A peanut cysteine-rich peptide, encoding gene, bacteriostatic agent and application thereof

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to preparation and application of peanut antibacterial peptide. We first identify and extract a novel antibacterial peptide family member from a peanut transcriptome library, and name it as peanut cysteine-rich peptide. The peanut cysteine-rich peptide prepared by using a solid-phase chemical synthesis method exhibits broad-spectrum antibacterial activity, and polypeptide fragments thereof exhibit strong inhibition and killing capacity on various common pathogenic bacteria and crop molds.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and specifically relates to a peanut-rich cysteine ​​peptide and its applications. Background Technology

[0002] Many fungal pathogens in nature affect the growth and yield of various crops, such as *Alternaria triticina*, the causal agent of wheat leaf blight, and *Fusarium oxysporum* f.sp. lycopersici, the causal agent of tomato wilt. Other important pathogens include *Rhizoctonia solani*, the causal agent of rice sheath blight, and *Sclerotium rolfsii*, the causal agent of potato wilt. If left unmanaged, these pathogens can lead to severe crop losses, highlighting the importance of understanding and mitigating them.

[0003] Many widely cultivated crops, such as wheat, rice, and potatoes, are threatened by these fungal pathogens. For example, *Pythium aphandidermatum* and *Sclerotinias clerotiorum* cause damping-off and head rot in tomatoes and sunflowers, respectively. *Alternaria solani* and *Alternaria alternate f.sp. lycopersici* both cause early blight in tomatoes and potatoes. *Ceratocystis paradoxa* causes pineapple disease in sugarcane, while *Phytophthorainfestans* is notorious for causing late blight in potatoes. The diversity of pathogens and affected crops underscores the critical need to research and develop effective disease management strategies.

[0004] These fungal pathogens not only reduce crop yields but also lower crop quality, and can even lead to the destruction of entire crops. For example, wheat leaf blight fungus (Alternaria triticina) forms lesions on leaves, severely affecting photosynthesis and ultimately leading to yield reduction. Tomato wilt fungus (Fusarium oxysporum f.sp. lycopersici) infects plants through the roots, causing plant wilting and death. Sheath blight fungus (Rhizoctonia solani) can cause disease throughout the entire growth period of rice, especially in hot and humid environments, easily causing large-scale yield reductions. Potato wilt fungus (Sclerotium rolfsii) forms numerous sclerotia, making the disease spread rapidly and difficult to control.

[0005] To combat these fungal diseases, agricultural scientists and farmers need to adopt integrated disease management strategies, including using resistant varieties, optimizing planting methods, and using chemical agents rationally. Control measures for *Pythium aphandidmatum* and *Sclerotinias clerotiorum*, the causal agent of sunflower head rot, include soil disinfection and crop rotation. Management of *Alternaria solani* and *Alternaria alternate* f.sp. lycopersici*, the causal agent of early blight, requires timely application of fungicides in the early stages of disease. For *Ceratocystis paradoxa*, the causal agent of pineapple rot, and *Phytophthorain festans*, disease monitoring and control should be strengthened, and timely measures should be taken to control the spread of the disease. Continuous research and innovation are crucial for developing new resistant varieties and environmentally friendly control measures to ensure the sustainable development of agricultural production.

[0006] To reduce chemical pollution and antibiotic resistance, green management methods are becoming increasingly important in agricultural disease control. Currently, the widespread use of chemical fungicides not only pollutes the environment but may also lead to pesticide residue problems, affecting food safety. At the same time, long-term use of antibiotics and chemical agents can cause pathogen resistance, making disease control more difficult. To address these issues, scientists and farmers are exploring more environmentally friendly and efficient alternatives, with peanut antimicrobial peptides becoming a focus of attention.

[0007] Peanuts (Arachis hypogaea) are leguminous plants widely distributed in Guangxi, Hainan, and other regions, and are an important economic crop in the southern coastal areas. Peanuts are not only an important food and oilseed crop, but also rich in protein, fat, vitamins, and minerals, possessing high nutritional value. Antimicrobial peptides (AMPs) are an important component of the innate immune system, exhibiting broad-spectrum antimicrobial activity. Common secondary structures include α-helices, β-sheets, and β-hairpins. In recent years, with the rapid development of aquaculture, water pollution and outbreaks of diseases have become major bottlenecks. Traditional antibiotic and chemical drug control methods have led to serious problems of drug residues and drug resistance. Peanut antimicrobial peptides, as a novel biosafety antibiotic, possess good thermal stability and water solubility, and are less prone to inducing drug resistance. They can not only replace traditional antibiotics in suppressing and killing diseases, but also enhance the immunity of farmed animals and improve production efficiency, showing great application potential in aquaculture.

[0008] Peanut antimicrobial peptides are naturally occurring antimicrobial substances with broad-spectrum antimicrobial activity, effective against a variety of fungal pathogens. Unlike traditional chemical agents, peanut antimicrobial peptides are environmentally friendly, harmless to humans, and do not cause drug resistance, making them an ideal alternative to chemical agents. They can effectively control diseases while reducing negative environmental impacts.

[0009] Using peanut antimicrobial peptides for disease control can effectively reduce the use of chemical pesticides and lower the risk of environmental pollution. Traditional chemical pesticides easily pollute soil, water, and air, impacting ecosystem health. As a natural substance, peanut antimicrobial peptides are rapidly degraded by the environment after application, leaving no residue in soil and water, thus reducing environmental harm. Furthermore, peanut antimicrobial peptides are harmless to human health and do not cause allergies or other adverse reactions, which is crucial for ensuring agricultural product safety and consumer health. Another important advantage is that peanut antimicrobial peptides do not develop drug resistance. Long-term use of chemical pesticides and antibiotics can lead to pathogen resistance, weakening or even rendering these pesticides ineffective. Peanut antimicrobial peptides, however, inhibit pathogen growth through a unique mechanism, making it difficult for pathogens to develop resistance. Therefore, peanut antimicrobial peptides maintain high efficacy with long-term use, which is of great significance for the sustainable control of agricultural diseases.

