Polypeptide ligand SR-22 targeting pathogen oomycete membrane protein and application of polypeptide ligand SR-22

Abstract

The invention discloses a polypeptide ligand SR-22 of a targeted pathogen oomycete membrane protein and an application of the polypeptide ligand SR-22. The amino acid sequence of the polypeptide ligand SR-22 is SRITRLLRRGSGSGSSRITRLLR. The polypeptide ligand SR-22 provided by the invention can remarkably inhibit the growth of various crop pathogenic oomycetes, can well inhibit pathogenic bacteria infection and prevent diseases in practical application, can be subsequently developed into a novel targeted polypeptide pesticide, and is used for green prevention and control of crop oomycetes diseases.

Description

Technical Field

[0001] The present invention relates to a polypeptide ligand SR-22 targeting a conserved key membrane protein of pathogenic oomycetes and application thereof, belonging to the field of crop disease prevention and control in the subject of agronomy plant protection. Background Art

[0002] Oomycetes belong to the class Oomycetes of the phylum Oomycota, kingdom Chromista. They are diploid eukaryotic microorganisms that are similar in morphology to fungi (haploid) but more closely related to diatoms and brown algae. Oomycetes currently include 15 orders, 24 families, nearly 100 genera, and about 1,500 species. Most of them are plant pathogens with a wide host range and strong harmfulness, harming important crops such as grains, vegetables, flowers, and fruit trees. For example, Phytophthora pathogenic fungi, soybean fungi, pepper fungi, camphor fungi, etc. can cause diseases such as rot and fruit shedding of potatoes, soybeans, fruits and vegetables, and trees. The annual crop yield loss caused by crop diseases in the world is as high as trillions of dollars; Pythium aphanidermatum, Pythium ultimum, Pythium bellii, etc. of the genus Pythium attack the roots of crops, causing crop death; Peronophythora litchii downy mildew harms litchi through air transmission, causing flower rot and fruit rot. These pathogenic oomycetes have caused significant losses to agricultural production. At present, my country's modern agriculture is developing rapidly, and the planting structure is transforming and upgrading. Oomycete diseases are a major challenge facing agricultural production and ecosystems.

[0003] Oomycetes can be spread through soil, rainwater, air flow and other channels. They have the characteristics of short incubation period, fast diffusion speed, strong infection ability and multi-site damage. After the pathogen invades the host, it can quickly form a large number of zoosporangia and release zoospores, forming a re-infection source, causing the disease to spread rapidly and making prevention and control very difficult. At present, the effects of chemical control and agricultural control of oomycete diseases are not ideal. On the one hand, it is because of the special characteristics of oomycetes in terms of transmission and infection. On the other hand, there are few types of fungicides for oomycetes. On the other hand, oomycetes mutate quickly in the field and have high genetic diversity, which can easily lead to problems such as drug resistance of pathogens and "loss of resistance" of varieties. Moreover, the large-scale use of single chemical pesticides has led to the emergence of the "3R" problem. These problems are not conducive to the implementation of my country's current policy of "double reduction" of pesticides and the sustainable development of modern agriculture. Therefore, it is particularly important to explore new, efficient and green methods for the prevention and control of oomycete diseases.

[0004] Studies have found that oomycetes and fungi have significant differences in cell wall structure, genetics, physiology and biochemistry, infection and pathogenicity, etc. Therefore, discovering key proteins in the growth and development of oomycetes and the pathogenicity process can be used as new targets for disease control agents and for the development of new drugs.

[0005] A polypeptide ligand is a short peptide (also called a polypeptide or small peptide) that is screened from an artificially synthesized random amino acid library and binds to a target protein with high specificity and affinity. It achieves a specific effect by interfering with and destroying the function of the target protein. Since its introduction in 1990, polypeptide ligands have been widely used as targeted drugs in the medical field for basic clinical research, drug design, and targeted disease treatment. The market prospects for polypeptide drugs are broad. In recent years, with the development of this technology, the application potential of polypeptide ligands in crop disease prevention and control has gradually been recognized. However, there are no polypeptide targeted drugs for the prevention and control of crop oomycete diseases available in the world, and no polypeptide targeted drugs developed in my country have been registered and reported. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a polypeptide ligand SR-22 targeting the conserved key membrane protein of pathogenic oomycetes and its application, which can significantly inhibit the occurrence of crop oomycete diseases.

[0007] Technical solution: In order to solve the above technical problems, the present invention provides a polypeptide ligand SR-22 targeting the conserved key membrane protein of pathogenic oomycetes, and its amino acid sequence is SRITRLLRGSGSGSSRITRLLR.

[0008] The present invention also provides a method for obtaining the polypeptide ligand SR-22, comprising the following steps:

[0009] 1. Screening of conserved proteins in oomycetes

[0010] The protein sequences of seven oomycetes (Phytophthora sojae, Phytophthora ramorum, Phytophthorainfestans, Phytophthora capsici, Pythium ultimum, Pythium aphanidermatum, Hyaloperonospora arabidopsidis) were downloaded, and the whole genome sequences of the seven oomycetes were compared with those of plants, animals, fungi, bacteria, viruses, and nematodes using bioinformatics methods. The oomycete-specific and highly conserved protein PcMEM1 was screened out, and the protein domain was predicted and analyzed.

