Application and preparation method of strawberry endogenous peptide

Abstract

The invention discloses application and a preparation method of strawberry endogenous peptide. Specifically, the invention relates to application of a peptide with an amino acid sequence of SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 in the aspects of regulation and control of strawberry fungal disease resistance and the like. The strawberry endogenous peptide disclosed by the invention is applied to inhibiting strawberry diseases caused by fungal pathogenic bacteria such as botrytis cinerea, colletotrichum gloeosporioides and fusarium oxysporum, and the resistance of strawberries to pathogenic bacteria infection can be effectively improved. And the strawberry endogenous peptide is a plant-friendly substance, is efficient and easy to control fungal diseases of strawberries, and is high in practicability.

Description

Technical Field

[0001] The present invention relates to the field of plant immune biotechnology, and in particular to the preparation of strawberry endogenous peptide FaPep5 and its application in fields such as improving strawberry resistance to fungal diseases. Background Art

[0002] strawberry( Fragaria × ananassa Duch. is a perennial herbaceous fruit tree of the genus Fragaria in the Rosaceae family. Fresh strawberry production in winter and spring is mainly carried out in solar greenhouses and plastic sheds, where the growing environment is characterized by airtightness, high temperature, and high humidity. Continuous cropping also deteriorates the physical and chemical properties of the soil, leading to increased accumulation of pathogens and frequent strawberry diseases, causing huge economic losses. Among them, Botrytis cinerea ( Botrytis cinerea ), Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ) and Fusarium oxysporum ( Fusarium oxysporum Fungal diseases caused by the fungus fusarium oxysporum are the main diseases affecting strawberry production. Gray mold, caused by Botrytis cinerea, is a major post-flowering disease of strawberries, primarily affecting fruit, causing them to become soft and rot or dry and fall. Anthracnose, caused by Colletotrichum gloeosporioides, primarily affects strawberry runners, petioles, leaves, and fruit, causing infected parts to wilt and die. Root rot, caused by Fusarium oxysporum, can cause root necrosis and eventually the entire plant to wither and die. Currently, chemical control is the primary method for strawberry disease prevention and control. In the early stages of the disease, pesticides such as procymidone, cyprodinil, pyrimethanil, captan, and polyoxin are used to control gray mold. Chemical pesticides such as mancozeb, metoclopramide, pyraclostrobin, and azoxystrobin are used to control anthracnose. Root rot is controlled by dimethomorph and iprodione. However, the extensive use of pesticides can lead to problems such as bacterial resistance, food safety, and environmental pollution. Therefore, exploring efficient and environmentally friendly strawberry disease prevention and control methods and systems is of great significance to 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, disease resistance-related genes (such as PR protein genes) are induced to express, and the multiple resistance of plants to bacteria, fungi and viruses is enhanced.

[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 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 leaves to Pseudomonas, 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 strawberry fungal resistance in the prior art, and there are few reports of obvious effects. Endogenous small peptides in the field of improving strawberry fungal disease resistance need more and deeper research. Among them, there is no report on the application of endogenous small peptide FaPEP in strawberry fungal disease resistance. SUMMARY

[0005] The purpose of the present application is to provide a preparation of strawberry endogenous peptide FaPep5 and its application in the field of improving strawberry fungal disease resistance. The purpose of the present application is achieved by the following technical scheme: Application of strawberry endogenous peptides. The plant in the embodiment of the present invention is octoploid 'Hongyan' strawberry. The application is any of the following: C1) Application of strawberry endogenous peptides in regulating strawberry fungal disease resistance; C2) Application of strawberry endogenous peptides in the preparation of products for regulating strawberry fungal disease resistance; C3) Application of strawberry endogenous peptides in preventing and controlling strawberry fungal diseases; C4) Application of strawberry endogenous peptides in the preparation of products for preventing and controlling strawberry fungal diseases; C5) Application of strawberry endogenous peptides in strawberry breeding; C6) Endogenous strawberry peptides activate strawberry disease resistance genes PR Application in expression; C7) Strawberry endogenous peptides in the preparation of activating strawberry disease resistance genes PR Application of the expressed product; The strawberry endogenous peptide is any one of the following: A1) The amino acid sequence is the small peptide of SEQ ID No. 1, namely the mature small peptide FaPep5 amino acid sequence: EDVVVVATGTARSTRALGVKRPIGTGSGPQIN.

