Plant defensin polypeptide, nucleotide and application

Abstract

The invention provides a plant defensin polypeptide, nucleotide and application, and relates to the technical field of plant defensin. The amino acid sequence of the plant defensin polypeptide is as shown in at least one of SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 5. The plant defensin polypeptide has efficient antibacterial and antifungal activity, can significantly enhance the disease resistance of plants, has good stability and biocompatibility, and has wide application prospects. The method has important application value in the fields of agriculture, medical treatment, food and the like due to the beneficial effects.

Description

Technical Field

[0001] The present invention relates to the technical field of plant defensins, and in particular to a plant defensin polypeptide, nucleotide and application. Background Art

[0002] Defensins are a class of naturally occurring antimicrobial peptides found in nearly all organisms, from plants and lower animals to mammals. These peptides have evolved through the long struggle against disease and constitute a crucial component of an organism's defense system. Defensins are typically encoded directly by genes and possess broad-spectrum antimicrobial activity, effectively protecting against invading bacteria, fungi, and some viruses. Advances in genetic engineering have expanded the production of defensins beyond natural extraction. They can now be efficiently expressed in microorganisms or plants through genetic engineering, paving the way for large-scale application.

[0003] In existing technologies, the application of defensins is primarily focused on antibacterial, antifungal, and insecticide applications. Research has shown that defensins can effectively inhibit the growth of a variety of pathogenic microorganisms, demonstrating great potential as new biopesticides or alternatives to antibiotics. Furthermore, by introducing defensin genes into crops through genetic engineering techniques, genetically modified crops with enhanced disease resistance can be cultivated, thereby reducing the use of chemical pesticides and lowering environmental pollution. However, despite the promising antimicrobial activity demonstrated in laboratory studies, defensins face several limitations in their practical application.

[0004] The main defects faced by defensins in the prior art in practical applications include poor antibacterial activity. Although defensins show certain antibacterial activity in laboratory environments, in practical applications, especially in complex biological environments and field conditions, their antibacterial effects are often unsatisfactory. For example, defensins may exhibit cytotoxicity and inflammatory reactions at high concentrations, limiting their direct application in vivo. In addition, defensins are sensitive to proteases and are easily rapidly degraded in the body, which further limits their stability and effectiveness in vivo. The delivery system of defensins is still imperfect and lacks effective pharmaceutical properties, resulting in limited absorption efficiency and metabolic cycle when systemically administered. These problems together limit the widespread promotion of defensins in practical applications.

[0005] In summary, despite the significant potential of defensins in antibacterial, antifungal, and anti-insect applications, existing technologies still suffer from numerous limitations that limit their widespread adoption in practical applications. These include poor antibacterial activity, insufficient in vivo stability, and imperfect delivery systems. These challenges require further research and technological advancements to fully realize the potential of defensins in biotechnology and medicine.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The present invention aims to provide a plant defensin polypeptide, nucleotide, and application thereof. The plant defensin polypeptide exhibits highly potent antibacterial and antifungal activity, significantly enhancing plant disease resistance, and possesses excellent stability and biocompatibility, with broad application prospects. These beneficial effects make it valuable for applications in agriculture, healthcare, and food.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0009] In a first aspect, the present invention provides a plant defensin polypeptide, wherein the amino acid sequence of the plant defensin polypeptide is shown in at least one of SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5.

[0010] In a second aspect, the present invention provides a nucleotide, the sequence of which is shown as at least one of SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6.

[0011] In an optional embodiment, the nucleotide sequence of SEQ ID NO.2 encodes the plant defensin polypeptide corresponding to the amino acid sequence shown in SEQ ID NO.1;

[0012] The nucleotide sequence of SEQ ID NO.4 encodes the plant defensin polypeptide corresponding to the amino acid sequence of SEQ ID NO.3;

[0013] The nucleotide sequence of SEQ ID NO.6 encodes the plant defensin polypeptide corresponding to the amino acid sequence shown in SEQ ID NO.5.

