Peanut aspartate-rich peptide, application and medicine thereof

Abstract

The application discloses an anti-fungal peanut aspartate-rich peptide and characterization of anti-fungal activity of the peanut aspartate-rich peptide. The peanut aspartate-rich peptide has high anti-fungal activity on peanut pathogens fusarium oxysporum and rhizoctonia solani.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological pesticides, and particularly relates to a kind of anti-fungal peanut rich aspartic acid peptide, and the anti-fungal activity of peanut pathogen fusarium oxysporum and rhizoctonia solani. BACKGROUND

[0002] Plants resist the infection of pathogenic bacteria by up-regulating the expression of genes including microbe-induced plant antitoxin (Darvill AG, Albersheim P (1984) Phytoalexins and their elicitors-a defense against microbial infection in plants. Annu Rev Plant Physiol 35:243-275.), lyase, protease inhibitor, etc. Therefore, the pathogenesis-related proteins have been the research focus in agriculture for a long time (Stintzi, Alain, et al. Plant‘pathogenesis-related’proteins and their role in defense against pathogens. Biochimie 75.8 (1993): 687-706; Jain, Deepti, and Jitendra Paul Khurana. Role of pathogenesis-related (PR) proteins in plant defense mechanism. Molecular aspects of plant-pathogen interaction. Springer, Singapore, 2018. 265-281.).

[0003] Peanut (Arachishypogaea) antimicrobial peptides represent a significant advancement in agricultural biotechnology and food safety, offering a novel and effective method for combating microbial pathogens (Zhao, Kai, et al. "Genome-wide investigation of defensin genes in peanut (Arachishypogaea L.) reveal AhDef2.2 conferring resistance to bacterial wilt." The Crop Journal 10.3 (2022):809-819; Zhao, Kai, et al. "Genome-wide investigation of defensin genes in peanut (Arachishypogaea L.) reveal AhDef2.2 conferring resistance to bacterial wilt." The Crop Journal 10.3 (2022):809-819.). These peptides, extracted from common legumes, offer a promising alternative to traditional antimicrobial agents, especially in the context of increasing antibiotic resistance (Mani, Saiprahalad, et al. "The updated review on plant peptides and their applications in human health." International Journal of Peptide Research and Therapeutics 28.5 (2022):135.).

[0004] The structural and functional properties of these peptides play a crucial role in their antibacterial efficacy. Typically, peanut antibacterial peptides are small, amphipathic molecules that enable them to effectively interact with microbial membranes. Their mode of action primarily involves disrupting the integrity of bacterial cell membranes, leading to cell lysis and death (Terea, Hafidha, et al. "Preparation and characterization of cellulose / ZnO nanoparticles extracted from peanut shells: Effects on antibacterial and antifungal activities." Biomass Conversion and Biorefinery (2023): 1-12). Aspergillus flavus is a saprophytic fungus often detected in oil-rich seeds. During colonization, this organism releases aflatoxins, posing a significant risk to food safety and human health. Therefore, an eco-friendly biological approach is needed to inhibit the pathogen. Experimental results showed that when the concentration of Sub3 exceeded 0.15 g L-1, A. flavus spores could not germinate in potato dextrose broth. Morphological assessments by flow cytometry and scanning electron microscopy showed that spores shrunk and developed pits after Sub3 exposure. Physiological assessments using propidium iodide, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide, 2,7-dichlorodihydrofluorescein diacetate, and 4',6-diamidino-2-phenylindole staining showed that cell membrane damage, decreased mitochondrial membrane potential, increased intracellular reactive oxygen species levels, nuclear condensation, and increased DNA fragmentation. Furthermore, mitochondrial dehydrogenase activity decreased by 29.42% and 45.48% after treatment with 0.1 and 0.15 g L-1 Sub3, respectively. Additionally, the colonization ability of peanuts was significantly reduced compared to the control group, and the number of spores on seeds treated with Sub3 decreased by 26.86% (0.1 g L-1) and 77.74% (0.15 g L-1), respectively. Sub3 may inhibit A. flavus by penetrating the cell wall and targeting the cell membrane, disrupting mitochondrial energy metabolism, inducing DNA damage, and causing spore death.Thus, Sub3 can provide a useful biocontrol strategy to control growth of A. flavus in peanuts (Zhang, Wei, et al. "Sub3 inhibits Aspergillus flavus growth by disrupting mitochondrial energy metabolism, and has potential biocontrol during peanut storage." Journal of the Science of Food and Agriculture 101.2 (2021): 486-496). This mechanism is quite different from the way traditional antibiotics work, which usually target specific bacterial enzymes or pathways, making these peptides potentially effective against a broader range of pathogens, including antibiotic-resistant strains.

