Polypeptide, plant immune inducer and method for improving disease resistance of plants

Abstract

The present invention provides a polypeptide, a plant immune elicitor and a method for improving the disease resistance of plants. The amino acid sequence of the polypeptide is as shown in SEQ ID NO: 9, or a polypeptide having a homology of more than 75% with the amino acid sequence shown in SEQ ID NO: 9. It can solve the problems of poor broad-spectrum property and poor thermal stability of the immune active peptides in the prior art, and is applicable to the field of plant immune elicitor agents.

Description

Technical Field

[0001] The present invention relates to the field of plant immune elicitor agents, and more particularly, to a polypeptide, a plant immune elicitor, and a method for improving the disease resistance of plants. Background Art

[0002] The prevention and control of crop diseases play a crucial role in global food security. However, the infection of pathogens causes huge economic losses to global agriculture every year, and the situation of disease prevention and control is urgent. Although traditional chemical pesticides are effective in controlling diseases in the short term, three major problems caused by their long-term use are becoming increasingly prominent: (1) the pathogens gradually accumulate resistance, resulting in a decreasing efficiency of chemical control year by year; (2) pesticide residues not only pollute the soil, leading to the accumulation of heavy metals, but also damage biodiversity and pose a threat to the ecological balance; (3) the residual chemical substances pose a potential hazard to food safety and directly threaten human health.

[0003] In view of this, exploring a green, environmentally friendly, efficient and sustainable disease resistance strategy has become an urgent issue to be solved in current agricultural science. Plant immune elicitors, a new type of biological pesticide that can activate the plant's own immune defense mechanism, show unique advantages because their action principle is similar to that of "plant vaccines". By simulating the plant's natural immune signals, these elicitors can activate the systemic resistance of plants and effectively resist the invasion of pests and diseases. Currently, researchers have identified and developed a variety of plant immune elicitors, including oligosaccharides, proteins, small molecule compounds and peptides. However, different types of elicitors have their own limitations: oligosaccharide elicitors such as chitosan oligosaccharide have a high activity threshold and a short induction time for immunity; protein elicitors such as some elicitor proteins are complex in preparation process and poor in stability; small molecule compound elicitors such as purine bases have certain activity, but their disease resistance spectra are limited and they are prone to cause plant metabolic disorders. In contrast, peptide elicitors show more ideal development potential because of their small molecular weight, strong activity (effective concentration at the 10-9M level), and easy large-scale production.

[0004] However, the development of peptide elicitors also faces several challenges:

[0005] 1. The types of peptides known to have immune activity are relatively limited, such as AtPep1, SlPep, and CEP14, etc., which limits their application scope in different crops and diseases;

[0006] 2. Some peptide elicitors have too high specificity for specific receptors, such as the PEPR2 receptor, resulting in a narrow disease resistance spectrum and difficulty in dealing with diverse pathogens;

[0007] 3. In actual field applications, peptide elicitors have poor thermal stability and anti-degradability, affecting their long-term effects and use efficiency.

[0008] The above problems highlight the deficiencies of existing plant immune elicitors in terms of broad-spectrum, receptor compatibility, and field stability. There is an urgent need for new breakthroughs to meet the requirements of modern agriculture for efficient and environmentally friendly disease resistance strategies, and to promote global food security and sustainable agricultural development. Summary of the Invention

[0009] The main object of the present invention is to provide a polypeptide, a plant immune elicitor, and a method for improving the disease resistance of plants, so as to solve the problems of poor broad-spectrum and poor thermal stability of immune active peptides in the prior art.

[0010] To achieve the above object, according to the first aspect of the present invention, there is provided a polypeptide, the amino acid sequence of which is as shown in SEQ ID NO: 9, or a polypeptide having more than 75% homology with the amino acid sequence shown in SEQ ID NO: 9.

[0011] To achieve the above object, according to the second aspect of the present invention, there is provided a plant immune elicitor, which comprises the above-mentioned polypeptide.

[0012] Further, the plant immune elicitor further comprises a surfactant.

[0013] Further, in the plant immune elicitor, the working concentration of the polypeptide is 1-10 μM; the volume content of the surfactant is 0.015-0.025%.

[0014] To achieve the above object, according to the third aspect of the present invention, there is provided a method for improving the disease resistance of plants, the method comprising applying the above-mentioned plant immune elicitor to a target plant.

[0015] Further, the application methods include one or more of spraying, smearing, soaking, or injecting.

[0016] Further, the target plants include solanaceous plants; solanaceous plants include tomatoes, peppers, eggplants, or potatoes.

[0017] To achieve the above object, according to the fourth aspect of the present invention, there is provided a method for improving the disease resistance of plants, the method comprising: expressing an exogenous polypeptide as described above in a plant, and / or increasing the expression level of the endogenous polypeptide as described above in the plant.

[0018] Furthermore, expressing exogenous polypeptides in plants includes: introducing exogenous genetic material capable of expressing polypeptides into plants, integrating the genetic material onto the plant genome and / or expressing it freely in the cells of the above plants; the genetic material includes nucleic acids capable of expressing the polypeptides; the nucleic acids include DNA molecules having the nucleotide sequence shown in SEQ ID NO: 2, or DNA molecules having a homology of more than 75% with the nucleotide sequence shown in SEQ ID NO: 2.

[0019] Furthermore, the introduction methods include particle bombardment, Agrobacterium infection, PEG-induced protoplast method, electroporation, silicon carbide fiber-mediated method or vacuum infiltration method.

[0020] Furthermore, the cells of plants include protoplast cells or suspension cells; the tissues of plants include callus, young embryos, mature embryos, leaves, shoot tips, young spikes or hypocotyls.

