A short peptide capable of enhancing the antiviral ability of plants and its application

Abstract

The present invention relates to the field of agricultural biology, and particularly to a short peptide capable of improving the antiviral ability of plants and its application. The present invention provides a short peptide capable of improving the antiviral ability of plants, and the amino acid sequence of the short peptide is any one or several of the sequences as set forth in SEQ ID NO.1 - SEQ ID NO.8. Through a large number of experiments, the inventors extracted 8 short peptides from cucumber, pepper, tomato and tobacco plants infected with virus diseases in the field; then, after externally spraying the 8 short peptides on the leaves of Nicotiana benthamiana and inoculating with virus TMV, the results showed that the external spraying of the 8 short peptides was not conducive to the infection of TMV; after externally spraying the 8 short peptides on the leaves of cucumber and inoculating with virus CMV, the results showed that the external spraying of the 8 short peptides was not conducive to the infection of CMV, indicating that these 8 short peptides can inhibit the infection of virus TMV and CMV to a certain extent.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biology, and particularly to a short peptide capable of improving the antiviral ability of plants and its application. Background Art

[0002] Plant virus diseases are the second largest category of plant diseases after plant fungal diseases. They have strong specificity, a wide variety, are mostly transmitted by insects, have a fast transmission speed, a high virus mutation rate, and are difficult to control. In recent years, the economic losses caused by plant virus infections in China's agricultural production have reached as high as $60 billion, seriously endangering the growth and development of crops and their economic value. The occurrence of plant virus diseases is affected by various factors, such as strain types, host plants, transmission vectors, environmental conditions, and human operations. The methods for controlling plant virus diseases are still being explored, and there is currently no specific medicine for virus diseases. At present, in agricultural production, comprehensive methods for controlling virus diseases are widely used, including plant quarantine, resistant varieties, crop rotation, control of transmission vectors (aphids, etc.), but the harm caused by virus diseases still cannot be completely solved. Therefore, using certain means to activate the plant's own immune response remains an important research direction for future plant virus disease control.

[0003] Plant small signaling peptides (SSPs), as novel and important signaling molecules, can not only transmit information over short distances between cells but also participate in the regulation process of plant responses to abiotic stress signals through long-distance information transmission. There are some plant endogenous secreted peptides in plants. They 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 secreted peptides can also regulate plant immunity through the hormone pathway. However, at present, the functional research on plant endogenous short peptides in plant antiviral responses is still very limited. Summary of the Invention

[0004] The object of the present invention is to provide a short peptide capable of improving the antiviral ability of plants and its application to solve the problems existing in the above-mentioned prior art. The present invention extracts 8 short peptides from cucumber, pepper, tomato, and tobacco plants infected with virus diseases in the field. After exogenous spraying of these 8 short peptides and then inoculating with the virus, the effects of each short peptide on plant antiviral diseases were studied, and it was found that these 8 short peptides can all enhance the resistance of plants to the virus and can be used for the preparation of antiviral products.

[0005] To achieve the above object, the present invention provides the following solution:

[0006] The present invention provides a short peptide capable of improving the antiviral ability of plants, and the amino acid sequence of the short peptide is any one or several of the sequences as set forth in SEQ ID NO.1 - SEQ ID NO.8;

[0007] The virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0008] The present invention provides a nucleic acid molecule encoding the above-mentioned short peptide.

[0009] The present invention provides the use of the above-mentioned short peptide or the above-mentioned nucleic acid molecule in the preparation of a product for improving the antiviral ability of plants, wherein the virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0010] Preferably, the product includes a virus inhibitor or an antiviral drug.

[0011] The present invention provides a virus inhibitor, which includes the above-mentioned short peptide; the virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0012] Preferably, the virus inhibitor further includes excipients.

[0013] The present invention provides the use of the above-mentioned short peptide, the above-mentioned nucleic acid molecule or the above-mentioned virus inhibitor in inhibiting virus infection, wherein the virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0014] The present invention provides a method for inhibiting plant virus infection, which includes the step of spraying the short peptide or the virus inhibitor on the plants to be treated; the virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0015] More preferably, the spraying site is the leaves of the plants to be treated.

