Application and preparation method of strawberry endogenous peptide

Abstract

This invention discloses the application and preparation method of an endogenous peptide from strawberry. Specifically, it describes the application of peptides with the amino acid sequences of SEQ ID No. 1, SEQ ID No. 2, or SEQ ID No. 3 in regulating resistance to strawberry fungal diseases. The strawberry endogenous peptide of this invention is applied to inhibit strawberry diseases caused by fungal pathogens such as Botrytis cinerea, Colletotrichum gloeosporioides, and Fusarium oxysporum, effectively improving the resistance of strawberries to pathogen infection. Furthermore, the strawberry endogenous peptide is a plant-friendly substance, highly effective and easy to use in controlling strawberry fungal diseases, and has strong practicality.

Description

Technical Field

[0001] This invention relates to the field of plant immune biotechnology, and in particular to the preparation of the strawberry endogenous peptide FaPep5 and its application in improving strawberry resistance to fungal diseases. Background Technology

[0002] strawberry( Fragaria × ananassa *Fragaria dichotoma* (Duch.) is a perennial herbaceous fruit tree belonging to the genus *Fragaria* in the family Rosaceae. Winter and spring strawberry production primarily relies on greenhouses and plastic tunnels, characterized by enclosed, high-temperature, and high-humidity environments. Continuous cropping further deteriorates soil physical and chemical properties, leading to increased pathogen accumulation and frequent strawberry diseases, resulting in significant economic losses. Among these, *Botrytis cinerea* (Glaucus spp.) is a major causative agent. Botrytis cinerea ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides ) and Fusarium oxysporum ( Fusarium oxysporum Fungal diseases caused by *Botrytis cinerea* are major diseases affecting strawberry production. Gray mold, caused by *Botrytis cinerea*, is one of the main diseases after strawberry flowering, primarily affecting the fruit, causing it to rot softly or dry and fall off. Anthracnose, caused by *Colletotrichum gloeosporioides*, mainly affects the strawberry runners, petioles, leaves, and fruit, causing the infected parts to wilt and die. Root rot, caused by *Fusarium oxysporum* infecting the roots, leads to root necrosis and eventually the death of the entire strawberry plant. Currently, strawberry disease control is mainly based on chemical control. At the early stage of disease, fungicides such as iprodione, pyraclostrobin, pyrimethanil, captan, and polyoxin are used to control gray mold; mancozeb, azoxystrobin, pyraclostrobin, and pyraclostrobin are used to control anthracnose; and dimethomorph and iprodione are used to control root rot. However, the extensive use of pesticides can lead to problems such as strain resistance, food safety, and environmental pollution. Therefore, exploring efficient and environmentally friendly methods and systems for the prevention and control of strawberry diseases is of great significance for promoting the healthy development of the strawberry industry.

[0003] Improving the resistance of crops to pathogenic bacteria by inducing plant innate immunity is an effective means to reduce strawberry diseases and pesticide application. When plants are infected by pathogenic bacteria, various pattern recognition receptors (PRRs) on the cell membrane can recognize pathogenic bacteria or small molecules released by plants, producing pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), and activating the defense response of pattern-triggered immunity (PTI). For example, bacterial flagellin flg22 can significantly trigger the immune response of plants, and exogenous application of Pseudomonas aeruginosa flg22 can improve the ability of plants to resist Pseudomonas syringae infection, Magnaporthe oryzae infection and Fusarium graminearum infection; fungal chitin is recognized by plant membrane receptors, activates downstream signal transduction, and induces plant disease resistance; exogenous application of systemin can induce the expression of protease inhibitors and activate plant systemic resistance. The application of bacterial Harpin protein is the first example of using induced plant immune response for commercial disease control. By spraying Harpin protein, the innate immune system of plants is activated, and disease-resistant genes (such as PR protein genes) are induced to express, thereby enhancing the multiple resistance of plants to bacteria, fungi and viruses.

[0004] Plant elicitor polypeptide (Peps) is a plant cell factor widely present in angiosperms, also classified as a peptide hormone, and is a typical DAMP molecule that can activate plant PTI and induce resistance gene expression. Peps are released from their precursor proteins PROPEPs by type II cysteine protease MC4 cleavage from the C-terminus to function. Previous studies have shown that Arabidopsis thaliana Pep and its homologues play a role in enhancing plant resistance to biotic stress such as pathogenic bacteria, insect feeding, etc. and abiotic stress such as salt stress. Exogenous treatment of Pep1 can enhance the resistance of Arabidopsis thaliana leaves to Pseudomonas syringae, ZmPep1 induces the production of salicylic acid and ethylene, regulates the expression of pathogen defense genes, and improves resistance to fungal pathogens. In Rosaceae, it was found that PpPep1, PdPep1 and PpPep2 pretreatment can improve the resistance of peach-almond hybrid leaves to Xanthomonas campestris in a dose-dependent manner; OsPep can improve the resistance of rice to piercing-sucking insects; StPep1 can improve the resistance of potato to root-knot nematodes. However, there is little research on endogenous small peptides for improving the resistance of strawberries to fungi in the prior art, and there are few reports of significant effects. Endogenous small peptides in the field of improving the resistance of strawberries to fungal diseases need more and deeper research. Among them, there is no report on the application of endogenous small peptides FaPEP in strawberries to fungal diseases. SUMMARY

