WO2021115392A1 - 融合蛋白、其氨基酸序列、编码核苷酸序列、制备方法和应用 - Google Patents

融合蛋白、其氨基酸序列、编码核苷酸序列、制备方法和应用 Download PDF

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WO2021115392A1
WO2021115392A1 PCT/CN2020/135367 CN2020135367W WO2021115392A1 WO 2021115392 A1 WO2021115392 A1 WO 2021115392A1 CN 2020135367 W CN2020135367 W CN 2020135367W WO 2021115392 A1 WO2021115392 A1 WO 2021115392A1
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polypeptide
fusion protein
flg22
pamp
immune
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French (fr)
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蔡易
李琦
郭晋雅
李雍
杨苹苹
周鑫琼
张丽梅
张怀渝
汪向荣
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四川农业大学
成都绿信诺生物科技有限公司
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Priority to US17/757,280 priority Critical patent/US20230036503A1/en
Publication of WO2021115392A1 publication Critical patent/WO2021115392A1/zh

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H3/00—Processes for modifying phenotypes, e.g. symbiosis with bacteria
    • A01H3/04—Processes for modifying phenotypes, e.g. symbiosis with bacteria by treatment with chemicals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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Definitions

  • the invention relates to the field of agricultural biotechnology, in particular to a fusion protein, its amino acid sequence, coding nucleotide sequence, preparation method and application.
  • biopesticide products In the agricultural ecosystem, compared with chemical pesticides, biopesticide products have the advantages of low toxicity, short degradation cycle, and greater environmental compatibility, and have been widely used in crop production.
  • traditional biological pesticides mainly focus on screening antibacterial and insecticidal substances from biological sources, and then develop biological pesticides to fight diseases and insect pests, often neglecting the role of plant self-immunity in the process of resisting diseases and insect pests.
  • plant immunity induction technology has become a new bright spot in the development of biological pesticides.
  • Plant immune antagonists are also called "plant vaccines", which stimulate the plant's own immune system to fight diseases, increase production, and improve quality.
  • plant vaccines will not cause resistance to pathogenic microorganisms and are more in line with the requirements of green and healthy agricultural production, which has attracted widespread attention and attention at home and abroad.
  • the PTI (PAMP-Triggered Immunity) immune mechanism of plants has broad prospects in the development of biological pesticides.
  • PAMP-Triggered Immunity The PTI (PAMP-Triggered Immunity) immune mechanism of plants has broad prospects in the development of biological pesticides.
  • Atelin and Harpin protein the products have considerable limitations: first, their receptors are not yet clear, and it is difficult to make scientific and effective guidance on their use. The scope of application and use concentration can only be judged by experimental experience; second, the plant The sensitivity to these plant-immune pesticides is low, the use concentration is large and the cost is high.
  • Axiom Harpin Protein is one of the most widely used biological protein pesticides in the world.
  • the unit price of its pure protein products is more than RMB 5,000 per gram, and the price of its 1% protein pesticide products is also more than RMB 140 per gram, which is extremely costly. Greatly restricts its application in agricultural production. Therefore, it is necessary to develop a biopesticide product with low price and good effect of improving plant's own immunity.
  • the present invention is dedicated to providing a fusion protein that assembles multiple PAMP molecular polypeptides into a fusion protein with multiple or multiple immune epitopes.
  • the fusion protein can interact with plant cell surfaces more quickly and widely. Receptor binding induces a variety of plants to produce an immune response, which significantly improves plant disease resistance.
  • the cost of preparing fusion proteins is lower than that of PAMP molecular polypeptides, and the unit yield of protein biopesticide products is increased. It is estimated that the cost of protein can be Reduced to 1 yuan/g, the use concentration is low, the effect is quick, and the application in agriculture can significantly reduce the agricultural production cost.
  • the first aspect of the present invention provides a fusion protein comprising or at least three, four, five, six, seven identical and/or different PAMP molecule polypeptides, and optionally, adjacent There is at least one linker or no linker between two PAMP molecule polypeptides
  • PAMPs Pathogen-associated patterns
  • PAMP molecules include polysaccharides, lipids, peptides and other molecular substances.
  • Polypeptide PAMP molecules are called PAMP molecular polypeptides.
  • Most PAMP molecules are essential components of the life cycle of pathogenic bacteria, and are often used by plants as signal molecules to perceive pathogenic bacteria. Studies have shown that the interaction of specific PAMP molecular polypeptides with receptor proteins on plant cell membranes can quickly trigger the plant’s defense response to resist infection by pathogenic bacteria.
  • the immune response of plant defense includes: inducing the rapid generation of active oxygen in plants and inducing the deposition of callose; the synthesis of plant hormones (such as salicylic acid, ethylene) in large quantities; the rapid expression of defense genes.
  • plant hormones such as salicylic acid, ethylene
  • certain autocrine peptides such as PIP1 and PEP1 produced by plants after stimulation are also PAMP molecular polypeptides, which can be used as second messengers to enhance immune signals and sustain immune effects.
  • the method of obtaining PAMP molecular peptides is usually by comparing and analyzing homologous sequences in a variety of pathogenic fungi or bacteria to find relatively conserved sequences in protein molecules, and then artificially synthesize the peptide sequences, and verify that they activate the plant immune response. ability.
  • PAMP molecular polypeptide mutants can maintain no less than 80% immunogenicity of wild-type PAMP molecular polypeptides, and can be activated the same as PAMP molecular polypeptides. And fixed immune receptors.
  • the fusion protein contains or consists of at least three, four, five, six, seven identical and/or different PAMP molecular polypeptides" can be understood as three interpretations:
  • the fusion protein comprises or consists of at least three, four, five, six, seven PAMP molecular polypeptides of the same kind, that is, including but not limited to or consisting of three, four, five, six One, seven or more peptides of the same kind of PAMP molecule;
  • the fusion protein comprises or consists of at least three, four, five, six, seven different kinds of PAMP molecular polypeptides, that is, including but not limited to or consisting of three, four, five, six One, seven and more different types of PAMP molecule peptide composition;
  • the fusion protein comprises or consists of at least three, four, five, six, seven peptides of the same and different types of PAMP molecules, including but not limited to or consisting of two identical and one different, two Two are the same and two different, two are the same and three different, two are the same and four different, two are the same and five different, two are the same and six different, or two are the same and seven different, and more
  • the same and different types of PAMP molecules are composed of peptides.
  • “Optionally” means there may or may not be present, 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 one or more amino acid residues, preferably at least 3 consecutive amino acid residues.
  • the linker connects two adjacent PAMP molecule polypeptides, so that at least three polypeptides are assembled into a fusion protein.
  • Linkers include but are not limited to: GAG, AGA, AAA, GGG, KRK, KKK, RRR, AKG.
  • the arrangement order of the PAMP molecular polypeptides constituting the fusion protein and the specific types of linkers are not specifically limited here, and appropriate changes, substitutions and adjustments can be made according to the types of plants or the types of pathogenic microorganisms targeted. As long as it can achieve the effect of inducing plant immune resistance.
  • the fusion protein has multiple and/or multiple immune epitopes. It can bind to the receptors on the surface of plant cells more quickly and widely, induce the immune response of plant defense, reduce the infection ability of pathogenic microorganisms, and obviously improve the disease resistance of plants.
  • the fusion protein as a biopesticide product also has the advantages of low toxicity, short degradation cycle, large environmental compatibility, and no resistance to pathogenic bacteria.
  • the fusion protein is mainly composed of at least three, four, five, six, seven mutants of the same and/or different PAMP molecule polypeptides, and two adjacent PAMP molecule polypeptides There is at least one linker between the mutants.
  • the PAMP molecule polypeptide includes the first polypeptide that activates the FLS2 immune receptor, the second polypeptide that activates the RLP23 immune receptor, the third polypeptide that activates the EFR immune receptor, and the fourth polypeptide that activates the RLK7 immune receptor, Activate the fifth polypeptide of PEPR1 immune receptor, activate the sixth polypeptide of CORE1 immune receptor, activate the seventh polypeptide of FLS3 immune receptor, activate the eighth polypeptide of FER receptor, and activate the ninth polypeptide of plant immune response Polypeptide pep13, the tenth polypeptide hrp24 which activates the plant immune response, and the eleventh polypeptide sys18 which activates the plant immune response.
  • the fusion protein contains multiple polypeptide components that activate different receptors, and has multiple advantages.
  • a variety of different signal pathways can activate the immune response of different plants, avoiding the lack of specific receptors in certain plants that cannot produce an immune response, and the scope of application to plants is wider.
  • the immune signals generated by them have a superimposed effect, and the immune activation is more sensitive and efficient.
  • the first polypeptide that activates the FLS2 immune receptor is the polypeptide flg15 and its homologous mutants; or the polypeptide flg22 and its homologous mutants.
  • flg15 The amino acid sequence of flg15 is shown in SEQ ID NO. 1: RINSAKDDAAGLQIA.
  • Homologous mutants of flg15 include a PAMP polypeptide that adds, deletes, or replaces one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO.1 and can activate the FLS2 immune receptor.
  • flg22 is a highly conserved area at the N-terminal of bacterial flagellin. Many studies have shown that flg22 can induce natural immunity in plants, activate FLS2 receptors, and act on signal pathways such as SA and MAPK, and is important for plant disease resistance. Impact.
  • the amino acid sequence of flg22 is shown in SEQ ID NO. 2: QRLSTGSRINSAKDDAAGLQIA.
  • Homologous mutants of flg22 include polypeptides that add, delete, or replace one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO. 2 and have the ability to activate FLS2 immune receptors.
  • the first polypeptide that activates the FLS2 immune receptor is preferably flg22 and its homologous mutants.
  • the homologous mutant flg22 m1 of flg22 has 4 amino acid substitution mutations at position 1, position 5, position 7, and position 8 of the amino acid sequence shown in SEQ ID NO. 2: Q1T, T5S , S7L, R8K;
  • the homologous mutant flg22 m2 of flg22 has 4 amino acid substitution mutations in the 5th, 7th, 20th, and 22nd positions of the amino acid sequence shown in SEQ ID NO.2: T5S, S7L, Q20A, A22S.
  • the first polypeptides with different amino acid sequences above are all homologous mutants with the same function. They can activate the FLS2 immune receptor and have been shown to have similar biological activities. For specific information, please refer to the article "Plants have a sensitive perception system for” The introduction of the peptides that activate the FLS2 immune receptor in the “most conserved domain of bacterial flagellin” and “CD2-1, the C-Terminal Region of Flagellin, Modulates the Induction of Immune Response in Rice”.
  • the second polypeptide that activates the RLP23 immune receptor is preferably the polypeptide nlp20 and its homologous mutants.
  • nlp20 is a polypeptide molecule composed of 20 amino acids, which is a characteristic amino acid sequence contained in the necrosis and ethylene-induced peptide-like protein family (NLPs). Studies have shown that nlp20 polypeptide can quickly cause plant immune response by activating the immune recognition receptor RLP23, and enhance the plant's immunity to microbial infections.
  • NLPs ethylene-induced peptide-like protein family
  • nlp20 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 replace one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO. 3 and have the ability to activate RLP23 immune receptors.
