Peptide aptamer and application thereof

Abstract

The present application relates to the use of a peptide aptamer comprising a variable peptide segment as shown in SEQ ID NO: 1 or comprising an amino acid sequence produced by substitution, deletion and / or addition of one or several amino acids from the amino acid sequence shown in SEQ ID NO: 1 in the preparation of a biological preparation, a pesticide or a protective agent for inhibiting, resisting or preventing a plant disease caused by G. cingulata; wherein the amino acid sequence produced by substitution, deletion and / or addition of one or several amino acids retains the activity or function of the variable peptide segment. The peptide aptamer of the present application shows obvious inhibitory effect on G. cingulata, can effectively inhibit the germination of spores, and can effectively prevent and control the infection of G. cingulata on the leaves of Stachys floridana, and reduce the infection symptoms of G. cingulata; at the same time, it does not produce toxic side effects on the leaves of Stachys floridana.

Description

TECHNICAL FIELD

[0001] The application relates to application of a peptide aptamer in inhibition of Stylosanthes anthracnose, in particular, prevention and treatment of Stylosanthes anthracnose, and belongs to the technical field of biotechnology. BACKGROUND

[0002] Stylosanthes guianensis, which is originally from Latin America, is a diversified tropical and subtropical forage legume (Zheng L, et al., 2014). Stylosanthes anthracnose, which is mainly caused by Colletotrichum gloeosporioides, has been the most important biological factor restricting the production, persistence and utilization of Stylosanthes guianensis (referring to Cai Z, Li G, Lin C, et al. Identifying pathogenicity genes in the rubber tree anthracnose fungus Colletotrichum gloeosporioides through random insertional mutagenesis [J]. Microbiological Research, 2013, 168(6): 340-350.), especially under the conditions of high temperature, high humidity and heavy rain, the disease develops extremely fast (referring to Wang H, Chen Z, Liu G, et al. Alterations of growth, antioxidant system and gene expression in Stylosanthes guianensis during Colletotrichum gloeosporioides infection [J]. Plant Physiology and Biochemistry, 2017, 118: 256-266). Since the introduction of Stylosanthes guianensis in China, the anthracnose has caused the yield of Stylosanthes guianensis in China to decrease by 21-40%, and the yield of seeds to decrease by 28-70%.

[0003] The current prevention and control of anthracnose of Gypsophila oldhamiana is mainly focused on germplasm resource collection, disease-resistant variety breeding, genetic engineering breeding, and chemical control (see Liang Y, Wu W, Xi J, et al. Biocontrol effect of biocontrol bacteria JNC2 on anthracnose of Gypsophila oldhamiana and its mechanism of action [J]. Genomics and Applied Biology, 2020, 39(12): 5567-5573.). Although disease-resistant varieties of Gypsophila oldhamiana have been continuously bred, most of them are not commercially valuable and cannot be widely applied. Due to the lack of research on the molecular mechanism of pathogen invasion into the host, the application of genetic engineering breeding of Gypsophila oldhamiana is still greatly limited. In addition, due to the pollution and harm of chemical pesticides to the environment, it is urgent to find alternative prevention and control means and strategies, especially environmentally friendly biological control means.

[0004] Application content

[0005] In view of the technical problems existing in the prior art, the present application provides the use of a peptide aptamer comprising an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence produced by substituting, deleting and / or adding one or more amino acids from the amino acid sequence as shown in SEQ ID NO: 1 in the preparation of a biological agent, a pesticide or a protective agent for inhibiting, resisting or preventing a plant disease caused by Glomerella cingulata; wherein the amino acid sequence produced by substituting, deleting and / or adding one or more amino acids retains the activity or function of the variable peptide segment.

[0006] Preferably, the peptide aptamer further comprises a staphylococcal nuclease as a scaffold protein, and the amino acid sequence of the staphylococcal nuclease is as shown in SEQ ID NO: 2.

[0007] Preferably, the plant is Gypsophila oldhamiana.

[0008] Preferably, the plant disease is anthracnose of the plant.

[0009] Preferably, the plant disease is anthracnose of the plant.

