Protein polypeptide drug screening method and application thereof

Abstract

The invention provides a screening method for screening a type of polypeptides which can be used for preventing and treating plant diseases and can be degraded by targeting proteins by combining polypeptide-protein molecular docking and ubiquitination detection from a genome of a microbiome. The polypeptide obtained by the screening method is a bifunctional peptide, can inhibit the activity of the plant PUB21 protein E3 ligase, can effectively reduce the number of pathogenic bacteria and relieve symptoms caused by plant diseases, and has an obvious treatment effect on the plant diseases.

Description

[0001] Cross - reference to related applications This application claims the priority of an international patent application with an application date of March 11, 2024 (application number PCT / CN2024 / 080958), and the entire content of this patent application is incorporated herein by reference. Technical field

[0002] The present invention belongs to the field of biotechnology, and particularly relates to a method for screening functional protein polypeptides as drugs, especially a method for screening polypeptides for target protein degradation, and particularly relates to a method for screening a class of polypeptides targeting protein degradation and its application in preventing and controlling plant diseases. Background art

[0003] Proteins are important components that make up all cell tissues of biological organisms. Protein homeostasis depends on a wide network regulated by molecular chaperones, protein degradation systems, endoplasmic reticulum stress, and their regulatory factors. It mainly includes three modules, namely protein synthesis, folding, and degradation. These three modules are closely connected to each other, and all components work together to maintain protein homeostasis. Proteins with normal activity usually have the correct high - level spatial conformation, and their biological activity is very important for normal living organisms. Intracellular protein degradation depends on two core mechanisms: the ubiquitin - proteasome system (UPS) and the autophagy - lysosome pathway. Among them, the UPS achieves the directional clearance of target proteins through specific covalent modification - the ubiquitin molecule composed of 76 amino acids forms a polyubiquitin chain to label the substrate under the cascade catalysis of E1 activating enzyme, E2 conjugating enzyme, and E3 ligase. The core regulation of this system stems from the spatio - temporal specific recognition function of E3 ligase, and its species diversity endows the advantage of precise substrate selection, making it an important target category for anti - cancer drug development.

[0004] In the field of new drug development, polypeptide molecules composed of 10 - 50 amino acids exhibit unique advantages: their low molecular weight, strong specificity, and controllable toxicity endow them with the potential to break through the limitations of traditional antibiotics in targeted therapy. Currently, polypeptide drugs have been widely used in many fields such as anti - infection, anti - tumor, metabolic diseases, cardiovascular diseases, immune regulation, nervous system diseases, vaccine development, and drug delivery.

[0005] The role of protein polypeptide drugs in infectious diseases is mainly reflected in the following aspects: 1. Antibacterial effect Antibacterial peptides: such as defensins, cathelicidins, etc., can damage the bacterial cell membrane, leading to bacterial death.

[0006] Lysozyme: directly kills bacteria by decomposing the peptidoglycan of the bacterial cell wall.

[0007] 2. Antiviral effect Interferon: Inhibits virus replication and enhances immune response.

[0008] Virus fusion inhibitors: Such as enfuvirtide, which prevents the virus from fusing with host cells and inhibits infection.

[0009] 3. Immunomodulation Cytokines: Such as IL-2 and IL-12, which regulate immune response and enhance the ability to resist infection.

[0010] Immunoglobulins: Neutralize pathogens and promote clearance.

[0011] 4. Antifungal effect Antifungal peptides: Disrupt the fungal cell membrane and inhibit its growth.

[0012] 5. Antiparasitic effect Antiparasitic peptides: Interfere with parasite metabolism or disrupt its cell structure.

[0013] 6. Vaccine development Polypeptide vaccines: Based on pathogen antigen epitopes, they induce specific immune responses.

[0014] 7. Drug delivery Targeted delivery: By modifying polypeptide drugs, precise delivery is achieved, improving efficacy and reducing side effects.

[0015] 8. Anti-inflammatory effect Anti-inflammatory peptides: Alleviate the inflammatory response caused by infection and avoid tissue damage.

[0016] The roles of protein and polypeptide drugs in chronic diseases are mainly reflected in the following aspects: 1. Antitumor Targeted therapy: Such as trastuzumab, which specifically binds to antigens on the surface of tumor cells and inhibits their growth.

[0017] Immunomodulation: Such as the cytokine IL-2, which activates the immune system and enhances the antitumor response.

[0018] 2. Metabolic diseases Diabetes treatment: Such as insulin and its analogs, which regulate blood glucose levels.

[0019] Obesity treatment: Such as GLP-1 receptor agonists, which control body weight by regulating appetite and metabolism.

[0020] 3. Cardiovascular diseases Antihypertensive drugs: Such as angiotensin-converting enzyme inhibitors (ACE inhibitors), which treat hypertension by regulating blood pressure.

[0021] Antithrombotic drugs: Such as bivalirudin, which prevents thrombus formation by inhibiting thrombin activity.

[0022] 4. Immunomodulation Cytokines: such as interferons, IL-2, regulate immune responses and are used in the treatment of viral infections and tumors.

[0023] Immunosuppressants: such as cyclosporine, are used to inhibit immune rejection after organ transplantation.

[0024] 5. Nervous System Diseases Analgesic drugs: such as ziconotide, relieve chronic pain by blocking calcium channels.

[0025] Neuroprotection: such as brain-derived neurotrophic factor (BDNF), promotes neuron survival and repair.

[0026] For a long time, traditional antibiotics have played an important role in the treatment of bacterial diseases. However, due to the irrational use of antibiotics, insufficient research on disease transmission between animals and humans, and the rapid emergence of new drug-resistant strains, the problem of antibiotic resistance has become increasingly serious. Research shows that many bacteria have developed resistance to existing antibiotics, leading to reduced or even ineffective treatment. This situation not only threatens human health but also has a negative impact on agricultural production and the ecosystem.