[0005] Fusarium solani is one of the pathogens that causes peanut root rot. It is a fungus that typically infects the roots of peanut plants, causing the roots to rot, reducing plant growth and yield. This pathogen can spread through the soil and rapidly multiply in warm, humid environments (Erazo, Jessica Gabriela, et al. "Biocontrol mechanisms of Trichoderma harzianum ITEM 3636 against peanut brown root rot caused by Fusarium solani RC 386." Biological Control 164 (2021): 104774.). Rhizoctonia solani is another fungus that causes problems for peanuts. It can produce toxins, one of which is called aflatoxin, which is harmful to humans and animals. Rhizoctonia solani typically thrives in warm, humid conditions and can contaminate peanuts and other grains if stored improperly, posing a threat to food safety (Abbas, Aqleem, et al. "Assessment of Genetic Variability and Evolutionary Relationships of Rhizoctonia solani Inherent in Legume Crops." Plants 12.13 (2023): 2515; and "Antibacterial effect of Bradyrhizobium spp., Pseudomonas spp., and Bacillus subtilis against pathogenic fungus Rhizoctonia solani on peanut plant." 18 (2022).) To control these pathogens, we attempted to extract the corresponding antimicrobial peptides from peanut meal and tested the antifungal effect.

[0006] Furthermore, the specificity and rapid action of these peptides are noteworthy. Unlike broad-spectrum antibiotics that can indiscriminately target both beneficial microorganisms and harmful bacteria, peanut antimicrobial peptides exhibit a degree of selectivity, minimizing negative impacts on beneficial microorganisms. This selectivity is crucial in maintaining the microbial balance in various environments, including soil, water, and the gastrointestinal tracts of plants and animals. Recent studies have explored the potential applications of these peptides in various fields. In agriculture, they can be used as biofungicides or biological control agents to protect crops from bacterial infections, thereby reducing reliance on chemical pesticides. In the food industry, incorporating them into food packaging materials or as food preservatives can enhance food safety by preventing the growth of foodborne pathogens. The biotechnological production and optimization of peanut antimicrobial peptides are hot areas of current research. Advances in genetic engineering and peptide synthesis techniques have facilitated the development of processes for efficiently producing these peptides in host organisms or in vitro systems. Additionally, efforts are being made to find low-abundance, highly active antifungal peptides through stepwise isolation methods.

[0007] In summary, peanut antimicrobial peptides represent a promising, environmentally friendly alternative to traditional antibiotics and chemicals for combating bacterial pathogens. Their unique mechanisms of action, specificity, and potential applications in agriculture and food safety make them valuable tools for ongoing efforts to combat fungal infections and antibiotic resistance. Further research and development in this area will undoubtedly expand their applications and practicality in various fields. SUMMARY

[0008] A highly active antifungal peptide was isolated from peanut meal, peanut asparagine-rich peptide, whose amino acid sequence is:

[0009] VDIKCTSIPRNSRVVTDGDPIRPAVLDGLAVTPPQNDYTNHNISHDAFTYV

[0010] PDASHPNKTSLPSVLGIACRCVGKFTMGYEKCTGARYELYGITIRHEHDP DNID.

[0011] The peanut asparagine-rich peptide has antifungal activity against the peanut pathogens Fusarium oxysporum and Rhizoctonia solani. The antifungal peanut asparagine-rich peptide is used for long-term stable application in one or more than two processes of inhibiting the peanut pathogens Fusarium oxysporum and Rhizoctonia solani.