[0006] A2) A small peptide whose amino acid sequence is SEQ ID No. 2: GPEF EDVVVVATGTARSTRALGVKRPIGTGSGPQIN (compared to SEQ ID No. 1, it contains a protease recognition sequence and a restriction enzyme cleavage site, a total of 4 amino acids) A3) The amino acid sequence of the polypeptide is SEQ ID No. 3, i.e., the amino acid sequence of the encoded FaPep5 precursor protein FaPROEPE5: MESSSSADQREEEEEERHPLLHSPCHFFEQLITSCLKCLGLDHHFSSSSSSAAADDGKTKADHHQQCPPQQTEMEMKLGEDVVVVATGTARSTRALGVKRPIGTGSGPQINRASS*; Application of a nucleotide sequence, wherein the application is any of the following: D1) Application in regulating resistance to strawberry fungal diseases; D2) Application in the preparation of products for regulating resistance to strawberry fungal diseases; D3) Application in the prevention and control of strawberry fungal diseases; D4) Application in the preparation of products for preventing and controlling strawberry fungal diseases; D5) Application in strawberry breeding; D6) Application in activating the expression of strawberry disease resistance gene PR; D7) Application in the preparation of products for activating the expression of strawberry disease resistance gene PR; The nucleotide sequence is any one of the following: B1) a nucleotide sequence encoding the strawberry endogenous peptide according to claim 1; B2) The sequence is the nucleotide sequence of SEQ ID No. 4, namely: FaPROPEP5 Gene nucleotide sequence: ATGGAGTCCTCGAGCTCAGCCGACCAGAGAGAAGAAGAAGAAGAAGAGCGACACCCTTTGTTGCACAGCCCCTGCCATTTCTTCGAGCAACTCATCACCTCATGCCTCAAGTGCCTCGGACTTGATCATCACTTCTCCTCCTCCTCCTCCGCCGCCGCCGACGATGGAAAGAC AAAGGCAGATCATCATCAACAGTGCCCTCCACAGCAGACTGAAATGGAAATGAAACTAGGAGAAAGATGTTGTTGTAGTAGCCACAGGTACTGCAAGGTCGACAAGAGCGTTGGGAGTGAAACGGCCTATAGGCACTGGTTCAGGTCCTCAAATTAACCGTGCTTCATCCTAG.

[0007] A method for preparing strawberry endogenous peptides comprises the following steps: E1) Cloning method of FaPep5 fragment: Prepare FaPROPEP5 The recombinant plasmid of the gene FaPROPEP5 The nucleotide sequence of the gene is shown in SEQ ID No.4; FaPROPEP5 The recombinant plasmid of the gene was used as a template for PCR amplification to obtain the amplified FaPep5 fragment; E2) Preparation of FaPep5 peptide: Prepare FaPep5 The recombinant plasmid containing the fragment FaPep5 The recombinant plasmid of the fragment was introduced into the engineered bacteria for expression, the bacterial solution was collected by centrifugation and purified to obtain the target protein, the obtained protein was proteolytically cleaved, separated by a column and freeze-dried to obtain the small peptide FaPep5, whose amino acid sequence is SEQ ID No.2.