[0027] Against this backdrop, in recent years, strict restrictions on the use of antibiotics in agriculture have been implemented globally to prevent abuse and reduce environmental pollution. According to national agricultural regulations (such as the European Union's "Animal Pharmacopoeia," the United States' USDA regulations, etc.), certain antibiotics are prohibited or restricted in agricultural production. These policies aim to balance the needs of agricultural production with the goals of environmental protection.

[0028] Due to its precise molecular recognition ability (such as its ability to specifically bind to specific glycoproteins or lipid molecules on the surface of higher organisms), polypeptides can act more targetedly on pathogenic bacteria, reduce damage to non-target cells, and still maintain efficacy in the face of increasing antibiotic resistance. They have been widely used in medicine, especially in anti-tumor, agriculture, and aquaculture, providing an innovative path for the development of precise, efficient, and environmentally friendly alternative treatment strategies.

[0029] However, in some fields (such as the prevention and control of plant diseases and pests), how to utilize the mild pharmacological properties, low toxicity, and persistent antibacterial effects of polypeptides to play a unique role in the treatment of plant bacterial diseases has not been effectively solved so far. How to use polypeptides to replace traditional chemical drugs such as antibiotics to address the increasingly severe challenge of plant bacterial diseases remains an urgent problem for humanity. Summary of the Invention The technical problem to be solved by the present invention is how to use big data analysis to screen candidate polypeptides for effectively preventing and treating plant diseases through targeted protein degradation pathways. The technical problems to be solved are not limited to the described technical themes, and those skilled in the art can clearly understand other technical themes not mentioned herein through the following description.

[0031] To solve the above technical problems, the present invention first provides a class of polypeptides for targeted protein degradation.

[0032] The polypeptides for targeted protein degradation provided by the present invention are any one of the following a1)-a6): a1) A polypeptide whose amino acid sequence contains the amino acid residues at positions 15-24 in SEQ ID No.1; a2) A polypeptide containing the amino acid sequence shown in SEQ ID No.1; a3) A polypeptide containing the amino acid sequence shown in SEQ ID No.2; a4) A polypeptide containing the amino acid sequence shown in SEQ ID No.3; a5) A polypeptide containing the amino acid sequence shown in SEQ ID No.4; a6) A polypeptide containing a sequence having more than 80% identity with the amino acid sequence described in any one of a1)-a5) and having the same function as the polypeptide described in any one of a)-a5).

[0033] In the polypeptide described in a6) above, the identity refers to the identity of the amino acid sequence. The identity can be determined by using a homology search site on the Internet to determine the identity of the amino acid sequence, such as the BLAST web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and performing a search to calculate the identity of a pair of amino acid sequences, and then the value of identity (%) can be obtained. The amino acid sequence with the identity has 80% or higher, or 85% or higher, or 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity.

[0034] In a specific embodiment of the present invention, the polypeptide is any one of the following A1)-A4): A1) A polypeptide with the amino acid sequence of SEQ ID No.1; A2) A polypeptide with the amino acid sequence of SEQ ID No.2; A3) A polypeptide with the amino acid sequence of SEQ ID No.3; A4) A polypeptide with the amino acid sequence of SEQ ID No.4; Any of the above-mentioned functions can be an antibacterial function (such as against the pathogen C Las).

[0035] Any of the above-mentioned polypeptides can be obtained by artificial synthesis or by biological expression after synthesizing its coding gene.

[0036] Any of the above-mentioned polypeptides can inhibit the E3 ligase activity of citrus PUB21 protein.

[0037] To solve the above technical problems, the present invention also provides a method for screening polypeptides with antibacterial activity, including the following steps: Based on human gut microbiome and citrus soil microbiome data, use AI to predict APP polypeptides with antibacterial activity; use homology modeling to predict the structures of PUB21 and APP polypeptides, predict the modeled structure of the PUB21-APP polypeptide complex based on the docking algorithm, and process the structural data; randomly select several APP polypeptides predicted by AI calculation to interact with PUB21 for chemical synthesis of polypeptides with potential targeted protein degradation; for the polypeptides obtained in step c), detect the level of their inhibition of the E3 ligase activity of PUB21 protein; screen out the polypeptides with antibacterial activity. The above homology modeling can use known homology detection models in the art, such as Modeller, Phyre2 network prediction, etc., to construct a three-dimensional protein model.

[0038] The above docking algorithm can use known algorithms in the art, such as ZDock, RosettaDock, or Autodock vina algorithm, etc.

[0039] The present invention also provides a method for constructing a potential PUB21 binding peptide library, including the following steps: a) Based on gut microbiome and soil microbiome data, use AI calculation to predict and identify polypeptides with antibacterial activity therefrom; b) Integrate the antibacterial peptide sequences publicly available on the network and analyzed in the laboratory; c) Construct a potential PUB21 binding peptide database containing approximately 8 million sequences.

[0040] Furthermore, the present invention also provides a potential PUB21-binding peptide library constructed by the above method for constructing a potential PUB21-binding peptide database. Each binding peptide contains a domain linked to PUB21, and the domain can interact with PUB21 and inhibit the E3 ligase activity of PUB21 protein.

[0041] The present invention also provides a biomaterial related to the above polypeptide. The biomaterial related to the above polypeptide provided by the present invention is any one of the following C1) to C16): C1) A nucleic acid molecule encoding the above polypeptide; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1); C4) A recombinant vector containing the expression cassette described in C2); C5) A recombinant microorganism containing the nucleic acid molecule described in C1); C6) A recombinant microorganism containing the expression cassette described in C2); C7) A recombinant microorganism containing the recombinant vector described in C3); C8) A recombinant microorganism containing the recombinant vector described in C4); C9) A transgenic animal cell line containing the nucleic acid molecule described in C1); C10) A transgenic animal cell line containing the expression cassette described in C2); C11) A transgenic animal cell line containing the recombinant vector described in C3); C12) A transgenic animal cell line containing the recombinant vector described in C4); C13) A transgenic plant cell line containing the nucleic acid molecule described in C1); C14) A transgenic plant cell line containing the expression cassette described in C2); C15) A transgenic plant cell line containing the recombinant vector described in C3); C16) A transgenic plant cell line containing the recombinant vector described in C4).