[0012] The peanut asparagine-rich peptide is an antifungal protein in peanut, and the peanut asparagine-rich peptide has antifungal activity against the peanut pathogens Fusarium oxysporum and Rhizoctonia solani.

[0013] The peanut aspartate-rich peptide has an amino acid sequence as shown in the sequence table SEQ ID NO. 1. The peanut aspartate-rich peptide is prepared by ion exchange chromatography and gel filtration chromatography step by step, and desalted by reverse phase column chromatography. It is a kind of peanut aspartate-rich peptide with high antifungal activity. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The peanut aspartate-rich peptide is prepared by ion exchange chromatography and gel filtration chromatography step by step, and desalted by reverse phase column chromatography. It is a kind of peanut aspartate-rich peptide with high antifungal activity.

[0015] Figure 2 The peanut aspartate-rich peptide is prepared by ion exchange chromatography and gel filtration chromatography step by step, and desalted by reverse phase column chromatography. It is a kind of peanut aspartate-rich peptide with high antifungal activity.

[0016] Figure 3 The peanut aspartate-rich peptide is prepared by ion exchange chromatography and gel filtration chromatography step by step, and desalted by reverse phase column chromatography. It is a kind of peanut aspartate-rich peptide with high antifungal activity.

[0017] Figure 4 The peanut aspartate-rich peptide is prepared by ion exchange chromatography and gel filtration chromatography step by step, and desalted by reverse phase column chromatography. It is a kind of peanut aspartate-rich peptide with high antifungal activity.

[0018] Figure 5 The peanut aspartate-rich peptide is prepared by ion exchange chromatography and gel filtration chromatography step by step, and desalted by reverse phase column chromatography. It is a kind of peanut aspartate-rich peptide with high antifungal activity.

[0019] Figure 6 The peanut aspartate-rich peptide is prepared by ion exchange chromatography and gel filtration chromatography step by step, and desalted by reverse phase column chromatography. It is a kind of peanut aspartate-rich peptide with high antifungal activity. DETAILED DESCRIPTION

[0020] The present application can be better understood according to the following examples. However, those skilled in the art will readily understand that the examples described are only for the purpose of illustrating the present application, and should not and will not limit the present application described in detail in the claims.

[0021] Example 1: Preparation of the peanut aspartate-rich peptide

[0022] Take 5.0 g of peanut meal (primary meal, which is the peanut residue left after the first pressing of oil extraction), repeatedly grind into fine powder (particle size below 0.5 mm) in liquid nitrogen, place in a 200 mL beaker, add 100 mL of protein extraction buffer (composition: 20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% (volume concentration) DMSO and 0.1% (volume concentration) β-mercaptoethanol in water), and mix well. Centrifuge at 10,000 g for 20 min, and take 60 ml of supernatant.