  • the homologous mutant nlp20 m1 of nlp20 has 3 amino acid substitution mutations in the 8th, 14th and 17th positions of the amino acid sequence shown in SEQ ID NO.3: F8M, V14S, L17I; nlp20
  • the homologous mutant nlp20 m2 has 3 amino acid substitution mutations at position 5, 14 and 15 of the amino acid sequence shown in SEQ ID NO.3: S5A, V14S, and T15P.
  • the above-mentioned second polypeptides with different amino acid sequences are all homologous mutants with the same function, which can activate the RLP23 immune receptor, and have been shown to have similar biological activities.
  • the third polypeptide that activates the EFR immune receptor is preferably the polypeptide elf18 and its homologous mutants.
  • elf18 is a polypeptide of 18 amino acids at the N-terminus of the bacterial protein elongation factor Tu (EF-Tu), which can activate the receptor EFR, induce the oxidation burst and biosynthesis of ethylene, and trigger resistance to subsequent infection with pathogenic bacteria.
  • EF-Tu bacterial protein elongation factor Tu
  • elf18 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 replace one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO. 4 and have the ability to activate EFR immune receptors.
  • the homologous mutant elf18 m1 of elf18 has 4 amino acid substitution mutations at position 1, position 3, position 8, and position 14 of the amino acid sequence shown in SEQ ID NO.4: S1A, E3S , T8N, V14I;
  • the homologous mutant elf18 m2 of elf18 produces 5 amino acids at position 1, 6, 8, 9, and 12 of the amino acid sequence shown in SEQ ID NO.4 Replacement mutations: S1V, E6D, T8S, K9L, V12C.
  • third polypeptides with different amino acid sequences are all homologous mutants with the same function, which can activate EFR immune receptors and have been shown to have similar biological activities.
  • the article “The N Terminus of Bacterial Elongation Factor Tu Introduction to peptides that activate EFR immune receptors in Elicits Innate Immunity in Arabidopsis Plants.
  • the fourth polypeptide that activates the RLK7 immune receptor is preferably the polypeptide pip1 and its homologous mutants.
  • pip1 is a 13-amino acid polypeptide that is secreted by prePIP1 into the extracellular space and cleaved in the conserved C-terminal region. pip1 sends a signal to activate receptor RLK7 through receptor-like kinase 7 (RLK7) on the cell surface, thereby activating the immune response of plants. Enhance the pathogen resistance of plants.
  • the amino acid sequence of pip1 is shown in SEQ ID NO. 5: RLASGPSPRGPGH.
  • Homologous mutants of pip1 include PAMP polypeptides that have added, deleted, or replaced one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO. 5 and have the ability to activate RLK7 immune receptors.
  • the homologous mutant pip1 m1 of pip1 adds two amino acid FVs between the first and second positions of the amino acid sequence shown in SEQ ID NO. 5, and the second, third, and ninth positions of the amino acid sequence shown in SEQ ID NO. There are 3 amino acid substitution mutations.
  • the fourth polypeptides with different amino acid sequences above are homologous mutants with the same function. They can activate RLK7 immune receptors and have been shown to have similar biological activities.
  • the Secreted Peptide PIP1 Amplifies Immunity Through Receptor- The introduction of peptides that activate RLK7 immune receptors in "Like Kinase 7".
  • the fifth polypeptide that activates the PEPR1 immune receptor is preferably the polypeptide pep1 and its homologous mutants.
  • Pep1 is an endogenous molecule derived from a peptide containing 23 amino acids at the C-terminus of the precursor protein proPEP1, which can activate the receptor PEPR1 and activate the autoimmunity of Arabidopsis plants.
  • pep1 The amino acid sequence of pep1 is shown in SEQ ID NO. 6: ATKVKAKQRGKEKVSSGRPGQHN.
  • Homologous mutants of pep1 include PAMP polypeptides that add, delete, or replace one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO. 6 and have the ability to activate PEPR1 immune receptors.
  • the homologous mutant pep1 m1 of pep1 is at position 1, position 4, position 5, position 6, position 8, position 10, and position 11 of the amino acid sequence shown in SEQ ID NO.6 ,
  • the 12th and 13th positions have 9 amino acid substitution mutations: A1E, V4A, K5R, A6G, Q8N, G10T, E11P, K12T, V13P;
  • the homologous mutant pep1 m2 of pep1 is shown in SEQ ID NO.6
  • the amino acid sequence shown has an eight amino acid deletion before the 9th position;
  • the homologous mutant pep1 m2 of pep1 has an eight amino acid deletion before the 9th position of the amino acid sequence shown in SEQ ID NO.
  • the fifth polypeptides with different amino acid sequences are all homologous mutants with the same function. They can activate PEPR1 immune receptors and have been shown to have similar biological activities. For specific information, please refer to the article "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 immune immune response” for the introduction of peptides that activate the PEPR1 immune receptor.
  • the sixth polypeptide that activates the CORE1 immune receptor may be the polypeptide csp15 and its homologous mutants; or the polypeptide csp22 and its homologous mutants.
  • the amino acid sequence of csp15 is shown in SEQ ID NO. 7: VKWFNAEKGFGFITP.
  • the homologous mutant of csp15 includes a PAMP polypeptide that adds, deletes, or replaces one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO. 7, and has a PAMP polypeptide that activates the CORE1 immune receptor.
  • Csp22 is a 22 amino acid polypeptide in the conserved domain of bacterial cold shock protein (CSP). It can activate the receptor CORE1 and efficiently induce tobacco defense responses.
  • the amino acid sequence of csp22 is shown in SEQ ID NO. 8: AVGTVKWFNAEKGFGFITPDDG.
  • Homologous mutants of csp22 include polypeptides that add, delete, or replace one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO. 8 and have the ability to activate CORE1 immune receptors.
  • the sixth polypeptide that activates the CORE1 immune receptor is preferably csp22 and its mutants.
  • the homologous mutant csp22 m1 of csp22 has an amino acid substitution mutation at position 11 of the amino acid sequence shown in SEQ ID NO. 8: E11A; the homologous mutant csp22 m2 of csp22 is in SEQ ID NO.
  • the sixth peptides with different amino acid sequences above are all homologous mutants with the same function. They can activate the CORE1 immune receptor and have been shown to have similar biological activities.
  • the highly conserved rna-binding motif rnp" -1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco” for the introduction of peptides that activate the CORE1 immune receptor.
  • the seventh polypeptide that activates the FLS3 immune receptor is preferably the polypeptide flgII-28 and its homologous mutants.
  • Both flgII-28 and flg22 are highly conserved regions at the N-terminus of bacterial flagellin. They are the main PAMP recognized by plants and can activate the receptor FLS3. flgII-28 can stimulate plants to increase the production of stress hormone ethylene and rapid ROS. Produce and activate the plant's immune response.
  • flgII-28 The amino acid sequence of flgII-28 is shown in SEQ ID NO. 9: ESTNILQRMRELAVQSRNDSNSATDREA.
  • Homologous mutants of flgII-28 include PAMP polypeptides that add, delete, or replace one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO. 9 and have the ability to activate FLS3 immune receptors.
  • the homologous mutant flgII-28 m1 of flgII-28 has 2 amino acid substitution mutations at positions 23 and 27 of the amino acid sequence shown in SEQ ID NO.9: A23S, E27D; flgII-28
  • the homologous mutant flgII-28 m2 has 3 amino acid substitution mutations at the 13th, 23rd, and 27th positions of the amino acid sequence shown in SEQ ID NO.9: A13V, A23S, E27D.
  • the seventh polypeptides with different amino acid sequences above are all homologous mutants with the same function. They can activate the FLS3 immune receptor and have been shown to have similar biological activities.
  • Allelic Variation in Two Distinct Pseudomonas Syringae Flagellin epitopes modulates the strength of plant immune responses but not bacterial motility and “Natural Variation for Responsiveness to flg22, flgII-28, and csp22 and Pseudomonas syringae pv.tomatoes in 3 Heirloom.
  • the eighth polypeptide that activates the FER immune receptor is preferably the polypeptide ralf17 and its homologous mutants.
  • ralf17 The amino acid sequence of ralf17 is shown in SEQ ID NO. 10: NSIGAPAMREDLPKGCAPGSSAGCKMQPANPYKPGCEASQRCRGG.
  • Homologous mutants of ralf17 include PAMP polypeptides that add, delete, or replace one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO. 10 and have the ability to activate FER immune receptors.
  • the homologous mutant ralf17 m1 of ralf17 has 4 amino acid substitution mutations at position 1, position 2, position 5, and position 12 of the amino acid sequence shown in SEQ ID NO.10: N1K, S2T , A5N, L12E.
  • the eighth polypeptides with different amino acid sequences above are all homologous mutants with the same function. They can activate FER immune receptors and have been shown to have similar biological activities.
  • the receptor kind FER is a RALF- The introduction of peptides that activate FER immune receptors in “regulated scaffold controlling plant immune signaling” and “How CrRLK1L receptor complexes perceive RALF signals”.
  • the PAMP molecular polypeptide also includes a ninth polypeptide that activates a plant immune response, preferably the polypeptide pep13 and its homologous mutants.
  • 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 influence on the recognition of oomycete pathogens and the activation of plant defense responses. Especially in parsley and potatoes, it can mediate the expression of defense genes and induce the synthesis of antibacterial phytoalexins.
  • 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 replace one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO. 11 and have the ability to activate plant immune responses.
  • the homologous mutant pep13 m1 of pep13 has a substitution mutation of 1 amino acid at the 12th position of the amino acid sequence shown in SEQ ID NO.11: Y12F; the homologous mutant pep13 m2 of pep13 is in SEQ ID NO.
  • the amino acid sequence shown in 11 has a substitution mutation of 1 amino acid at position 12: Y12A.
  • the PAMP molecular polypeptide also includes a tenth polypeptide that activates a plant immune response, preferably the polypeptide hrp15 and its homologous mutants.
  • 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 replace one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO. 12 and have the ability to activate plant immune responses.
  • the homologous mutant hrp15 m1 of hrp15 has 9 amino acid substitution mutations in the amino acid sequence shown in SEQ ID NO.
  • the homologous mutant hrp24 is based on the amino acid sequence shown in SEQ ID NO.12, with 9 amino acids added.
  • the tenth polypeptide is preferably a homologous mutant hrp24 of polypeptide hrp15.
  • the homologous mutant hrp15, hrp24 added 9 amino acids to the amino acid sequence shown in SEQ ID NO.12.
  • the amino acid sequence of hrp24 is shown in SEQ ID NO. 13: PNQDLGQLLGGLLQKGLEATLQDA.
  • the two tenth polypeptides with different amino acid sequences are homologous mutants with the same function, and have been proved to have similar biological activities.
  • the PAMP molecular polypeptide also includes the eleventh polypeptide that activates the plant immune response, preferably the polypeptide sys18 and its homologous mutants.
  • the amino acid sequence of sys18 is shown in SEQ ID NO. 14: AVQSKPPSKRDPPKMQTD.
  • Homologous mutants of sys18 include PAMP polypeptides that add, delete, or replace one or more (for example, 1-10) amino acids in the amino acid sequence shown in SEQ ID NO. 14 and have the ability to activate plant immune responses.
