Peanut aspartic acid-rich peptide, application and medicine

Abstract

The invention discloses an antifungal peanut aspartic acid-rich peptide and characterization of the antifungal activity of the antifungal peanut aspartic acid-rich peptide. The peanut aspartic acid-rich peptide has the antifungal activity on peanut pathogens, namely fusarium oxysporum and rhizoctonia solani, and is the peanut aspartic acid-rich peptide with relatively high antifungal activity.

Description

Technical Field

[0001] The invention belongs to the technical field of biological pesticides, and specifically relates to a chemically synthesized antifungal peanut aspartic acid-rich peptide having antifungal activity against peanut pathogens Fusarium oxysporum and Rhizoctonia solani. Background Art

[0002] Plants resist infection by upregulating the expression of genes including microbial-induced phytoalexins (Darvill AG, Albersheim P (1984) Phytoalexins and their elicitors-a defense against microbial infection in plants. Annu Rev Plant Physiol 35: 243–275.), lyases, and protease inhibitors. Therefore, pathogenesis-related proteins have long been a hot topic in agricultural research (Stintzi, Alain, et al. Plant'pathogenesis-related'proteins and their role indefense againstpathogens." Biochimie 75.8 (1993): 687-706; Jain, Deepti, and Jitendra Paul Khurana. "Role ofpathogenesis-related (PR) proteins in plantdefense mechanism. Molecular aspects of plant-pathogen interaction. Springer, Singapore, 2018. 265-281.).

[0003] Peanut (Arachishypogaea) antimicrobial peptides are an important advance in agricultural biotechnology and food safety, representing a novel and effective approach to combat microbial pathogens (Zhao, Kai, et al. "Genome-wide investigation of defensin genes in peanut (Arachishypogaea L.) reveals 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.) reveals 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 key role in their antimicrobial efficacy. Typically, peanut antimicrobial peptides are small, amphiphilic molecules that enable them to effectively interact with microbial membranes. Their mode of action mainly 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 that is often detected in oil-rich seeds. During colonization, this organism releases aflatoxins, posing a serious risk to food safety and human health. Therefore, an eco-friendly biological approach is needed to inhibit pathogens. Experimental results showed that when the Sub3 concentration exceeded 0.15 g L-1, Aspergillus flavus spores could not germinate in potato dextrose broth medium. Morphological evaluation by flow cytometry and scanning electron microscopy showed that spores shrank and pitted after Sub3 exposure. Physiological evaluation using propidium iodide, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide, 2,7-dichlorodihydrofluorescein diacetate, and 4',6-diamidino-2-phenylindole staining showed damaged cell membranes, decreased mitochondrial membrane potential, increased intracellular reactive oxygen species levels, nuclear condensation, and increased DNA fragmentation. In addition, mitochondrial dehydrogenase activity decreased by 29.42% and 45.48% after treatment with 0.1 and 0.15 g L-1 Sub3, respectively. In addition, the colonization ability of peanut was significantly reduced compared with the control group, and the number of spores on seeds treated with Sub3 was reduced 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.Therefore, Sub3 may provide a useful biocontrol strategy to control the growth of Aspergillus 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 very different from the mode of action of traditional antibiotics, which usually target specific bacterial enzymes or pathways, making these peptides potentially effective against a wider range of pathogens, including antibiotic-resistant strains.

[0005] Fusarium oxysporum is one of the pathogens that causes peanut root rot. It is a fungus that usually infects the roots of peanut plants, causing root rot and reducing plant growth and yield. This pathogen can be spread through the soil and multiply rapidly in warm and humid environments (Erazo, Jessica Gabriela, et al. "Biocontrol mechanisms of Trichodermaharzianum ITEM 3636 against peanut brown root rot caused by Fusariumsolani RC 386." Biological Control 164 (2021): 104774.). Rhizoctonia solani is another fungus that causes problems in peanuts. It can produce toxins, one of which is called aflatoxin, which is harmful to humans and animals. Rhizoctonia solani usually thrives in warm and 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).). In order to control these pathogens, we tried to extract the corresponding antimicrobial peptides from peanut bran and tested the antifungal effect.