[0010] To promote the application of peanut antimicrobial peptides, scientists are continuously researching and optimizing their production and application technologies. Through genetic engineering and biotechnology, high-purity peanut antimicrobial peptides can be produced on a large scale, and formulations suitable for different crops and diseases can be developed. Scientists are also exploring the synergistic effects of peanut antimicrobial peptides with other biological control measures to improve their effectiveness. For example, combining peanut antimicrobial peptides with beneficial microorganisms can create a more comprehensive disease control system, enhancing crop disease resistance. Promoting and applying green management practices such as peanut antimicrobial peptides is crucial for reducing the use of chemical agents, mitigating environmental pollution risks, and improving the quality of agricultural products.

[0011] In this invention, we obtained a novel antimicrobial peptide family member from a peanut transcriptome library for the first time, named peanut cysteine-rich peptide. We then prepared the cysteine-rich peptide using solid-phase chemical synthesis, resulting in a polypeptide fragment with broad-spectrum antimicrobial activity. This polypeptide fragment exhibits strong inhibitory and toxic activity against various agricultural pathogens, including *Alternaria triticina* (wheat leaf blight), *Fusarium oxysporum* f.sp. lycopersici (tomato wilt), *Rhizoctonia solani* (sheath blight), *Sclerotium rolfsii* (potato wilt), *Pythium aphandidermatum* (damping-off), *Sclerotinias clerotiorum* (sunflower head rot), *Alternaria solani* (early blight), *Alternaria alternate* f.sp. lycopersici (tomato early blight), *Ceratocystis paradoxa* (pineapple wilt), and *Phytophthorainfestans* (late blight). Summary of the Invention

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] This invention provides a peanut cysteine-rich peptide, the nucleotide sequence of which is shown in SEQ ID NO.1; specifically:

[0014] GATAATGCCATCAACTTTATCAAAACTGGGCAGTGCCTTGGTAAATGT

[0015] GCAGACGACATTGTTTGCTGCGGGATGAAAGATTGCTACATTAATTAC

[0016] ATGCAGCACTTCTTGCCTTGTGCGGTGAAATGTGAAAACTTGGGGTTC

[0017] CTGTGCATCCCATCATGCAGGGATATCGAGAATCCTCTTACTAATGCTT

[0018] TGACAGGGTTATCCTCTCACAAGGTAGGACCATCTCACAAGGTAAAACCGGGTCACCGGGTACCGCCATCTCACCGGCCA.

[0019] The amino acid sequence is shown in SEQ ID NO.2; specifically:

[0020] DNAINFIKTGQCLGKCADDIVCCGMKDCYINYMQHFLPCAVKCENLGFL CIPSCRDIENPLTNALTGLSSHKVGPSHKVKPGHRVPPSHRP.

[0021] Another object of the present invention is to provide a peanut cysteine-rich peptide, which is a polypeptide fragment selected from cysteine-rich peptides with antibacterial activity. The three-dimensional structure of this peptide sequence reveals several key structures of a complex protein. Figure 1 Peanut-rich cysteine ​​peptides are characterized by their unique helical structure. This peptide, composed of multiple coiled helices, is predominantly blue and yellow, indicating different types of secondary structures or functional regions. The red portions in the image may be located at one end of the peptide, indicating specific functional groups or active sites, possibly indicating the N-terminus or a particular biochemically active region. This structural characterization helps visualize the molecule's potential interactions with other biomolecules or is used to study its properties in the biochemical environment.

[0022] A polypeptide or protein containing the above-mentioned antimicrobial peptide is also within the scope of protection of this invention.

[0023] The present invention provides a cysteine-rich peptide prepared by solid-phase chemical synthesis, which exhibits broad-spectrum antibacterial activity. Its polypeptide fragments show strong inhibitory and killing abilities against a variety of common pathogenic bacteria and crop molds.

[0024] The present invention has the following beneficial effects:

[0025] This invention provides for the first time a member of the cysteine-rich peptide family with broad-spectrum antibacterial activity derived from peanuts. This antibacterial peptide active fragment can be prepared in large quantities through solid-phase chemical synthesis. Furthermore, this antibacterial peptide active fragment has strong inhibitory and bactericidal effects on common pathogenic Vibrio bacteria and foodborne pathogenic Vibrio bacteria in the aquaculture industry. It can be used as an ecologically safe antibacterial additive in aquaculture and has broad application prospects. Attached Figure Description

[0026] Figure 1 This is a tertiary protein structure diagram of a mature peptide rich in cysteine ​​from peanuts;

[0027] Figure 2 HPLC and mass spectrometry identification of cysteine-rich peptides and control peptides; A, HPLC of cysteine-rich peptides; B, HPLC of control peptides; C, mass spectrometry of cysteine-rich peptides; D, mass spectrometry of control peptides.

[0028] Figure 3Time curves of growth inhibition rate of peanut cysteine-rich peptide and control peptide against wheat leaf blight pathogen (Alternaria triticina);

[0029] Figure 4 Time curves of inhibition rate of peanut cysteine-rich peptides and control peptides against the growth of Fusarium oxysporum f. sp. lycopersici, the pathogen of tomato;

[0030] Figure 5 Time curves showing the inhibition rates of peanut cysteine-rich peptides and control peptides against the growth of Rhizoctonia solani.

[0031] Figure 6 Antimicrobial peptide hemolysis test; A, peanut cysteine-rich peptide hemolysis test; B, control peptide hemolysis test. Detailed Implementation

[0032] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0033] Example 1: Obtaining the gene sequence of peanut cysteine-rich peptide

[0034] We purchased peanut meal from Zhengzhou Huaguan Biotechnology Development Co., Ltd. Its physicochemical properties (mass content) are as follows:

[0035] - Crude protein content: ≥46%, - Crude fat content: ≤2%, - Crude ash content: ≤8%, - Crude fiber content: ≤10%, and - Moisture content: ≤12%.