[0023] A saturated solution of ammonium sulfate (100%) was prepared by slowly adding 767 g of (NH4)2SO4 to 1 liter of distilled water while stirring, and adjusting to pH 7.0 with 25-28% ammonia by mass (and / or 95% sulfuric acid by mass). An equal volume of the saturated ammonium sulfate solution (100%) was added to the supernatant described above while stirring. The resulting solution was left to stir overnight on a magnetic stirrer (4°C, 12 hours) to allow the proteins to precipitate fully. The protein solution was centrifuged at 10,000 g for 30 min (4°C). The supernatant was discarded and the pellet was retained. The pellet was dissolved in 20 ml of protein extraction buffer (composition: 20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% DMSO by volume, 0.1% β-mercaptoethanol by volume, and 0.2 g / L sodium azide in water). The solution was placed in a 40 ml dialysis bag (2 kDa molecular weight cut-off) and dialyzed against 1 liter of protein extraction buffer for 24 hours (4°C), changing the dialysis buffer every 6 hours to remove the ammonium sulfate completely. The dialyzed solution was collected in the dialysis bag. A DEAE-Sepharose Fast Flow column (2.6 x 100 cm in diameter and length, Merk, type 17-0709-10) was equilibrated with three column volumes of protein extraction buffer (composition: 20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% DMSO by volume, and 0.1% β-mercaptoethanol by volume in water) at a flow rate of 0.5 ml / min. The 25 ml dialyzed solution was loaded onto the DEAE-Sepharose Fast Flow column (2.6 x 100 cm), which was again equilibrated with three column volumes of protein extraction buffer (composition: 20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% DMSO by volume, and 0.1% β-mercaptoethanol by volume in water) at a flow rate of 0.5 ml / min. The column was eluted for 0.5 hours using a 0.5 M NaCl gradient (linear gradient of NaCl concentration from 0.15-0.5 M in the elution buffer) at a flow rate of 1.0 ml / min. The column was prepared as follows: first, the protein extraction buffer was prepared by mixing all the components in water and adjusting the pH to 8.0. Then, the column was prepared according to the type of experiment and was ensured to be equilibrated with the appropriate buffer.Afterwards, the sample containing the target protein is slowly loaded, allowing the sample to fully contact the packing material. Next, a 0.5 M NaCl solution is prepared and mixed with the extraction buffer to form a gradient, and eluted through the column at a flow rate of 1.0 mL / min. During this process, the eluate is collected in 2 mL fractions and monitored for protein content using a UV detector or similar method. Finally, the collected protein is subjected to the necessary subsequent analysis. From the protein detector, the protein eluate is collected starting from the point at which the detector can no longer detect protein and ending at the point at which the detector can detect protein, for a total of 45 mL. The collected material is then applied to a gel filtration column (Sephacryl S-200, 5 x 90 cm in diameter and length, GE, USA, model 17-0584-01) equilibrated with 3 column volumes of 20 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl, and collected in 3 mL fractions. Elution is performed at a flow rate of 0.5 mL / min using 20 mM Tris-HCl buffer (pH 8.0) containing 0.35 M NaCl. From the point at which the detector can detect protein, 18 fractions are collected every 3 mL. Figure 1 A: Forming A1 peak (eluted in the 2nd tube), A2 peak (eluted in the 6th tube), A3 peak (eluted in the 8th tube), A4 peak (eluted in the 12th tube), and A5 peak (eluted in the 14th tube).

[0024] The 5 parts of protein forming A1 peak (eluted in the 2nd tube), A2 peak (eluted in the 6th tube), A3 peak (eluted in the 8th tube), A4 peak (eluted in the 12th tube), and A5 peak (eluted in the 14th tube) are analyzed for antifungal activity, respectively. Potato glucose agar plates (9 cm in diameter, 0.3 cm in thickness) are used (formula: 20 g of potato to make an infusion, 2 g of glucose, 1.5 g of agar, 100 mL of water, no pH adjustment (pH natural). Preparation method: Take 20 g of fresh potato, wash and peel, cut into small pieces (0.1-0.5 cm long rectangular), add 80 mL of water and boil for 30 min, filter with four layers of gauze (50 mesh), add 2 g of glucose and 1.5 g of agar, continue to heat (70-80°C) and stir evenly, slightly cool (50-60°C) and make up to 100 mL, sterilize at 121°C for 20 min). Add 10 μL of frozen bacteria solution (Fusarium oxysporum (purchased from China General Microbiological Culture Collection Center, CGMCC No. 3.6864) and Rhizoctonia solani (purchased from China Industrial Microbial Culture Collection Center, CICC No. 40529) with a density of 1 x 10 5 ~ 2 x 10 5cfu / g lyophilized powder, each gram of sample was dissolved in 10 ml of 0.85% physiological saline by mass concentration, and a bacterial solution was obtained). The anti-fungal activity of the eluted peanut proteins in tubes 2, 6, 8, 12, and 14 was evaluated, respectively. A 1 cm diameter circular sterile filter paper disc was drawn with a "cross" line in the center of the plate (i.e., the center, as the center of the "cross" line), a paper disc was placed at the intersection of the center, and a paper disc was placed at a distance of 1.5 cm from the center along each of the two "cross" lines in the direction away from the center, and a total of 5 paper discs were placed in the 9 cm diameter plate. 10 μL of the above eluted test protein in 20 mM PBS (pH 7.5) buffer was added to the paper disc, and the control was added with only 10 μL of 20 mM PBS buffer (pH 7.5). After incubation at 25°C for 72 hours, the mycelium grew around the peripheral disc containing the control, and a transparent ring was formed around the disc containing the anti-fungal sample. The larger the area of the transparent ring, the higher the inhibition rate of the protein. The antibacterial experiment showed that the A2 peak (eluted in tube 6) of the eluted peanut aspartate-rich peptide had obvious antibacterial activity against Fusarium oxysporum (inhibition ring area about 9 cm 2 ) and Rhizoctonia solani (inhibition ring area about 7.4 cm 2 ), and the A1 peak (tube 2), A3 peak (tube 8), A4 peak (tube 12), and A5 peak (tube 14) had weak antibacterial activity against Fusarium oxysporum and Rhizoctonia solani, less than 0.5 cm 2 ( Figure 1 B). Therefore, the A2 peak (eluted in tube 6) is likely to be the ideal antibacterial peanut protein, which is named peanut aspartate-rich peptide, for subsequent experiments.

