Fusion protein, its amino acid sequence, encoding nucleotide sequence, preparation method and application

Abstract

The present invention provides a fusion protein, its amino acid sequence, encoding nucleotide sequence, preparation method and application; it relates to the field of agricultural biotechnology. The fusion protein comprises or consists of at least three, four, five, six, seven, or eight identical and / or different PAMP molecule polypeptides, and optionally, there is at least one linker or no linker between adjacent two PAMP molecule polypeptides. Assembling multiple PAMP molecule polypeptides into a fusion protein with multiple and / or various immune epitopes can rapidly induce an immune response of plant defense, reduce the infection ability of pathogenic microorganisms, and significantly improve the disease resistance of plants. The preparation method of the fusion protein combines the PTI immune mechanism and genetic engineering techniques to obtain a fusion protein with multiple immune epitopes that does not exist in nature, effectively solving the problems of high production cost of plant immune PAMP molecule polypeptides and their long-term inability to be applied to agricultural production.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and particularly relates to a fusion protein, its amino acid sequence, coding nucleotide sequence, preparation method and application. Background Art

[0002] In the agricultural ecosystem, compared with chemical pesticides, biopesticide products have the advantages of low toxicity, short degradation period, and high environmental compatibility, and have been widely used in crop production. However, the research and development of traditional biopesticides mainly focus on screening antibacterial and insecticidal substances of biological origin, and then developing biopesticides to combat pests and diseases, often ignoring the role of plant's own immunity in the process of combating pests and diseases. In recent years, plant immune induction technology has become a new highlight in the development of biopesticides. Plant immune elicitors, also known as "plant vaccines", can stimulate the plant's own immune system to achieve the effects of disease resistance, yield increase, and quality improvement. Compared with traditional biopesticides, "plant vaccines" will not cause resistance in pathogenic microorganisms, and are more in line with the requirements of green and healthy agricultural production, which has attracted extensive attention and emphasis at home and abroad.

[0003] The PTI (PAMP-Triggered Immunity) immune mechanism of plants has broad prospects in the development of biopesticides. At present, there are few types of plant immune pesticides on the market, only a few types such as Atailing and Harpin protein. However, these products have quite limitations: First, their receptors are not clear, and it is difficult to make scientific and effective guidance in use, and only the scope of application and concentration can be judged through experimental experience; Second, plants have low sensitivity to these plant immune pesticides, with high application concentration and high cost. Axiom Harpin Protein is a widely used biological protein pesticide internationally. However, the unit price of its pure protein product is higher than 5000 yuan per gram, and the selling price of its 1% protein pesticide product is also greater than 140 yuan per gram. The high cost greatly limits its application in agricultural production. Therefore, it is necessary to develop a biopesticide product with low price and good effect of improving the plant's own immunity. Summary of the Invention

[0004] In view of this, the present invention is committed to providing a fusion protein, assembling multiple PAMP molecular polypeptides into a fusion protein with multiple or various immune epitopes. The fusion protein can bind to the receptors on the surface of plant cells more quickly and widely, induce immune responses in various plants, significantly improve the disease resistance of plants, and the preparation cost of the fusion protein is lower than that of PAMP molecular polypeptides, increasing the unit yield of protein-based biopesticide products. It is expected that the protein cost can be reduced to 1 yuan / g, with low application concentration and fast onset, and can significantly reduce the agricultural production cost when applied in agriculture.

[0005] In the first aspect of the present invention, a fusion protein is provided. The fusion protein comprises or consists of at least three, four, five, six, seven identical and / or different PAMP molecule polypeptides. Optionally, there is at least one linker between adjacent PAMP molecule polypeptides or there is no linker.

[0006] Pathogen-associated molecular patterns (PAMPs) refer to some molecules that are highly conserved in evolution and are characterized by being able to rapidly trigger the immune defense response of plants. There are various types of PAMP molecules, including polysaccharides, lipids, polypeptide molecules, etc. The polypeptide-type PAMP molecules are called PAMP molecule polypeptides. Most PAMP molecules are essential components of the pathogen life cycle and are often used by plants as signal molecules to sense pathogen invasion. Research shows that specific PAMP molecule polypeptides interact with receptor proteins on the plant cell membrane and can rapidly trigger the plant's defense response to resist pathogen infection. The immune responses of plant defense include: inducing the rapid generation of reactive oxygen species in plants and inducing the deposition of callose; the large synthesis of plant hormones (such as salicylic acid, ethylene); the rapid expression of defense genes. Moreover, some autocrine peptides produced by plants after receiving stimuli, such as PIP1 and PEP1, are also PAMP molecule polypeptides and can act as second messengers to enhance the immune signal and sustain the immune effect.

[0007] The method for obtaining PAMP molecule polypeptides is often to conduct homology sequence comparison and analysis in a variety of pathogenic fungi or bacteria, find the relatively conserved sequences in the protein molecules, then artificially synthesize the polypeptide sequences, and verify their ability to activate the plant immune response.

[0008] Due to different pathogen species and naturally occurring mutations, there are naturally multiple mutants of PAMP molecule polypeptides. The PAMP molecule polypeptide mutants can maintain an immunogenicity not less than 80% of the wild-type PAMP molecule polypeptides and can activate the same and fixed immune receptors as the PAMP molecule polypeptides.

[0009] "The fusion protein comprises or consists of at least three, four, five, six, seven identical and / or different PAMP molecule polypeptides" can be understood in three interpretations:

[0010] (1) The fusion protein comprises or consists of at least three, four, five, six, seven of the same type of PAMP molecule polypeptides, that is, it includes but is not limited to or consists of three, four, five, six, seven and more of the same type of PAMP molecule polypeptides;

[0011] (2) The fusion protein comprises or consists of at least three, four, five, six, seven different kinds of PAMP molecule polypeptides, that is, including but not limited to or consisting of three, four, five, six, seven and more different kinds of PAMP molecule polypeptides;

[0012] (3) The fusion protein comprises or consists of at least three, four, five, six, seven identical and different kinds of PAMP molecule polypeptides, that is, including but not limited to or consisting of two identical and one different, two identical and two different, two identical and three different, two identical and four different, two identical and five different, two identical and six different or two identical and seven different and other more identical and different kinds of PAMP molecule polypeptides.

[0013] "Optionally" means that it can be present or absent, that is, there may be no linker between two adjacent PAMP molecule polypeptides constituting the fusion protein, or there may be at least one linker. The linker is a linking region having 1 or more amino acid residues, preferably at least 3 consecutive amino acid residues. The linker connects two adjacent PAMP molecule polypeptides, thereby assembling at least three polypeptides into a fusion protein.

[0014] The linker includes but is not limited to: GAG, AGA, AAA, GGG, KRK, KKK, RRR, AKG.

[0015] It should be noted that the specific arrangement order of the PAMP molecule polypeptides constituting the fusion protein and the specific types of the linker are not specifically limited here, and can be appropriately changed, replaced and adjusted according to the type of plant or the type of pathogenic microorganism targeted, as long as the effect of inducing plant immune resistance can be achieved.

[0016] In the present invention, by studying the PTI immune mechanism of plants, at least three specific PAMP molecule polypeptides are formed into a fusion protein through molecular design and directional assembly with the help of genetic engineering technology. The fusion protein has multiple and / or various immune epitopes, can bind to the receptors on the surface of plant cells more quickly and more widely, induce the immune response of plant defense, reduce the infectivity of pathogenic microorganisms, and significantly improve the disease resistance of plants. Moreover, when the fusion protein is used as a biological pesticide product, it also has the advantages of low toxicity, short degradation period, large environmental compatibility, and no drug resistance of pathogenic bacteria.

[0017] In one embodiment of the present invention, the fusion protein mainly consists of at least three, four, five, six, seven identical and / or different PAMP molecule polypeptide mutants, and there is at least one linker between two adjacent PAMP molecule polypeptide mutants.

[0018] Furthermore, the PAMP molecular polypeptide includes a first polypeptide that activates the FLS2 immune receptor, a second polypeptide that activates the RLP23 immune receptor, a third polypeptide that activates the EFR immune receptor, a fourth polypeptide that activates the RLK7 immune receptor, a fifth polypeptide that activates the PEPR1 immune receptor, a sixth polypeptide that activates the CORE1 immune receptor, a seventh polypeptide that activates the FLS3 immune receptor, an eighth polypeptide that activates the FER receptor, a ninth polypeptide pep13 that activates the plant immune response, a tenth polypeptide hrp24 that activates the plant immune response, and an eleventh polypeptide sys18 that activates the plant immune response.

[0019] The fusion protein contains multiple polypeptide components that activate different receptors and has multiple advantages. First, multiple different signaling pathways can activate the immune responses of different plants, avoiding the situation where immune responses cannot be generated due to the absence of specific receptors in some plants, and the range of applicable plants is wider. Second, when multiple signaling pathways activate the same plant immune response simultaneously, the immune signals generated have a superimposed effect, and immune activation is more sensitive and efficient.

[0020] Furthermore, the first polypeptide that activates the FLS2 immune receptor is polypeptide flg15 and its homologous mutants; or polypeptide flg22 and its homologous mutants.

[0021] The amino acid sequence of flg15 is shown in SEQ ID NO.1: RINSAKDDAAGLQIA. The homologous mutants of flg15 include PAMP polypeptides that add, delete, or substitute one or more (e.g., 1 - 10) amino acids in the amino acid sequence shown in SEQ ID NO.1 and can activate the FLS2 immune receptor.

