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

Abstract

The invention provides a polypeptide, a plant immune resistance inducer and a method for improving the disease resistance of plants. The amino acid sequence of the polypeptide is shown as SEQ ID NO: 9, or the polypeptide has more than 75% of homology with the amino acid sequence shown as SEQ ID NO: 9. The problems of poor broad spectrum and poor thermal stability of immunoactive peptides in the prior art can be solved, and the immunoactive peptides are suitable for the field of plant immune anti-inducing peptide agents.

Description

Technical Field

[0001] The present invention relates to the field of plant immunity inducing peptide agents, in particular to a polypeptide and a plant immunity inducing agent and a method for improving plant disease resistance. Background Art

[0002] Crop disease control plays a vital role in global food security. However, pathogen infection causes huge economic losses to global agriculture every year, and the situation of disease 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) Pathogens gradually accumulate resistance to drugs, resulting in a year-on-year decline in the efficiency of chemical control; (2) Pesticide residues not only pollute the soil and cause heavy metal accumulation, but also destroy biodiversity and pose a threat to ecological balance; (3) Residual chemicals pose a hidden danger 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 in current agricultural science. Plant immune inducers, a new type of biological pesticide that can activate the plant's own immune defense mechanism, show unique advantages because its working principle is similar to that of "plant vaccines". By simulating the natural immune signals of plants, this type of inducer can activate the systemic resistance of plants and effectively resist the invasion of pests and diseases. At present, researchers have identified and developed a variety of plant immune inducers, including oligosaccharides, proteins, small molecule compounds and peptides. However, different types of inducers have their own limitations: oligosaccharide inducers such as chitosan oligosaccharides have a high activity threshold and a short time to induce immunity; protein inducers, such as some elicitor proteins, have a complex preparation process and poor stability; small molecule compound inducers, such as purine bases, have certain activity, but their disease resistance spectrum is limited and they are prone to cause plant metabolic disorders. In contrast, peptide inducers have a more ideal development potential because of their small molecular weight, strong activity (effective concentration of 10-9M level) and easy large-scale production.

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

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

[0006] 2. Some peptide inducers have too high specificity for certain receptors, such as the PEPR2 receptor, which results in a narrow spectrum of resistance and makes it difficult to cope with a variety of pathogens;

[0007] 3. In actual field applications, peptide inducers have poor thermal stability and resistance to degradation, which affects their long-term effects and efficiency of use.

[0008] The above problems highlight the shortcomings of existing plant immune inducers in terms of broad spectrum, receptor compatibility and field stability. New breakthroughs are urgently needed to meet the needs of modern agriculture for efficient and environmentally friendly disease resistance strategies and promote global food security and sustainable agricultural development. Summary of the invention

[0009] The main purpose of the present invention is to provide a polypeptide and a plant immune inducer and a method for improving plant disease resistance, 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 a first aspect of the present invention, a polypeptide is provided, the amino acid sequence of the polypeptide being 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] In order to achieve the above object, according to the second aspect of the present invention, a plant immunity inducer is provided, and the plant immunity inducer comprises the above polypeptide.

[0012] Furthermore, the plant immunity inducer also includes a surfactant.

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

[0014] In order to achieve the above object, according to a third aspect of the present invention, a method for improving plant disease resistance is provided, the method comprising applying the above plant immunity inducer to a target plant.

[0015] Further, the application method includes: one or more of spraying, painting, soaking or injection.

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

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

[0018] Furthermore, expressing an exogenous polypeptide in a plant includes: introducing exogenous genetic material capable of expressing the polypeptide into the plant, integrating the genetic material into the genome of the plant and / or expressing it freely in the cells of the above-mentioned plant; the genetic material includes a nucleic acid capable of expressing the polypeptide; the nucleic acid includes a DNA molecule having a 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.

[0019] Furthermore, the introduction method includes gene gun method, Agrobacterium infection method, PEG-induced protoplast method, electrode method, silicon carbide fiber-mediated method or vacuum infiltration method.

[0020] Furthermore, the plant cells include protoplast cells or suspension cells; the plant tissues include callus, immature embryos, mature embryos, leaves, stem tips, young ears or hypocotyls.