[0036] Total RNA extraction: Take 1 gram of peanut meal, ensuring the sample is clean and uncontaminated. Place the peanut meal in a test tube containing TRIzol reagent (total RNA extraction reagent, Life Sciences, USA), and incubate at room temperature for 5 minutes with 1 mL of TRIzol reagent. Then, add 200 μL of chloroform, vortex vigorously for 15 seconds, and incubate for 10 minutes. Centrifuge at 12,000 x g for 15 minutes to separate the supernatant. Transfer the supernatant to a new test tube, add 500 μL of isopropanol, mix well, and incubate for 10 minutes. Centrifuge again at 12,000 x g for 10 minutes, discard the supernatant, and retain the precipitate (RNA). Wash the RNA precipitate twice with 1 mL of 75% ethanol, centrifuging at 7,500 x g for 5 minutes each time, discard the supernatant, and air-dry the precipitate. RNA precipitate was dissolved in 30 μL of RNase-free water (DEPC water), and its concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). A260 = 1.8, A280 = 1.0, concentration = 180 ng / μL. An A260 / A280 ratio between 1.8 and 2.0 indicates good RNA purity and the absence of significant protein or other contaminants.

[0037] Quality control and transcriptome library sequencing:

[0038] RNA integrity assessment

[0039] First, RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Ensuring a RNA integrity (RIN value) greater than 7.0 is a prerequisite for high-quality transcriptome sequencing. In this experiment, the RIN value of our extracted RNA sample was 8.2, indicating good RNA quality and high integrity, suitable for subsequent library construction and sequencing.

[0040] Library Construction

[0041] Library construction was performed using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, USA), strictly following the kit's instructions. The specific steps are as follows: First, the total RNA sample was fragmented by sonication to obtain RNA fragments approximately 200-300 bp in length, ensuring uniform fragment distribution and smooth operation of subsequent reverse transcription and amplification. Then, the fragmented RNA was reverse transcribed using random primers to generate first-strand cDNA, covering the entire transcriptome and ensuring representativeness. Next, the cDNA ends were repaired, and an A-tail was added to the 3' end for adapter ligation, ensuring end consistency across all cDNA fragments and promoting efficient adapter ligation. Subsequently, adapters containing sequencing primer sequences were ligated; these adapters contained the primer sequences required for the Illumina sequencing platform, preparing for subsequent sequencing. Next, PCR amplification was performed using random primers to obtain the final cDNA library, ensuring sufficient library volume for high-throughput sequencing. Finally, the library quality was again assessed using an Agilent 2100 Bioanalyzer to ensure appropriate fragment length and concentration. The target fragment lengths were distributed between 200-300 bp, with a concentration of 28 nM. Specific detection results showed that the library fragment lengths were between 200-300 bp, with a concentration of 28 nM, meeting the standards for high-quality sequencing libraries.

[0042] High-throughput sequencing

[0043] High-throughput sequencing was performed using the Illumina NovaSeq 6000 platform, employing a paired-end 150bp (PE150) sequencing mode. The specific steps were as follows: Library samples were loaded onto the Illumina NovaSeq 6000 sequencing platform, with each sample labeled using a different barcode to ensure sample identification during multi-sample medley sequencing. The sequencer was started, and paired-end 150bp sequencing was performed. By reading the sequences at both ends of the DNA fragments, sequencing errors were reduced, improving sequencing accuracy and data coverage. After sequencing, raw reads were collected. In this experiment, the sequencing depth reached 35 million reads, ensuring data coverage and accuracy. Preliminary data processing was performed using Illumina's official software, including the removal of low-quality reads and adapter sequences. The processed data was used for subsequent bioinformatics analysis. The results show that each step from RNA extraction and library construction to high-throughput sequencing strictly followed standard operating procedures, ensuring high-quality and reliable data. Specific data examples are as follows: RNA integrity assessment RIN value of 8.2, library quality detection fragment length of 200-300bp, concentration of 28nM, and high-throughput sequencing depth of 35 million reads.

[0044] Transcript Assembly: FastQC: FastQC is a widely used open-source tool for evaluating the quality of high-throughput sequencing data. From Babraham Bioinformatics, UK, it can be legally downloaded and used from their official website. Trinity: Trinity is an open-source software for de novo transcript assembly, provided by the Broad Institute. The software is available for free on their official GitHub page. BUSCO: BUSCO (Benchmarking Universal Single-Copy Orthologs) is a tool for evaluating assembled and annotated genomes. A free version is available for academic purposes. It can be obtained from their official website or GitHub page. BLAST: BLAST is an open-source toolkit provided by the National Center for Biotechnology Information (NCBI), which can be legally downloaded and used for free.

[0045] Raw data were quality controlled using FastQC (Babraham Bioinformatics, UK) to remove low-quality reads and adapter sequences. Denovo transcript assembly was performed using Trinity software (v2.8.5, Broad Institute, USA). The memory settings were 50GB and the number of processing threads were 16. Finally, the integrity of the assembly was assessed using BUSCO (Benchmarking Universal Single-Copy Orthologs). Finally, a peanut antimicrobial peptide DNA sequence (SEQ ID NO.1) was identified for the first time using blastp software through homology alignment. Its gene sequence is as follows:

[0046] GATAATGCCATCAACTTTATCAAAACTGGGCAGTGCCTTGGTAAATGT

[0047] GCAGACGACATTGTTTGCTGCGGGATGAAAGATTGCTACATTAATTAC

[0048] ATGCAGCACTTCTTGCCTTGTGCGGTGAAATGTGAAAACTTGGGGTTC

[0049] CTGTGCATCCCATCATGCAGGGATATCGAGAATCCTCTTACTAATGCTT

[0050] TGACAGGGTTATCCTCTCACAAGGTAGGACCATCTCACAAGGTAAAA

[0051] CCGGGTCACCGGGTACCGCCATCTCACCGGCCA

[0052] (a) Sequence characteristics:

[0053] ●Length: 273

[0054] ●Type: Nucleotide sequence

[0055] ●Chain type: Single chain

[0056] ●Topology: Linear

[0057] (b) Molecular type: Nucleotide

[0058] (c) Assumption: No

[0059] (d) Antonym: No

[0060] (e) Original source: peanut

[0061] The full-length amino acid sequence of the peanut cysteine-rich peptide is SEQ ID NO.2, as follows: DNAINFIKTGQCLGKCADDIVCCGMKDCYINYMQHFLPCAVKCENLGFL CIPSCRDIENPLTNALTGLSSHKVGPSHKVKPGHRVPPSHRP