[0022] flg22 is a highly conserved region at the N - terminus of bacterial flagellin. Many studies have shown that flg22 can induce the innate immunity of plants, can activate the FLS2 receptor, act on signal pathways such as SA and MAPK, and has an important impact on plant disease resistance. The amino acid sequence of flg22 is shown in SEQ ID NO.2: QRLSTGSRINSAKDDAAGLQIA. The homologous mutants of flg22 include polypeptides that add, delete, or substitute one or more (e.g., 1 - 10) amino acids in the amino acid sequence shown in SEQ ID NO.2 and have the ability to activate the FLS2 immune receptor.

[0023] Furthermore, the first polypeptide that activates the FLS2 immune receptor is preferably flg22 and its homologous mutants. Specifically, the homologous mutant of flg22, flg22 m1At positions 1, 5, 7, and 8 of the amino acid sequence shown in SEQ ID NO.2, four amino acid substitution mutations occurred: Q1T, T5S, S7L, R8K; a homologous mutant of flg22, flg22 m2 At positions 5, 7, 20, and 22 of the amino acid sequence shown in SEQ ID NO.2, four amino acid substitution mutations occurred: T5S, S7L, Q20A, A22S.

[0024] The first polypeptides with different amino acid sequences above all belong to homologous mutants with the same function, can all activate the FLS2 immune receptor, and have been proven to have similar biological activities. Specific information can be referred to the introduction of polypeptides that activate the FLS2 immune receptor in the articles "Plants have a sensitive perception system for the most conserved domain of bacterial flagellin" and "CD2-1, the C-Terminal Region of Flagellin, Modulates the Induction of Immune Responses in Rice".

[0025] Furthermore, the second polypeptide that activates the RLP23 immune receptor is preferably polypeptide nlp20 and its homologous mutants.

[0026] nlp20 is a polypeptide molecule composed of 20 amino acids and is a characteristic amino acid sequence contained in the necrosis and ethylene-inducing peptide-like protein family (NLPs). Research shows that the nlp20 polypeptide can rapidly induce an immune response in plants by activating the immune recognition receptor RLP23 and enhance the plant's immune ability against microbial infection.

[0027] The amino acid sequence of nlp20 is shown in SEQ ID NO.3: AIMYSWYFPKDSPVTGLGHR. Homologous mutants of nlp20 include PAMP polypeptides that add, delete, or substitute one or more (such as 1 - 10) amino acids in the amino acid sequence shown in SEQ ID NO.3 and have the ability to activate the RLP23 immune receptor. Specifically, the homologous mutant of nlp20, nlp20 m1 At positions 8, 14, and 17 of the amino acid sequence shown in SEQ ID NO.3, three amino acid substitution mutations occurred: F8M, V14S, L17I; the homologous mutant of nlp20, nlp20 m2At positions 5, 14, and 15 of the amino acid sequence shown in SEQ ID NO.3, three amino acid substitution mutations occurred: S5A, V14S, and T15P.

[0028] The second polypeptides with different amino acid sequences described above all belong to homologous mutants with the same function. They can all activate the RLP23 immune receptor and have been proven to have similar biological activities. For specific information, refer to the introduction of the polypeptides that activate the RLP23 immune receptor in the article "A Conserved Peptide Pattern from a Widespread Microbial Virulence Factor Triggers Pattern-Induced Immunity in Arabidopsis".

[0029] Furthermore, the third polypeptide that activates the EFR immune receptor is preferably the polypeptide elf18 and its homologous mutants.

[0030] elf18 is a polypeptide consisting of 18 amino acids at the N-terminus of the bacterial protein elongation factor Tu (EF-Tu). It can activate the receptor EFR, induce the oxidative burst and biosynthesis of ethylene, and trigger resistance to subsequent pathogen infections.

[0031] The amino acid sequence of elf18 is shown in SEQ ID NO.4: SKEKFERTKPHVNVGTIG. Homologous mutants of elf18 include PAMP polypeptides that add, delete, or substitute one or more (such as 1 - 10) amino acids in the amino acid sequence shown in SEQ ID NO.4 and have the ability to activate the EFR immune receptor. Specifically, the homologous mutant elf18 of elf18 m1 At positions 1, 3, 8, and 14 of the amino acid sequence shown in SEQ ID NO.4, four amino acid substitution mutations occurred: S1A, E3S, T8N, and V14I; the homologous mutant elf18 of elf18 m2 At positions 1, 6, 8, 9, and 12 of the amino acid sequence shown in SEQ ID NO.4, five amino acid substitution mutations occurred: S1V, E6D, T8S, K9L, and V12C.

[0032] The third polypeptides with different amino acid sequences above all belong to homologous mutants with the same function, can all activate the EFR immune receptor, and have been proven to have similar biological activities. For specific information, refer to the introduction of the polypeptides that activate the EFR immune receptor in the article "The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants".

[0033] Furthermore, the fourth polypeptide that activates the RLK7 immune receptor is preferably polypeptide pip1 and its homologous mutants.

[0034] pip1 is a 13-amino acid polypeptide formed by the secretion of prePIP1 into the extracellular space and cleavage in the conserved C-terminal region. pip1 signals through the receptor-like kinase 7 (RLK7) on the cell surface to activate the receptor RLK7, thereby activating the plant's immune response and enhancing the plant's pathogen resistance.

[0035] The amino acid sequence of pip1 is shown in SEQ ID NO.5: RLASGPSPRGPGH. The homologous mutants of pip1 include PAMP polypeptides that add, delete, or substitute one or more (such as 1-10) amino acids in the amino acid sequence shown in SEQ ID NO.5 and have the ability to activate the RLK7 immune receptor. Specifically, the homologous mutant pip1 of pip1 m1 has added two amino acids FV between the 1st and 2nd positions of the amino acid sequence shown in SEQ ID NO.5, and has three amino acid substitution mutations at the 2nd, 3rd, and 9th positions.

[0036] The fourth polypeptides with different amino acid sequences above all belong to homologous mutants with the same function, can all activate the RLK7 immune receptor, and have been proven to have similar biological activities. For specific information, refer to the introduction of the polypeptides that activate the RLK7 immune receptor in the article "The Secreted Peptide PIP1 Amplifies Immunity through Receptor-Like Kinase 7".

[0037] Furthermore, the fifth polypeptide that activates the PEPR1 immune receptor is preferably polypeptide pep1 and its homologous mutants.

[0038] pep1 is an endogenous molecule, a polypeptide containing 23 amino acids from the C-terminus of the precursor protein proPEP1, and can activate the receptor PEPR1 to activate the autoimmunity of Arabidopsis plants.

[0039] The amino acid sequence of pep1 is shown in SEQ ID NO.6: ATKVKAKQRGKEKVSSGRPGQHN. The homologous mutants of pep1 include PAMP polypeptides with the addition, deletion, substitution of one or more (e.g., 1 - 10) amino acids in the amino acid sequence shown in SEQ ID NO.6 and having the ability to activate the PEPR1 immune receptor. Specifically, the homologous mutant of pep1, pep1 m1 Nine amino acid substitution mutations occurred at positions 1, 4, 5, 6, 8, 10, 11, 12, and 13 in the amino acid sequence shown in SEQ ID NO.6: A1E, V4A, K5R, A6G, Q8N, G10T, E11P, K12T, V13P; the homologous mutant of pep1, pep1 m2 Eight amino acids were deleted before position 9 in the amino acid sequence shown in SEQ ID NO.6; the homologous mutant of pep1, pep1 m2 Eight amino acids were deleted before position 9 and a substitution mutation of 1 amino acid occurred at position 10 in the amino acid sequence shown in SEQ ID NO.6: G10A.

[0040] The above fifth polypeptides with different amino acid sequences all belong to homologous mutants with the same function, can all activate the PEPR1 immune receptor, and have been proven to have similar biological activities. For specific information, refer to the introduction of the polypeptides that activate the PEPR1 immune receptor in the articles "Structure–activity studies of AtPep1, a plant peptide signal involved in the innate immune response" and "An endogenous peptide signal in Arabidopsis activates components of the innate immune response".

[0041] Furthermore, the sixth polypeptide that activates the CORE1 immune receptor may be polypeptide csp15 and its homologous mutants; or polypeptide csp22 and its homologous mutants.

[0042] The amino acid sequence of csp15 is shown in SEQ ID NO.7: VKWFNAEKGFGFITP. The homologous mutants of csp15 include PAMP polypeptides with the addition, deletion, substitution of one or more (e.g., 1 - 10) amino acids in the amino acid sequence shown in SEQ ID NO.7 and having the ability to activate the CORE1 immune receptor.

[0043] Csp22 is a 22-amino acid polypeptide on the conserved domain of bacterial cold shock protein (CSP), which can activate the receptor CORE1 and efficiently induce the defense response of tobacco. The amino acid sequence of csp22 is shown in SEQ ID NO.8: AVGTVKWFNAEKGFGFITPDDG. The homologous mutants of csp22 include polypeptides with the ability to activate the CORE1 immune receptor by adding, deleting, or substituting one or more (such as 1-10) amino acids in the amino acid sequence shown in SEQ ID NO.8.