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

[0022] In order to achieve the above-mentioned purpose, according to the fifth aspect of the present invention, there is provided the above-mentioned polypeptide, or the above-mentioned plant immune inducer, or the above-mentioned method for improving plant disease resistance, or the use of the above-mentioned method for improving plant disease resistance in improving plant immunity and disease resistance.

[0023] Applying the technical solution of the present invention, the present application provides a polypeptide having an amino acid sequence as shown in SEQ ID NO: 9, or a polypeptide having more than 75% homology with the amino acid sequence as shown in SEQ ID NO: 9. The protein expressed by the polypeptide has immune activity, enriches the types of such immune active peptides, overcomes the problem of narrow disease resistance caused by excessive specificity of the target receptor; and has a broad spectrum, and when present in the plant inducer, it has high thermal stability, is not easy to degrade, and can be applied to a variety of plants, significantly improving the practicality and durability of plant immune inducers, and opening up a new way for green prevention and control of agricultural diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary 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 The schematic diagram of the transcriptome analysis and TPS2 gene expression verification of tomato infected by pathogens in Example 1 of the specification of the present invention is shown; wherein, Figure 1 A in the middle is a schematic diagram of the RNA-seq dataset of tomato leaves treated with bacterial pathogens and PAMPs; Figure 1 Figure B shows the result that pathogen treatment significantly induced the up-regulated expression of multiple secretory peptide encoding genes.

[0026] Figure 2 The conservative structural domains of tomato secretory peptide genes preTPS1-4 and TPS2 immunoreactive small peptides in Example 1 of the specification of the present invention are shown; Figure 2 A in the middle is a schematic diagram of the evolutionary tree analysis of tomato secretory peptide genes; Figure 2 Middle B is the result of the amino acid sequence motif analysis of preTPS1-4 prerequisite proteins; Figure 2 Figure C is a schematic diagram of the core amino acid sequence (54-69aa) of preTPS2.

[0027] Figure 3 The result graph of qRT-PCR data of TPS2 gene of tomato infected by pathogen in Example 1 of the specification of the present invention is shown.

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

[0029] Figure 5 The results of ROS detection on leaves of various crops after TPS2 treatment in Example 2 of the specification of the present invention are shown.

[0030] Figure 6 The results of tomato TPS2 MAPK detection in Example 2 of the present specification are shown.

[0031] Figure 7 The results of MAPK detection in leaves of multiple crops treated with TPS2 in Example 2 of the present specification are shown.

[0032] Figure 8 The results of in vitro thermal stability test of the immunologically active small peptide TPS2 in Example 2 of the present specification are shown.

[0033] Fig. 9 The results of disease resistance testing of DC3000 pathogen inoculated after TPS2 treatment in Example 3 of the specification of the present invention are shown.

[0034] Fig.10 The results of disease resistance testing after TPS2 treatment in Example 3 of the present specification and inoculation with gray mold pathogens are shown. DETAILED DESCRIPTION

[0035] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0036] Terminology explanation:

[0037] Plant immune inducers: a type of substance that can induce plants to produce immune defense responses against pests and diseases. At low concentrations, they can be recognized by plants as signal substances, inducing and enhancing the plant's own immune response, thereby improving the plant's ability to resist diseases and delaying or alleviating the occurrence and development of diseases. Its mechanism of action can be divided into: activating defense gene expression, regulating hormone balance, inducing disease resistance protein synthesis, and improving secondary metabolic processes. Its types include: proteins, oligosaccharides, lipids, small molecule metabolites, and microorganisms.

[0038] As mentioned in the background technology, the development of peptide inducers in the prior art faces the following technical bottlenecks, namely, insufficient molecular diversity of immune peptides, inconsistency between receptor compatibility and broad spectrum, 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 inducers, and thus propose a series of protection schemes of this application.

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

[0040] The polypeptide (TSP2) with the amino acid sequence shown in SEQ ID NO: 9 in the present application is a plant secretory peptide, which is a class of small molecule polypeptides (usually 2-100 amino acids) synthesized by plant cells and released to the extracellular space through the secretory pathway, and plays an important role in signal transduction and regulation in plant growth and development, stress resistance and immune response. Secretory peptides are usually encoded and synthesized by plant genes: in plant cells, peptide chains are synthesized according to specific gene sequence information through the action of organelles such as ribosomes; then, after a series of post-translational modifications, such as glycosylation and phosphorylation, secretory peptides with biological activity are formed; these secretory peptides can be stored in vesicles in plant cells, and when subjected to external stimuli (such as pathogen infection or environmental stress signals), the vesicles fuse with the cell membrane and release the secretory peptides into the extracellular space.