[0010] The use of a peptide aptamer comprising an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence produced by substituting, deleting and / or adding one or more amino acids from the amino acid sequence as shown in SEQ ID NO: 3 in the preparation of a biological agent, a pesticide or a protective agent for inhibiting, resisting or preventing a plant disease caused by Glomerella cingulata; wherein the amino acid sequence produced by substituting, deleting and / or adding one or more amino acids retains the activity or function of the peptide aptamer.

[0011] Use of an amino acid or an amino acid fragment encoding a peptide aptamer in the manufacture of a biological preparation, a pesticide or a protective agent for inhibiting, resisting or preventing a plant disease caused by G. glooeum, wherein the peptide aptamer consists of a scaffold protein and a variable peptide segment, and wherein the amino acid sequence of the amino acid or the amino acid fragment encoding the variable peptide segment is set forth in SEQ ID NO: 1; an amino acid sequence after one or several amino acids of the amino acid sequence set forth in SEQ ID NO: 1 are substituted, deleted and / or added; or an amino acid sequence specifically hybridizing with the amino acid sequence set forth in SEQ ID NO: 1 or with the amino acid sequence after one or several amino acids of the amino acid sequence set forth in SEQ ID NO: 1 are substituted, deleted and / or added, wherein the amino acid sequence after one or several amino acids are substituted, deleted and / or added or the amino acid sequence specifically hybridizing therewith or specifically hybridizing with the amino acid sequence set forth in SEQ ID NO: 1 retains the activity or function of the amino acid sequence set forth in SEQ ID NO: 1.

[0012] Use of an amino acid or an amino acid fragment encoding a peptide aptamer in the manufacture of a biological preparation, a pesticide or a protective agent for inhibiting, resisting or preventing a plant disease caused by G. glooeum, wherein the amino acid sequence of the peptide aptamer is set forth in SEQ ID NO: 3; an amino acid sequence after one or several amino acids of the amino acid sequence set forth in SEQ ID NO: 3 are substituted, deleted and / or added; or an amino acid sequence specifically hybridizing with the amino acid sequence set forth in SEQ ID NO: 3 or with the amino acid sequence after one or several amino acids of the amino acid sequence set forth in SEQ ID NO: 3 are substituted, deleted and / or added, wherein the amino acid sequence after one or several amino acids are substituted, deleted and / or added or the amino acid sequence specifically hybridizing therewith or specifically hybridizing with the amino acid sequence set forth in SEQ ID NO: 3 retains the activity or function of the amino acid sequence set forth in SEQ ID NO: 3.

[0013] Use of a nano-peptide aptamer complex in preparation of a biological agent, pesticide or protective agent for inhibiting, resisting or preventing plant diseases caused by G. cingulata; the nano-peptide aptamer complex is composed of a peptide aptamer and a medium, and wherein the amino acid sequence of the peptide aptamer is shown as SEQ ID NO: 3; an amino acid sequence after substitution, deletion and / or addition of one or several amino acids of the amino acid sequence shown as SEQ ID NO: 3; or an amino acid sequence specifically hybridized with the amino acid sequence shown as SEQ ID NO: 3 or with the amino acid sequence after substitution, deletion and / or addition of one or several amino acids of the amino acid sequence shown as SEQ ID NO: 3, wherein the amino acid sequence after substitution, deletion and / or addition of one or several amino acids or the amino acid sequence specifically hybridized therewith or specifically hybridized with the amino acid sequence shown as SEQ ID NO: 3 retains the activity or function of the amino acid sequence shown as SEQ ID NO: 3.

[0014] Preferably, the medium is montmorillonite K-10.

[0015] A peptide aptamer with a sequence shown as SEQ ID NO: 3 is configured to prepare a biological agent, pesticide or protective agent for inhibiting, resisting or preventing plant diseases caused by G. cingulata.

[0016] A nano-peptide aptamer complex comprising: a peptide aptamer with a sequence shown as SEQ ID NO: 3; and a medium; the nano complex is configured to prepare a biological agent, pesticide or protective agent for inhibiting, resisting or preventing plant diseases caused by G. cingulata.

[0017] A biological agent, pesticide or protective agent for inhibiting, resisting or preventing plant diseases caused by G. cingulata, comprising a peptide aptamer as described above, or a nano-peptide aptamer complex as described above.