[0021] Furthermore, the target plants include Solanaceae plants; Solanaceae plants include tomatoes, peppers, eggplants or potatoes.

[0022] To achieve the above object, according to the fifth aspect of the present invention, there is provided an application of the above polypeptide, or the above plant immune elicitor, or the above method for improving plant disease resistance, or the above method for improving plant disease resistance in improving plant immunity and disease resistance.

[0023] Applying the technical solution of the present invention, this application provides a polypeptide with the amino acid sequence shown in SEQ ID NO: 9, or a polypeptide having a homology of more than 75% with the amino acid sequence shown in SEQ ID NO: 9. The protein expressed by this polypeptide has immunological activity, enriching the types of such immunologically active peptides, overcoming the problem of narrow disease resistance spectrum caused by overly strong target receptor specificity; moreover, it has broad-spectrum properties. When present in plant elicitors, it has high thermal stability and is not easily degraded, and can be applied to a variety of plants, significantly enhancing the practicality and effect persistence of plant immune elicitors, and opening up a new way for the green prevention and control of agricultural diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The specification drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0025] Figure 1 Shows a schematic diagram of transcriptome analysis of pathogen-infected tomatoes and verification of TPS2 gene expression in Example 1 of the present invention's specification; wherein, Figure 1 A in it is a schematic diagram of the RNA-seq dataset of tomato leaves treated with bacterial pathogens and PAMPs;Figure 1 Figure B shows the results of significant induction of the upregulated expression of multiple secreted peptide-encoding genes by pathogen treatment.

[0026] Figure 2 Figure shows the conserved domains of the immunologically active peptides of the tomato secreted peptide genes preTPS1-4 and TPS2 according to Example 1 of the specification of the present invention. Figure 2 Figure A shows a schematic diagram of the phylogenetic tree analysis of tomato secreted peptide genes. Figure 2 Figure B shows the results of motif analysis of the amino acid sequences of the preTPS1-4 precursor proteins. Figure 2 Figure C shows a schematic diagram of the core amino acid sequence (54-69aa) of preTPS2.

[0027] Figure 3 Figure shows the qRT-PCR data results of the TPS2 gene of pathogenic bacteria infecting tomatoes according to Example 1 of the specification of the present invention.

[0028] Figure 4 Figure shows the results of the detection of tomato TPS2 ROS according to Example 1 of the specification of the present invention.

[0029] Figure 5 Figure shows the results of the detection of ROS after treatment with TPS2 on the leaves of various crops according to Example 2 of the specification of the present invention.

[0030] Figure 6 Figure shows the results of the detection of tomato TPS2 MAPK according to Example 2 of the specification of the present invention.

[0031] Figure 7 Figure shows the results of the detection of MAPK after treatment with TPS2 on the leaves of multiple crops according to Example 2 of the specification of the present invention.

[0032] Figure 8 Figure shows the results of the in vitro thermal stability detection of the immunologically active peptide TPS2 according to Example 2 of the specification of the present invention.

[0033] Figure 9 Figure shows the results of the disease resistance detection after inoculation with the DC3000 pathogenic bacteria after treatment with TPS2 according to Example 3 of the specification of the present invention.

[0034] Figure 10 Figure shows the results of the disease resistance detection after inoculation with the Botrytis cinerea pathogenic bacteria after treatment with TPS2 according to Example 3 of the specification of the present invention. Detailed implementation manners

[0035] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0036] Glossary of terms:

[0037] Plant immune elicitors: A class of substances that can induce plants to produce immune defense responses against pests and diseases. They can be recognized as signal substances by plants at low concentrations, induce and enhance the plant's own immune response, thereby improving the plant's resistance to diseases and delaying or reducing the occurrence and development of diseases. Their mechanisms of action can be divided into: activating the expression of defense genes, regulating hormone balance, inducing the synthesis of disease-resistant proteins, and improving secondary metabolic processes. Their types include: protein-based, oligosaccharide-based, lipid-based, small molecule metabolite-based, and microbial-based.

[0038] As mentioned in the background art, the development of peptide elicitors in the prior art faces the following technical bottlenecks, namely, insufficient molecular diversity of immune peptides, contradictions between receptor compatibility and broad-spectrum properties, and insufficient stability and field applicability. Therefore, in this application, the inventors attempt to explore a new immune polypeptide that can be applied to the development of peptide elicitors, and thus propose a series of protection schemes for this application.

[0039] In the first typical embodiment of this application, a polypeptide is provided. The amino acid sequence of this polypeptide is as shown in SEQ ID NO: 9, or a polypeptide having a homology of more than 75% (including but not limited to 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) with the amino acid sequence shown in SEQ ID NO: 9.

[0040] The polypeptide (TSP2) with the amino acid sequence as shown in SEQ ID NO: 9 in this application is a plant-secreted peptide, a class of small polypeptides (usually 2 - 100 amino acids) synthesized by plant cells and released extracellularly through the secretory pathway, which play important signal transduction and regulatory roles in plant growth and development, stress resistance, and immune responses. Secreted peptides are usually encoded and synthesized by plant genes: inside plant cells, peptide chains are synthesized according to specific gene sequence information through the action of organelles such as ribosomes; then, through a series of post-translational modifications, such as glycosylation, phosphorylation, etc., bioactive secreted peptides are formed; these secreted peptides can be stored in vesicles within plant cells, and when stimulated by external stimuli (such as pathogen infection or environmental stress signals), the vesicles fuse with the cell membrane to release the secreted peptides into the extracellular space.

