Short peptide capable of improving antiviral capability of plants and application of short peptide

Abstract

The invention relates to the field of agricultural biology, in particular to an oligopeptide capable of improving the antiviral capacity of plants and application of the oligopeptide. The invention provides an oligopeptide capable of improving the antiviral ability of plants. The amino acid sequence of the oligopeptide is any one or more of SEQ ID NO.1-SEQ ID NO.8. The invention also provides a preparation method of the oligopeptide. Through a large number of experiments, eight oligopeptides are extracted from cucumber, pepper, tomato and tobacco plants infected with virus diseases in the field; then, 8 oligopeptides are sprayed to leaves of nicotiana benthamiana exogena exogenously, then virus TMV is inoculated, and the result shows that after the 8 oligopeptides are sprayed exogenously, TMV infection is not facilitated; after 8 oligopeptides are respectively sprayed to leaves of cucumbers in an exogenous manner, the cucumbers are inoculated with the virus CMV, and the result shows that the 8 oligopeptides are not beneficial to infection of the CMV after being sprayed in the exogenous manner, which indicates that the 8 oligopeptides can inhibit infection of the viruses TMV and CMV to a certain extent.

Description

Technical Field

[0001] The invention relates to the field of agricultural biology, and in particular to a short peptide capable of improving the antiviral ability of plants and an application thereof. Background Art

[0002] Plant virus diseases are the second largest category of plant diseases after plant fungal diseases. They are highly specialized, diverse, and mostly transmitted by insects. They spread quickly, have high virus mutation rates, and are difficult to prevent and control. The occurrence of plant virus diseases is affected by many factors, such as strain type, host plant, transmission vector, environmental conditions, and human manipulation. Methods for preventing and controlling plant virus diseases are still being explored, and there is currently no specific medicine for viral diseases. At this stage, comprehensive methods for preventing and controlling viral diseases are widely used in agricultural production, including plant quarantine, resistant varieties, crop rotation, and prevention and control of infectious agents (aphids, etc.), but they still cannot completely solve the harm caused by viral diseases. Therefore, using certain means to activate the plant's own immune response is still an important research direction for the prevention and control of plant virus diseases in the future.

[0003] Plant small signaling peptides (SSPs) are new and important signal molecules that can transmit information between cells over short distances and participate in the regulation of plant responses to abiotic stress signals through long-distance information transmission. There are some endogenous secretory peptides in plants that are released from cells attacked by pathogens such as fungi and bacteria into the extracellular space and can trigger plant immune responses. In addition, some secretory peptides can also regulate plant immunity through hormone pathways. However, there are currently few studies on the functions of endogenous short peptides in plant antiviral responses. Summary of the invention

[0004] The purpose of the present invention is to provide a short peptide capable of improving the antiviral ability of plants and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention uses cucumber, pepper, tomato and tobacco plants infected with viral diseases in the field to extract 8 short peptides. After exogenous spraying of these 8 short peptides and inoculation of viruses, the effect of each short peptide on plant antiviral disease is studied, and it is found that these 8 short peptides can enhance the resistance of plants to viruses and can be used for the preparation of antiviral products.

[0005] To achieve the above object, the present invention provides the following solutions: The present invention provides a short peptide capable of improving the antiviral ability of plants, wherein the amino acid sequence of the short peptide is any one or more of the sequences described in SEQ ID NO.1 to SEQ ID NO.8; The virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0006] The present invention provides a nucleic acid molecule encoding the short peptide.

[0007] The present invention provides the use of the short peptide or the nucleic acid molecule in preparing a product for improving the antiviral ability of plants, wherein the virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0008] Preferably, the product comprises a viral inhibitor or an antiviral drug.

[0009] The present invention provides a virus inhibitor, which comprises the above-mentioned short peptide; the virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0010] Preferably, the virus inhibitor further comprises an auxiliary material.

[0011] The present invention provides the use of the above short peptide, the above nucleic acid molecule or the above virus inhibitor in inhibiting virus infection, wherein the virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0012] The invention provides a method for inhibiting plant virus infection, comprising the step of spraying the plants to be treated with the short peptide or the virus inhibitor; the virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0013] Further preferably, the spraying site is the leaves of the plant to be treated.