[0011] 2. Functional analysis of PcMEM1

[0012] The full-length PcMEM1 gene of Phytophthora capsici was positively cloned into the vector pGFPN and transferred into the P. capsici strain by protoplast transformation to obtain PcMEM1 overexpression transformants. The morphological characteristics and pathogenicity changes of the overexpression transformants at different growth and development stages (hyphae, zoosporangium, zoospore) were observed.

[0013] At the same time, the PcMEM1 gene was transiently expressed in Nicotiana benthamiana leaves to observe the leaf phenotype.

[0014] 3. Screening and identification of targeting peptides

[0015] Randomly composed oligonucleotide fragments were assembled in the pGADT7-derived plasmid pLIB2 and then amplified in Escherichia coli to obtain a polypeptide ligand library; the coding gene of the membrane protein PcMEM1 was cloned in pGBKT7 as bait, and the polypeptide library was screened by yeast two-hybridization; plasmid DNA was extracted from positive colonies and sequenced to obtain polypeptide sequences; the polypeptide plasmid and the bait protein plasmid were co-transformed into yeast and re-verified by yeast two-hybridization.

[0016] The present invention also provides a reagent or medicine containing the polypeptide ligand SR-22.

[0017] The present invention also provides the use of the polypeptide ligand SR-22 or the reagent or drug in inhibiting the growth of oomycetes.

[0018] The present invention also provides the use of the polypeptide ligand SR-22 or the reagent or drug in preventing and controlling oomycete diseases of crops.

[0019] Among them, the oomycete is an oomycete that uses the conserved key membrane protein PcMEM1 as a key factor for growth, development, infection and pathogenicity.

[0020] Wherein, the oomycetes include fungi of the genus Phytophthora, Peronophythora, Phytopythium or Pythium.

[0021] Wherein, the oomycetes include Phytophthora capsici, Phytophthora infestans, Phytophthora sojae, Phytophthora cactorum, Peronophythora litchii, Phytopythium helicoides, Pythium aphanidermatum, Pythium vexans or Pythium ultimum.

[0022] The effective inhibitory concentration of the polypeptide ligand SR-22 is 0.0003-0.3517 mM.

[0023] Wherein, the crops include potatoes, peppers or litchi.

[0024] The present invention also provides a plate antibacterial test of the polypeptide ligand SR-22: the polypeptide SR-22 is added to a rye V8 solid culture medium or a PDA culture medium to prepare a drug-containing plate with a polypeptide concentration of 200, 300, and 500 μM, respectively. Nine plant pathogenic oomycetes of different nutritional types (Phytophthora capsici, Phytophthora infestans, Phytophthora sojae, Phytophthora cactorum, Peronophythora litchii, Phytopythium helicoides, Pythium ultimum, Pythium vexans, and Pythium aphanidermatum) are inoculated to determine the inhibitory effect of SR-22 on their growth.

[0025] The present invention also provides a plant leaf spraying test: a potato (Solanum tuberosum) variety Désirée grown in a greenhouse pot is selected, a 500 μM SR-22 polypeptide solution is sprayed on the leaf surface, and a suspension of zoospores of Phytophthora infestans is inoculated 12 hours later to determine the control effect of SR-22 on potato late blight.

[0026] The present invention also provides a plant root irrigation test: a pepper (Capsicum annuum) variety "Hanyu Special Horn Pepper" is selected. A 500 μM polypeptide solution is irrigated to the roots of pepper seedlings planted in a greenhouse, and then a suspension of Phytophthora capsici zoospores is inoculated to the roots 8 hours later to determine the effect of SR-22 in preventing and controlling pepper root blight.

[0027] The present invention also provides a fruit infiltration test: the fruit of the litchi (Litchi chinensis) variety "Guiwei" is selected, the litchi fruit is fully infiltrated with a 500 μM SR-22 polypeptide solution, and litchi downy mildew is inoculated 2.5 hours later to determine the effect of SR-22 in preventing and controlling litchi downy mildew.

[0028] The present invention also provides a polypeptide antibacterial specificity analysis: non-target fungi - Magnaporthe grisea and non-target bacteria - Escherichia coli DH5α strain were selected. SR-22 was added to their culture media to a final concentration of 200, 300, and 500 μM, respectively, and fungi or bacteria were inoculated. The colony growth inhibition rate of Magnaporthe grisea was measured, and the absorbance value OD of Escherichia coli was measured every 1 hour.600 , analyze whether the peptides have an inhibitory effect on the growth of non-target microorganisms (fungi and bacteria).