  • the homologous mutant sys18 m1 of sys18 has a substitution mutation of 1 amino acid at position 6 of the amino acid sequence shown in SEQ ID NO.14: P6A; the homologous mutant sys18 m2 of sys18 is in SEQ ID NO.
  • the 10th position of the amino acid sequence shown in 14 has a substitution mutation of 1 amino acid: R10A.
  • the two eleventh polypeptides with different amino acid sequences are homologous mutants with the same function, and have been proved to have similar biological activities.
  • mutants are not their own biological names, but are named uniformly for the convenience of writing and understanding of this patent.
  • nlp20 m1 its biological name is Pyanlp20, and other mutants are named in the same way.
  • the biological name of the specific mutant is based on the amino acid sequence and can be found in the article or database.
  • each PAMP molecule polypeptide that activates different receptors is based on the disclosed amino acid sequence, and the rest of the amino acid sequence is composed of polypeptide mutants, such as additions or deletions compared with the original amino acid sequence. , Substituting one or more PAMP molecular polypeptide mutants that have the function of activating the same receptor are all within the protection scope 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 six polypeptide is csp22
  • the seventh polypeptide is flgII-28
  • the eighth polypeptide is ralf17
  • the ninth polypeptide is pep13
  • the ten polypeptide is hrp24
  • the eleventh polypeptide is sys18.
  • the fusion protein is composed of three identical and/or different PAMP molecular polypeptides.
  • the fusion protein is composed of four identical and/or different PAMP molecular polypeptides.
  • the fusion protein is composed of five identical and/or different PAMP molecular polypeptides.
  • the fusion protein is composed of six identical and/or different PAMP molecule polypeptides.
  • the fusion protein comprises or consists of seven different PAMP molecule polypeptides; optionally, there is at least one linker or no linker between two adjacent PAMP molecule polypeptides. .
  • the fusion protein is composed of 7 different PAMP molecular polypeptides and at least 6 linkers.
  • the seven different PAMP molecular polypeptides are selected from combinations of any seven of flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, ralf17, pep13, hrp24 or sys18.
  • the arrangement order of the seven different PAMP molecular polypeptides and the specific types of linkers are not specifically limited. They can be adjusted appropriately according to the types of plants or the types of pathogenic microorganisms, as long as they have the effect of inducing plant immune resistance. Moreover, fusion proteins with more immune epitopes, better effects, and lower cost of use obtained through adjustment and optimization are within the protection scope of the present invention.
  • Protein tag refers to a polypeptide that is expressed by fusion with the target protein by using DNA in vitro recombination technology to facilitate the expression, detection and purification of the target protein.
  • the protein tags that can be connected to the fusion protein of the present invention include but are not limited to: HIS, GST, Flag, MBP, HA, c-Myc, eGFP, eYFP, eCFP.
  • the amino acid sequence of the fusion protein composed of 7 different PAMP molecular polypeptides are flg22, nlp20, elf18, pip1, pep1, csp22, and flgII-28 as shown in SEQ ID NO.15 .
  • the amino acid sequence shown in SEQ ID NO. 15 shows that the fusion protein is connected to the HIS protein tag, and the sequence of the PAMP molecular polypeptide is elf18, csp22, flg22, flgII-28, nlp20, pep1, pip1. There are linkers between adjacent PAMP molecular polypeptides, and GAG and AGA are used as linkers.
  • the amino acid sequence of the fusion protein composed of 7 different PAMP molecular polypeptides, respectively, flg22, nlp20, elf18, pip1, pep1, csp22, and flgII-28 is shown in SEQ ID NO.15.
  • the amino acid sequence shown has a functional homologous sequence with at least 80% sequence identity.
  • the functional homologous sequence of identity includes, but is not limited to, the amino acid shown in SEQ ID NO. 15 has about 80% or more, 85% or more, 90% or more, 95% or more, 98% or more , Amino acid sequences with 99% or more identity.
  • the fusion protein composed of the above seven different PAMP molecular polypeptides, combined with the appropriate linker and tag protein, assembled into a fusion protein, compared with a single PAMP molecular polypeptide has more immunologically active epitopes, and can be used in plants. Faster and more sensitive binding to the receptors on the surface of plant cells, quickly inducing the immune response of plant defense, resisting pathogenic microorganisms, improving plant disease resistance, and has the advantages of low toxicity, short degradation cycle and large environmental compatibility. .
  • the second aspect of the present invention provides a nucleotide sequence encoding the above-mentioned fusion protein.
  • the composition of the above-mentioned fusion protein is variable, there is at least one linker or no linker between two adjacent PAMP molecule polypeptides, and the number and types of linkers are variable. Therefore, the corresponding nucleotide sequence encoding it is also variable. Therefore, the sequence of bases in the nucleotide sequence and the number of bases are not specifically limited here.
  • the nucleotide sequence capable of encoding the fusion protein, and its complementary sequence, degenerate sequence or homologous sequence are all in the present invention. Within the scope of protection.
  • nucleotide sequence of the fusion protein encoding the amino acid sequence shown in SEQ ID NO. 15 is shown in SEQ ID NO. 16.
  • the nucleotide sequence of the fusion protein encoding the amino acid sequence shown in SEQ ID NO. 15 hybridizes with the nucleotide sequence of SEQ ID NO. 16 under stringent conditions, and can encode The nucleotide sequence of the fusion protein.
  • stringent conditions refer to conditions under which the probe will hybridize to its target sequence to a detectable extent beyond hybridization with other sequences (such as at least 2 times the background). Stringent conditions are sequence-dependent and vary from environment to environment. By controlling the stringency of hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified.
  • the nucleotide sequence of the fusion protein encoding the amino acid sequence shown in SEQ ID NO. 15 is a degenerate sequence of the nucleotide sequence shown in SEQ ID NO. 16.
  • the described degenerate sequence after changing one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 16, changes the position of the nucleotide sequence corresponding to the same type of amino acid encoded, and the encoded fusion protein The amino acid sequence of is unchanged.
  • the nucleotide sequence of the fusion protein encoding the amino acid sequence shown in SEQ ID NO. 15 is a homologous sequence with the nucleotide sequence shown in SEQ ID NO. 16.
  • the homologous sequence is a polynucleotide having at least 85% or more identity with the nucleotide sequence shown in SEQ ID NO. 16.
  • the homologous sequence includes, but is not limited to, the nucleotides shown in SEQ ID NO. 16 have about 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 98% Or above, 99% or above identity, and having a polynucleotide encoding the fusion protein.
  • the third aspect of the present invention provides a vector into which the nucleotide sequence encoding the fusion protein is introduced.
  • the specific vector type is not limited, and a recombinant expression vector can be constructed after the above-mentioned nucleotide sequence and the vector are successfully connected, and the expression vector can normally express the fusion protein in the host cell.
  • 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.
  • the fourth aspect of the present invention provides microorganisms or cells into which the above-mentioned nucleotide sequence encoding the fusion protein and/or the above-mentioned vector are introduced.
  • the microorganism or cell alone contains the nucleotide sequence encoding the fusion protein; 2.
  • the microorganism or cell alone contains the above-mentioned vector, and the vector contains the nucleus encoding the fusion protein.
  • the microorganism or cell contains the nucleotide sequence encoding the fusion protein, and at the same time contains the above-mentioned vector, and the vector contains the nucleotide sequence encoding the fusion protein at the same time.
  • the microorganism can be any prokaryotic or eukaryotic cell that can normally express the fusion protein.
  • a microorganism or cell refers to a specific microorganism or cell into which a nucleotide sequence encoding a fusion protein is introduced, and also includes the progeny of this microorganism carrying the vector.
  • the microorganisms include one or more of Escherichia coli, Agrobacterium or Bacillus subtilis; preferably Escherichia coli.
  • strains of Escherichia coli include but are not limited to BL21(DE3), ⁇ DE3, RosettaTM, K-12, HMS174, NovaBlue, Tuner, OrigamiB.
  • strains of Agrobacterium include but are not limited to EH101, EHA105, C58C1, LBA4404.
  • strains of Bacillus subtilis include but are not limited to pMA5, PUB110, pE194, and pWB.
  • methods for transforming the above-mentioned nucleotide sequence encoding the fusion protein and/or the above-mentioned vector into the host microorganism in vivo and/or in vitro include, but are not limited to: heat shock, heat shock, electroporation, calcium phosphate precipitation, Polyethylene glycol (PEG) transformation, lipofection and microinjection.
  • heat shock heat shock
  • electroporation calcium phosphate precipitation
  • PEG Polyethylene glycol
  • the fifth aspect of the present invention provides a plant immune inducer comprising the fusion protein, or the carrier, or the microorganism or cell.
  • Plant immune inducers refer to exogenous organisms or molecules that can induce or activate the immune response of plants and increase the resistance of plants to certain pathogenic microorganisms.
  • the fusion protein, or the vector containing the nucleotide sequence encoding the fusion protein, or the microorganism can be used as the raw material for preparing the plant immune inducer, and then the plant immune inducer is used as a biological pesticide product in agriculture In production, it also has the advantages of low concentration, quick onset, application in agricultural production cost, low toxicity, short degradation cycle, and high environmental compatibility.
  • the plant immune inducer also includes one or more agronomically acceptable carriers, excipients, diluents or solvents.
  • the plant immune inducer In the preparation process of the plant immune inducer, it not only includes the above-mentioned fusion protein, or the carrier, or the microorganism as the main material, but also includes a variety of agronomically acceptable carriers, excipients, diluents or solvents. Excipients to obtain more dosage forms, more stable effects, and more convenient use of plant immune inducers.
  • the dosage form of the plant immune inducer is selected from the group consisting of powder, soluble powder, wettable powder, granule, water, microemulsion, suspension and water dispersible granules.
  • plant immune inducers There are various formulations of plant immune inducers, which can expand the scope of their use to better apply to different kinds of plants.
  • the sixth aspect of the present invention provides a method for preparing the fusion protein, including the step of culturing the microorganism or cell containing the nucleotide, or the step of artificially synthesizing the fusion protein;
  • the method includes the following steps:
  • the above-mentioned preparation method of fusion protein combines the PTI immune mechanism and genetic engineering technology, and at least three identical and/or different PAMP molecular polypeptides that have been discovered are used to construct a new genetic recombination fusion through genetic engineering technology. Proteins, fusion proteins with multiple immune epitopes that do not exist in nature are obtained through protein expression technology.
  • the above preparation method has simple preparation process, short time-consuming, and low investment economic cost, and effectively solves the problem of high production cost for preparing plant immune PAMP molecular polypeptides and long-term application in agricultural production.
  • the method for preparing the fusion protein includes the following steps:
  • the seventh aspect of the present invention provides that the fusion protein, or the plant immune inducer, or the fusion protein prepared by the preparation method of the fusion protein can improve plant disease resistance, induce plant defense response and/ Or to resist the application of pathogenic microorganisms.
  • plants include but are not limited to: Arabidopsis, corn, wheat, rice, tomato, and tobacco.
  • pathogenic microorganisms include but are not limited to: Pseudomonas syringae, Fusarium graminearum, Magnaporthe grisea, and tobacco mosaic virus.