[0025] Figure 1 Sephacryl S-200 chromatography column elution curve of peanut protein, forming A1 peak (eluted in tube 2), A2 peak (eluted in tube 6), A3 peak (eluted in tube 8), A4 peak (eluted in tube 12), and A5 peak (eluted in tube 14). Elution was performed at a flow rate of 0.5 ml / min with 20 mM Tris-HCl buffer (pH 8.0) containing 0.35 M NaCl. Protein was detected from the detector, and one tube was collected every 5 ml, for a total of 18 tubes. By measuring the absorbance at 280 nm, the peaks with higher protein concentration were concentrated to form A1 peak (eluted in tube 2), A2 peak (eluted in tube 6), A3 peak (eluted in tube 8), A4 peak (eluted in tube 12), and A5 peak (eluted in tube 14).

[0026] Figure 1Isolation and antibacterial identification of peanut antimicrobial peptides. A. Elution curves of peanut peptides on a Sephacryl S-200 column, showing peaks A1 (elution tube 2), A2 (elution tube 6), A3 (elution tube 8), A4 (elution tube 12), and A5 (elution tube 14). B. Antibacterial effect of each elution peak. C. Area of ​​the clear zone of the isolated antifungal peanut aspartic acid-rich peptides (A1, A2, A3, A4, and A5) against Fusarium oxysporum and Rhizoctonia solani. The diameter of each inhibition zone was measured three times, and the average diameter of the inhibition zone was calculated. The area of ​​the clear zone was calculated using the area formula (S = π * (d / 2)², where π represents pi, r represents radius, d represents diameter, and S represents area).

[0027] Example 2: Identification of the molecular weight and sequence of the antifungal peanut aspartic acid-rich peptide obtained in Example 1

[0028] 1 ml of antifungal peanut aspartic peptide (A2 peak obtained in Example 1 (eluting tube 6)) was passed through a reverse-phase liquid chromatography (HPLC) column (Welch XB, Welch Materials, model number 6). XB-C18 (inner diameter and length C18 4.6 × 150 mm) was purified by chromatographic desalting. The mobile phase was 40% v / v pure water (containing 0.1% trifluoroacetic acid (TFA)) - 60% v / v acetonitrile (ACN) (containing 0.1% TFA), the flow rate was 1 mL / min, and the detection was performed at 220 nm. The elution peak was collected, lyophilized, and purified peanut aspartic acid peptide was obtained. The purified peptide was then resuspended in 0.1% formic acid aqueous solution for analysis by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The molecular weight of the synthesized antifungal peanut aspartic acid peptide was identified by electrospray mass spectrometry. Samples were injected using a liquid chromatography system with a mobile phase of 50% H₂O / 50% CAN at a flow rate of 0.2 mL / min, a protective gas nitrogen flow rate of 1.5 L / min, a collision energy of 4.5 kV, and cation mode.