[0008] The specific operations of step E1) include: E11) using strawberry cDNA as a template and a first set of specific primers to perform PCR amplification, wherein the nucleotide sequences of the first set of specific primers FaPROPEP5-F and FaPROPEP5-R are shown in SEQ ID No. 5 (ATGGAGTCCTCGAGCTCA) and SEQ ID No. 6 (CTAGGATGAAGCACGGTTAA), respectively; E12) The obtained PCR product was connected with the pClone007 cloning vector to obtain a ligation product. pClone007- FaPROPEP5 ; E13) Connect the product pClone007-FaPROPEP5 Transform E. coli DH5α and spread on LB solid medium containing ampicillin. Pick a single colony, shake the bacteria, extract the plasmid, perform plasmid PCR, and then send the plasmid for sequencing to obtain the recombinant plasmid with the correct sequence. E14) to sequence the correct pClone007-FaPROPEP5 The recombinant plasmid was used as a template and a second set of specific primers were used to perform PCR amplification to obtain the FaPep5 fragment; 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.

[0009] The specific operations of step E2) include: E21) will expand FaPep5 Fragments and double enzyme digestion ( BamH Ⅰ. EcoR Ⅰ) The pTac-His-MBP plasmid was ligated and transformed into Escherichia coli DH5α and plated on LB solid medium containing ampicillin. A single colony was picked and shaken to extract the plasmid. After plasmid PCR, the plasmid was sequenced to obtain the recombinant plasmid with the correct sequence. E22) The recombinant plasmid obtained in E21) was introduced into the E. coli BL21 (DE3) engineering bacteria expression system for expression. The bacterial solution was shaken at 37 °C and 220 rpm until the OD 600 When the concentration of 0.5 μg / L IPTG was 0.6-0.8, 0.1 mg / L IPTG was added and cultured at 28°C and 220 rpm for 16 h. The bacterial precipitate was collected by centrifugation, resuspended in PBS and ultrasonically disrupted, the supernatant was collected, and the target protein was purified by His-tag column; E23) The protein obtained in E22) is digested with HRV 3C protease at 4 ℃ overnight, the flow-through liquid containing small peptide FaPep5 is collected by His-tag column and freeze-dried into powder to obtain small peptide FaPep5.

[0010] A method for improving the resistance of strawberry to fungal diseases, wherein 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, and the diluent is driped on the roots of strawberry.

[0011] The fungal disease is any one of gray mold, anthracnose, and root rot.

[0012] When the fungal disease is gray mold, the concentration of the diluent is not less than 200 nM.

[0013] When the fungal disease is anthracnose, the concentration of the diluent is not less than 100 nM.

[0014] When the fungal disease is root rot, the concentration of the diluent is not less than 400 nM.

[0015] The advantages and beneficial effects of the present application are: 1) The present application applies the endogenous peptide of strawberry to inhibit the strawberry diseases caused by fungal pathogens such as Botrytis cinerea, Colletotrichum gloeosporioides and Fusarium oxysporum, which can effectively improve the resistance of strawberry to pathogen infection, and provides a new idea for the prevention and control of strawberry pathogens.

[0016] 2) The endogenous peptide of strawberry in the preparation of the present application is a plant-friendly substance, which can be degraded in the environment and organism, and has no residue after use, and is harmless to human body and environment.

[0017] 3) The preparation of the present application is efficient, easy and low in cost for preventing and controlling the fungal diseases of strawberry. BRIEF DESCRIPTION OF DRAWINGS

[0018] The present application will be further described below in combination with the drawings and examples.

[0019] Figure 1 For examples 1, 2 and 3 FaPROPEP5 respond to different fungal pathogen infections, wherein Figure 1 A is FaPROPEP5 responding to Botrytis cinerea infection; Figure 1 B is FaPROPEP5 responding to Colletotrichum gloeosporioides infection; Figure 1 C is FaPROPEP5 responding to Fusarium oxysporum infection.

[0020] Figure 2 is FaPep5Schematic diagram of the recombinant plasmid structure in which the fragment was connected to pTac-His-MBP.

[0021] Figure 3 This is a staining diagram of the His-MBP-FaPep5 induced expression under different temperature conditions in Example 4, where M is a protein marker; 1 is the supernatant induced at 16°C; 2 is the supernatant induced at 19°C; 3 is the supernatant induced at 22°C; 4 is the supernatant induced at 25°C; 5 is the supernatant induced at 28°C; 6 is the supernatant induced at 31°C; 7 is the supernatant induced at 34°C; and 8 is the supernatant induced at 37°C.