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention targets a conserved key membrane protein PcMEM1 involved in the growth, development and pathogenicity of oomycetes, and uses biotechnology to develop for the first time a polypeptide ligand SR-22 that targets and chelates it; 2. The polypeptide ligand SR-22 significantly inhibits the growth of oomycetes, but does not inhibit the growth of non-target fungi and bacteria, and has good specificity; 3. The polypeptide ligand SR-22 has good antibacterial and disease resistance effects, and is expected to be used as a new, safe and environmentally friendly polypeptide pesticide for the green prevention and control of crop oomycete diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 To analyze the conservation of PcMEM1 in oomycetes;

[0031] Figure 2 For the domain analysis of PcMEM1 protein;

[0032] Figure 3 Subcellular localization of PcMEM1 in the hyphae of overexpressed transformants; A, hyphae under laser confocal microscope; B, Western blot detection of PcMEM1 protein expression;

[0033] Figure 4 Overexpression of PcMEM1 affects the growth and pathogenicity of pepper Phytophthora; A, observation of growth and development characteristics of different strains; B, comparison of growth rates of different strains; C, comparison of the number of zoosporangia of different strains (under 10x objective); D, comparison of zoospore concentration of different strains; E, comparison of resting spore germination rate of different strains; F, comparison of pathogenicity of different strains; G, comparison of lesion area caused by different strains;

[0034] Figure 5 PcMEM1 and its four oomycete homologous proteins cause cell death in Nicotiana benthamiana leaves; A, expression of PcMEM1 and its four oomycete homologous proteins 4 days after Agrobacterium injection, GFP as negative control; B, Western blot detection of protein expression;

[0035] Figure 6 The peptide SR-22 was obtained by yeast two-hybrid screening; A, the peptide ligand library was transferred into the yeast strain AH109 containing the bait protein PcMEM1, and the yeast was grown on a defective medium without tryptophan (W), leucine (L), adenine (A) and histidine (H); B, the yeast colony was streaked and grown on the above four-deficient medium containing x-α-gal for 2 days;

[0036] Figure 7 To verify the interaction between the peptide SR-22 and the bait protein PcMEM1 (i.e. Pc127259);

[0037] Figure 8 It is a synthetic SR-22 object;

[0038] Fig. 9 is the mass spectrometry data of peptide SR-22;

[0039] Fig.10 HPLC data of peptide SR-22;

[0040] Fig.11 Different concentrations of peptide SR-22 inhibited the growth of 9 oomycetes;

[0041] Fig.12 Foliar spraying of SR-22 prevents the occurrence of potato late blight; A, the disease condition of the leaves after spraying 500μM SR-22 solution and control ddH2O on potato leaves and then inoculating with Phytophthora infestans for 3 days; B, quantitative comparison of leaf lesion area after spraying 500μM SR-22 solution and control ddH2O and inoculating with Phytophthora infestans for 3 days; C, comparison of the biomass of Phytophthora infestans in potato leaves after spraying 500μM SR-22 solution and control ddH2O and inoculating with Phytophthora infestans for 6 days (asterisk *** indicates significant difference P<0.001);

[0042] Fig.13 SR-22 prevents the occurrence of pepper root blight; A, the disease of pepper after irrigating the pepper root with 500μM peptide SR-22 solution and control ddH2O and then inoculating pepper Phytophthora zoospores for 3 days; B, the statistical diagram of the lesion length of pepper root after inoculation with pepper Phytophthora 4 days after ddH2O treatment and SR-22 treatment (asterisk * indicates significant difference P<0.01);

[0043] Fig.14 The SR-22 solution was used to infiltrate the fruit to prevent the occurrence of litchi downy mildew; A, the disease status of litchi fruit after inoculating litchi downy mildew 68 hours after infiltrating the litchi surface with 500 μM SR-22 solution and control ddH2O for 3 times; B, the statistical graph of the disease grade of litchi fruit after inoculation with litchi downy mildew 68 hours after treatment with SR-22 and ddH2O. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0045] Example 1 Screening of Oomycete Conservative Proteins

[0046] 1. Download all protein sequences encoded by the genomes of seven oomycetes (Phytophthora sojae, Phytophthora ramorum, Phytophthora infestans, Phytophthoracapsici, Pythium ultimum, Pythium aphanidermatum, Hyaloperonosporaarabidopsidis) from the JGI database (https: / / genome.jgi.doe.gov / portal);

[0047] 2. All protein sequences of the above 7 oomycetes were self-aligned by blastP (E-value < 1e-5) using seqhunter software, and then MCL (Markov Cluster Algorithm) clustering was performed using TribeMCL software to cluster homologous sequences into the same orthogroup, and then only those orthogroups containing homologous sequences of the 7 oomycetes were selected;