[0029] Purified antifungal peanut aspartic peptide was obtained by high performance liquid chromatography (HPLC) (Welch XBC, Welch Materials, model number 100000). The purity of XB-C18 (inner diameter and length 184.6 x 250 mm) was determined, and its molecular weight was analyzed using electrospray mass spectrometry. The purity determination results by high-performance liquid chromatography (HPLC) are as follows: Figure 3 As shown: the antifungal peanut aspartic peptide showed a single peak at 28.6 min. Figure 2 ).

[0030] Figure 2 High-performance liquid chromatography (HPLC) analysis of the purity of antifungal peanut aspartic acid peptides.

[0031] The identification result of the electrospray mass spectrum is shown in Figure 3 The molecular weight of the protein is close to the theoretical value 11.55 kDa.

[0032] Figure 3 The electrospray mass spectrum identifies the anti-fungal peanut aspartate-rich peptide. The labeled molecular weight is 11554.38 Da. Considering the cation mode, it is close to the theoretical molecular weight 11554.94 Da.

[0033] Example 3: Sequencing of the anti-fungal peanut aspartate-rich peptide obtained in Example 1

[0034] The anti-fungal peanut aspartate-rich peptide was sequenced using the phenyl isothiocyanate method (Edman) degradation method according to the previous literature [Sels J., Mathys J., De Coninck B. M., Cammue B. P., De Bolle M. F. Plant pathogenesis-related (pr) proteins: A focus on pr peptides. Plant Physiol. Biochem. 2008; 46: 941-950.]. The purified peanut aspartate-rich peptide (20 μg) obtained in Example 2 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 automatic Edman degradation of the S-carboxamide methylated peptide and the detection of the phenylthioacetal derivative were carried out on an automatic protein sequencer (Applied Biosystems, Model 476A). In the presence of 6 mol / L HCl, cleavage occurs at the first peptide bond, resulting in a peptide segment minus the first base and the released residue in the form of the first anilinothiazolinone (ATZ). The shortened peptide segment can be released by another round of coupling and cleavage of the second residue, and so on, until the last amino acid residue is released. The sequencing result is determined as the peanut anti-fungal peanut aspartate-rich peptide.

[0035] The sequence table SEQ ID No. 1 is:

[0036] VDIKCTSIPRNSRVVTDGDPIRPAVLDGLAVTPPQNDYTNHNISHDAF TYVPDASHPNKTSLPSVLGIACRCVGKFTMGYEKCTGARYELYGITIRHE HDPDNID (105 aa)

[0037] (a) Sequence characteristics:

[0038] ●Length: 105

[0039] ●Type: Amino acid sequence

[0040] ●Chain type: Single chain

[0041] ●Topology: Linear

[0042] (b) Molecular type: protein

[0043] (c) Assumption: No

[0044] (d) Antonym: No

[0045] (e) Original source: peanut

[0046] Example 4: Control of fungal infections in rice by the aspartic acid-rich peptide obtained in Example 1