[0042] The nucleic acid molecule described in the above C1) may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

[0043] In the nucleic acid molecule described in C2) above, the identity refers to the sequence similarity with the natural nucleic acid sequence. The identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. The identity of more than 80% above can be 80%, 85%, 90% or more than 95% identity. The identity of more than 80% can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The identity of more than 85% can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The identity of more than 90% can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The identity of more than 95% can be at least 95%, 96%, 97%, 98% or 99% identity.

[0044] In the biological material described above, the expression cassette can be DNA capable of expressing the above polypeptide in a host cell. This DNA can not only include a promoter that initiates the transcription of the polypeptide-encoding gene, but also include a terminator that terminates the transcription of the polypeptide-encoding gene. The expression cassette can also include an enhancer sequence.

[0045] In the biological material described above, the vector refers to a vector capable of transporting the above polypeptide-encoding gene into a host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), Ti plasmids, viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). The recombinant vector refers to a recombinant DNA molecule constructed by ligating the above polypeptide-encoding gene and the vector in vitro.

[0046] Among the above-mentioned biological materials, the microorganism can be a bacterium, a fungus, an actinomycete, a protozoan, an alga or a virus. Among them, the bacterium can be from the genus Escherichia (Escherichia sp.), the genus Erwinia (Erwinia sp.), the genus Agrobacterium (Agrobacterium sp.), the genus Flavobacterium (Flavobacterium sp.), the genus Alcaligenes (Alcaligenes sp.), the genus Pseudomonas (Pseudomonas sp.), the genus Bacillus (Bacillus sp.), etc., but not limited to this. For example, the bacterium can be Escherichia coli, Bacillus subtilis or Bacillus pumilus. The fungus can be yeast, and the yeast can be from the genus Saccharomyces (such as Saccharomyces cerevisiae), the genus Kluyveromyces (such as Kluyveromyces lactis), the genus Pichia (such as Pichia pastoris), the genus Schizosaccharomyces (such as Schizosaccharomyces pombe), the genus Hansenula (such as Hansenula polymorpha), etc., but not limited to this. The fungus can also be from the genus Fusarium (Fusarium sp.), the genus Rhizoctonia (Rhizoctonia sp.), the genus Verticillium (Verticillium sp.), the genus Penicillium (Penicillium sp.), the genus Aspergillus (Aspergillus sp.), the genus Cephalosporium (Cephalosporium sp.), etc., but not limited to this. The actinomycete can be from the genus Streptomyces (Streptomycessp.), the genus Nocardia (Nocardia sp.), the genus Micromonospora (Micromonospora sp.), the genus Streptosporangium (Streptosporangium sp.), the genus Actinoplanes (Actinoplanes sp.), the genus Thermoactinomyces (Thermoactinomyces sp.), etc., but not limited to this. The alga can be from the genus Fucus (Fucus sp.), the genus Achnanthes (Achnanthes sp.), the genus APPhiprora (APPhiprora sp.), the genus APPhora (APPhora sp.), the genus Ankistrodesmus (Ankistrodesmus sp.), the genus Asteromonas (Asteromonas sp.), the genus Boekelovia (Boekelovia sp.), etc., but not limited to this. The virus can be a rotavirus, a herpes virus, an influenza virus, an adenovirus, etc., but not limited to this.

[0047] The recombinant host cell (e.g., recombinant microorganism) refers to a host cell of interest (e.g., microorganism of interest) whose genes are manipulated and modified to obtain a recombinant host cell with changed functions (e.g., recombinant microorganism with changed functions). For example, a recombinant host cell (e.g., recombinant microorganism) obtained after introducing the above expression cassette or the above recombinant vector into a host cell of interest (e.g., microorganism of interest). The recombinant host cell (e.g., recombinant microorganism) can be understood to refer not only to a specific recombinant host cell (e.g., recombinant microorganism), but also to the progeny of such a cell, and due to natural, accidental or intentional mutations and / or alterations, the progeny need not be identical to the original parental cell, but is still included within the scope of the recombinant host cell (e.g., recombinant microorganism).

[0048] To solve the above technical problems, the present invention also provides a new use of the above polypeptide or biological material.

[0049] The present invention provides an application of the above polypeptide or biological material in endowing or enhancing the bacterial resistance of plants, plant parts or plant cells.

[0050] The present invention also provides an application of the above polypeptide or biological material in the preparation of a product for endowing or enhancing the bacterial resistance of plants, plant parts or plant cells.

[0051] The endowing or enhancing of the bacterial resistance of plants, plant parts or plant cells is: reducing the number of pathogenic bacteria in plants infected with bacterial diseases caused by pathogens and / or promoting the lysis of pathogenic bacteria cells in plants infected with the bacterial diseases.

[0052] Wherein the pathogenic bacteria include non-culturable or difficult-to-culture pathogenic bacteria.

[0053] Wherein the pathogenic bacteria are selected from the group consisting of Liberibacter, Phytoplasma, Spiroplasma, and Candidatus Phytoplasma asteris, etc.

[0054] To solve the above technical problems, the present invention also provides a method for endowing or enhancing the bacterial resistance of plants, plant parts or plant cells, wherein the method comprises the steps of inhibiting the E3 ligase activity of PUB21 protein in the plants, plant parts or plant cells and / or endowing, stabilizing or enhancing the level of MYC2.

[0055] The method for endowing or enhancing the bacterial resistance of plants, plant parts or plant cells provided by the present invention comprises the step of applying the above polypeptide or the above biological material to the plants.