[0005] The purpose of the present application is to provide a preparation of a strawberry endogenous peptide FaPep5 and its application in the field of improving the resistance of strawberries to fungal diseases. The purpose of the present application is achieved by the following technical scheme:

[0006] The application of the strawberry endogenous peptide, the plant in the embodiment of the application is octoploid 'Hongyan' strawberry, and the application is any one of the following:

[0007] C1) Application of the strawberry endogenous peptide in regulating the resistance of strawberry to fungal diseases;

[0008] C2) Application of the strawberry endogenous peptide in preparing a product for regulating the resistance of strawberry to fungal diseases;

[0009] C3) Application of the strawberry endogenous peptide in preventing and treating fungal diseases of strawberry;

[0010] C4) Application of the strawberry endogenous peptide in preparing a product for preventing and treating fungal diseases of strawberry;

[0011] C5) Application of the strawberry endogenous peptide in breeding of strawberry;

[0012] C6) Application of the strawberry endogenous peptide in activating the expression of a disease-resistant gene of strawberry; PR

[0013] C7) Application of the strawberry endogenous peptide in preparing a product for activating the expression of a disease-resistant gene of strawberry; PR

[0014] The strawberry endogenous peptide is any one of the following:

[0015] A1) A small peptide with an amino acid sequence of SEQ ID No. 1, i.e., a mature small peptide FaPep5 amino acid sequence: EDVVVVATGTARSTRALGVKRPIGTGSGPQIN.

[0016] A2) A small peptide with an amino acid sequence of SEQ ID No. 2: GPEF EDVVVVATGTARSTRALGVKRPIGTGSGPQIN (compared with SEQ ID No. 1, containing a protease recognition sequence and an enzyme cutting site, a total of 4 amino acids)

[0017] A3) A polypeptide with an amino acid sequence of SEQ ID No. 3, i.e., a FaPep5 precursor protein FaPROEPE5 amino acid sequence encoded:

[0018] MESSSSADQREEEEEERHPLLHSPCHFFEQLITSCLKCLGLDHHFSSSSSAAADDGKTKADHHQQCPPQQTEMEMKLGEDVVVVATGTARSTRALGVKRPIGTGSGPQINRASS*;

[0019] The application of the nucleotide sequence, and the application is any one of the following:

[0020] ​​D1) Application in regulating resistance to fungal diseases in strawberries;

[0021] D2) Application in the preparation of products that regulate resistance to fungal diseases in strawberries;

[0022] D3) Application in the prevention and control of fungal diseases in strawberries;

[0023] D4) Application in the preparation of products for the prevention and control of strawberry fungal diseases;

[0024] Application of D5 in strawberry breeding;

[0025] Application of D6 in activating the expression of the strawberry disease resistance gene PR;

[0026] D7) Application in the preparation of products that activate the expression of the strawberry disease resistance gene PR;

[0027] The nucleotide sequence is any one of the following:

[0028] B1) The nucleotide sequence encoding the strawberry endogenous peptide of claim 1;

[0029] B2) The sequence is the nucleotide sequence of SEQ ID No. 4, namely: FaPROPEP5 Gene nucleotide sequence:

[0030] ATGGAGTCCTCGAGCTCAGCCGACCAGAGAGAAGAAGAAGAAGAAGAGCGACACCCTTTGTTGCACAGCCCCTGCCATTTCTTCGAGCAACTCATCACCTCATGCCTCAAGTGCCTCGGACTTGATCATCACTTCTCCTCCTCCTCCTCCGCCGCCGCCGACGATGGAAAGAC AAAGGCAGATCATCATCAACAGTGCCCTCCACAGCAGACTGAAATGGAAATGAAACTAGGAGAAAGATGTTGTTGTAGTAGCCACAGGTACTGCAAGGTCGACAAGAGCGTTGGGAGTGAAACGGCCTATAGGCACTGGTTCAGGTCCTCAAATTAACCGTGCTTCATCCTAG.