[0041] The mechanism of action of plant secretory peptides mainly depends on their interaction with receptor kinases, namely signal recognition. Secretory peptides bind to receptor kinases 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 signaling pathways (such as MAPK cascade reactions) to regulate gene expression and cellular responses → Physiological effects: ultimately manifested as promoting growth, enhancing stress resistance or activating immune responses.

[0042] The present invention aims at the technical bottlenecks of "lack of molecular diversity, contradiction between receptor compatibility and broad spectrum, and lack of stability and field applicability" in the field of plant immune-inducing peptide agents, and uses a reverse genetics strategy to mine new functional peptides from the tomato endogenous secretory peptide library. Its core amino acid sequence (located at positions 54-69) SEQ ID NO: 9 (TSP2) has both cross-species conservation and plant immune activation functions, and TPS2 maintains its biological activity well after being treated at 50°C for 4 hours. The corresponding immune inducer is developed based on TPS2. When it is applied to crop planting, 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, which can meet the multiple needs of crops in terms of safety, quality, high yield, effectiveness and long-term disease prevention.

[0043] "Homology" in this specification refers to similarity or identity, and particularly refers to 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 is the total ratio of nucleotides of the same type in the nucleotide sequence. "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 properties of the side chains. 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, amino acid residues are abbreviated 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] Substitution and replacement rules. Generally speaking, the effects of replacing amino acids with similar properties are similar. For example, conservative amino acid replacements may occur in the above homologous proteins. "Conservative amino acid replacements" 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] Substitution of hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) 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] A person skilled in the art may 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 a second typical embodiment of the present application, a plant immune inducer is provided, which comprises the above-mentioned polypeptide.

[0052] Reactive oxygen species (ROS) are byproducts of biological aerobic metabolism and are a general term for a class of substances that contain oxygen and are active in nature. In cellular immune responses, ROS can also induce the activation of various signaling pathways, promote cell migration and differentiation, and thus enhance the immune function of cells. In immune responses, 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 plays a key role in responding to environmental stimuli and immune responses. The activation of the MAPK pathway is usually accompanied by its phosphorylation.

[0054] In the present application, the protein homopolymer of the above-mentioned polypeptide (immune active small peptide TPS2) is prepared into a working solution using ultrapure water as a solvent, which can be used to prepare a plant immune inducer having the immune function of the polypeptide. The plant immune inducer can increase the activity of ROS, induce the response of the MAPK signaling pathway, promote cell migration and differentiation, induce activation of tomato immune activity, and enhance the immune function of cells.

[0055] In a preferred embodiment, the plant immune inducer also includes a surfactant; preferably, the surfactant includes 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 1~10μM, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10μM; 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 immunity inducer of the present application also includes the above-mentioned surfactant, which is a substance that can significantly reduce the surface tension of a liquid. Adding a surfactant to the plant immunity inducer of the present application can improve the adhesion ability of the agent, so that the plant immunity inducer can be attached to the leaves containing wax, 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 inside of plant organs and tissues, thereby more effectively activating the immune system of the plant. The present application accurately controls the protein concentration and the surfactant within the above-mentioned range, so that the two can work synergistically, so that the above-mentioned plant immunity inducer can better play a role and improve the immune disease resistance of the target plant.

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

[0058] In a preferred embodiment, the application method includes: one or more of spraying, painting, soaking or injection; preferably, the application method also includes mixing the plant immunity inducer into the planting soil of the target plant.

[0059] When the plant immune inducer of the present application is applied to the target plant through an exogenous source, the immune disease resistance of the target plant can be significantly improved. The present application has been tested and found that when the above-mentioned plant immune inducer is applied to the organ tissue of the target plant, it can increase ROS activity and activate the response of the MAPK signaling pathway, further improving the immune ability of the plant, and can effectively prevent and control bacterial diseases and fungal diseases, and promote the development of crop disease prevention and control. The above-mentioned application method can be flexibly selected by those skilled in the art for any conventional exogenous application method to treat the organs of the target plant according to actual conditions, and the effect of the present application can be achieved.

