Antifungal alpha helix-loop peptides and preparation and use thereof

Abstract

The application discloses peanut meal treated by liquid nitrogen, and can obtain peanut alpha helix-loop peptides with broad-spectrum antifungal activity through separation. The peanut alpha helix-loop peptides with antifungal activity can effectively inhibit various plant pathogenic fungi (rust fungi, fusarium, colletotrichum and sordaria). The peanut alpha helix-loop peptides can effectively improve the antifungal activity of plants.

Description

Technical Field

[0001] This invention belongs to the field of biopesticide technology, specifically relating to the preparation of antifungal peanut α-helix-cyclic peptide and its application in the preparation of antifungal agents. Background Technology

[0002] Since the discovery of antibiotics, the treatment of fungal infections has been fundamentally improved, and their use has saved agricultural economies and increased crop yields. However, the widespread use or abuse of antibiotics, especially in developing countries, has led to the emergence and spread of drug-resistant bacteria, including some highly virulent pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae. Therefore, finding safe, effective antimicrobial drugs that are less likely to induce resistance has become a focus of competition and effort among scientists worldwide.

[0003] Higher plants possess a wide range of defense mechanisms to cope with physical, chemical, and biological stresses, such as drought, cold, heavy metals, pollutants, and pathogen attacks from fungi, bacteria, and viruses. To combat infection by a variety of pathogens, plants exhibit upregulation of a group of genes associated with systemically acquired resistance [Stintzi A., Heitz T., Prasad V., Wiedemann-Merdinoglu S., Kauffmann S., Geoffroy P., Legrand M., Fritig B. Plant “pathogenesis-related” proteins and their role in defense against pathogens. Biochimie. 1993; 75:687–706.]. General resistance is achieved through the release of secondary metabolites such as phytoalexins, tannins, and polyphenolic compounds, as well as the production of pathogenesis-related (PR) proteins. PR protein was first discovered in tobacco leaves in the early 1970s in response to tobacco mosaic virus infection and was later defined as an inducible protein released during pathogenic outbreaks [Stintzi A., Heitz T., Prasad V., Wiedemann-Merdinoglu S., Kauffmann S., Geoffroy P., Legrand M., Fritig B. Plant “pathogenesis-related” proteins and their role in defense against pathogens. Biochimie. 1993; 75:687–706; Sinha M., Singh R.P., Kushwaha GS, Iqbal N., Singh A., Kaushik S., Kaur P., Sharma S., Singh T.P. Current overview of allergens of plant pathogenesis related protein families. Sci. World J. 2014; 2014:543195.].According to a recent review, at least 17 families have been detected and isolated, possessing a wide range of defense-related properties, including antibacterial, antifungal, antiviral, antioxidant, chitinase, and protease inhibitory activities [Stintzi A., Heitz T., Prasad V., Wiedemann-Merdinoglu S., Kauffmann S., Geoffroy P., Legrand M., Fritig B. Plant “pathogenesis-related” proteins and their role in defense against pathogens. Biochimie. 1993; 75:687–706; Sinha M., Singh RP, Kushwaha GS, Iqbal N., Singh A., Kaushik S., Kaur P., Sharma S., Singh T. Current overview of allergens of plant pathogenesis-related protein families. Sci. World J. 2014; 2014:543195; Ebrahim S., Usha K., Singh B. Pathogenesis-related (pr)proteins in plant defense mechanism. Sci. Against Microb. Pathog. 2011; 2:1043–1054; 4. Sels J., Mathys J., DeConinck BM, Cammue BP, De Bolle MF. Plant pathogenesis-related (pr)proteins: A focus on peptides. Plant Physiol. Biochem. 2008; 46:941–950.], including peptides with antibacterial activity, namely thioxins (PR-13 family), defensins (PR-12 family), hevein-like peptides, knottin, α-hairpin proteins, lipid transfer proteins (PR-14 family), and snake proteins.

[0004] Aspergillus niger and Penicillium chrysogenum are biotrophic plant pathogens that depend on the metabolism of living plant cells, such as grapevines. These fungi belong to the biotrophic fungal group. They directly penetrate the epidermis of plant cells, causing the infiltrated cells to subsequently die. After penetration, the fungus transitions to an obligate biotrophic lifestyle. A subgroup of biotrophic fungal pathogens that primarily follow this infection strategy is the hemitrophic fungal group. Compared to hemitrophic pathogens, hemitrophic pathogens live in a biotrophic state for a short period before beginning to kill host cells and / or the host organism.