[0031] A method for preparing strawberry endogenous peptides includes the following steps:

[0032] E1) Cloning method for the FaPep5 fragment: Preparation of a fragment containing... FaPROPEP5 Recombinant plasmids of genes, the FaPROPEP5 The nucleotide sequence of the gene is shown in SEQ ID No. 4; containing FaPROPEP5Using the recombinant plasmid of the gene as a template, PCR amplification was performed to obtain the amplified gene. FaPep5 Fragment;

[0033] E2) Preparation of polypeptide FaPep5: Preparation of polypeptide containing FaPep5 The recombinant plasmid of the fragment will contain FaPep5 The recombinant plasmid fragment was introduced into engineered bacteria for expression. The bacterial culture was collected by centrifugation and purified to obtain the target protein. The obtained protein was digested with protease, separated by column, and freeze-dried to obtain the small peptide FaPep5, whose amino acid sequence is SEQ ID No. 2.

[0034] The specific operations of step E1 include:

[0035] E11) Using strawberry cDNA as a template, PCR amplification was performed using the first set of specific primers. The nucleotide sequences of the first set of heterogeneous primers FaPROPEP5-F and FaPROPEP5-R are shown in SEQ ID No. 5 (ATGGAGTCCTCGAGCTCA) and SEQ ID No. 6 (CTAGGATGAAGCACGGTTAA), respectively.

[0036] E12) The obtained PCR product was ligated with the pClone007 cloning vector to obtain the ligation product. pClone007- FaPROPEP5 ;

[0037] E13) Connecting products pClone007-FaPROPEP5 Transformed Escherichia coli DH5α and plated on LB solid medium containing ampicillin. Single colonies were picked, shaken, and plasmids were extracted. Plasmid PCR was performed, and the plasmids were sent for sequencing to obtain recombinant plasmids with correct sequences.

[0038] E14) with correct sequencing pClone007-FaPROPEP5 Using the recombinant plasmid as a template, the FaPep5 fragment was obtained by PCR amplification using the second set of specific primers. The nucleotide sequences of the second set of specific primers, FaPep5-F and FaPep5-R, are shown in SEQ ID No. 7 (TGTTCCAGGGGCCCGAATTCGAAGATGTTGTTGTAGTAGCCACAG) and SEQ ID No. 8 (AGCCATGGGGTACCGGATCCCTAGTTAATTTGAGGACCTGAACCA), respectively.

[0039] The specific operations in step E2 include:

[0040] E21) will amplify FaPep5 Fragments and double enzyme digestion ( BamH I. EcoRThe pTac-His-MBP plasmid of I) is linked to transform E. coli DH5a and coated on LB solid medium containing ampicillin, a single colony is picked, the plasmid is extracted after shaking, the plasmid is sequenced after plasmid PCR, and the correct sequence of the recombinant plasmid is obtained;

[0041] E22) The recombinant plasmid obtained in E21) is introduced into an E. coli BL21 (DE3) engineering bacteria expression system for expression, and the bacterial liquid is shaken at 37 ℃ and 220 rpm until the OD 600 is 0.6-0.8, 0.1 mg / L IPTG is added, 28 ℃, 220 rpm culture for 16 h, centrifugal collection of bacterial liquid precipitate, resuspended with PBS, ultrasonic broken, collection supernatant, purified by His-tag column to obtain the target protein;

[0042] E23) The protein obtained in E22) is cut by HRV 3C protease at 4 ℃ overnight, the flow-through liquid containing small peptide FaPep5 is collected by His-tag column separation, and the small peptide FaPep5 is obtained by freeze-drying into powder.

[0043] A method for improving the resistance of strawberry fungal diseases, a small peptide with an amino acid sequence of SEQ ID No. 1 or SEQ ID No. 2 is dissolved in water (ddH2O) to obtain a preparation, and the preparation is diluted with water to obtain a diluent which is driped on the roots of strawberries.

[0044] The fungal disease is any one of gray mold, anthracnose, and root rot.

[0045] When the fungal disease is gray mold, the concentration of the diluent is not less than 200 nM.

[0046] When the fungal disease is anthracnose, the concentration of the diluent is not less than 100 nM.

[0047] When the fungal disease is root rot, the concentration of the diluent is not less than 400 nM.

[0048] The advantages and beneficial effects of the present application are:

[0049] 1) The present application applies strawberry endogenous peptides to inhibit strawberry diseases caused by fungal pathogens such as Botrytis cinerea, Colletotrichum gloeosporioides and Fusarium oxysporum, which can effectively improve the resistance of strawberries to pathogenic fungi, and provide a new idea for the prevention and control of strawberry pathogens.

[0050] 2) The strawberry endogenous peptides in the preparation are plant-friendly substances, which can be degraded in the environment and organisms, and have no residues after use, and are harmless to the human body and the environment.

[0051] 3) The preparation of the present application is efficient and easy to use for preventing and treating strawberry fungal diseases, and has low cost. BRIEF DESCRIPTION OF DRAWINGS

[0052] The application will be further described below in connection with the drawings and examples.