[0016] More preferably, the plants to be treated are tobacco and cucumber.

[0017] The present invention provides the use of the above-mentioned short peptide, the above-mentioned nucleic acid molecule or the above-mentioned virus inhibitor in improving the antiviral ability of plants, wherein the virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0018] The present invention provides a method for improving the antiviral ability of plants, which includes the step of spraying the short peptide or the virus inhibitor on the plants to be treated; the virus is tobacco mosaic virus and / or cucumber mosaic virus.

[0019] More preferably, the spraying site is the leaves of the plants to be treated.

[0020] More preferably, the plants to be treated are tobacco and cucumber.

[0021] The present invention discloses the following technical effects:

[0022] The present invention provides a short peptide capable of enhancing the antiviral ability of plants, and the amino acid sequence of the short peptide is any one or several of the sequences described in SEQ ID NO.1 - SEQ ID NO.8. Through 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, after externally spraying the 8 short peptides on the leaves of Nicotiana benthamiana respectively and inoculating with virus TMV, the results showed that the external spraying of the 8 short peptides was not conducive to the infection of TMV; after externally spraying the 8 short peptides on the leaves of cucumber respectively and inoculating with virus CMV, the results showed that the external spraying of the 8 short peptides was not conducive to the infection of CMV, indicating that these 8 short peptides can inhibit the infection of virus TMV and CMV to a certain extent. In summary, the results of the specific embodiments of the present invention show that by externally spraying short peptides on plants, the defense response of plants against viral diseases can be activated and the infection of viruses to plants can be inhibited. Therefore, these 8 short peptides can all be used to prepare antiviral products to inhibit the infection of viruses to plants; moreover, compared with chemical pesticides, the short peptides provided by the present invention are non-toxic, pollution-free, highly efficient and convenient, and have important application values. Brief Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 Diagram of the infection situation of Nicotiana benthamiana inoculated with TMV after externally spraying 8 short peptides respectively; among them, A is the statistical chart of the fluorescence accumulation of the virus in the systemic leaves of Nicotiana benthamiana; B is the result chart of the accumulation level of the virus protein in the systemic leaves of Nicotiana benthamiana;

[0025] Figure 2 Diagram of the infection situation of cucumber inoculated with CMV after externally spraying 8 short peptides respectively; among them, A is the symptom diagram of cucumber infected with the virus; B is the result chart of the accumulation level of the virus protein in the systemic leaves of cucumber;

[0026] Figure 3 Diagram of the disease situation of Nicotiana benthamiana inoculated with TMV 10 days after externally spraying 8 short peptides respectively; among them, A is the statistical chart of the incidence rate investigation result of Nicotiana benthamiana inoculated with TMV for 10 days; B is the statistical chart of the disease index investigation result of Nicotiana benthamiana inoculated with TMV for 10 days;

[0027] Figure 4The figure shows the disease incidence of cucumbers sprayed with 8 different exogenous short peptides after inoculation with CMV for 15 days. Among them, A is the statistical chart of the disease incidence of cucumbers after inoculation with CMV for 15 days, and B is the statistical chart of the disease index of cucumbers after inoculation with CMV for 15 days. Detailed implementation manners

[0028] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0029] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0031] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0032] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0033] Example 1 Discovery of 8 short peptides

[0034] Through a large number of experiments, the inventors of the present invention discovered 8 short peptides from cucumbers, peppers, tomatoes, and tobacco plants infected with virus diseases in the field. The specific steps are as follows:

[0035] 1. Extraction of intercellular fluid proteins from cucumber, pepper, tomato, and tobacco leaves

[0036] (1)Take cucumber, pepper, tomato, and tobacco leaves infected with virus disease in the field, wash the surface with ddH2O, and blot the surface moisture with absorbent paper.

[0037] (2)After completing step (1), soak the washed leaves in Cockail containing 0.1% protease inhibitor, and use a vacuum pump to completely immerse the solution into the leaves.