  • the fusion protein or the plant immune inducer can be applied to plants, but the specific application method and application amount are not limited, and reasonable selection can be made according to the plant species and the types of diseases and insect pests. .
  • the fusion protein and/or plant immune inducer can interact with the receptor protein on the plant cell membrane, and can quickly trigger the defense response of the plant to resist infection by pathogenic bacteria. Therefore, the fusion protein can be used as a biological pesticide product in agricultural production to activate the disease resistance of the plant itself, thereby improving the ability of the plant to resist pathogenic microorganisms and reducing the use of chemical pesticides.
  • the fusion protein provided by the present invention has multiple or multiple immune epitopes, has a low concentration, has a quick onset, can bind to receptors on the surface of plant cells more quickly and widely, and has a broad spectrum and High efficiency, can induce a variety of plants to produce an immune response, reduce the ability of pathogenic microorganisms to infect, and significantly improve plant disease resistance;
  • the preparation cost of the fusion protein provided by the present invention is lower than that of the PAMP molecular polypeptide, and it can significantly reduce the agricultural production cost when used as a biopesticide product in agriculture;
  • the fusion protein provided by the present invention does not cause plant drug resistance, has the advantages of low toxicity, short degradation cycle, and high environmental compatibility;
  • the preparation method of the fusion protein provided by the present invention has a simple preparation process, short time-consuming, and low investment economic cost, and effectively solves the problem of high production cost for preparing plant immune PAMP molecular polypeptides and long-term application in agricultural production.
  • Figure 1 shows the SDS-PAGE electrophoresis diagram of the heptapeptide fusion protein His-MP7;
  • Figure 2 shows the accumulation of callose in Arabidopsis plants treated with different concentrations of the fusion protein His-MP7 and PAMP molecular polypeptide flg22;
  • Figure 3 shows the accumulation of callose in Arabidopsis plants under the treatment of 100nM fusion protein His-MP7 and different PAMP molecular polypeptides
  • Figure 4 shows the production of reactive oxygen species in corn plants induced by the His-MP7 fusion protein
  • Figure 5 shows the experimental results of the fusion protein His-MP7 enhancing the resistance of Arabidopsis plants to the DC3000 pathogen
  • Figure 6 shows the experimental results of the fusion protein His-MP7 enhancing the resistance of corn plants to Fusarium graminearum; among them, a is the fusion protein His-MP7 inhibiting the growth of corn infected by Fusarium graminearum; b is the fusion Bar graph of experimental results of protein His-MP7 inhibiting Fusarium graminearum infection in corn;
  • Figure 7 shows the experimental results of the fusion protein His-MP7 enhancing the resistance of rice plants to Magnaporthe grisea; among them, a is the fusion protein His-MP7 inhibiting the growth of rice blast infection by the actual test comparison; b is the fusion protein His -Histogram of experimental results of MP7 inhibiting rice blast infection to rice.
  • PAMP molecular polypeptide refers to the comparative analysis of homology sequences in the secreted proteins of various pathogenic fungi or bacteria or plants themselves to find polypeptide fragments with relatively conservative sequences and immune activation capabilities in the protein molecules. It is often used by plants as a signal molecule polypeptide that senses the invasion of pathogenic bacteria.
  • linker refers to having at least one amino acid residue, preferably at least two consecutive amino acid residues.
  • plant immune inducer refers to an exogenous organism or molecule capable of inducing or activating the immune response of plants and improving the resistance of plants to certain pathogenic microorganisms.
  • PAMP immune mechanism the full name of pathogen-related molecular pattern triggered immunity (PAMP-Triggered Immunity) mechanism, refers to the mechanism by which PAMP signal molecules are recognized by plant cell receptors to activate plant immune response.
  • protein tag refers to a polypeptide expressed by fusion with the target protein by using DNA in vitro recombination technology, so as to facilitate the expression, detection and purification of the target protein.
  • Plant materials rice (Oryzasativa L.) is a short-grain japonica rice variety Nipponbare (NPB) (NPB is an international variety that has been sequenced through the whole genome), corn is Jundan 20, and both rice and corn are purchased from the market; cultivated tomato (Solanum lycopersicum) ), Colombian wild-type Arabidopsis col-0, N89 tobacco strains are derived from the research group of Professor Cai Yi, College of Life Sciences, Sichuan Agricultural University.
  • Pseudomonas syringae DC3000, rice blast race ZB15, tobacco mosaic virus, and Fusarium graminearum all come from the research group of Professor Cai Yi, College of Life Sciences, Sichuan Agricultural University.
  • His tag protein purification kit was purchased from Kangwei Century Company, product number CW0894; BCA method protein concentration determination kit was purchased from Solarbio Company, product catalog number PC0020-500 Micropore (50T).
  • the supernatant is purified by His-tag protein purification kit (soluble protein).
  • His-tag protein purification kit soluble protein
  • the specific operation is as follows: first load 5 mL of Ni-Agarose filler into the empty column of the affinity column, then slowly flow the supernatant through the affinity column, and then use Elute 6 column volumes with PBS buffer containing 10 mM imidazole to remove impurities, and finally eluate 5 column volumes with PBS buffer containing 500 mM imidazole and collect the post-column eluate, which is the fusion protein His-MP7 solution;
  • the concentration of the fusion protein His-MP7 was measured to be 0.2 mg/mL; after SDS-PAGE detection, as shown in Figure 1, purified to obtain a molecular weight of about 23kDa containing histamine Acid expression protein (His-MP7).
  • the accumulation of corpus callosin occurs in the immune response of plant cells, which can strengthen the mechanical strength of plant cell walls and block the passage of pathogens from spreading between cells, thereby limiting the invasion of pathogenic microorganisms.
  • the model plant Arabidopsis thaliana was used as the material, and callose accumulation was used as the immune index.
  • the immune activation ability of the fusion protein His-MP7 at different concentrations was analyzed and compared with a single PAMP molecular peptide. The specific experiment operation is as follows:
  • the fusion protein His-MP7 induces plants to produce reactive oxygen burst immune response
  • the burst of reactive oxygen species is considered to be one of the earliest responses of plants to pathogenic microorganisms, and plays an important role in plant defense responses. Studies have shown that active oxygen can be directly used as an antibacterial agent in plants to have direct toxicity to pathogenic microorganisms and inhibit their growth. After plants are infected by pathogenic microorganisms, active oxygen bursts out and accumulates in the body. Using plant corn as the material and reactive oxygen species as the immune index, the immune activation ability of 100nM fusion protein His-MP7 was analyzed. The specific experiment operation is as follows:
  • the fusion protein His-MP7 can enhance the resistance of plants to DC3000 pathogens
  • T3 5 days per two leaves as a sample take the same small round punch, added 250 ⁇ L 10mM MgCl 2, ground in a tube and 1.5mL ep stepwise diluted with MgCl 2 to 1 ⁇ 10 - 5 .
  • the fusion protein His-MP7 can enhance plant resistance to Fusarium graminearum
  • Fig. 6 The results of the experiment are shown in Fig. 6: a shows the comparison of corn leaf growth measured, and the bar chart shown in b shows: the percentage of diseased spots on the leaves without the His-MP7 fusion protein treatment is 12.3%, and the leaves with the His-MP7 fusion protein are diseased. The spot area percentage is 3.6%. It shows that the fusion protein His-MP7 significantly improves the resistance of corn to Fusarium graminearum and reduces the infection rate of Fusarium graminearum by 70.8%.
  • the fusion protein His-MP7 can enhance plant resistance to rice blast
  • Magnaporthe grisea was inoculated on CM solid medium, cultured at 28°C for 13-15 days, scraped off all the hyphae with a pipette tip, rinsed with a small amount of 5-10mL sterile water, filtered with gauze and centrifuged at 50mL Collect the spores by centrifugation at 10000rpm/10min in the tube, count the number of spores on a hemocytometer, adjust the concentration to 1 ⁇ 10 6 , and store at room temperature (use within one week);
  • auxin 6BA 1 ⁇ g/mL auxin 6BA solution
  • Fig. 7 A shows the comparison of the actual growth of rice leaves, and the histogram shown in b shows: the percentage of diseased spots on the leaves without the His-MP7 fusion protein treatment is 11.6%, and the leaves with the His-MP7 fusion protein are diseased. The spot area percentage is 1.9%. It shows that the fusion protein His-MP7 significantly improves the resistance of rice to rice blast, reduces the infection rate of rice blast fungus by 83.6%, and reduces the damage of rice blast to rice.
  • the fusion protein His-MP7 can enhance tobacco resistance to tobacco mosaic virus (TMV)
  • the experiment has an experimental group and a control group, each with 20 strains of tobacco.
  • the experimental group is inoculated with virus after injection of recombinant protein
  • the control group is inoculated with virus after injection of sterile water.
  • the inoculation method is as follows:
  • Grade 1 A small amount of chlorotic yellow spots appear along the veins at the base of the heart leaf, without curling;
  • Grade 5 New leaves have a lot of chlorosis stripes parallel to the veins, and the leaves are curled and thin;
  • Grade 7 The plant is dwarfed, the leaves appear yellow and white stripes rolled up, the new leaves are twisted and drooped, and they cannot open normally;
  • Control effect (%) [(control disease index-treatment disease index)/control disease index] ⁇ 100.
  • the experimental results showed that the disease index of the experimental group after treatment with the fusion protein His-MP7 decreased to 21.7, and the control effect reached 72.5%.
  • (2) Inducing the expression of the above-mentioned E. coli includes the following steps: inoculating the expression strain in LB liquid medium, culturing overnight at 37°C and shaking at 200 rpm/min to obtain the first bacterial solution; Transfer the volume ratio to LB liquid medium containing 100 ⁇ g/mL ampicillin, continue shaking at 37°C and 200rpm/min, cultivate until the concentration of the bacterial solution OD600nm is 0.6, and then add the final concentration of 0.3mmol/L IPTG, Incubate with shaking at 25°C and 200 rpm/min for 12 hours to obtain the second bacterial solution; centrifuge the second bacterial solution at 12000 rpm/min, collect the bacteria and add PBS buffer, after ultrasonically disrupting the bacteria, centrifuge at 4°C, 12000 rpm/min, Collect the supernatant;
  • the supernatant is purified with a GST-tagged protein purification kit (soluble protein) to obtain a GST-TP fusion protein solution.
  • the BCA method protein concentration determination kit was used to quantify the protein solution.
  • the method for obtaining the nucleotide sequences encoding the above-mentioned 20 tetrapeptide fusion proteins is the same as in Example 1.
  • the method for obtaining the nucleotide sequences encoding the above 20 pentapeptide fusion proteins is the same as in Example 1.
  • the method for obtaining the nucleotide sequences encoding the above 20 hexapeptide fusion proteins is the same as in Example 1.
  • the method for obtaining the nucleotide sequences encoding the above-mentioned 20 heptapeptide fusion proteins is the same as in Example 1.
  • the model plant Arabidopsis thaliana was used as the material, callose accumulation was used as the immune index, and water was used as the blank control.
  • the final concentration of all the fusion protein adjustment solutions obtained in the above Examples 5-9 was 100nM, and it was infected by syringe infiltration method for four weeks.