[0018] The peptide aptamer of the present application shows obvious inhibitory effect on G. cingulata, can effectively inhibit spore germination, and can effectively prevent and control the infection of G. cingulata on leaves of Gossypium arboreum, and reduce the infection symptoms of G. cingulata; at the same time, no toxic and side effects are generated on the leaves of Gossypium arboreum.

[0019] The foregoing merely is illustrative and is in no way limiting. Further aspects, implementations, and features will become more readily apparent upon consideration of the following detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0020] Hereinafter, preferred embodiments of the present application will be further described in detail with reference to the accompanying drawings, in which:

[0021] Figure 1 is a full-length amino acid sequence of a peptide aptamer according to an embodiment of the present application as shown in SEQ ID NO: 3;

[0022] Figure 2 is a result of resazurin detection of spore viability of G. cingulate according to an embodiment of the present application;

[0023] Figure 3 is an inhibitory effect of a peptide aptamer on spores of G. cingulate according to an embodiment of the present application;

[0024] Figure 4 is a membrane damage of a peptide aptamer on spores of G. cingulate according to an embodiment of the present application;

[0025] Figure 5 is an effect of different concentrations of a peptide aptamer on spore killing rate according to an embodiment of the present application;

[0026] Figure 6 is a damaging effect of a peptide aptamer on spores of G. cingulate according to an embodiment of the present application; (A-C) images of normal spores; (D-F) images of spores after SNP-16 treatment. CW: cell wall, PM: plasma membrane, ER: endoplasmic reticulum, V: vacuole, NU: nucleus;

[0027] Figure 7 is an inhibitory effect of a nano-peptide aptamer complex on spores of G. cingulate according to an embodiment of the present application; and

[0028] Figure 8 is an inhibition of infection of G. cingulate on leaves of P. acutifolius by a nano-peptide aptamer complex according to an embodiment of the present application. DETAILED DESCRIPTION

[0029] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0030] In the following detailed description, reference will be made to the accompanying drawings, which form a part of this description, illustrating certain embodiments of the present application. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The embodiments of the present application will be described in sufficient detail to enable those skilled in the art to make and use it, and it is to be understood that other embodiments can be utilized and that structural, logical, and electrical changes can be made without departing from the scope of the present application.

[0031] In one embodiment, the peptide aptamer described in the present application is a short peptide that can bind to a target with high specificity and high affinity, which is screened from a library of randomly synthesized amino acids (Hopper S F, Crnkovic M I, Tomai E, et al. "Peptide aptamers: specific inhibitors of protein function," [J]. Curr. Mol. Med., 2004, 4(5): 529-538).

[0032] The main structure of the peptide aptamer includes a stable scaffold protein (SN) and a variable peptide segment fixed on both ends of the scaffold protein (see Groner B, Borghouts C, Kunz C. "Peptide aptamer libraries," [J]. Comb. Chem. High Throughput Screen, 2008, 11(2): 135-145). The variable peptide segment usually consists of 8-20 amino acids and has the function of recognizing and binding to target proteins.

[0033] The peptide aptamer has the following three characteristics: first, it has extremely high specificity for homologous targets and can distinguish different members of the same protein family; second, compared with antibodies, the peptide aptamer has small molecular weight and comparable ability to recognize and bind to target proteins; third, the peptide aptamer can fold into a stable tertiary conformation in vivo, which is more conducive to maintaining high biological activity compared with general free amino acids (see Jiang L, Lan XP. "A new member of the aptamer family: peptide aptamer," [J]. Chinese Journal of Biochemistry and Molecular Biology, 2009, 2(1): 99-103).

[0034] The present application provides a peptide aptamer, wherein the amino acid sequence of the peptide aptamer is as shown in SEQ ID NO: 1 or is generated by substituting, deleting and / or adding one or more amino acids from the amino acid sequence as shown in SEQ ID NO: 1; wherein the amino acid sequence generated by substituting, deleting and / or adding one or more amino acids retains the activity or function of the variable peptide segment or the variable peptide segment is composed of the above-mentioned amino acid sequence.

[0035] In one embodiment, the peptide aptamer of the present application further comprises a scaffold protein. Preferably, the scaffold protein is staphylococcal nuclease, i.e., the peptide aptamer of the present application takes staphylococcal nuclease as the basic skeleton.