[0044] Furthermore, the sixth polypeptide that activates the CORE1 immune receptor is preferably csp22 and its mutants. Specifically, the homologous mutant csp22 m1 has a substitution mutation of 1 amino acid at position 11 in the amino acid sequence shown in SEQ ID NO.8: E11A; the homologous mutant csp22 m2 has a substitution mutation of 1 amino acid at position 14 in the amino acid sequence shown in SEQ ID NO.8: F14Y.

[0045] The above sixth polypeptides with different amino acid sequences all belong to homologous mutants with the same function, can all activate the CORE1 immune receptor, and have been proven to have similar biological activities. For specific information, refer to the introduction of the polypeptide that activates the CORE1 immune receptor in the article "The highly conserved rna-binding motif rnp-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco".

[0046] Furthermore, the seventh polypeptide that activates the FLS3 immune receptor is preferably the polypeptide flgII-28 and its homologous mutants.

[0047] Both flgII-28 and flg22 are highly conserved regions at the N-terminus of bacterial flagellin and are the main PAMPs recognized by plants, which can activate the receptor FLS3. FlgII-28 can stimulate plants to increase the production of the stress hormone ethylene and the rapid production of ROS, activating the immune response of plants.

[0048] The amino acid sequence of flgII-28 is shown in SEQ ID NO.9: ESTNILQRMRELAVQSRNDSNSATDREA. The homologous mutants of flgII-28 include PAMP polypeptides that add, delete, or substitute one or more (e.g., 1-10) amino acids in the amino acid sequence shown in SEQ ID NO.9 and have the ability to activate the FLS3 immune receptor. Specifically, the homologous mutant of flgII-28, flgII-28 m1 produced two amino acid substitution mutations at positions 23 and 27 in the amino acid sequence shown in SEQ ID NO.9: A23S, E27D; the homologous mutant of flgII-28, flgII-28 m2 produced three amino acid substitution mutations at positions 13, 23, and 27 in the amino acid sequence shown in SEQ ID NO.9: A13V, A23S, E27D.

[0049] The above-mentioned seventh polypeptides with different amino acid sequences all belong to homologous mutants with the same function, can all activate the FLS3 immune receptor, and have been proven to have similar biological activities. For specific information, refer to the introduction of polypeptides that activate the FLS3 immune receptor in the articles "Allelic variation intwo distinct Pseudomonas syringae flagellin epitopes modulates the strengthof plant immune responses but not bacterial motility" and "Natural Variation forResponsiveness to flg22,flgII-28,and csp22 and Pseudomonas syringae pv.tomatoin Heirloom Tomatoes".

[0050] Furthermore, the eighth polypeptide that activates the FER immune receptor is preferably polypeptide ralf17 and its homologous mutants.

[0051] The amino acid sequence of ralf17 is shown in SEQ ID NO.10: NSIGAPAMREDLPKGCAPGSSAGCKMQPANPYKPGCEASQRCRGG. The homologous mutants of ralf17 include PAMP polypeptides that add, delete, or substitute one or more (e.g., 1-10) amino acids in the amino acid sequence shown in SEQ ID NO.10 and have the ability to activate the FER immune receptor. Specifically, the homologous mutant of ralf17, ralf17 m1Four amino acid substitution mutations, N1K, S2T, A5N, and L12E, occurred at positions 1, 2, 5, and 12 of the amino acid sequence shown in SEQ ID NO.10.

[0052] The eighth polypeptides with different amino acid sequences above all belong to homologous mutants with the same function, can all activate the FER immune receptor, and have been proven to have similar biological activities. For specific information, refer to the introduction of the polypeptides that activate the FER immune receptor in the articles "The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling" and "How CrRLK1L receptor complexes perceive RALF signals".

[0053] Furthermore, the PAMP molecular polypeptide further includes a ninth polypeptide that activates plant immune responses, preferably polypeptide pep13 and its homologous mutants.

[0054] pep13 is a conserved polypeptide fragment in the cell wall glycoprotein GP42. Cell wall glycoproteins are widely present in oomycetes, so pep13 has an important impact on oomycete pathogen recognition and activation of plant defense responses. Especially in parsley and potatoes, it can mediate defense gene expression and induce the synthesis of antibacterial phytoalexins.

[0055] The amino acid sequence of pep13 is shown in SEQ ID NO.11: VWNQPVRGFKVYE. Homologous mutants of pep13 include PAMP polypeptides that add, delete, or substitute one or more (e.g., 1 - 10) amino acids in the amino acid sequence shown in SEQ ID NO.11 and have the ability to activate plant immune responses. Specifically, the homologous mutant pep13 m1 had a single amino acid substitution mutation at position 12 of the amino acid sequence shown in SEQ ID NO.11: Y12F; the homologous mutant pep13 m2 had a single amino acid substitution mutation at position 12 of the amino acid sequence shown in SEQ ID NO.11: Y12A.

[0056] The above-mentioned ninth polypeptides with different amino acid sequences all belong to homologous mutants with the same function and have been proven to have similar biological activities. For specific information, please refer to the introduction in the article "Pep-13, a plant defense-inducing pathogen associated pattern from Phytophthora transglutaminases".

[0057] Furthermore, the PAMP molecular polypeptide further includes a tenth polypeptide that activates the plant immune response, preferably polypeptide hrp15 and its homologous mutants.

[0058] The amino acid sequence of hrp15 is shown in SEQ ID NO.12: DLGQLLGGLLQKGLE. Homologous mutants of hrp15 include PAMP polypeptides that add, delete, or substitute one or more (e.g., 1-10) amino acids in the amino acid sequence shown in SEQ ID NO.12 and have the ability to activate the plant immune response. Specifically, the homologous mutant hrp15 m1 in the amino acid sequence shown in SEQ ID NO.12 has 9 amino acid substitution mutations: D1Q, G3D, G7T, G8Q, L10I, Q11M, K12A, G13L, E15Q; the homologous mutant hrp24 of hrp15 has 9 amino acids added on the basis of the amino acid sequence shown in SEQ ID NO.12.

[0059] Furthermore, the tenth polypeptide is preferably the homologous mutant hrp24 of polypeptide hrp15.

[0060] The homologous mutant hrp24 of hrp15 has 9 amino acids added on the basis of the amino acid sequence shown in SEQ ID NO.12. The amino acid sequence of hrp24 is shown in SEQ ID NO.13: PNQDLGQLLGGLLQKGLEATLQDA.

[0061] The above-mentioned tenth polypeptides with different amino acid sequences all belong to homologous mutants with the same function and have been proven to have similar biological activities. For specific information, please refer to the introduction in the article "Functional mapping of harpin hrpZ of Pseudomonas syringae reveals the sites responsible for protein oligomerization, lipid interactions and plant defence induction".

[0062] Furthermore, the PAMP molecular polypeptide further includes an eleventh polypeptide that activates the plant immune response, preferably polypeptide sys18 and its homologous mutants.

[0063] The amino acid sequence of sys18 is shown in SEQ ID NO.14: AVQSKPPSKRDPPKMQTD. The homologous mutants of sys18 include PAMP polypeptides that add, delete, or substitute one or more (such as 1-10) amino acids in the amino acid sequence shown in SEQ ID NO.14 and have the ability to activate the plant immune response. Specifically, the homologous mutant sys18 of sys18 m1 has a substitution mutation of 1 amino acid at the 6th position in the amino acid sequence shown in SEQ ID NO.14: P6A; the homologous mutant sys18 of sys18 m2 has a substitution mutation of 1 amino acid at the 10th position in the amino acid sequence shown in SEQ ID NO.14: R10A.

[0064] The above two eleventh polypeptides with different amino acid sequences both belong to homologous mutants with the same function and have been proven to have similar biological activities. For specific information, refer to the introduction in the article "Structure-activity of deleted andsubstituted systemin.an 18-amino acid polypeptide inducer of plant defensivegenes".

[0065] It should be noted that the names of all the above mutants are not their biological names, but are uniformly named for the convenience of writing and understanding this patent. For example, nlp20 m1 , whose biological name is Pyanlp20, and the naming methods of other mutant names are the same. The biological names of specific mutants can be obtained by querying in articles or databases based on the amino acid sequence.

[0066] It should be noted that due to naturally occurring mutations, for each PAMP molecular polypeptide that activates different receptors, based on the above-mentioned publicly disclosed amino acid sequence, polypeptide mutants composed of the remaining amino acid sequences, such as PAMP molecular polypeptide mutants that add, delete, substitute one or more and have the function of activating the same receptor, are all within the protection scope of the present invention.

[0067] In a preferred embodiment of the present invention, the first polypeptide is flg22, the second polypeptide is nlp20, the third polypeptide is elf18, the fourth polypeptide is pip1, the fifth polypeptide is pep1, the sixth polypeptide is csp22, the seventh polypeptide is flgII-28, the eighth polypeptide is ralf17, the ninth polypeptide is pep13, the tenth polypeptide is hrp24, and the eleventh polypeptide is sys18.

[0068] In a preferred embodiment of the present invention, the fusion protein is composed of three identical and / or different PAMP molecule polypeptides. Optionally, there is at least one linker between adjacent two PAMP molecule polypeptides or there is no linker.

[0069] In a preferred embodiment of the present invention, the fusion protein is composed of four identical and / or different PAMP molecule polypeptides. Optionally, there is at least one linker between adjacent two PAMP molecule polypeptides or there is no linker.