[0005] In recent years, fungal diseases, such as Aspergillus niger, have become increasingly important in agriculture, such as grape production. For example, among rust fungi, wheat stripe rust caused by Puccinia striiformis is one of the most important wheat diseases worldwide [Chen, XMEpidemiology and control of stripe rust Pucciniastriiformis f.sp.tritici on wheat. Canadian Journal of Plant Pathology 2005; 27.3:314-337.]. Among Fusarium fungi, Fusarium oxysporum is highly representative of the rhizosphere microbiota. Almost all strains exist saprophytically and are known for inducing wilt or root rot in plants [Fravel, D., Chantal Olivain, and Claude Alabouvette. Fusarium oxysporum and its biocontrol. New Phytologist 2003; 157.3:493-502.]. Anthracnose species that induce anthracnose are found in tropical and subtropical regions worldwide. It is a pathogen of berry diseases, infecting leaves and berries at any stage of their development. It is also the pathogen of brown blight in crops, frequently infecting mature berries [Chen Z, Franco CF, Baptista RP, Cabral JM, Coelho AV, Rodrigues CJ, Melo EP. Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides. Applied microbiology and biotechnology. 2007; 73:1306-13.]. Among Rhizoctonia fungi, the Rhizoctonia solani complex represents an economically important group of soil-borne basidiomycete pathogens, occurring in many plant species worldwide [Cubeta, MA, and R. Vilgalys. "Population biology of the Rhizoctonia solani complex." Phytopathology 1997; 87.4:480-484.].

[0006] For a long time, the prevention and control of crop diseases and pests in my country has mainly relied on chemical pesticides, and pesticide pollution has become an urgent problem to be solved in food safety production. Compared with chemical pesticides, biological pesticides have advantages such as safety, environmental protection, long-lasting effect, and no residue. They emphasize low toxicity, high efficiency, and strong selectivity, and have become one of the current research hotspots.

[0007] Therefore, there is a need in this field to develop biological agents for controlling fungi and methods for providing antifungal plants. Peanut meal is a product obtained from peanut kernels after pressing and refining for oil. Peanut meal is typically classified into primary and secondary meal. Primary meal refers to the peanut residue remaining after the first pressing, while secondary meal is the residue after two pressings. Peanut meal yields can usually reach over 44%, making it a rich by-product resource. Peanut meal is rich in protein and carbohydrates. We obtained a series of proteins from the soaking liquid of peanut meal. We isolated these proteins and found that some had antifungal activity; one protein showed resistance to multiple plant pathogenic fungi, including *Rust*, *Fusarium*, *Anthracnose*, and *Rhizoctonia*. Summary of the Invention

[0008] The purpose of this invention is to provide a novel antifungal peanut α-helical-cyclic peptide for crops and its preparation.

[0009] An antifungal peanut α-helix-cyclic peptide, wherein the amino acid sequence of the antifungal peanut α-helix-cyclic peptide is as follows.

[0010] A method for preparing the antifungal peanut α-helical-cyclic peptide as described above involves using ion exchange, where the peanut α-helical-cyclic peptide is separated stepwise by ion exchange and gel filtration chromatography, and then purified by reverse-phase column chromatography for desalting.

[0011] The application of the aforementioned antifungal peanut α-helical-cyclic peptide is characterized in the preparation of antifungal agents against anthrax fungi, rust fungi, Fusarium fungi, and Rhizoctonia fungi.

[0012] The beneficial effects of this invention are as follows: The purified antifungal peanut α-helix-cyclic peptide of this invention has advantages such as moderate molecular weight, storage stability, and strong antifungal ability. The antifungal peanut α-helix-cyclic peptide has strong antibacterial activity against anthrax fungi, rust fungi, Fusarium fungi, and Rhizoctonia fungi, but its antibacterial activity against probiotics, such as Bacillus subtilis, is weak, and its toxicity is low, indicating that the antifungal peanut α-helix-cyclic peptide has good antibacterial selectivity and few toxic side effects. Attached Figure Description

[0013] Figure 1Elution curves of peanut protein on a Sephacryl S-200 column, showing peaks A1 (tube 2), A2 (tube 5), and A4 (tube 10). Elution was performed with 20 mM PBS buffer (pH 8.0) (containing 0.35 M NaCl) at a flow rate of 5 ml / min. Ten tubes were collected, one 5 ml tube at a time, starting from when protein was detected by the detector. Absorbance was measured at 280 nm, and the peaks showing the highest protein concentrations were A1 (tube 2), A2 (tube 5), A3 (tube 8), and A4 (tube 10). Figure 2 The area of ​​the inhibition zone of the isolated antifungal peanut α-helical-cyclic peptide (A1 (tube 2), A2 (tube 5), A3 (tube 8), and A4 (tube 10)) was measured. The diameter of each inhibition zone was measured three times, and the average diameter of the inhibition zone was calculated. The area of ​​the inhibition zone was then calculated using the area formula (S=π*(d / 2)2, where π represents pi, r represents the radius, d represents the diameter, and S represents the area).

[0014] Figure 3 High-performance liquid chromatography (HPLC) analysis of the purity of antifungal peanut α-helical-cyclic peptide.

[0015] Figure 4 Electrospray mass spectrometry identification of antifungal peanut α-helical-cyclic peptide.

[0016] Figure 5 Antifungal activity of peanut α-helical-cyclic peptide against the rust-causing fungus *Puccinia striiformis*. (A) Antifungal activity against *Puccinia striiformis* in solutions with different pH values. (B) Antifungal activity against *Puccinia striiformis* after heat treatment. The diameter of the transparent ring is represented by a thermogram, corresponding to the numerical value of the transparent ring diameter on the scale bar. Brightness represents a larger transparent ring diameter, and darkness represents a smaller transparent ring diameter.