[0041] The mechanism of action of plant-secreted peptides mainly depends on their interaction with receptor kinases, namely signal recognition. The secreted peptide binds to the receptor kinase on the cell membrane, such as the binding of PSK to PSKR → Signal transduction: After the receptor kinase is activated, it transmits signals through downstream signal pathways (such as the MAPK cascade reaction) to regulate gene expression and cell responses → Physiological effects: Eventually, it is manifested as promoting growth, enhancing stress resistance, or activating immune responses.

[0042] In view of the technical bottlenecks in the field of plant immune elicitor peptides, namely "insufficient molecular diversity, contradiction between receptor compatibility and broad spectrum, and insufficient stability and field applicability", the present invention uses a reverse genetics strategy to excavate novel functional peptides from the tomato endogenous secretory peptide library. The core amino acid sequence (located at positions 54-69) SEQ ID NO: 9 (TSP2) has both cross-species conservation and plant immune activation function, and TPS2 can well maintain its biological activity after being treated at 50 °C for 4 h. Based on TPS2, a corresponding immune elicitor is developed. When it is applied to crop cultivation, it can enhance the disease resistance of crops, prevent the occurrence of diseases such as gray mold and bacterial leaf spot, and has strong thermal stability, meeting the multiple requirements of crops for safe, high-quality, high-yield, effective and long-term disease prevention.

[0043] As used herein, "homology" refers to similarity or identity, especially identity. "Homology of amino acid sequences" refers to homology relative to the entire amino acid sequence. "Homology" between amino acid sequences refers to the total ratio of amino acid residues of the same type in these amino acid sequences. "Homology" between nucleotide sequences refers to the total ratio of nucleotides of the same type in the nucleotide sequences. "Similarity" between amino acids refers to the total ratio of amino acid residues of the same type in these amino acid sequences and the ratio of amino acid residues with similar side chain properties. The homology of amino acid sequences and nucleotide sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool).

[0044] As used herein, the abbreviations of amino acid residues are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

[0045] Regarding rules such as substitution and replacement, generally, the effects are similar after mutual substitution between amino acids with similar properties. For example, in the above homologous proteins, conservative amino acid substitutions can occur. "Conservative amino acid substitutions" include, but are not limited to:

[0046] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0047] Hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with bulky side chains;

[0048] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0049] Amino acids with polar uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar uncharged side chains.

[0050] Those skilled in the art can also perform conservative substitutions on amino acids according to amino acid substitution rules well-known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0051] In the second typical embodiment of the present application, a plant immune inducer is provided, and the plant immune inducer includes the above-mentioned polypeptide.

[0052] Reactive Oxygen Species (ROS) are by-products of biological aerobic metabolism and are a general term for a class of oxygen-containing and reactive substances. In the cellular immune response, ROS can also induce the activation of various signal pathways, promote cell migration and differentiation, thereby enhancing the immune function of cells. In the immune response, immune cells attack pathogenic microorganisms or abnormal cells by releasing cytotoxins and capturing cells, playing a very important role in the bactericidal effect of immune cells.

[0053] The MAPK (Mitogen-Activated Protein Kinase) signaling pathway regulates multiple physiological and biochemical processes in plants, especially playing a key role in response to environmental stimuli and immune responses. The activation of the MAPK pathway is usually accompanied by its phosphorylation.

[0054] The working solution prepared by using the protein pure compound of the above-mentioned polypeptide (immunologically active small peptide TPS2) with ultrapure water as a solvent in the present application can be used to prepare a plant immune inducer with the immunological function of this polypeptide. This plant immune inducer can improve the activity of ROS, induce the response of the MAPK signaling pathway, promote cell migration and differentiation, induce the activation of tomato immune activity, and enhance the immune function of cells.

[0055] In a preferred embodiment, the plant immune inducer further comprises a surfactant; preferably, the surfactant comprises one or more of Silwet L-77, Tween 20, Tween 80, Sodium Lauryl Sulfate (SLS), Kinetic or Breaker 735; preferably, in the plant immune inducer, the working concentration of the polypeptide is 1-10 μM, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μM; preferably, the volume content of the surfactant is 0.015-0.025%, including but not limited to 0.015%, 0.020% or 0.0025%.

[0056] The plant immune inducer of the present application further comprises the above-mentioned surfactant, and the surfactant is a substance that can significantly reduce the surface tension of the liquid. Adding the surfactant to the plant immune inducer of the present application can improve the adhesion ability of the medicament, so that the plant immune inducer can adhere to the waxy leaves, thereby improving the utilization rate of the immune inducer of the present application, and can also promote the immune inducer of the present application to better penetrate into the interior of plant organs and tissues, thereby more effectively activating the plant's immune system. The present application precisely controls the protein concentration and the surfactant within the above ranges, enabling the two to act synergistically, so that the above-mentioned plant immune inducer can better play its role and improve the immune disease resistance of the target plant.

[0057] In the third typical embodiment of the present application, a method for improving the disease resistance of plants is provided, and the method includes applying the above-mentioned polypeptide or the above-mentioned plant immune inducer to the target plant. Preferably, the disease resistance includes resistance to bacterial diseases and / or fungal diseases.

[0058] In a preferred embodiment, the application method includes one or more of spraying, smearing, soaking or injecting; preferably, the application method further includes mixing the plant immune inducer into the planting soil of the target plant.

[0059] When the plant immune inducer of the present application is externally applied to the target plant, it can significantly improve the immune disease resistance of the target plant. The present application has detected that when the above-mentioned plant immune inducer is applied to the organ tissues of the target plant, it can increase the ROS activity, activate the response of the MAPK signaling pathway, further improve the immune ability of the plant, and can effectively prevent and control bacterial diseases and fungal diseases, promoting the development of crop disease prevention and control. Those skilled in the art can flexibly select any conventional external application method according to the actual situation to treat the organs of the target plant, and the effects of the present application can be achieved.