[0062] (a) Sequence characteristics:

[0063] ●Length: 91

[0064] ●Type: Amino acid sequence

[0065] ●Chain type: Single chain

[0066] ●Topology: Linear

[0067] (b) Molecular type: protein

[0068] (c) Assumption: No

[0069] (d) Antonym: No

[0070] (e) Original source: peanut

[0071] The peptide structure was analyzed online using Alphafold2. The peptide chain primarily exhibits an α-helix, a common secondary structure in proteins and peptide chains. This right-handed helix structure is stabilized by hydrogen bonds between the amide and carbonyl groups in the main chain. The α-helix is ​​tightly wound, with the amino acid side chains protruding outwards. Different colors were used in the image, possibly to distinguish specific regions or residues within the helix, showing an ordered conformation without obvious disordered regions or β-sheet structures. This α-helix structure typically plays a crucial role in protein stability and function.

[0072] Example 2: Identification of peanut cysteine-rich peptides and control peptides

[0073] The control peptide (GenBank No. XM_021104843.1) showed 78% homology to the amino acid sequence of the currently known peanut cysteine-rich peptide. The peanut cysteine-rich peptide and the control peptide (GenBank No. XP_020960502.1) were synthesized by Changzhou Kanglong Biotechnology Co., Ltd. This HPLC analysis examined the synthesized peanut cysteine-rich peptide and the control peptide. The chromatographic column used was a Gemini-NX 5μC18110A (model 00B-4454-B0), with dimensions of 4.6*250 mm (inner diameter*length). Mobile phase A was an acetonitrile solution with a final volume concentration of 0.1% trifluoroacetic acid, and mobile phase B was an aqueous solution with a final volume concentration of 0.1% trifluoroacetic acid. The gradient elution program (v / v) is as follows: Initially (0.0 min), phase A accounts for 10% of the volume and phase B accounts for 90%; at 25.0 min, phase A reaches 100% of the volume in a linear gradient, and phase B is 0%; from 25.0 min, phase A is maintained at 100% and phase B at 0% until 30.0 min, at which point the elution is stopped. The injection volume is 20 μl, the detection wavelength is 220 nm, and the flow rate is 1.0 ml / min.

[0074] In the analysis, the sample was first injected into the HPLC system at a volume of 20 μL. A Gemini-NX5μC18110A column was used for detection, with the detection wavelength set to 220 nm and the mobile phase flow rate at 1.0 mL / min. This configuration ensured adequate separation and detection of the target compounds in the sample within the HPLC system. Mobile phase A was a solution of 0.1% trifluoroacetic acid in acetonitrile, and mobile phase B was a solution of 0.1% trifluoroacetic acid in water. Separation was performed using the following gradient elution program: starting at 0.0 min, phase A was 10% and phase B was 90%; at 25.0 min, phase A was gradually increased to 100% and phase B decreased to 0%; this ratio was maintained (phase A 100%, phase B 0%).

[0075] The mobile phase was stopped at 30.0 minutes. The flow rate was set to 1.0 ml / min. This method effectively separated and detected the target compound in the sample. The results showed that the main peak of the peanut cysteine-rich peptide appeared at 15.4 minutes, indicating that the purity of this component was high. Figure 2 A). As can be seen from the provided chromatogram, the main peaks of the synthesized peanut cysteine-rich peptide and the control peptide appear at approximately 15.4 minutes ( ). Figure 2 A) and 17.2 minutes ( Figure 2 Regarding retention time (B), this peak is the most prominent and likely represents the peanut-rich cysteine ​​peptide and the control peptide. To determine purity, the area of ​​this major peak needs to be compared to the total area of ​​all peaks. The purity percentage is calculated as: major peak area divided by the total area of ​​all peaks, then multiplied by 100. From the chromatogram, the major peak is significantly larger than any other peak, indicating high sample purity. If minor peaks are very small compared to the major peak, a purity exceeding 90% can be reasonably inferred. Based on visual estimation, the purity of this sample does indeed exceed 95%.

[0076] Mass Spectrometry Report

[0077] The samples were peanut-rich cysteine ​​peptides and control peptides.

[0078] Instrument: SHIMADZU LCMS-2020

[0079] Probe type: ESI

[0080] Probe bias: +4.5kV

[0081] Sample batch number: P240422-WY181666

[0082] Molecular weight: Peanut cysteine-rich peptide 9968.63 Da, control peptide 12986.33 Da

[0083] Experimental procedure and detailed parameters

[0084] In this mass spectrometry analysis, peanut cysteine-rich peptides and control peptides were used, and the analysis date was May 27, 2024. The mass spectrometer used was a SHIMADZU LCMS-2020, the probe type was electrospray ionization (ESI), and the probe bias voltage was set to +4.5 kV.

[0085] Sample preparation and testing conditions

[0086] 1. Sample preparation

[0087] The sample concentration was 1 mg / mL, dissolved in a solution of 50% water and 50% acetonitrile (v / v). The prepared sample was injected into the mass spectrometer at a flow rate of 0.2 mL / min.

[0088] 2. Instrument settings

[0089] - Atomizing gas flow rate: 1.5 L / min (protective gas is nitrogen)

[0090] - Detector voltage: 1.2kV

[0091] -CDL voltage: -20.0V

[0092] -CDL temperature: 250℃

[0093] - Heating module temperature: 400℃

[0094] - Mobile phase composition: 50% water and 50% acetonitrile (v / v)

[0095] Detailed experimental procedures

[0096] 1. Sample injection

[0097] The prepared sample was injected into the mass spectrometry system at a flow rate of 0.2 mL / min to ensure stable injection and uniform distribution of the sample.

[0098] 2. Electrospray ionization (ESI) process

[0099] The sample was ionized using an ESI probe with a bias voltage set to +4.5 kV, generating charged droplets. The nebulizer gas flow rate was set to 1.5 L / min, using cation mode to aid in sample nebulization and improve ionization efficiency.

[0100] 3. Ion transport and detection

[0101] The ionized sample ions pass through the neutral dissociation region (CDL), with the CDL voltage set at -20.0V and the CDL temperature at 250℃ for effective ion transport and cooling. The detector voltage is set at 1.2kV for detecting and amplifying the ion signal.