[0038] (3)After completing step (2), blot the liquid on the leaf surface dry with absorbent paper, place it in a syringe (specification: 20 mL), then place it in a centrifuge tube (specification: 50 mL), centrifuge at 4°C and 1000 g for 1 min, and collect the liquid phase.

[0039] (4)Add the liquid phase collected in step (3) to a MerckMillipore ultrafiltration tube (specification: 0.5 mL, 3KDa), centrifuge at 4°C and 6000 g for 1 min; collect the effluent in a new centrifuge tube (specification: 2 mL), then use a freeze-concentration dryer to concentrate the liquid to a dry powder; finally, dissolve it with 200 μL of Cockail containing 0.1% protease inhibitor to obtain the intercellular fluid proteins of cucumber, pepper, tomato, and tobacco leaves.

[0040] 2. Desalting

[0041] Take a centrifuge tube (specification: 2 mL), add 100 μL of the intercellular fluid proteins of cucumber, pepper, tomato, and tobacco leaves and 200 μL of an aqueous solution containing 0.5% TFA and 0.5% acetonitrile to obtain a sample.

[0042] First, activate the C 18 desalting column with 200 μL of an aqueous solution containing 0.1% TFA and 80% acetonitrile, and then balance the desalting column with 400 - 600 μL of an aqueous solution containing 0.1% TFA and 1% acetonitrile; then add the sample (the intercellular fluid proteins of cucumber, pepper, tomato, and tobacco leaves obtained in step "1. Extraction of intercellular fluid proteins of cucumber, pepper, tomato, and tobacco leaves") to the C 18 desalting column, and let the sample slowly flow through the C 18 desalting column. The polypeptide is trapped by the C 18 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 wash the C 18 desalting column to wash away the residual salts; finally, add 300 μL of an aqueous solution containing 0.1% TFA and 80% acetonitrile, and let the liquid slowly flow through the C 18 desalting column to elute the polypeptide, collect the elution solution with a new EP tube, and freeze-dry the eluate to obtain a dry powder.

[0043] 3. LCMS / MS analysis of polypeptides

[0044] The detection system is a combination of Easy-nLC 1200 (Thermo Scientic, P / N LC140) and Orbitrap Exploris 480 (Thermo Scientic, P / N BRE725533) from Thermoelectric Company. The dried powder obtained in step "2. Desalting" was dissolved with 10 μL of mobile phase A (0.1% formic acid aqueous solution), and then 5 μL was injected. The peptides were trapped by a trapping column (PepMap C 18 , 100 μm × 2 cm) for 3 min at a flow rate of 10 μL / min. Subsequently, the peptides were subjected to gradient elution chromatography separation on a nano-scale analytical column (PepMap C 18 , 75 μm x 25 cm). The separation gradient was that mobile phase B (acetonitrile solution containing 0.1% formic acid) increased from 5% to 30% within 60 min. The chromatographic flow rate was 200 nL / min, and the column temperature of the chromatographic column was 55 °C. The spray voltage of the ion source was 2.0 kV, the heated capillary of the mass spectrometer was set at 320 °C, and data-dependent mode was used to automatically switch between MS and MS / MS for acquisition. Full-scan MS was performed with Orbitrap for the first-level scan, the scan range was m / z 100 - 1600, and the resolution was set at 70000 (at m / z 200). The maximum ion injection time was 50 ms, and the automatic gain control (AGC) was set at 5×10 5 . Subsequently, higher energy C-trap dissociation (HCD) was used to fragment the top 15 precursor ions that met the tandem (MS / MS) fragmentation conditions and scanned with orbitrap. The scan resolution was set at 17500. The scan range was automatically controlled according to the precursor ion mass-to-charge ratio, the lowest scan range was fixed at m / z = 100, and the highest reached 2000. The minimum ion intensity value for MS / MS was set at 13000. The maximum ion injection time for MS / MS was 100 ms, the AGC control was set at 2.0×10 5 , and the precursor ion selection window was set at 1.6 Da. MS / MS acquisition was performed for ions with 1, 2, 3, and 4 charges, and dynamic exclusion was set to perform MS / MS on each precursor ion once within 10 s, and then excluded for 40 s with 30% collision energy.

