A knot1 domain-containing peptide or arachidonic knot1 domain-containing peptide nucleic acid complex and uses thereof

Abstract

The present application discloses an anti-fungal peanut Knot1 domain-containing peptide nucleic acid complex and characterization of its anti-fungal activity. The anti-fungal activity of the peanut Knot1 domain-containing peptide nucleic acid complex against peanut pathogens Fusarium oxysporum and Rhizoctonia solani. The Knot1 domain-containing peptide is crucial for anti-fungal activity, but the stability of the protein is limited. Peptide nucleic acid can increase the resistance of Knot1 domain-containing peptide to protease attack. Therefore, the present patent designs a peanut Knot1 domain-containing peptide nucleic acid complex with high stability and anti-fungal activity.

Description

Technical Field

[0001] This invention belongs to the field of biopesticide technology, specifically involving the chemical synthesis of an antifungal peanut peptide-nucleic acid complex containing the Knot1 domain, exhibiting antifungal activity against peanut pathogens *Fusarium oxysporum* and *Rhizoctonia solani*. It also possesses sufficient biostability, reducing the degradation of the Knot1 domain-containing peptide. Background Technology

[0002] Plants resist pathogen infection by upregulating the expression of genes including microbial-induced plant antitoxins (Darvill AG, Albersheim P (1984) Phytoalexins and their elicitors-a defense against microbial infection in plants. Annu Rev Plant Physiol 35:243–275.), hydroxyproline-rich glycoproteins (Hahn MG, Buchell P, Cervone F, Doares SH, O'Neil RA, Darvill AG, Albersheim P (1989) Roles of cell wall constituents in plant–pathogen interactions. In: Kosuge T, Nester EW (eds) Plant–microbe interactions—molecular and genetic perspectives, 1st edn, vol 3. McGraw-Hill, New York, pp 131–181.), lyases, and protease inhibitors. Therefore, proteins related to pathogenesis have long been a research hotspot in agriculture (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] Plants possess an active defense system, protecting themselves from pathogen invasion by secreting various small antifungal peptides. We identified a Knot1-domain-containing peptide with antifungal activity in the economic crop peanut; however, this protein has limited stability and is easily degraded by proteases (Shigeru Oota. Decomposition of Antibacterial Peptide and α-Choni in the Treatment of Kojibacco Proteases. Japan Soybean Industry Association Journal 95, no. 10 (2000): 776-779.). Peptide nucleic acids are oligonucleotide analogs in which the phosphodiester backbone is replaced by a polyamide structure. Peptide nucleic acids have attracted much attention due to their numerous beneficial properties, including resistance to nuclease and protease digestion, stability in serum and cell extracts, and enhanced resistance to proteases (Rathee, Permender, Dharmender Rathee, Ashima Hooda, Vikash Kumar, and Sushila Rathee. "Peptide Nucleic Acids: An Overview." The Pharma Innovation 1, no. 7, Part A (2012): 25.). Therefore, peptide nucleic acids possess sufficient biostability to reduce the degradation of peptides containing the Knot1 domain. The antifungal activity of peptide nucleic acid complexes containing the Knot1 domain was also explored. Summary of the Invention

[0004] Transcriptome studies and analyses of peanut meal were conducted, and antimicrobial peptides containing the Knot1 domain were identified and chemically synthesized. A highly stable peptide nucleic acid was synthesized (Patil, Nitin A., Varsha J. Thombare, Rong Li, Xiaoji He, Jing Lu, Heidi H. Yu, Hasini Wickremasinghe et al. "An Efficient Approach for the Design and Synthesis of Antimicrobial Peptide-Peptide Nucleic Acid Conjugates." Frontiers in Chemistry 10 (2022).). Simultaneously, a peanut-containing Knot1 domain peptide nucleic acid complex was prepared chemically, with the following amino acid sequence:

[0005] ERHCSGTHRFKGMCGHERNCVRNLEGFSFGCGGLGRNCGCKYCH. Peptides containing the Knot1 domain exhibit antifungal activity against peanut pathogens *Fusarium oxysporum* and *Rhizoctonia solani*. However, the stability of these Knot1 domain-containing peptides is limited. The present invention aims to synthesize a peptide nucleic acid (Cys-PEG4-Cyt-Thy-Cyt-Ade-Thy-Ade-Cyt-Thy-Cyt-Thy-Thy-Gua-Lys-NH2) to increase the stability of the antifungal peanut Knot1 domain-containing peptide.