[0053] Figure 1 For Example 1, 2, 3 FaPROPEP5 Response to different fungal pathogen infection, wherein Figure 1 A is FaPROPEP5 Response to Botrytis cinerea infection; Figure 1 B is FaPROPEP5 Response to Colletotrichum gloeosporioides infection; Figure 1 C is FaPROPEP5 Response to Fusarium oxysporum f. sp. cucumeris infection.

[0054] Figure 2 For FaPep5 The schematic diagram of recombinant plasmid structure of the fragment connected with pTac-His-MBP.

[0055] Figure 3 For Example 4, the SDS-PAGE diagram of induced expression of His-MBP-FaPep5 under different temperature conditions, wherein M is protein Marker; 1 is 16 ℃ induced supernatant; 2 is 19 ℃ induced supernatant; 3 is 22 ℃ induced supernatant; 4 is 25 ℃ induced supernatant; 5 is 28 ℃ induced supernatant; 6 is 31 ℃ induced supernatant; 7 is 34 ℃ induced supernatant; 8 is 37 ℃ induced supernatant.

[0056] Figure 4 For Example 4, the SDS-PAGE diagram of the flow-through fluid containing FaPep5 collected after His-MBP-FaPep5 was cut by HRV 3C protease overnight.

[0057] Figure 5 For Example 4, the protein mass spectrum result diagram of FaPep5 protein collected after purification and cutting by protease.

[0058] Figure 6 For Example 5, strawberry fruits were treated with different concentrations of FaPep5 and water control for 24 h, then inoculated with Botrytis cinerea cake, and the disease incidence of strawberry fruits was compared, wherein Figure 6 A is the lesion area diagram of strawberry fruits inoculated with Botrytis cinerea cake for 3 days; Figure 6 B is the disease incidence diagram of strawberry fruits inoculated with Botrytis cinerea cake for 3 days.

[0059] Figure 7 For Example 6, strawberry fruits were treated with different concentrations of FaPep5 and water control for 24 h, then inoculated with Colletotrichum gloeosporioides cake, and the disease incidence of strawberry fruits was compared, wherein Figure 7 A is the lesion area diagram of strawberry fruits inoculated with Colletotrichum gloeosporioides cake for 3 days; Figure 7B is the disease incidence of strawberry plants 3 days after inoculation with a piece of P. fragariae.

[0060] Figure 8 Example 7, strawberry plants were inoculated with F. oxysporum after 2 days of pre-treatment with FaPep5 or water. The disease incidence of strawberry plants was compared, wherein Figure 8 A is the plant status of strawberry plants 7 days after being mixed with F. oxysporum mycelium in a vermiculite substrate; Figure 8 B is the disease index of strawberry plant stems infected with F. oxysporum (disease index: 0 for no disease, 1 for 0-20 % of the total length of the stem, 2 for 20-40 %, 3 for 40-60 %, 4 for 60-80 %, and 5 for 80-100 % of the total length of the stem). Disease index = 100 x ∑ (number of leaves at each level x representative value at each level) / (total number of leaves surveyed x highest representative value)

[0061] Figure 9 Example 8, the response of strawberry resistance genes to FaPep5 treatment. DETAILED DESCRIPTION

[0062] Example 1

[0063] In strawberry fruits FaPROPEP5 Response to infection with Botrytis cinerea

[0064] 1. Inoculation of strawberry fruits with Botrytis cinerea

[0065] Strawberry fruits were harvested for this example. Botrytis cinerea was inoculated onto PDA medium and incubated at 23 °C for 2-3 weeks. A puncher was used to take a piece of the fungus and inoculate it onto the strawberry fruits, which were incubated at 23 °C in the dark. The fruit around the inoculated fungus was taken at 0, 12, 24, 48, 72, and 96 h of incubation, and immediately frozen in liquid nitrogen for later use.

[0066] 2. Extraction of total RNA and reverse transcription

[0067] (1) Sample grinding: 100 mg of sample was placed in a mortar pre-cooled in liquid nitrogen, and liquid nitrogen was added to grind the sample to a silk state;

[0068] (2) Lysis: The ground plant sample was added to a 1.5 mL RNase-free EP tube pre-cooled in liquid nitrogen with a small spoon pre-cooled in liquid nitrogen, 500 μL of RCL lysis solution, 70 μL of Plantaid, and 10 μL of mercaptoethanol were added, and vortexed for 2 min, 2 min in a 60 °C water bath, vortexed / water bathed repeatedly for 3-4 times;

[0069] (3) Centrifugation: Centrifuge at 12500 r / s for 12 min;

[0070] (4) DNA removal: Add the supernatant after centrifugation to the DNA adsorption column and centrifuge at 12500 r / s for 1 min;