[0045] 4. Database Search and Peptide Identification

[0046] The raw data obtained from the step of "3. LCMS / MS analysis of polypeptides" was processed and the spectra were analyzed using PEAKS software. PEAKS can perform De Novo sequencing and protein identification (PEAKS DB). The parameters were set as follows when performing de novo sequencing and database searching: deep-sea actinomycete protein database, non-enzymatic digestion, the tolerance of the first-level mass spectrometry was 10 ppm, the tolerance of the second-level mass spectrometry was 0.02 Da, there was no fixed modification, the variable modification was set to methionine oxidation and N-terminal acetylation, and the charge was set to +1, +2, +3, +4. The false discovery rate (FDR) of peptide identification was set to 1%. For the results of de novo sequencing, the full name of ALC (%) is average local confidence, which is the confidence of the de novo sequencing data results. Generally, a confidence greater than 80% is more credible, and a confidence greater than 95% is very credible. Finally, 8 short peptides SJPep1 - SJPep8 were found. The amino acid sequences of the short peptides SJPep1 - SJPep8 are shown in SEQ ID NO.1 - SEQ ID NO.8, as follows:

[0047] SJPep1: KHSGPSPSGDGH, SEQ ID NO.1;

[0048] SJPep2: KHASGPSMRGPGH, SEQ ID NO.2;

[0049] SJPep3: AVQSKPPSKRDPPKMQTD, SEQ ID NO.3;

[0050] SJPep4: AVHSTPPSKRPPPKMQTD, SEQ ID NO.4;

[0051] SJPep5: EAHLDY(SO3H)IY(SO3H)TQHHNHP, SEQ ID NO.5;

[0052] SJPep6: HLDY(SO3H)IY(SO3H)TQSINNNHP, SEQ ID NO.6;

[0053] SJPep7: ECLMRRTLEAHLDY(SO3H)IY(SO3H)TQRHK, SEQ ID NO.7;

[0054] SJPep8: CLKRRMVAEAHLDY(SO3H)IY(SO3H)TQHKPK, SEQ ID NO.8;

[0055] 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].

[0056] Example 2 Application of 8 short peptides SJPep1 - SJPep8 in plant antiviral

[0057] Preparation of the dilutions of short peptides SJPep1 - SJPep8: The 8 short peptides SJPep1 - SJPep8 in this example were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The dilutions of the 8 short peptides SJPep1 - SJPep8 were obtained by diluting the short peptides SJPep1 - SJPep8 with sterile water, and the concentration of the short peptides SJPep1 - SJPep8 in the dilutions of the short peptides SJPep1 - SJPep8 was 300 μg / mL for each.

[0058] 1. Take 30 Nicotiana benthamiana seedlings grown for 3 weeks, randomly divide them into 2 groups, namely the experimental group and the control group, with 15 Nicotiana benthamiana seedlings in each group, and set 3 replicates. Take 30 cucumber seedlings grown for 3 weeks, randomly divide them into 2 groups, namely the experimental group and the control group, with 15 cucumber seedlings in each group, and set 3 replicates. Then, the above groups were treated as follows:

[0059] Experimental group: Use one of the dilutions of the short peptides SJPep1 - SJPep8 to spray the whole Nicotiana benthamiana seedlings or cucumber seedlings until the whole plant is wet, and record them as SJPep1 - SJPep8 in sequence. Then spray the short peptide dilution again after an interval of 3 - 5 days, and spray a total of 3 times.

[0060] Control group: Use sterile water to spray the whole Nicotiana benthamiana seedlings or cucumber seedlings until the whole plant is wet, and record it as CK. Then spray sterile water again after an interval of 3 - 5 days, and spray a total of 3 times.