  • the specific experimental operation is the same as that of Example 3.
  • the obtained corpus callosin accumulation fluorescence image is calculated by the image processing software ImageJ and the fluorescence density is used to quantify the immune activation ability of the fusion protein to the plant.
  • the calculation method is as follows:
  • Measurement Area picture area

Abstract

提供了一种融合蛋白，包含或由至少三个、四个、五个、六个、七个、或八个相同和/或不同的PAMP分子多肽组成，任选地，相邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子。该融合蛋白可以诱导植物防御的免疫反应，降低病原微生物侵染能力，提高植物抗病性。该融合蛋白的制备方法包括，将PTI免疫机制和基因工程的技术联合运用，获取了自然界不存在的多免疫表位的融合蛋白，解决了制备植物免疫PAMP分子多肽生产成本高，长期无法应用于农业生产的问题。

Description

融合蛋白、其氨基酸序列、编码核苷酸序列、制备方法和应用 技术领域
本发明涉及农业生物技术领域，具体涉及一种融合蛋白、其氨基酸序列、编码核苷酸序列、制备方法和应用。
背景技术
在农业生态系统中，相较于化学农药而言，生物农药产品具有毒性低，降解周期短，环境相容性大等优点，已经被广泛运用于农作物生产。然而，传统的生物农药的研究开发，主要是针对生物来源的抑菌和杀虫物质进行筛选，进而开发出生物农药对抗病虫害，往往忽略了植物自身免疫力在抗病虫害过程中的作用。近年来，植物免疫诱导技术成为生物农药发展中的一个新亮点。植物免疫诱抗剂也称为“植物疫苗”，通过激发植物自身的免疫系统以达到抗病、增产、改善品质的作用。与传统的生物农药相比，“植物疫苗”不会引起病原微生物的抗性，更加符合绿色健康的农业生产要求，已引起国内外的广泛关注和重视。
植物的PTI(PAMP-Triggered Immunity)免疫机制，在生物农药开发上具有广阔前景。目前市场上的植物免疫类农药种类较少，仅有阿泰灵，Harpin蛋白等几种类型。然而这些产品存在着相当的局限性：第一，他们的受体尚不清晰，在使用上难以做出科学有效的指导，只能通过实验经验来判断其适用范围和使用浓度；第二，植物对这些植物免疫类农药的敏感性较低，使用浓度大和成本高昂。Axiom Harpin Protein是国际上使用较为广泛的生物蛋白农药，然而其纯蛋白产品每克单价高于5000元人民币，其1％含量的蛋白农药产品每克售价也大于140元人民币，高昂的成本极大的限制了它在农业生产上的应用。因此，有必要开发一种价格低廉，且提高植物自身免疫力的效果好的生物农药产品。
发明内容
有鉴于此，本发明致力于提供一种融合蛋白，将多个PAMP分子多肽组装成具有多个或多种免疫表位的融合蛋白，融合蛋白可以更快速、更广泛地的与植物细胞表面的受体结合，诱导多种植物产生免疫反应，明显提高植物抗病性，融合蛋白的制备成本相较于PAMP分子多肽的制备成本低，提高了蛋白类生物农药产品的单位产量，预计蛋白成本可以降低至1元/g，使用浓度低，起效快，应用于农业中可以明显降低农业生产成本。
本发明第一方面提供了一种融合蛋白，所述融合蛋白包含或至少三个、四个、五个、六个、七个相同和/或不同的PAMP分子多肽组成，任选地，相邻两个PAMP分子多肽之间具有至少一 个连接子或者没有连接子
病原相关模式(PAMPs)是指在进化上具有高度的保守性的一些分子，其特征是能够快速触发植物体的免疫防御反应。PAMP分子种类多样，包括多糖类，脂质类，多肽类等分子物质。多肽类的PAMP分子称为PAMP分子多肽，大多数PAMP分子是病原菌生活周期的必需成分，常被植物作为感知病原菌侵害的信号分子。研究表明，特异性的PAMP分子多肽与植物细胞膜上的受体蛋白相互作用，能够快速触发植物体的防御反应来抵抗病原菌的感染。植物防御的免疫反应包括：诱导植物体内活性氧快速生成和诱导胼胝质的沉积；植物激素(例如水杨酸、乙烯)大量合成；防御基因的快速表达。而且植物接收刺激后产生的某些自分泌肽如PIP1、PEP1，也是PAMP分子多肽，可以作为第二信使增强免疫信号，持续免疫效应。
PAMP分子多肽的获取方法，常常是通过在多种病原真菌或细菌中进行同源性序列对比分析，找到蛋白分子中相对保守的序列，然后人工合成该多肽序列，并验证其激活植物免疫反应的能力。
由于病原菌种类不同和自然存在的突变，PAMP分子多肽天然存在多种突变体，PAMP分子多肽突变体可以维持不低于野生型PAMP分子多肽80％的免疫原性，且能够与PAMP分子多肽激活相同且固定的免疫受体。
“融合蛋白包含或由至少三个、四个、五个、六个、七个相同和/或不同的PAMP分子多肽组成”可以理解为三种解释：
(1)所述融合蛋白包含或由至少三个、四个、五个、六个、七个相同种类的PAMP分子多肽组成，即包括但不限于或由三个、四个、五个、六个、七个及更多个相同种类的PAMP分子多肽组成；
(2)所述融合蛋白包含或由至少三个、四个、五个、六个、七个不同种类的PAMP分子多肽组成，即包括但不限于或由三个、四个、五个、六个、七个及更多个不同种类的PAMP分子多肽组成；
(3)所述融合蛋白包含或由至少三个、四个、五个、六个、七个相同和不同种类的PAMP分子多肽组成，即包括但不限于或由两个相同且一个不同、两个相同且两个不同、两个相同且三个不同、两个相同且四个不同、两个相同且五个不同、两个相同且六个不同或两个相同且七个不同及其它更多个相同和不同种类的PAMP分子多肽组成。
“任选地”表示可以有或者没有，即组成融合蛋白的相邻两个PAMP分子多肽之间可以没有连接子，或者有至少一个连接子。连接子为具有1个及1个以上氨基酸残基，优选为至少3个连续的氨基酸残基的连接区域。连接子将相邻的两个PAMP分子多肽连接，进而使至少三个多肽组装成融合蛋白。
连接子包括但不限于：GAG、AGA、AAA、GGG、KRK、KKK、RRR、AKG。
需要说明的是，此处对于组成融合蛋白的PAMP分子多肽的排列顺序和连接子的具体种类 不做具体限定，可以根据植物的种类或者针对的病原微生物的种类进行适当的改变，替换和调整，只要能够达到诱导植物免疫抗性的效果即可。
本发明通过研究植物的PTI免疫机制，借助基因工程的技术将至少三个特异性的PAMP分子多肽经过分子设计和定向组装形成融合蛋白，融合蛋白具有多个和/或多种免疫表位，可以更快速、更广泛地的与植物细胞表面的受体结合，诱导植物防御的免疫反应，降低病原微生物侵染能力，明显提高植物抗病性。而且将该融合蛋白作为生物农药产品，还具有毒性低，降解周期短，环境相容性大，不会引起病原菌的耐药性等优点。
本发明的一种实施方式中，所述融合蛋白主要由至少三个、四个、五个、六个、七个相同和/或不同的PAMP分子多肽突变体组成，相邻两个PAMP分子多肽突变体之间具有至少一个连接子。
进一步，PAMP分子多肽包括激活FLS2免疫受体的第一多肽，激活RLP23免疫受体的第二多肽，激活EFR免疫受体的第三多肽，激活RLK7免疫受体的第四多肽，激活PEPR1免疫受体的第五多肽，激活CORE1免疫受体的第六多肽，激活FLS3免疫受体的第七多肽，激活FER受体的第八多肽，激活植物免疫反应的第九多肽pep13，激活植物免疫反应的第十多肽hrp24，激活植物免疫反应的第十一多肽sys18。
所述融合蛋白含有激活不同受体的多种多肽成分，具有多种优势。首先，多种不同的信号通路可激活不同植物的免疫反应，避免了由于某些植物中特定受体的缺失无法产生免疫响应的情况，适用植物的范围更加广泛。其次，多种信号通路同时激活同一植物免疫反应时，其产生的免疫信号具有叠加效应，免疫激活更加灵敏高效。
进一步，激活FLS2免疫受体的第一多肽为多肽flg15及其同源突变体；或者为多肽flg22及其同源突变体。
flg15的氨基酸序列如SEQ ID NO.1所示：RINSAKDDAAGLQIA。flg15的同源突变体包括在SEQ ID NO.1所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且能激活FLS2免疫受体的PAMP多肽。
flg22是细菌鞭毛蛋白N端的一段保守性极高的区域，许多研究证明flg22能够诱导植物的天然免疫性，可以激活FLS2受体，对SA、MAPK等信号途径产生作用，对植物抗病性有着重要的影响。flg22的氨基酸序列如SEQ ID NO.2所示：QRLSTGSRINSAKDDAAGLQIA。flg22的同源突变体包括在SEQ ID NO.2所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且具有激活FLS2免疫受体能力的多肽。
进一步，激活FLS2免疫受体的第一多肽优选为flg22及其同源突变体。具体的，flg22的同源突变体flg22 m1在SEQ ID NO.2所示的氨基酸序列的第1位，第5位、第7位、第8位产生了4个氨基酸的替换突变：Q1T、T5S、S7L、R8K；flg22的同源突变体flg22 m2在SEQ ID NO.2所示的氨基酸序列的第5位，第7位、第20位、第22位，产生了4个氨基酸的替换突变：T5S、 S7L、Q20A、A22S。
上述氨基酸序列不同的第一多肽均属于具有相同功能的同源突变体，均能激活FLS2免疫受体，且被证明具有相近的生物活性，具体信息可以参考文章“Plants have a sensitive perception system for the most conserved domain of bacterial flagellin”和“CD2-1,the C-Terminal Region of Flagellin,Modulates the Induction of Immune Responses in Rice”中对于激活FLS2免疫受体的多肽的介绍。
进一步，激活RLP23免疫受体的第二多肽优选为多肽nlp20及其同源突变体。
nlp20是由20个氨基酸组成多肽分子，是坏死和乙烯诱导肽样蛋白家族(NLPs)中包含的特征性氨基酸序列。研究表明，nlp20多肽能通过激活免疫识别受体RLP23，迅速的引起植物的免疫反应，增强植物对微生物感染的免疫能力。
nlp20的氨基酸序列如SEQ ID NO.3所示：AIMYSWYFPKDSPVTGLGHR。nlp20的同源突变体包括在SEQ ID NO.3所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且具有激活RLP23免疫受体能力的PAMP多肽。具体的，nlp20的同源突变体nlp20 m1在SEQ ID NO.3所示的氨基酸序列的第8位，第14位、第17位产生了3个氨基酸的替换突变：F8M、V14S、L17I；nlp20的同源突变体nlp20 m2在SEQ ID NO.3所示的氨基酸序列的第5位，第14位、第15位产生了3个氨基酸的替换突变：S5A、V14S、T15P。