[0017] Figure 6 Antifungal activity of peanut α-helical-cyclic peptide against the rust-causing fungus *Fusarium oxysporum*. (A) Antifungal activity against *Fusarium oxysporum* in solutions with different pH values. (B) Antifungal activity against *Fusarium oxysporum* after heat treatment. The diameter of the transparent ring is represented by a thermogram, corresponding to the numerical value of the transparent ring diameter on the scale bar. Brightness represents a larger transparent ring diameter, and darkness represents a smaller transparent ring diameter.

[0018] Figure 7Antifungal activity of peanut α-helical-cyclic peptide against the rust-causing fungus *Colletotrichum gloeosporioides*. (A) Antifungal activity against *Colletotrichum gloeosporioides* in solutions with different pH values. (B) Antifungal activity against *Colletotrichum gloeosporioides* after heat treatment. The diameter of the transparent ring is represented by a thermogram, corresponding to the numerical value of the transparent ring diameter on the scale bar; brightness represents a larger transparent ring diameter, and darkness represents a smaller transparent ring diameter.

[0019] Figure 8 Antifungal activity of peanut α-helical-cyclic peptide against the rust-causing fungus *Rhizoctonia solani*. (A) Antifungal activity against *Rhizoctonia solani* in solutions with different pH values. (B) Antifungal activity against *Rhizoctonia solani* after heat treatment. The diameter of the transparent ring is represented by a thermogram, corresponding to the numerical value of the transparent ring diameter on the scale bar. Brightness represents a larger transparent ring diameter, and darkness represents a smaller transparent ring diameter. Detailed Implementation

[0020] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0021] Example 1: Preparation of antifungal peanut α-helical-cyclic peptide

[0022] Weigh 10.0g of peanut meal and grind it repeatedly into a fine powder in liquid nitrogen. In a 200mL beaker, add 100mL of protein extraction buffer (composed of: 20mM phosphate (PBS), pH 7.5, 10mM EDTA, 150mM NaCl, 1% dimethyl sulfoxide (DMSO), and 1mM dithiothreitol (DTT) in water) and mix thoroughly. Centrifuge at 10000g for 20min and collect 50mL of the supernatant.

[0023] While stirring, slowly add 767g of (NH4)2SO4 to 1 liter of distilled water, and adjust the pH to 7.0 with 25%-28% ammonia or 95% sulfuric acid. This will give you a 100% saturated ammonium sulfate solution (4.1mol / L, 25℃).

[0024] While stirring, slowly add an equal volume of 100% saturated ammonium sulfate solution to the supernatant. Stir the solution overnight (4°C) on a magnetic stirrer to allow the protein to precipitate completely. Centrifuge the protein solution at 10000g for 30 minutes (4°C). Discard the supernatant and retain the precipitate. Dissolve the precipitate in 20 ml of protein extraction buffer (composition: 20 mM PBS, pH 7.5, 10 mM EDTA, 150 mM NaCl, 1% DMSO, 1 mM DTT, and 0.2 g / L sodium azide in water). Place the buffer in a 40 ml dialysis bag (molecular weight cutoff 2 kDa) and dialyze against 1 L of protein extraction buffer for 24 hours (4°C), changing the dialysis buffer every 6 hours to completely remove the ammonium sulfate. Equilibrate a fast-flow DEAE-Sepharose Fast Flow column (2.6 × 100 cm inner diameter and length, Merck, model 17-0709-10) with three column volumes of protein extraction buffer (composition: 20 mM PBS pH 7.5, 10 mM EDTA, 150 mM NaCl, 1% DMSO, and 1 mM DTT in water) at a flow rate of 0.5 ml / min. Load 20 ml of crude extraction solution onto the DEAE-Sepharose Fast Flow column (2.6 × 100 cm), and equilibrate the DEAE-Sepharose Fast Flow column (2.6 × 100 cm) again with three column volumes of protein extraction buffer (composition: 20 mM PBS, pH 7.5, 10 mM EDTA, 150 mM NaCl, 1% DMSO, and 1 mM DTT in water) at a flow rate of 0.5 ml / min. Finally, the column was eluted with protein extraction buffer (composition: 20 mM PBS, pH 7.5, 10 mM EDTA, 150 mM NaCl, 1% DMSO and 1 mM DTT in water) and a NaCl gradient (0.35 M) at a flow rate of 5.0 mL / min. Protein eluent was collected from the point when protein was detected by the detector until it was no longer detected, for a total of 50 mL. The collected sample was added to a gel filter column (Sephacryl S-200, 5 x 90 cm, GE Healthcare, model 17-0584-01) equilibrated with 3 column volumes of 20 mM Tris-HCl buffer (pH 8.0) (containing 0.35 M NaCl). Elution was performed with 20 mM PBS buffer (pH 8.0) (containing 0.35 M NaCl) at a flow rate of 5 mL / min. One tube was collected every 5 mL from the point when protein was detected by the detector, for a total of 10 tubes. By measuring absorbance at 280 nm, peaks with higher protein concentrations were observed. Figure 1A1 (tube 2), A2 (tube 5), A3 (tube 8), and A4 (tube 10).