[0006] In addition, the specificity and rapid action of these peptides are also noteworthy. Unlike broad-spectrum antibiotics that may indiscriminately target both beneficial and harmful microorganisms, peanut antimicrobial peptides exhibit a degree of selectivity that minimizes negative effects on beneficial microorganisms. This selectivity is crucial in maintaining the microbial ecological balance in various environments, including soil, water, and the gastrointestinal tract 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 biocontrol agents to protect crops from bacterial infections, thereby reducing dependence on chemical pesticides. In the food industry, their addition to food packaging materials or as food preservatives can improve food safety and prevent the growth of foodborne pathogens. The biotechnological production and optimization of peanut antimicrobial peptides are currently hot areas of research. Advances in genetic engineering and peptide synthesis technologies have facilitated processes for the efficient production of these peptides in host organisms or in vitro systems. In addition, efforts are being made to find low-abundance, highly active antifungal peptides through stepwise isolation methods.

[0007] In conclusion, peanut antimicrobial peptides represent a promising, environmentally friendly alternative to conventional antibiotics and chemicals for combating bacterial pathogens. Their unique mechanism of action, specificity, and potential applications in agriculture and food safety make them valuable tools in the ongoing fight against fungal infections and antibiotic resistance. Further research and development in this area will undoubtedly expand their application and utility in a variety of fields. Summary of the invention

[0008] A highly active antifungal peptide was isolated from peanut bran and identified as peanut aspartic acid-rich peptide by protein sequencing: its amino acid sequence is:

[0009] VDIKCTSIPRNSRVVTDGDPIRPAVLDGLAVTPPQNDYTNHNISHDAFTYV

[0010] PDASHPNKTSLPSVLGIACRCVGKFTMGYEKCTGARYELYGITIRHEHDP DNID.

[0011] The peanut aspartic acid-rich peptide has antifungal activity against peanut pathogens Fusarium oxysporum and Rhizoctonia solani. The antifungal peanut aspartic acid-rich peptide has long-term stable application in the process of inhibiting one or more of the peanut pathogens Fusarium oxysporum and Rhizoctonia solani.

[0012] Peanut aspartic acid-rich peptide is an antifungal protein in peanut, and peanut aspartic acid-rich peptide has antifungal activity against peanut pathogens Fusarium oxysporum and Rhizoctonia solani.

[0013] The peanut aspartic acid-rich peptide has an amino acid sequence as shown in the sequence table SEQ ID NO. 1. The peanut aspartic acid-rich peptide is prepared by stepwise separation through ion exchange chromatography and gel filtration chromatography and desalting and purification through reverse phase column chromatography. It is a peanut aspartic acid-rich peptide with high antifungal activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 .Separation and preliminary identification of the activity of peanut antibacterial peptides. The elution curve of peanut protein on Sephacryl S-200 column forms A1 (eluted in the second tube), A2 (eluted in the sixth tube), and A3 peak (eluted in the eighth tube), A4 peak (eluted in the twelfth tube) and A5 peak (eluted in the fourteenth tube).

[0015] Figure 2 :Inhibitory clearing zone areas of isolated antifungal peanut aspartic acid-rich peptides (A1, A2, A3, A4 and A5) against Fusarium oxysporum and Rhizoctonia solani.

[0016] Figure 3 :Analysis of the purity of antifungal peanut aspartate-rich peptides by high pressure liquid chromatography.

[0017] Figure 4 :Identification of antifungal peanut aspartate-rich peptides by electrospray ionization mass spectrometry.

[0018] Figure 5 :Comparison of antibacterial effects of peanut aspartic acid-rich peptides.

[0019] Figure 6 :Comparison of the inhibitory effects of peanut aspartic acid-rich peptides on glycosynthase activity (GS). DETAILED DESCRIPTION

[0020] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0021] Example 1: Preparation of antifungal peanut aspartic acid-rich peptide

[0022] Weigh 5.0g peanut meal (primary meal, which is the peanut residue left after the initial pressing and oil extraction), grind it repeatedly in liquid nitrogen into fine powder (particle size less than 0.5 mm), put it in a 200mL beaker, add 100mL protein extraction buffer (composed of: 20mM tris-HCl, pH8.0, 10mM ethylenediaminetetraacetic acid (EDTA), 150mM NaCl, 1% dimethyl sulfoxide (DMSO) and 0.1% β-mercaptoethanol in water), stir and mix. Centrifuge at 10000g for 20min, and take 60ml supernatant.