[0048] 3. The highly conserved orthogroups proteins in oomycetes were compared with the proteins encoded by the genomes of 19 plants, 13 animals, 32 fungi, 17 bacteria, 5 viruses, and 8 nematodes in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) (Table 1) by blastP (E-value < 1e-5), and orthogroups with different origins in non-oomycete species were selected as the final candidate orthogroups. Based on this, the oomycete-specific and highly conserved protein PcMEM1 (protein ID Pc127259 in the JGI pepper phytophthora database, NCBI protected unpublished accession number PQ277303) was screened out. The evolutionary clustering relationship is as follows: Figure 1As shown: Among them, PcMEM1, Pc008439, DVH05_005936, DV H05_024782 are from Phytophthora capsici, PITG22036 and PITG08183 are from Phytophthora in festans, PAG1G010589 is from Pythium aphanidermatum, KAK1930365.1 is from Phytophthora citrophthora, KAG7379021.1 is from Phytophthora pseudosyringae, KUG01621.1 is from Phytophthora ranicotianae, KAG2764292.1 is from Phytophthora cactorum, KAH7482102.1 is from Phytophthora aramorum, KAE8899104.1 is from Phytophthora fragariae, POM76769.1 from Phytophthorapalmivora, CAH0493029.1 from Peronospora farinosa, CAI5703558.1 from Peronosporaeffusa, RLN94750.1 from Nothophytophthora sp., KAI9916111.1 from Peronosclerosporasorghi, CAI5741223.1 from Hyaloperonospora brassicae, TDH72102.1 from Bremialactucae, KAF1329390.1 from Globisporangium splendens, XP_024582811.1 from Plasmopara halstedii; large red dots indicate proteins that can induce cell death in Nicotiana benthamiana leaves;

[0049] Table 1 Names of other organisms whose genomes were compared with those of oomycetes

[0050]

[0051]

[0052]

[0053]

[0054] 4. Use SMART software to predict the protein domain of PcMEM1. The results are as follows: Figure 2) showed that PcMEM1 contained 5 transmembrane domains, among which TM was the transmembrane domain; ICD (intracellular domain) was the intracellular part, and the numbers indicated the amino acid positions.

[0055] Example 2 PcMEM1 gene function analysis

[0056] The full-length PcMEM1 gene of Phytophthora capsici (sequence ID 127259 in the JGI Phytophthora capsici database https: / / mycocosm.jgi.doe.gov / Phyca11 / Phyca11.home.html, NCBI protected unpublished accession number PQ277303) was forward cloned into the universal vector pGFPN (enzyme digestion system: 0.5 μL each of PacI and NheI, 5 μL of 10×Buffer, 1 μg of DNA, and sterile water to 50 μL. The enzyme digestion reaction was incubated at 37°C for 3 h. Ligation conditions: 0.1 μL of T4 DNA Ligase, 0.15 pmol of gene fragment, 0.015 pmol of linearized vector, 10×T4 DNA LigaseBuffer 1μL, and sterile water was added to 10μL. The ligation reaction was incubated at 16℃ for 12h), and it was placed behind the promoter of the downy mildew gene Ham34 (NCBI accession number X16984) of lettuce, fused with GFP protein at the C-terminus, and transformed into protoplasts mediated by CaCl2-PEG (for specific methods, refer to the literature published by Chen Xiaoren et al. Chen XR, Zhang Y, Li HY, Zhang ZH, Sheng GL, Li YP, Xing YP, Huang SX, Tao H, Kuan T, Zhai Y, Ma W. The RXLR Effector PcAvh1 Is Required for Full Virulence of Phytophthora capsici. Mol Plant Microbe Interact.2019,32(8):986-1000.doi:10.1094 / MPMI-09-18-0251-R.) was transformed into the protoplasts of the pepper phytophthora strain Pc537, and G418-resistant transformants were obtained through protoplast regeneration and 37.5μg / mL G418 screening; the GFP fluorescence of the transformants was observed under a fluorescence microscope, and positive transformants OE1, OE2 and OE3 expressing green fluorescence were obtained; the expression level of PcMEM1 in the overexpression transformants was further detected by Western blot, and it was confirmed that it was a PcMEM1 overexpression transformant, among which the expression level of the protein in the OE2 transformant was the highest. The hyphae fluorescence of these pepper phytophthora PcMEM1 gene overexpression transformants was observed, and the results ( Figure 3 ) shows that the protein is an oomycete membrane protein. Figure 3WT in the figure is the wild-type strain; Ctrl is the transformation control; OE1, OE2, and OE3 are all PcMEM1 overexpression transformant strains; the excitation wavelength is 488 nm, bar=10 μm; the antibody is GFP antibody; the morphological characteristics of the growth and development stages (hyphae, zoosporangia, zoospores) of the gene overexpression transformants were observed under a microscope, and their infection and pathogenicity were observed after inoculation with Nicotiana benthamiana ( Figure 4 ); 42 hours after inoculation, it was observed that gene overexpression significantly weakened the pathogenicity of pepper phytophthora, and 87 hours after inoculation, it was observed that gene overexpression seriously affected the growth and development of pepper phytophthora; the growth rates of the three overexpression transformant strains were significantly reduced, the mycelium had more branching, twisting and swelling, and other deformations, a higher zoosporangium deformation rate, and the size of the zoosporangium was significantly smaller. The number of zoospores and the germination rate were significantly reduced, and the pathogenicity on Nicotiana benthamiana leaves was significantly reduced. These results indicate that PcMEM1 is a key factor in the growth and development and infection and pathogenicity of oomycetes. CRISPR / Cas9 technology was used to attempt to knock out this gene in pepper phytophthora, but no transformants were obtained, further indicating that this protein is a conserved key protein in oomycetes, and knocking out this gene will lead to the death of the strain. Among them, Figure 4 Figure A shows the colony characteristics of the wild type (WT), transformation control (Ctrl) and PcMEM1 overexpressing transformants (OE1, OE2, OE3) (first row; photographed 87 hours after inoculation, bar = 1 cm), as well as hyphae morphology (second row; bar = 50 μm), zoospore production (third row; bar = 100 μm), and resting spore germination (fourth row; bar = 50 μm); in Figure F, the zoospore inoculation amount of WT, Ctrl, OE1, and OE2 was adjusted to 1500, and the inoculation amount of OE3 was 20.