[0060] It should be noted that this improvement in the disease resistance of plants is a broad-spectrum disease resistance. By increasing the ROS activity of plants and activating the response of the MAPK signaling pathway, the plant immune ability is enhanced, thereby achieving an improvement in the resistance to various diseases, including but not limited to the ability to resist bacterial diseases and fungal diseases. Preferably, the above-mentioned bacterial diseases include but are not limited to one or more of Pst DC3000, Pseudomonas syringae pv. tomato; preferably, the above-mentioned fungal diseases include but are not limited to one or more of Botrytis cinerea, Phytophthora infestans, Alternaria solani.

[0061] In a preferred embodiment, the target plant includes Solanaceae plants; preferably, the Solanaceae plants include tomato, pepper, eggplant or potato.

[0062] In the fourth typical embodiment of the present application, a method for improving the disease resistance of plants is provided, and the method includes: expressing the exogenous polypeptide in the plant and / or increasing the expression level of the endogenous polypeptide in the plant.

[0063] In a preferred embodiment, expressing the exogenous polypeptide in the plant includes: introducing the exogenous genetic material capable of expressing the polypeptide into the plant, and the genetic material is integrated into the genome of the plant and / or freely expressed in the cells of the plant; the genetic material includes a DNA molecule containing the nucleotide sequence shown in SEQ ID NO: 2, or a DNA molecule having more than 75% homology with the nucleotide sequence shown in SEQ ID NO: 2.

[0064] It should be noted that this improvement in the disease resistance of plants is a broad-spectrum disease resistance. By increasing the ROS activity of plants and activating the response of the MAPK signaling pathway, the plant immune ability is enhanced, thereby achieving an improvement in the resistance to various diseases, including but not limited to the ability to resist bacterial diseases and fungal diseases. Preferably, the above-mentioned bacterial diseases include but are not limited to one or more of Pst DC3000, Pseudomonas syringae pv. tomato; preferably, the above-mentioned fungal diseases include but are not limited to one or more of Botrytis cinerea, Phytophthora infestans, Alternaria solani. In a preferred embodiment, the introduction methods include gene gun method, Agrobacterium infection method, PEG-induced protoplast method, electrode method, silicon carbide fiber-mediated method or vacuum infiltration method.

[0065] In a preferred embodiment, the cells of the target plant include protoplast cells or suspension cells; preferably, the tissues of the target plant include callus, immature embryo, mature embryo, leaf, shoot tip, young spike or hypocotyl.

[0066] In the fifth typical embodiment of the present application, there is provided an application of the above-mentioned polypeptide, or the above-mentioned plant immune inducer, or the above-mentioned method for improving plant disease resistance in improving plant disease resistance; preferably, improving plant immune ability includes increasing the ROS activity of plants and / or increasing the phosphorylation activity of the MAPK pathway; preferably, the disease resistance includes, but is not limited to, the ability to resist bacterial diseases and the ability to resist fungal diseases. More preferably, the above-mentioned bacterial diseases include, but are not limited to, one or more of Pst DC3000, Pseudomonas syringae pv. tomato or the first type; preferably, the above-mentioned fungal diseases include, but are not limited to, one or more of Botrytis cinerea, Phytophthora infestans, Alternaria solani.

[0067] When the above-mentioned polypeptide TPS2 is applied to the preparation of a plant immune inducer in the present application, the prepared plant immune inducer has broad-spectrum and compatibility, and the immunologically active TPS2 can significantly induce immune responses in solanaceous plants such as tomatoes, peppers, eggplants, and potatoes, and has potential application value in preventing solanaceous plant diseases. When the above-mentioned plant immune inducer is externally sprayed on tomatoes, it enhances the ability of tomatoes to resist Botrytis cinerea and bacterial leaf spot, can prevent the occurrence of tomato diseases in agricultural production, and at the same time ensures that tomatoes are not contaminated by pesticides.

[0068] The beneficial effects of the present application will be further explained in detail below with specific examples.

[0069] Example 1 Screening of the TPS2 immunologically active peptide sequence

[0070] 1. Analysis of tomato pathogen-responsive transcriptome data

[0071] 1.1 Data acquisition and analysis

[0072] Obtain the tomato leaf RNA-seq data set treated with bacterial pathogens and PAMPs from the Tomato Functional Genomics Database (TFGD, http: / / ted.bti.cornell.edu / ), as shown in Figure 1 Figure A. Among them, "mock PAMPs" is translated as "simulated PAMPs", "mock bacteria" is translated as "simulated bacteria", "Agrobacteriun tumefaciens" is translated as "Agrobacterium tumefaciens", "Pseudomonas fluorescens" is translated as "Pseudomonas fluorescens", and "Pseudomonas putida" is translated as "Pseudomonas putida".

[0073] DESeq2 software (v1.30.1) was used for differential gene analysis, and the screening conditions were set as log2FoldChange>1 and padj<0.05.

[0074] The results showed that pathogen treatment significantly induced the up-regulation of multiple secretory peptide encoding genes, such as Figure 1 As shown in B, the English meaning is as follows Figure 1 The meaning of A in Chinese.

[0075] 1.2 Candidate gene identification

[0076] Phylogenetic tree analysis (MEGA11 software, neighbor-joining method, Bootstrap=1000) revealed that four unannotated secretory peptide genes had evolutionary homology, which were named preTPS1-4 (e.g. Figure 2 As shown in A, "Eggplant" is translated as "eggplant", "Solanum lycopersicum" is translated as "tomato", "Capsicum annuum" is translated as "pepper", and "Nicotiana attenuate" is translated as "small-leaf tobacco". The alphanumeric characters after the species name refer to the name of its peptide gene).