[0071] (5) Precipitate RNA: Add an equal volume of RCB to the filtered supernatant and mix well by pipetting;

[0072] (6) Transfer the RNA precipitate to the RNA collection column, centrifuge at 12500 r / s for 1 min, retain the column and discard the filtrate;

[0073] (7) Removal of macromolecules such as proteins: Add 500 μL of RWF to the column, centrifuge at 12500 r / s for 1 min, discard the filtrate, and repeat once;

[0074] (8) Removal of small molecules such as sugars and phenols: Add 500 μL of Buffer II to the column, centrifuge at 12500 r / s for 1 min, discard the filtrate, and repeat once;

[0075] (9) RNA drying: Centrifuge the RNA collection column at 12500 r / s for 2 min;

[0076] (10) RNA collection: Place the column into a new 1.5 ml RNase-free EP tube, take 40 μl of DEPC water preheated to 60℃, drop it vertically into the center of the column, let stand for 2 min, centrifuge at 12000 r / s for 1 min, and the filtrate is the final RNA sample.

[0077] Total RNA extracted from strawberry pulp was reverse transcribed into cDNA. Specifically, the reagents were added to a 200 μL RNase-free PCR tube according to the reverse transcription system described in the Novizan (R223) instructions. The tube was then placed in a PCR instrument and reacted at 37°C for 15 min, then at 85°C for 5 s to obtain strawberry pulp cDNA. Finally, the sample was stored at -20°C for subsequent RT-qPCR experiments. 3. FaPROPEP5 Response to Botrytis cinerea infection like Figure 1 As shown in Figure A, real-time quantitative PCR technology was used to... FaPROPEP5 The transcription level was detected. FaPROPEP5 The expression level was significantly upregulated 48 h after inoculation with Botrytis cinerea cake, and it participated in the response of strawberry fruit to Botrytis cinerea infection.

[0078] Example 2

[0079] In strawberry fruitFaPROPEP5 Response to G. cingulata infection

[0080] 1. Inoculation of strawberry fruits with G. cingulata plugs

[0081] Strawberry fruits were harvested for this experiment. G. cingulata was inoculated on PDA medium and incubated at 23 °C for 2-3 weeks. The plugs were inoculated on strawberry fruits using a puncher and incubated at 23 °C in the dark. The fruit tissue surrounding the inoculated plugs was harvested at 0, 12, 24, 48, 72, 96 h of incubation and immediately frozen in liquid nitrogen for later use.

[0082] 2. Total RNA extraction and reverse transcription

[0083] Total RNA was extracted from all strawberry fruit samples using Omega E.Z.N.A plant RNA kit. The extracted total RNA from strawberry fruits was reverse transcribed into cDNA using Superscript® III Reverse Transcriptase (R223). The procedure was the same as in Example 1.

[0084] 3. FaPROPEP5 Response to G. cingulata infection

[0085] As shown in Figure B, the transcriptional level of Figure 1 was detected using real-time quantitative PCR, FaPROPEP5 was significantly up-regulated at 48 h after inoculation with G. cingulata plugs, indicating its involvement in the resistance response of strawberry fruits to G. cingulata infection. FaPROPEP5 Example 3

[0086] Response to F. oxysporum infection in strawberry roots

[0087] FaPROPEP5 Strawberry plantlets were used for this experiment. F. oxysporum mycelia were inoculated in PDB liquid medium and incubated at 23 °C for 2 days. The mycelia were collected by centrifugation and mixed with vermiculite. The strawberry plantlets were transplanted into the vermiculite medium containing F. oxysporum mycelia and incubated at 23 °C under 12 h-12 h light-dark cycles. The roots of the plantlets were harvested at 0, 12, 24, 48, 72, 96 h of incubation and immediately frozen in liquid nitrogen for later use.

[0088] 1. Inoculation of strawberry fruits with G. cingulata plugs

[0089] Strawberry fruits were harvested for this experiment. G. cingulata was inoculated on PDA medium and incubated at 23 °C for 2-3 weeks. The plugs were inoculated on strawberry fruits using a puncher and incubated at 23 °C in the dark. The fruit tissue surrounding the inoculated plugs was harvested at 0, 12, 24, 48, 72, 96 h of incubation and immediately frozen in liquid nitrogen for later use.

[0090] 2. Total RNA extraction and reverse transcription

[0091] Total RNA was extracted from all strawberry fruit samples using Omega E.Z.N.A plant RNA kit. The extracted total RNA from strawberry fruits was reverse transcribed into cDNA using Superscript® III Reverse Transcriptase (R223). The procedure was the same as in Example 1.​

[0092] 3. FaPROPEP5 Response to Fusarium scutellaria infection

[0093] like Figure 1 As shown in C, real-time quantitative PCR technology was used to... FaPROPEP5 The transcription level was detected. FaPROPEP5 The expression level was significantly upregulated 72 h after inoculation with Fusarium oxysporum, and it is involved in the resistance response of strawberries to anthracnose.