[0057] At the same time, the PcMEM1, Pc008439, PITG22036, PITG08183, and PAG1_G010589 genes were cloned into the universal plasmid pBin (PcMEM1, Pc008439, PITG22036, and PITG08183 enzyme digestion system: BamHI and KpnI 0.5 μL each, 10×Buffer 5 μL, DNA 1 μg, and sterile water to 50 μL. The enzyme digestion reaction was incubated at 30°C and 37°C for 2 h each. The enzyme digestion system of PAG1_G010589: SalI and KpnI 0.5 μL each, 10×Buffer 5 μL, DNA 1 μg, and sterile water to 50 μL. The enzyme digestion reaction was incubated at 37°C for 3 h. Unified connection conditions: T4 DNA Ligase 0.1 μL, 0.15 pmol of gene fragment, 0.015 pmol of linearized vector, 1 μL of 10×T4 DNA Ligase Buffer, and sterile water to 10 μL. The ligation reaction was incubated at 16°C for 12 h, and then transformed into Agrobacterium GV3101 strain. 600 When the concentration of 1% morpholineethanesulfonic acid (MFA), 0.1 mmol / L acetosyringone (ACET), and 10 mmol / L MgCl2 (MgCl2) was 0.6, the mixture was incubated for 3 h with 10 mmol / L 4-morpholineethanesulfonic acid, 0.1 mmol / L acetosyringone, and 10 mmol / L MgCl2, and then injected into Nicotiana benthamiana leaves. The plants were cultured in a greenhouse at 20-22°C, the phenotype of the plant leaves was observed with the naked eye, and the protein expression was detected by Western blot. Results ( Figure 5 ) showed that PcMEM1 and its homologous proteins induced cell death in Nicotiana benthamiana leaves, and the proteins were expressed normally.

[0058] Example 3 Screening and identification of targeting polypeptides

[0059] 1. Use BamHI and ApaI restriction sites (restriction system: BamHI and ApaI 0.5μL each, 10× Buffer 5μL, DNA 1μg, sterile water to 50μL. The restriction reaction was incubated at 30℃ and 37℃ for 2h each. Connection conditions: T4 DNA Ligase 0.1μL, fragment 0.15pmol, linearized vector 0.015pmol, 10×T4 DNA Ligase Buffer 1 μL, and sterile water was added to 10 μL. The ligation reaction was incubated at 16°C for 12 h), and the oligonucleotide fragment (5'-GGCAGAGTGGATCCAA (NNK) 8GGCTCCGGTTCTGGCTCT (NNK) 8AAGGGGCCCCTT-3') was inserted into the universal plasmid pLIB2 (AD vector for short) derived from pGADT7, and then amplified in E. coli DH5α cells, and 1×10 6The plasmids were purified by SDS alkaline lysis method to obtain the peptide ligand library;

[0060] 2. Clone the gene encoding the membrane protein PcMEM1 into the universal plasmid pGBKT7 (BD vector for short);

[0061] 3. Using PcMEM1 as bait protein, the peptide ligands that can specifically target and bind to the bait protein PcMEM1 were screened from the peptide ligand library by yeast two-hybrid technology ( Figure 6 ); wherein, the plasmid was isolated from the blue positive colonies indicated by the box and sequenced to obtain the sequence of the polypeptide SR-22;

[0062] 4. Extract plasmid DNA from positive colonies grown on yeast four-deficient medium (-LWHA) and sequence them to obtain the specific sequence of polypeptide SR-22;

[0063] 5. The obtained polypeptide plasmid SR-22 and the bait protein PcMEM1 plasmid were co-transformed into yeast and yeast two-hybrid verification was performed to confirm that SR-22 could interact with the target protein PcMEM1 ( Figure 7 ). The text on the left indicates different combinations of yeast two-hybridization, BD is the abbreviation of vector pGBKT7, AD is the abbreviation of vector pLIB2, and the combination of RecT-AD+Lam-BD is the reciprocal positive control; the top is the four-deficient culture medium used, 3-AT is the competitive inhibitor of yeast HIS3 protein (His3p), 3-amino-1,2,4-triazole, and the four colonies in each group in the small picture are 10-fold gradient diluted from left to right, and the colonies were grown on the culture medium for 4 days and then photographed. SR-22 was synthesized by Shanghai Jiepeptide Biotechnology Co., Ltd. ( Figure 8 , Fig. 9 , Fig.10 ), the amino acid sequence of the obtained polypeptide SR-22 is SRITRLLRGSGSGSSRITRLLR.