上述氨基酸序列不同的第二多肽均属于具有相同功能的同源突变体，均能激活RLP23免疫受体，且被证明具有相近的生物活性，具体信息参考文章“A Conserved Peptide Pattern from a Widespread Microbial Virulence Factor Triggers Pattern-Induced Immunity in Arabidopsis”中对于激活RLP23免疫受体的多肽的介绍。
进一步，激活EFR免疫受体的第三多肽，优选为多肽elf18及其同源突变体。
elf18是细菌蛋白延伸因子Tu(EF-Tu)N末端的18个氨基酸的多肽，能够激活受体EFR，诱导乙烯的氧化爆发和生物合成，并引发对随后感染病原菌的抵抗力。
elf18的氨基酸序列如SEQ ID NO.4所示：SKEKFERTKPHVNVGTIG。elf18的同源突变体包括在SEQ ID NO.4所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且具有激活EFR免疫受体能力的PAMP多肽。具体的，elf18的同源突变体elf18 m1在SEQ ID NO.4所示的氨基酸序列的第1位、第3位、第8位、第14位产生了4个氨基酸的替换突变：S1A、E3S、T8N、V14I；elf18的同源突变体elf18 m2在SEQ ID NO.4所示的氨基酸序列的第1位、第6位、第8位、第9位、第12位产生了5个氨基酸的替换突变：S1V、E6D、T8S、K9L、V12C。
上述氨基酸序列不同的第三多肽均属于具有相同功能的同源突变体，均能激活EFR免疫受体，且被证明具有相近的生物活性，具体信息参考文章“The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants”中对于激活EFR免疫受体的多肽的介绍。
进一步，激活RLK7免疫受体的第四多肽，优选为多肽pip1及其同源突变体。
pip1是prePIP1分泌到细胞外空间并在保守C末端区域切割形成的13个氨基酸的多肽，pip1通过细胞表面的类受体激酶7(RLK7)发出信号激活受体RLK7，从而激活植物的免疫应答，增强植物的病原体抗性。
pip1的氨基酸序列如SEQ ID NO.5所示：RLASGPSPRGPGH。pip1的同源突变体包括在SEQ ID NO.5所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且具有激活RLK7免疫受体能力的PAMP多肽。具体的，pip1的同源突变体pip1 m1在SEQ ID NO.5所示的氨基酸序列的第1位、第2位之间增加了两个氨基酸FV，并且第2位，第3位，第9位产生了3个氨基酸的替换突变。
上述氨基酸序列不同的第四多肽均属于具有相同功能的同源突变体，均能激活RLK7免疫受体，且被证明具有相近的生物活性，具体信息参考文章“The Secreted Peptide PIP1Amplifies Immunity through Receptor-Like Kinase 7”中对于激活RLK7免疫受体的多肽的介绍。
进一步，激活PEPR1免疫受体的第五多肽，优选为多肽pep1及其同源突变体。
pep1是一种内源性分子，源自前体蛋白proPEP1C端包含23个氨基酸的多肽，能够激活受体PEPR1，激活拟南芥植物的自身免疫。
pep1的氨基酸序列如SEQ ID NO.6所示：ATKVKAKQRGKEKVSSGRPGQHN。pep1的同源突变体包括在SEQ ID NO.6所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且具有激活PEPR1免疫受体能力的PAMP多肽。具体的，pep1的同源突变体pep1 m1在SEQ ID NO.6所示的氨基酸序列的第1位、第4位、第5位、第6位、第8位、第10位、第11位、第12位、第13位产生了9个氨基酸的替换突变：A1E、V4A、K5R、A6G、Q8N、G10T、E11P、K12T、V13P；pep1的同源突变体pep1 m2在SEQ ID NO.6所示的氨基酸序列的第9位之前产生了八个氨基酸的缺失；pep1的同源突变体pep1 m2在SEQ ID NO.6所示的氨基酸序列的第9位之前产生了八个氨基酸的缺失和第10位产生了1个氨基酸的替换突变：G10A。
上述氨基酸序列不同的第五多肽均属于具有相同功能的同源突变体，均能激活PEPR1免疫受体，且被证明具有相近的生物活性，具体信息参考文章“Structure–activity studies of AtPep1,a plant peptide signal involved in the innate immune response”和“An endogenous peptide signal in Arabidopsis activates components of the innate immune response”中对于激活PEPR1免疫受体的多肽的介绍。
进一步，激活CORE1免疫受体的第六多肽，其可能为多肽csp15及其同源突变体；或者为多肽csp22及其同源突变体。
csp15的氨基酸序列如SEQ ID NO.7所示：VKWFNAEKGFGFITP。csp15的同源突变体包括在SEQ ID NO.7所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且具有激活CORE1免疫受体的PAMP多肽。
csp22是细菌冷休克蛋白(CSP)的保守结构域上的22个氨基酸的多肽，能够激活受体 CORE1，可高效诱导烟草防御反应。csp22的氨基酸序列如SEQ ID NO.8所示：AVGTVKWFNAEKGFGFITPDDG。csp22的同源突变体包括在SEQ ID NO.8所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且具有激活CORE1免疫受体能力的多肽。
进一步，激活CORE1免疫受体的第六多肽优选为csp22及其突变体。具体的，csp22的同源突变体csp22 m1在SEQ ID NO.8所示的氨基酸序列的第11位产生了1个氨基酸的替换突变：E11A；csp22的同源突变体csp22 m2在SEQ ID NO.8所示的氨基酸序列的第14位产生了1个氨基酸的替换突变：F14Y。
上述氨基酸序列不同的第六多肽均属于具有相同功能的同源突变体，均能激活CORE1免疫受体，且被证明具有相近的生物活性，具体信息参考文章“The highly conserved rna-binding motif rnp-1of bacterial cold shock proteins is recognized as an elicitor signal in tobacco”中对于激活CORE1免疫受体的多肽的介绍。
进一步，激活FLS3免疫受体的第七多肽，优选为多肽flgII-28及其同源突变体。
flgII-28与flg22都是细菌鞭毛蛋白N端的一段保守性极高的区域，是植物主要识别的PAMP，能够激活受体FLS3，flgII-28能刺激植物增加应激激素乙烯的产生和ROS的快速产生，激活植物的免疫反应。
flgII-28的氨基酸序列如SEQ ID NO.9所示：ESTNILQRMRELAVQSRNDSNSATDREA。flgII-28的同源突变体包括在SEQ ID NO.9所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且具有激活FLS3免疫受体能力的PAMP多肽。具体的，flgII-28的同源突变体flgII-28 m1在SEQ ID NO.9所示的氨基酸序列的第23位、第27位产生了2个氨基酸的替换突变：A23S，E27D；flgII-28的同源突变体flgII-28 m2在SEQ ID NO.9所示的氨基酸序列的第13位、第23位、第27位产生了3个氨基酸的替换突变：A13V，A23S，E27D。
上述氨基酸序列不同的第七多肽均属于具有相同功能的同源突变体，均能激活FLS3免疫受体，且被证明具有相近的生物活性，具体信息参考文章“Allelic variation in two distinct Pseudomonas syringae flagellin epitopes modulates the strength of plant immune responses but not bacterial motility”和“Natural Variation for Responsiveness to flg22,flgII-28,and csp22 and Pseudomonas syringae pv.tomato in Heirloom Tomatoes”中对于激活FLS3免疫受体的多肽的介绍。
进一步，激活FER免疫受体的第八多肽，优选为多肽ralf17及其同源突变体。
ralf17的氨基酸序列如SEQ ID NO.10所示：NSIGAPAMREDLPKGCAPGSSAGCKMQPANPYKPGCEASQRCRGG。ralf17的同源突变体包括在SEQ ID NO.10所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且具有激活FER免疫受体能力的PAMP多肽。具体的，ralf17的同源突变体ralf17 m1在SEQ ID NO.10所示的氨基酸序列的第1位，第2位，第5位，第12位产生了4个氨基酸的替换突变：N1K，S2T，A5N，L12E。
上述氨基酸序列不同的第八多肽均属于具有相同功能的同源突变体，均能激活FER免疫受体，且被证明具有相近的生物活性，具体信息参考文章“The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling”和“How CrRLK1L receptor complexes perceive RALF signals”中对于激活FER免疫受体的多肽的介绍。
进一步，所述PAMP分子多肽还包括激活植物免疫反应的第九多肽，优选为多肽pep13及其同源突变体。
pep13是细胞壁糖蛋白GP42中的一个保守的多肽片段。细胞壁糖蛋白在卵菌中广泛存在，因此pep13对卵菌病原体识别和植物的防御反应激活有重要影响。特别是在欧芹和马铃薯中，能够介导防御基因表达和诱导抗菌性植保素合成。
pep13的氨基酸序列如SEQ ID NO.11所示：VWNQPVRGFKVYE。pep13的同源突变体包括在SEQ ID NO.11所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且具有激活植物免疫反应能力的PAMP多肽。具体的，pep13的同源突变体pep13 m1在SEQ ID NO.11所示的氨基酸序列的第12位产生了1个氨基酸的替换突变：Y12F；pep13的同源突变体pep13 m2在SEQ ID NO.11所示的氨基酸序列的第12位产生了1个氨基酸的替换突变：Y12A。
上述两个氨基酸序列不同的第九多肽均属于具有相同功能的同源突变体，且被证明具有相近的生物活性，具体信息参考文章“Pep-13,a plant defense-inducing pathogen associated pattern from Phytophthora transglutaminases”中的介绍。
进一步，所述PAMP分子多肽还包括激活植物免疫反应的第十多肽，优选为多肽hrp15及其同源突变体。