[0070] In a preferred embodiment of the present invention, the fusion protein is composed of five identical and / or different PAMP molecule polypeptides. Optionally, there is at least one linker between adjacent two PAMP molecule polypeptides or there is no linker.

[0071] In a preferred embodiment of the present invention, the fusion protein is composed of six identical and / or different PAMP molecule polypeptides. Optionally, there is at least one linker between adjacent two PAMP molecule polypeptides or there is no linker.

[0072] Furthermore, in an embodiment of the present invention, the fusion protein comprises or is composed of seven different PAMP molecule polypeptides; optionally, there is at least one linker between adjacent two PAMP molecule polypeptides or there is no linker.

[0073] Preferably, the fusion protein is composed of 7 different PAMP molecule polypeptides and at least 6 linkers. Preferably, the seven different PAMP molecule polypeptides are selected from any combination of seven of flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, ralf17, pep13, hrp24 or sys18.

[0074] There is no specific limitation on the arrangement order of the 7 different PAMP molecule polypeptides and the specific types of the linkers. It can be appropriately adjusted according to the types of plants or pathogenic microorganisms, as long as it has the effect of inducing plant immune resistance. Moreover, fusion proteins obtained by adjustment and optimization with more immune epitopes, better effects and lower usage costs are all within the protection scope of the present invention.

[0075] Fusion proteins usually need to be expressed as a fusion with a protein tag. A protein tag refers to a polypeptide that is expressed as a fusion with the target protein using in vitro DNA recombination technology, facilitating the expression, detection, and purification of the target protein.

[0076] The protein tags that can be linked to the fusion protein in the present invention include, but are not limited to: HIS, GST, Flag, MBP, HA, c-Myc, eGFP, eYFP, eCFP.

[0077] In one embodiment of the present invention, the amino acid sequence of the fusion protein composed of 7 different PAMP molecular polypeptides, namely flg22, nlp20, elf18, pip1, pep1, csp22, and flgII-28, is shown in SEQ ID NO.15.

[0078] The amino acid sequence shown in SEQ ID NO.15 indicates that the fusion protein is linked with a HIS protein tag, and the arrangement order of the PAMP molecular polypeptides is elf18, csp22, flg22, flgII-28, nlp20, pep1, and pip1 in sequence. There are linkers between adjacent PAMP molecular polypeptides, and the linkers are selected as GAG and AGA.

[0079] In one embodiment of the present invention, the amino acid sequence of the fusion protein composed of 7 different PAMP molecular polypeptides, namely flg22, nlp20, elf18, pip1, pep1, csp22, and flgII-28, is a functional homologous sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.15.

[0080] The above-mentioned functional homologous sequences of identity include, but are not limited to, amino acid sequences having about 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more identity with the amino acids shown in SEQ ID NO.15.

[0081] The fusion protein composed of the above seven different PAMP molecular polypeptides, assembled with appropriate linkers and tag proteins, has more immune active epitopes compared to a single PAMP molecular polypeptide. When used in plants, it can bind to the receptors on the surface of plant cells more quickly and sensitively, rapidly induce the immune response of plant defense, resist pathogenic microorganisms, improve plant disease resistance, and also has the advantages of low toxicity, short degradation period, and high environmental compatibility.

[0082] The second aspect of the present invention provides a nucleotide sequence encoding the above fusion protein.

[0083] It should be noted that since the composition of the above-mentioned fusion protein is variable, there is at least one linker or no linker between adjacent PAMP molecule polypeptides, and the number and type of linkers are variable. Therefore, the corresponding nucleotide sequence encoding it is also variable. Therefore, the arrangement order and the number of bases in the nucleotide sequence are not specifically limited herein, and the nucleotide sequence capable of encoding the fusion protein, as well as its complementary sequence, degenerate sequence or homologous sequence, are all within the protection scope of the present invention.

[0084] In one embodiment of the present invention, the nucleotide sequence encoding the fusion protein with the amino acid sequence shown in SEQ ID NO.15 is as shown in SEQ ID NO.16.

[0085] In one embodiment of the present invention, the nucleotide sequence encoding the fusion protein with the amino acid sequence shown in SEQ ID NO.15 is a nucleotide sequence that hybridizes with the nucleotide sequence of SEQ ID NO.16 under stringent conditions and is capable of encoding the said fusion protein.

[0086] Exemplarily, as used herein, "stringent conditions" refer to conditions under which a probe will hybridize to its target sequence to a detectable degree that exceeds hybridization to other sequences (such as at least 2-fold over background). Stringent conditions are sequence-dependent and vary with the environment. By controlling the stringency of the hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified.

[0087] In one embodiment of the present invention, the nucleotide sequence encoding the fusion protein with the amino acid sequence shown in SEQ ID NO.15 is a degenerate sequence of the nucleotide sequence shown in SEQ ID NO.16. After changing one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.16, the type of amino acid encoded at the corresponding position of the nucleotide sequence remains unchanged, and the amino acid sequence of the encoded fusion protein remains unchanged.

[0088] In one embodiment of the present invention, the nucleotide sequence encoding the fusion protein with the amino acid sequence shown in SEQ ID NO.15 is a homologous sequence of the nucleotide sequence shown in SEQ ID NO.16. Preferably, the homologous sequence is a polynucleotide having at least 85% or more identity with the nucleotide sequence shown in SEQ ID NO.16.

[0089] The said homologous sequence includes, but is not limited to, polynucleotides having about 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 98% or more, 99% or more identity with the nucleotide shown in SEQ ID NO.16 and being capable of encoding the said fusion protein.

[0090] The third aspect of the present invention provides a vector into which the nucleotide sequence encoding the fusion protein is introduced.

[0091] There is no limitation on the specific type of vector, as long as it can successfully ligate the above nucleotide sequence with the vector to construct a recombinant expression vector, and the expression vector can normally express the fusion protein in the host cell.

[0092] Furthermore, the vector includes but is not limited to pET-28b(+), pETBlue-1, pETBlue-2, pET-32, pET-34b(+), pET-35b(+), pET-30EK / LIC, pET-32EK / LIC, pET-34EK / LIC, pET-36EK / LIC.

[0093] The fourth aspect of the present invention provides a microorganism or cell into which the above nucleotide sequence encoding the fusion protein and / or the above vector is introduced.

[0094] It should be noted that the "and / or" here has three interpretations: 1. The microorganism or cell separately contains the nucleotide sequence encoding the fusion protein; 2. The microorganism or cell separately contains the above vector, and the vector contains the nucleotide sequence encoding the fusion protein; 3. The microorganism or cell contains the nucleotide sequence encoding the fusion protein and at the same time contains the above vector, and the vector also contains the nucleotide sequence encoding the fusion protein. The microorganism can be any prokaryotic or eukaryotic cell that can normally express the fusion protein.

[0095] In a preferred embodiment, the microorganism or cell refers to a specific microorganism or cell into which the nucleotide sequence encoding the fusion protein is introduced, and also includes the progeny of such a microorganism carrying the vector.

[0096] In one embodiment of the present invention, the microorganism includes one or more of Escherichia coli, Agrobacterium, or Bacillus subtilis; preferably Escherichia coli.

[0097] Furthermore, the strains of Escherichia coli include but are not limited to BL21(DE3), λDE3, Rosetta TM, K-12, HMS174, NovaBlue, Tuner, OrigamiB.

[0098] Furthermore, the strains of Agrobacterium include but are not limited to EH101, EHA105, C58C1, LBA4404.

[0099] Furthermore, the strains of Bacillus subtilis include but are not limited to pMA5, PUB110, pE194, pWB.

[0100] Furthermore, in planta and / or in vitro, the method for transforming the nucleotide sequence encoding the fusion protein and / or the above-mentioned vector into a host microorganism includes, but is not limited to: heat activation, heat shock, electroporation, calcium phosphate precipitation, polyethylene glycol (PEG) transformation, lipofection, and microinjection.

[0101] The fifth aspect of the present invention provides a plant immune inducer containing the fusion protein, or the above-mentioned vector, or the microorganism or cell.

[0102] A plant immune inducer refers to an exogenous organism or molecule that can induce or activate the immune response of plants and improve the resistance of plants to certain pathogenic microorganisms. In the present invention, the fusion protein, or a vector containing the nucleotide sequence encoding the fusion protein, or the microorganism can be used as a raw material for preparing a plant immune inducer, and then the plant immune inducer can be used as a biological pesticide product in agricultural production, which also has the advantages of low use concentration, fast onset, low cost in agricultural application, low toxicity, short degradation period, and high environmental compatibility.

[0103] Furthermore, the plant immune inducer further includes one or more agriculturally acceptable carriers, excipients, diluents or solvents.

[0104] In the preparation process of the plant immune inducer, not only the above-mentioned fusion protein, or the vector, or the microorganism is used as the main ingredient, but also various agriculturally acceptable carriers, excipients, diluents or solvents are used as auxiliary ingredients to obtain a plant immune inducer with more dosage forms, more stable effects, and more convenient use.

[0105] Furthermore, the dosage form of the plant immune inducer is selected from the group consisting of powder, soluble powder, wettable powder, granule, aqueous solution, microemulsion, suspension, and water dispersible granule. The dosage forms of the plant immune inducer are diverse, which can expand its scope of use to better apply to different types of plants.