[0036] In one embodiment, the staphylococcal nuclease as the scaffold protein of the present application has an amino acid sequence as shown in SEQ ID NO: 2 or comprises an amino acid sequence generated by substituting, deleting and / or adding one or more amino acids from the amino acid sequence as shown in SEQ ID NO: 2, provided that the amino acid sequence generated by substituting, deleting and / or adding one or more amino acids retains the activity or function of the peptide aptamer or the peptide aptamer is composed of the above-mentioned amino acid sequence.

[0037] In one embodiment, the peptide aptamer of the present application is composed of a scaffold protein and a variable peptide segment of the present application, preferably, the amino acid sequence of the variable peptide segment is as shown in SEQ ID NO: 1 or the amino acid sequence thereof is as shown in SEQ ID NO: 3. In a preferred embodiment, the scaffold protein is staphylococcal nuclease.

[0038] In one embodiment, the amino acid sequence encoding the variable peptide segment is as shown in SEQ ID NO: 3, or is generated by substituting, deleting and / or adding one or more amino acids from the amino acid sequence as shown in SEQ ID NO: 3, or specifically hybridizes with the amino acid sequence as shown in SEQ ID NO: 3 or the amino acid sequence generated by substituting, deleting and / or adding one or more amino acids from the amino acid sequence as shown in SEQ ID NO: 3, provided that the amino acid sequence generated by substituting, deleting and / or adding one or more amino acids or the amino acid sequence specifically hybridized with or specifically hybridized with the amino acid sequence as shown in SEQ ID NO: 3 retains the activity or function of the amino acid sequence as shown in SEQ ID NO: 3 or the amino acid sequence encoding the variable peptide segment is composed of the above-mentioned amino acid sequence.

[0039] In one embodiment, the variable peptide segment of the peptide aptamer described herein comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homologous to the amino acid sequence of SEQ ID NO: 1.

[0040] Optionally, the amino acid sequence of the scaffold protein encoding the peptide aptamer described herein comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homologous to the amino acid sequence of SEQ ID NO: 2.

[0041] In one embodiment, the peptide aptamer described herein comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homologous to the amino acid sequence of SEQ ID NO: 3. Optionally, the amino acid sequence encoding the peptide aptamer described herein comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homologous to the amino acid sequence of SEQ ID NO: 4.

[0042] Figure 1 is the full-length amino acid sequence of the peptide aptamer of the amino acid sequence set forth in SEQ ID NO: 3, according to one embodiment of the present application, wherein the helices of the phycochrome represent a- helices and the blue arrows represent the beta-sheets of the protein. In some embodiments, the peptide aptamer of the present application is denoted SNP-16 and the scaffold protein of the peptide aptamer is denoted SN.

[0043] In one embodiment, the amino acid sequence or amino acid sequence of the variable peptide segment and / or peptide aptamer described herein can be modified or labeled by methods commonly used in the art. For example, the amino acid sequence or amino acid sequence of the variable peptide segment and / or peptide aptamer described herein can be modified by substitution, deletion and / or insertion of one or more amino acids, or modified by any modification means known in the art, so as to impart additional properties to the variable peptide segment and / or peptide aptamer while maintaining its original function. For example, the amino acid sequence or amino acid sequence of the variable peptide segment and / or peptide aptamer described herein can be detectably labeled, so as to determine the presence or amount of the target protein. Labeling methods are well known in the art, and the labels include those commonly used in the art, including but not limited to fluorescent labels, isotope labels, etc.

[0044] According to another aspect of the present application, the use of the peptide aptamer or the amino acid segment encoding the same described above in the preparation of a biological agent, pesticide or protective agent for inhibiting or resisting G. cingulata, or in the preparation of a biological agent, pesticide or protective agent for inhibiting, resisting or preventing plant diseases caused by G. cingulata is proposed. In view of the fact that G. cingulata can infect plants, trees, vegetables, flowers, forest trees, etc., thereby causing anthracnose, the use of the peptide aptamer described above in the preparation of a biological agent, pesticide or protective agent for inhibiting or resisting G. cingulata against the above-mentioned plants is also proposed.