[0056] A method for producing plants, plant parts or plant cells with increased bacterial resistance provided by the present invention comprises the following steps: a) Introduce the polypeptide or biological material into a plant, a plant part or a plant cell; b) Generate a genetically modified plant, a genetically modified plant part or a genetically modified plant cell from the plant, plant part or plant cell obtained in a); and c) The genetically modified plant cell, the genetically modified plant part or the genetically modified plant has a reduced or attenuated expression of PUB21 protein and a stable or enhanced level of MYC2; wherein MYC2 induces the expression of PUB21 protein through promoter interaction.

[0057] In the method for conferring or enhancing bacterial resistance of a plant, a plant part or a plant cell, the polypeptide is applied to the plant through an aqueous polypeptide solution.

[0058] The present invention also provides a product, the active ingredient of which is the polypeptide or the biological material.

[0059] The concentration of the aqueous polypeptide solution can be 0.1 - 1 μM.

[0060] In a specific embodiment of the present invention, the concentration of the aqueous polypeptide solution is 0.1 μM.

[0061] In another specific embodiment of the present invention, the concentration of the aqueous polypeptide solution is 1 μM.

[0062] In the method for conferring or enhancing bacterial resistance of a plant, a plant part or a plant cell, the application method can be injection.

[0063] In a specific embodiment of the present invention, the injection method is vacuum injection.

[0064] In another specific embodiment of the present invention, the injection method is trunk infusion.

[0065] To solve the above technical problems, the present invention finally provides a composition for conferring or enhancing bacterial resistance of a plant, a plant part or a plant cell.

[0066] The active ingredient of the composition for conferring or enhancing bacterial resistance of a plant, a plant part or a plant cell provided by the present invention is the polypeptide or the biological material.

[0067] In the composition for controlling citrus huanglongbing, the composition further includes excipients, such as water.

[0068] In a specific embodiment of the present invention, the composition is an aqueous polypeptide solution. The concentration of the aqueous polypeptide solution can be 0.1 - 1 μM.

[0069] In a preferred embodiment of the present invention, the concentration of the polypeptide aqueous solution is 0.1 μM.

[0070] In another preferred embodiment of the present invention, the concentration of the polypeptide aqueous solution is 1 μM.

[0071] In any of the above-mentioned applications, methods or products, the imparting or enhancing of bacterial resistance in plants, plant parts or plant cells is manifested as reducing the number of pathogenic bacteria in plants infected with huanglongbing and / or promoting the lysis of pathogenic bacteria cells in plants infected with huanglongbing.

[0072] In a specific embodiment of the present invention, the pathogenic bacterium is Candidatus Liberibacter asiaticus (CLas), the pathogen of huanglongbing.

[0073] In any of the above-mentioned applications, methods or products, the plant is any one of the following P1)-P5): P1) Monocotyledonous plants or dicotyledonous plants; P2) Plants of the order Rutales; P3) Plants of the family Rutaceae; P4) Plants of the genus Citrus; P5) Citrus (such as red orange).

[0074] The present invention also provides a plant, a plant part or a plant cell in which the E3 ligase activity of the PUB21 protein is inhibited and the level of MYC2 is imparted, stabilized or enhanced, and the plant, the plant part or the plant cell contains the polypeptide or the biomaterial.

[0075] The plant is any one of the following P1)-P5): P1) Monocotyledonous plants or dicotyledonous plants; P2) Plants of the order Rutales; P3) Plants of the family Rutaceae; P4) Plants of the genus Citrus; P5) Citrus (such as red orange).

[0076] Combined with bioinformatics methods, the present invention has developed a screening method for obtaining a class of polypeptides targeting protein degradation that can be used to control plant diseases by screening from the genomes of microbiomes and combining polypeptide-protein molecular docking and ubiquitination detection. Combining with experimental results shows that the polypeptides APP3, APP9, and APP17 can inhibit the E3 ligase activity of citrus PUB21 protein, can effectively reduce the number of pathogenic bacteria, and relieve the symptoms caused by plant diseases. They are a class of bifunctional peptides with obvious therapeutic effects on plant diseases and have very good potential application value for controlling plant diseases (such as citrus huanglongbing). BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1To screen for polypeptides that inhibit the E3 ligase activity of the Huanglongbing susceptibility protein PUB21. Figure 1 A shows a schematic diagram of the drug screening process for identifying polypeptides that inhibit the E3 ligase activity of the Huanglongbing susceptibility protein PUB21. Figure 1 B shows that the bifunctional peptides APP3, APP9, and APP17 can reduce the autoubiquitination level of PUB21 and the ubiquitination level of the substrate protein MYC2 when the Huanglongbing effector SDE5 and the citrus protein PUB21 coexist.

[0078] Figure 2 The bifunctional peptides APP3, APP9, APP17, and the APP3 truncation APP3-14 that were screened can reduce the C Las titer in infected citrus leaves. Figure 2 A shows the detection of the content of Las (relative content in 50 ng of plant DNA) by quantitative PCR after treating citrus leaves infected with Huanglongbing with the bifunctional peptides APP1 - APP12 (1 μM) obtained through the screening process for 48 hours. C The content of Las (relative content in 50 ng of plant DNA). Treatment with BSA was used as a negative control. Figure 2 B shows the detection of the content of Las (relative content in 50 ng of plant DNA) by quantitative PCR after treating citrus leaves infected with Huanglongbing with 1 μM APP1, APP3, APP9, APP17, APP20, and the APP3 truncations (APP3-α and APP3-14), and 100 nM APP3-14 for 48 hours. C The content of Las (relative content in 50 ng of plant DNA). Treatments with 1 μM and 10 μM tetracycline (Tet) were used as positive controls, and treatment with BSA was used as a negative control. Values are mean ± SEM (n = 6). According to one-way ANOVA and Duncan's multiple range test, lowercase letters indicate significant differences between different columns (P < 0.05).