[0106] The sixth aspect of the present invention provides a method for preparing the fusion protein, including the step of culturing a microorganism or cell containing the nucleotide, or including the step of artificially synthesizing the fusion protein;

[0107] Preferably, the method includes the following steps:

[0108] (a) Synthesize the nucleotide, preferably analyze and design the nucleotide sequence spliced into the nucleotide sequence encoding the fusion protein before synthesis;

[0109] (b) Transform the synthesized nucleotide sequence (preferably through a vector transformation) into a microorganism or cell, and culture the microorganism or cell to express the fusion protein; and,

[0110] (c) Optionally, collect and purify the expressed fusion protein.

[0111] The method for preparing the above-mentioned fusion protein combines the PTI immune mechanism and genetic engineering techniques. At least three identical and / or different PAMP molecule polypeptides that have been discovered are used to construct a new genetically recombinant fusion protein through genetic engineering techniques, and a fusion protein with multiple immune epitopes that does not exist in nature is obtained through protein expression techniques. The above-mentioned preparation method has a simple preparation process, short time consumption, and low economic cost, effectively solving the problem of high production cost of plant immune PAMP molecule polypeptides and their long-term inability to be applied to agricultural production.

[0112] In a preferred embodiment, the method for preparing the fusion protein includes the following steps:

[0113] (a) Select the nucleotide sequences of seven different PAMP molecule polypeptides, and use the bioinformatics software Geneious R9 combined with online analysis and design of Swiss-model to splice and form the nucleotide sequence encoding the fusion protein as shown in SEQ ID NO.16, and artificially synthesize the nucleotide sequence; the seven different PAMP molecule polypeptides are flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28.

[0114] (b) Using genetic engineering methods, after connecting the synthesized nucleotide sequence with the pET-28b(+) expression vector, transform it into Escherichia coli BL21(DE3) for induced expression of the fusion protein;

[0115] (c) Collect and purify the expressed fusion protein;

[0116] (d) Sequence the expressed fusion protein, and find that the amino acid sequence of the fusion protein is as shown in SEQ ID NO.15.

[0117] The seventh aspect of the present invention provides the application of the above-mentioned fusion protein, or the above-mentioned plant immune inducer, or the fusion protein prepared by the method for preparing the fusion protein in improving plant disease resistance, inducing plant defense responses and / or resisting pathogenic microorganisms.

[0118] Furthermore, the plants include but are not limited to: Arabidopsis thaliana, maize, wheat, rice, tomato, tobacco.

[0119] Furthermore, the pathogenic microorganisms include but are not limited to: Pseudomonas syringae, Fusarium graminearum, Magnaporthe oryzae, Tobacco mosaic virus.

[0120] In the specific use process, the fusion protein or the plant immune inducer can be applied to plants, but the specific application method and the amount applied are not limited and can be reasonably selected according to the plant species and the type of pests and diseases.

[0121] The fusion protein and / or the plant immune inducer can interact with the receptor protein on the plant cell membrane and can quickly trigger the plant's defense response to resist the infection of pathogenic bacteria. Therefore, this fusion protein can be used as a biological pesticide product in agricultural production to activate the plant's own disease resistance, thereby improving the plant's ability to resist pathogenic microorganisms and reducing the use of chemical pesticides.

[0122] The present invention has the following beneficial effects by adopting the above technical solutions:

[0123] (1) The fusion protein provided by the present invention has multiple or various immune epitopes, has a low use concentration and a fast onset of action, can bind to the receptors on the plant cell surface more quickly and more widely, has broad-spectrum and high efficiency, can induce multiple plants to produce immune responses, reduce the infection ability of pathogenic microorganisms, and significantly improve the disease resistance of plants;

[0124] (2) The preparation cost of the fusion protein provided by the present invention is lower than that of the PAMP molecular polypeptide. As a biological pesticide product used in agriculture, it can significantly reduce the agricultural production cost;

[0125] (3) The fusion protein provided by the present invention does not cause plant drug resistance and has the advantages of low toxicity, short degradation period, and large environmental compatibility;

[0126] (4) The preparation method of the fusion protein provided by the present invention has a simple preparation process, short time consumption, and low input economic cost, effectively solving the problems of high production cost of preparing plant immune PAMP molecular polypeptides and long-term inability to be applied to agricultural production. Description of the Drawings

[0127] Figure 1 Shown is the SDS-PAGE electrophoresis pattern of the heptapeptide fusion protein His-MP7;

[0128] Figure 2 Shown is the callose accumulation of Arabidopsis plants under treatment with different concentrations of the fusion protein His-MP7 and the PAMP molecular polypeptide flg22;

[0129] Figure 3 Shown is the callose accumulation of Arabidopsis plants under treatment with 100 nM of the fusion protein His-MP7 and different PAMP molecular polypeptides;

[0130] Figure 4 Shown is the production of reactive oxygen species in maize plants induced by the fusion protein His-MP7;

[0131] Figure 5 Shown are the experimental results of the fusion protein His-MP7 enhancing the resistance of Arabidopsis plants to the pathogen DC3000;

[0132] Figure 6 Shown are the experimental results of the fusion protein His-MP7 enhancing the resistance of maize plants to Fusarium graminearum; wherein, a is the actual measured comparison of the growth of Fusarium graminearum infecting maize inhibited by the fusion protein His-MP7; b is the bar chart of the experimental results of the fusion protein His-MP7 inhibiting the infection of maize by Fusarium graminearum;

[0133] Figure 7 Shown are the experimental results of the fusion protein His-MP7 enhancing the resistance of rice plants to Magnaporthe oryzae; wherein, a is the actual measured comparison of the growth of Magnaporthe oryzae infecting rice inhibited by the fusion protein His-MP7; b is the bar chart of the experimental results of the fusion protein His-MP7 inhibiting the infection of rice by Magnaporthe oryzae. Detailed implementation manners

[0134] In the present invention, the term "PAMP molecular polypeptide" refers to a polypeptide fragment that is relatively conserved in sequence and has immune activation ability found by performing homologous sequence comparison analysis in secreted proteins of various pathogenic fungi, bacteria, or plants themselves, and is often used by plants as a signal molecular polypeptide for sensing pathogen invasion.

[0135] In the present invention, the term "linker" refers to having at least 1 amino acid residue, preferably at least 2 consecutive amino acid residues.

[0136] In the present invention, the term "plant immune inducer" refers to an exogenous organism or molecule that can induce or activate the immune response of plants and improve the resistance of plants to certain pathogenic microorganisms.

[0137] In the present invention, the term "PTI immune mechanism", the full name is pathogen-associated molecular pattern-triggered immunity mechanism, refers to the mechanism by which the PAMP signal molecule activates the plant immune response after recognizing the plant cell receptor.

[0138] In the present invention, "protein tag" refers to a polypeptide that is fused and expressed together with the target protein by using DNA in vitro recombination technology for the expression, detection, and purification of the target protein.

[0139] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0140] In the following embodiments, the introduction of some plant materials, bacterial strains, and virus sources.

[0141] Plant materials: Rice (Oryza sativa L.) is the short-grain japonica variety Nipponbare (NPB) (NPB is an internationally common variety that has been whole-genome sequenced), and maize is Xundan 20. Both rice and maize are purchased from the market; cultivated tomato (Solanum lycopersicum), Columbia wild-type Arabidopsis thaliana col-0, and N89 tobacco lines are all from the research group of Professor Cai Yi of the College of Life Sciences, Sichuan Agricultural University.

[0142] Bacterial strains and viruses: Pseudomonas syringae pv. tomato DC3000, Magnaporthe oryzae race ZB15, Tobacco mosaic virus, and Fusarium graminearum are all from the research group of Professor Cai Yi of the College of Life Sciences, Sichuan Agricultural University.

[0143] In addition, materials, reagents, consumables, etc. not mentioned in the embodiments can all be purchased from the market.

[0144] The His-tag protein purification kit is purchased from CW Biotech Co., Ltd., product number CW0894; the BCA protein concentration assay kit is purchased from Solarbio Science & Technology Co., Ltd., product catalog number PC0020-500 microwells (50T).

[0145] Example 1 Molecular design and nucleotide sequence acquisition of the heptapeptide fusion protein His-MP7

[0146] (1) Among 11 different PAMP molecular polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15, sys18 and their mutants, 7 different PAMP molecular polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28 are selected to form a fusion protein, and the linkers are AGA and GAG;

[0147] (2) Use the online analysis platform ExPASy (https: / / www.expasy.org) to analyze the basic properties such as molecular mass, amino acid composition (protparam), and hydrophobicity of the above protein sequence (protscale); use the online platform Phyre2 (http: / / www.sbg.bio.ic.ac.uk / phyre2 / html / page.cgi? id=index) to model the above protein structure;

[0148] (3) Combine the analysis results, screen out 1 design scheme, name this fusion protein MP7, and the amino acid sequence is shown in SEQ ID NO:15;

[0149] (4) Use the bioinformatics software Geneious R9 software and the online analysis platform Jcat (http: / / www.jcat.de) to design the nucleotide sequence encoding the fusion protein MP7, obtain the nucleotide sequence shown in SEQ ID NO:16, and artificially synthesize the said nucleotide sequence.