[0102] 4. Data recording and analysis

[0103] During mass spectrometry analysis, the mass-to-charge ratio (m / z) and intensity signal of the sample are recorded to ensure the accuracy and reliability of the analytical results. The experimental run time is set according to the properties of the sample and the analytical requirements, typically ranging from several minutes to more than ten minutes.

[0104] Experimental results

[0105] Mass spectrometry analysis revealed that peanuts contain 9968.27 cysteine ​​peptides. Figure 2 C), control peptide 12986.25 ( Figure 2D) The experimental data meticulously records the mass-to-charge ratio (m / z) and corresponding signal intensity of each component, helping analysts accurately identify and quantify the components in the sample. This detailed data and parameters ensure the repeatability and reliability of the mass spectrometry experiments, guaranteeing the accuracy and credibility of the analytical results.

[0106] Example 3: Analysis of the minimum inhibitory concentration (MIC) and growth inhibition time curves of peanut cysteine-rich peptide and control peptide (GenBank No. XP_015936675.1) against wheat leaf blight pathogen (Alternaria triticina).

[0107] The MIC values ​​of antimicrobial peptides were determined using the microdilution method [Saporito P., Mojsoska B., Olesen AL, Jenssen H. Antibacterial mechanisms of GN-2-derived peptides and peptoids against Escherichia coli. Biopolymers. 2019; 110:e23275; Cui H., Zhang C., Li C., Lin L. Antimicrobial mechanism of clove oil on Listeria monocytogenes. FoodControl.]. First, under aseptic conditions, 10 mg of *E. coli* mycelium was weighed and inoculated into 50 mL of YPD liquid medium (Zeye Biotechnology, ZY130895, hereinafter the same). The medium was then transferred to a 250 mL Erlenmeyer flask to ensure adequate aeration. The flask was placed in a 28°C constant-temperature shaker with a shaker speed set to 200 rpm. The culture time was 48 hours, during which OD600 values ​​were measured periodically to ensure that the spore concentration was within the required range (OD600≈0.5-1). Indicator bacterial suspension was obtained and added to 96-well titration plates, 100 μL per well. Then, serial dilutions of equal volumes of peptide solution (100 μL peptide aqueous solution per well) were performed, resulting in peptide concentrations of 0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 16 μM, 18 μM, and 20 μM in the wells, and incubated at 28°C for 48 hours. The lowest concentration of antimicrobial peptide at which no visible microbial growth was observed was determined using the ELISA (SPARK) method. The purified antimicrobial peptide concentration was adjusted to the MIC level with water for time-kill analysis, and the same volume of peptide aqueous solution as the indicator bacterial suspension (obtained as above) was applied to 96-well plates (at least three wells per concentration). The plates were then incubated at 28°C for 2, 4, 6, 8, 10, 12, and 24 hours. Afterward, the OD value was measured at 600 nm.

[0108] The inhibition assays of peanut cysteine-rich peptides and control peptides were performed using a modified method as previously described [Guangming S., Yang Y., Yuan Q., Shi G., Wu L., Lou Z., Huo R., Wu H., Borriss R., Gao X. Bacillomycin D Produced by Bacillus amyloliquefaciens Is Involved in the Antigonistic Interaction with the Plant-Pathogenic Fungus Fusarium graminearum. Appl. Environ. Microbiol. 2017; 83:e01075–e01117.]. 200 μL (1 mg / mL) of the antimicrobial peptide was uniformly mixed with 10 mL of wheat leaf blight spores (10... 7 ( / mL) and incubated at 28℃. The spore number inhibition rate was observed and recorded every 2 hours. Figure 3 To calculate the spore count inhibition rate every 2 hours, the spore counts of the control group (a homogeneous mixture of 200 μL water and 10 mL of *Wheat blight* spores) and the treatment group (a homogeneous mixture of 200 μL (1 mg / mL) antimicrobial peptide and 10 mL of *Wheat blight* spores) need to be recorded first. The control group consisted of the untreated spore count (a homogeneous mixture of 200 μL water and 10 mL of *Wheat blight* spores incubated at 28°C; the spore count inhibition rate was observed and recorded every 2 hours), while the treatment group consisted of the inhibited spore count (a homogeneous mixture of 200 μL (1 mg / mL) antimicrobial peptide and 10 mL of *Wheat blight* spores incubated at 28°C; the spore count inhibition rate was observed and recorded every 2 hours). 7 ( / mL) and incubated at 28°C. Spore number inhibition rate was observed and recorded every 2 hours from 0 to 10 hours.

[0109] The formula for calculating the inhibition rate is:

[0110]

[0111] Where, N control N represents the number of spores in the control group. treatment This represents the number of spores in the treatment group. Using this formula, the percentage reduction in the number of spores in the treatment group relative to the control group can be calculated, i.e., the inhibition rate.

[0112] The specific operating steps are as follows: 1. Record the spore count of the control group and the treatment group every 2 hours. 2. Calculate the inhibition rate at each recording using the formula above.

[0113] For example, at a certain time point, if the number of spores in the control group is 200 and the number of spores in the treatment group is 150, then the inhibition rate is calculated as follows:

[0114]

[0115] This means that at that point in time, the number of spores in the treatment group was reduced by 25% compared to the control group.

[0116] The experimental results showed that the MIC for the wheat leaf blight pathogen was 9.5 μM peanut cysteine-rich peptide and 13.7 μM control peptide.

[0117] To prepare spore inoculum of *Wheat Leaf Blight*, the following steps were performed: First, under aseptic conditions, 1 mg of the inoculum (*Wheat Leaf Blight*) was weighed and inoculated into 50 mL of YPD liquid medium (OD600 ≈ 0.02-0.05). The medium was transferred to a 250 mL Erlenmeyer flask to ensure adequate aeration. The flask was placed in a 25°C shaker at a speed of 200 rpm. The culture time was 48 hours, during which the OD600 value was measured periodically to ensure the spore concentration remained within the desired range. After culture, the culture medium was filtered through sterile gauze or filter paper, and the spore suspension was collected and stored in a suitable preservation solution for later use.