[0025] The antifungal activity of each fraction of the peanut protein eluted in the above buffer solution was analyzed in tubes 2, 5, 8, and 10. Potato dextrose agar plates (9 cm in diameter, 0.3-0.4 cm thick) were used (formulation as follows: 20 g potato extract, 2 g glucose, 1.5 g agar, 100 mL water, pH unadjusted (natural pH). Preparation method: Take 20 g fresh potato, wash, peel, and cut into small pieces, add 80 mL water and boil for 30 min, filter through four layers of gauze, add 2 g glucose and 1.5 g agar, continue heating and stirring, cool slightly, add water to 100 mL, and sterilize at 121℃ for 20 min).

[0026] The antifungal activity of peanut protein from tubes 2, 5, 8, and 10 was evaluated. 10 μL of frozen bacterial suspensions (anthrax fungi, rust fungi, Fusarium fungi, Rhizoctonia solani fungi, and Bacillus subtilis, respectively, with a cell density of 1 × 10⁻⁶) were added to the center of each plate. 5 ~2×10 5 CFU / g lyophilized powder (each gram of sample dissolved in 10 mL of 0.85% physiological saline). Using a 1 cm diameter sterile circular paper disc, draw a cross shape around the center of the disc, placing one disc at the central intersection. Place another disc every 1.5 cm away from the center along the two lines, for a total of 5 discs on a 9 cm diameter disc. Add 10 μL of protein in 20 mM PBS, pH 8.0 buffer to the disc. For the control, add only 10 μL of 20 mM PBS, pH 7.5 buffer. Incubate at 25°C for 72 hours. Mycelial growth will surround the outer disc containing the control, and a clear zone of inhibition will form around the disc containing the antifungal sample. A larger clear zone indicates a higher protein inhibition rate. Antibacterial experiments showed that A3 (eluted tube 8) of the eluted peanut α-helical-cyclic peptides had significant antibacterial activity against rust fungi (rust pathogens), Fusarium, Anthracnose spp., and Rhizoctonia solani, while A1 (tube 2), A2 (tube 5), and A4 (tube 10) showed very weak antibacterial activity against rust fungi (rust pathogens), Fusarium, Anthracnose spp., and Rhizoctonia solani. Figure 2 All peanut proteins have very weak antibacterial activity against Bacillus subtilis. Figure 2 Therefore, A3 (the 8th eluted tube) is likely the ideal antibacterial peanut α-helix-cyclic peptide, named the antifungal peanut α-helix-cyclic peptide, for subsequent experiments.

[0027] Figure 1Elution curves of peanut protein on a Sephacryl S-200 column, showing peaks A1 (tube 2), A2 (tube 5), and A4 (tube 10). Elution was performed with 20 mM PBS buffer (pH 8.0) (containing 0.35 M NaCl) at a flow rate of 5 ml / min. One tube was collected every 5 ml from the point where protein was detected by the detector, for a total of 10 tubes. Absorbance was measured at 280 nm, and the peaks showing the highest protein concentrations are A1 (tube 2), A2 (tube 5), A3 (tube 8), and A4 (tube 10).

[0028] Figure 2 The area of ​​the inhibition zone of the isolated antifungal peanut α-helical-cyclic peptide (A1 (tube 2), A2 (tube 5), A3 (tube 8), and A4 (tube 10)) was measured. The diameter of each inhibition zone was measured three times, and the average diameter of the inhibition zone was calculated. The area of ​​the inhibition zone was then calculated using the area formula (S=π*(d / 2)2, where π represents pi, r represents the radius, d represents the diameter, and S represents the area).

[0029] Example 2: Identification of the molecular weight and sequence of the antifungal peanut α-helical-cyclic peptide obtained in Example 1

[0030] In Example 1, a complete experimental dataset was recorded and preserved during the molecular weight and sequence identification of the antifungal peanut α-helical-cyclic peptide. This included details of the injected 100 μL peptide sample (10 μg / μL), chromatographic separation at a column temperature of 35 °C and a flow rate of 0.3 mL / min, and all mass spectrometry data captured in cation mode from 500 to 14000 m / z. Furthermore, all MS / MS data used for sequence validation were also retained. This complete recording and preservation of data ensured thorough documentation of experimental conditions and results, facilitating accurate verification and further analysis of the peptide's properties and its antifungal mechanism, achieving the theoretical molecular weight of 12.3 kDa.

[0031] One ml of the antifungal peanut α-helical-cyclic peptide was purified by desalting using a reverse-phase liquid chromatography (HPLC) column (Welch XB C18, inner diameter and length 4.6 × 150 mm). The mobile phase was 40% v / v pure water (containing 0.1% trifluoroacetic acid (TFA)) - 60% v / v acetonitrile (ACN) (containing 0.1% TFA), at a flow rate of 1 mL / min, detected at 220 nm. The elution peak was collected, lyophilized, and then resuspended in 0.1% formic acid for analysis using HPLC-MS. The molecular weight of the synthesized antifungal peanut α-helical-cyclic peptide was identified using electrospray ionization mass spectrometry (ESI-MS). The sample was injected into the HPLC system using a mobile phase of 50% H₂O / 50% CAN at a flow rate of 0.2 mL / min, with a protective gas nitrogen flow rate of 1.5 L / min and a collision energy of 4.5 kV, in anion mode.