[0014] Further preferably, the plants to be processed are tobacco and cucumber.

[0015] The present invention provides the use of the short peptide, the nucleic acid molecule or the virus inhibitor in improving the antiviral ability of plants, wherein the virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0016] The present invention provides a method for improving the antiviral ability of plants, comprising the step of spraying the plants to be treated with the short peptide or the virus inhibitor; the virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0017] Further preferably, the spraying site is the leaves of the plant to be treated.

[0018] Further preferably, the plants to be processed are tobacco and cucumber.

[0019] The present invention discloses the following technical effects: The present invention provides a short peptide capable of improving the antiviral ability of plants, wherein the amino acid sequence of the short peptide is any one or more of the sequences described in SEQ ID NO.1-SEQ ID NO.8. After a large number of experiments, the inventor extracted 8 short peptides from cucumber, pepper, tomato and tobacco plants infected with viral diseases in the field; then, the 8 short peptides were sprayed exogenously on the leaves of Nicotiana benthamiana and then inoculated with the virus TMV. The results showed that the exogenous spraying of the 8 short peptides was not conducive to the infection of TMV; the exogenous spraying of the 8 short peptides on the leaves of cucumber was inoculated with the virus CMV. The results showed that the exogenous spraying of the 8 short peptides was not conducive to the infection of CMV, indicating that the 8 short peptides can inhibit the infection of the viruses TMV and CMV to a certain extent. In summary, the results of the specific embodiments of the present invention show that by spraying short peptides exogenously on plants, the defense response of plants to viral diseases is activated, and the infection of viruses to plants is inhibited. Therefore, these 8 short peptides can be used to prepare antiviral products, thereby inhibiting viral infection of plants; and compared with chemical pesticides, the short peptides provided by the present invention are non-toxic and pollution-free, efficient and convenient, and have important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The following are the infection status of Nicotiana benthamiana leaves sprayed with 8 short peptides respectively after inoculation with TMV; A is the statistical diagram of virus fluorescence accumulation in Nicotiana benthamiana leaves; B is the result diagram of virus protein accumulation level in Nicotiana benthamiana leaves; Figure 2 The following are the infection status diagrams of cucumbers inoculated with CMV after being sprayed with 8 short peptides respectively; A is the symptom diagram of cucumbers infected with the virus; B is the result diagram of the accumulation level of virus protein in cucumber systemic leaves; Figure 3 The following are the disease incidence diagrams of N. benthamiana sprayed with 8 short peptides respectively after inoculation with TMV 10 days; A is the statistical diagram of the disease incidence survey results of N. benthamiana 10 days after inoculation with TMV; B is the statistical diagram of the disease index survey results of N. benthamiana 10 days after inoculation with TMV; Figure 4 The figures are the disease incidence diagrams of cucumbers sprayed with 8 short peptides respectively 15 days after inoculation with CMV; among them, A is the statistical graph of the incidence survey results of cucumbers 15 days after inoculation with CMV; B is the statistical graph of the disease index survey results of cucumbers 15 days after inoculation with CMV. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Example 1 Discovery of 8 short peptides After a large number of experiments, the inventors of the present invention found 8 short peptides from cucumbers, peppers, tomatoes and tobacco infected with virus diseases in the field. The specific steps are as follows: 1. Extraction of intercellular fluid protein of Nicotiana benthamiana (1) Take cucumber, pepper, tomato and tobacco leaves infected with virus diseases in the field, clean their surfaces with ddH2O, and dry the surface moisture with absorbent paper.

[0028] (2) After completing step (1), soak the cleaned leaves in Cockail containing 0.1% protease inhibitor and use a vacuum pump to completely immerse the solution into the leaves.