[0023] 767 g (NH 4 ) 2 SO 4Slowly add to 1 liter of distilled water while stirring, and adjust to pH 7.0 with 25%-28% ammonia water (and / or 95% sulfuric acid), which is a 100% saturated ammonium sulfate solution (4.1mol / L, 25℃). Add an equal volume of 100% saturated ammonium sulfate solution to the above supernatant while stirring. Stir the resulting solution on a magnetic stirrer overnight (4℃, 12 hours) to fully precipitate the protein. Centrifuge the protein solution at 10000g for 30min (4℃). Discard the supernatant and retain the precipitate. Dissolve the precipitate in 20ml protein extraction buffer (composed of: 20mMTris-HCl, pH8.0, 10mMEDTA, 150mMNaCl, 1% DMSO by volume, 0.1% β-mercaptoethanol by volume and 0.2g / L sodium azide in water). Place in a 40 ml dialysis bag (molecular weight cutoff 2 kDa), dialyze with 1 liter of protein extraction buffer for 24 hours (4°C), change the dialysis buffer every 6 hours to completely remove ammonium sulfate, and collect the crude extract in the dialysis bag. Use three column volumes of protein extraction buffer (composed of: 20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% DMSO and 0.1% β-mercaptoethanol in water) to balance the DEAE-Sepharose FastFlow column (high flow rate ionic (DEAE) agarose gel column, inner diameter and length 2.6×100 cm, Merck, model 17-0709-10), flow rate 0.5 ml / min. Load 25 ml of the crude extract solution onto a 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 (composed of: 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 specific process of eluting the column at a flow rate of 1.0 mL / min was as follows: First, prepare the protein extraction buffer, mix all the components required for the protein extraction buffer in water and adjust the pH to 8.0. Then, prepare the corresponding column according to the experimental type and ensure that it has been equilibrated with a suitable buffer. After that, slowly load the sample containing the target protein to allow the sample to fully contact the filler.Next, prepare a 0.5M NaCl solution and mix it with the extraction buffer to form a gradient, and elute through the column at a flow rate of 1.0mL / min. During this process, collect the eluate in every 2 ml tube, and monitor the absorbance using a UV detector or other methods to identify the protein. Finally, perform necessary subsequent analysis on the collected protein. Collect the protein eluate from the time when the protein detector can detect the protein, and end when the detector cannot detect the protein, and collect 45ml of protein in total. Add the collection to a gel filtration column (Sephacryl S-200, inner diameter and length 5x90cm, GE, USA, model 17-0584-01) balanced with 3 column volumes of 20mMTris-HCl buffer (pH 8.0) (containing 0.15MNaCl), and collect a portion of the peak protein solution every 3ml. Elute with 20mMTris-HCl buffer (pH 8.0) (containing 0.35M NaCl) at 0.5ml / min. From the time the detector can detect the protein, collect one tube every 3ml, and collect a total of 18 tubes. By monitoring the absorbance at 280 nm, peaks showing higher protein concentrations were concentrated. Figure 1 A: A1 peak (the second tube eluted), A2 peak (the sixth tube eluted), A3 peak (the eighth tube eluted), A4 peak (the twelfth tube eluted) and A5 peak (the fourteenth tube eluted) are formed.