[0032] The purity of the purified antifungal peanut α-helical-cyclic peptide was determined by high-performance liquid chromatography (HPLC) (Welch XBC 18, inner diameter and length 4.6 x 250 mm), and molecular weight was determined by electrospray ionization mass spectrometry. The HPLC purity determination results are as follows: Figure 3 As shown: the antifungal peanut α-helical-cyclic peptide exhibits a single peak at 22.6 min ( Figure 3 ).

[0033] Figure 3 High-performance liquid chromatography (HPLC) analysis of the purity of antifungal peanut α-helical-cyclic peptide.

[0034] Electrospray mass spectrometry identification results are as follows Figure 4 As shown: The protein's molecular weight is close to the theoretical value of 12.3 kDa. Figure 4 ).

[0035] Figure 4 Electrospray mass spectrometry identification of antifungal peanut α-helical-cyclic peptide.

[0036] Example 3: Sequencing of the antifungal peanut α-helical-cyclic peptide obtained in Example 1

[0037] Following previous literature, the Edman degradation method of phenyl isothiocyanate was used to sequence the antifungal peanut α-helical-cyclic peptide

[16] . The purified peptide (20 μg) was dissolved in 50 μl of 0.2 M ammonium bicarbonate (pH 8.0, containing 4 M guanidine hydrochloride) and mixed with 5 ml of 45 mM dithiothreitol. The automated Edman degradation of the S-carboxamide methylated peptide and the detection of phenylthioacetal derivatives were performed on an automated protein sequencer (Applied Biosystems, Model 476A). In the presence of 6 mol / L HCl, cleavage occurred at the first peptide bond, resulting in a peptide fragment with the first base removed and the release of the first anilinothiazolinone (ATZ) residue. Other reactants and released residues were washed away with 20 mM PBS (pH 7.0) buffer, and the shortened peptide fragment could be released by another round of coupling and cleavage to release the second residue (process and conditions as above), and so on, until the last amino acid residue was released. Sequencing results confirmed it to be peanut α-helical-cyclic peptide, an antifungal agent derived from peanuts.

[0038] The sequence number SEQ ID No. 1 is:

[0039] HSKHLRVSSYHTASEDLSACDGLKDYGKSMDAKLSSRDLHARRNTSAERRKKCRQQSILLYSDPEQVCRHHLSPFKKTYGPVKGQSEGQFPKYPNSPAWCVAKYIQSQ

[0040] (a) Sequence characteristics:

[0041] ●Length: 108

[0042] ●Type: Amino acid sequence

[0043] ●Chain type: Single chain

[0044] ●Topology: Linear

[0045] (b) Molecular type: protein

[0046] (c) Assumption: No

[0047] (d) Antonym: No

[0048] (e) Original source: peanut

[0049] This peptide chain structure exhibits a combination of various secondary structure elements. It contains β-sheets, which form a stable planar structure through hydrogen bonding interactions between backbone atoms. Additionally, the main component is an α-helix structure, maintained by internal hydrogen bonds. The remaining portion consists of random coil cyclic peptide structures, which are flexible and disordered parts of the peptide chain. These diverse structural elements—β-sheets, α-helices, and cyclic peptides—are interconnected to form a compact and functionally important three-dimensional structure that plays a crucial role in protein stability and interactions.

[0050] Example 4: Sequencing of the antifungal peanut α-helical-cyclic peptide obtained in Example 1

[0051] trypsin digestion

[0052] 1 ml of the antifungal peanut α-helical-cyclic peptide solution (1 mg / ml) obtained in Example 1 was treated with 10 mM dithiothreitol (DTT) to reduce disulfide bonds at 55 °C for 20 min. After cooling to room temperature, 20 mM iodoacetamide (IAA) was added, and protein alkylation was induced at room temperature in the dark for 30 min. The peptides were digested with trypsin (enzyme:substrate solution = 1:50 (w / v, mg / ml)) at 37 °C for 16 h with gentle shaking. The digested peptides were desalted using an octyl nonpolar C18 solid-phase extraction column (Phenomenex Jupiter, packing particle size 15 μm, Phenomenex, model 00G-4053-P0) and dried in a vacuum desiccator for 2 h.

[0053] The peanut α-helical-cyclic peptide digested by trypsin obtained in Example 4 was desalted using a desalting column with 100 μL of binding buffer AT (80% acetonitrile (CAN) and 1% trifluoroacetic acid (TFA)). The specific steps were as follows: The hydrophilic column (Merck ZIC-HILIC, model: 1.50478.0001) was activated with 100 μL of AT binding buffer and equilibrated with 300 μL of AT binding buffer. 100 μL of the peanut α-helical-cyclic peptide digested by trypsin obtained in Example 4 was loaded onto the ZIC-HILIC hydrophilic column and allowed to stand for 5 minutes. The column was then washed with 800 μL of AT binding buffer. Finally, the N-glycopeptide was eluted with 300 μL of 0.1% TFA, followed by 100 μL of 50 mM NH4HCO3. The eluent was dried in a vacuum desiccator and resuspended in 100 μL of 0.1% TFA.