[0060] It should be noted that this improvement in plant disease resistance is a broad-spectrum disease resistance, which improves plant immunity by increasing plant ROS activity and activating the response of the MAPK signaling pathway, thereby achieving improved resistance to a variety of diseases, including but not limited to resistance to bacterial diseases and resistance to fungal diseases. Preferably, the above-mentioned bacterial diseases include but are not limited to Pst DC3000, Pseudomonas syringae pv. tomato or one or more thereof; preferably, the above-mentioned fungal diseases include but are not limited to one or more of Botrytis cinerea, Phytophthora blight, and Alternaria solani.

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

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

[0063] In a preferred embodiment, expressing an exogenous polypeptide in a plant comprises: introducing exogenous genetic material capable of expressing the polypeptide into the plant, integrating the genetic material into the genome of the plant and / or expressing it freely in the cells of the plant; the genetic material comprises 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 plant disease resistance is a broad-spectrum disease resistance, which improves plant immunity by increasing plant ROS activity and activating the response of the MAPK signaling pathway to achieve an improvement in resistance to a variety of diseases, including but not limited to resistance to bacterial diseases and resistance to fungal diseases. Preferably, the above-mentioned bacterial diseases include but are not limited to Pst DC3000, Pseudomonas syringae pv. tomato or one or more thereof; preferably, the above-mentioned fungal diseases include but are not limited to one or more of gray mold, phytophthora, and Alternaria solani. In a preferred embodiment, the introduction method includes 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 embryos, mature embryos, leaves, stem tips, young panicles or hypocotyls.

[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 immunity inducer, or the above-mentioned method for improving plant disease resistance in improving plant disease resistance; preferably, improving plant immunity includes improving plant ROS activity and / or improving the phosphorylation activity of MAPK pathway; preferably, 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 Pst DC3000, Pseudomonas syringae pv. tomato or one or more thereof; preferably, the above-mentioned fungal diseases include but are not limited to one or more of gray mold, phytophthora, and Alternaria solani.

[0067] In this application, when the polypeptide TPS2 is used to prepare a plant immune inducer, the prepared plant immune inducer has a broad spectrum and compatibility, and the immunologically active TPS2 can significantly induce immune responses in Solanaceae plants such as tomatoes, peppers, eggplants, and potatoes, and has potential application value in preventing Solanaceae plants from resisting diseases. When the plant immune inducer is sprayed exogenously on tomatoes, the tomato's resistance to gray mold and bacterial leaf spot is enhanced, which can prevent the occurrence of tomato diseases in agricultural production, while protecting tomatoes from pesticide contamination.

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

[0069] Example 1 Screening of TPS2 immunologically active small peptide sequences

[0070] 1. Transcriptome data analysis of tomato pathogen response

[0071] 1.1 Data acquisition and analysis

[0072] The RNA-seq dataset of tomato leaves treated with bacterial pathogens and PAMPs was obtained from the Tomato Functional Genomics Database (TFGD, http: / / ted.bti.cornell.edu / ), as Figure 1 As shown in A. Among them, "mock PAMPs" is translated as "simulated PAMPs", "mock bacteria" is translated as "simulated bacteria", "Agrobacteriun tumefaciens" is translated as "Agrobacterium", "Pseudomonas fluorescens" is translated as "fluorescing Pseudomonas", and "Pseudomonas putida" is translated as "Put Pseudomonas".

[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] 。

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

[0085]

[0086] 2. Prediction of immunologically active small peptide sequences

[0087] 2.1 Amino acid sequence analysis of preTPS1-4 precursor proteins

[0088] According to the nucleotide sequence of preTPS1-4 gene, the amino acid sequence of the small peptide precursor protein encoded by it is:

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

[0090] MKSQSRIILMLILIFLISLRSNEAARILDDNNEKEEVDFLKGGKYLLLSSLQWNSVRPPAPNPGTNARTQITSQVAERNFAGRKEFAHPPPPYNAQTKIALDVAMD.

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

[0092] MKSHLACSNTLLMILLIVLISITLNEAARILKDNNEKEVDVLKGGRYLLLPSLQWRPVRSPTPNPGTNVPNQITSQVTQRNFVGRKEFAHPPPPPYNEYPQTKIAFGVATD.

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

[0094] MAAKNYSNIIFATSYCVIFILFLFLSMQTSEATRNLQHKEDQDFMGKDNLLLPSLQWRPVRSPKSNPGTNVPTNIASQVSERNFVGRKELIAHPPSLSNSFQ.