[0006] The stability of the peptide containing the Knot1 domain is limited. Peptide nucleic acids enhance the resistance of the Knot1 domain-containing peptide to protease attack and reduce its degradation.

[0007] Peptide-nucleic acid enhances the resistance of peptides containing the Knot1 domain to protease attack, and possesses sufficient biostability to reduce the degradation of peptides containing the Knot1 domain. This demonstrates the long-term stable application of the antifungal peanut peptide-nucleic acid complex containing the Knot1 domain in inhibiting one or more of the peanut pathogens *Fusarium oxysporum* and *Rhizoctonia solani*.

[0008] Knot1-domain-containing peptides are antifungal proteins found in peanuts, exhibiting antifungal activity against peanut pathogens *Fusarium oxysporum* and *Rhizoctonia solani*. However, this protein is unstable and easily attacked by proteases. Peptide nucleic acids enhance the stability of peanut-containing Knot1-domain-containing peptides in their antifungal activity. An antifungal peanut-containing Knot1-domain-containing peptide-nucleic acid complex is presented. This complex consists of two parts: a Knot1-domain-containing peptide with the amino acid sequence shown in SEQ ID NO. 1, and a peptide nucleic acid with a nucleotide and amino acid hybrid sequence shown in SEQ ID NO. 2. The peanut-containing Knot1-domain-containing peptide-nucleic acid complex is prepared by chemical synthesis. Attached Figure Description

[0009] Figure 1 Mass spectrometry analysis of the molecular weight of chemically synthesized peptides containing the Knot1 domain.

[0010] Figure 2 Electrospray mass spectrometry analysis (cation mode) of chemically synthesized peptide nucleic acids containing the Knot1 domain.

[0011] Figure 3 Peptides containing the Knot1 domain and peptide nucleic acids inhibit mycelial growth, where A is Fusarium oxysporum and B is a solanaceous pathogen.

[0012] Figure 4Peptides containing the Knot1 domain improve the stability of the antifungal activity of peptides containing the Knot1 domain. Detailed Implementation

[0013] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0014] Example 1: Source of peanut-containing Knot1 domain peptides

[0015] Peanut meal was purchased from Shandong Provincial Feed Industry Co., Ltd. Its physicochemical properties (mass content) are as follows:

[0016] - Crude protein content: ≥46%, - Crude fat content: ≤2%, - Crude ash content: ≤8%, - Crude fiber content: ≤10%, and - Moisture content: ≤12%.

[0017] 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 Technologies, USA), add 1 mL of TRIzol reagent, and incubate at room temperature for 5 minutes. 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. The 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.

[0018] Quality control and transcriptome library sequencing:

[0019] RNA integrity assessment

[0020] 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.

[0021] Library Construction

[0022] 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.

[0023] High-throughput sequencing

[0024] 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.

[0025] 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.

[0026] 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. 4) was identified for the first time using blastp software through homology alignment. This sequence showed 78% homology with the defensin in Genbank No. XM_025818275.2. The gene sequence is as follows:

[0027] GAGAGACACTGTTCGGGGACCCACCGGTTCAAAGGAATGTGCGGGC

[0028] ACGAACGCAACTGTGTCCGTAACTTAGAAGGTTTCTCCTTTGGTTGCG

[0029] GCGGCTTGGGTCGAAACTGCGGCTGCAAGTATTGCCAT

[0030] (a) Sequence characteristics:

[0031] ●Length: 132

[0032] ●Type: Nucleotide sequence

[0033] ●Chain type: Single chain

[0034] ●Topology: Linear

[0035] (b) Molecular type: Nucleotide

[0036] (c) Assumption: No

[0037] (d) Antonym: No

[0038] (e) Original source: peanut

[0039] The full-length amino acid sequence of the cysteine-rich peptide is SEQ ID NO.1, as follows: ERHCSGTHRFKGMCGHERNCVRNLEGFSFGCGGLGRNCGCKYCH

[0040] (a) Sequence characteristics:

[0041] ●Length: 44

[0042] ●Type: Amino acid sequence

[0043] ●Chain type: Single chain

[0044] ●Topology: Linear

[0045] (b) Molecular type: protein

[0046] (c) Assumption: No

[0047] (d) Antonym: No

[0048] (e) Original source: peanut

[0049] The Knot1 domain typically exhibits a complex folding pattern, forming a tight, knot-like structure. This structure contributes to increased stability and resistance to enzymatic degradation. The Knot1 domain is rich in specific amino acids, such as cysteine, which are usually linked by disulfide bonds, thereby enhancing the peptide's stability.

[0050] Peanuts contain a Knot1 domain peptide (sequence:

[0051] The chemical synthesis of ERHCSGTHRFKGMCGHERNCVRNLEGFSFGCGGLGRNCGCKYCH was commissioned to Changzhou Kanglong Biotechnology Co., Ltd. Alternatively, the standard procedure for solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl (FMOC) can be referenced. The basic principle is that in FMOC solid-phase peptide synthesis, the peptide chain is assembled stepwise, one amino acid at a time, simultaneously linked to an insoluble resin support. This allows for the removal of reaction byproducts at each step through simple washing. The amino terminus of the amino acid is protected by an FMOC (9-fluorenylmethoxycarbonyl) group and coupled to the growth chain after activation at the carboxylic acid terminus. The FMOC group is then removed by piperidine treatment, and this process is repeated. After peptide assembly, it is removed from the resin by treatment with trifluoroacetic acid (TFA). Simultaneously, the protecting groups on the amino acid side chains are also removed, yielding a crude linear peptide. One-step purification by reversed-phase HPLC is usually sufficient to obtain peptides with a purity >95%.

[0052] Analysis of Knot1-domain-containing peptides by reversed-phase liquid chromatography-electrospray ionization mass spectrometry (RPLC-ESI-MS / MS): Peanut Knot1-domain-containing peptides (100 μL, 1 mg / ml protein) were analyzed using a reversed-phase liquid chromatography-electrospray ionization mass spectrometry system operated in positive ion mode. A C18 column (Waters, [missing information]) was used. Chromatographic separation was performed on a 5μm, 4.6mm x 250mm lens (model 186001265) at a flow rate of 300 nL / min. The mobile phase consisted of 99.8% H₂O and 0.2% TFA(A) at v / v, and 95% ACN, 4.8% H₂O, and 0.2% TFA(B) at v / v. The LC gradients were as follows: 2% B, 10 min; linear 2%-40% B, 190 min; linear 40%-95% B, 10 min; 95% B, 5 min; linear 95%-2% B, 5 min; 2% B, 20 min. The ESI conditions were as follows: spray voltage 2.8 kV, capillary temperature 320 °C, and high-pressure ring iontophoresis device (S-lens RF) level 75 V. Full-scan mass spectrometry was acquired in the range of 500-14000 m / z. The main ESI source settings were: microscan 1, mass resolution 70 k, automatic gain control (AGC) target 2e5, and maximum ion implantation time 50 ms. MSMS spectra were acquired in Top20 data correlation acquisition mode with the following settings: microscan 1, mass resolution 17.5 k, AGC target 5e5, maximum ion implantation time 250 ms, isolation window 3 m / z, higher energy collisional dissociation (HCD), step normalization of collision energies 20%, 30%, and 40%, dynamic exclusion for 20 seconds, and charge inclusion 2-6.