[0094] Example 4

[0095] Cloning of the FaPep5 fragment:

[0096] Using strawberry cDNA as a template, PCR amplification was performed using the first set of specific primers. The nucleotide sequences of the first set of heterozygous primers FaPROPEP5-F and FaPROPEP5-R are shown in SEQ ID No. 5 (ATGGAGTCCTCGAGCTCA) and SEQ ID No. 6 (CTAGGATGAAGCACGGTTAA), respectively. The obtained PCR product was ligated into the pClone007 cloning vector to obtain the ligation product. pClone007-FaPROPEP5 ; Connect the products pClone007-FaPROPEP5 Transformed Escherichia coli DH5α and plated on LB solid medium containing ampicillin. Single colonies were picked, shaken, and plasmids were extracted. Plasmid PCR was performed, and the plasmids were sent for sequencing to obtain recombinant plasmids with correct sequences.

[0097] With correct sequencing pClone007-FaPROPEP5 Using the recombinant plasmid as a template, and with the second set of specific primers, PCR amplification was performed to obtain... FaPep5 The nucleotide sequences of the second set of specific primers FaPep5-F and FaPep5-R are shown in SEQ ID No. 7 (TGTTCCAGGGGCCCGAATTCGAAGATGTTGTTGTAGTAGCCACAG) and SEQ ID No. 8 (AGCCATGGGGTACCGGATCCCTAGTTAATTTGAGGACCTGAACCA), respectively.

[0098] Preparation of FaPep5:

[0099] Amplification FaPep5 Fragments and double enzyme digestion ( BamH I. EcoRThe pTac-His-MBP plasmid of (I) is linked to transform E. coli DH5a and coated on LB solid medium containing ampicillin, a single colony is picked, and the plasmid is extracted after shaking, and the plasmid is sequenced after plasmid PCR, and the sequence-corrected recombinant plasmid is obtained. The obtained recombinant plasmid is introduced into the E. coli BL21 (DE3) engineering bacteria expression system for expression, and the bacterial liquid is shaken at 37°C, 220 rpm, until the OD 600 of the bacterial liquid is 0.6-0.8, 0.1 mg / L IPTG is added, 28°C (optimal induction temperature), and the bacterial liquid is cultured at 220 rpm for 16 h. The bacterial liquid is collected by centrifugation, resuspended with PBS, and then broken by ultrasonic, and the supernatant is collected, and the target protein is obtained by His-tag column purification, and the induction result is as shown in Figure 3 . The obtained protein is cut by HRV 3C protease at 4°C overnight, and the flow-through liquid containing the polypeptide FaPep5 is collected by His-tag column separation. After Coomassie brilliant blue staining Figure 4 and protein spectrum confirmation Figure 5 , the flow-through liquid containing FaPep5 is freeze-dried into powder to prepare the small peptide FaPep5 of the application, and the amino acid sequence is SEQ ID No. 2

[0100] GPEF EDVVVVATGTARSTRALGVKRPIGTGSGPQIN (containing a protease recognition sequence and a cleavage site, a total of 4 amino acids). The FaPep5 short peptide sequence provided in this embodiment consists of 36 amino acids, which is the C-terminal part of the full-length protein.

[0101] Example 5

[0102] Application of FaPep5 in strawberry fruit resistance to gray mold

[0103] In this embodiment, the small peptide FaPep5 prepared in Example 4 is used to stimulate the resistance to gray mold by feeding to simulate the absorption of strawberry fruit, so as to control the strawberry gray mold. The main steps include:

[0104] 1. Preparation of FaPep5 small peptide solution

[0105] The FaPep5 small peptide is diluted with water to prepare FaPep5 solutions with concentrations of 100 nM, 200 nM and 400 nM. Prior to the present application, there is no report on the function of the small peptide FaPep5.

[0106] 2. Treatment of FaPep5 small peptide on strawberry

[0107] The plant treatment method of the FaPep5 small peptide is feeding. The strawberry stalk model fruit is fed with a water solution of the FaPep5 small peptide with a concentration of 100 nM, 200 nM, and 400 nM to simulate fruit absorption, and water is used as a control. After 24 hours of feeding, the strawberry fruit of the treated plant is inoculated with a Botrytis cinerea cake, and is cultured at 23°C in the dark for 3 days. The incidence of strawberry fruit disease is observed and counted. The results are shown in Table 1. Figure 6 The strawberry fruit treated with the above solution has obviously stronger resistance to gray mold than the control strawberry fruit. The fruit resistance to gray mold has a FaPep5 concentration dependence, and the higher the FaPep5 concentration, the stronger the fruit resistance.