[0014] In a third aspect, the present invention provides a construct comprising the nucleotides described in the aforementioned embodiments.

[0015] In a fourth aspect, the present invention provides a recombinant vector comprising the nucleotides described in the aforementioned embodiments.

[0016] In a fifth aspect, the present invention provides a recombinant cell comprising the nucleotide described in the above embodiment.

[0017] In a sixth aspect, the present invention provides a composition comprising the plant defensin polypeptide as described in the above embodiments.

[0018] In a seventh aspect, the present invention provides a functional product comprising the plant defensin polypeptide according to the aforementioned embodiment;

[0019] In a preferred embodiment, the functional product comprises at least one of a drug, a detection reagent, and a detection kit;

[0020] In a preferred embodiment, the drug comprises at least one of an antibacterial drug, an antifungal drug, and an antiinsect drug.

[0021] In an eighth aspect, the present invention provides a transgenic crop comprising the plant defensin polypeptide as described in the aforementioned embodiment.

[0022] In a ninth aspect, the present invention provides a use of the plant defensin polypeptide as described in the aforementioned embodiment in the preparation of antibacterial, antifungal or antiinsect drugs.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a plant defensin polypeptide, the amino acid sequence of which is shown in at least one of SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5. The polypeptide has significant antibacterial and antifungal activity, can effectively inhibit the growth of various plant pathogens at low concentrations, and reduce the occurrence of diseases. It has high stability in vivo, low sensitivity to proteases, can maintain activity for a long time in complex biological environments, and significantly improves effectiveness and safety. After being introduced into crops through genetic engineering, it can enhance plant disease resistance and reduce the use of chemical pesticides. In addition, the polypeptide has broad application prospects in the fields of agriculture, medical care, and food preservation. It can be used as a new type of biological pesticide, antibiotic substitute, and natural preservative, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 The antibacterial effect of defensin ABC at different time periods in the examples of this application;

[0027] Figure 2 This is the charge distribution diagram of the defensin in Example 1 of this application. DETAILED DESCRIPTION

[0028] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all commercially available conventional products.

[0029] The present invention provides a plant defensin polypeptide, wherein the amino acid sequence of the plant defensin polypeptide is shown in at least one of SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5.

[0030] Plant defensins are a class of cationic peptides with a molecular weight of approximately 5 kDa. They are a major component of the plant immune system and participate in a variety of plant physiological and biochemical activities. They are typically composed of 45-54 amino acids and contain eight conserved cysteine ​​residues, forming four pairs of intrachain disulfide bonds that stabilize three antiparallel β-sheets and one α-helix, forming the so-called Csαβ motif structure. Plant defensins exhibit broad-spectrum antimicrobial activity at very low concentrations, inhibiting the growth of various plant pathogens, fungi, and some viruses. Plant defensins also have the ability to inhibit enzyme activity, inhibit cancer cell proliferation, and act as ion channel blockers.

[0031] The plant defensin polypeptides provided in the application examples are three different new plant defensin polypeptides obtained through design, screening, and activity verification. They can be any one of them or a combination of multiple ones. Specifically, in this example, they can be named defensin A, defensin B, and defensin C, respectively. Specific examples are shown in Table 1:

[0032] Table 1. Names and sequences of plant defensin polypeptides

[0033]

[0034] In the table, No. represents the number of the amino acid sequence, i.e., SEQ ID No. 1 corresponds to defensin A, SEQ ID No. 3 corresponds to defensin B, and SEQ ID No. 5 corresponds to defensin C.

[0035] In a second aspect, the present invention provides a nucleotide, the sequence of which is shown as at least one of SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO. 6.