[0029] (3) After completing step (2), wipe the liquid on the surface of the leaf with absorbent paper, place it in a syringe (specification: 20 mL), and then place it in a centrifuge tube (specification: 50 mL). Centrifuge at 4°C and 1000 g for 1 min to 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 Take a centrifuge tube (2 mL in size), add 100 μL of Nicotiana benthamiana intercellular fluid protein and 200 μL of an aqueous solution containing 0.5% TFA and 0.5% acetonitrile to obtain a sample.

[0032] First, C was activated with 200 μL of an aqueous solution containing 0.1% TFA and 80% acetonitrile. 18 The desalting column was then equilibrated with 400-600 μL of an aqueous solution containing 0.1% TFA and 1% acetonitrile; the sample (the Nicotiana benthamiana intercellular fluid protein obtained in step "1. Extraction of Nicotiana benthamiana intercellular fluid protein") was then added to C 18 In the desalting column, allow the sample to flow slowly through the C 18 Desalting column, peptides were C 18 The desalting column is used to capture the non-hydrophobic small molecules such as salt, which are then discarded. Then, 200 μL of an aqueous solution containing 0.1% TFA and 0.5% acetonitrile is added to wash the C 18 Desalting column to wash away residual salts; finally, add 300 μL of aqueous solution containing 0.1% TFA and 80% acetonitrile, and let the liquid flow slowly through the C 18 Desalting column, eluting the polypeptide, collecting the elution solution in a new EP tube, and freeze-drying the eluate to obtain dry powder.

[0033] 3. LCMS / MS analysis of peptides The detection system was Easy-nLC 1200 (Thermo Scientic, P / N LC140) and Orbitrap Exploris 480 (Thermo Scientic, P / N BRE725533) from Thermo Scientific. The dry powder obtained in step "2, desalting" was dissolved in 10 μl of mobile phase A (0.1% formic acid in water), and then 5 μl was loaded. The peptides were captured on a column (PepMap C at a flow rate of 10 μL / min). 18, 100 μm × 2 cm) for 3 min, and then the peptides were analyzed on a nanoliter column (PepMap C 18 , 75μmx 25cm) was used for gradient elution chromatography. The separation gradient was from 5% to 30% of mobile phase B (acetonitrile solution containing 0.1% formic acid) within 60min. The chromatographic flow rate was 200nL / min, and the column temperature of the chromatographic column was 55°C. The ion source spray voltage was 2.0kV, the mass spectrometer heated capillary was set to 320°C, and the data-dependent mode was used to automatically switch between MS and MS / MS acquisition. The full scan MS used Orbitrap for primary scanning with a scanning range of m / z 100-1600 and a resolution of 70000 (at m / z 200). The maximum ion introduction time was 50ms, and the automatic gain control (AGC) was set to 5×10 5 Then, the top 15 parent ions that met the MS / MS fragmentation conditions were fragmented using high energy C-trap dissociation (HCD) and scanned using orbitrap, with the scan resolution set to 17500. The scan range was automatically controlled according to the parent ion mass-to-charge ratio, with the minimum scan range fixed at m / z = 100 and the maximum at 2000. The minimum ion intensity value for MS / MS was set to 13000. The maximum ion introduction time for MS / MS was 100 ms, and the AGC control was set to 2.0×10 5 The precursor ion selection window was set to 1.6 Da. MS / MS acquisition was performed for ions with charge numbers of 1, 2, 3, and 4, and dynamic exclusion was set to perform MS / MS once for each precursor ion within 10 seconds, followed by exclusion for 40 seconds and 30% collision energy.