[0022] Figure 4 This is a staining image of the flow-through containing FaPep5 collected after His-MBP-FaPep5 was cleaved by HRV 3C protease overnight in Example 4.

[0023] Figure 5 This is the protein spectrum result of FaPep5 after proteolytic cleavage that was purified and collected in Example 4.

[0024] Figure 6 The strawberry fruits in Example 5 were treated with different concentrations of FaPep5 and water as a control for 24 h and then inoculated with Botrytis cinerea cake to compare the disease incidence of the strawberry fruits. Figure 6 A is the lesion area of ​​strawberry fruit 3 days after inoculation with Botrytis cinerea cake; Figure 6 B is a picture of the disease condition of strawberry fruit 3 days after inoculation with Botrytis cinerea cake.

[0025] Figure 7 The strawberry fruits in Example 6 were treated with different concentrations of FaPep5 and water as a control for 24 h and then inoculated with anthrax cakes to compare the incidence of the strawberry fruits. Figure 7 A is the lesion area of ​​strawberry fruit 3 days after inoculation with anthracnose cake; Figure 7 B is a diagram showing the disease condition of strawberry fruit 3 days after inoculation with anthrax cake.

[0026] Figure 8 The strawberry plants in Example 7 were pretreated with FaPep5 and water for 2 days before being inoculated with Fusarium scutellariae. The comparison of the disease incidence of the strawberry plants was performed. Figure 8 A is a picture of the status of strawberry plants after 7 days in a vermiculite matrix mixed with Fusarium mycelium; Figure 8 B is the disease index of strawberry plant stems infected with Fusarium oxysporum (disease index: no disease is 0, the length of the diseased part accounts for 0-20% of the total stem length is 1, 20%-40% is 2, 40%-60% is 3, 60%-80% is 4, and 80%-100% is 5). Disease index = 100 × ∑ (number of diseased leaves at each level × representative value of each level) / (total number of leaves surveyed × highest representative value) Figure 9 This is the response of strawberry resistance genes to FaPep5 treatment in Example 8. DETAILED DESCRIPTION

[0027] Example 1 Strawberry fruit FaPROPEP5 Response to Botrytis cinerea infection 1. Strawberry fruit inoculation with Botrytis cinerea cake Strawberry fruits were harvested for this study. Botrytis cinerea was inoculated onto PDA medium and cultured at 23°C for 2-3 weeks. A bacterial cake was then inoculated onto strawberry fruits using a borer and cultured in the dark at 23°C. The fruit pulp surrounding the inoculated cake was collected at 0, 12, 24, 48, 72, and 96 hours of incubation and rapidly frozen in liquid nitrogen for later use.

[0028] 2. Total RNA extraction and reverse transcription Using the OmegaE.ZNA plant RNA kit, total RNA was extracted from all strawberry pulp samples. The specific steps are as follows: RNA extraction: (1) Sample grinding: Place 100 mg of sample in a liquid nitrogen pre-cooled mortar, add liquid nitrogen and freeze-grind until smooth; (2) Lysis: Add the ground plant sample to a 1.5 mL RNase-free EP tube pre-cooled with liquid nitrogen using a small spoon pre-cooled with liquid nitrogen, add 500 μL of RCL lysis buffer, 70 μL of Plantaid, and 10 μL of mercaptoethanol, vortex for 2 min, and place in a 60°C water bath for 2 min. Repeat the vortex / water bath 3-4 times. (3) Centrifugation: 12500 rpm for 12 min; (4) DNA removal: aspirate the supernatant after centrifugation and add it to the DNA adsorption column, and centrifuge at 12500 r / s for 1 min; (5) Precipitate RNA: Add an equal volume of RCB to the filtered supernatant and pipette to mix thoroughly. (6) RNA collection column: Transfer the RNA precipitate to the RNA collection column, centrifuge at 12500 r / s for 1 min, and discard the filtrate; (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; (8) Removal of small molecules such as glycophenols: Add 500 μL of Buffer II to the column, centrifuge at 12500 r / s for 1 min, discard the filtrate, and repeat once; (9) RNA drying: centrifuge the RNA collection column at 12500 r / s for 2 min; (10) RNA collection: Place the column in a new 1.5 ml RNase-free EP tube, draw 40 μl of DEPC water preheated at 60°C, drop the column membrane vertically in the center, let it stand for 2 minutes, centrifuge at 12000 r / s for 1 minute, and the filtrate is the final RNA sample.