[0064] Example 4 Plate antibacterial test

[0065] 1. Inoculate a 6 mm diameter mycelium block of Phytophthora infestans on a rye V8 solid culture medium, place it in a constant temperature environment of 18°C ​​and culture it in the dark for 14 days before use; inoculate pepper Phytophthora capsici, Phytophthora sojae, Phytophthora cactorum, Peronophythora litchii, Phytopythium helicoides, Pythium ultimum, Pythium vexans, and Pythium aphanidermatum on a PDA culture medium, place it in a constant temperature environment of 25°C and culture it in the dark for 7 days before use;

[0066] 2. Dissolve the SR-22 polypeptide powder in sterile water to prepare a 50mM mother liquor for standby use. Add 28, 42, and 70 μL of the polypeptide mother liquor to the PDA (applicable to pepper phytophthora, soybean phytophthora, phytophthora scabra, litchi peronospora, hanging stalk plant pythium, ultimate pythium, bell pythium, icariin pythium) or rye V8 medium (applicable to pathogenic phytophthora) that has been melted and cooled to about 50°C, shake well and pour into a sterilized culture dish (diameter 60mm), and 7mL of the culture medium poured into each culture dish is quantitatively poured. Finally, drug-containing plates with polypeptide concentrations of 200, 300, and 500 μM are prepared, and the culture medium plate without polypeptide is used as a negative control group;

[0067] 3. Use a sterile puncher to punch a 6mm diameter bacterial cake at the edge of the aforementioned freshly cultured colony, and use a sterile inoculation needle to inoculate a 6mm diameter bacterial cake into the center of the drug-containing plate (with the mycelium side of the bacterial cake facing downward). After sealing the culture dish with a sealing film, invert the infestation phytophthora plate at 18°C, and invert the other oomycete plates in a 25°C constant temperature incubator for dark culture;

[0068] 4. Observe and take photos of the colony growth within 1 to 7 days after inoculation, measure the colony diameter using the cross method and calculate the colony growth inhibition rate. The colony growth inhibition rate is calculated according to the following formula: colony growth inhibition rate = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%. According to the results of the antibacterial test, the toxicity equation and effective inhibition concentration of SR-22 to oomycetes were calculated using the probability analysis method.

[0069] Depend on Fig.11It can be found that polypeptide SR-22 has good inhibitory effects on pepper Phytophthora, infestans Phytophthora, soybean Phytophthora, Phytophthora caerulea, litchi downy mildew, plant rotten rot, aphaniderma, bell-shaped rot and ultimum rot. These 9 pathogens come from 4 different genera of Oomycetes, namely Phytophthora, Downy Mildew, Plant Pythium and Pythium. It can be seen that polypeptide SR-22 has a broad-spectrum effect in inhibiting the growth of oomycetes.

[0070] SR-22 can significantly inhibit the mycelial growth of 9 kinds of oomycetes, and its inhibitory effect is dose-dependent. At a concentration of 500μM, SR-22 has a colony growth inhibition rate of 84.47%, 78.58%, 86.00%, 69.67%, 92.40%, 78.95%, 70.65%, 79.17%, and 82.61% on pepper phytophthora, infestation phytophthora, soybean phytophthora, phytophthora caerulea, litchi peronophyte, phytophthora aphanidermatum, pythium aphanidermatum, pythium ultimum, respectively, indicating that the polypeptide has a very significant antibacterial effect on oomycetes. Among them, Fig.11 The text on the left is the Latin species name of the tested oomycetes, and the top is the SR-22 concentration used. In the picture, Phytophthora capsici was photographed 2 days after inoculation, Phytophthora infestans was photographed 7 days after inoculation, Phytophthora sojae was photographed 84 hours after inoculation, Phytophthora cactorum was photographed 2 days after inoculation, Peronophythora litchii was photographed 78 hours after inoculation, Phytopythium helicoides was photographed 20 hours after inoculation, Pythium aphanidermatum was photographed 2 days after inoculation, Pythium vexans was photographed 1 day after inoculation, and Pythium ultimum was photographed 20 hours after inoculation.

[0071] According to the results of the antibacterial test, the toxicity equation and effective inhibitory concentration of SR-22 to oomycetes were calculated using the probability analysis method. The results are shown in Table 2.

[0072] Table 2 Antibacterial activity of peptide SR-22 against 9 oomycetes

[0073]

[0074] Example 5 Plant leaf spraying test

[0075] 1. Select potato (Solanum tuberosum) variety Désirée, which is one of the staple foods. Transplant the germinated potato tubers into plastic pots containing sterile soil, and cultivate them in a greenhouse at 22-25℃ and 16h / 8h light-dark alternation. Grow them for 4-6 weeks before use.