[0118] Figure 3 The inhibition time curves of peanut cysteine-rich peptide and control peptide against the growth of *Alternaria triticina*, the wheat leaf blight pathogen, are shown. According to the data in the figure, both peanut cysteine-rich peptide and control peptide showed significant inhibitory effects on the growth of *Alternaria triticina* at different time points. The inhibitory effect gradually increased over time, especially after 4 hours. Specific analysis results are as follows: 1. Early inhibition effect: Within 2 hours, the inhibitory effect was relatively weak, but some growth inhibition was still observed. 2. Mid-term inhibition effect: After 4 hours, the inhibitory effect significantly increased, showing that peanut cysteine-rich peptide has a strong inhibitory effect on the pathogen. 3. Long-term inhibition effect: The inhibitory effect reached its peak at 10 hours, showing that both peanut cysteine-rich peptide and control peptide have sustained and effective antibacterial activity. The inhibitory effect of peanut cysteine-rich peptide was higher than that of the control peptide (…). Figure 3 ).

[0119] Overall, peanut-rich cysteine ​​peptides and the control peptides significantly inhibited the growth of wheat leaf blight pathogens, with the inhibitory effect becoming more pronounced over time. This suggests that peanut-rich cysteine ​​peptides may have potential application value in plant disease resistance.

[0120] Example 4: Analysis of the minimum inhibitory concentration (MIC) and growth inhibition rate of peanut cysteine-rich peptide and control peptide (GenBank No. XP_015936675.1) against Fusarium oxysporum f.sp. lycopersici, the causal agent of tomato wilt.

[0121] The MIC values ​​of antimicrobial peptides were determined using the microdilution method [Saporito P., Mojsoska B., Olesen AL, Jenssen H. Antibacterial mechanisms of GN-2-derived peptides and peptoids against Escherichia coli. Biopolymers. 2019; 110:e23275; Cui H., Zhang C., Li C., Lin L. Antimicrobial mechanism of clove oil on Listeria monocytogenes. FoodControl.]. First, under aseptic conditions, 10 mg of Fusarium oxysporum f.sp. lycopersici mycelium was weighed and inoculated into 50 mL of YPD liquid medium. The medium was then transferred to a 250 mL Erlenmeyer flask to ensure adequate aeration. The flask was incubated in a shaker at 28°C with a shaking speed of 200 rpm. The culture time was 48 hours, during which OD600 values ​​were measured periodically to ensure that the spore concentration was within the required range (OD600 ≈ 0.5-1), and 100 μL of the peptide was added to each well of a 96-well titration plate. Then, serial dilutions of equal volumes of peptide were performed (0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 16 μM, 18 μM, and 20 μM), and incubated at 28°C for 48 hours. The lowest concentration of antimicrobial peptide at which no visible microbial growth was observed was determined using the ELISA (SPARK) method. The purified antimicrobial peptide concentrations were adjusted to the MIC level for time-kill assays, and the same volume of peptide as the indicator bacteria was applied to each well of a 96-well plate (at least three wells per concentration). The plates were then incubated at 28°C for 2, 4, 6, 8, 10, 12, and 24 hours. OD values ​​were then measured at 600 nm.The inhibition assay of peanut cysteine-rich peptide and control peptide was performed using a modified method as previously described [Guangming S., Yang Y., Yuan Q., Shi G., Wu L., Lou Z., Huo R., Wu H., Borriss R., Gao X. Bacillomycin D Produced by Bacillus amyloliquefaciens Is Involved in the Antagonistic Interaction with the Plant-Pathogenic Fungus Fusarium graminearum. Appl. Environ. Microbiol. 2017; 83:e01075–e01117.]. Approximately 200 μL (1 mg / mL) of the antimicrobial peptide was homogeneously mixed with 10 mL of Fusarium oxysporum f.sp. lycopersici spores (10). 7 ( / mL) and incubated at 28°C. The inhibition rate of spore count was observed and recorded every 2 hours from 0 to 10 hours. Figure 4 To calculate the spore count inhibition rate every 2 hours, the spore counts in the control and treatment groups must first be recorded. The control group contains the number of spores not treated for inhibition, while the treatment group contains the number of spores treated for inhibition.

[0122] The formula for calculating the inhibition rate is:

[0123]

[0124] Where, N control N represents the number of spores in the control group. treatment This represents the number of spores in the treatment group. Using this formula, the percentage reduction in the number of spores in the treatment group relative to the control group can be calculated, i.e., the inhibition rate.

[0125] The specific operating steps are as follows: 1. Record the spore count of the control group and the treatment group every 2 hours. 2. Calculate the inhibition rate at each recording using the formula above.

[0126] For example, at a certain time point, if the number of spores in the control group is 200 and the number of spores in the treatment group is 150, then the inhibition rate is calculated as follows:

[0127]

[0128] This means that at that point in time, the number of spores in the treatment group was reduced by 25% compared to the control group.

[0129] The experimental results showed that the MIC for *Fusarium oxysporum* f. sp. lycopersici, the causal agent of tomato wilt, was 11.6 μM peanut-rich cysteine ​​peptide and 16.8 μM control peptide. To prepare the spore inoculum for *Fusarium oxysporum* f. sp. lycopersici, the following steps were performed: First, under aseptic conditions, 1 mg of the inoculum was weighed and inoculated into 50 mL of YPD liquid medium (OD600 ≈ 0.02–0.05). The medium was transferred to a 250 mL Erlenmeyer flask to provide sufficient aeration. The flask was placed in a 25°C constant-temperature shaker at a speed of 200 rpm. The culture time was 48 hours, during which the OD600 value was measured periodically to ensure the spore concentration remained within the required range. After the culture was completed, the culture medium was filtered using sterile gauze or filter paper, and the spore suspension was collected and stored in a suitable preservation solution for later use.