[0034] 4. Database search and peptide identification The raw data obtained in step "3, LCMS / MS analysis of peptides" was processed and analyzed by PEAKS software. PEAKS can perform De Novo sequencing and protein identification (PEAKS DB). The parameters set for de novo sequencing and database retrieval are as follows: deep-sea actinomycete protein database, non-enzymatic digestion, primary mass spectrometry tolerance of 10ppm, secondary mass spectrometry tolerance of 0.02Da, no fixed modification, variable modification set to methionine oxidation, N-terminal acetylation, charge set to +1, +2, +3, +4. The false positive rate (FDR) of peptide identification was set to 1%. For the results of de novo sequencing, ALC (%) stands for average local confidence. The confidence of the results of de novo sequencing data is generally more reliable than 80%, and more reliable than 95%. Finally, 8 short peptides SJPep1-SJPep8 were found. The amino acid sequences of short peptides SJPep1-SJPep8 are shown in SEQ ID NO.1-SEQ ID NO.8, as follows: SJPep1: KHSGPSPSGDGH, SEQ ID NO.1; SJPep2: KHASGPSMRGPGH, SEQ ID NO.2; SJPep3:AVQSKPPSKRDPPKMQTD, SEQ ID NO.3; SJPep4:AVHSTPPSKRPPPKMQTD, SEQ ID NO.4; SJPep5:EAHLDY(SO3H)IY(SO3H)TQHHNHP, SEQ ID NO.5; SJPep6: HLDY(SO3H)IY(SO3H)TQSINNNHP, SEQ ID NO.6; SJPep7:ECLMRRTLEAHLDY(SO3H)IY(SO3H)TQRHK, SEQ ID NO.7; SJPep8: CLKRRMVAEAHLDY(SO3H)IY(SO3H)TQHKPK, SEQ ID NO.8; Among them, the structural formula of Y(SO3H)IY(SO3H)TQ (SEQ ID NO.9) is [H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH].

[0035] Example 2 Application of 8 short peptides SJPep1-SJPep8 in plant antiviral Preparation of short peptide SJPep1-SJPep8 diluents: The eight short peptides SJPep1-SJPep8 in this example were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The eight short peptide SJPep1-SJPep8 diluents were prepared by diluting the short peptide SJPep1-SJPep8 with sterile water, and the concentration of the short peptide SJPep1-SJPep8 in the short peptide SJPep1-SJPep8 diluents was 300 μg / mL.

[0036] 1. Take 30 Nicotiana benthamiana seedlings grown to 3 weeks old and randomly divide them into 2 groups, the experimental group and the control group, with 15 Nicotiana benthamiana seedlings in each group, and set 3 replicates. Take 30 cucumber seedlings grown to 3 weeks old and randomly divide them into 2 groups, the experimental group and the control group, with 15 cucumber seedlings in each group, and set 3 replicates. Then, the above groups are treated as follows: Experimental group: Spray the whole plant of Nicotiana benthamiana seedlings or cucumber seedlings with one of the dilutions of the short peptide SJPep1-SJPep8 until the whole plant is moistened, which are recorded as SJPep1-SJPep8. Then, the short peptide dilution is sprayed again at intervals of 3-5 days, for a total of 3 times.

[0037] Control group: Spray the entire plant of Nicotiana benthamiana or cucumber seedlings with sterile water until the entire plant is moistened, which is recorded as CK. Then, spray sterile water again after an interval of 3-5 days, for a total of 3 times.

[0038] 2. After completing step 1, inoculate TMV virus (tobacco mosaic virus) or CMV virus (cucumber mosaic virus) by Agrobacterium infiltration. The specific steps are as follows: (1) A TMV-GFP infectious clone (provided by the Institute of Plant Virology, Ningbo University, and disclosed in the document "Agrobacterium inoculation as a simple method for infection of plant virus vectors" (Jia Hongge, Pang Yongqi, Fang Rongxiang. Agrobacterium inoculation as a simple method for infection of plant virus vectors (English) [J]. Acta Botanica Sinica, 2003, (07): 770-773.), in which the TMV-GFP infectious clone in the present invention is named Agrobacterium containing p35S-30B::GFP in the document, and is promised to be released to the public for 20 years) was inoculated into 10 mL of YEP liquid culture medium (containing 100 μg / mL kanamycin sulfate and 20 μg / mL rifampicin), cultured with shaking at 28°C for 12-16 h, and then centrifuged at 10,000 rpm for 1 min to collect the precipitate. Then resuspend it with 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) and dilute to obtain OD 600nm It is 0.0004 TMV virus solution.