[0045] In one embodiment, the plant disease caused by G. cingulata is plant anthracnose. In one embodiment, the plant includes various plants such as cucurbits, solanaceae, bananas, cotton plants, legumes and flowers, for example, columnflower, peach, apricot, plum, plum, cotton, sugarcane, banana, tomato, cucumber, cantaloupe, watermelon, soybean, cowpea, etc. Preferably, the plant is banana, and the blight is banana anthracnose.

[0046] In the present application, the "variant" of an amino acid sequence or amino acid sequence refers to an amino acid sequence or amino acid sequence that can maintain its function or activity after substitution, deletion and / or addition of one or more amino acids or amino acids to the amino acid sequence or amino acid sequence, or an amino acid sequence or amino acid sequence that can maintain its function or activity and has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology with the amino acid sequence or amino acid sequence, or an amino acid sequence or amino acid sequence that can maintain its function or activity after modification or labeling of the amino acid sequence or amino acid sequence by means known in the art.

[0047] The purpose of the present application is to inhibit Colletotrichum gloeosporioides using an artificial peptide aptamer, and then to improve the bacteriostatic effect of the peptide aptamer by introducing nano material montmorillonite K-10 for loading, forming a nano peptide aptamer complex, and further developing a green spray pesticide for the daily prevention and control of Colletotrichum gloeosporioides.

[0048] In order to achieve the above purpose, the technical scheme of the present application is as follows:

[0049] From the previously constructed peptide aptamer library, the present application screens the specific peptide aptamer SNP-16 through the crude extract bacteriostatic experiment, which has an inhibitory effect on Colletotrichum gloeosporioides. However, as a biologically active protein molecule, SNP-16 is prone to inactivation and degradation in actual agricultural application, and its antibacterial performance is not outstanding compared with some pesticides, and it is not worth popularization and application. Therefore, by loading SNP-16 into a nano carrier, the antibacterial effect and the durability of SNP-16 in practical application are improved.

[0050] In one embodiment, the bactericidal ability of the peptide aptamer of the present application on Colletotrichum gloeosporioides spores is concentration-dependent, and the minimum inhibitory concentration is 8 μM. When the concentration of the peptide aptamer is 8 μM, the spore germination can be delayed, but the spores cannot be completely killed, and the killing effect can only reach about 30% of the pesticide carbendazim. After treatment with the peptide aptamer, the overall morphology of the spores appears irregular shrinkage, the cell membrane wrinkles, the endoplasmic reticulum is fragmented, the nucleus is disintegrated, and a large number of vesicles appear in the cytoplasm, showing a damaged state. These implementation results show that the peptide aptamer as a biologically active peptide exhibits certain bactericidal activity on Colletotrichum gloeosporioides.

[0051] In one embodiment, the mixture of the peptide aptamer and montmorillonite K-10 of the present application is incubated at room temperature. The nano peptide aptamer complex can be obtained.

[0052] The technical scheme of the present application will be described in detail below through examples.

[0053] Among them, the experimental materials and peptide aptamer sequences involved in the present application are shown as follows: if not specified, the technical means used in the examples are conventional means familiar to those skilled in the art; if not specified, the experimental reagents used in the examples are commercially available.

[0054] 1. Test strains, plasmids and pathogenic bacteria

[0055] Test strains: E. coli competent cells, E. coli BL21 are laboratory preserved strains.

[0056] Plasmids: pET-28a, pTRG are laboratory preserved plasmids. The peptide aptamer library pTRG-SNP is constructed by the laboratory.

[0057] Pathogenic bacteria: Plant pathogenic bacteria: Colletotrichum gloeosporioides penz. HK-04 was provided by Ms. Luo Lijuan.

[0058] 2. Culture medium

[0059] LB liquid medium formula: tryptone 1.0 g, yeast extract 0.5 g, NaCl 0.5 g, distilled water 100 mL.

[0060] LB solid medium formula: tryptone 1.0 g, yeast extract 0.5 g, NaCl 0.5 g, agarose 1.5 g, distilled water 100 mL.

[0061] PDA medium formula: potato dextrose agar medium (purchased from Solarbio Company) 3.7 g, distilled water 100 ml.