[0130] Figure 4 The inhibition time curves of peanut cysteine-rich peptides and control peptides on the growth of Fusarium oxysporum f. sp. lycopersici, the pathogen of tomato, are shown. Figure 3 The effects were similar. According to the data in the figure, both peanut-rich cysteine ​​peptide and the control peptide showed significant inhibitory effects on the growth of *Fusarium oxysporum* f.sp. lycopersici, the causal agent of tomato wilt, at different time points. The antibacterial effect gradually increased over time, especially after 4 hours. Specific analysis results are as follows: 1. Early inhibition effect: Within 2 hours, the antibacterial effect was relatively weak, but some growth inhibition could still be observed. 2. Mid-term inhibition effect: After 4 hours, the antibacterial effect significantly increased, showing that both peanut-rich cysteine ​​peptide and the control peptide had strong inhibitory effects on the pathogen. 3. Long-term inhibition effect: At 10 hours, the antibacterial effect reached its peak, showing that peanut-rich cysteine ​​peptide has sustained and effective antibacterial activity. The antibacterial effect of peanut-rich cysteine ​​peptide was higher than that of the control peptide (…). Figure 4 ).

[0131] Overall, peanut-rich cysteine ​​peptides significantly inhibited the growth of Fusarium oxysporum f. sp. lycopersici, the causal agent of tomato wilt, and the inhibitory effect became more pronounced over time. This suggests that peanut-rich cysteine ​​peptides may have potential application value in plant disease resistance.

[0132] Example 5: Analysis of the minimum inhibitory concentration (MIC) and growth inhibition time curves of peanut cysteine-rich peptides and control peptides (GenBank No. XP_015936675.1) against Rhizoctonia solani.

[0133] The MIC values ​​of antimicrobial peptides were determined using the microdilution method [Saporito P., Mojsoska B., Olesen AL, Jenssen H. Antibacterial mechanisms of GN-2-derived peptides and peptoids against Escherichia coli. Biopolymers. 2019; 110:e23275; Cui H., Zhang C., Li C., Lin L. Antimicrobial mechanism of clove oil on Listeria monocytogenes. FoodControl.]. First, under aseptic conditions, 10 mg of Rhizoctonia solani was weighed.

[0134] Mycelia were collected and inoculated into 50 mL of YPD liquid medium. The medium was then transferred to 250 mL Erlenmeyer flasks to ensure adequate aeration. The flasks were incubated in a shaker at 28 °C at 200 rpm for 48 hours. OD600 values ​​were measured periodically to ensure spore concentration was within the desired range (OD600 ≈ 0.5–1), and 100 μL of the solution was added to each well of a 96-well titration plate. Serial dilutions of the peptide were then performed at equal volumes (0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 16 μM, 18 μM, and 20 μM) and incubated at 28 °C for 48 hours. The lowest concentration of antimicrobial peptide at which no visible microbial growth was observed was determined using the ELISA (SPARK) method. The purified antimicrobial peptide concentrations were adjusted to the MIC level for time-kill assays, and the same volume of peptide as the indicator bacteria was applied to 96-well plates (at least three wells per concentration). The plates were then incubated at 28°C for 2, 4, 6, 8, 10, 12, and 24 hours. OD values ​​were then measured at 600 nm. Inhibition assays of peanut cysteine-rich peptides and control peptides were performed using a modified method as previously described [Guangming S., Yang Y., Yuan Q., Shi G., Wu L., Lou Z., Huo R., Wu H., Borriss R., Gao X. Bacillomycin D Produced by Bacillus myloliquefaciens Is Involved in the Antagonistic Interaction with the Plant-Pathogenic Fungus Fusarium graminearum. Appl. Environ. Microbiol. 2017; 83:e01075–e01117.]. Approximately 200 μL (1 mg / mL) of antimicrobial peptide was uniformly mixed with 10 mL of Rhizoctonia solani spores. 7 ( / mL) and incubated at 28°C. The inhibition rate of spore count was observed and recorded every 2 hours from 0 to 10 hours. Figure 5 To calculate the spore count inhibition rate every 2 hours, the spore counts in the control and treatment groups must first be recorded. The control group contains the number of spores not treated for inhibition, while the treatment group contains the number of spores treated for inhibition.

[0135] The formula for calculating the inhibition rate is:

[0136]

[0137] Where, N control N represents the number of spores in the control group. treatment This represents the number of spores in the treatment group. Using this formula, the percentage reduction in spore count in the treatment group relative to the control group can be calculated, i.e., the inhibition rate.

[0138] The specific operating steps are as follows: 1. Record the spore count of the control group and the treatment group every 2 hours. 2. Calculate the inhibition rate at each recording using the formula above.

[0139] For example, at a certain time point, if the number of spores in the control group is 200 and the number of spores in the treatment group is 150, then the inhibition rate is calculated as follows:

[0140]

[0141] This means that at that point in time, the number of spores in the treatment group was reduced by 25% compared to the control group.

[0142] The experimental results showed that the MIC for *Rhizoctonia solani*, the causal agent of rice sheath blight, was 10.6 μM peanut cysteine-rich peptide and 14.5 μM control peptide. To prepare the spore inoculum for *Rhizoctonia solani*, the following steps were performed: First, under aseptic conditions, 10 mg of mycelium was weighed and inoculated into 50 mL of YPD liquid medium. The medium was transferred to a 250 mL Erlenmeyer flask to provide sufficient aeration. The flask was placed in a 25°C constant-temperature shaker at a speed of 200 rpm. The culture time was 48 hours, during which the OD600 value was measured periodically to ensure the spore concentration was within the required range (OD600 = 1). After the culture was completed, the culture medium was filtered using sterile gauze or filter paper, and the spore suspension was collected and stored in a suitable preservation solution for later use.

[0143] Figure 5The inhibition time curves of peanut cysteine-rich peptides against the growth of *Rhizoctonia solani* are shown. According to the data in the figure, both the peanut cysteine-rich peptide and the control peptide showed significant inhibitory effects on the growth of *Rhizoctonia solani* at different time points. The inhibitory effect gradually increased over time, especially after 6 hours. Specific analysis results are as follows: 1. Early inhibition effect: Within 4 hours, the inhibitory effect was relatively weak, but some growth inhibition was still observed. 2. Mid-term inhibition effect: After 6 hours, the inhibitory effect significantly increased, showing that the peanut cysteine-rich peptide control peptide had a strong inhibitory effect on the pathogen. 3. Long-term inhibition effect: The inhibitory effect reached its peak at 10 hours, showing that the peanut cysteine-rich peptide has a sustained and effective antibacterial activity. Overall, peanut cysteine-rich peptides have a significant inhibitory effect on the growth of *Rhizoctonia solani*, and the inhibitory effect becomes more pronounced with prolonged time. This suggests that peanut cysteine-rich peptides may have potential application value in plant disease resistance. Peanut-rich cysteine ​​peptides showed higher antibacterial effects than control peptides. Figure 5 ).