[0024] The five parts of protein formed A1 peak (eluted in the second tube), A2 peak (eluted in the sixth tube), A3 peak (eluted in the eighth tube), A4 peak (eluted in the twelfth tube) and A5 peak (eluted in the fourteenth tube), and the antifungal activity of each part was analyzed. Potato glucose agar plate (diameter 9 cm, thickness 0.3 cm) was used (the formula is as follows: 20g of potato was used to make an extract, 2g of glucose, 1.5g of agar, 100mL of water, and the pH was not adjusted (natural pH). Preparation method: Take 20g of fresh potato, wash and peel it, cut it into small pieces (rectangles with a side length of 0.1-0.5 cm), add 80ml of water and boil for 30min, filter it with four layers of gauze (50 mesh), add 2g of glucose and 1.5g of agar, continue heating (70-80℃) and stir well, cool it slightly (50-60℃), add water to 100mL, and sterilize it at 121℃ for 20 minutes). 10 μL of frozen bacterial solution (Fusarium oxysporum (purchased from China General Microbiological Culture Collection, CGMCC No. 3.6864) and Rhizoctonia solani (purchased from China Industrial Microbiological Culture Collection, CICC No. 40529)) were added to the center of the plate, and the bacterial cell density was 1×10 5 ~2×10 5cfu / g lyophilized powder, each gram of sample was dissolved in 10 ml of 0.85% saline to obtain bacterial solution). The antifungal activity of the 2nd, 6th, 8th, 12th and 14th tubes of eluted peanut protein was evaluated respectively. A round sterile filter paper disk with a diameter of 1 cm was drawn with a "cross" line at the center of the plate (i.e., the center, as the center of the "cross" line), and a paper disk was placed at the intersection of the center. A paper disk was placed every 1.5 cm away from the center along the two "cross" lines. A total of 5 paper disks were placed on a plate with a diameter of 9 cm. 10 μL of the above eluted test protein in 20 mM PBS (pH 7.5) buffer was dripped onto the paper disk, and only 10 μL of 20 mM PBS buffer (pH 7.5) was dripped onto the control. After culturing at 25°C for 72 hours, mycelium growth surrounded the peripheral disk containing the control, and a transparent circle of inhibition was produced around the disk with the antifungal sample. The larger the area of ​​the transparent circle, the higher the inhibition rate of the protein. The antibacterial experiment showed that the A2 peak (eluted in the 6th tube) in the eluted peanut aspartic acid-rich peptide was effective against Fusarium oxysporum (the inhibition zone area was about 9 cm 2 ) and Rhizoctonia solani (inhibition zone area of ​​about 7.4 cm 2 ) had obvious antibacterial activity, while peaks A1 (tube 2), A3 (tube 8), A4 (tube 12), and A5 (tube 14) had weak antibacterial activity against Fusarium oxysporum and Rhizoctonia solani, with a diameter of less than 0.5 cm 2 ( Figure 1 B). Therefore, peak A2 (eluted in tube 6) may be the ideal antibacterial peanut protein, named peanut aspartic acid-rich peptide, for subsequent experiments.

[0025] Figure 1 .The elution curve of peanut protein on Sephacryl S-200 column forms A1 peak (2nd tube of elution), A2 peak (6th tube of elution), A3 peak (8th tube of elution), A4 peak (12th tube of elution) and A5 peak (14th tube of elution). Elution was performed with 20mMTris-HCl buffer (pH 8.0) (containing 0.35MNaCl) at 0.5ml / min. From the time when the detector can detect protein, one tube was collected for every 5ml, and a total of 18 tubes were collected. By measuring the absorbance at 280nm, the peaks with higher protein concentration were formed as A1 peak (2nd tube of elution), A2 peak (6th tube of elution), A3 peak (8th tube of elution), A4 peak (12th tube of elution) and A5 peak (14th tube of elution).

[0026] Figure 1: Isolation and antibacterial identification of peanut antimicrobial peptides. A, Elution curve of peanut peptides on Sephacryl S-200 column, forming peak A1 (eluted in the second tube), peak A2 (eluted in the sixth tube), peak A3 (eluted in the eighth tube), peak A4 (eluted in the twelfth tube) and peak A5 (eluted in the fourteenth tube). B, Antibacterial effect of each elution peak. B, Antibacterial transparent zone area of ​​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 3 times, and the average value of the diameter of the inhibition zone was calculated. The area of ​​the transparent zone was calculated according to the area formula (S = π*(d / 2)2, π represents pi, r represents radius, d represents diameter, and S represents area).

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

[0028] 1 ml of antifungal peanut aspartic acid-rich peptide (A2 peak obtained in Example 1 (eluted in the 6th tube)) was passed through a liquid chromatography (HPLC) reverse phase column (WelchXB, Welch Materials, model XB-C18, inner diameter and length C18 4.6×150mm) chromatography desalting purification, the mobile phase is 40% v / v pure water (containing 0.1% trifluoroacetic acid (TFA) by volume)-60% v / v acetonitrile (ACN) (containing 0.1% trifluoroacetic acid by volume), the flow rate is 1mL / min, and the detection is at a wavelength of 220nm. The effluent peak is collected and lyophilized to obtain the purified peanut aspartic acid-rich peptide, which is then resuspended in a 0.1% formic acid aqueous solution by volume for liquid chromatography-mass spectrometry (HPLC-MS) analysis. The molecular weight of the synthesized antifungal peanut aspartic acid-rich peptide was identified using electrospray mass spectrometry. The sample was injected using a liquid phase system, and the mobile phase was 50% H 2 O / 50% CAN, flow rate of 0.2 mL / min, protective gas nitrogen flow rate of 1.5 L / min, collision energy of 4.5 kV, and positive ion mode.