[0108] Example 6

[0109] Application of FaPep5 in strawberry fruit resistance to anthracnose

[0110] In the embodiment of the present application, the FaPep5 small peptide prepared in Example 4 is used to simulate strawberry fruit absorption by feeding to stimulate the production of resistance to anthracnose bacteria, thereby controlling strawberry anthracnose. The main steps include:

[0111] 1. Preparation of a FaPep5 small peptide solution

[0112] The FaPep5 small peptide is diluted with water to a FaPep5 solution with a concentration of 100 nM, 200 nM, and 400 nM.

[0113] 2. Treatment of strawberry seedlings with FaPep5 small peptides

[0114] The plant treatment method of the FaPep5 small peptide is feeding. The strawberry fruit stalk is fed with a water solution of the FaPep5 small peptide with a concentration of 100 nM, 200 nM, and 400 nM to simulate fruit absorption, and water is used as a control. After 24 hours of feeding, the strawberry fruit of the treated plant is inoculated with a Botrytis cinerea cake, and is cultured at 23°C in the dark for 3 days. The incidence of strawberry fruit disease is observed and counted. The results are shown in Table 1. Figure 7 The strawberry fruit treated with the above solution has obviously stronger resistance to gray mold than the control strawberry fruit. The fruit resistance to gray mold has a FaPep5 concentration dependence, and the higher the FaPep5 concentration, the stronger the fruit resistance.

[0115] Example 7

[0116] Application of FaPep5 in strawberry root resistance to sharp knife fusarium

[0117] In the embodiment of the present application, the FaPep5 small peptide prepared in Example 4 is used to simulate strawberry fruit absorption by feeding to stimulate the production of resistance to anthracnose bacteria, thereby controlling strawberry anthracnose. The main steps include:

[0118] 1. Preparation of FaPep5 small peptide solution

[0119] FaPep5 small peptide was diluted with water to a FaPep5 solution with a concentration of 400 nM.

[0120] 2. Treatment of strawberry seedlings with FaPep5 small peptide

[0121] The plant treatment method of FaPep5 small peptide was root treatment. Strawberry tissue culture seedlings with 3 weeks of rooting were cultured in rooting medium (MS + 30 g / L sucrose + 8 g / L agar + 400 nM FaPep5) with a concentration of 400 nM of FaPep5, and the control was rooting medium without FaPep5. After 7 days of pretreatment at 23°C with 12 h-12 h dark-light alternation, the strawberry plants were transplanted after the fungal hyphae of Fusarium oxysporum were mixed evenly with vermiculite, and the plants were cultured at 23°C with 12 h-12 h light-dark alternation. The results are shown in Figure 8 The strawberry plants treated with the above preparation had significantly stronger resistance to Fusarium oxysporum than the control strawberry plants.

[0122] Example 8

[0123] FaPep5 activates the disease resistance genes of strawberry

[0124] Strawberry tissue culture seedlings with 3 weeks of rooting were selected, and FaPep5 prepared in Example 4 was added to the rooting medium (MS + 30 g / L sucrose + 8 g / L agar + 400 nM FaPep5), and the control was ordinary rooting medium. The plants were cultured with 12 h-12 h dark-light alternation, and root samples of the plants treated for 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min were quickly frozen in liquid nitrogen for standby. Total RNA was extracted from the samples and reverse transcribed for subsequent RT-qPCR experiments.

[0125] The results are shown in Figure 9 FaPep5 can activate the expression of disease resistance genes PR of strawberry.

[0126] Finally, it should be noted that the above is only used to illustrate the technical solutions of the present application and is not limiting. Although the present application has been described in detail with reference to the preferred arrangement, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. Use of a strawberry endogenous peptide, characterized in that, The application is any of the following: C1) application of the strawberry endogenous peptide in regulating the resistance of strawberry to fungal diseases; C2) application of the strawberry endogenous peptide in preparing a product for regulating the resistance of strawberry to fungal diseases; C3) application of the strawberry endogenous peptide in preventing and treating fungal diseases of strawberry; C4) application of the strawberry endogenous peptide in preparing a product for preventing and treating fungal diseases of strawberry; C5) application of the strawberry endogenous peptide in breeding of strawberry; C6) Use of a strawberry endogenous peptide in activating a strawberry disease resistance gene PR application in expression; C7) Use of a strawberry endogenous peptide in the preparation of an activated strawberry disease resistance gene PR application in the products expressed; The strawberry endogenous peptide is any of the following: A1) a small peptide with an amino acid sequence of SEQ ID No. 1; A2) a small peptide with an amino acid sequence of SEQ ID No. 2; The fungal disease is any of the following: gray mold, anthracnose, and root rot.