[0039] According to the above steps, the TMV-GFP infectious clone was replaced with the CMV-GFP infectious clone (provided by the Institute of Plant Virology, Ningbo University, and published in the document "Construction of the Trans-Genetic Replication System of Cucumber Mosaic Virus" (Wang Lin, Cheng Xiaodong, Lu Ran, et al. Construction of the Trans-Genetic Replication System of Cucumber Mosaic Virus [J]. Journal of Zhejiang Sci-Tech University (Natural Science Edition), 2020, 43(02): 262-266.), specifically, the Agrobacterium containing plasmid pCB301-R1, Agrobacterium containing pCB301-R2, and Agrobacterium containing pCB301-R3-gfp in the document were mixed in a volume ratio of 1:1:1, and promised to be released to the public for 20 years), and the OD was obtained. 600nm It is 0.06 CMV virus liquid.

[0040] (2) Take the Nicotiana benthamiana seedlings and cucumber seedlings that have completed step 1 and inject 0.2 mL of virus solution (TMV-GFP virus solution or CMV-GFP virus solution) into the plant leaves using a sterile syringe. The injection area is about 1 cm 2 The injection location is the second whorl of leaves of Nicotiana benthamiana seedlings or the cotyledons of cucumber seedlings.

[0041] 3. After completing step 2, culture the Nicotiana benthamiana seedlings and cucumber seedlings at 25°C for 10-15 days.

[0042] 4. After completing step 3, observe the virus infection (TMV-GFP can observe the virus fluorescence under UV light, but because the fluorescence of CMV-GFP is not obvious, the virus fluorescence was not observed under UV light, and the bright field symptom photos were taken) and phenotype. The observation results are as follows: Figure 1 A and Figure 2 As shown in A. The results showed that compared with the control group, the fluorescence accumulation of tobacco virus in the experimental group was significantly less, the height of cucumber seedlings in the experimental group was higher, and the virus infection symptoms were significantly weaker. It can be seen that the application of short peptides SJPep1-SJPep8 can reduce the accumulation of tobacco virus and alleviate the symptoms of virus infection.

[0043] 5. After completing step 3, perform Western-blotting on N. benthamiana seedlings and cucumber seedlings, and calibrate the sample loading using Ponceau Staining (PSS) or Coomassie Brilliant Blue Staining (CBB). The specific steps are as follows: (1) Extraction of total protein After completing step 3, randomly select inoculated leaves or system leaves (system leaves are the upper leaves of inoculated leaves) of 3 seedlings of the control group and the experimental group (Nicotiana benthamiana seedlings and cucumber seedlings), grind them into powder in liquid nitrogen; then take 0.2g of powder in a 2mL sterile centrifuge tube, add 0.2mL of cell lysis buffer (containing 200mM NaCl, 1.0mM EDTA, 1.0mM DTT (pH 7.4) and 20mM Tris-HCl buffer), vortex mix, and stand on ice for 20min; then centrifuge at 4℃ and 14000g for 10min, collect the supernatant; mix 0.2mL of supernatant with 50μL of loading buffer (GenScript, Beijing) in a 1.5mL sterile centrifuge tube to obtain a mixture; boil the mixture in a boiling water bath for 5min, then stand on ice for 5min, and centrifuge at 4℃ and 14000g for 10min to obtain a treated protein mixed sample.

[0044] (2) Western blotting (2-1) The treated protein mixed sample obtained in step (1) was electrophoresed in SDS-PAGE gel (GenScript, Beijing) until the marker (Takara, Shanghai) showed the separation of the target band (GFP molecular weight is about 27 kD).

[0045] (2-2) When using the Coomassie Brilliant Blue staining method (CBB) to calibrate the sample loading amount, two SDS-PAGE gels with the same sample loading in step (2-1) should be electrophoresed, one for transfer and the other for Coomassie Brilliant Blue staining. The gel to be stained should be stained using a protein stainer (GenScript, Beijing). After staining, remove the SDS-PAGE gel and take a photo.

[0046] (2-3) After completing step (2-1), transfer the membrane. Before transfer, soak the nitrocellulose membrane in transfer buffer, and place the sponge pad, protein gel, nitrocellulose membrane, and sponge pad in the transfer apparatus (GenScript, Beijing) from the negative electrode to the positive electrode, taking care to remove the bubbles in the gap. Transfer the membrane at a constant current of 0.3A for 14 minutes.