[0079] The amino acid sequences of each bifunctional peptide and truncation are as follows: APP3: MKKVKNIFHKIANADPMIWGYVMLSESK (SEQ ID No.1) APP9: MAVISGSAFGPGGEGFIRCCYATSMKDIAEALTRMDNFLTNLRKKQAREG (SEQ ID No.2) APP17: MKKQIISTKNAPSAVGPYVQGIKAGNTVLCIGPAWH (SEQ ID No.3) APP3-14: DPMIWGYVMLSESK (SEQ ID No.4) APP3-α: MKKVKNIFHKIVNA (SEQ ID No.5) Figure 3 For field application of the bifunctional peptides APP3-14, APP9, and APP17 to treat diseased citrus plants. Figure 3 A shows an aerial view of a diseased citrus orchard before treatment with 1 μM APP3-14, APP9, and APP17 in the field. BSA treatment was used as a negative control group, and 1 μM tetracycline (Tet) treatment was used as a positive control group. Figure 3 B shows an overall comparison of citrus plants 6 months after treatment with 1 μM APP3-14, APP9, and APP17 for diseased citrus. BSA treatment was used as a negative control group, and 1 μM tetracycline (Tet) treatment was used as a positive control group.

[0080] Embodiments of the present invention The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0081] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0082] The citrus variety used in the following examples is Guangdong Lianjiang Red Orange ( Citrus sinensis L.).

[0083] The vectors pACYCDuet-CDS-Myc-AtUBC8-S, pGEX-DC, pCDFDuet-AtUBA1-S, and pET-28a-FLAG-UBQ involved in the Escherichia coli ubiquitination detection system in the following examples are all described in the literature "Yufang Han, Jianhang Sun, Jun Yang, Zhaoyun Tan, Jijing Luo, Dongping Lu. Reconstitution of the plant ubiquitination cascade in bacteria using a synthetic biology approach. The Plant Journal 91, 766 - 776 (2017).".

[0084] The main reagents and their sources in the following examples are as follows: SYBR qPCR Mix is a product of TOYOBO; Cocktail protease inhibitor is a product of Roche; IPTG is a product of Inolco; 40% Acrylamide is a product of Sigma; the antibodies of the primary antibody and the secondary antibody are both products of Beijing TransGen Biotech Co., Ltd.; the prestained protein molecular weight marker is a product of Bioeasy Gene Technology Co., Ltd.; the ECL luminescent solution is a product of GE healthcare; the MYC2 antibody is a product of Wuhan ABclonal Technology Co., Ltd.; the competent cells DH5α and BL21 are products of Tsingke Biotechnology Co., Ltd.; the Ni-NTA magnetic beads are products of Bioeasy Gene Technology Co., Ltd.; the PD-10 column is a product of cytiva.

[0085] The primers used in the following examples were synthesized by Tsingke Biotechnology Co., Ltd. and relevant sequencing work was carried out.

[0086] The polypeptides used in the following examples were synthesized by GenScript Biotech Corporation.

[0087] The amino acid sequence of the PUB21 protein (the PUB21 protein is a citrus Huanglongbing susceptible protein) in the following examples is shown in SEQ ID No.6; the amino acid sequence of the SDE5 protein (the SDE5 protein is a secreted protein of the Huanglongbing pathogen C Las) is shown in SEQ ID No.7.

[0088] Example 1. Screening of polypeptides targeting protein degradation I. Inhibition of the E3 ligase activity of PUB21 by APP3, APP9 and APP17 polypeptides 1. Vector construction First, construct vectors for in vitro ubiquitination detection in Escherichia coli: Clone the full length of PUB21 into the pACYCDuet-CDS-Myc-AtUBC8-S vector by double digestion and ligation method to obtain the pACYCDuet-PUB21-Myc-AtUBC8-S vector; Clone the full length of SDE5 into the pGEX-DC vector by double digestion and ligation method to obtain the pGEX-SDE5 vector. The primers for vector construction are as follows: PUB21-Fw(BamHI): CAAGGGATCCATGATTTTGTCATGGAAAAGAC (SEQ ID No.8); PUB21-Rv(StuI): CAAGAGGCCTAAACGGCCTTTTCAGGTCCT (SEQ ID No.9); SDE5-Fw(KpnI): CAAGGGTACCATGCTTAATTGCAACGAAAC (SEQ ID No.10); SDE5-Rv(XhoI): CAAGCTCGAGCAATTATTTATAAATGGGCA (SEQ ID No.11).

[0089] 2. Construction of an E. coli ubiquitination detection system (1) Co-transform the pACYCDuet-PUB21-Myc-AtUBC8-S vector, pGEX-SDE5 vector, pCDFDuet-AtUBA1-S vector, and pET-28a-FLAG-UBQ vector into BL21(DE3) competent cells. Among them, PUB21 serves as the E3 ubiquitin ligase, AtUBC8 serves as the E2 ubiquitin-conjugating enzyme, AtUBA1 serves as the E1 ubiquitin-activating enzyme, and UBQ is the small ubiquitin protein Ubiqutin.

[0090] (2) The strain containing the expression vector is cultured at 37°C until the OD 600nm reaches 0.4 - 0.6. After adding 500 nM isopropyl β-D-thiogalactoside (IPTG), protein induction expression is carried out at 28°C for 10 hours, and then the reaction is carried out overnight at 4°C.

[0091] (3) The crude protein is separated by an 8% SDS-PAGE gel, and the anti-c-myc tag antibody is used for immunoblot analysis of the self-ubiquitination level of PUB21.

[0092] 3. Screening of polypeptides by targeting and inhibiting the E3 ligase activity of PUB21 (1) Small molecule inhibitors targeting ubiquitination are effective in the treatment of certain diseases. Since the E3 ligase activity of PUB21 is crucial for the susceptibility of Huanglongbing HLB, small molecule inhibitors are identified by targeting and inhibiting the E3 ligase activity of PUB21 to treat Huanglongbing HLB disease.

[0093] (2) In the present invention, AI is empowered for computational prediction to identify novel polypeptides with antibacterial activity from gut microbiome and soil microbiome data, and the antibacterial peptide sequences publicly available on the network and laboratory analysis are integrated to construct a database containing approximately 8 million sequences, and potential bifunctional peptides are screened from this library ( Figure 1 A).