[0053] The molecular weight of this protein was determined to be approximately 4905.41 kDa, consistent with the theoretical molecular weight. Figure 1 The sequence is identified as a short peptide containing the Knot1 domain, and its SEQ ID No. 1 is: ERHCSGTHRFKGMCGHERNCVRNLEGFSFGCGGLGRNCGCKYCH(44aa).

[0054] (a) Sequence characteristics:

[0055] ●Length: 44

[0056] ●Type: Amino acid sequence

[0057] ●Chain type: Single chain

[0058] ●Topology: Linear

[0059] (b) Molecular type: protein

[0060] (c) Assumption: No

[0061] (d) Antonym: No

[0062] (e) Original source: peanut

[0063] Figure 1 Mass spectrometry analysis of the molecular weight of chemically synthesized peptides containing the Knot1 domain.

[0064] Example 2: Chemical Synthesis of Peptide Nucleic Acid Containing Knot1 Domain

[0065] The Knot1-domain-containing peptide was synthesized by means of a disulfide bond (-ss-) formed between the Knot1-domain-containing peptide synthesized in Example 1 and the peptide nucleic acid synthesized in Example 2, and the Knot1-domain-containing peptide (ERHCSGTHRFKGMCGHERNCVRNLEGFSFGCGGLGRNCGCKYCH) on the C-terminal cysteine ​​residue of the Knot1-domain-containing peptide and the thiol group of the cysteine ​​side chain of the peptide nucleic acid.

[0066] -ss-peptide nucleic acid (C-PEG4-ctcatactcttg-K-NH2)). Chemical synthesis was outsourced to Changzhou Kanglong Biotechnology Co., Ltd. Alternatively, refer to the tutorial on peptide nucleic acid synthesis, Vol. 2002, by Peter E. Nielsen (Editor), ASIN: 0896039765, published by Humana; July 23, 2002; Language: English; ISBN-10: 9780896039766.

[0067] ISBN-13:978-0896039766).

[0068] Referring to pages 43-48 of the textbook *Peptide Nucleic Acids: Methods and Protocols* (Methods in Molecular Biology, 208), 2002 nd Edition, Peter E. Nielsen (Editor), ASIN: 0896039765, Publisher: Humana; 2002 edition (July 23, 2002); Language: English; ISBN-10: 9780896039766; ISBN-13: 978-0896039766), the redox chemistry of sulfur can be used to form disulfide (-ss-) bridging conjugates. We utilize the Cys peptide containing the Knot1 domain at the C-terminus of the peptide nucleic acid to couple it with a disulfide bond.

[0069] The process is briefly described as follows:

[0070] 1. The Knot1 domain-containing peptide (1.0 μmole) synthesized in Example 1 and dithiothreitol (DTT) (1 mg) were dissolved in 100 μL of 0.1 M NH4HCO3 and left to stand at room temperature for 2 hours.

[0071] 2. Add 11 mg of dipyridyl disulfide to 100 μL of DMF and let stand at room temperature for 2 hours.

[0072] 3. Immediately react the reaction mixture with the peptide nucleic acid (1 μmole) synthesized in Example 2 for 3 h.

[0073] 4. Refer to page 40 of the tutorial (Peptide Nucleic Acids: Methods and Protocols (Methods in Molecular Biology, 208) 2002 nd Edition, Peter E. Nielsen (Editor), ASIN: 0896039765, Publisher: Humana; 2002 edition (July 23, 2002); Language: English; ISBN-10: 9780896039766; ISBN-13: 978-0896039766) to analyze and purify the product using HPLC, and characterize it using the electrospray mass spectrometry method described below.