hrp15的氨基酸序列如SEQ ID NO.12所示：DLGQLLGGLLQKGLE。hrp15的同源突变体包括在SEQ ID NO.12所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且具有激活植物免疫反应能力的PAMP多肽。具体的，hrp15的同源突变体hrp15 m1在SEQ ID NO.12所示的氨基酸序列产生了9个氨基酸的替换突变：D1Q、G3D、G7T、G8Q、L10I、Q11M、K12A、G13L、E15Q；hrp15的同源突变体hrp24在SEQ ID NO.12所示的氨基酸序列基础上，添加了9个氨基酸。
进一步，所述第十多肽优选为多肽hrp15的同源突变体hrp24。
hrp15的同源突变体hrp24在SEQ ID NO.12所示的氨基酸序列基础上，添加了9个氨基酸。hrp24的氨基酸序列如SEQ ID NO.13所示：PNQDLGQLLGGLLQKGLEATLQDA。
上述两个氨基酸序列不同的第十多肽均属于具有相同功能的同源突变体，且被证明具有相近的生物活性，具体信息参考文章“Functional mapping of harpin hrpZ of Pseudomonas syringae reveals the sites responsible for protein oligomerization,lipid interactions and plant defence induction”中的介绍。
进一步，所述PAMP分子多肽还包括激活植物免疫反应的第十一多肽，优选为多肽sys18 及其同源突变体。
sys18的氨基酸序列如SEQ ID NO.14所示：AVQSKPPSKRDPPKMQTD。sys18的同源突变体包括在SEQ ID NO.14所示的氨基酸序列中添加、缺失、替换一个或多个(例如1-10个)氨基酸且具有激活植物免疫反应能力的PAMP多肽。具体的，sys18的同源突变体sys18 m1在SEQ ID NO.14所示的氨基酸序列的第6位产生了1个氨基酸的替换突变：P6A；sys18的同源突变体sys18 m2在SEQ ID NO.14所示的氨基酸序列的第10位产生了1个氨基酸的替换突变：R10A。
上述两个氨基酸序列不同的第十一多肽均属于具有相同功能的同源突变体，且被证明具有相近的生物活性，具体信息参考文章“Structure-activity of deleted and substituted systemin.an 18-amino acid polypeptide inducer of plant defensive genes”中的介绍。
需要说明的是，以上所有突变体的名称并不是其本身的生物学名称，而是为了方便本专利写作与理解进行了统一命名。例如nlp20 m1，其生物学名称为Pyanlp20，其他突变体名称命名方式相同。具体突变体的生物学名称，因以氨基酸序列为准，在文章或者数据库中查询获得。
需要说明的是，由于自然存在的突变，每种激活不同受体的PAMP分子多肽在公开的上述氨基酸序列的基础上，其余氨基酸序列组成的多肽突变体，如与原氨基酸序列相比添加、缺失、替换一个或多个且具有激活相同受体功能的PAMP分子多肽突变体，都在本发明的保护范围之内。
在本发明的一种优选地实施方式中，所述第一多肽为flg22，第二多肽为nlp20，第三多肽为elf18，第四多肽为pip1，第五多肽为pep1，第六多肽为csp22，第七多肽为flgII-28，第八多肽为ralf17，第九多肽为pep13，十多肽为hrp24，第十一多肽为sys18。
在本发明的一种优选地实施方式中，所述融合蛋白由三个相同和/或不同的PAMP分子多肽组成，任选地，相邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子。
在本发明的一种优选地实施方式中，所述融合蛋白由四个相同和/或不同的PAMP分子多肽组成，任选地，相邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子。
在本发明的一种优选地实施方式中，所述融合蛋白由五个相同和/或不同的PAMP分子多肽组成，任选地，相邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子。
在本发明的一种优选地实施方式中，所述融合蛋白由六个相同和/或不同的PAMP分子多肽组成，任选地，相邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子。
进一步，在本发明的一种实施方式中，所述融合蛋白包含或由七个不同的PAMP分子多肽组成；任选地，相邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子。
优选地，所述融合蛋白由7个不同的PAMP分子多肽和至少6个连接子组成。优选所述七个不同的PAMP分子多肽选自flg22、nlp20、elf18、pip1、pep1、csp22、flgII-28、ralf17、pep13、hrp24或sys18中的任意七种的组合。
对于7个不同的PAMP分子多肽的排列顺序和连接子的具体种类不做具体限定，可以根据 植物的种类或者针对病原微生物的种类进行适当的调整，只要具有诱导植物免疫抗性的效果即可，而且通过调整优化获得免疫表位更多、效果更好、使用成本更低的融合蛋白都在本发明的保护范围之内。
融合蛋白通常需要与蛋白标签一起融合表达。蛋白标签是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽，以便于目的蛋白的表达、检测和纯化。
本发明中的融合蛋白可以连接的蛋白标签包括但不限于：HIS、GST、Flag、MBP、HA、c-Myc、eGFP、eYFP、eCFP。
在本发明的一种实施方式中，由7个不同的PAMP分子多肽分别为flg22、nlp20、elf18、pip1、pep1、csp22、flgII-28组成的融合蛋白的氨基酸序列如SEQ ID NO.15所示。
SEQ ID NO.15所示的氨基酸序列表明，融合蛋白连接了HIS蛋白标签，PAMP分子多肽的排列顺序依次为elf18、csp22、flg22、flgII-28、nlp20、pep1、pip1。相邻的PAMP分子多肽之间具有连接子，连接子选用GAG和AGA。
在本发明的一种实施方式中，由7个不同的PAMP分子多肽分别为flg22、nlp20、elf18、pip1、pep1、csp22、flgII-28组成的融合蛋白的氨基酸序列为与SEQ ID NO.15所示的氨基酸序列具有至少80％序列同一性的功能性同源序列。
所述的同一性的功能性同源序列包括但不限于SEQ ID NO.15所示的氨基酸具有约80％或以上、85％或以上、90％或以上、95％或以上、98％或以上、99％或以上同一性的氨基酸序列。
由上述七个不同的PAMP分子多肽组成的融合蛋白，配合使用合适的连接子和标签蛋白，组装成的融合蛋白，相较于单个PAMP分子多肽具有更多免疫活性表位，用于植物中可以更快速、更灵敏的与植物细胞表面的受体结合，快速诱导植物防御的免疫反应，抵抗病原微生物，提高植物抗病性，而且还具有毒性低，降解周期短和环境相容性大等优点。
本发明第二方面提供了编码上述融合蛋白的核苷酸序列。
需要说明的是，由于上述融合蛋白的组成是可变的，相邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子，连接子的数目和种类是可变的。所以对应的编码其的核苷酸序列也是可变的。所以此处对核苷酸序列中碱基的排列顺序和碱基数量不作具体限定，能够编码所述融合蛋白的核苷酸序列，及其互补序列、简并序列或同源序列都在本发明的保护范围之内。
在本发明的一种实施方式中，编码SEQ ID NO.15所示的氨基酸序列的融合蛋白的核苷酸序列如SEQ ID NO.16所示。
在本发明的一种实施方式中，编码SEQ ID NO.15所示的氨基酸序列的融合蛋白的核苷酸序列为在严格条件下与SEQ ID NO.16的核苷酸序列杂交，且能够编码所述的融合蛋白的核苷酸序列。
示例性地，在本文中所述“严格条件”是指探针将与其靶序列杂交至可探测程度超过与其它序列杂交(如至少2倍于背景)的条件。严格条件具有序列依赖性，且因环境的不同而不同。 通过控制杂交和/或洗涤条件的严格性，可以鉴定与探针100％互补的靶序列。
在本发明的一种实施方式中，编码SEQ ID NO.15所示的氨基酸序列的融合蛋白的核苷酸序列为与SEQ ID NO.16所示的核苷酸序列的简并序列。所述的简并序列，改变SEQ ID NO.16所示的核苷酸序列中的某个或多个核苷酸后，改变核苷酸序列位置对应编码的氨基酸种类不变，编码的融合蛋白的氨基酸序列不变。
在本发明的一种实施方式中，编码SEQ ID NO.15所示的氨基酸序列的融合蛋白的核苷酸序列为与SEQ ID NO.16所示的核苷酸序列的同源序列。优选地，所述同源序列为与SEQ ID NO.16所示的核苷酸序列具有至少85％或以上同一性的多核苷酸。
所述的同源序列包括但不限于SEQ ID NO.16所示的核苷酸具有约85％或以上、88％或以上、90％或以上、93％或以上、95％或以上、98％或以上、99％或以上同一性且具有编码所述的融合蛋白的多核苷酸。
本发明第三方面提供了导入了编码所述融合蛋白的核苷酸序列的载体。
对具体的载体种类不作限定，能够将上述核苷酸序列与载体连接成功后构建重组的表达载体，且该表达载体能够通过在宿主细胞内正常表达融合蛋白即可。
进一步地，载体包括但不限于pET-28b(+)、pETBlue-1、pETBlue-2、pET-32、pET-34b(+)、pET-35b(+)、pET-30EK/LIC、pET-32EK/LIC、pET-34EK/LIC、pET-36EK/LIC。
本发明第四方面提供了导入了上述编码融合蛋白的核苷酸序列，和/或上述载体的的微生物或细胞。
需要说明的是，此处“和/或”有三种解释：1.微生物或细胞单独包含编码融合蛋白的核苷酸序列；2.微生物或细胞单独包含上述的载体，载体包含编码融合蛋白的核苷酸序列；3.微生物或细胞包含编码融合蛋白的核苷酸序列，同时包含上述载体，且载体中同时包含编码融合蛋白的核苷酸序列。微生物可以是任何能够正常表达所述融合蛋白的原核或真核细胞。
在一种优选地实施方式中，微生物或细胞是指引入编码融合蛋白的核苷酸序列的特定微生物或细胞，并且还包括携带所述载体的这种微生物的子代。
在本发明一种实施方式中，所述微生物包括大肠杆菌、农杆菌或枯草芽孢杆菌中的一种或几种；优选大肠杆菌。
进一步，大肠杆菌的菌种包括但不限于BL21(DE3)、λDE3、Rosetta TM、K-12、HMS174、NovaBlue、Tuner、OrigamiB。
进一步，农杆菌的菌种包括但不限于EH101、EHA105、C58C1、LBA4404。
进一步，枯草芽孢杆菌的菌种包括但不限于pMA5、PUB110、pE194、pWB。
进一步，在植物体内和/或体外，将上述编码融合蛋白的核苷酸序列和/或上述载体转化入宿主微生物中的方法包括但不限于：热激化、热休克、电穿孔、磷酸钙沉淀、聚乙二醇(PEG)转化、脂质转染和显微注射。
本发明第五方面提供了包含所述融合蛋白，或所述的载体，或所述的微生物或细胞的植物免疫诱抗剂。
植物免疫诱抗剂指能够诱导或激活植物的免疫反应，提高植物对某些病原微生物的抗性的外源生物或分子。本发明中，融合蛋白，或包含编码融合蛋白核苷酸序列的载体，或所述的微生物可以作为制备植物免疫诱抗剂的原料，进而使用该植物免疫诱抗剂作为生物农药产品应用于农业生产中，也具有使用浓度低，起效快，应用于农业生产成本，毒性低，降解周期短，环境相容性大等优点。
进一步，植物免疫诱抗剂还包括一种或多种农学上可接受的载体、赋形剂、稀释剂或溶剂。