[0061] 2. After completing step 1, inoculate TMV virus (Tobacco mosaic virus) or CMV virus (Cucumber mosaic virus) by the method of agroinfiltration. The specific steps are as follows:

[0062] (1) Inoculate the TMV-GFP infectious clone (provided by the Institute of Plant Virology, Ningbo University, publicly available in the literature "Agrobacterium-mediated inoculation as a simple method for infecting plant virus vectors" (Jia Hongge, Pang Yongqi, Fang Rongxiang. Agrobacterium-mediated inoculation as a simple method for infecting plant virus vectors (English) [J]. Acta Botanica Sinica, 2003, (07): 770-773.). In this invention, the name of the TMV-GFP infectious clone in this literature is Agrobacterium containing p35S-30B∶∶GFP, and it is promised to be distributed externally for 20 years) into 10 mL of YEP liquid medium (containing 100 μg / mL kanamycin sulfate and 20 μg / mL rifampicin), culture with shaking at 28 °C for 12 - 16 h, then centrifuge at 10000 rpm for 1 min to collect the precipitate. Then resuspend with tobacco infiltration solution (10 mL of tobacco infiltration solution consists of 1 mL of MES solution with a concentration of 100 mM, 0.1 mL of MgCl2 solution with a concentration of 1 M, 20 μL of As solution with a concentration of 2 mM, and 8.88 mL of ddH2O), and dilute to obtain a TMV virus solution with an OD 600nm of 0.0004.

[0063] According to the above steps, replace the TMV-GFP infectious clone with the CMV-GFP infectious clone (provided by the Institute of Plant Virology, Ningbo University, publicly available in the literature "Construction of a reverse genetic replication system for Cucumber mosaic virus" (Wang Lin, Cheng Xiaodong, Lu Ran, et al. Construction of a reverse genetic replication system for Cucumber mosaic virus [J]. Journal of Zhejiang Sci-Tech University (Natural Science Edition), 2020, 43(02): 262-266.). Specifically, mix the Agrobacterium containing plasmid pCB301-R1, Agrobacterium containing pCB301-R2, and Agrobacterium containing pCB301-R3-gfp in the literature according to a volume ratio of 1:1:1, and it is promised to be distributed externally for 20 years), to obtain a CMV virus solution with an OD 600nm of 0.06.

[0064] (2) Take the Nicotiana benthamiana seedlings and cucumber seedlings that have completed step 1, and use a sterile syringe to inject 0.2 mL of the virus solution (TMV-GFP virus solution or CMV-GFP virus solution) into the plant leaves, with an injection area of approximately 1 cm 2 , and the injection position is the second round of leaves of Nicotiana benthamiana seedlings or the cotyledons of cucumber seedlings.

[0065] 3. After completing step 2, place the Nicotiana benthamiana seedlings and cucumber seedlings at 25 °C for 10 - 15 days respectively.

[0066] 4. After completing step 3, observe the virus infection situation (TMV-GFP can observe virus fluorescence under UV light. Since the fluorescence of CMV-GFP is not obvious, the virus fluorescence was not observed under UV light, and bright-field symptom photos were taken) and phenotypes. The observation results are as shown in Figure 1 A in Figure 2 and A in

[0067] shown. The results show that: compared with the control group, the fluorescence accumulation of tobacco virus in the experimental group is significantly less, the plant height of cucumber seedlings in the experimental group is higher, and the virus infection symptoms are significantly weaker. Thus, it can be seen that after applying short peptides SJPep1-SJPep8, the accumulation of tobacco virus can be reduced and the virus infection symptoms can be alleviated.