[0074] Peptide nucleic acids (PNAs) are DNA synthesis analogs based on the N-(2-aminoethyl)glycine pseudopeptide backbone. Bases (corresponding to A, G, C, T) are linked to the backbone via methylene carbonyl linkers, resulting in PN oligomers that can form highly stable complexes with complementary DNA and RNA. They also possess the significant advantage of resistance to nucleases (and proteases), making them potentially ideal for antisense and antigene applications. Numerous methods for modifying oligonucleotides with peptides have been developed for therapeutic and diagnostic applications or for the assembly of nanostructures. We have developed a method for constructing peptides based on Knot1 domain-containing peptides using PNAs as a scaffold. The formation of disulfide bonds between the Knot1 domain-containing peptide and the PNAs enhances their stability, thereby expanding the applications of functional PNAs. Electrospray ionization mass spectrometry (ESMS) was used to identify the molecular weight of the synthesized Knot1 domain-containing PN peptides. The ESMS experiments were performed on a XevoTQ MS at Waters-USA. ESMS spectra were recorded in positive ion mode. One mg of the peptide nucleic acid peptide containing the Knot1 domain was dissolved in 1 mL of a 1:1 methanol-water solution. 10 μL of this solution was then introduced into the mass spectrometer at a solvent flow rate of 5 μL / min. The diluted sample was sprayed using two ionization modes within the m / z range of 50-5000, with an applied voltage of 5 kV. The ESI source parameters were set to a capillary voltage of 3500 V, nitrogen as the nebulizer gas at a pressure of 30.00 psi and a flow rate of 650 L / h. The capillary temperature was set to 250 °C. Figure 2 The cationic ESIMS spectrum is shown, indicating the presence of a peptide-nucleic acid-peptide conjugate containing the Knot1 domain, with a molecular weight of 9384.94 Da. As can be seen from SEQ ID No. 2, it is flanked by cysteine ​​and lysine residues. Therefore, the addition of the Knot1 domain-containing peptide binds to one side of the cysteine ​​residue of the peptide-nucleic acid to form an amide bond (peptide bond), resulting in SEQ ID No. 3.

[0075] Prepare a peptide-nucleic acid complex containing the Knot1 domain. The protein has a specific sequence length of 47 natural amino acids, as shown in SEQ ID No. 1 of the sequence listing.

[0076] (a) Sequence characteristics:

[0077] ●Length: 44

[0078] ●Type: Amino acid sequence

[0079] ●Chain type: Single chain

[0080] ●Topology: Linear

[0081] (b) Molecular type: protein

[0082] (c) Assumption: No

[0083] (d) Antonym: No

[0084] (e) Original source: peanut

[0085] The prepared peptide nucleic acid is a complex sequence of bases and amino acids, as shown in the sequence listing SEQ ID No. 2: C-PEG4-ctcatactcttg-K-NH2 (C, cysteine; K, lysine; PEG4, tetraethylene glycol; lowercase letters represent the corresponding nucleotide bases, adenine a, thymine t, guanine g, and cytosine c).

[0086] (a) Sequence characteristics:

[0087] ●Length: 15

[0088] ●Type: Peanut protein isolation

[0089] ●Chain type: Single chain

[0090] ●Topology: Linear

[0091] (b) Molecular type: peptide nucleic acid

[0092] (c) Assumption: No

[0093] (d) Antonym: No

[0094] (e) Original source: Reference Patil, Nitin A., Varsha J. Thombare, Rong Li, Xiaoji He, Jing Lu, Heidi H. Yu, Hasini Wickremasinghe et al. "An Efficient Approach for the Design and Synthesis of Antimicrobial Peptide-Peptide Nucleic Acid Conjugates." Frontiers in Chemistry 10 (2022).

[0095] A peptide-nucleic acid protein complex was prepared, as detailed in SEQ ID No. 3 of the sequence listing. A disulfide (-SS-) bridging conjugate was formed using sulfur redox chemistry. We coupled the cysteine ​​residue at the C-terminus of the peptide containing the Knot1 domain to the cysteine ​​residue of the peptide nucleic acid via a disulfide bond. The molecular weight of the peptide-nucleic acid complex was 9384.94 Da. Figure 2 )

[0096] (a) Sequence characteristics:

[0097] ●Length: 59

[0098] ●Type: Base and amino acid complex sequence

[0099] ●Chain type: Single chain

[0100] ●Topology: Linear

[0101] (b) Molecular type: peptide-nucleic acid-protein complex

[0102] (c) Assumption: No

[0103] (d) Antonym: No

[0104] (e) Original source: Self-synthesized chemically

[0105] Figure 2 Electrospray mass spectrometry analysis (cation mode) of chemically synthesized peptide nucleic acids containing the Knot1 domain.