[0150] Example 2 Expression and purification of heptapeptide fusion protein His-MP7

[0151] (1) Clone the nucleotide sequence shown in SEQ ID NO:16 into the HindIII and XholI sites of the pET-28b(+) expression vector (Novagen), transform it into Escherichia coli DH5α by heat shock, pick positive clones, shake the bacteria and extract the plasmid. After enzymatic digestion and sequencing verification, transform it into Escherichia coli BL21(DE3) by heat shock. The Escherichia coli containing the recombinant plasmid pET-28b-MP7 is named BL21(DE3) / pET-28b-MP7;

[0152] (2) Induce the expression of BL21(DE3) / pET-28b-MP7, including the following steps: inoculate the expression strain into LB liquid medium, culture it overnight at 37°C with shaking at 200 rpm / min to obtain the first bacterial solution; transfer the overnight bacterial solution to LB liquid medium containing 100 μg / mL kanamycin at a volume ratio of 1:100, continue to shake at 37°C and 200 rpm / min until the OD600nm value of the bacterial solution reaches 0.6, then add IPTG with a final concentration of 0.5 mmol / L, and culture it at 28°C and 200 rpm / min for 12 h to obtain the second bacterial solution; centrifuge the second bacterial solution at 12000 rpm / min, collect the bacterial cells, add PBS buffer, ultrasonically disrupt the bacterial cells, and then centrifuge at 4°C and 12000 rpm / min to collect the supernatant;

[0153] (3) The supernatant was purified using a His-tag protein purification kit (soluble protein), and the specific operation was as follows: First, 5 mL of Ni-Agarose packing was loaded into an empty affinity column, and the supernatant was slowly passed through the affinity column. Then, it was eluted with PBS buffer containing 10 mM imidazole for 6 column volumes to remove impurities. Finally, it was eluted with PBS buffer containing 500 mM imidazole for 5 column volumes, and the eluate after passing through the column was collected, which was the His-MP7 solution of the fusion protein;

[0154] (4) It was detected using a BCA protein concentration assay kit, and the concentration of the fusion protein His-MP7 was measured to be 0.2 mg / mL; After SDS-PAGE detection, as Figure 1 shown, an expressed protein containing histidine (His-MP7) with a molecular weight of approximately 23 kDa was obtained by purification.

[0155] Example 3 Potency study on the immune activation of the heptapeptide fusion protein His-MP7

[0156] During the immune response of plant cells, callose accumulates. Callose can enhance the mechanical strength of the plant cell wall and block the channels for the spread of pathogens between cells, thereby restricting the invasion of pathogenic microorganisms. Using the model plant Arabidopsis thaliana as the material and callose accumulation as the immune index, the immune activation ability of the fusion protein His-MP7 at different concentrations was analyzed and compared with the single PAMP molecule polypeptide. The specific experimental operation was as follows:

[0157] I. Comparison of Callose Accumulation in Arabidopsis Induced by Fusion Protein His-MP7 at Different Concentrations

[0158] The final concentrations of the fusion protein His-MP7 and the polypeptide flg22 solution were adjusted to 1 μM, 100 nM, and 10 nM respectively, and water was used as the blank control. Four-week-old Arabidopsis thaliana leaves were infected by syringe infiltration method. After 12 hours of treatment, the treated leaves were collected and placed in a six-well plate, and an appropriate amount of eluent was added for horizontal incubation for 4 hours. The eluent was replaced with aniline blue staining solution with a final concentration of 0.1 mg / mL, and stained at room temperature in the dark for 1 hour. The callose accumulation was observed under a 10× objective lens of a fluorescence microscope. Each treatment had three biological replicates, and the experiment was repeated three times.

[0159] The experimental results were as Figure 2 shown: 100 nM of the fusion protein His-MP7 could significantly cause callose accumulation in Arabidopsis thaliana, and the effect was equivalent to that of the 1 μM PAMP molecule polypeptide flg22.

[0160] II. Comparison of Immune Activation Abilities of Fusion Protein His-MP7 and Different Polypeptides at the Same Concentration

[0161] The final concentrations of the fusion protein His-MP7 and the polypeptides flg22, nlp20, elf18, pip1, pep1, csp22, and flgII-28 were all adjusted to 100 nM. Using csp22 and flgII-28 as negative controls, four-week-old Arabidopsis leaves were infiltrated by syringe infiltration method, and the sample treatment method was the same as above.

[0162] The experimental results are as Figure 3 shown: The immune activation effect of 100 nM fusion protein His-MP7 is better than that of all PAMP molecular polypeptides.

[0163] III. Fusion Protein His-MP7 Induces the Immune Response of Plants with Reactive Oxygen Species Burst

[0164] The burst of reactive oxygen species (ROS) is considered to be one of the earliest responses of plants to pathogenic microorganisms and plays an important role in plant defense responses. Existing studies have shown that ROS can directly act as an antibacterial agent in plants, having direct toxicity to pathogenic microorganisms and inhibiting their growth. After being infected by pathogenic microorganisms, plants will produce and accumulate ROS bursts. Using maize as the plant material and ROS as the immune index, the immune activation ability of 100 nM fusion protein His-MP7 was analyzed. The specific experimental procedures are as follows:

[0165] Take two-week-old maize leaves, cut the middle part into 5 cm lengths and immerse them in 1 μg / mL auxin 6BA solution, add the fusion protein His-MP7 with a final concentration of 100 nM, and use the aqueous solution of auxin 6BA as the blank control. Soak for 48 hours, stain with 1 mg / mL DAB solution for 12 hours, elute with the eluent (ethanol: acetic acid: glycerol = 3:1:1) for 12 h, and observe after standing for 30 minutes.

[0166] The experimental results are as Figure 4 shown: 100 nM fusion protein His-MP7 can induce plants to produce an ROS burst immune response.

[0167] The above three experiments prove that the fusion protein His-MP7 at a relatively low concentration (100 nM) can efficiently activate plant immunity, and the immune activation ability of the fusion protein His-MP7 at the same concentration is better than that of a single PAMP molecular polypeptide.

[0168] Example 4 Detection of the ability of the heptapeptide fusion protein His-MP7 to improve plant disease resistance

[0169] I. Fusion Protein His-MP7 Can Enhance the Resistance of Plants to Pathogen DC3000

[0170] Experimental procedure:

[0171] (1) Inoculate the Pseudomonas syringae DC3000 strain into 20 mL of SOC + str (streptomycin) liquid medium, culture overnight at 28 °C for 14 - 16 hours, measure the OD600 value, and dilute serially until OD600 = 0.00005 to obtain the bacterial solution;

[0172] (2) Add the fusion protein His - MP7 to the bacterial solution to a final concentration of 100 nM, using the bacterial solution with water added as the positive control. Inject the leaves of four - week - old healthy Arabidopsis thaliana at the same time, four leaves per plant (T0), two leaves per of five plants (T3). Sample on the 0th day and the third day respectively. Punch holes in each leaf with a hole puncher, take one small round piece of the punched leaf. Add 500 μL of 10 mM MgCl2 on the 0th day, grind it in a 1.5 mL ep tube. For four samples of the same treatment, take 50 μL from each and spot them on the same SOC + str (streptomycin) solid plate (the plate must be dried to maintain the spotting shape), culture at 28 °C for 16 - 24 hours, take pictures, and observe and count the colony growth;

[0173] (3) Use two leaves per of five plants on the T3 day as a sample. Similarly, punch holes to take small round pieces, add 250 μL of 10 mM MgCl2, grind it in a 1.5 mL ep tube and dilute it step - by - step with MgCl2 to 1×10 -5 . For five samples of the same treatment, including each diluted sample (30 samples for one treatment), take 10 μL from each and spot them on the same SOC + str (streptomycin) solid plate (use a square dish, the plate must be dried to maintain the spotting shape), culture at 28 °C for 16 - 24 hours, take pictures, and observe and count the colony growth.

[0174] The experimental results are as shown in the bar chart of Figure 5 : The bacterial growth index of the control group is 5.38, and the bacterial growth index of Arabidopsis thaliana treated with 100 nM of the fusion protein His - MP7 is 4.05. The growth amount of Pseudomonas syringae DC3000 has decreased by more than 10%. His - MP7 can effectively enhance the immune ability of plants against pathogenic bacteria.

[0175] II. Fusion Protein His-MP7 Can Enhance the Resistance of Plants to Fusarium graminearum

[0176] Experimental Procedure:

[0177] (1) Transfer the mycelium of Fusarium graminearum from the plate to CMC liquid medium, culture in the dark at 25 °C for 3 - 7 days, filter with gauze, centrifuge at 10000 rpm / 10 min to collect spores, count the spore number with a hemocytometer, and adjust the concentration to 2×10 5 , and store it at 4 °C (for use within one month);

[0178] (2) Take corn leaves at two weeks old, cut the middle part into 5 cm lengths and immerse them in a 1 μg / mL auxin 6-BA solution. Take 12 - 13 leaves for each treatment, add the fusion protein His-MP7 at a final concentration of 100 nM, and use the aqueous solution of auxin 6-BA as the blank control;

[0179] (3) Uniformly spot the Fusarium graminearum spore liquid on the leaves respectively, and culture them for 3 - 4 days at 28℃ under the conditions of 12-hour light and 12-hour darkness. Observe the disease incidence, and statistically analyze the percentage of lesion area through the imageJ software.