[0036] In an optional embodiment, the nucleotide sequence of SEQ ID NO.2 encodes the plant defensin polypeptide corresponding to the amino acid sequence shown in SEQ ID NO.1;

[0037] The nucleotide sequence of SEQ ID NO.4 encodes the plant defensin polypeptide corresponding to the amino acid sequence of SEQ ID NO.3;

[0038] The nucleotide sequence of SEQ ID NO.6 encodes the plant defensin polypeptide corresponding to the amino acid sequence shown in SEQ ID NO.5.

[0039] Specifically, in this embodiment, corresponding to the aforementioned plant defensin polypeptides, the encodings are 709, 376 and 478. The details are shown in Table 1:

[0040] Table 2. Names and sequences of nucleotides

[0041]

[0042] In the table, No. represents the number of the amino acid sequence, i.e., SEQ ID No. 2 corresponds to defensin A, SEQ ID No. 4 corresponds to defensin B, and SEQ ID No. 6 corresponds to defensin C.

[0043] This coding relationship ensures the specific function of the nucleotide sequence in practical applications, namely the ability to express plant defensin polypeptides with specific antibacterial, antifungal, or anti-insect activities. This is of great significance for the development of genetically engineered products based on these nucleotide sequences (such as recombinant vectors and transgenic plants).

[0044] In the field of biotechnology, nucleotide sequences (such as DNA or mRNA) are the basis for encoding proteins (such as plant defensin polypeptides). Through genetic engineering techniques, specific nucleotide sequences can be introduced into host cells to express the target protein. This example clarifies the coding relationship between specific nucleotide sequences and plant defensin polypeptides, providing a basis for the application and protection of related technologies.

[0045] The present application provides a construct in the examples, comprising the nucleotides described in the above embodiments.

[0046] The construct provided in this example may comprise the aforementioned nucleotides. The construct in this example can be used to effectively transform recombinant cells, and the recombinant cells obtained by culturing the transformed cells can quickly and efficiently produce the aforementioned plant defensin polypeptides.

[0047] The present application provides a recombinant vector in an embodiment, comprising the nucleotides described in the above embodiment.

[0048] As mentioned above, a recombinant vector is a genetically engineered DNA molecule used to introduce exogenous genes (e.g., genes encoding plant defensin polypeptides) into host cells for expression or research. In this example, the recombinant vector contains the nucleotide sequences mentioned in the previous embodiments, which encode plant defensin polypeptides with specific antibacterial, antifungal, or antiinsect activity.

[0049] Functionally, recombinant vectors can ensure that the genes of plant defensin polypeptides are efficiently expressed in host cells, thereby producing defensin polypeptides with biological activity; recombinant vectors can introduce target genes into various host cells (such as bacteria, yeast, mammalian cells, etc.) for gene function research or protein production; in addition, in some cases, recombinant vectors can also be used for gene editing, such as the CRISPR-Cas9 system, to achieve precise modification of the host cell genome.

[0050] The present application provides a recombinant cell in an embodiment, comprising the nucleotides described in the aforementioned embodiment.

[0051] Recombinant cells are cells that have been genetically modified. These cells have acquired new genetic properties and functions by introducing a recombinant vector or directly introducing an exogenous gene. In this example, the recombinant cells contain the nucleotide sequences mentioned in the previous embodiments, which encode plant defensin polypeptides with specific antibacterial, antifungal, or anti-insect activities.

[0052] As mentioned above, recombinant cells can efficiently express plant defensin peptides, which can be used to produce biologically active defensin peptides. This can be used to study the biological functions of plant defensin peptides, such as their antibacterial, antifungal, and insect resistance mechanisms. Recombinant cells can also be used to produce plant defensin peptides for the development of new biopesticides, reducing the use of chemical pesticides.

[0053] The present application provides a composition in an embodiment, comprising the plant defensin polypeptide as described in the above embodiments.

[0054] As mentioned above, the composition may include plant defensin polypeptides and other ingredients according to different functions.