[0054] Reversed-phase liquid chromatography-electrospray ionization mass spectrometry (RPLC ESI-MS / MS) analysis

[0055] Peanut α-helical-cyclic peptide (100 μL 1 mg / ml protein) was analyzed using a reversed-phase liquid chromatography-electrospray ionization mass spectrometry system, with the spectrometer operating in positive ion mode. A C18 column (Waters, Chromatographic separation was performed on a 5μm, 4.6mm x 250mm lens (model 186001265) at a flow rate of 300 nL / min. The mobile phase consisted of 99.8% H₂O and 0.2% TFA(A) at v / v, and 95% ACN, 4.8% H₂O, and 0.2% TFA(B) at v / v. The LC gradients were as follows: 2% B, 10 min; linear 2%-40% B, 190 min; linear 40%-95% B, 10 min; 95% B, 5 min; linear 95%-2% B, 5 min; 2% B, 20 min. The ESI conditions were as follows: spray voltage 2.8 kV, capillary temperature 320 °C, and high-pressure ring iontophoresis device (S-lens RF) level 75 V. Full-scan mass spectrometry was acquired in the range of 500-14000 m / z. The main ESI source settings were: microscan 1, mass resolution 70 k, automatic gain control (AGC) target 2e5, and maximum ion implantation time 50 ms. MSMS spectra were acquired in Top20 data correlation acquisition mode with the following settings: microscan 1, mass resolution 17.5 k, AGC target 5e5, maximum ion implantation time 250 ms, isolation window 3 m / z, higher energy collisional dissociation (HCD), step normalization of collision energies 20%, 30%, and 40%, dynamic exclusion for 20 seconds, and charge inclusion 2-6.

[0056] In conjunction with Examples 3 and 4, an antifungal peanut α-helical-cyclic peptide is disclosed, the amino acid sequence of which is SEQ ID NO.1. The sequence contains 108 amino acid residues, has a molecular weight of 12.3 kDa, and an isoelectric point of 9.59.

[0057] Example 5: Experiment on the antifungal effect of the peanut α-spiral-cyclic peptide obtained in Example 1 against the rust-causing fungus *Puccinia striiformis*.

[0058] The solid culture medium used in the antifungal studies included potato dextrose agar (PDA) for fungi (potato, 200 g; glucose, 20 g; agar, 18 g; and distilled water, 1 L). To test antifungal activity, 1 mL of a suspension of fungal spores (Puccinia striiformis) (1 x 10⁻⁶) was prepared. 7Spores (per ml) were evenly added to 100 ml of LPDA medium and incubated at 40-50°C to prepare solid plates (90 mm in diameter, 3-4 mm thick). After the medium solidified, Oxford cups (stainless steel tubes, 6 mm inner diameter, 8 mm outer diameter, 10 mm height) were placed between them, spaced 2 cm apart. Then, 5 μL of different concentrations of antifungal peanut α-helical-cyclic peptide (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / ml) were added to the center of each Oxford cup, and the cups were incubated at 30°C for 48 h. The diameter of the antifungal clear zone was measured. For the control group, an equal volume of 5 μL of physiological saline was added, and the same procedure and conditions were followed for incubation. Simultaneously, the antifungal activity of peanut α-helical-cyclic peptide was tested at different temperatures (20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃) and different pH values ​​(0-12, respectively) for treatment. The antibacterial activity was then tested repeatedly using the same method and conditions as above.

[0059] The Oxford cup method was used to directly antagonize the growth of certain fungi and other microorganisms on agar plates, determining the antifungal activity of peanut α-helix-cyclic peptide against *Puccinia striiformis*, the causal agent of wheat stripe rust. In this study, peanut α-helix-cyclic peptide showed significant resistance to *Puccinia striiformis* spores after 48 hours (clear zone diameter <6 mm at 2 mg / L, >6 mm at concentrations above 2 mg / L). Within a pH range of 2.0 to 10.0, the clear zone diameter increased with increasing concentration. Antifungal activity against *Puccinia striiformis* was observed across a pH range of 2.0 to 10.0, with the highest activity near pH 8. Figure 5 A) It exhibits high activity at temperatures between 30-60℃, but its activity decreases sharply at temperatures above 70℃. Figure 5 B).

[0060] Figure 5 Antifungal activity of peanut α-helical-cyclic peptide against the rust-causing fungus *Puccinia striiformis*. (A) Antifungal activity against *Puccinia striiformis* in solutions with different pH values. (B) Antifungal activity against *Puccinia striiformis* after heat treatment. The diameter of the transparent ring is represented by a thermogram, corresponding to the numerical value of the transparent ring diameter on the scale bar. Brightness represents a larger transparent ring diameter, and darkness represents a smaller transparent ring diameter.

[0061] Example 6: Experiment on the antifungal effect of peanut α-spiral-cyclic peptide obtained in Example 1 against Fusarium oxysporum, the fungus causing Fusarium wilt.