[0062] 3. Peptide aptamer sequence

[0063] VTFLVNTYPNGVQSRAS (EQ ID NO: 1)

[0064] MGYPYDVPDYASLPGIQPATSTKKLHKEPATLIKAIDGTTVKLMYKGQPMTFRLLLVDTPEFRSKYGPEASAFTKKMVENAKKIEVELDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPKNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQVDID (EQ ID NO: 2)

[0065] MGYPYDVPDYASLPGIQPATSTKKLHKEPATLIKAIDGTTVKLMYKGQPMTFRLLLVDTPEFGGVTFLVNTYPNGVQSRAGGRSKYGPEASAFTKKMVENAKKIEVELDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPKNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQVDIDHHHHHH* (EQ ID NO: 3)

[0066] Example 1 Evaluation of the bacteriostatic effect of peptide aptamer

[0067] (1) Minimum bacteriostatic concentration determination of peptide aptamer SNP-16 on Colletotrichum gloeosporioides

[0068] The minimum inhibitory concentration (MIC) of SNP-16 was determined by detecting spore activity using resazurin. 45 μL of PBS buffer and 45 μL of spore suspension (4 × 10⁵ spores / mL) were mixed in a 96-well plate, followed by the addition of 20 μL of resazurin solution (20 μg / mL). SNP-16 solution at concentrations of 2, 4, 6, 8, 10, and 20 μM was added, with three replicates for each concentration. The plates were then incubated at 28°C for 48 hours. Samples were then analyzed at 595 nm. To eliminate the influence of the scaffold protein SN, SN treatment was also included as a control.

[0069] Figure 2 The results of detecting the spore viability of *Colletotrichum candida* according to one embodiment of this application are shown. Figure 2 A- Figure 2 As shown in Figure C, it is evident that the inhibitory effect of SNP-16 on Colloidal anthracnose increases with increasing SNP-16 concentration.

[0070] (2) Antibacterial experiment of peptide aptamer SNP-16

[0071] After determining the SNP-16 concentration, dilute to 8 μM, and add 10 μL of protein solution and 10 μL of 4×10 5 The spore suspension of *Colletotrichum gloeosporioides* was mixed thoroughly, with PBS treatment as a blank control, 0.2% carbendazim as a positive control, and SN protein treatment as an exclusion control. All treatments were incubated at 28℃ for 6 h, 12 h, and 24 h, and spore germination was observed under a 40× objective lens using an optical microscope. Three different fields of view were taken from each group.

[0072] Figure 3 This describes the inhibitory effect of a peptide aptamer according to one embodiment of this application on the germination of *Colletotrichum gloeosporioides* spores. For example... Figure 3 As shown, compared with the blank control group treated with PBS and the exclusion control that excludes the influence of scaffold protein SN, the germination of *Colletotrichum gloeosporioides* spores treated with 8 μM peptide aptamer SNP-16 was significantly inhibited, and the inhibitory effect was comparable to that of the commercially available antibacterial agent carbendazim.

[0073] (3) Flow cytometry determination of the antibacterial effect of peptide aptamer SNP-16

[0074] Take 400 μL 4×10 5 Spore suspension at 1 / mL was co-incubated with 400 μL of 8 μM SNP-16 protein at 28°C for 3 h. PBS buffer was used as a blank control, and 0.2% carbendazim was used as a positive control. After treatment, the spore suspension was centrifuged at 4000 rpm for 10 min, the supernatant was removed, and the spores were resuspended in 400 μL of PBS buffer. Finally, 40 μL of PI staining solution (100 μg / mL) was added for staining for 5 min. Samples were then analyzed by flow cytometry.

[0075] Figure 4 The damage effect of the peptide aptamer pair according to an embodiment of the present application on the cell membrane of the G. cingulata spores. Figure 4 As can be clearly seen in the figure, the cell membrane of the G. cingulata spores treated with the peptide aptamer SNP-16 at 8 μM has a significant damage effect compared with the PBS control, and the damage degree of the peptide aptamer on the cell membrane of the G. cingulata spores is equivalent to that of the commercial bacteriostatic agent.

[0076] (4) Spore dilution plate regrowth experiment

[0077] The spore suspension (1×10 4 μL of the purified SNP-16 protein (8 μM, 20 μM) was incubated under the optimal conditions for 24 h. After the treatment, the supernatant was removed by centrifuging the suspension at 4000 rpm for 10 min to remove the protein, and finally 50 μL of ddH2O was added to resuspend the spores. The spore solution was continuously diluted and plated on PDA plates, which were cultured at 28°C for 3 d, and the number of viable conidia was counted. PBS was used as a blank control, and carbendazim was used as a positive control.