[0029] Purified antifungal peanut aspartate-rich peptides were purified by HPLC (Welch XBC, Welch Materials, Model XB-C18, inner diameter and length 184.6 x 250 mm) to identify its purity, and the molecular weight was determined by electrospray mass spectrometry. The results of HPLC purity identification are as follows Figure 3 As shown: The antifungal peanut aspartic acid-rich peptide showed a single peak at 28.6 min ( Figure 2 ).

[0030] Figure 2:Analysis of the purity of antifungal peanut aspartate-rich peptides by high pressure liquid chromatography.

[0031] The electrospray ionization mass spectrometry results are as follows Figure 3 As shown: The molecular weight of the protein is close to the theoretical value of 11.55 kDa.

[0032] Figure 3 : Identification of antifungal peanut aspartate-rich peptide by electrospray mass spectrometry. The marker molecular weight is 11554.38 Da. Considering the positive ion mode, it is close to the theoretical molecular weight of 11554.94 Da.

[0033] Example 3: Sequencing of the antifungal peanut aspartic acid-rich peptide obtained in Example 1

[0034] The antifungal peanut aspartic acid-rich peptides were sequenced using the phenyl isothiocyanate method (Edman) degradation method with reference to 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 aspartic acid-rich peptides (20 μg) obtained in Example 2 were 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 S-carboxamidomethylated peptides were automatically Edman-degraded and the phenylthioacetal derivatives were detected 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 minus the first base and the first anilinothiazolinone (ATZ) residue released. Other reactants and released residues were washed away with 20 mM TrisHCl (pH 8.0) buffer, and the shortened peptide could be released through another round of coupling and cleavage to release the second residue, and so on, until the last amino acid residue was released. The sequencing results confirmed that it was a peanut antifungal peanut aspartic acid-rich peptide.

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

[0036] VDIKCTSIPRNSRVVTDGDPIRPAVLDGLAVTPPQNDYTNHNISHDAF TYVPDASHPNKTSLPSVLGIACRCVGKFTMGYEKCTGARYELYGITIRHE HDPDNID(105aa)

[0037] (a) Sequence characteristics:

[0038] Length: 105

[0039] ●Type: amino acid sequence

[0040] ●Chain type: Single chain

[0041] ●Topology: Linear

[0042] (b) Molecule type: protein

[0043] (c) Assumption: No

[0044] (d) Antonym: No

[0045] (e) Original source: Peanut

[0046] Example 4: Prevention and treatment of rice-infecting fungi using the aspartic acid-rich peptide obtained in Example 1

[0047] We first focus on the selection and treatment of rice root samples. Healthy, mature rice plants (25-35 weeks) were selected to ensure that each plant was of uniform size and color and had no obvious damage from pests and diseases. The treatment method included soaking 5-10 cm of roots in 200 ml of saline solution containing 0.1 mg / ml peanut aspartic acid-rich peptide (A2 peak obtained in Example 1 (eluted tube 6)) for 1 minute, followed by surface drying under a sterile environment (20°C) (10 root segment treatment groups). The rice roots of the control group were treated with only 200 ml of saline (immersion for 1 minute and then drying) under the same conditions. The treated samples were stored in suitable containers and kept at 10-15°C to simulate field conditions. Next, the fungal contamination of rice roots was measured every 5 days. A portion of the roots (0.5-0.6 cm) were randomly selected from each rice plant, washed, and soaked in 50 mL of sterile mass concentration 0.1% peptone solution for 5 minutes to remove surface fungi and obtain a washing solution (original washing solution). The washing solution was serially diluted with 0.85 NaCl, and the dilution multiples (volume multiples) were 1 (original washing solution volume): 10 (liquid volume after dilution), 1 (original washing solution volume): 100 (liquid volume after dilution) and 1 (original washing solution volume): 1000 (liquid volume after dilution). Each dilution solution (0.1 mL) was inoculated on YPD (yeast extract powder peptone dextrose agar) plate medium (diameter 9 cm, medium thickness 3-4 mm (here 3.5 mm)), and incubated at 25°C for 2 days. The colony forming units (CFU) on each plate were counted and the CFU number was expressed as the number per gram of rice root sample (CFU / g). The colony characteristics were observed under a microscope to distinguish different types of hyphae. Finally, the CFU counts of the treatment group and the control group were compared, and the differences between the two groups were analyzed using statistical methods. Data recording and analysis: The number of fungal infections on the rice roots of the treatment group and the control group were recorded and compared, and different types of hyphae were observed and distinguished under a microscope. The figure shows the results of analyzing the number of fungal infections in rice using the plate culture method. Figure 4 It can be seen that during the 26-day storage period (storage period) (storage temperature 10-15°C), the number of fungal infections in the treatment group and the control group showed significantly different trends. The number of fungal infections in the control group (treated with saline) remained relatively stable throughout the storage period, and the number of CFU / g (colony forming units per gram of sample) was very low, showing a good antibacterial effect. In contrast, the number of fungal infections in the treatment group (treated with aspartic acid-rich peptides) also remained at a low level within the first 20 days, but after about 20 days, the number of infections in the control group rose sharply, while the experimental group (treated group) remained in a suppressed state, indicating that after long-term storage, the antibacterial effect of aspartic acid-rich peptides was significant.