[0077] The gene sequences of preTPS1-4 are as follows:

[0078] preTPS1 (SEQ ID NO: 1):

[0079] ATGAAATCTCAAAGTAGAATAATATTAATGTTAATTCTTATATTCCTAATTAGCCTCAGATCAAATGAAGCTGCTCGAATTCTTGACGATAACAATGAAAAAGAAGAAGTAGATTTTTTGAAGGGAGGTAAATATCTTTTGTTATCATCTCTCCAATGGAA TTCTGTTCGTCCACCAGCTCCAAACCCTGGCACCAATGCTCGTACTCAAATAACAAGCCAAGTTGCTGAAAGAAACTTTGCAGGTCGCAAAGAGTTTGCTCATCCTCCTCCTCCTTAACGCGCAGACTAAAATTGCATTGGACGTGGCTATGGATTGA.

[0080] preTPS2 (SEQ ID NO: 2):

[0081] ATGAAATCTCATTTAGCATGCAGCAATACATTACTGATGATACTTCTTATAGTTCTAATCAGCATCACATTAAATGAGGCTGCTCGAATTCTTAAAGATAACAATGAAAAAGAAGTAGATGTTTTGAAGGGAGGCAGATATCTTTTGTTACCATCTCTACAATGGCGTCCTGTTCGTTCACCAACTCCAAATCCAGGCACTAATGTTCCTAATCAAATAACAAGCCAAGTGACTCAAAGAAACTTTGTAGGTCGCAAAGAGTTTGCTCATCCTCCTCCTCCTCCTTATAACGAATACCCACAAACTAAAATTGCATTTGGTGTGGCTACGGATTGA。

[0082] preTPS3 (SEQ ID NO: 3):

[0083] ATGTTGAATAGGACTCGAATCGCGTTACAAGATGATAGAATACTTCGAGCCCTTCTTTGGTATCAAGGAGAGACTGACACTCTGAATGACGATGCTAAATTGTACAAATCAAGATTGTATAAATTCTTCACTGATGTGCGCAATGACTTGAATACACCTACATTACCGATTATTCAGGTAACTTGAACGTCTTACTCATAATTATCAGTAAAATAACTATCTTTTCACGATATCTTACTTCTTGTTAAATTTGAATAAGTTTAAGTGATGTAATGTAATAGTGTAAATTAATGTCCAAAAAGGTGGAATTTAAGTTTTATGAAAAAACTACTTAATTAGTTAGACAAACATTCATACCAAAATTCATGTCAGACACACCTAGATACATGTATTTTTTCTATCCGAGCGGGATAGTCTATGTATCTCATACATATGAATGACACTACGTATATGTATATAGGATCCCAGATAAGGGGAGACAAGAGAGTTATGAACGAGACAGTCAGATACGTGTGAATCCACTTAGATATTATGTATATCGAATAAATTACATATAATTTTGACCACATGTATCTAAAGTCTCAACTCTGACAAGATTAATAATATTGCAAAGTAACGTGAATCCAATCAATTAGCTCATAAACTAGTGAGATCTCTATAAATTACCCGTAAGTCATATACGCTAATAGTGGGCTTTGTTTGTGGATACACAGGTGGCATTGGCATCAAAATTTGGGCCTTATACAGAGGAAATAAGACAGGCCCAATTAGAAACTCAGCTTCAAAATGTGAAAACAGTTGATGCTAATGGGCTCAAAATAGGCCCAGATTTTGTTTATCTCAATACTCCATCAGAGGTCCAACCCTTTCTGGGCTTTGGTTCAGCCCATAATTTATTATAA。

[0084] preTPS4 (SEQ ID NO: 4):

[0085]

[0086] 2. Prediction of Immunologically Active Peptide Sequences

[0087] 2.1 Analysis of the Amino Acid Sequences of preTPS1-4 Precursor Proteins

[0088] Based on the nucleotide sequences of the preTPS1-4 genes, the amino acid sequences of the peptide precursor proteins they encode are as follows:

[0089] Amino acid sequence of the protein encoded by preTPS1 (SEQ ID NO: 5):

[0090] MKSQSRIILMLILIFLISLRSNEAARILDDNNEKEEVDFLKGGKYLLLSSLQWNSVRPPAPNPGTNARTQITSQVAERNFAGRKEFAHPPPPYNAQTKIALDVAMD。

[0091] Amino acid sequence of the protein encoded by preTPS2 (SEQ ID NO: 6):

[0092] MKSHLACSNTLLMILLIVLISITLNEAARILKDNNEKEVDVLKGGRYLLLPSLQWRPVRSPTPNPGTNVPNQITSQVTQRNFVGRKEFAHPPPPPYNEYPQTKIAFGVATD。

[0093] Amino acid sequence of the protein encoded by preTPS3 (SEQ ID NO: 7):

[0094] MAAKNYSNIIFATSYCVIFILFLFLSMQTSEATRNLQHKEDQDFMGKDNLLLPSLQWRPVRSPKSNPGTNVPTNIASQVSERNFVGRKELIAHPPSLSNSFQ。

[0095] Amino acid sequence of the protein encoded by preTPS4 (SEQ ID NO: 8):

[0096] MVAKSYNIFYATYCCIVLLLLFFISMETSEGARFLEHKEEGVWRENNAQYLLLPSLQWRSVRSPGSNPGTNSLTDATSQISERNFAGRKEVAHPPPLPFSNNKYISTVDTKNCW。

[0097] 2.2 Identification of Conserved Domains in preTPS1-4 Precursor Proteins

[0098] The motif analysis of the preTPS1-4 precursor protein amino acid sequence was performed using the online tool MEME 5.5.7, and three typical domains were identified: the N-terminal signal peptide (1-27aa), the middle variable region (28-75aa), and the C-terminal conserved active domain (75-98aa) (the results are shown as Figure 2 shown in B). Among them, the core amino acid sequence of preTPS2 (54-69aa) is TPS2 (the results are shown as Figure 2 shown in C).