[0047] We first focus on the selection and treatment of rice root samples. Select healthy, mature rice plants (25-35 weeks), ensure that each plant is uniform in size and color, and has no obvious damage from pests and diseases. The treatment method includes immersing 5-10 cm root system in 200 ml of physiological saline solution containing 0.1 mg / ml peanut aspartate-rich peptide (A2 peak obtained in Example 1 (elution of the 6th tube)) for 1 minute, and then surface drying in a sterile environment (20°C) (10 root segments treatment group). The control group of rice roots is only treated with 200 ml of physiological saline (soaked for 1 minute and then dried), under the same conditions. The treated samples are stored in suitable containers and stored at 10-15°C to simulate field conditions. Next, fungal contamination of rice roots is measured every 5 days. Randomly select part of the roots (0.5-0.6 cm) from each rice plant, wash and immerse in 50 mL of sterile 0.1% protein peptone solution for 5 minutes to remove surface fungi, and obtain the wash solution (original wash solution). Dilute the wash solution with 0.85 NaCl, with dilution factors (volume factors) of 1 (original wash solution volume): 10 (volume of diluted solution), 1 (original wash solution volume): 100 (volume of diluted solution), and 1 (original wash solution volume): 1000 (volume of diluted solution). Each dilution solution (0.1 mL) is inoculated onto YPD (yeast extract powder peptone glucose agar) flat plate medium (9 cm in diameter, medium thickness 3-4 mm (3.5 mm in this case)), and incubated at 25°C for 2 days. Count the colony forming units (CFU) on each plate, and express the CFU number as the number of CFU per gram of rice root sample (CFU / g), and observe the colony characteristics under a microscope to distinguish different types of mycelium. Finally, compare the CFU counts of the treatment and control groups, and use statistical methods to analyze the differences between the two groups. Data recording and analysis: Record and compare the number of fungal infections in the rice roots of the treatment and control groups, and use a microscope to observe and distinguish different types of mycelium. The results of using plate culture method to analyze the number of fungal infections in rice are shown in the figure. From Figure 4 It can be seen that during the 26-day preservation period (storage period) (preservation temperature 10-15°C), the number of fungal infections in the treatment and control groups showed significantly different trends. The number of fungal infections in the control group (treated with physiological saline) remained relatively stable throughout the preservation period, with a low CFU / g (colony forming units per gram of sample) number, showing good antibacterial effect. In contrast, the number of fungal infections in the treatment group (treated with aspartate-rich peptide) also remained low in the first 20 days, but after about 20 days, the number of infections in the control group increased sharply, while the test group (treatment group) remained in the inhibition state, indicating that the antibacterial effect of aspartate-rich peptide is obvious after long-term preservation.

[0048] Figure 4. Plate culture method was used to analyze the fungal infection of rice. Rice was stored at 10-15°C for 26 days. Treatment group, aspartate-rich peptide. Control group, physiological saline.

[0049] Example 5: Inhibition of Fusarium oxysporum and Rhizoctonia solani by aspartate-rich peptide obtained in Example 1

[0050] The solid medium used in the antifungal study included potato dextrose agar (PDA) for fungi (potato, 200 g; dextrose, 20 g; agar, 18 g; and distilled water, 1 L). To test the antifungal activity, 1 mL of fungal spore suspension (1 x 10 7 The spores were added to 100 mL of PDA medium at a temperature of 40-50°C to prepare solid plates (plate thickness 3-4 mm (3.5 mm in this case)) and placed in an Oxford cup stainless steel tube (two open ends, inner diameter 6 mm, outer diameter 8 mm, height 10 mm). Then 5 μL of different concentrations of aspartate-rich peptide (A2 peak (eluted from tube 6) obtained in Example 1) (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / mL in physiological saline) were added to the center of different Oxford cups, and the plates were incubated at 30°C for 48 h. The diameter of the antibacterial circle was measured. The control group was treated with 5 μL of physiological saline in the same volume, and the same conditions were used for incubation. The preliminary screening of the antifungal activity of the aspartate-rich peptide by the formation of a transparent circle showed that the IC50 values of the aspartate-rich peptide for Fusarium oxysporum and Rhizoctonia solani were 13.6 and 82 μg / mL, respectively. Figure 5 ).

[0051] Figure 5 . Comparison of antibacterial effect. Control, physiological saline. Treatment group, physiological saline-dissolved antifungal peanut aspartate-rich peptide solution obtained in Example 1.

[0052] Example 6: Effect of aspartate-rich peptide obtained in Example 1 and amphotericin B on glycoside synthase activity of Fusarium oxysporum and Rhizoctonia solani