[0068] 5. After completing step 3, perform Western-blotting on Nicotiana benthamiana seedlings and cucumber seedlings respectively, and calibrate the sample loading amount using Ponceau S staining method (PSS) or Coomassie Brilliant Blue staining method (CBB). The specific steps are as follows:

[0069] (1) Extract total proteins

[0070] After completing step 3, randomly take 3 seedlings (Nicotiana benthamiana seedlings and cucumber seedlings) from the control group and the experimental group respectively, and grind the inoculated leaves or systemic leaves (systemic leaves are the upper leaves of the inoculated leaves) into powder in liquid nitrogen; then take 0.2 g of the powder into a 2 mL sterilized centrifuge tube, add 0.2 mL of cell lysis buffer (containing 200 mM NaCl, 1.0 mM EDTA, 1.0 mM DTT (pH 7.4) and 20 mM Tris-HCl buffer), vortex and mix well, and let it stand on ice for 20 min; then centrifuge at 4 °C and 14,000 g for 10 min to collect the supernatant; mix 0.2 mL of the supernatant with 50 μL of sample loading buffer (GenScript, Beijing) in a 1.5 mL sterilized centrifuge tube to obtain a mixture; boil the mixture in a water bath for 5 min, then let it stand on ice for 5 min, and centrifuge at 4 °C and 14,000 g for 10 min to obtain the processed protein mixed sample.

[0070] (2) Western-blotting

[0071] (2-1) Electrophorese the processed protein mixed sample obtained in step (1) in an SDS-PAGE gel (GenScript, Beijing) until the target band separation is shown by the marker (Takara, Shanghai) (the molecular weight of GFP is about 27 kD).

[0072] When calibrating the sample loading amount using Coomassie Brilliant Blue staining (CBB) in step (2-2), it is necessary to electrophorese two SDS-PAGE gels with the same sample loading in step (2-1). One is for membrane transfer and the other is for Coomassie Brilliant Blue staining. Stain the gel to be stained using a protein staining instrument (GenScript, Beijing). After staining, take out the SDS-PAGE gel and take a photo.

[0073] After completing step (2-1), perform membrane transfer. Soak the nitrocellulose membrane in the membrane transfer buffer before membrane transfer. Place a sponge pad, a protein gel, a nitrocellulose membrane, and a sponge pad from the negative electrode to the positive electrode in the membrane transfer instrument (GenScript, Beijing). Pay attention to expelling the air bubbles in the gap, and transfer the membrane at a constant current of 0.3 A for 14 minutes.

[0074] After the membrane transfer is completed, place the nitrocellulose membrane in the pre-prepared Ponceau S staining solution (the preparation method of 1 L of Ponceau S staining solution is as follows: mix 0.1 g of Ponceau S, 5 mL of acetic acid, and 95 mL of ddH2O, and then make up the volume to 1 L with ddH2O). Shake it on a horizontal shaker until obvious red bands appear. Discard the Ponceau S, and then 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 that the sample loading amount is consistent, and take a photo. Then wash the membrane with pure water 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 1 h.

[0075] After blocking, discard the blocking solution, wash the membrane three times with 1×PBS, 10 minutes each time; add 5% (m / V) skim milk containing the GFP antibody, and the ratio of the antibody to skim milk is 1:40000. Incubate it on a horizontal shaker for 1 h; discard the primary antibody, wash the membrane three times with 1×PBS, 10 minutes each time; add 5% (m / V) skim milk containing the corresponding secondary antibody, and the ratio of the antibody to skim milk is 1:40000. Incubate it on a horizontal shaker for 1 h; then discard the secondary antibody, wash the membrane three times with 1×PBS, 10 minutes each time.

[0076] Discard the PBS, put the membrane into a developing bag, add the developing solution. After the developing solution is evenly distributed, take it out and develop it in the dark. Perform exposure on the AI600 instrument, and the detection results are as shown in B in Figure 1 and B in Figure 2 . The results show that compared with the control group, the accumulation amount of the virus in the experimental group is significantly reduced.

[0077] 6. After completing step 3, count the disease incidence of each group of treatments respectively, and calculate the disease index and control effect.