[0094] (3)Protein structure prediction and polypeptide-PUB21 protein complex structure modeling: To further understand the polypeptides that can effectively inhibit the E3 ligase activity of PUB21, the structures of PUB21 and APP polypeptides were predicted using Modeller or Phyre2 networks known in the art. Based on docking algorithms such as ZDOCK, RosettaDock, or Autodock vina, the modeled structure of the PUB21-APP polypeptide complex was predicted, and the structural data was processed using the program PyMOL software.

[0095] (4)Twenty-two polypeptides that can interact with PUB21 and potentially target protein degradation were randomly selected for chemical synthesis (APP1-APP22 are derived from the human gut microbiome) to detect whether they can inhibit the ubiquitination level of PUB21. The polypeptides and their amino acid sequences are as follows: APP1: MTKIDRANFGSKLGVILASAGSAVGLGKHLEISL (SEQ ID No.12) APP2: MSEIESKVKAIIVDKLGVDEAEVVGPSKFHKRFRC (SEQ ID No.13) APP3: MKKVKNIFHKIANADPMIWGYVMLSESK (SEQ ID No.1) APP4: MTVKKGDKVITGKFAGQELKLDGEDYVICKLADILAIVD (SEQ ID No.14) APP5: MKNFKKIISLVLAVCLMASRAIAYRTGERASVPLKF (SEQ ID No.15) APP6: MISTDLKECYEFLGEIIGKTYKDDIIDRLFQDFCVGK (SEQ ID No.16) APP7: MIGEISGMLNDIKPVKTIIEDIVSGLPGVIQTIENDCK (SEQ ID No.17) APP8: MANYVFGIDVGGTSVKCGLFQTDGTLLEKWEIPTRT (SEQ ID No.18) APP9: MAVISGSAFGPGGEGFIRCCYATSMKDIAEALTRMDNFLTNLRKKQAREG (SEQ ID No.2) APP10: MKEVIHTSNAPAAIGPYSQAIKAGGVPEEPFVLWKV (SEQ ID No.19) APP11: MEFSSIFETIKGFFEANVLPTLKPIIDAIMDLIGGIIGGK (SEQ ID No.20) APP12: MYSISIALGVNSIALGDNSKAYGDNSKGYGDRIDAYKKV (SEQ ID No.21) APP13: MHKYINEQIQEEDTAQDIIDKFTFAGESTSAKYSIDKELGGRIN (SEQ ID No.22) APP14: MMCITAKEPNMYDLMRKLPIGIQTFEDIRRKNYLYVDKTALV (SEQ ID No.23) APP15: MEEKAEYLGANAIIGLKISYDNLGGTMGNTILVTAYGTAIKYE (SEQ ID No.24) APP16: MEKRIIPISRELAEKEPCVIVGRCADFILAASPLIRTSR (SEQ ID No.25) APP17: MKKQIISTKNAPSAVGPYVQGIKAGNTVLCIGPAWH (SEQ ID No.3) APP18: MGKLDNKVAIITGGNAGVGKEIAKLFASEGAKVVISA (SEQ ID No.26) APP19: MNLMGMNVGPLRMPLCEMEEDTKAALAKEIEKFGLKLAK (SEQ ID No.27) APP20: MKIGLGFEGGIAVVILAIILDRITQGMAGRKNKKG (SEQ ID No.28) APP21: MTITVEPGIYIEGLGGVRIEDCCIMTEDGYINPVTSPKELIIIE (SEQ ID No.29) APP22: MLPKVQAAVSFAESKPGRTALITLLEKAKDGIAGKTGTAVH (SEQ ID No.30) (5) Using the one without adding E2 and polypeptide as a control, it was found that polypeptides APP3, APP9, and APP17 could significantly inhibit the autoubiquitination level of PUB21 and also reduce the ubiquitination and degradation ability of the substrate protein MYC2 in the presence of the effector SDE5 of Xanthomonas citri subsp. citri ( Figure 1 B).

[0096] Example 2: Use of symptomatic leaves in vitro for rapid evaluation of the treatment effect of Huanglongbing I. Establishment of a system for using symptomatic leaves in vitro for rapid evaluation of the treatment effect of Huanglongbing 1. Identification of citrus leaves infected with Huanglongbing Collect leaves of citrus (red orange) infected with Huanglongbing, collect midrib samples of the leaves, extract leaf DNA by the CTAB method, and then detect the C Las titer in the midrib of the leaves by fluorescence quantitative PCR. At the same time, use healthy citrus (red orange) leaves as a control and COX as an internal reference gene. The primer sequences are as follows: Pathogen of Huanglongbing C Specific detection primer HLBas for Las: GTCGAGCGCGTATGCAATACG (SEQ ID No. 31); Pathogen of Huanglongbing C Specific detection primer HLBr for Las: GCGTTATCCCGTAGAAAAAGGTAG (SEQ ID No. 32); Primer COXf for detecting citrus internal reference gene: GGTATGCCACGTCGCATTCCAGA (SEQ ID No. 33); Primer COXr for detecting citrus internal reference gene: GCCAAAACTGCTAAGGGCATTC (SEQ ID No. 34).

[0097] In symptomatic leaves C The detection result of Las concentration is as Figure 2 shown in B. In healthy leaves C the Las titer is 0, and the presence of Las cannot be detected C while in symptomatic citrus leaves C the Las titer reaches 16x10 3 or so, which is significantly different from that in healthy samples, indicating that the symptomatic leaves have a high C Las content.