[0078] Figure 5 The effect of different concentrations of the peptide aptamer on the bactericidal rate of the spores according to an embodiment of the present application. As shown in the figure, the peptide aptamer has a significant damage inhibition effect on the G. cingulata spores, and the effect at a concentration of 20 μM is equivalent to that of the commercial bacteriostatic agent. Figure 5

[0079] (5) Transmission electron microscope observation of the effect of the peptide aptamer SNP-16 on the ultrastructure of the spores

[0080] An equal amount of the spore suspension (4×10 5 μL) was incubated with 8 μM of the SNP-16 protein at 28°C for 3 h. After the treatment, the spore suspension was centrifuged at 4000 rpm for 10 min, the supernatant was removed, and the spore precipitate was collected to a size visible to the naked eye, and the sample was prepared. Meanwhile, the normal spores were also treated in the same manner. The sample was photographed and observed by transmission electron microscope.

[0081] Figure 6 The damage effect of the peptide aptamer on the G. cingulata spores according to an embodiment of the present application; wherein Figure 6 A- Figure 6 C is a transmission electron microscope image of the normal spores; Figure 6 D-F are transmission electron microscope images of the spores after the SNP-16 treatment; in the figure, CW: cell wall, PM: plasma membrane, ER: endoplasmic reticulum, V: vacuole, NU: nucleus. As shown in the figure, Figure 6 A- Figure 6 ​As shown in FIG. F, the peptide aptamer has obvious damaging effect on the spores of Colletotrichum gloeosporioides.

[0082] Example 2 Montmorillonite K-10 loaded peptide aptamer

[0083] The montmorillonite K-10 was ultrasonically dispersed in ddH2O (double distilled water) to prepare a stock solution of 4 mg / mL, and the peptide aptamer solution after dialysis was diluted to 20 μM for standby;

[0084] Take 250 μL of the peptide aptamer solution, and mix with 3.6, 3.2, 2.8, 2.4, 2, 1.6, 1.2, 0.8, 0.4, 0.2, 0.1 mg / mL of MMT solution, respectively, 250 μL each. After 2 h of shaking incubation, the mixture was centrifuged at 8000 rpm for 10 minutes, and the supernatant and the precipitate resuspended in PBS were taken for protein concentration determination. Through the results, the optimal adsorption concentration of montmorillonite K-10 was selected under the condition that SNP-16 of 8 μM could be completely adsorbed to form a nano-peptide aptamer complex.

[0085] Example 3 Evaluation of antibacterial effect of nano-peptide aptamer complex

[0086] (1) Observation of spore germination by optical microscope

[0087] The method is as in Example 1.

[0088] (2) Detection of antibacterial effect on leaves of Anthocephalus chinensis

[0089] The cleaned and disinfected leaves of Anthocephalus chinensis were divided and placed in a humidified environment, and 5 μL of SNP-16 (4 μM, 8 μM), montmorillonite K-10 (0.1 mg / mL) and nano-peptide aptamer complex (0.1 mg / mL, 4 μM) were spotted on the leaves, followed by inoculation of 10 μL of spore suspension (1 × 10 5 The leaves treated with carbendazim (0.2%) were used as positive control. After incubation at room temperature for 48 h, the leaves were photographed and the size of the lesion was measured and calculated.

[0090] Figure 6 Inhibition of spores of Colletotrichum gloeosporioides by nano-peptide aptamer complex according to an embodiment of the present application; and Figure 6 Inhibition of infection of leaves of Anthocephalus chinensis by Colletotrichum gloeosporioides by nano-peptide aptamer complex according to an embodiment of the present application. As shown in FIG. F, the nano-peptide aptamer complex of the present application has obvious inhibitory effect on the spores of Colletotrichum gloeosporioides. Figure 7 Figure 8 Figures 7-8