[0076] 2. Cultivate the pathogenic fungus for 14 days in the above manner, rinse the culture plate with sterile distilled water, and scrape the surface of the plate with a sterile spreader to collect sporangia, and then place the collected sporangium suspension in a 4°C refrigerator for 15-30 minutes and an 18°C ​​constant temperature box for 20-30 minutes to stimulate the sporangia to release zoospores;

[0077] 3. Prepare SR-22 peptide stock solution (50mM), use a pipette to draw 30μL, dilute to 3mL with sterile water to make 500μM peptide working solution for standby use;

[0078] 4. Cut potato leaves of uniform size, rinse the leaves with sterile water, remove impurities on the leaves and carefully wipe off the remaining water stains on the leaves. Turn it over and spray 3mL of polypeptide working solution evenly on the back of the leaves. Spray it 2 to 3 times. Spray the control group with an equal amount of sterile water. Treat 4 leaves in each group. Place the leaves in a 25℃ greenhouse for about 12 hours to dry them, then inoculate 10μL of zoospore suspension (concentration of 60 spores / μL) on the back of the leaves. After inoculation, place the leaves in a sterile plastic box with a moist filter paper pad, seal the plastic box with plastic wrap, and place the box in an 18℃ incubator. Continue to observe the results of pathogen infection within 2 to 6 days after inoculation and take photos to record;

[0079] 5. The biomass of Phytophthora infestans in leaves was measured by qPCR (reaction system and procedure are shown in Tables 3 and 4, and primer sequences are shown in Table 5). Total genomic DNA was extracted from equal areas of the leaf inoculation site 6 days after inoculation, and the relative expression of the housekeeping gene Pief2α (NCBI accession number XM_002901697) of Phytophthora infestans was analyzed with the potato housekeeping gene StEF1α (NCBI accession number NM_001288491) as a reference control to measure the biomass of Phytophthora infestans per unit area of ​​leaves.

[0080] Table 3 qPCR reaction system (20 μL)

[0081]

[0082] Table 4 qPCR reaction procedure

[0083]

[0084] The primer sequences used (Table 5) are as follows:

[0085] Table 5 Primer sequences

[0086]

[0087]

[0088] 6. After spraying 500 μM of peptide SR-22 solution on potato leaves, observe the disease situation 3 days after inoculation of Phytophthora infestans, measure the relative biomass of Phytophthora infestans in the leaves at 6 days, and evaluate the prevention and control effect of potato late blight.

[0089] Spray 500 μM of peptide SR-22 solution on potato leaves and then inoculate with Phytophthora infestans. The disease situation after 3 days is as follows: Fig.12 As shown in A and 12B, the relative biomass of Phytophthora infestans in leaves at 6 days is as follows Fig.12 C. Fig.12 It can be seen that SR-22 can achieve good effects in inhibiting the infection of pathogenic Phytophthora and controlling the occurrence of potato late blight when sprayed on leaves.

[0090] Example 6 Plant root irrigation test

[0091] 1. Select pepper (Capsicum annuum) variety "Hanyu Extra Large Horn Pepper", after germination, transplant it into a plastic pot containing sterile soil, place it in a greenhouse at 22-25°C and 16h / 8h light and dark alternation, and grow it for 4-6 weeks before use;

[0092] 2. Pour 10% V8 culture solution into a sterile culture dish (9cm in diameter), 15-20mL per dish, transfer 8-10 pieces of freshly cultured pepper phytophthora hyphae into the culture solution, and culture in a 25℃ constant temperature incubator in the dark for 2-3 days. After the mycelium is formed, pour out the culture solution, add 15-20mL of sterilized water, resuspend the mycelium, and continue to culture at a constant temperature. After that, change the water every 12-24 hours, for a total of 2-3 times, until a large number of zoosporangia are formed. To obtain zoospores, place the culture dish in a 4℃ refrigerator for 10-15min and a 25℃ constant temperature incubator for 10-30min in turn to stimulate the release of zoospores;

[0093] 3. Prepare 50mM SR-22 peptide stock solution, take 500μL with a pipette, dilute to 50mL with sterile water to make 500μM peptide solution for later use;

[0094] 4. Irrigate the pepper roots with 8 mL of 500 μM peptide solution, and irrigate the plants with the same amount of sterile water as a control. Six plants were treated and six plants were controlled. After being placed in the greenhouse for 8 hours, 6 mL of pepper Phytophthora zoospore suspension (concentration of 50 zoospores / μL) was irrigated to the pepper roots. The disease of peppers was continuously observed and photographed within 3-6 days after root irrigation. Fig.13As shown, compared with the control group, the degree of infection of pepper roots by Phytophthora capsici was significantly inhibited after pre-treatment with SR-22, and the occurrence of pepper blight was significantly controlled. It can be seen that the polypeptide SR-22 can be used to inhibit the infection of pathogenic oomycetes in the roots of peppers and even other plants.