[0062] The solid culture medium used in the antifungal studies included potato dextrose agar (PDA) for fungi (potato, 200 g; glucose, 20 g; agar, 18 g; and distilled water, 1 L). To test antifungal activity, 1 mL of a suspension of the fungal spore *Fusarium oxysporum* (1 x 10⁻⁶) was prepared. 7 Spores (per ml) were evenly added to 100 mL of PDA medium and incubated at 40-50°C to prepare solid plates (90 mm in diameter, 3-4 mm thick). After the medium solidified, the plates were placed on solid plates made from stainless steel tubes in Oxford cups (6 mm inner diameter, 8 mm outer diameter, 10 mm height). Then, 5 μL of different concentrations of antifungal peanut α-helical-cyclic peptide (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / mL) were added to the center of each Oxford cup, and the plates were incubated at 30°C for 48 h. The diameter of the antifungal clear zone was measured. For the control group, an equal volume of 5 μL of physiological saline was added, and the plates were incubated under the same conditions. The antifungal activity of the peanut α-helical-cyclic peptide was tested at different temperatures (20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C) and different pH values ​​(0-12, 0, 2, 4, 6, 8, 10, 12), and the above method was repeated for testing.

[0063] The Oxford cup method was used to directly antagonize the growth of certain fungi and other microorganisms on agar plates, determining the antifungal activity of peanut α-helix-cyclic peptide against *Fusarium oxysporum*. In this study, peanut α-helix-cyclic peptide showed significant resistance to fungal spores (*Puccinia striiformis*) after 48 hours (clear zone diameter <5 mm at 2 mg / L, >5 mm at concentrations above 2 mg / L). Within a pH range of 2.0 to 10.0, the clear zone diameter increased with increasing concentration. Antifungal activity against *Fusarium oxysporum* was observed across a pH range of 2.0 to 10.0, with the highest activity near pH 8. Figure 6 A) It exhibits higher activity at temperatures between 30-60℃, but its activity decreases sharply above 70℃. Figure 6 B).

[0064] Figure 6Antifungal activity of peanut α-helical-cyclic peptide against the rust-causing fungus *Fusarium oxysporum*. (A) Antifungal activity against *Fusarium oxysporum* in solutions with different pH values. (B) Antifungal activity against *Fusarium oxysporum* after heat treatment. The diameter of the transparent ring is represented by a thermogram, corresponding to the numerical value of the transparent ring diameter on the scale bar. Brightness represents a larger transparent ring diameter, and darkness represents a smaller transparent ring diameter.

[0065] Example 7: Experiment on the antifungal effect of peanut α-helical-cyclic peptide obtained in Example 1 against Colletotrichum gloeosporioides.

[0066] The solid culture medium used in the antifungal studies included potato dextrose agar (PDA) for fungi (potato, 200 g; glucose, 20 g; agar, 18 g; and distilled water, 1 L). To test antifungal activity, 1 mL of anthracnose fungal spore suspension (Colletotrichum gloeosporioides) (1 x 10⁻⁶) was used. 7 Spores (per ml) were evenly added to 100 ml of LPDA medium and incubated at 40-50°C to prepare solid plates (90 mm in diameter, 3-4 mm thick). After the medium solidified, the plates were placed on solid plates made from stainless steel tubes in Oxford cups (6 mm inner diameter, 8 mm outer diameter, 10 mm height). Then, 5 μL of different concentrations of antifungal peanut α-helical-cyclic peptide (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / ml) were added to the center of each Oxford cup, and the plates were incubated at 30°C for 48 h. The diameter of the antifungal circle was measured. For the control group, an equal volume of physiological saline was added, and the plates were incubated under the same conditions. The antifungal activity of the peanut α-helical-cyclic peptide was tested simultaneously at different temperatures (20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C) and different pH values ​​(0-12, 0, 2, 4, 6, 8, 10, 12). The antifungal activity was then tested repeatedly using the same method and conditions.

[0067] The Oxford cup method was used to directly antagonize the growth of certain fungi and other microorganisms on agar plates, determining the antifungal activity of peanut α-helix-cyclic peptides against *Colletotrichum gloeosporioides*. In this study, peanut α-helix-cyclic peptides showed significant resistance to *Colletotrichum gloeosporioides* spores after 48 hours (clear zone diameter <4 mm at 2 mg / L, and >4 mm at concentrations above 2 mg / L). Within a pH range of 2.0 to 10.0, the clear zone diameter increased with increasing concentration. Antifungal activity against *Colletotrichum gloeosporioides* was observed across a pH range of 2.0 to 10.0, with the highest activity near pH 8. Figure 7 A) It exhibits higher activity at temperatures between 30-60℃, but its activity decreases sharply above 70℃. Figure 7 B).

[0068] Figure 7 Antifungal activity of peanut α-helical-cyclic peptide against the rust-causing fungus *Colletotrichum gloeosporioides*. (A) Antifungal activity against *Colletotrichum gloeosporioides* in solutions with different pH values. (B) Antifungal activity against *Colletotrichum gloeosporioides* after heat treatment. The diameter of the transparent ring is represented by a thermogram, corresponding to the numerical value of the transparent ring diameter on the scale bar; brightness represents a larger transparent ring diameter, and darkness represents a smaller transparent ring diameter.