[0098] 2. Evaluation of the treatment effect of candidate polypeptides on symptomatic leaves in vitro by vacuum injection method To test the treatment ability of candidate polypeptides against Huanglongbing HLB disease, symptomatic leaves in vitro were used for rapid evaluation of the treatment effect of Huanglongbing. The specific operation is as follows: First, the leaves identified as infected in step 1 CA few wounds were gently scratched on the back of citrus leaves infected with Las using a 1Ml syringe needle. At the same time, the main veins of the leaves were punctured with the needle. The treated leaves were immersed in a petri dish containing 30 mL of 1 μM polypeptide solution (with water as the solvent), placed in a vacuum pump instrument, and vacuum was applied to expel the gas inside the leaves. After keeping still for 3 minutes, the air was slowly released, allowing the solution to enter the mesophyll and leaf veins under the action of internal and external pressures. At the same time, a 1 μM BSA solution (with water as the solvent) was used as a control. The leaves after vacuum treatment were continuously immersed in the original solution. After being placed at room temperature for 48 h, the midrib samples of the leaves were collected, and the leaf DNA was extracted by the CTAB method. Subsequently, the C Las titer C The detection of Las is shown in step 1. By comparing C the Las titer to judge the treatment ability of the polypeptide against the HLB disease of Huanglongbing.

[0099] Example 3: Polypeptides APP3, APP9, and APP17 can reduce the pathogen of Huanglongbing C Las titer I. Polypeptides APP3, APP9, and APP17 can reduce C Las titer 1. The artificially synthesized polypeptides APP3, APP9, and APP17 can reduce C Las titer The treatment ability of the polypeptide obtained by detecting according to the method for rapidly evaluating the treatment effect of Huanglongbing using the detached diseased leaves in Example 2 above on C Las. After treatment with various different polypeptides C the Las titer detection results are as Figure 2 shown in A and B. 1 μM APP3, APP9, and APP17 can significantly reduce C the Las titer. Compared with the antibiotic tetracycline at the same concentration, the effects of these 1 μM APP polypeptides are better, equivalent to the application effect of 10 μM tetracycline. Combining the above, APP3, APP9, and APP17 can effectively inhibit the autoubiquitination level of PUB21 and also reduce the ubiquitination degradation ability of the substrate protein MYC2, indicating that APP3, APP9, and APP17 are a kind of bifunctional peptides.

[0100] 2. The APP3 truncation APP3-14 has a more significant effect on reducing C Las titer in diseased citrus leaves Structural modeling revealed that the APP3 polypeptide consists of an α-helix (APP3-α) and a β-sheet (APP3-14), and the interaction interface with PUB21 is located in the β-sheet structural region. To determine the therapeutic ability of APP3-α and APP3-14 against Huanglongbing HLB disease, the inhibitory effects of APP3-α and APP3-14 on C Las in detached leaves were also detected using the method described in Example 2 above. The results are shown in Figure 2 Figure B. Both 1 μM APP3-α and APP3-14 were able to significantly reduce C the Las titer, but APP3-14, which only contains a β-sheet, had a more significant effect on reducing C the Las titer, equivalent to the application effect of 10 μM tetracycline, and 100 nM of APP3-14 was able to significantly reduce C the Las titer in the infected leaf veins.

[0101] Example 4. Field application of bifunctional peptides to treat infected citrus plants I. Bifunctional peptides can effectively treat infected citrus plants To further detect the therapeutic effect of bifunctional peptides on citrus plants infected with Huanglongbing, in an outdoor citrus orchard, 1 μM APP3-14, APP9, APP17 bifunctional peptide solutions (solvent is water), 1 μM BSA solution (solvent is water), and 1 μM tetracycline were injected into citrus plants identified as infected with C Las in Example 1 by trunk infusion using a vacuum pressure bottle, as shown in Figure 3 Figure A. Six months later, the changes in the status of citrus trees were photographed and recorded. As shown in Figure 3 Figures A and B, before treatment, the overall leaves of the citrus orchard trees were sparse and there were many yellow shoots. After 6 months of BSA treatment, the infected citrus plants still had sparse leaves and obvious leaf yellowing. The symptoms of the infected citrus plants treated with tetracycline were alleviated to a certain extent and the fruit setting rate increased, but the overall growth of the whole plant was not good. However, the leaf yellowing symptoms of the infected plants treated with bifunctional peptides disappeared, the plants were lush, and the fruit setting rate and yield were significantly increased, indicating that bifunctional peptides can effectively treat infected citrus plants.

[0102] To more clearly quantify the therapeutic effect of bifunctional peptides on citrus plants infected with Huanglongbing, the same number of citrus plants were further selected, and the fruit setting rate and yield of diseased citrus plants after being treated with different drugs in the field experiment for 6 months were measured. The measurement results are shown in Table 1. The use of 1 μM APP3-14, APP9, and APP-17 can significantly increase the fruit setting rate and yield of citrus plants. The citrus yield increased by more than 22% compared to the plants treated with 10 μM antibiotics and increased several to more than ten times compared to the plants treated with 1 μM BSA solution. Specifically, after being treated with 1 μM APP-9 and APP-17 for 6 months respectively, the average fruit setting weight of diseased citrus plants reached 16.199 Kg and 18.835 Kg per plant respectively, exceeding the fruit setting weight of citrus plants treated with 10 μM tetracycline for 6 months by 22.69% and 42.66% respectively. The application effect of 1 μM APP3-14 was the most significant, and the fruit setting weight of diseased citrus plants exceeded the fruit setting weight of those treated with 10 μM tetracycline by 95.28%.

[0103] Table 1 Record table of fruit setting yield of diseased citrus plants

[0104] To date, there have been more than 300 bacterial diseases caused by unculturable or difficult-to-culture pathogens, including liberibacters, phytoplasmas, spiroplasmas, and Candidatus Liberibacter asiaticus. The screening and application strategies of the polypeptides targeting protein degradation developed in the present invention can help solve these diseases by targeting pathogen effectors or host immune protein stabilization.

[0105] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application. Some basic features can be applied according to the scope of the following appended claims.