[0091] ​The above examples are only for illustrating the present application and are not intended to limit the present application. Those skilled in the art can make various changes and modifications without departing from the scope of the present application. Therefore, all equivalent technical solutions shall belong to the scope of the present application. SEQUENCE LISTING <110> Hainan University <120> A peptide aptamer and application thereof <130> 2022.6.15 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 17 <212> PRT <213> Artificial Sequence (unknown) <400> 1 Val Thr Phe Leu Val Asn Thr Tyr Pro Asn Gly Val Gln Ser Arg Ala 1 5 10 15 Ser <210> 2 <211> 165 <212> PRT <213> Artificial Sequence (unknown) <400> 2 Met Gly Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Pro Gly Ile 1 5 10 15 Gln Pro Ala Thr Ser Thr Lys Lys Leu His Lys Glu Pro Ala Thr Leu 20 25 30 Ile Lys Ala Ile Asp Gly Thr Thr Val Lys Leu Met Tyr Lys Gly Gln 35 40 45 Pro Met Thr Phe Arg Leu Leu Leu Val Asp Thr Pro Glu Phe Arg Ser 50 55 60 Lys Tyr Gly Pro Glu Ala Ser Ala Phe Thr Lys Lys Met Val Glu Asn 65 70 75 80 Ala Lys Lys Ile Glu Val Glu Leu Asp Lys Gly Gln Arg Thr Asp Lys 85 90 95 Tyr Gly Arg Gly Leu Ala Tyr Ile Tyr Ala Asp Gly Lys Met Val Asn 100 105 110 Glu Ala Leu Val Arg Gln Gly Leu Ala Lys Val Ala Tyr Val Tyr Lys 115 120 125 Pro Lys Asn Thr His Glu Gln His Leu Arg Lys Ser Glu Ala Gln Ala 130 135 140 Lys Lys Glu Lys Leu Asn Ile Trp Ser Glu Asp Asn Ala Asp Ser Gly 145 150 155 160 Gln Val Asp Ile Asp 165 <210> 3 <211> 191 <212> PRT <213> Artificial Sequence (unknown) <400> 3 Met Gly Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Pro Gly Ile 1 5 10 15 Gln Pro Ala Thr Ser Thr Lys Lys Leu His Lys Glu Pro Ala Thr Leu 20 25 30 Ile Lys Ala lie Asp Gly Thr Thr Val Lys Leu Met Tyr Lys Gly Gin 35 40 45 Pro Met Thr Phe Arg Leu Leu Leu Val Asp Thr Pro Glu Phe Gly Gly 50 55 60 Val Thr Phe Leu Val Asn Thr Tyr Pro Asn Gly Val Gin Ser Arg Ala 65 70 75 80 Gly Gly Arg Ser Lys Tyr Gly Pro Glu Ala Ser Ala Phe Thr Lys Lys 85 90 95 Met Val Glu Asn Ala Lys Lys lie Glu Val Glu Leu Asp Lys Gly Gin 100 105 110 Arg Thr Asp Lys Tyr Gly Arg Gly Leu Ala Tyr lie Tyr Ala Asp Gly 115 120 125 Lys Met Val Asn Glu Ala Leu Val Arg Gin Gly Leu Ala Lys Val Ala 130 135 140 Tyr Val Tyr Lys Pro Lys Asn Thr His Glu Gin His Leu Arg Lys Ser 145 150 155 160 Glu Ala Gin Ala Lys Lys Glu Lys Leu Asn lie Trp Ser Glu Asp Asn 165 170 175 Ala Asp Ser Gly Gin Val Asp lie Asp His His His His His His 180 185 190

Claims

1. Use of a peptide aptamer with the amino acid sequence shown in SEQ ID NO:3 in the preparation of a biological agent, pesticide or protectant for inhibiting, resisting or preventing plant diseases caused by Colletotrichum gloeosporioides.

2. The use according to claim 1, wherein, The plant in question is Stylosanthes.

3. The use according to claim 1, wherein, The plant disease mentioned is anthracnose.

4. The use according to claim 1, wherein, The plant disease mentioned is collodion anthracnose.

5. Use of a nanopeptide aptamer complex in the preparation of a biological agent, pesticide, or protectant for inhibiting, resisting, or preventing plant diseases caused by *Colletotrichum gloeosporioides*; wherein the nanopeptide aptamer complex comprises a peptide aptamer and a medium, and wherein the amino acid sequence of the peptide aptamer is as shown in SEQ ID NO:

3.

6. The use according to claim 5, wherein the medium is montmorillonite K-10.

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