment group was found to have a lower viral Ct, indicating a high viral solubility in the plant. The height of rice seedlings treated with PBS and RdAVP7 was significantly lower than that of rice seedlings treated with other antiviral peptides ( Figure 2For rice stripe mosaic virus RSMV, except for RdAVP3, the other antiviral peptides have significant effects on rice stripe mosaic virus ( Figure 3 ), the height of rice seedlings treated with PBS and RdAVP3 was significantly lower than that of rice seedlings treated with other antiviral peptides ( Figure 4 ).

[0078] Example 2 Detecting the antiviral ability of antiviral peptides using the electric leafhopper strain carrying rice virus disease virus

[0079] The electric leafhopper is the main vector of rice virus disease in rice fields. The virus can replicate and proliferate in the body of the electric leafhopper, so we also use the virus-carrying electric leafhopper to verify the antiviral ability of the antiviral peptide.

[0080] Preparation of antiviral peptide injection: The purified antiviral peptide obtained by prokaryotic expression in Example 1 was added to 1×PBS to prepare an antiviral peptide injection with a concentration of 1 mg / ml.

[0081] Reared electric leafhoppers identified as carriers of rice virus disease were frozen unconscious using an ice box. 4.6 nL of antiviral peptide injection solution was injected into the thorax of the electric leafhopper using an insect microinjector. After 72 hours, the amount of virus in the electric leafhopper was detected: RNA was extracted from the electric leafhopper before and after injection of the antiviral peptide, and the Ct value of rice virus disease was detected by real-time fluorescence quantitative PCR using the one-step high-efficiency RT-PCR kit HiScript II One Step RT-PCR Kit (Novozymes) (using leafhopper EF-1α as an internal reference). The primer sequences are shown in Table 4.

[0082] The relative viral load was calculated using a formula, and the changes in viral titer (Ct) values in the leafhoppers were measured at the nucleic acid level before and after injection of the antiviral peptide. Data were statistically analyzed and graphed using Excel software, and GraphPad Prism 8.0.

[0083] The results are as follows Figure 5 and Figure 6 As shown, except for RdAVP7, all antiviral peptides exerted significant inhibitory effects on RGDV virus after injection into the electric leafhopper; except for RdAVP1 and RdAVP2, all antiviral peptides exerted significant inhibitory effects on RSMV virus after injection into the electric leafhopper.

[0084] Example 3: Testing the antiviral ability of antiviral peptides using white-backed planthopper strains carrying rice virus disease virus

[0085] White-backed planthopper (Sogatellafurcifera) is also the main vector of rice virus diseases in rice fields and can spread southern rice black-streaked dwarf virus SRBSDV. Therefore, we also use the virus-carrying white-backed planthopper to verify the antiviral ability of antiviral peptides.

[0086] Preparation of antiviral peptide injection: The purified antiviral peptide obtained by prokaryotic expression in Example 1 was added to 1×PBS to prepare an antiviral peptide injection with a concentration of 1 mg / ml.

[0087] Reared white-backed planthoppers, identified as carriers of southern rice black-streaked dwarf virus, were frozen unconscious in an ice box. 4.6 nL of the antiviral peptide injection solution was injected into the thorax of the white-backed planthoppers using an insect microinjector. After 72 hours, the virus load in the white-backed planthoppers was detected. RNA was extracted from the electric leafhoppers before and after injection of the antiviral peptide, and the Ct value of rice virus disease was detected by real-time fluorescence quantitative PCR using the HiScript II One Step RT-PCR Kit (Novozymes) (using white-backed planthopper actin as an internal reference). The primer sequences are shown in Table 4.

[0088] The relative viral load was calculated using a formula, and the changes in viral titer (Ct) values in white-backed planthoppers before and after injection of antiviral peptides were measured at the nucleic acid level. Data were statistically analyzed and graphed using Excel software, and GraphPad Prism 8.0.

[0089] The results are as follows Figure 7 As shown, the three antiviral peptides RdAVP3, RdAVP4, and RdAVP6 could not inhibit SRBSDV, while the other antiviral peptides exerted a significant inhibitory effect on SRBSDV after injection into white-backed planthoppers.

Claims

1. An antiviral peptide, wherein the antiviral peptide is any of the following polypeptides: The antiviral peptide RdAVP1 whose amino acid sequence is shown in SEQ ID NO.25; The antiviral peptide RdAVP2 whose amino acid sequence is shown in SEQ ID NO.26; The antiviral peptide RdAVP3 whose amino acid sequence is shown in SEQ ID NO.27; The antiviral peptide RdAVP4 whose amino acid sequence is shown in SEQ ID NO.28; The antiviral peptide RdAVP5 whose amino acid sequence is shown in SEQ ID NO.29; The antiviral peptide RdAVP6 whose amino acid sequence is shown in SEQ ID NO.30; The antiviral peptide RdAVP7 whose amino acid sequence is shown in SEQ ID NO.31; The amino acid sequence of the antiviral peptide RdAVP8 is shown in SEQ ID NO.

32.

2. A biomaterial related to the antiviral peptide according to claim 1, wherein the biomaterial is any one of the following: (1) A nucleic acid molecule encoding the antiviral peptide according to claim 1; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule described in (1); (4) a recombinant vector containing the expression cassette described in (2); (5) A recombinant microorganism containing the nucleic acid molecule described in (1); (6) a recombinant microorganism containing the expression cassette described in (2); (7) a recombinant microorganism containing the recombinant vector described in (3); (8) A recombinant microorganism containing the recombinant vector described in (4).

3. The biomaterial according to claim 2, characterized in that: in, (1) A nucleic acid molecule encoding the antiviral peptide according to claim 1, wherein the nucleotide sequences of the nucleic acid molecule encoding the antiviral peptides shown in SEQ ID NOs. 25 to 32 are shown in SEQ ID NOs. 17 to 24, respectively.

4. A product for preventing and controlling rice viral diseases or resisting rice viral disease pathogens, characterized by: The active ingredient is the antiviral peptide according to claim 1 or the biomaterial according to claim 2.

5. The product according to claim 4, characterized in that: The rice virus diseases include rice gall dwarf disease, rice stripe mosaic disease and southern rice black streaked dwarf disease.

6. The product according to claim 4, characterized in that: The rice virus disease pathogens include rice gall dwarf virus RGDV, rice stripe mosaic virus RSMV and southern rice black streaked dwarf virus SRBSDV.

7. Use of the antiviral peptide according to claim 1, the biomaterial according to claim 2, or the product according to any one of claims 4 to 6 in any of the following: (1) Resistance to rice virus disease pathogens; (2) Preparation of products resistant to rice virus disease pathogens; (3) Prevent and control rice virus diseases; (4) Preparation of products for preventing and treating rice viral diseases.

8. The use according to claim 7, characterized in that: The rice virus diseases include rice gall dwarf disease, rice stripe mosaic disease and southern rice black streaked dwarf disease; The rice virus disease pathogens include rice gall dwarf virus RGDV, rice stripe mosaic virus RSMV and southern rice black streaked dwarf virus SRBSDV.

9. The use according to claim 8, characterized in that: The product is applied to diseased rice plants by soaking the roots of rice seedlings with the antiviral peptide solution or by soaking the seedlings before transplanting.

10. The use according to claim 9, characterized in that: The product is an antiviral peptide solution with a concentration of 0.5 to 4 mg / ml, preferably 2 to 3 mg / ml.

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