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

[0096] MVAKSYNIFYATYCCIVLLLLFFISMETSEGARFLEHKEEGVWRENNAQYLLLPSLQWRSVRSPGSNPGTNSLTDATSQISERNFAGRKEVAHPPPLPFSNNKYISTVDTKNCW.

[0097] 2.2 Identification of conserved domains of preTPS1-4 precursor proteins

[0098] The amino acid sequences of preTPS1-4 precursor proteins were analyzed using the MEME 5.5.7 online tool, 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) (results are shown in Figure 2 The core amino acid sequence of preTPS2 (54-69aa) is TPS2 (as shown in Figure 2B). Figure 2 (as 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 Test tomato materials

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

[0103] 3.2 Inoculation and induction of pathogen Pst DC3000

[0104] The pathogen of tomato bacterial leaf spot was Pseudomonas syringae pv. tomato Pst DC3000. PstDC3000 was diluted to 25 mg mL −1 King's B liquid medium containing rifampicin was cultured at 28°C overnight. The bacteria 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 induction treatment. The ddH2O spraying treatment was used as a blank control. Tomato leaves were taken 24h and 48h after treatment and quickly frozen in liquid nitrogen for later use.

[0105] 3.3 Induction of flg22 and flgⅡ-28

[0106] Flg22 (Flagellin 22) is a highly conserved 22-amino acid peptide at the N-terminus of bacterial flagellin, and flgII-28 is a conserved 28-amino acid fragment in bacterial flagellin. They can act as pathogen-associated molecular patterns (PAMPs) to activate plant immune responses. Studies have shown that flgII-28 only causes PTI (Pattern-Triggered Immunity) responses in Solanaceae plants such as tomatoes, while flg22 can cause 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 retrieved from a previously reported article (Veluchamy S, Hind SR, Dunham DM, MartinGB, Panthee DR. Natural variation for responsiveness to flg22, flgII-28, andcsp22 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 peptides were synthesized by Qiangyao Company and prepared into a working solution with a concentration of 5 μM using ddH2O as solvent.

[0111] Take 4-week-old tomato leaves and use a hole punch to obtain a hole with a diameter of 0.6 mm. 2 The leaf disc was placed in a cell culture dish containing 1 mL of ddH2O and incubated overnight at room temperature. The leaf discs were treated with flg22 and flgⅡ-28 peptides using the working solution, and sterile water was used as a blank control. Leaf disc samples were taken 0.5 h and 6 h after treatment and quickly frozen in liquid nitrogen for later use.

[0112] 3.4 Gene expression analysis

[0113] The total RNA of leaves / leaf discs treated with Pst DC3000 and flg22 and flgⅡ-28 was extracted using the trizol method and reverse transcribed using HiScript III 1stStrand cDNA Synthesis Kit (+gDNA wiper) (Novozymes, Nanjing, China) according to the manufacturer's instructions.

[0114] Quantitative real-time PCR (qRT-PCR) was performed using the QuantStudio™ 3 Real-time Fluorescence Quantitative PCR System. Each reaction system (20 µl) contained 2 x Q3 SYBR qPCR Master Mix (Universal) 5 μL, Primer F (10 μM) 0.2 μL, Primer R (10 μM) 0.2 μL, Template DNA / cDNA 2 μL, and ddH2O to a total volume of 10 μL. PCR conditions were as follows: 95°C for 30 seconds, followed by 40 cycles, each cycle at 95°C for 10 seconds and 60°C for 30 seconds. EF1α (Elongation factor 1α) was used as an internal control, and real-time fluorescence quantitative PCR (qRT-PCR) analysis was performed with 3 biological replicates. Real-time quantitative PCR and 2 -∆Ct The relevant gene expression data were analyzed by the method.

[0115] qRT-PCR analysis was performed according to a previous report (Livak, KJ & Schmittgen, TD Analysis of 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] 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): CCGTAGCCACACAAATGC.

[0124] qRT-PCR data Figure 3 As shown in the figure, the results show that the secreted peptide TPS2 can be upregulated by the pathogen Pst DC3000. Under the induction treatment of flg22 and flgⅡ-28, the expression of the secreted peptide TPS2 gene was upregulated. Figure 3 shown.