[0106] Example 3: Identification of nucleic acid activity of peanut-containing Knot1 domain peptides

[0107] The antifungal activity of peanut Knot1 domain peptide nucleic acid was determined by the radial growth inhibition method described by Schlumbaum et al. (Schlumbaum A, Mooch F, U, Boller T (1986) Plant chitinases are potent inhibitors of offungal growth. Nature 324:365–367.) and microspectrophotometry (Broekaert WF, Frankt RG, Terras Bruno PA, Cammue, Jos Vanderleyden (1990) An automated quantitative assay for fungal growth inhibition. FEMS Microbiol Lett 69:55–60.). *Solanum lycopersicum* and *Fusarium oxysporum* were used sequentially to examine the antifungal activity of peptide nucleic acids containing the Knot1 domain. For spectrophotometric determination, 10 μl of peanut nucleic acid samples containing Knot1 domain peptides at different concentrations (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / mL) were mixed with 90 μl of samples containing fungal spores (1 x 10^6 mg / mL). 7The spores were mixed with Sabouraud glucose broth (SDB, HiMedia) at 37°C for 16 hours in 96-well microtiter plates. Fungal growth was estimated by observing absorbance at 595 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, USA). IC50 values ​​were calculated using a double dilution step (Terras FR, Schoofs HM, De Bolle MF, Van Leuven F, Rees SB, Vanderleyden J, Cammue BP, Broekart WF (1992) Analysis of two novel classes of plant antifungal proteins from rad-ish (Raphanus sativus L.) seeds. J Biol Chem 267:15301–15309.).

[0108] The solid culture medium used in the study of antifungal activity of peptides containing the Knot1 domain of *Fusarium oxysporum* and *Solanum lycopersicum* consisted of potato dextrose agar (PDA) for fungi (potato, 200 g; glucose, 20 g; agar, 18 g; and distilled water, 1 L). To test antifungal activity, 1 mL of fungal spore suspensions (1 x 10⁻⁶) of *Fusarium oxysporum* and *Solanum lycopersicum* were separately added. 7 Spores (per ml) were evenly added to 100 mL of PDA medium. Solid plates (3-4 mm thick) were prepared at 40-50℃, and stainless steel tubes (6 mm inner diameter, 8 mm outer diameter, 10 mm height) were placed in each plate. Then, 5 μL of peptides containing the Knot1 domain (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / mL) were added to the Oxford cups. The plates were incubated at 30℃ for 48 h, and the diameter of the antibacterial zone was measured. A control group was prepared by adding an equal volume of physiological saline and incubating under the same conditions. Preliminary screening of the antifungal activity of peptides containing the Knot1 domain by clear zone formation showed that the IC50 values ​​of peptides containing the Knot1 domain against *Fusarium oxysporum* and *Solanum lycopersicum* were 10.2 (…). Figure 3 A) and 67 μg / ml ( Figure 3 B), the IC50 values ​​of peptides containing the Knot1 domain against Fusarium oxysporum and Solanum lycopersicum were 15.8 μg / ml, respectively. Figure 3 A) and 109 μg / ml ( Figure 3 B).

[0109] Figure 3 Peptides containing the Knot1 domain and peptide nucleic acids inhibit mycelial growth. A. *Fusarium oxysporum*. B. *Solanum lycopersicum*.