[0055] For example, in a composition for agricultural use, it may include but is not limited to agriculturally acceptable carriers, such as water, organic solvents, soil conditioners, etc.; adjuvants, such as surfactants, stabilizers, preservatives, etc., for improving the stability and dispersibility of the composition; and other active ingredients, such as other antibacterial agents, pesticides, plant growth regulators, etc., for enhancing the control effect of the composition.

[0056] For example, a composition for medical use, for treating bacterial, fungal, or viral infections, may include a plant defensin polypeptide, a pharmaceutically acceptable carrier, such as physiological saline, buffer, or water for injection; excipients, such as stabilizers, preservatives, and excipients, to improve the stability and bioavailability of the composition; and other active ingredients, such as antibiotics, antifungal drugs, and immunomodulators, to enhance the therapeutic effect of the composition.

[0057] For another example, a composition for food preservation, used to extend the shelf life of food and prevent spoilage, may include plant defensin polypeptides; food-grade carriers such as water, cooking oil, and emulsifiers; excipients such as antioxidants, preservatives, and thickeners to enhance the stability and preservation of the composition; and other active ingredients such as natural preservatives (e.g., citric acid, sorbic acid, etc.) and antimicrobial agents to enhance the composition's preservation effect.

[0058] The present application provides a functional product in the examples, including the plant defensin polypeptide according to the aforementioned embodiment.

[0059] In some embodiments, the functional product includes at least one of a drug, a detection reagent, and a detection kit.

[0060] The above-mentioned medicine refers to a substance used to prevent, treat or diagnose a disease. In this embodiment, the medicine may include at least one of an antibacterial drug, an antifungal drug and an anti-insect drug.

[0061] As mentioned above, the detection reagent refers to a reagent used to detect a specific biomarker or pathogen. In this embodiment, the detection reagent can be used to detect a biomarker or pathogen related to a plant defensin polypeptide.

[0062] As mentioned above, the detection kit refers to a set of detection reagents and auxiliary materials for rapid and convenient detection. In this embodiment, the detection kit can be used to detect biomarkers or pathogens related to plant defensin polypeptides.

[0063] Functional products can include at least one of drugs, test reagents, and test kits. These products have a wide range of applications in healthcare, agriculture, and environmental monitoring. Drugs treat bacterial, fungal, and insect pest infections; test reagents detect pathogens or biomarkers; and test kits provide a rapid and convenient testing solution. These functional products can effectively improve the efficiency of disease diagnosis and treatment while reducing reliance on traditional chemical drugs.

[0064] In some embodiments, the drug comprises at least one of an antibacterial drug, an antifungal drug, and an antiinsect drug.

[0065] In an embodiment of the present application, a transgenic crop is provided, wherein the transgenic crop comprises the plant defensin polypeptide as described in the aforementioned embodiment.

[0066] As mentioned above, genetically modified crops are crops in which exogenous genes (such as insect-resistant, disease-resistant, and stress-resistant genes) are introduced into the plant genome through genetic engineering techniques and stably expressed within the plant, thereby imparting new desirable traits (such as insect resistance, disease resistance, stress tolerance, high yield, and high quality). Genetic modification technology breaks down the natural barriers to hybridization between species, expands the range of accessible genes, and enables plant traits to be improved that are difficult to achieve with traditional breeding techniques.

[0067] Genetically modified crops are created by introducing foreign genes into the plant genome through genetic engineering techniques, imparting new and desirable traits. These crops offer significant advantages in insect resistance, disease resistance, stress tolerance, improved quality, and high yields, and are widely used in agriculture, medicine, and environmental protection. Genetic modification not only increases crop yield and quality but also reduces the use of pesticides and fertilizers, thus having significant implications for environmental protection and sustainable development.

[0068] The present application provides an example of a use of the plant defensin polypeptide described in the above embodiment in the preparation of antibacterial, antifungal or antiinsect drugs.

[0069] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0070] Example 1: Sequence design, generation and optimization

[0071] In this example, the design, generation, and screening of target sequences were performed.