[0099] The amino acid sequence of TPS2 is: QWRPVRSPTPNPGTN (SEQ ID NO: 9).

[0100] 3. Verification of TPS2 gene expression

[0101] 3.1 Tested tomato materials

[0102] The tested tomato variety is the cultivated tomato variety (line) Ailsa Craig (AC), which is a susceptible variety to tomato bacterial leaf spot; it was sown in nutrient pots filled with a substrate (a mixture of peat and vermiculite in a volume ratio of 3:1), and placed in a growth chamber with a light cycle of 12 hours / 12 hours (day / night) and a temperature of 25°C. Approximately 4 weeks after sowing, 4-week-old tomato seedlings were used as test materials.

[0103] 3.2 Inoculation and induction treatment with the pathogen Pst DC3000

[0104] The tested pathogen of tomato bacterial leaf spot is Pseudomonas syringae pv. tomato Pst DC3000; Pst DC3000 was cultured overnight at 28°C in King's B liquid medium containing 25 mg mL −1 rifampicin. The bacterial cells were collected, and Pst DC3000 was diluted to OD600 = 0.2 with sterile water containing 0.02% (w / v) Silwet L-77, and sprayed on the back of tomato leaves for inoculation and induction treatment, with ddH 2 O spraying treatment as the blank control. Tomato leaves at 24 h and 48 h after treatment were quickly frozen in liquid nitrogen for later use.

[0105] 3.3 Induction treatment with flg22 and flgⅡ-28

[0106] flg22 (Flagellin 22) is a highly conserved 22 - amino - acid small peptide at the N - terminus of bacterial flagellin. flgII - 28 is a conserved 28 - amino - acid fragment in bacterial flagellin. They can act as pathogen - associated molecular patterns (PAMPs, Pathongen - Associated Molecular Pattern) to activate the immune response of plants. Research shows that flgII - 28 only induces PTI (Pattern - Triggered Immunity) responses in Solanaceae plants such as tomatoes, while flg22 can induce PTI responses in a variety of plants.

[0107] flg22 (SEQ ID NO: 10): QRLSTGSRINSAKDDAAGLQIA.

[0108] flgⅡ - 28 (SEQ ID NO: 11): ESTNIQRMRELAVQSRNDSNSATDREA.

[0109] The sequence information was queried from a previously reported article (Veluchamy S, Hind SR, Dunham DM, Martin GB, Panthee DR. Natural variation for responsiveness to flg22, flgII - 28, and csp22 and Pseudomonas syringae pv. tomato in heirloom tomatoes. PLoS One. 2014 Sep 2;9(9):e106119. doi: 10.1371 / journal.pone.0106119.).

[0110] The flg22 and flgⅡ - 28 small peptides were synthesized and prepared by Qiangyao Company, and a working solution with a concentration of 5 μM was prepared using ddH 2 O as the solvent.

[0111] Take 4 - week - old tomato leaves, and use a punch to obtain leaf discs with a diameter of 0.6 mm 2 The leaf discs were placed in a cell culture dish containing 1 mL of ddH 2 O and left to stand at room temperature and incubated overnight. The leaf discs were treated with the flg22 and flgⅡ - 28 small peptides using the working solution, and sterile water treatment was used as a blank control. Take the leaf disc samples at 0.5 h and 6 h after treatment and quickly freeze them in liquid nitrogen for later use.

[0112] 3.4 Gene expression analysis

[0113] Take the leaf / leaf disc samples after inoculation and induction treatment with the pathogen Pst DC3000 and induction treatment with flg22 and flgⅡ-28. Extract the total RNA of the treated samples using the trizol method, and perform reverse transcription according to the manufacturer's instructions using the HiScript III 1stStrand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China).

[0114] Quantitative real-time PCR (qRT-PCR) was performed using a QuantStudio™ 3 real-time fluorescence quantitative PCR system. Each reaction system (20 μl) contained 5 μL of 2 x Q3 SYBR qPCR Master Mix (Universal), 0.2 μL of Primer F (10 μM), 0.2 μL of Primer R (10 μM), 2 μL of Template DNA / cDNA, and ddH 2 O was added to make the total volume of the system up to 10 μL. The PCR conditions were as follows: reaction at 95°C for 30 seconds, followed by 40 cycles, each cycle at 95°C for 10 seconds and 60°C for 30 seconds. Using EF1α (Elongation factor 1α) as an internal reference control, real-time fluorescence quantitative PCR (qRT-PCR) analysis was performed with 3 biological replicates. Analyze the relevant gene expression data using real-time quantitative PCR and the 2 -∆Ct method.

[0115] The qRT-PCR analysis referred to a previously reported article (Livak, K. J. & Schmittgen, T. D. Analysisof relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif.) 25, 402-408, doi:10.1006 / meth.2001.1262 (2001).