[0029] Reverse transcribe the extracted strawberry pulp total RNA into cDNA using the reverse transcription system specified in the Novozyme (R223) instructions. Add all reagents to a 200 μL RNase-free PCR tube. Place the tube in a PCR instrument and perform the reaction at 37°C for 15 minutes and 85°C for 5 seconds to obtain strawberry pulp cDNA. Finally, store the sample in a -20°C freezer for subsequent RT-qPCR experiments. 3. FaPROPEP5 Response to Botrytis cinerea infection Figure 1 As shown in A, using real-time fluorescence quantitative PCR technology, FaPROPEP5 The transcription level of FaPROPEP5 The expression level was significantly upregulated 48 hours after inoculation with Botrytis cinerea cake, and was involved in the response of strawberry fruit to Botrytis cinerea infection.

[0030] Example 2 Strawberry fruit FaPROPEP5 Response to infection with Colletotrichum gloeosporioides 1. Inoculation of strawberry fruit with Colletotrichum gloeosporioides cake Strawberry fruits were harvested for this study. Colletotrichum gloeosporioides was inoculated onto PDA medium and cultured at 23°C for 2-3 weeks. A bacterial cake was then inoculated onto strawberry fruits using a borer and cultured in the dark at 23°C. The fruit pulp surrounding the inoculated cake was collected at 0, 12, 24, 48, 72, and 96 hours of incubation and rapidly frozen in liquid nitrogen for later use.

[0031] 2. Total RNA Extraction and Reverse Transcription Total RNA was extracted from all strawberry pulp samples using the OmegaE.ZNA plant RNA kit. The extracted total RNA was reverse transcribed into cDNA using Novozyme (R223). The specific process was the same as in Example 1.

[0032] 3. FaPROPEP5 Response to infection with Colletotrichum gloeosporioides like Figure 1 As shown in B, using real-time fluorescence quantitative PCR technology, FaPROPEP5 The transcription level of FaPROPEP5 The expression level was significantly upregulated 48 hours after inoculation with Colletotrichum gloeosporioides cake, and it was involved in the resistance response of strawberry fruit to Colletotrichum gloeosporioides.

[0033] Example 3 Strawberry root system FaPROPEP5 Response to Fusarium acephate infection 1. Transplant the strawberry plant roots into the vermiculite culture medium containing Fusarium scabra mycelium Strawberry tissue culture seedlings were used in this case study. Fusarium lanceolatum hyphae were inoculated into PDB liquid medium and cultured at 23°C for 2 days. The pellet was collected by centrifugation, mixed evenly with vermiculite, and then transplanted into strawberry plants. The plants were cultured at 23°C under a 12-hour light-dark cycle. Roots were harvested at 0, 12, 24, 48, 72, and 96 hours of incubation and quickly frozen in liquid nitrogen for later use.

[0034] 2. Total RNA Extraction and Reverse Transcription Total RNA was extracted from all strawberry root samples using the OmegaE.ZNA plant RNA kit. The extracted strawberry pulp total RNA was reverse transcribed into cDNA using Novozyme (R223). The specific process was the same as in Example 1.

[0035] 3. FaPROPEP5 Response to Fusarium acerifolium infection like Figure 1 As shown in C, using real-time fluorescence quantitative PCR technology, FaPROPEP5 The transcription level of FaPROPEP5 The expression level was significantly upregulated 72 hours after inoculation with Fusarium oxysporum, and was involved in the resistance response of strawberry to anthracnose.