[0072] Experimental methods:

[0073] In this experiment, the sequence design of defensin (PDB: 1BK8) was performed.

[0074] The overall process includes:

[0075] (1) obtaining a protein sequence to be analyzed and constructing a sequence data set including the protein sequence;

[0076] (2) constructing a multi-attribute condition vector of different functional attributes based on the protein sequence to be analyzed in the sequence data set; wherein the functional attribute includes at least one of solubility, enzyme activity, and thermal stability;

[0077] (3) constructing a protein sequence generation model based on a deep learning model with an attention mechanism, and obtaining the trained protein sequence generation model through training;

[0078] (4) encoding the protein sequence to be analyzed in the sequence data set into a discrete representation and inputting the discrete representation into the protein sequence generation model;

[0079] (5) Use the multi-attribute conditional vector to guide the generation process and generate the target protein sequence based on the target attribute.

[0080] Based on the mechanism of defensins themselves, there are four pairs of key disulfide bonds that stabilize the structure: CYS2-CYS50, CYS12-CYS35, CYS20-CYS44, and CYS24-CYS46. Among them, the structure of plant defensins is composed of three antiparallel β-pleated sheets (Lys2→Arg6; His33→Tyr38; His43→Pro50) and an α-helix (Asn18→Leu28). With the help of four pairs of disulfide bonds, they form a very stable β-α-β-β structure.

[0081] Among the tested samples, CYS2-CYS50 was the most easily destroyed structure, and sequence screening was performed based on the hydrophobic mechanism of plant defensin R32 and R40 residues.

[0082] Scoring systems were constructed based on structural similarity and sequence homology, and the top 50 sequences with the highest secondary structure similarity among 1,000 sequences were screened to construct a phylogenetic tree. The phylogenetic tree included plant defensins from the same family (PDB: 1BK8, 2N2Q, 1AYJ, 2M8B, 2N2R). The top 20 sequences were screened based on their characteristic sites, among which the secondary structure similarity was 82-92%, the sequence similarity was 60-70%, and the soluble expression met the requirements for Escherichia coli expression (score>0.5).

[0083] Based on the defensin mechanism, the intermolecular interaction force of four key disulfide bonds was screened and the charge distribution was predicted. The sequences defensin A, defensin B, and defensin C were ranked as the top three based on structural similarity and soluble expression. Figure 2 In the charge distribution, the red part is positive charge and the blue part is positive charge. The more positive charge distribution, the more conducive it is for defensins to destroy the fungal cell membrane.

[0084] The novel plant defensin protein sequence is derived from the European horse chestnut Aesculus hippocastanum, and its protein sequence is shown in SEQ ID NOs: 1, 3, and 5, and its nucleic acid sequence is shown in SEQ ID NOs: 2, 4, and 6, as shown in Tables 1 and 2.

[0085] Example 2: Activity Test

[0086] In this example, the novel plant defensins obtained in Example 1, namely, defensin A, defensin B, and defensin C, were subjected to gene cloning, expression, purification, and antibacterial activity testing.

[0087] Experimental methods:

[0088] The novel defensin sequence was directly cloned between the NcoI and XhoI restriction sites of pET-28a(+) to construct a double-copy system. The flexible linker peptide was used to obtain the recombinant plasmid pET-28a-defensin-6×his tag (pET-28a plasmid was obtained from General Motors). Heat shock was used to transform the recombinant plasmid into Escherichia coli BL21(DE3) competent cells, and positive clones were screened.

[0089] The bacteria were inoculated into 50 mL TB medium and cultured at 37°C overnight. 600 When the cell volume grows to 0.6-0.8 mL, add isopropyl β-d-thiogalactoside at a final concentration of 0.1 mM and induce at 20°C for 24 hours. The resulting cells are suspended in a potassium phosphate buffer solution at pH 7, placed on ice, and disrupted using an ultrasonic disruptor for 10 minutes. Subsequently, centrifuge at 12,000 rpm for 10 minutes under low-temperature conditions to obtain the defensin supernatant crude enzyme solution. Defensins A, B, and C are added to this area, and other crude enzyme solutions are used as blank controls and applied to a culture medium containing Aspergillus niger for an antibacterial ring reaction at 28 degrees Celsius for 24 hours. The plate inhibition zone effect is observed.