[0047] (2-4) After the transfer, place the nitrocellulose membrane in the prepared Ponceau stain solution (the preparation method of 1L Ponceau stain solution is as follows: mix 0.1g Ponceau, 5mL acetic acid and 95mL ddH2O, and then dilute to 1L with ddH2O), shake on a horizontal shaker until obvious red bands appear, discard the Ponceau, and wash 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 the consistency of the sample loading amount, and take a photo. Wash the membrane with pure water again until the red bands almost disappear, place the membrane in 5% (m / V) skim milk prepared with 1× PBS, and block it on a horizontal shaker for 1h.

[0048] (2-5) After blocking, discard the blocking solution and wash the membrane three times with 1× PBS, each time for 10 min; add 5% (m / V) skim milk containing GFP antibody, the ratio of antibody to skim milk is 1:40,000, and incubate on a horizontal shaker for 1 h; discard the primary antibody and wash the membrane three times with 1× PBS, each time for 10 min; add 5% (m / V) skim milk containing the corresponding secondary antibody, the ratio of antibody to skim milk is 1:40,000, and incubate on a horizontal shaker for 1 h; discard the secondary antibody and wash the membrane three times with 1× PBS, each time for 10 min.

[0049] (2-6) Discard PBS, place the membrane in a color development bag, add color development solution, take it out after the color development solution is evenly distributed, develop in the dark, and expose it in the AI600 instrument. The test results are as follows: Figure 1 B and Figure 2 As shown in B. The results showed that the accumulation of virus in the experimental group was significantly reduced compared with the control group.

[0050] 6. After completing step 3, statistically analyze the incidence of each treatment group and calculate the disease index and prevention and treatment effect.

[0051] (1) Virus disease classification standards are based on the tobacco virus disease severity classification standards (national standard GB / T23222-2008) and are graded and investigated on a strain-by-strain basis. The details are as follows: Level 0: The whole plant is disease-free; Level 1: The heart leaf veins are clear or the leaves are mosaic and twisted, and the diseased plants have no obvious dwarfing; Level 3: 1 / 3 of the leaves are mosaic or twisted, or the diseased plant is dwarfed to more than 3 / 4 of the normal plant height; Level 5: 1 / 3 to 1 / 2 of the leaves are mosaic, or a few leaves are deformed, or the main veins turn black, or they wilt slightly, or the diseased plants are dwarfed to 2 / 3 to 3 / 4 of the normal plant height; Level 7: 1 / 2 to 2 / 3 of the leaves are mosaic, or deformed or the main and lateral veins are necrotic, wilting, and the diseased plants are stunted to 1 / 2 to 2 / 3 of the normal plant height; Level 9: The leaves of the entire plant are mosaic, severely deformed, wilted or necrotic, or the diseased plant is shrunk to more than 1 / 2 of the normal plant height.

[0052] (2) The protective effect of each short peptide against TMV or CMV Calculate according to the following formula.

[0053] Disease index = 100 × ∑ (number of diseased plants at each level × relative level) / (total number of plants investigated × 9); Relative protective effect (%) = 100 × (disease index of control group - disease index of treatment group) / disease index of control group.

[0054] The morbidity survey of each treatment group after 10 days of inoculation of TMV virus in Nicotiana benthamiana seedlings is as follows Figure 3As shown in A. The results showed that the incidence of TMV decreased by 69.77% after SJPep1 treatment, 62.79% after SJPep2 and SJPep5 treatment, 65.12% after SJPep3 and SJPep8 treatment, 72.09% after SJPep4 treatment, and 58.14% after SJPep6 and SJPep7 treatment.