[0053] Fusarium oxysporum and Rhizoctonia solani frozen cell suspension 0.1 mL was plated on 20 mL PDA medium plates (9 cm in diameter, 3-4 mm (3.5 mm in this case) in medium thickness) and incubated at 25 °C for 5 days. Mycelia of both fungi were washed down with 20 mL distilled water and the washed down mycelia were lyophilized. The dried mycelia (1.0 g) were added to 4 mL sodium acetate buffer (50 mmol / L, pH = 5) and ground in a pre-cooled (0-4 °C) mortar to obtain a homogenate. The homogenate was then centrifuged at 10,000 g for 15 min to obtain the supernatant containing crude glycosyl synthetase (GS). The protein content of the supernatant containing crude enzyme was determined using a BCA assay kit. Enzyme activity was expressed as units / mg protein. In performing the glycosyl synthetase (GS) activity assay, a common method is to use a biochemical assay with color change. For example, using p-Nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate for the enzymatic reaction, where the released p-Nitrophenol (pNP) has absorbance at 405 nm, which can be used for quantitative analysis. The specific experimental procedure includes: in a buffer containing appropriate pH and buffer strength, 5-10 (6 in this case) mmol / L pNPG and 50 μL of enzyme sample were added, the total volume was adjusted to 1 mL, and then incubated at 37 °C for 30 min. The equipment needed in the experiment includes a spectrophotometer (such as Shimadzu UV-1280) to determine the absorbance of the sample, a high-speed centrifuge (Eppendorf 5810R) to remove unreacted particles in the sample, and a constant temperature water bath or incubator (Precision General Purpose water bath from Thermo Fisher Scientific) to keep the reaction at a constant temperature. After the reaction is completed, the sample should be immediately transferred to a 100 °C water bath for 10 min to stop the reaction. The reaction mixture was centrifuged at 10,000 g for 10 min at 4 °C to remove unreacted particles and residues. The absorbance of the supernatant was determined at 405 nm using a spectrophotometer to determine the concentration of pNP. Finally, the GS activity was calculated according to the standard curve, which is usually expressed as the amount of substrate converted per mg of protein per minute (units / mg protein / min). The results show that the anti-fungal peanut aspartate-rich peptide obtained in Example 1 (A2 peak obtained in Example 1 (eluted in the 6th tube)) has good inhibitory activity on the glycosyl synthetase of the mold Figure 6 Figure 6 ​In this case, we see the results of the experiment to determine the effect of the peanut aspartate-rich peptide on the activity of the glycosynthase enzyme (GS). The two histograms represent the GS enzyme activity (in U / mg) for the control group (using saline) and the treatment group (using 0.1 mg / ml of the antifungal peanut aspartate-rich peptide solution).

[0054] From the histograms, it can be observed that the GS enzyme activity in the control group is significantly higher than in the treatment group. Specifically, under the experimental conditions, the peanut aspartate-rich peptide was able to significantly reduce the activity of the GS enzyme, indicating the potential of this compound to inhibit the enzyme. The asterisks indicate statistical significance, with four asterisks (****) indicating a p-value less than 0.0001, meaning that the difference between the two groups is highly significant. Overall, these data indicate that, at the concentration given in the experiment, the peanut aspartate-rich peptide was able to significantly inhibit the activity of the glycosynthase enzyme, which can be of great importance for controlling biological processes that depend on the activity of this enzyme, such as metabolic pathways in certain fungal organisms. Therefore, this peptide can be a promising candidate for the development of new antifungal agents.

[0055] Figure 6 . Comparison of the inhibitory effect of the peanut aspartate-rich peptide on the activity of the glycosynthase enzyme (GS). Control, saline. Treatment group, saline solution of the antifungal peanut aspartate-rich peptide obtained in Example 1 (0.1 mg / ml).

Claims

1. A peanut-rich aspartic peptide, characterized by: The peanut-rich aspartic peptide has the amino acid sequence composition shown in SEQ ID NO.

1.

2. An application of the peanut-enriched aspartic peptide according to claim 1, characterized in that, The application of the peanut-rich aspartic peptide as an antifungal active ingredient in the preparation of one or two drugs that inhibit peanut pathogens Fusarium oxysporum and / or Rhizoctonia solani.

3. A drug for treating one or two of Fusarium oxysporum and / or Rhizoctonia solani, wherein the active ingredient is the antifungal peanut-rich aspartic peptide as described in claim 1.

4. The medicament according to claim 3, further comprising a pharmaceutically acceptable carrier or excipient.

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