[0090] Experimental results:

[0091] refer to Figure 1 It can be seen that the bar graph shows the antibacterial effect of the new defensin on Aspergillus niger at different time points (0 hours, 12 hours and 24 hours).

[0092] The three different filling patterns in the figure represent three different defensins (defensins A, B and C), and the ordinate represents the diameter of the inhibition zone (unit: mm).

[0093] (1) Defensin A showed consistent antibacterial effects at all time points (0, 12, and 24 hours), with the diameter of the inhibition zone being approximately 10 mm. This indicates that defensin A has stable and effective antibacterial activity, and its effect is not affected by time.

[0094] (2) Defensin B also showed stable antibacterial activity at 0, 12, and 24 hours, with the diameter of the inhibition zone being approximately 12-13 mm. This suggests that defensin B not only has a good antibacterial effect but also can continue to work within the experimental observation time frame.

[0095] (3) The antibacterial effect of defensin C at 0 and 12 hours was similar to that of A and B, and the diameter was also approximately 10 mm. However, at 24 hours, its antibacterial effect decreased, and the diameter of the inhibition zone decreased to approximately 8 mm. This may indicate that the antibacterial effect of defensin C weakened over time, or that its stability decreased after 24 hours.

[0096] Both defensins A and B showed good stability and sustained antibacterial effects, while the antibacterial effect of defensin C decreased after 24 hours.

[0097] Among the three defensins tested, defensin B had a relatively strong antibacterial effect, followed by A. The effect of defensin C weakened after 24 hours.

[0098] Defensins A and B, due to their stable antimicrobial effects, may be more suitable for the development of long-lasting antimicrobial products. Although defensin C showed promising initial results, its stability requires further study and improvement. These results are important for the development of novel plant defensin-based antimicrobial agents, particularly in the search for natural alternatives to traditional antibiotics and pesticides.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A plant defensin polypeptide, characterized in that The amino acid sequence of the plant defensin polypeptide is shown as at least one of SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.

5.

2. A nucleotide, characterized in that The nucleotide sequence is shown as at least one of SEQ ID NO. 2, SEQ ID NO. 4 and SEQ ID NO.

6.

3. The nucleotide according to claim 2, wherein The nucleotide sequence of SEQ ID NO.2 encodes the plant defensin polypeptide corresponding to the amino acid sequence of SEQ ID NO.1 in claim 1; The nucleotide sequence of SEQ ID NO.4 encodes the plant defensin polypeptide corresponding to the amino acid sequence of SEQ ID NO.3 in claim 1; The nucleotide sequence of SEQ ID NO.6 encodes the plant defensin polypeptide corresponding to the amino acid sequence of SEQ ID NO.5 in claim 1.

4. A construct, characterized in that Comprising the nucleotide according to claim 2 or 3.

5. A recombinant vector, characterized in that Comprising the nucleotide according to claim 2 or 3.

6. A recombinant cell, characterized in that Comprising the nucleotide according to claim 2 or 3.

7. A composition, characterized in that The invention comprises the plant defensin polypeptide according to claim 1.

8. A functional product, characterized in that: comprising the plant defensin polypeptide according to claim 1; Preferably, the functional product comprises at least one of a drug, a detection reagent, and a detection kit; Preferably, the drug includes at least one of an antibacterial drug, an antifungal drug and an antiinsect drug.

9. A genetically modified crop, characterized in that: The transgenic crop comprises the plant defensin polypeptide as claimed in claim 1.

10. Use of the plant defensin polypeptide according to claim 1 in the preparation of antibacterial, antifungal or antiinsect drugs.

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