[0116] Kanai, M., Yamada, T., Hayashi, M., Mano, S. & Nishimura, M. Soybean (Glycine max L.) triacylglycerol lipase GmSDP1 regulates the quality and quantity of seed oil. Scientific reports 9, 8924, doi:10.1038 / s41598-019-45331-8 (2019).)。

[0117] The primers were designed by Primer 3Plus software, and the reaction primers are as follows:

[0118] EF1α:

[0119] F (SEQ ID NO: 12): ACAGGCGTTCAGGTAAGGAA.

[0120] R (SEQ ID NO: 13): GAGGGTATTCAGCAAAGGTCTC.

[0121] TPS2:

[0122] F (SEQ ID NO: 14): GGCGTCCTGTTCGTTCAC.

[0123] R (SEQ ID NO: 15): CCGTAGCCACACCAAATGC.

[0124] The qRT-PCR data are as Figure 3 shown. The results show that the secreted peptide TPS2 can be up-regulated by the pathogen Pst DC3000. Under the induction of flg22 and flgⅡ-28, the gene expression level of the secreted peptide TPS2 is up-regulated, and the results are as Figure 3 shown.

[0125] Example 2 Induction analysis of the immune activity of TPS2 peptide

[0126] Transcriptome and gene expression analysis confirmed that the gene preTPS2 encoding the precursor protein of the TPS2 peptide was up-regulated by Pst DC3000. Further research was carried out to verify the induction of the immune activity of the TPS2 peptide.

[0127] 1. Synthesis of TPS2 peptide and preparation of working solution

[0128] Synthesize the immunologically active peptide according to the TPS2 amino acid sequence SEQ ID NO: 9, and prepare the immunologically active peptide TPS2 protein pure compound with a purity > 95% (synthesized and prepared by entrusting Shanghai Qiangyao Biotechnology Co., Ltd.).

[0129] Preparation of the mother liquor and working solution of the immunologically active peptide: Take the immunologically active peptide TPS2 protein pure compound and prepare a mother liquor with a concentration of 1 mM using ultrapure water as the solvent, and dilute it to a working solution with a concentration of 1 μM before use for experimental treatment.

[0130] 2. Detection of TPS2-induced ROS activity

[0131] Take the leaves of 4-5-week-old tomatoes (cultured by the same method as described above: tomato cultivation), prepare leaf discs with a radius of 0.3 mm, and after static incubation in the dark at 25 °C for 12 h, treat them with 1 μM TPS2 (experimental group), 1 μM flg22 (positive control), and ddH 2 O (negative control), and add luminol (50 μM) + horseradish peroxidase (10 μg / mL) to detect the ROS activity with a GloMax® Navigator chemiluminescence detector.

[0132] The results are as Figure 4 shown. The TPS2 treatment group induced ROS burst within 30 min, and the accumulation amount was significantly higher than that of the negative control and significantly higher than that of the flg22 positive control (p < 0.01).

[0133] Further verify the broad-spectrum application of TPS2 in solanaceous crops (peppers, eggplants, tobacco). And perform the treatment using the aforementioned method to detect the ROS in the leaves. The results are as Figure 5 shown. The TPS2 treatment can significantly induce ROS burst in each species, confirming that TPS2 has broad-spectrum immune induction characteristics in solanaceae.

[0134] 3. Detection of TPS2-induced MAPK phosphorylation activity

[0135] The study evaluated the activation status of tomato cell immune activity by detecting the phosphorylation of MAPK.

[0136] Take the leaves of 5-week-old tomatoes, cut them into leaf discs with a radius of 0.3 mm and place them in a cell culture dish containing 1 mL of ddH 2 O, and incubate overnight at room temperature.

[0137] Extract total protein: Use TPS2 (1 μM), flg22 (1 μM), and ddH 2After treatment with O for 15 minutes, it was placed in a 2 mL centrifuge tube, frozen in liquid nitrogen and then homogenized. 100 μL of buffer (50 mM Tris-HCl, pH 7.5, 100 mM sodium chloride, 15 mM EGTA, 10 mM magnesium chloride, 1 mM sodium fluoride, 1 mM Na 2 MoO 4 ·2H 2 O, 0.5 mM NaVO 3 , 30 mM β-glycerophosphate, 0.1% IGEPAL CA 630, 100 nM calyculin A [CST], 0.5 mM PMSF and 1% protease inhibitor) were added and mixed well, and centrifuged at 12,000 rpm at 4°C for 5 minutes. 80 μL of the supernatant was taken and placed in a 1.5 mL centrifuge tube, and 20 μL of 5×SDS loading was added and boiled in a metal bath at 95°C for 5 minutes. Samples were loaded on a 10% SDS-PAGE, and the gel was run using a running system of 90V (20 min) followed by 140V (50 min). After transferring the membrane, the PVDF membrane was blocked in 5% BSA at room temperature for 1 hour, then incubated overnight at 4°C with pER1 / 2 antibody. The next day, it was washed three times with 1×TPST at room temperature for 5 minutes each time, incubated with rabbit secondary antibody at room temperature for 1 hour, washed three times again with 1×TPST, and then developed to verify the immune induction activity of the plant defense response.

[0138] The results are as Figure 6 shown. Compared with the control, TPS2 could significantly induce the MAPK response in tomatoes and induce the activation of tomato immune activity.

[0139] Using TPS2 to treat peppers, eggplants and tobacco, the results are as Figure 7 shown. Treatment with TPS2 could significantly activate the MAPK pathway in peppers, eggplants and tobacco, causing a wide range of MAPK responses in solanaceous crops, further verifying the broad-spectrum nature of its immune induction function.