[0055] The disease index of each treatment group 10 days after the tobacco seedlings were inoculated with TMV virus is as follows Figure 3 As shown in B, the disease index can be used to obtain the relative protective efficacy of each short peptide against TMV virus. The results showed that the relative protective efficacy of SJPep1 against TMV was 70.59%, the relative protective efficacy of SJPep2 against TMV was 63.64%, the relative protective efficacy of SJPep3 against TMV was 65.24%, the relative protective efficacy of SJPep4 against TMV was 79.68%, the relative protective efficacy of SJPep5 against TMV was 62.57%, the relative protective efficacy of SJPep6 against TMV was 60.43%, the relative protective efficacy of SJPep7 against TMV was 59.36%, and the relative protective efficacy of SJPep8 against TMV was 60.96%.

[0056] The incidence of each treatment group was investigated 15 days after the cucumber seedlings were inoculated with CMV virus. Figure 4 As shown in A. The results showed that the incidence of CMV decreased by 69.05% after SJPep1 treatment, 64.29% after SJPep2 treatment, 52.38% after SJPep3 treatment, 61.90% after SJPep4 treatment, 59.52% after SJPep5 treatment, 54.76% after SJPep6 treatment, 57.14% after SJPep7 treatment, and 59.52% after SJPep8 treatment.

[0057] The disease index of each treatment group 15 days after cucumber seedlings were inoculated with CMV virus is as follows Figure 4 As shown in B, the disease index can be used to obtain the relative protective efficacy of each short peptide against CMV virus. The results showed that the relative protective efficacy of SJPep1 against CMV was 71.35%, the relative protective efficacy of SJPep2 against CMV was 66.15%, the relative protective efficacy of SJPep3 against CMV was 55.21%, the relative protective efficacy of SJPep4 against CMV was 63.54%, the relative protective efficacy of SJPep5 against CMV was 63.02%, the relative protective efficacy of SJPep6 against CMV was 59.90%, the relative protective efficacy of SJPep7 against CMV was 61.46%, and the relative protective efficacy of SJPep8 against CMV was 64.06%.

[0058] In summary, the short peptides SJPep1-SJPep8 can inhibit TMV infection when sprayed on Nicotiana benthamiana exogenously; the short peptides SJPep1-SJPep8 can inhibit CMV infection when sprayed on cucumber exogenously. That is, the short peptides SJPep1-SJPep8 can inhibit virus infection (such as TMV and CMV) to a certain extent.

[0059] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A short peptide capable of improving the antiviral ability of plants, characterized in that: The amino acid sequence of the short peptide is any one or more of the sequences described in SEQ ID NO.1 to SEQ ID NO.8; The virus is tobacco mosaic virus and / or cucumber mosaic virus.

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

3. Use of the short peptide according to claim 1 or the nucleic acid molecule according to claim 2 in the preparation of a product for improving the antiviral ability of plants, characterized in that: The virus is tobacco mosaic virus and / or cucumber mosaic virus.

4. The use according to claim 3, characterized in that: The products include viral inhibitors or antiviral drugs.

5. A virus inhibitor, characterized in that: The virus inhibitor comprises the short peptide according to claim 1; the virus is tobacco mosaic virus and / or cucumber mosaic virus.

6. The virus inhibitor according to claim 5, characterized in that The viral inhibitor also includes auxiliary materials.

7. Use of the short peptide according to claim 1, the nucleic acid molecule according to claim 2 or the virus inhibitor according to claim 5 or 6 in inhibiting virus infection, characterized in that: The virus is tobacco mosaic virus and / or cucumber mosaic virus.

8. A method for inhibiting plant virus infection, characterized in that: The method comprises the step of spraying the plants to be treated with the short peptide according to claim 1 or the virus inhibitor according to claim 5 or 6; the virus is tobacco mosaic virus and / or cucumber mosaic virus.

9. Use of the short peptide according to claim 1, the nucleic acid molecule according to claim 2, or the virus inhibitor according to claim 5 or 6 in improving the antiviral ability of plants, characterized in that: The virus is tobacco mosaic virus and / or cucumber mosaic virus.

10. A method for improving plant antiviral ability, characterized in that: The method comprises the step of spraying the plants to be treated with the short peptide according to claim 1 or the virus inhibitor according to claim 5 or 6; the virus is tobacco mosaic virus and / or cucumber mosaic virus.

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