[0078] (1)The grading standard for virus diseases was investigated by grading in units of plants according to the grading standard for the severity of tobacco virus diseases (National Standard GB / T 23222-2008), as follows:

[0079] Grade 0: The whole plant is disease-free;

[0080] Grade 1: The veins of the heart leaf are clear or mosaic, distorted, and the diseased plant shows no obvious dwarfing;

[0081] Grade 3: 1 / 3 of the leaves are mosaic, distorted, or the diseased plant is dwarfed to more than 3 / 4 of the normal plant height;

[0082] Grade 5: 1 / 3 to 1 / 2 of the leaves are mosaic, or a few leaves are deformed, or the main vein turns black, or there is slight wilting, or the diseased plant is dwarfed to 2 / 3 to 3 / 4 of the normal plant height;

[0083] Grade 7: 1 / 2 to 2 / 3 of the leaves are mosaic, or deformed or the main and lateral veins are necrotic, wilted, and the diseased plant is dwarfed to 1 / 2 to 2 / 3 of the normal plant height;

[0084] Grade 9: All the leaves of the whole plant are mosaic, severely deformed, wilted or necrotic, or the diseased plant is dwarfed to more than 1 / 2 of the normal plant height.

[0085] (2)The control efficacy of each short peptide against TMV or CMV

[0086] It was calculated according to the following formula.

[0087] Disease index = 100×∑(number of diseased plants at each level × relative level) / (total number of plants surveyed × 9);

[0088] Relative control efficacy (%) = 100×(disease index of the control group - disease index of the treatment group) / disease index of the control group.

[0089] The incidence of each treatment group after inoculating Nicotiana benthamiana seedlings with TMV virus for 10 days is shown in Figure 3 A as shown. The results showed that after treatment with SJPep1, the incidence of TMV decreased by 69.77%, after treatment with SJPep2 and SJPep5, the incidence of TMV decreased by 62.79%, after treatment with SJPep3 and SJPep8, the incidence of TMV decreased by 65.12%, after treatment with SJPep4, the incidence of TMV decreased by 72.09%, and after treatment with SJPep6 and SJPep7, the incidence of TMV decreased by 58.14%.

[0090] The disease index of each treatment group after inoculating Nicotiana benthamiana seedlings with TMV virus for 10 days is shown in Figure 3As shown in B of , the relative control efficacy of each short peptide against TMV virus can be obtained through this disease index. The results show that the relative control efficacy of SJPep1 against TMV is 70.59%, that of SJPep2 against TMV is 63.64%, that of SJPep3 against TMV is 65.24%, that of SJPep4 against TMV is 79.68%, that of SJPep5 against TMV is 62.57%, that of SJPep6 against TMV is 60.43%, that of SJPep7 against TMV is 59.36%, and that of SJPep8 against TMV is 60.96%.

[0091] The incidence of each treatment group after inoculating cucumber seedlings with CMV virus for 15 days is investigated as shown in Figure 4 A of . The results show that the incidence of CMV decreased by 69.05% after treatment with SJPep1, 64.29% after treatment with SJPep2, 52.38% after treatment with SJPep3, 61.90% after treatment with SJPep4, 59.52% after treatment with SJPep5, 54.76% after treatment with SJPep6, 57.14% after treatment with SJPep7, and 59.52% after treatment with SJPep8.

[0092] The disease index of each treatment group after inoculating cucumber seedlings with CMV virus for 15 days is investigated as shown in Figure 4 B of . The relative control efficacy of each short peptide against CMV virus can be obtained through this disease index. The results show that the relative control efficacy of SJPep1 against CMV is 71.35%, that of SJPep2 against CMV is 66.15%, that of SJPep3 against CMV is 55.21%, that of SJPep4 against CMV is 63.54%, that of SJPep5 against CMV is 63.02%, that of SJPep6 against CMV is 59.90%, that of SJPep7 against CMV is 61.46%, and that of SJPep8 against CMV is 64.06%.

[0093] In summary, exogenous spraying of Nicotiana benthamiana with short peptides SJPep1 - SJPep8 can inhibit the infection of TMV; exogenous spraying of cucumber with short peptides SJPep1 - SJPep8 can inhibit the infection of CMV, that is, short peptides SJPep1 - SJPep8 can inhibit the infection of viruses (such as TMV and CMV) to a certain extent.

[0094] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention's design, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall 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 as shown in SEQ ID NO.1 or SEQ ID NO.2; 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 product is selected from 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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