[0106] Industrial applicability The present invention provides a method for screening polypeptides targeting protein degradation. Using this method and targeting the E3 ligase activity of the disease-susceptible gene PUB21 in the protein degradation pathway, a series of polypeptides were obtained, and the polypeptides screened by this method were verified by an in vitro diseased leaf rapid evaluation system for Huanglongbing treatment effect to be able to reduce the pathogen titer in citrus infected with Huanglongbing. After treating diseased citrus trees in the field, it can significantly relieve leaf yellowing, enhance tree growth, and improve the fruit setting rate.

Claims

1. A polypeptide targeting protein degradation, characterized in that: The polypeptide is selected from any one of the following a1) to a6): a1) a polypeptide whose amino acid sequence comprises amino acid residues 15 to 24 of SEQ ID No.1; a2) a polypeptide comprising the amino acid sequence shown in SEQ ID No.1; a3) a polypeptide comprising the amino acid sequence shown in SEQ ID No.2; a4) a polypeptide comprising the amino acid sequence shown in SEQ ID No.3; a5) a polypeptide comprising the amino acid sequence shown in SEQ ID No.4; a6) A polypeptide comprising a sequence having 80% or more identity with the amino acid sequence of any one of a1) to a5) and having the same function as the polypeptide of any one of a1) to a5).

2. The polypeptide according to claim 1, characterized in that: The polypeptide is obtained by artificial synthesis, or by synthesizing its encoding gene and then expressing it biologically.

3. A method for screening polypeptides having antibacterial activity, comprising the following steps: a) Using AI to predict peptides with antibacterial activity based on gut microbiome and soil microbiome data; b) predicting the structures of PUB21 and the above-mentioned peptides using homology modeling, predicting the modeled structure of the PUB21-peptide complex based on the docking algorithm, and processing the structural data; c) Randomly select several peptides that interact with PUB21 from the peptides predicted by AI, and chemically synthesize potential peptides that target protein degradation; d) detecting the level of the polypeptide obtained in step c) in inhibiting the activity of PUB21 protein E3 ligase; e) Screening out the polypeptide having antibacterial activity as claimed in any one of claims 1 to 2.

4. A biological material selected from any one of the following C1) to C16): C1) A nucleic acid molecule encoding the polypeptide according to claim 1 or 2; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1); C4) a recombinant vector containing the expression cassette described in C2); C5) a recombinant microorganism containing the nucleic acid molecule described in C1); C6) a recombinant microorganism containing the expression cassette described in C2); C7) a recombinant microorganism containing the recombinant vector described in C3); C8) a recombinant microorganism containing the recombinant vector described in C4); C9) a transgenic animal cell line containing the nucleic acid molecule described in C1); C10) a transgenic animal cell line containing the expression cassette described in C2); C11) A transgenic animal cell line containing the recombinant vector described in C3); C12) a transgenic animal cell line containing the recombinant vector described in C4); C13) A transgenic plant cell line containing the nucleic acid molecule described in C1); C14) a transgenic plant cell line containing the expression cassette described in C2); C15) A transgenic plant cell line containing the recombinant vector described in C3); C16) A transgenic plant cell line containing the recombinant vector described in C4).

5. Use of the polypeptide according to any one of claims 1 to 2 or the biomaterial according to claim 4 in conferring or enhancing bacterial resistance to plants, plant parts or plant cells; or, Use of the polypeptide according to any one of claims 1 to 2 or the biomaterial according to claim 4 in the preparation of a product that confers or enhances bacterial resistance in plants, plant parts or plant cells.

6. The use according to claim 5, characterized in that: The said imparting or enhancing bacterial resistance to plants, plant parts or plant cells is: reducing the number of pathogens in plants infected with bacterial diseases caused by pathogens and / or promoting the lysis of pathogenic bacteria cells in plants infected with said bacterial diseases; Preferably, the pathogenic bacteria include pathogenic bacteria that are unculturable or difficult to culture; More preferably, the pathogenic bacteria are selected from bacillus, phytoplasma, spirochetes and Trichoderma.

7. A method for conferring or enhancing bacterial resistance in a plant, plant part or plant cell, wherein the method comprises the step of inhibiting PUB21 protein E3 ligase activity and / or conferring, stabilizing or enhancing MYC2 levels in the plant, plant part or plant cell.

8. The method according to claim 7, comprising the step of applying the polypeptide according to any one of claims 1 to 2 or the biological material according to claim 4 to the plant.

9. A method for producing a plant, plant part or plant cell having increased bacterial resistance, comprising the steps of: a) introducing the polypeptide according to any one of claims 1 to 2, or the biological material according to claim 4, into a plant, a plant part or a plant cell, b) producing genetically modified plants, genetically modified plant parts or genetically modified plant cells from the plants, plant parts or plant cells obtained in a); and c) the genetically modified plant cell, genetically modified plant part or genetically modified plant having suppressed or attenuated PUB21 protein expression and stabilized or enhanced MYC2 level; The polypeptide is applied to the plant via an aqueous polypeptide solution.

10. A product, the active ingredient of which is the polypeptide according to any one of claims 1 to 2 or the biomaterial according to claim 4.

11. The product according to claim 10, characterized in that: The concentration of the polypeptide is 0.1 μM-1 μM.

12. The use according to claim 5 or 6, or the method according to any one of claims 7 to 9, characterized in that: The plant is a monocot or a dicot; Preferably, the dicotyledonous plant is a Rutaceae plant; More preferably, the Rutales plant is a Rutaceae plant; More preferably, the Rutaceae plant is a citrus plant; More preferably, the citrus plant is citrus.

13. A plant, plant part or plant cell in which PUB21 protein E3 ligase activity is inhibited and MYC2 level is conferred, stabilized or enhanced, the plant, plant part or plant cell comprising the polypeptide according to any one of claims 1 to 2, or the biomaterial according to claim 4, wherein: The plant is a monocot or a dicot; Preferably, the dicotyledonous plant is a Rutaceae plant; More preferably, the Rutales plant is a Rutaceae plant; More preferably, the Rutaceae plant is a citrus plant; More preferably, the citrus plant is citrus.

Images