2. Use of a nucleotide sequence, characterized in that, The application is any of the following: D1) application in regulating the resistance of strawberry to fungal diseases; D2) application in preparing a product for regulating the resistance of strawberry to fungal diseases; D3) application in preventing and treating fungal diseases of strawberry; D4) application in preparing a product for preventing and treating fungal diseases of strawberry; D5) application in breeding of strawberry; D6) Use of activating raspberry disease resistance genes PR application in expression; D7) Use of the products expressed in the preparation of activated raspberry disease resistance genes PR in the preparation of vaccines. The nucleotide sequence is a nucleotide sequence encoding the strawberry endogenous peptide of claim 1, and the fungal disease is any of the following: gray mold, anthracnose, and root rot.

3. A method of preparing a strawberry endogenous peptide, characterized by, The method comprises the following steps: E1) preparing a recombinant plasmid containing FaPROPEP5 the nucleotide sequence of the gene is shown as SEQ ID No. 4; and FaPROPEP5 E2) preparing a recombinant plasmid containing FaPROPEP5 the nucleotide sequence of the gene is shown as SEQ ID No. 4; and FaPep5 E3) preparing a recombinant plasmid containing E2) Preparation containing FaPep5 The recombinant plasmid of the fragment will contain FaPep5 The recombinant plasmid fragment was introduced into engineered bacteria for expression. The bacterial culture was collected by centrifugation and purified to obtain the target protein. The obtained protein was digested with protease, separated by column, and freeze-dried to obtain the small peptide FaPep5, whose amino acid sequence is SEQ ID No.

2.

4. The method for preparing strawberry endogenous peptides according to claim 3, characterized in that, The specific operation of step E1) comprises the following steps: E11) using the first group of specific primers, taking the cDNA of strawberry as a template, and performing PCR amplification, wherein the nucleotide sequences of the first group of specific primers FaPROPEP5-F and FaPROPEP5-R are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively; E12) Ligation of the obtained PCR product with pClone007 cloning vector, to obtain a ligation product pClone007- FaPROPEP5 ; E13) ligating the products pClone007-FaPROPEP5 The transformed E. coli DH5α was spread on LB solid medium containing ampicillin, single colonies were picked, and the plasmid was extracted after shaking culture. After plasmid PCR, the plasmid was sent for sequencing, and the recombinant plasmid with correct sequence was obtained. E14) with correct sequencing pClone007-FaPROPEP5 Using the recombinant plasmid as a template, and with the second set of specific primers, PCR amplification was performed to obtain... FaPep5 The nucleotide sequences of the second set of specific primers FaPep5-F and FaPep5-R are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively.

5. The method of claim 3, wherein the strawberry endogenous peptide is prepared by, The method comprises the following steps: The specific operation of step E2) comprises the following steps: E21) Amplification of the fragment and ligation with the double enzyme cut (EcoRI and Notl) of the pTac-His-MBP plasmid FaPep5 BamH I, EcoR I) of the pTac-His-MBP plasmid. E. coli DH5a was transformed and plated on LB solid medium containing ampicillin. Single colonies were picked, and the plasmid was extracted after shaking. After plasmid PCR, the plasmid was sent for sequencing, and the recombinant plasmid with the correct sequence was obtained.​ E22) The recombinant plasmid obtained in E21) is introduced into an E. coli BL21 (DE3) engineering bacteria expression system for expression, and the bacterial liquid is shaken at 37 °C and 220 rpm until the OD 600 is 0.6-0.8, 0.1 mg / L IPTG is added, and the culture is carried out at 28 °C and 220 rpm for 16 h. The bacterial liquid is collected by centrifugation, resuspended in PBS, and then ultrasonically broken. The supernatant is collected and purified by a His-tag column to obtain the target protein; E23) using HRV 3C protease to enzymatically digest the protein obtained in E22) at 4 ℃, collecting the flow-through liquid containing the small peptide FaPep5 through a His-tag column, and freeze-drying the small peptide FaPep5 into a powder.

6. A method of increasing fungal disease resistance in strawberry, characterized by: The small peptide with the amino acid sequence of SEQ ID No. 1 or SEQ ID No. 2 is dissolved in water to obtain a preparation, and the preparation is diluted with water to obtain a diluent, which is fed to the strawberry peduncle or drip irrigated on the roots of the strawberry; and the fungal disease is any of the following: gray mold, anthracnose, and root rot.

7. The method of claim 6, wherein: When the fungal disease is gray mold, the concentration of the diluent is not less than 200 nM.

8. The method of claim 6, wherein: When the fungal disease is anthracnose, the concentration of the diluent is not less than 100 nM.

9. The method of claim 6, wherein: When the fungal disease is root rot, the concentration of the diluent is not less than 400 nM.

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