[0110] Example 4: The stability of antifungal activity of peptide-containing nucleic acids with Knot1 domains was improved. 1 mL of either a peptide-containing nucleic acid with Knot1 domains or a peptide containing Knot1 domains (5 mg / mL, in physiological saline) was taken and incubated at 30°C for 20 days. 5 μL of either a peptide-containing nucleic acid with Knot1 domains or a peptide containing Knot1 domains (5 mg / mL, in physiological saline) was taken daily for the following antifungal experiment. The solid culture 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 antifungal activity, 1 mL of a suspension of Fusarium oxysporum spores (1 x 10⁻⁶) was added. 7 Spores (per ml) were evenly added to 100 mL of PDA medium. Solid plates (3-4 mm thick, 3.5 mm in this case) were prepared at 40-50℃. Oxford cups (open at both ends, 6 mm inner diameter, 8 mm outer diameter, 10 mm height) were then placed on the plates (2 cm apart, 2-5 mm deep). 5 μL of peptide nucleic acid containing the Knot1 domain or peptide containing the Knot1 domain (5 mg / mL in physiological saline) was added to the center of each Oxford cup. The plates were incubated at 30℃ for 48 h, and the diameter of the antimicrobial zone was measured. A control group was prepared by adding an equal volume of physiological saline and incubating under the same conditions. The relative antifungal activity of the peptide nucleic acid containing the Knot1 domain after 24 hours was considered 100%. The antimicrobial zone diameters of peptide nucleic acids containing the Knot1 domain at other incubation times were compared with those after 24 hours to determine the corresponding activity.

[0111] Figure 4 Peptides containing the Knot1 domain improve the stability of the antifungal activity of peptides containing the Knot1 domain.

[0112] Using the skeletal structure of peptide nucleic acids, the results showed that peptides containing the Knot1 domain improved the stability of the antifungal activity of peptides containing the Knot1 domain, extending it from 10 days to 20 days. Figure 4 This method is feasible and we will consider promoting it in production in the future.

Claims

1. A peptide containing a Knot1 domain, characterized in that, The amino acid sequence of the peptide containing the Knot1 domain is shown in SEQ ID NO.1 of the sequence listing.

2. A peanut-based peptide-nucleic acid complex containing a Knot1 domain, characterized in that, The peanut-containing Knot1 domain peptide-nucleic acid complex comprises a portion of the Knot1 domain-containing peptide as described in claim 1, and a portion of the peptide-nucleic acid. The peanut-containing Knot1 domain peptide-nucleic acid complex is linked together by a disulfide bond formed through an oxidation reaction between the thiol groups of the cysteine ​​residues at the 3' end of the peptide-nucleic acid and the cysteine ​​residues at the C-terminus of the Knot1 domain-containing peptide. The sequence of the Knot1 domain-containing peptide-nucleic acid complex is as follows: ERHCSGTHRFKGMCGHERNCVRNLEGFSFGCGGLGRNCGCKYCH-ssC-PEG4-ctcatactcttg-K-NH2.

3. The application of a Knot1 domain-containing peptide or peanut Knot1 domain-containing peptide nucleic acid complex as described in claim 1 or 2, characterized in that, The peanut-containing Knot1 domain peptide and / or peanut-containing Knot1 domain peptide nucleic acid complex has antifungal activity against the peanut pathogen Fusarium oxysporum and can be used as an antifungal drug or antifungal active ingredient against Fusarium oxysporum. The application is for non-disease diagnosis and treatment purposes.

4. The application of a Knot1 domain-containing peptide or peanut Knot1 domain-containing peptide nucleic acid complex as described in claim 1 or 2, characterized in that, The application of the peanut Knot1 domain peptide and / or peanut Knot1 domain peptide nucleic acid complex in inhibiting the peanut pathogen Fusarium oxysporum, wherein the application is for non-disease diagnosis and treatment purposes.

5. The application of the Knot1 domain-containing peptide or peanut antifungal peanut Knot1 domain-containing peptide nucleic acid complex as described in claim 1 or 2, characterized in that, The application of the peanut Knot1 domain peptide and / or peanut Knot1 domain peptide nucleic acid complex in the preparation of a drug to inhibit the peanut pathogen Fusarium oxysporum.

6. A drug for treating Fusarium oxysporum, wherein the active ingredient is the Knot1 domain-containing peptide or peanut Knot1 domain-containing peptide nucleic acid complex as described in claim 1 or 2.

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