[0125] Example 2 Analysis of TPS2 small peptide immune activity induction

[0126] Transcriptome and gene expression analysis confirmed that the gene preTPS2 encoding the TPS2 peptide precursor protein was upregulated by PstDC3000. Further studies were conducted to verify the induction of TPS2 peptide immune activity.

[0127] 1. TPS2 peptide synthesis and working solution preparation

[0128] An immunologically active small peptide was synthesized according to the TPS2 amino acid sequence SEQ ID NO: 9, and an immunologically active small peptide TPS2 protein pure compound with a purity of > 95% was prepared (commissioned to Shanghai Qiangyao Biotechnology Co., Ltd. for synthesis and preparation).

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

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

[0131] Leaf discs with a radius of 0.3 mm were prepared from 4-5 week-old tomato leaves (same as above: tomato culture). After incubation at 25°C in the dark for 12 h, they were treated with 1 μM TPS2 (experimental group), 1 μM flg22 (positive control) and ddH2O (negative control), respectively. Luminol (50 μM) + horseradish peroxidase (10 μg / mL) were added to detect ROS activity in a GloMax® Navigator chemiluminescence detector.

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

[0133] The broad spectrum of TPS2 application was further verified in Solanaceae crops (pepper, eggplant, tobacco). The above method was used to detect leaf ROS. Figure 5 As shown, TPS2 treatment can significantly induce ROS burst in various species, confirming that TPS2 has broad-spectrum immune-inducing properties 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 5-week-old tomato leaves, cut them into leaf disks with a radius of 0.3 mm, place them in a cell culture dish containing 1 mL ddH2O, and incubate them at room temperature overnight.

[0137] Extraction of total protein: After treatment with TPS2 (1 μM), flg22 (1 μM) and ddH2O for 15 minutes, the mixture was placed in a 2 mL centrifuge tube, frozen in liquid nitrogen, and 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 Na2MoO4·2H2O, 0.5 mM NaVO3, 30 mMβ-glycerophosphate, 0.1% IGEPAL CA 630, 100 nM calyculin A [CST], 0.5 mM PMSF and 1% protease inhibitor) was added, mixed thoroughly, and centrifuged at 12000 rpm at 4°C for 5 minutes. 80 μL of the supernatant was placed in a 1.5 mL centrifuge tube, and 20 μL of 5×SDS loding was added and boiled in a 95°C metal bath for 5 minutes. Spot the sample on 10% SDS-PAGE and run the gel using a 90V (20min) followed by a 140V (50min) operating system. After transfer, the PVDF membrane was blocked in 5% BSA at room temperature for 1 hour, then incubated with pER1 / 2 antibody at 4°C overnight, washed three times with 1×TPST at room temperature for five minutes each, incubated with rabbit secondary antibody at room temperature for 1 hour, washed three times with 1×TPST again, and then developed to verify the immune-inducing activity of the plant defense response.

[0138] The results are as follows Figure 6 As shown, compared with the control, TPS2 can significantly induce tomato MAPK response and activate tomato immune activity.

[0139] The results of using TPS2 to treat pepper, eggplant and tobacco are as follows: Figure 7 As shown, TPS2 treatment can induce significant activation of the MAPK pathway in pepper, eggplant and tobacco, causing a wide range of MAPK responses in Solanaceae crops, further verifying the broad spectrum of its immune-inducing function.

[0140] 4. TPS2 thermal stability analysis

[0141] The thermal stability of immunologically active peptides is one of the important factors that restrict their practical application. Excessively high temperatures often lead to the breakage or denaturation of the peptides, thereby reducing their biological activity. The invention analyzes the thermal stability of TPS2 immunologically active peptides in detail. The results of treating TPS2 at different temperatures show that its activity fluctuates to a certain extent, but under different heat treatment conditions of 25-50°C, it can maintain its biological activity well. The results are as follows: Figure 8 This indicates that TPS2 can maintain stable immune-inducing activity under normal to high temperature conditions.

[0142] Thermal stability test: The synthesized immunoactive peptide TPS2 mother solution was placed in an incubator at different temperatures of 25°C, 37°C, 42°C, and 50°C for heat treatment. The TPS2 mother solution was taken out 1h, 2h, and 4h after heat treatment to prepare the working solution, and the ROS in tomato leaves was tested to detect its immune activity.