[0069] Example 8: Experiment on the antifungal effect of peanut α-helical-cyclic peptide against Rhizoctonia solani

[0070] The solid culture medium used in the antifungal studies included potato dextrose agar (PDA) for fungi (potato, 200 g; glucose, 20 g; agar, 18 g; and distilled water, 1 L). To test antifungal activity, 1 mL of a suspension of Rhizoctonia solani spores (1 x 10⁻⁶) was prepared. 7Spores (per ml) were evenly added to 100 mL of PDA medium and incubated at 40-50°C to prepare solid plates (90 mm in diameter, 3-4 mm thick). After the medium solidified, stainless steel tubes (6 mm inner diameter, 8 mm outer diameter, 10 mm height) were placed in Oxford cups. Then, 5 μL of different concentrations of antifungal peanut α-helical-cyclic peptide (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / ml) were added to the center of each Oxford cup, and the plates were incubated at 30°C for 48 h. The diameter of the antifungal circle was measured. For the control group, an equal volume of physiological saline was added, and the plates were incubated under the same conditions. The antifungal activity of the peanut α-helical-cyclic peptide was tested simultaneously at different temperatures (20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C) and different pH values ​​(0-12, 0, 2, 4, 6, 8, 10, 12). The antifungal activity was then tested repeatedly using the same method and conditions.

[0071] The Oxford cup method was used to directly antagonize the growth of certain fungi and other microorganisms on agar plates, determining the antifungal activity of peanut α-helix-cyclic peptide against Rhizoctonia solani. In this study, peanut α-helix-cyclic peptide showed significant resistance to Rhizoctonia solani spores after 48 hours (clear zone diameter <4 mm at 2 mg / L, and >4 mm at concentrations above 2 mg / L). Within a pH range of 2.0 to 10.0, the clear zone diameter increased with increasing concentration. Antifungal activity against Rhizoctonia solani was observed across a pH range of 2.0 to 10.0, with the highest activity around pH 8. Figure 8 A) It exhibits higher activity at temperatures between 30-60℃, but its activity decreases sharply at temperatures above 70℃. Figure 8 B).

[0072] Figure 8 Antifungal activity of peanut α-helical-cyclic peptide against the rust-causing fungus *Rhizoctonia solani*. (A) Antifungal activity against *Rhizoctonia solani* in solutions with different pH values. (B) Antifungal activity against *Rhizoctonia solani* after heat treatment. The diameter of the transparent ring is represented by a thermogram, corresponding to the numerical value of the transparent ring diameter on the scale bar. Brightness represents a larger transparent ring diameter, and darkness represents a smaller transparent ring diameter.

Claims

1. An antifungal α-helical-cyclic peptide, characterized in that, The antifungal agent is a peanut α-helical-cyclic peptide, the amino acid sequence of which is shown in SEQ ID NO.1 of the sequence listing.

2. A method for preparing the antifungal peanut α-helical-cyclic peptide according to claim 1, wherein peanut seeds are used as raw material, and the peanut α-helical-cyclic peptide is separated stepwise by ion exchange chromatography and gel filtration chromatography, and then purified by reversed-phase column chromatography for desalting.

3. The application of the antifungal peanut α-helical-cyclic peptide according to claim 1, characterized in that, The application of the antifungal peanut α-helical-cyclic peptide in inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi are one or more of Puccinia striiformis, Fusarium oxysporum, Colletotrichum gloeosporioides, and Rhizoctonia solani, and the application is for non-disease diagnosis and treatment purposes.

4. The application of the antifungal peanut α-helical-cyclic peptide according to claim 1, characterized in that, The application of the antifungal peanut α-helical-cyclic peptide in the preparation of drugs that inhibit plant pathogenic fungi, wherein the plant pathogenic fungi are one or more of Puccinia striiformis, Fusarium oxysporum, Colletotrichum gloeosporioides, and Rhizoctonia solani.

5. The application of the antifungal peanut α-helical-cyclic peptide according to claim 1, characterized in that, The application of the antifungal peanut α-helical-cyclic peptide in the preparation of drug formulations that inhibit plant pathogenic fungi, wherein the plant pathogenic fungi are one or more of Puccinia striiformis, Fusarium oxysporum, Colletotrichum gloeosporioides, and Rhizoctonia solani.

6. A drug for combating plant pathogenic fungi, comprising the antifungal peanut α-helical-cyclic peptide of claim 1 as an active ingredient, wherein the plant pathogenic fungi are one or more of Puccinia striiformis, Fusarium oxysporum, Colletotrichum gloeosporioides, and Rhizoctonia solani.

7. A pharmaceutical preparation for combating plant pathogenic fungi, comprising the antifungal peanut α-helical-cyclic peptide of claim 1 as an active ingredient, wherein the plant pathogenic fungi are one or more of Puccinia striiformis, Fusarium oxysporum, Colletotrichum gloeosporioides, and Rhizoctonia solani.

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