[0048] Figure 4.Analysis of fungal infection number of rice by plate culture method. Rice storage period 10-15℃, 26 days. Treatment group, aspartic acid-rich peptide. Control group, physiological saline.

[0049] Example 5: Inhibition of Fusarium oxysporum and Rhizoctonia solani by the aspartic acid-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; glucose, 20 g; agar, 18 g; and distilled water, 1 L). To test the antifungal activity, 1 mL of the fungal spore suspension (1×10 7 Spores / ml) were evenly added into 100mL PDA medium at a temperature of 40-50°C, a solid plate was prepared (plate thickness 3-4mm (here 3.5mm)), and an Oxford cup stainless steel tube (open at both ends, inner diameter 6mm, outer diameter 8mm, height 10mm) was placed. Then, 5 μL of different concentrations (A2 peak obtained in Example 1 (eluted 6th tube)) of aspartic acid-rich peptides (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / mL, in normal saline) were added to the midpoints of different Oxford cups, respectively, and cultured at 30°C for 48h, and the diameter of the antibacterial circle was measured. In the control group, 5 μL of an equal volume of normal saline was added to the Oxford cup and cultured under the same conditions. Preliminary screening of the antifungal activity of aspartic acid-rich peptides by transparent circle formation showed that the IC50 values ​​of aspartic acid-rich peptides against Fusarium oxysporum and Rhizoctonia solani were 13.6 and 82 μg / ml, respectively ( Figure 5 ).

[0051] Figure 5 Comparison of antibacterial effects. Control group: physiological saline. Treatment group: antifungal peanut aspartic acid-rich peptide solution obtained in Example 1 dissolved in physiological saline.

[0052] Example 6: Effects of the aspartic acid-rich peptides obtained in Example 1 and amphotericin B on the glycoside synthase activities of Fusarium oxysporum and Rhizoctonia solani

[0053] Take 0.1 ml of frozen bacterial suspension of Fusarium oxysporum and Rhizoctonia solani and spread them on 20 ml PDA medium plates (9 cm in diameter, 3-4 mm in thickness (3.5 mm in this case)) and culture them at 25°C for 5 days. The hyphae of both molds were rinsed with 20 ml of distilled water, and the rinsed hyphae were freeze-dried. The dried hyphae (1.0 g) were added to 4 mL of sodium acetate buffer (50 mmol / L, pH = 5) and ground in a precooled (0-4°C) mortar to obtain a homogenate. The homogenate was then centrifuged at 10,000 g for 15 minutes to obtain a supernatant containing crude glycoside synthase (GS). The protein content of the supernatant containing the crude enzyme was determined using a BCA assay kit. Enzyme activity is expressed as enzyme units / mg protein. When performing glycoside synthase (GS) activity detection, a common method is a biochemical detection using color change. For example, the enzymatic reaction using p-Nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate, in which the released p-Nitrophenol (pNP) has an absorbance at 405nm, can be used for quantitative analysis. The specific experimental steps include: in a buffer containing appropriate pH and buffer strength, add 5-10 (here 6) mmol / L pNPG and 50μL of enzyme sample, adjust the total volume to 1mL, and then incubate at 37℃ for 30 minutes. The equipment needed in the experiment includes a spectrophotometer (such as Shimadzu UV-1280) to measure the absorbance of the sample, a high-speed centrifuge (Eppendorf5810R) 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℃ water bath for 10 minutes to stop the reaction. The reaction mixture was centrifuged at 10,000 g for 10 minutes at 4°C to remove unreacted particles and residues. The absorbance of the supernatant was measured at 405 nm using a spectrophotometer to determine the concentration of pNP. Finally, the GS activity was calculated based on the standard curve, usually expressed as the amount of substrate converted per milligram of protein per unit time (unit / mg protein / minute). The results showed that the antifungal peanut aspartic acid-rich peptide obtained in Example 1 (A2 peak obtained in Example 1 (eluted tube 6)) had a good inhibitory activity on mycoside synthase ( Figure 6 , P<0.0001). Figure 6In the figure, we can see the experimental results of the effect of peanut aspartic acid-rich peptide on glycoside synthase activity (GS). The two bar graphs represent the GS enzyme activity (unit: U / mg) of the control group and the treatment group, respectively. The control group used normal saline, while the treatment group was treated with 0.1mg / ml antifungal peanut aspartic acid-rich peptide solution.