[0143] Example 3 Application of TPS2 immunoreactive peptides for immune induction

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

[0145] Here are the steps:

[0146] 1. Preparation of TPS2 Inducer

[0147] Preparation scheme: The immunologically active peptide TPS2 protein pure compound is prepared into a 5 μM working solution using ultrapure water as a solvent, and Silwet L-77 is added to the working solution to make the volume percentage of Silwet L-77 0.02%, thereby preparing a plant disease resistance inducer.

[0148] 2. Evaluation of TPS2-induced plant immune responses

[0149] Using ddH2O 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] Disease resistance test of tomato Pst DC3000: Pst DC3000 was cultured overnight in KB medium at 28°C. The bacteria were collected and diluted to OD600=0.2 with sterile water containing 0.02% (w / v) Silwet L-77, and sprayed on the back of tomato leaves. 24 hours after the treatment group plants were sprayed with TPS2 inducer, the bacterial suspension was inoculated on the back of the leaves. The lesion area and severity were recorded 72 hours after inoculation, and the leaf colony count (CFU / cm²) was determined 24 hours and 72 hours after inoculation by gradient dilution plate counting method.

[0152] like Fig. 9 As shown in the figure, TPS2 treatment significantly inhibited the proliferation of pathogens, and the number of colonies at 72 h was significantly reduced compared with the control (p<0.01). Its immune activation effect was stronger than that of the positive control flg22 (commissioned by Shanghai Qiangyao Biotechnology Co., Ltd.), indicating that TPS2 can effectively activate the immune pathway of tomato plants.

[0153] (2) Botrytis cinerea infection defense experiment

[0154] Tomato resistance to gray mold testing: Cultivate gray mold spores, collect gray mold pathogen B05.1 spores, dilute to 5×10 5 The surface of tomato leaves was treated with 10000 colonies / mL, and the leaves were cultured in the dark for 24 h before switching to the normal light cycle. The lesion expansion area was determined by image analysis after 48 h.

[0155] The results are as follows Fig.10 As shown in the figure, TPS2 treatment significantly reduced the gray mold spot area compared with the control. Compared with the positive control application of systemin (CAS NO: 137181-56-7, a common plant growth regulator that can induce plant immune response, commissioned by Shanghai Qiangyao Biotechnology Co., Ltd. for synthesis and preparation), tomato plant immune activation was improved and the area infected by pathogens was significantly reduced.

[0156] The above experiments prove that exogenous application of TPS2 immune inducer can significantly enhance the dual resistance of tomatoes to Gram-negative bacteria (Pst DC3000) and necrotic 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: This application is based on the polypeptide TPS2 (i.e., immune peptide) to prepare a plant immune inducer that can improve the disease resistance of plants, which can increase the ROS activity of plants and activate the response of the MASK signaling pathway, further improving the immune disease resistance of plants, so that when they are affected by bacterial or fungal diseases, they can have a certain resistance and prevent pathogens from causing excessive damage to crops. The discovery and application of immune peptides in this application have broken through the bottleneck of few types of peptide plant immune inducers, poor broad spectrum, and poor thermal stability, which is conducive to promoting the development of crop disease prevention and breeding.

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in 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, or a polypeptide having more than 75% homology with the amino acid sequence 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.

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 6, characterized in that The target plants include plants of the Solanaceae family.

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

9. A method for improving plant disease resistance, the method comprising: Expressing the exogenous polypeptide of claim 1 in a plant, and / or increasing the expression level of the endogenous polypeptide of claim 1 in a plant.

10. The method according to claim 9, characterized in that The polypeptides expressed in plants include: Introducing exogenous genetic material capable of expressing the polypeptide into the plant, wherein the genetic material is integrated into the genome of the plant and / or expressed episomally in cells of the plant; The genetic material includes a nucleic acid capable of expressing the polypeptide; The nucleic acid includes a DNA molecule having a nucleotide sequence as shown in SEQ ID NO: 2, or a DNA molecule having a homology of more than 75% with the nucleotide sequence as shown in SEQ ID NO:

2.

11. The method according to claim 10, characterized in that The target plants include plants of the Solanaceae family.

12. The method according to claim 11, characterized in that The Solanaceae plant includes tomato, pepper, eggplant or potato.

13. 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 8, or the method for improving plant disease resistance of any one of claims 9 to 12 in improving plant immunity and disease resistance.

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