[0036] Example 4 Cloning of FaPep5 fragment: PCR amplification was performed using strawberry cDNA as a template and a first set of specific primers. The nucleotide sequences of the first set of heterosexual 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 with the pClone007 cloning vector to obtain a ligation product. pClone007-FaPROPEP5 ; Connect the product pClone007-FaPROPEP5 Transform E. coli DH5α and spread on LB solid medium containing ampicillin. Pick a single colony, shake the bacteria, extract the plasmid, perform plasmid PCR, and then send the plasmid for sequencing to obtain the recombinant plasmid with the correct sequence. Correct sequencing pClone007-FaPROPEP5 The recombinant plasmid was used as a template and a second set of specific primers was used to perform PCR amplification to obtain FaPep5fragment; 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.

[0037] Preparation of FaPep5: The amplified FaPep5 fragment was double-digested with BamH I, EcoR I) and ligated to the pTac-His-MBP plasmid to transform E. coli DH5a and 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 sequenced to obtain a recombinant plasmid with correct sequence. The obtained recombinant plasmid was introduced into the E. coli BL21 (DE3) engineering bacteria expression system for expression. The bacterial liquid was shaken at 37 °C and 220 rpm until the OD 600 was 0.6-0.8, 0.1 mg / L IPTG was added, and the culture was incubated at 28 °C (the optimal induction temperature) and 220 rpm for 16 h. The bacterial liquid was collected by centrifugation, resuspended with PBS, and broken by ultrasonic. The supernatant was collected, and the target protein was obtained by His-tag column purification. The induction results are shown in Figure 3 . The obtained protein was digested with HRV 3C protease at 4 °C overnight, and the polypeptide FaPep5-containing flow-through liquid was collected by His-tag column separation. After Coomassie brilliant blue staining Figure 4 and protein spectrum confirmation Figure 5 , the FaPep5-containing flow-through liquid was freeze-dried into powder to prepare the small peptide FaPep5 of the present application, and the amino acid sequence thereof is SEQ ID No. 2 GPEF EDVVVVATGTARSTRALGVKRPIGTGSGPQIN (containing a protease recognition sequence and a digestion 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.

[0038] Example 5 Application of FaPep5 in strawberry fruit resistance to gray mold In this example, the small peptide FaPep5 prepared in Example 4 was used to stimulate the resistance to gray mold of the strawberry fruit by feeding to simulate the absorption of the strawberry fruit, so as to control the strawberry gray mold. The main steps include: 1. Preparation of FaPep5 small peptide solution The FaPep5 small peptide is diluted with water to form a FaPep5 solution with a concentration of 100 nM, 200 nM, or 400 nM. Prior to the present application, there is no report on the function of the small peptide FaPep5.

[0039] 2. Treatment of strawberry with FaPep5 small peptide The plant treatment method of the FaPep5 small peptide is feeding. The strawberry peduncle is fed with a water solution of the FaPep5 small peptide with a concentration of 100 nM, 200 nM, or 400 nM to simulate fruit absorption, and water is used as a control. After 24 hours of feeding, the strawberry fruits of the treated plants are inoculated with a Botrytis cinerea cake, and are 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 As shown in Table 1, the strawberry fruits treated with the above solutions have a significantly stronger resistance to gray mold than the control strawberry fruits. The fruit resistance to gray mold has a FaPep5 concentration dependence, and the higher the FaPep5 concentration, the stronger the fruit resistance.

[0040] Example 6 Application of FaPep5 in strawberry fruit resistance to anthracnose In the present application, the FaPep5 small peptide prepared in Example 4 is used to stimulate the production of resistance to anthracnose bacteria by feeding to simulate strawberry fruit absorption, so as to control strawberry anthracnose. The main steps include: 1. Preparation of FaPep5 small peptide solution The FaPep5 small peptide is diluted with water to form a FaPep5 solution with a concentration of 100 nM, 200 nM, or 400 nM.