[0180] The experimental results are as Figure 6 shown in a of the figure, which shows the measured comparison of corn leaf growth, and b shows the bar chart: the percentage of lesion area of the leaves without the treatment of the fusion protein His-MP7 is 12.3%, and the percentage of lesion area of the leaves treated with the fusion protein His-MP7 is 3.6%. It shows that the fusion protein His-MP7 significantly improves the resistance of corn to Fusarium graminearum, reducing the infection rate of Fusarium graminearum by 70.8%.

[0181] III. Fusion Protein His-MP7 Can Enhance the Resistance of Plants to Magnaporthe oryzae

[0182] Experimental Procedure:

[0183] (1) Inoculate Magnaporthe oryzae on the CM solid medium, culture it upright at 28℃ for 13 - 15 days, scrape all the mycelia with a pipette tip, take a small amount and rinse it with 5 - 10 mL of sterile water, filter it with gauze and place it in a 50 mL centrifuge tube, centrifuge at 10000 rpm / 10 min to collect the spores, count the number of spores with a hemocytometer, and adjust the concentration to 1×10 6 , and store it at room temperature (for use within one week);

[0184] (2) Take rice leaves at four weeks old, cut the middle part into 5 cm lengths and immerse them in a 1 μg / mL auxin 6-BA solution. Take 12 - 13 leaves for each treatment, add the fusion protein His-MP7 at a final concentration of 100 nM, and use the aqueous solution of auxin 6-BA as the blank control;

[0185] (3) Uniformly spot the Magnaporthe oryzae spore liquid on the leaves respectively, and culture them for 3 - 4 days at 28℃ under the conditions of 12-hour light and 12-hour darkness. Observe the disease incidence, and statistically analyze the percentage of lesion area through the imageJ software.

[0186] The experimental results are as Figure 7In Figure a, the measured comparison of rice leaf growth is shown, and in Figure b, the bar graph shows that: the percentage of leaf lesion area in the group without treatment with the fusion protein His-MP7 was 11.6%, and the percentage of leaf lesion area in the group treated with the fusion protein His-MP7 was 1.9%. This indicates that the fusion protein His-MP7 significantly improved the resistance of rice to rice blast, reduced the infection rate of Magnaporthe oryzae by 83.6%, and reduced the damage of rice blast to rice.

[0187] IV. The fusion protein His-MP7 can enhance the resistance of tobacco to tobacco mosaic virus (TMV)

[0188] The experiment was set up with an experimental group and a control group, with 20 tobacco plants in each group. In the experimental group, the virus was inoculated after injecting the recombinant protein, and in the control group, the virus was inoculated after injecting sterile water. The inoculation method is as follows:

[0189] (1) Add a small amount of sterilized phosphate buffer (1:200) to fresh TMV-infected leaves and grind them in a mortar. Filter the leaf debris with sterilized gauze, take out the fresh juice to make an inoculum, and adjust the concentration of the TMV virus inoculum to obtain an aqueous solution of the TMV virus;

[0190] (2) When the tobacco seedlings are at the stage of having 4 - 5 true leaves, select fully expanded true leaves, evenly sprinkle an appropriate amount of quartz sand on the leaf surface, dip a degreased cotton ball in the aqueous solution of the TMV virus and gently rub it 1 - 2 times, and then immediately rinse the leaf surface with water;

[0191] (3) 21 days after inoculation, observe the disease incidence of tobacco plants. Taking each plant as a unit, the grading standard is as follows:

[0192] Grade 0: Disease-free;

[0193] Grade 1: A small amount of chlorotic yellow spots appear along the veins at the base of the heart leaf, without curling;

[0194] Grade 3: Yellow-green stripes parallel to the veins appear on the newly emerged leaves, with slight curling;

[0195] Grade 5: A large number of chlorotic stripes parallel to the veins appear on the newly emerged leaves, with the leaves curling and becoming thin and weak;

[0196] Grade 7: The plants are dwarfed, the leaves show yellow-white stripes and curl up, the newly emerged leaves are twisted and drooping, and cannot open normally;

[0197] Grade 9: The plants are severely dwarfed, chlorotic or dead.

[0198] Calculate the disease index and control effect. The calculation methods for the disease index and control effect are as follows:

[0199] Disease index = [Σ(number of diseased plants at each level × relative level value) / (total number of plants surveyed × 9)] × 100

[0200] Control effect (%) = [(control disease index - treated disease index) / control disease index] × 100.

[0201] The results are shown in Table 1 and Table 2:

[0202] Table 1 Number of tobacco diseased plants and disease grading

[0203] Unit (plant) Grade 0 Grade 1 Grade 3 Grade 5 Grade 7 Grade 9 <![CDATA[H2O]]> 0 0 2 2 9 7 His-MP7 1 11 6 2 0 0

[0204] Table 2 Tobacco disease index and control effect

[0205]

[0206] The experimental results show that after treatment with the fusion protein His-MP7, the disease index of the experimental group decreased to 21.7, and the control effect reached 72.5%. The experiment proved that the fusion protein His-MP7 can enhance the resistance of tobacco to TMV.

[0207] Example 5 Molecular design, expression and purification of multiple tripeptide fusion proteins

[0208] I. Molecular Design of Multiple Tripeptide Fusion Proteins

[0209] (1) Among 11 different PAMP molecular polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15, sys18 and their homologous mutants, three polypeptides were randomly selected to form multiple fusion proteins, with the linker being AKG, to obtain multiple design schemes;

[0210] (2) Use the online analysis platform ExPASy to analyze the basic properties such as molecular mass, amino acid composition, and hydrophobicity of the above protein sequences; use the Phyre2 online platform to model the structures of the above fusion proteins;

[0211] (3) Combining the analysis results, 20 design schemes were screened out and named TP1, TP2, TP3, TP4, TP5, TP6, TP7, TP8, TP9, TP10, TP11, TP12, TP13, TP14, TP15, TP16, TP17, TP18, TP19, TP20 respectively. The composition design schemes of multiple tripeptide fusion proteins are shown in Table 3:

[0212] Table 3 Design schemes of tripeptide fusion proteins

[0213] Name Sequence (Linker AKG) Name Sequence (Linker AKG) TP1 flg22-csp22-pep13 TP11 <![CDATA[flg22 m2 -flg22 m2 -flg22 m2 > TP2 flg22-elf18-pep1 TP12 <![CDATA[flg22 m2 -nlp20 m1 -hrp15]]> TP3 flg22-elf18-pip1 TP13 flg22-nlp20-csp22 TP4 flg22-flg22-flg22 TP14 <![CDATA[flg22-nlp20 m1 -pep1]]> TP5 flg22-flgII-28-csp22 TP15 <![CDATA[flg22-nlp20 m2 -csp22 m1 > TP6 flg22-flgII-28-nlp20 TP16 flg22-pep1-pip1 TP7 flg22-hrp15-sys18 TP17 <![CDATA[flg22-ralf17 m1 -hrp15]]> TP8 <![CDATA[flg22 m1 -flgII-28 m1 -flg22 m2 > TP18 flg22-ralf17-hrp24 TP9 <![CDATA[flg22 m1 -flg22 m1 -flg22 m1 > TP19 flg22-ralf17-pip1 TP10 <![CDATA[flg22 m1 -ralf17 m1 -csp22 m1 > TP20 flg22-ralf17-sys18

[0214] (4) The method for obtaining the nucleotide sequences encoding the above 20 tripeptide fusion proteins is the same as that in Example 1;

[0215] II. Expression and Purification of Multiple Tripeptide Fusion Proteins

[0216] (1) The nucleotide sequences encoding the above 20 kinds of tripeptide fusion proteins were respectively cloned into the BamHI and XholI sites of the pEGX-4T-1 expression vector, heat-shock transformed into Escherichia coli DH5α, positive clones were picked, cultured in a shaker and the plasmids were extracted. After digestion with enzymes and correct sequencing verification, it was heat-shock transformed into Escherichia coli BL21(DE3). The Escherichia coli containing the recombinant plasmid pEGX-4T-1-TP was named BL21(DE3) / pEGX-4T-1-TP;

[0217] (2) The above Escherichia coli was induced for expression, including the following steps: The expression strain was inoculated into LB liquid medium and cultured overnight at 37°C with shaking at 200 rpm / min to obtain the first bacterial solution; The overnight bacterial solution was transferred to LB liquid medium containing 100 μg / mL ampicillin at a volume ratio of 1:100, and continued to shake at 37°C and 200 rpm / min until the OD600nm value of the bacterial solution concentration was 0.6. Then, IPTG with a final concentration of 0.3 mmol / L was added, and it was cultured with shaking at 25°C and 200 rpm / min for 12 h to obtain the second bacterial solution; The second bacterial solution was centrifuged at 12000 rpm / min, the cells were collected and added with PBS buffer. After ultrasonic disruption of the cells, it was centrifuged at 4°C and 12000 rpm / min, and the supernatant was collected;

[0218] (3) The supernatant was purified using a GST-tagged protein purification kit (soluble protein) to obtain a GST-TP fusion protein solution. The protein concentration of the protein solution was quantified using a BCA method protein concentration assay kit.