[0054] As can be observed from the bar graph, the GS enzyme activity in the control group was significantly higher than that in the treated group. Specifically, under the experimental conditions, the peanut aspartic acid-rich peptide was able to significantly reduce the activity of the GS enzyme, indicating that the compound has the potential to inhibit the enzyme. Asterisks indicate statistical significance, and four asterisks (****) indicate that the p-value is less than 0.0001, which means that the difference between the two groups is very significant. Overall, these data indicate that at the concentrations given in the experiment, the peanut aspartic acid-rich peptide is able to significantly inhibit the activity of glycoside synthase, which may be of great significance for controlling biological processes that rely on GS enzyme activity, such as the metabolic pathways of certain fungal organisms. Therefore, this peptide may be a promising candidate for the development of new antifungal agents.

[0055] Figure 6 Comparison of the inhibitory effect of peanut aspartic acid-rich peptide on glycoside synthase activity (GS). Control: physiological saline. Treatment group: solution of the antifungal peanut aspartic acid-rich peptide (0.1 mg / ml) obtained in Example 1 dissolved in physiological saline.

Claims

1. A peanut aspartic acid-rich peptide, characterized in that: The invention comprises or consists of the amino acid sequence shown in SEQ ID NO.1 in the sequence listing.

2. The peanut aspartic acid-rich peptide according to claim 1, characterized in that: Its amino acid sequence is: VDIKCTSIPRNSRVVTDGDPIRPAVLDGLAVTPPQNDYTNHNISHDAFTYV PDASHPNKTSLPSVLGIACRCVGKFTMGYEKCTGARYELYGITIRHEHDP DNID.

3. The peanut aspartic acid-rich peptide according to claim 1 or 2, characterized in that: The distribution of aspartic acid (D) in the amino acid sequence shows some unique features; in the given peptide sequence, a total of 105 amino acids are contained; the distribution of aspartic acid (D) in the sequence has a certain regularity, and it appears at the 2nd, 17th, 19th, 27th, 37th, 46th, 53rd, 100th, 102nd and 105th positions respectively; these positions indicate that aspartic acid is relatively dispersed in the entire sequence, but shows a trend of concentrated appearance at the end of the sequence.

4. A use of the peanut aspartic acid-rich peptide according to any one of claims 1 to 3, characterized in that: The peanut aspartic acid-rich peptide has antifungal activity against peanut pathogens Fusarium oxysporum and / or Rhizoctonia solani, and can be used as an antifungal drug or antifungal active ingredient for Fusarium oxysporum and / or Rhizoctonia solani.

5. An application of the peanut aspartic acid-rich peptide according to any one of claims 1 to 3, characterized in that: The peanut aspartic acid-rich peptide is used in the process of inhibiting one or both of peanut pathogens Fusarium oxysporum and / or Rhizoctonia solani.

6. An application of the peanut aspartic acid-rich peptide according to any one of claims 1 to 3, characterized in that: The peanut aspartic acid-rich peptide is used as an antifungal active ingredient in the preparation of one or two drugs or drug preparations for inhibiting peanut pathogens Fusarium oxysporum and / or Rhizoctonia solani.

7. A drug for resisting one or both of Fusarium oxysporum and / or Rhizoctonia solani, comprising the antifungal peanut aspartic acid-rich peptide according to any one of claims 1 to 3 as an active ingredient.

8. The medicine according to claim 7, further comprising a pharmaceutically acceptable carrier or excipient.

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