[0041] 2. Treatment of strawberry seedlings with FaPep5 small peptide The plant treatment method of the FaPep5 small peptide is feeding. The strawberry peduncle is fed with a water solution of the FaPep5 small peptide with a concentration of 100 nM, 200 nM, or 400 nM to simulate fruit absorption, and water is used as a control. After 24 hours of feeding, the strawberry fruits of the treated plants are inoculated with a Botrytis cinerea cake, and are 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 As shown in Table 1, the strawberry fruits treated with the above solutions have a significantly stronger resistance to gray mold than the control strawberry fruits. The fruit resistance to gray mold has a FaPep5 concentration dependence, and the higher the FaPep5 concentration, the stronger the fruit resistance.

[0042] Example 7 Application of FaPep5 in strawberry root resistance to Ophiostoma japonicum The present invention utilizes the FaPep5 peptide prepared in the example to simulate root irrigation in production by adding FaPep5 to the rooting medium, thereby stimulating the plant's resistance to Fusarium spp., thereby controlling Fusarium spp. in strawberry. The main steps include: 1. Preparation of FaPep5 Peptide Solution The FaPep5 peptide was diluted with water to a 400 nM concentration of FaPep5 solution.

[0043] 2. Treatment of strawberry seedlings with FaPep5 peptide The plant treatment method of FaPep5 peptide is root treatment. Strawberry tissue culture seedlings that have been rooted for 3 weeks were cultured in rooting medium (MS + 30 g / L sucrose + 8 g / L agar + 400 nM FaPep5) with a concentration of 400 nM FaPep5. The rooting medium without FaPep5 was used as the control. After pretreatment at 23 ℃ and 12 h-12 h dark-light alternation for 7 days, the mycelium of Fusarium lanceolatum was evenly mixed with vermiculite and transplanted into the strawberry plants. The plants were cultured at 23 ℃ and 12 h-12 h light-dark alternation. The results are as follows: Figure 8 As shown, the strawberry plants treated with the above preparations had significantly stronger resistance to Fusarium spp. than the control strawberry plants.

[0044] Example 8 FaPep5 activates disease resistance genes in strawberry Three-week-old strawberry tissue culture seedlings were selected and FaPep5 prepared in Example 4 was added to a rooting medium (MS + 30 g / L sucrose + 8 g / L agar + 400 nM FaPep5). Standard rooting medium was used as a control. The plants were cultured under a 12-hour dark-light cycle. Root samples were collected from plants treated for 5, 10, 15, 20, 25, and 30 minutes and rapidly frozen in liquid nitrogen for later use. Total RNA was extracted and reverse transcribed for subsequent RT-qPCR experiments.

[0045] The results are as follows Figure 9 As shown, FaPep5 can activate the expression of strawberry disease resistance gene PR.

[0046] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention and is not limiting. Although the present invention is described in detail with reference to the preferred arrangement scheme, ordinary technicians in this field should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. An application of strawberry endogenous peptide, characterized in that: The application is any of the following: C1) Application of strawberry endogenous peptides in regulating strawberry fungal disease resistance; C2) Application of strawberry endogenous peptides in the preparation of products for regulating strawberry fungal disease resistance; C3) Application of strawberry endogenous peptides in preventing and controlling strawberry fungal diseases; C4) Application of strawberry endogenous peptides in the preparation of products for preventing and controlling strawberry fungal diseases; C5) Application of strawberry endogenous peptides in strawberry breeding; C6) Endogenous strawberry peptides activate strawberry disease resistance genes PR Application in expression; C7) Strawberry endogenous peptides in the preparation of activating strawberry disease resistance genes PR Application of the expressed product; The strawberry endogenous peptide is any one of the following: A1) a small peptide whose amino acid sequence is SEQ ID No. 1; A2) a small peptide whose amino acid sequence is SEQ ID No. 2; A2) A polypeptide whose amino acid sequence is SEQ ID No.

3.