[0095] Example 7 Fruit Infiltration Test

[0096] 1. Cultivate litchi downy mildew for 4-6 days and set aside;

[0097] 2. Choose fresh and healthy lychee (Litchi chinensis) varieties "Guiwei";

[0098] 3. Prepare SR-22 peptide stock solution (50mM), take 50μL with a pipette, dilute to 5mL with sterile water to make 500μM peptide working solution for later use;

[0099] 4. Rinse the surface of fresh lychee fruit with sterile water, and then use absorbent paper to absorb any residual moisture on the fruit surface. Soak the lychee fruit in 500μM SR-22 polypeptide solution several times, and use the fruit soaked in an equal amount of sterile water as a control. There are 8 fruits in each of the experimental and control groups. Leave the fruit at room temperature for 2.5 hours to dry, use a 6mm diameter sterilized puncher to punch a block of lychee downy mildew hyphae at the edge of the colony, place the side with hyphae close to the surface of the fruit, seal the plastic box with plastic wrap to keep it moist, and place it in a 25℃ incubator for 2 to 4 days. During this period, observe the disease of the lychee and take photos to record. The results obtained are as follows: Fig.14 As shown, compared with the control, the incidence of litchi downy mildew was significantly reduced after litchi fruit was treated with 500 μM SR-22 polypeptide solution, indicating that the polypeptide SR-22 can inhibit the infection of pathogenic oomycetes on litchi and other plant fruits by spraying or infiltration. Among them, the disease grade is divided into Grade 0 (Grade0, no lesions), Grade 1 (Grade1, lesion area <10%), Grade 2 (Grade2, lesion area 10% to 30%), Grade 3 (Grade3, lesion area 30% to 50%), and Grade 4 (Grade4, lesion area 50% to 100%).

[0100] Example 8 Analysis of Antibacterial Specificity of Peptides

[0101] 1. Inoculate the rice blast fungus (Magnaporthe grisea) on a PDA culture medium plate and place it in a constant temperature environment of 25℃ for dark culture for 1 to 7 days before use. According to the previous method, drug-containing plates with peptide concentrations of 200, 300, and 500μM were prepared, and the culture medium plate without peptide was used as a negative control group. Use a sterile puncher to punch a 6mm diameter bacterial cake at the edge of the freshly cultured colony, and use a sterile inoculation needle to inoculate a piece of bacterial cake into the center of the drug-containing plate (with the mycelium side of the bacterial cake facing down). After sealing the culture dish with a sealing film, invert it in a 25℃ constant temperature incubator for dark culture. Calculate the colony growth inhibition rate and effective inhibition concentration according to the previous method. The results showed that when the concentration was as high as 500μM, the inhibition rate of SR-22 on rice blast fungus was only 11.31%, and the EC50 value was 319.869mg / mL, which shows that SR-22 has no inhibitory effect on the growth of rice blast fungus.

[0102] 2. Cultivate Escherichia coli DH5α strain in LB liquid medium at 37°C and 200 rpm overnight (12-13 h). Add LB medium and increase the OD 600 Dilute to 0.1, and then add the peptide solution to make the final concentrations 200, 300, and 500 μM respectively. Continue to cultivate E. coli and measure OD every 1 hour. 600 The results showed that the growth of E. coli was not significantly affected after SR-22 was added to the liquid culture medium. When the culture time reached 5 hours, the OD of the bacteria in the control and 500μM SR-22 treatment groups was 600 The values ​​of SR-22 and SR-22 were 1.6485 and 1.448, respectively, and there was no significant difference between the two, indicating that SR-22 had no inhibitory effect on the growth of E. coli. In summary, SR-22 had no significant inhibitory effect on non-target microorganisms (i.e., fungi and bacteria).

Claims

1. A polypeptide ligand SR-22 targeting pathogenic oomycete membrane protein, characterized in that: Its amino acid sequence is SRITRLLRGSGSGSSRITRLLR.

2. A reagent or drug containing the polypeptide ligand SR-22 according to claim 1.

3. Use of the polypeptide ligand SR-22 according to claim 1 or the reagent or drug according to claim 2 in inhibiting the growth of oomycetes.

4. Use of the polypeptide ligand SR-22 according to claim 1 or the reagent or drug according to claim 2 in preventing and controlling oomycete diseases of crops.

5. The use according to any one of claims 3 to 4, characterized in that: The oomycete is an oomycete that uses membrane protein PcMEM1 as a key factor for growth, development, infection and pathogenicity.

6. The use according to any one of claims 3 to 4, characterized in that: The oomycetes include fungi of the genus Phytophthora, Peronophythora, Phytopythium or Pythium.

7. The use according to any one of claims 3 to 4, characterized in that: The oomycetes include Phytophthora capsici, Phytophthora infestans, Phytophthora sojae, Phytophthora cactorum, Peronophythora litchii, Phytopythium helicoides, Pythium aphanidermatum, Pythium vexans or Pythium ultimum.

8. The use according to any one of claims 3 to 4, characterized in that: The effective inhibitory concentration of the polypeptide ligand SR-22 is 0.0003-0.3517 mM.

9. The use according to claim 4, characterized in that: The crops include potatoes, peppers or litchi.

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