[0144] Example 6: Hemolysis Test

[0145] The hemolytic activity of two antimicrobial peptides (control peptide GenBank No. XP_015936675.1 and peanut cysteine-rich peptide) was evaluated according to the previously described method [Gautam A., Chaudhary K., Singh S., Joshi A., Anand P., Tuknait A., Mathur D., Varshney GC, Raghava G. Hemolytik: A database of experimentally determined hemolytic and non-hemolytic peptides. Nucleic Acids Res.]. Briefly, fresh pig blood was centrifuged at 1500 × g for 10 minutes at 4°C, and red blood cells were collected. The cells were washed three times with pre-cooled 0–4°C 0.1M PBS buffer (pH = 7.2, hereinafter the same), and then diluted with 0–4°C 0.1M PBS buffer to a 1% red blood cell suspension (V / V). Prepare equal volumes of diluted erythrocytes and antimicrobial peptide solutions (aqueous solutions) at concentrations of 1×MIC (20 μM), 1.5×MIC (30 μM), 2×MIC (40 μM), 2.5×MIC (50 μM), and 3×MIC (60 μM). Mix 100 μL of erythrocyte suspension with 100 μL of the corresponding concentration of antimicrobial peptide solution and incubate at 37°C for 1 hour. After incubation, centrifuge the mixture at 4000×g for 10 minutes and measure the absorbance of the supernatant to assess the erythrocyte hemolysis rate. Using an ELISA (SPARK) plate reader, transfer the supernatant to a 96-well plate and measure the absorbance at 385 nm. A Triton X-100 (Sigma, model MFCD00128254) erythrocyte suspension (V / V) was used as the control group (ATriton), while incubation with only 1% blood cells in 20 nM 0.1M PBS buffer (V / V) served as the negative control (APBS). Furthermore, the percentage of hemolysis using 0.1% Triton X-100 in PBS was also measured. The percentage of hemolytic activity was calculated as follows: [(Apeptide-APBS) / (ATriton-APBS)] × 100.

[0146] The final formula is used to calculate the percentage of hemolytic activity in erythrocytes, that is, the extent to which erythrocytes rupture and release hemoglobin due to the antimicrobial peptide solution. The formula is as follows:

[0147]

[0148] Explain the meaning of each variable:

[0149] A peptide : Absorbance value of the sample supernatant containing antimicrobial peptide solution. -A PBSAbsorbance value of the supernatant from the negative control group (red blood cell suspension treated only with PBS buffer). -A Triton Positive control group (using 0.1 mM Tritonx-100, catalog number 9036-19-5, Sigma).

[0150] Formula calculation steps:

[0151] 1. Calculate the absorbance change caused by the antimicrobial peptide: A peptide -A PBS - This step represents the change in absorbance caused by hemolysis of erythrocytes (release of hemoglobin) induced by the antimicrobial peptide solution.

[0152] 2. Calculate the absorbance change caused by Triton X-100: A Triton -A PBS This step represents the change in absorbance caused by using 0.1 mM Triton x-100.

[0153] 3. Calculate the relative percentage of hemolysis caused by antimicrobial peptides: - By comparing the absorbance change caused by the antimicrobial peptide with the absorbance change under complete hemolysis, the relative percentage of hemolysis caused by the antimicrobial peptide is determined.

[0154] The final results represent the hemolytic activity of the antimicrobial peptide solution at different concentrations against erythrocytes, presented as a percentage. This helps in assessing the hemolytic toxicity of the antimicrobial peptide.

[0155] Figure 6 The results of hemolysis experiments on peanut cysteine-rich peptides and control peptides are shown. Based on the data in the figure, the hemolysis rates of peanut cysteine-rich peptides and control peptides at different concentrations on erythrocytes were determined. The results showed that at lower concentrations (e.g., 1 μg / mL and 100 μg / mL), the hemolysis rates were low, close to those of the control group, indicating that peanut cysteine-rich peptides and control peptides did not have a significant destructive effect on erythrocytes at these concentrations. However, when the concentration was increased to 1000 μg / mL, the highest hemolytic activity of 2% was observed. This indicates that peanut cysteine-rich peptides and control peptides have low hemolytic toxicity to erythrocytes at high concentrations, but are relatively safe at low concentrations. Therefore, when using peanut cysteine-rich peptides as an antibacterial agent, its concentration should be carefully controlled to avoid unnecessary damage to host cells.

Claims

1. A peanut-rich cysteine ​​peptide, which is a peanut antimicrobial peptide, characterized in that: The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO.

2.

2. A gene encoding the peanut cysteine-rich peptide of claim 1, the nucleotide sequence of which is shown in SEQ ID NO.

1.

3. An antibacterial agent, characterized in that: It uses the peanut cysteine-rich peptide described in claim 1 as its active ingredient.

4. The antibacterial agent according to claim 3, characterized in that: The antibacterial agent is a fungal antibacterial agent used for the prevention and control of plant fungal pathogens; The antibacterial agent inhibits one or more of the following pathogens: wheat leaf blight fungus (Alternaria triticina), tomato wilt fungus (Fusarium oxysporum f. sp. lycopersici), and sheath blight fungus (Rhizoctonia solani).

5. The application of the peanut cysteine-rich peptide of claim 1 in the preparation of an antibacterial agent, characterized in that: The antibacterial agent is a fungal antibacterial agent used for the prevention and control of plant fungal pathogens; The antibacterial agent inhibits one or more of the following pathogens: wheat leaf blight fungus (Alternaria triticina), tomato wilt fungus (Fusarium oxysporum f. sp. lycopersici), and sheath blight fungus (Rhizoctonia solani).

Images