2. Application of nucleotide sequence, characterized in that, The application is any of the following: D1) Application in regulating resistance to strawberry fungal diseases; D2) Application in the preparation of products for regulating resistance to strawberry fungal diseases; D3) Application in the prevention and control of strawberry fungal diseases; D4) Application in the preparation of products for preventing and controlling strawberry fungal diseases; D5) Application in strawberry breeding; D6) Activating disease-resistant genes in strawberries PR Application in expression; D7) Activating disease resistance genes in strawberries PR Application of the expressed product; The nucleotide sequence is any one of the following: B1) a nucleotide sequence encoding the strawberry endogenous peptide according to claim 1; B2) The sequence is the nucleotide sequence of SEQ ID No.

4.

3. A method for preparing strawberry endogenous peptides, characterized in that: The following steps are involved: E1) Prepare FaPROPEP5 The recombinant plasmid of the gene FaPROPEP5 The nucleotide sequence of the gene is shown in SEQ ID No. 4; FaPROPEP5 The recombinant plasmid of the gene was used as a template for PCR amplification to obtain the amplified FaPep5 fragment; E2) Prepare FaPep5 The recombinant plasmid containing the fragment FaPep5 The recombinant plasmid of the fragment was introduced into the engineered bacteria for expression, the bacterial solution was collected by centrifugation and purified to obtain the target protein, the obtained protein was proteolytically cleaved, separated by a 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 peptide according to claim 3, wherein The following steps are involved: The specific operations of step E1) include: E11) using strawberry cDNA as a template and a first set of specific primers to perform PCR amplification, wherein the nucleotide sequences of the first set of specific primers FaPROPEP5-F and FaPROPEP5-R are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively; E12) The obtained PCR product was connected with the pClone007 cloning vector to obtain a ligation product. pClone007- FaPROPEP5 ; E13) Connect the product pClone007-FaPROPEP5 Transform E. coli DH5α and spread on LB solid medium containing ampicillin. Pick a single colony, shake the bacteria, extract the plasmid, perform plasmid PCR, and then send the plasmid for sequencing to obtain the recombinant plasmid with the correct sequence. E14) to sequence the correct pClone007-FaPROPEP5 The recombinant plasmid was used as a template and a second set of specific primers was used to perform PCR amplification to obtain FaPep5 fragment; 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 for preparing strawberry endogenous peptide according to claim 3, characterized in that: The following steps are involved: The specific operations of step E2) include: E21) will expand FaPep5 Fragments and double enzyme digestion ( BamH Ⅰ. EcoR Ⅰ) The pTac-His-MBP plasmid was ligated and transformed into Escherichia coli DH5α and plated on LB solid medium containing ampicillin. A single colony was picked and shaken to extract the plasmid. After plasmid PCR, the plasmid was sequenced to obtain the recombinant plasmid with the correct sequence. E22) The recombinant plasmid obtained in E21) was introduced into the E. coli BL21 (DE3) engineering bacteria expression system for expression. The bacterial solution was shaken at 37 °C and 220 rpm until the OD 600 When the concentration of 0.5 μg / L IPTG was 0.6-0.8, 0.1 mg / L IPTG was added and cultured at 28°C and 220 rpm for 16 h. The bacterial precipitate was collected by centrifugation, resuspended in PBS and ultrasonically disrupted, the supernatant was collected, and the target protein was purified by His-tag column. E23) The protein obtained in E22) was cleaved with HRV 3C protease at 4°C, and the flow-through containing the small peptide FaPep5 was collected by separation with a His-tag column and lyophilized into powder to obtain the small peptide FaPep5.

6. A method for improving resistance to fungal diseases in strawberries, 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 dilution to feed strawberry cobs or drip irrigate the strawberry roots.

7. The method according to claim 6, characterized in that: The fungal disease is any one of gray mold, anthracnose, and root rot.

8. The method according to claim 7, wherein: When the fungal disease is gray mold, the concentration of the dilution solution is not less than 200 nM.

9. The method according to claim 7, wherein: When the fungal disease is anthracnose, the concentration of the dilution solution is not less than 100 nM.

10. The method according to claim 7, wherein: When the fungal disease is root rot, the concentration of the dilution solution is not less than 400 nM.

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