[0140] 4. Thermal stability analysis of TPS2

[0141] The thermal stability of immune-active peptides is one of the important factors restricting their practical application. Excessive temperature often leads to the cleavage or denaturation of peptides, thereby reducing their biological activity. The invention analyzed in detail the thermal stability of the TPS2 immune-active peptide. The treatment results of TPS2 at different temperatures showed that its activity fluctuated to some extent, but it could maintain its biological activity well under different heat treatment conditions of 25 - 50°C. The results are as Figure 8 shown. It indicates that TPS2 can maintain stable immune induction activity from room temperature to high temperature.

[0142] Thermal stability detection: The synthesized immune-active small peptide TPS2 stock solution was placed in incubators at different temperatures of 25°C, 37°C, 42°C, and 50°C for heat treatment. The TPS2 stock solution was taken at 1 h, 2 h, and 4 h after heat treatment to prepare a working solution for detecting ROS in tomato leaves to detect its immune activity.

[0143] Example 3 Immune-induced resistance application of TPS2 immune-active small peptide

[0144] Based on the activation characteristics of the TPS2 gene on the plant immune system, the present invention discloses an application method of mature small peptide TPS2 as a plant immune inducer in disease prevention and control. The specific implementation scheme is as follows:

[0145] The steps are as follows:

[0146] 1. Preparation of TPS2 inducer

[0147] Preparation scheme: The immune-active small peptide TPS2 protein pure compound was formulated into a 5 μM working solution with ultrapure water as the solvent, and Silwet L-77 was added to the working solution so that the volume percentage content of Silwet L-77 was 0.02% to prepare a plant disease-resistant inducer.

[0148] 2. Evaluation of plant immune response induced by TPS2

[0149] Using ddH 2 O as a blank control, the inducer was applied to tomato plants by foliar spraying to systematically evaluate its immune activation effect:

[0150] (1)Pst DC3000 bacterial disease prevention and control experiment

[0151] Tomato Pst DC3000 disease resistance detection: Pst DC3000 was cultured overnight at 28°C using KB medium. The bacterial cells were collected, and Pst DC3000 was diluted to OD600 = 0.2 with sterile water containing 0.02% (w / v) Silwet L-77 and sprayed on the back of tomato leaves. After 24 h of spraying the TPS2 inducer on the treatment group plants, the bacterial suspension was inoculated on the back of the leaves. After 72 h of inoculation, the lesion area and severity were recorded, and the leaf colony count (CFU / cm²) at 24 h and 72 h after inoculation was measured by gradient dilution plate counting method.

[0152] As Figure 9 shown, the treatment with TPS2 significantly inhibited the proliferation of pathogenic bacteria, and the colony count at 72 h was significantly lower than that of the control (p < 0.01). Its immune activation effect was stronger than that of the positive control flg22 (synthesized and prepared by Shanghai Qiangyao Biotechnology Co., Ltd.) treatment group, indicating that TPS2 can effectively activate the immune pathway of tomato plants.

[0153] (2)Botrytis cinerea Infection Defense Experiment

[0154] Detection of tomato resistance to Botrytis cinerea: Culture Botrytis cinerea spores, collect the spores of Botrytis cinerea pathogen B05.1, dilute them to 5×10 5 colony-forming units / mL, treat the surface of tomato leaves, and transfer them to a normal photoperiod after 24 h of dark culture. After 48 h, measure the lesion expansion area by image analysis method.

[0155] The results are as Figure 10 shown. Treatment with TPS2 significantly reduced the Botrytis cinerea lesion area compared with the control. Compared with the positive control of applying systemin (CAS NO: 137181-56-7, a common plant growth regulator that can induce plant immune responses, synthesized and prepared by Shanghai Qiangyao Biotechnology Co., Ltd.), the immune activation of tomato plants was improved, and the area infected by pathogenic bacteria decreased significantly.

[0156] The above experiments prove that exogenous application of the TPS2 immune inducer can significantly enhance the dual resistance of tomatoes to Gram-negative bacteria (Pst DC3000) and necrotrophic fungi (B. cinerea) by activating the plant's basal immune mechanism.

[0157] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: Based on the polypeptide TPS2 (i.e., immune peptide), the present application prepares a plant immune inducer that can improve the disease resistance and immune performance of plants, can increase the ROS activity of plants, and activate the response of the MASK signaling pathway, further improving the immune disease resistance ability of plants, enabling them to have certain resistance when affected by bacterial or fungal diseases, and preventing excessive damage to crops by pathogenic bacteria. The discovery and application of the immune peptide in the present application break through the bottleneck of few types, poor broad-spectrum, and poor thermal stability of peptide-based plant immune inducers, which is beneficial to promoting the development of crop disease control and breeding.

[0158] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO:

9.

2. A plant immune inducer, characterized in that: The plant immunity inducer comprises the polypeptide according to claim 1.

3. The plant immunity inducer according to claim 2, characterized in that The plant immunity inducer also includes a surfactant.

4. The plant immunity inducer according to claim 3, characterized in that In the plant immunity inducer, the working concentration of the polypeptide is 1-10 μM; The volume content of the surfactant is 0.015-0.025%.

5. A method for improving plant disease resistance, characterized in that: The method comprises applying the polypeptide of claim 1 or the plant immunity inducer of any one of claims 2 to 4 to a target plant; The target plant is a plant of the Solanaceae family.

6. The method according to claim 5, characterized in that The application method includes: one or more of spraying, painting, soaking or injecting.

7. The method according to claim 5, characterized in that The Solanaceae plant includes tomato, pepper, eggplant or potato.

8. Use of the polypeptide of claim 1, or the plant immunity inducer of any one of claims 2 to 4, or the method for improving plant disease resistance of any one of claims 5 to 7 in improving plant immunity and disease resistance; The plant is a plant of the Solanaceae family.

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