[0219] Example 6 Molecular Design, Expression and Purification of Multiple Kinds of Tetrapeptide Fusion Proteins

[0220] I. Molecular Design of Multiple Tetrapeptide Fusion Proteins

[0221] Among 11 different PAMP molecular polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15, sys18 and their homologous mutants, four polypeptides were randomly selected to form a fusion protein, the linker was AKG, and the design method was the same as that in Example 5. Twenty design schemes were screened and named FP1, FP2, FP3, FP4, FP5, FP6, FP7, FP8, FP9, FP10, FP11, FP12, FP13, FP14, FP15, FP16, FP17, FP18, FP19, FP20 respectively. The composition design schemes of multiple kinds of tetrapeptide fusion proteins are shown in Table 4:

[0222] Table 4 Design schemes of tetrapeptide fusion proteins

[0223] Name Sequence (Linker AKG) Name Sequence (Linker AKG) FP1 flg22-csp22-pep13-ralf17 FP11 <![CDATA[flg22 m1 -elf18 m1 -nlp20 m2 -csp22 m1 > FP2 flg22-elf18-cap22-nlp20 FP12 <![CDATA[flg22 m1 -flg22 m1 -flg22 m1 -elf18]]> FP3 flg22-elf18-pep1-pip1 FP13 <![CDATA[flg22 m1 -nlp20 m2 -csp22 m1 -pip1 m1 > FP4 flg22-elf18-pip1-pep1 FP14 <![CDATA[flg22 m1 -ralf17 m1 -csp22-ralf17]]> FP5 <![CDATA[flg22-elf18-nlp20 m2 -csp22 m1 > FP15 <![CDATA[flg22 m2 -flg22 m2 -flg22 m2 -pip1 m1 > FP6 flg22-flg22-flg22-flg22 FP16 <![CDATA[flg22 m2 -nlp20 m1 -csp22-hrp15]]> FP7 flg22-flgII-28-csp22-hrp24 FP17 <![CDATA[flg22-ralf17 m1 -hrp15-flg22]]> FP8 flg22-flgII-28-nlp20-hrp24 FP18 flg22-ralf17-pep1-csp22 FP9 <![CDATA[flg22-flgII-28-nlp20 m1 -csp22 m1 > FP19 flg22-ralf17-pip1-pep1 FP10 <![CDATA[flg22-hrp15-sys18-sys18 m1 > FP20 flg22-ralf17-sys18-hrp15

[0224] The method for obtaining the nucleotide sequences encoding the above 20 tetrapeptide fusion proteins is the same as that in Example 1.

[0225] II. Expression and Purification of Multiple Tetrapeptide Fusion Proteins, the same as Example 5

[0226] Example 7 Molecular design, expression and purification of various pentapeptide fusion proteins

[0227] I. Molecular Design of Multiple Tetrapeptide Fusion Proteins

[0228] Among 11 different PAMP molecular polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15, sys18 and their mutants, five polypeptides were randomly selected to form a fusion protein, with the linker being AKG. The design method is the same as that in Example 5. Twenty design schemes were screened and named MP5-1, MP5-2, MP5-3, MP5-4, MP5-5, MP5-6, MP5-7, MP5-8, MP5-9, MP5-10, MP5-11, MP5-12, MP5-13, MP5-14, MP5-15, MP5-16, MP5-17, MP5-18, MP5-19, MP5-20 respectively. The composition design schemes of various pentapeptide fusion proteins are shown in Table 5:

[0229] Table 5 Design schemes of pentapeptide fusion proteins

[0230]

[0231]

[0232] The method for obtaining the nucleotide sequences encoding the above 20 pentapeptide fusion proteins is the same as that in Example 1.

[0233] II. Expression and Purification of Multiple Pentapeptide Fusion Proteins, the same as Example 5

[0234] Example 8 Molecular design of various hexapeptide and heptapeptide fusion proteins

[0235] Among 11 different PAMP molecular polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15, sys18 and their mutants, six were randomly selected to form a variety of fusion proteins with the linker AKG, and the design method was the same as in Example 5. Twenty design schemes were screened and named MP6-1, MP6-2, MP6-3, MP6-4, MP6-5, MP6-6, MP6-7, MP6-8, MP6-9, MP6-10, MP6-11, MP6-12, MP6-13, MP6-14, MP6-15, MP6-16, MP6-17, MP6-18, MP6-19, MP6-20 respectively. The composition design schemes of a variety of heptapeptide fusion proteins are shown in Table 6:

[0236] Table 6 Design schemes of heptapeptide fusion proteins

[0237]

[0238]

[0239] The method for obtaining the nucleotide sequences encoding the above 20 heptapeptide fusion proteins was the same as in Example 1.

[0240] II. Multiple Six Peptide Fusion Protein Expression and Purification, the same as Example 5

[0241] Example 9 Molecular design, expression and purification of a variety of heptapeptide fusion proteins

[0242] Among 11 different PAMP molecular polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15, sys18 and their mutants, seven polypeptides were randomly selected to form a fusion protein with the linker AKG, and the design method was the same as in Example 5. Twenty design schemes were screened and named MP7-1, MP7-2, MP7-3, MP7-4, MP7-5, MP7-6, MP7-7, MP7-8, MP7-9, MP7-10, MP7-11, MP7-12, MP7-13, MP7-14, MP7-15, MP7-16, MP7-17, MP7-18, MP7-19, M7-20 respectively. The composition design schemes of a variety of heptapeptide fusion proteins are shown in Table 7:

[0243] Table 7 Design schemes of heptapeptide fusion proteins

[0244]

[0245] The method for obtaining the nucleotide sequences encoding the above 20 heptapeptide fusion proteins is the same as that in Example 1.

[0246] II. Expression and purification of various heptapeptide fusion proteins, which are the same as in Example 5 。

[0247] Example 10 Detection of the Immune Responses of Different Fusion Proteins

[0248] Using the model plant Arabidopsis thaliana as the material, with callose accumulation as the immune index and water as the blank control, the final concentrations of the solutions of all the fusion proteins obtained in Examples 5 - 9 above were adjusted to 100 nM, and the four-week-old Arabidopsis thaliana leaves were infected by syringe infiltration method. The specific experimental operation was the same as that in Example 3. For the obtained fluorescence images of callose accumulation, the fluorescence density was calculated by the image processing software ImageJ to quantify the immune activation ability of the fusion proteins on plants. The calculation method is as follows:

[0249] Measure IntDen = integrated density = integrated optical density;

[0250] Measure Area = picture area;

[0251] MD (mean optical density) = IntDen / Area.

[0252] The experimental results are shown in Table 8:

[0253] Table 8: Identification of the Immune Response Intensities of Multiple Fusion Proteins

[0254]

[0255]

[0256] From the experimental results in Table 8, it can be seen that compared with the blank control group, various fusion proteins such as various tripeptides, various tetrapeptides, various pentapeptides, various hexapeptides, and various heptapeptides provided in Examples 5 - 9 all have immune activation ability.

Claims

1. A fusion protein, characterized in that, The fusion protein consists of seven different PAMP molecular polypeptides, and there is at least one linker or no linker between adjacent two PAMP molecular polypeptides; the seven different PAMP molecular polypeptides are selected from flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28; the amino acid sequence of the fusion protein is as shown in SEQ ID NO.

15.

2. Nucleotides encoding the fusion protein according to claim 1.

3. The nucleotide encoding the fusion protein according to claim 2, wherein The nucleotides are as shown in SEQ ID NO.

16.

4. A vector into which the nucleotides according to any one of claims 2-3 are introduced.

5. A microorganism or cell into which the nucleotides according to any one of claims 2-3, and / or the vector according to claim 4 are introduced.

6. The microorganism or cell according to claim 5, characterized in that, The microorganism or cell includes one or more of Escherichia coli, Agrobacterium, Lactobacillus, Saccharomyces cerevisiae or Bacillus subtilis.

7. The microorganism or cell according to claim 6, characterized in that, The microorganism or cell is Escherichia coli.

8. A plant immune inducer containing the fusion protein according to claim 1, or the vector according to claim 4, or the microorganism or cell according to any one of claims 5-7.

9. The plant immune inducer according to claim 8, wherein The plant immune inducer further includes one or more agriculturally acceptable carriers, excipients, diluents or solvents.

10. The plant immune elicitor according to claim 8, characterized in that, The dosage form of the plant immune inducer is selected from the group consisting of powder, granule, aqueous solution, microemulsion, suspension and water dispersible granule.

11. A method for preparing the fusion protein according to claim 1, characterized in that, It includes the step of culturing the microorganism or cell according to any one of claims 5-7, or includes the step of artificially synthesizing the fusion protein according to claim 1.

12. The preparation method according to claim 11, wherein, The method includes the following steps: (a) Synthesize the nucleotides according to claims 2-3; (b) Transform the synthesized nucleotides into a microorganism or cell, and culture the microorganism or cell to express the fusion protein; and, (c) Collect and purify the expressed fusion protein.

13. The preparation method according to claim 12, wherein, Before synthesis, the nucleotides in step (a) are analyzed and designed to splice into nucleotides encoding the fusion protein according to claim 1.

14. The preparation method according to claim 12, wherein, The sequence transformation of the nucleotides in step (b) is carried out by vector transformation.

15. Use of the fusion protein according to claim 1, or the plant immune inducer according to any one of claims 8-10, or the fusion protein prepared by the preparation method of the fusion protein according to any one of claims 11-14 in improving plant disease resistance, inducing plant defense responses and / or resisting plant pathogenic microorganisms; the plants are selected from Arabidopsis thaliana, maize, rice, tobacco; the pathogenic microorganisms are selected from Pseudomonas syringae, Fusarium graminearum, Magnaporthe oryzae, Tobacco mosaic virus.

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