植物免疫诱抗剂在制备过程中，不仅包括上述融合蛋白，或所述载体，或所述微生物作为主料，还需包括多种农学上可接受的载体、赋形剂、稀释剂或溶剂的辅料，以获得更多剂型，效果更稳定，使用更加方便的植物免疫诱抗剂。
进一步，植物免疫诱抗剂的剂型选自由粉剂、可溶粉剂、可湿性粉剂、颗粒剂、水剂、微乳剂、悬浮剂和水分散粒剂组成的组。植物免疫诱抗剂的剂型种类多样，可以扩展其使用的范围，以更好地应用于不同种类的植物。
本发明第六方面提供了一种所述融合蛋白的制备方法，包括培养包含所述核苷酸的微生物或细胞的步骤，或包括人工合成所述融合蛋白的步骤；
优选地，所述方法包括以下步骤：
(a)合成所述核苷酸，优选地在合成之前分析和设计拼接成编码融合蛋白的核苷酸序列；
(b)将合成的核苷酸序列转化(优选通过载体转化)至微生物或细胞中，并培养所述微生物或细胞，以表达融合蛋白；和，
(c)任选地，收集和纯化所表达的融合蛋白。
上述融合蛋白的制备方法，将PTI免疫机制和基因工程的技术联合运用，将已经发现的至少三个相同和/或不同的PAMP分子多肽，通过基因工程的技术构建了一个新的基因重组的融合蛋白，通过蛋白质表达技术获取了自然界不存在多免疫表位的融合蛋白。上述制备方法，制备过程简单，耗时短，投入经济成本低，有效解决了制备植物免疫PAMP分子多肽生产成本高，长期无法应用于农业生产的问题。
在一种优选地实施方式中，所述融合蛋白的制备方法，包括以下步骤：
(a)选取七个不同的PAMP分子多肽的核苷酸序列，利用生物信息学软件Geneious R9结合Swiss-model在线分析和设计拼接成如SEQ ID NO.16所示的编码融合蛋白的核苷酸序列，人工合成所述核苷酸序列；七个不同的PAMP分子多肽分别为flg22、nlp20、elf18、pip1、pep1、csp22、flgII-28。
(b)运用基因工程的方法，将合成的核苷酸序列与pET-28b(+)表达载体连接后，转化入大肠杆菌BL21(DE3)中进行诱导表达融合蛋白；
(c)收集和纯化所表达的融合蛋白；
(d)将所表达的融合蛋白进行测序，发现融合蛋白的氨基酸序列如SEQ ID NO.15所示。
本发明第七方面提供了所述的融合蛋白，或所述的植物免疫诱抗剂，或所述的融合蛋白的制备方法制备得到的融合蛋白在提高植物抗病性、诱导植物防御反应和/或抵抗病原微生物中的应用。
进一步，所述植物包括但不限于：拟南芥、玉米、小麦、水稻、番茄、烟草。
进一步，所述病原微生物包括但不限于：丁香假单胞菌、禾谷镰刀菌、稻瘟病菌、烟草花叶病毒。
具体的使用过程中，可以将所述的融合蛋白，或所述的植物免疫诱抗剂施用至植物，但对具体施用方法和施用的量不作限定，可以根据植物种类以及病虫害类型进行合理的选择。
融合蛋白和/或植物免疫诱抗剂可以与植物细胞膜上受体蛋白相互作用，能够快速触发植物体的防御反应来抵抗病原菌的感染。由此，可以将该融合蛋白作为生物农药产品应用于农业生产中，激活植物自身的抗病性，以此提高植物抵抗病原微生物的能力，减少化学农药的使用。
本发明采用上述技术方案具有以下有益效果：
(1)本发明提供的融合蛋白，具有多个或多种免疫表位，使用浓度低，起效快，可以更快速、更广泛地的与植物细胞表面的受体结合，具有广谱性和高效性，可以诱导多种植物产生免疫反应，降低病原微生物侵染能力，明显提高植物抗病性；
(2)本发明提供的融合蛋白的制备成本相较于PAMP分子多肽的制备成本低，作为生物农药产品用于农业中可以明显降低农业生产成本；
(3)本发明提供的融合蛋白不会引起植物耐药性，具有毒性低，降解周期短，环境相容性大等优点；
(4)本发明提供的融合蛋白的制备方法，制备过程简单，耗时短，投入经济成本低，有效解决了制备植物免疫PAMP分子多肽生产成本高，长期无法应用于农业生产的问题。
附图说明
图1所示为七肽融合蛋白His-MP7的SDS-PAGE电泳图；
图2所示为拟南芥植物在不同浓度融合蛋白His-MP7和PAMP分子多肽flg22处理下的胼胝质积累情况；
图3所示为拟南芥植物在100nM融合蛋白His-MP7与不同PAMP分子多肽处理下的胼胝质积累情况；
图4所示为玉米植物在融合蛋白His-MP7诱导下活性氧产生情况；
图5所示为融合蛋白His-MP7增强拟南芥植物对DC3000病原菌的抗性的实验结果；
图6所示为融合蛋白His-MP7增强玉米植物对禾谷镰刀菌的抗性的实验结果；其中，a为融 合蛋白His-MP7抑制禾谷镰刀菌侵染玉米的生长实测对比；b为融合蛋白His-MP7抑制禾谷镰刀菌侵染玉米的实验结果柱状图；
图7所示为融合蛋白His-MP7增强水稻植物对稻瘟病菌的抗性的实验结果；其中，a为融合蛋白His-MP7抑制稻瘟病菌侵染水稻的生长实测对比；b为融合蛋白His-MP7抑制稻瘟病菌侵染水稻的实验结果柱状图。
具体实施方式
本发明中，术语“PAMP分子多肽”指通过在多种病原真菌或细菌或植物本身的分泌蛋白中进行同源性序列对比分析，找到蛋白分子中序列相对保守且具有免疫激活能力的多肽片段，常被植物作为感知病原菌侵害的信号分子多肽。
本发明中，术语“连接子”指具有至少1个氨基酸残基，优选为至少2个连续的氨基酸残基。
本发明中，术语“植物免疫诱抗剂”指能够诱导或激活植物的免疫反应，提高植物对某些病原微生物的抗性的外源生物或分子。
本发明中，术语“PTI免疫机制”，全称病原物相关分子模式触发免疫(PAMP‐Triggered Immunity)机制，指PAMP信号分子与植物细胞受体识别后激活植物免疫反应的机制。
本发明中，“蛋白标签”是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽，以便于目的蛋白的表达、检测和纯化。
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下述实施例中，关于部分植物材料与菌种、病毒来源的介绍。
植物材料：水稻(OryzasativaL.)为短粒粳稻品种日本晴(NPB)(NPB为已经全基因组测序的国际通用品种)、玉米为浚单20，水稻和玉米均通过市场购入；栽培番茄(Solanum lycopersicum)、哥伦比亚野生型拟南芥col-0、N89烟草株系均来源于四川农业大学生命科学学院蔡易教授课题组。
菌种与病毒：丁香假单胞菌DC3000，稻瘟病ZB15小种，烟草花叶病毒、禾谷镰刀菌均来源于四川农业大学生命科学学院蔡易教授课题组。
此外，实施例中没有提及来源的材料、试剂和耗材等均可以从市场购入。
His标签蛋白纯化试剂盒购自康为世纪公司产品，产品货号CW0894；BCA法蛋白质浓度测定试剂盒购自Solarbio公司产品，产品目录号PC0020-500微孔(50T)。
实施例1 七肽融合蛋白His-MP7的分子设计与核苷酸序列获取
(1)在11种不同的PAMP分子多肽：flg22、nlp20、elf18、pip1、pep1、csp22、flgII-28、elf18、pep13、ralf17、hrp15、sys18及其突变体中，选取7种不同的PAMP分子多肽：flg22、nlp20、elf18、pip1、pep1、csp22、flgII-28组成融合蛋白，连接子为AGA和GAG；
(2)使用在线分析平台ExPASy(https://www.expasy.org)对上述蛋白质序列进行分子质量、氨基酸组成(protparam)和疏水性等基本性质分析(protscale)；使用Phyre2在线平台(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi？id＝index)对上述蛋白结构进行建模；
(3)结合分析结果，筛选出1种设计方案，命名该融合蛋白为MP7，氨基酸序列如SEQ ID NO:15所示；
(4)利用生物信息学软件Geneious R9软件，在线分析平台Jcat(http://www.jcat.de)设计编码融合蛋白MP7的核苷酸序列，得到如SEQ ID NO:16所示的核苷酸序列，人工合成所述核苷酸序列。
实施例2 七肽融合蛋白His-MP7表达和纯化
(1)将SEQ ID NO:16所示的核苷酸序列克隆到pET-28b(+)表达载体(Novagen)的HindIII和XholI位点，热激转化到大肠杆菌DH5α中，挑取阳性克隆，摇菌并提取质粒，酶切并测序验证正确后，热激转化到大肠杆菌BL21(DE3)中，得到含有重组质粒pET-28b-MP7的大肠杆菌命名为BL21(DE3)/pET-28b-MP7；
(2)对BL21(DE3)/pET-28b-MP7进行诱导表达，包括以下步骤：表达菌株接种于LB液体培养基中，37℃，200rpm/min振荡培养过夜，得到第一菌液；将过夜的菌液以1:100的体积比转接至含100μg/mL卡那霉素的LB液体培养基中，37℃、200rpm/min继续振荡，培养至菌液的浓度OD600nm值为0.6，然后加入终浓度为0.5mmol/L IPTG，于28℃、200rpm/min振荡培养12h，得到第二菌液；将第二菌液12000rpm/min离心，收集菌体加入PBS缓冲液，经超声破碎菌体后，4℃，12000rpm/min离心，收集上清液；
(3)上清液采用His标签蛋白纯化试剂盒纯化(可溶性蛋白)，具体操作如下：先将5mLNi-Agarose填料装入亲和柱空柱，将上清液缓慢流过亲和柱，再用含有10mM咪唑的PBS缓冲液洗脱6个柱体积以去杂质，最后用含500mM咪唑的PBS缓冲液洗脱5个柱体积并收集过柱后洗脱液，即为融合蛋白His-MP7溶液；
(4)利用BCA法蛋白质浓度测定试剂盒检测，测得融合蛋白His-MP7的浓度为0.2mg/mL；经过SDS-PAGE检测，如图1所示，纯化获得一个分子量约23kDa的包含组氨酸的表达蛋白(His-MP7)。
实施例3 七肽融合蛋白His-MP7的免疫激活的效价研究
植物细胞免疫反应中会产生胼胝质的积累，胼胝质可以加强植物细胞壁的机械强度和堵塞病原体在细胞间扩散的通道，从而限制病原微生物的入侵。以模式植物拟南芥为材料，以胼胝质积累为免疫指标，分析了不同浓度的融合蛋白His-MP7的免疫激活能力，并与单一的PAMP分 子多肽做对比。具体实验操作如下：
一、不同浓度融合蛋白His-MP7引起拟南芥胼胝质积累的对比
调整融合蛋白His-MP7与多肽flg22溶液终浓度分别为1μM，100nM，10nM，并以水为空白对照。通过注射器渗透法侵染四周龄的拟南芥叶片，处理12小时后，采集处理过的叶片放入六孔板，加入适量洗脱液水平孵育4小时。用终浓度为0.1mg/mL的苯胺蓝染色液替换洗脱液，避光室温染色1小时，在荧光显微镜10倍物镜下观察胼胝质积累情况。每个处理三个生物学重复，重复三次实验。
实验结果如图2所示：100nM融合蛋白His-MP7能明显引起拟南芥胼胝质积累，且与1μM PAMP分子多肽flg22效果相当。
二、 同一浓度下融合蛋白His-MP7与不同多肽的免疫激活能力对比
调整融合蛋白His-MP7与多肽flg22，nlp20，elf18，pip1，pep1，csp22，flgII-28溶液终浓度均为100nM，并以csp22和flgII-28为阴性对照，通过注射器渗透法侵染四周龄的拟南芥叶片，样品处理方法与上述相同。
实验结果如图3所示：100nM融合蛋白His-MP7免疫激活效果优于所有PAMP分子多肽。
三、 融合蛋白His-MP7诱导植物产生活性氧爆发免疫反应
活性氧迸发被认为是植物对病原微生物应答的最早期的反应之一，在植物防卫反应中起到重要作用。已有研究证明，活性氧在植物中可以直接作为抗菌剂对病原微生物有直接的毒性和抑制其生长的作用，植物受到病原微生物侵染后体内会产生并积累活性氧迸发。以植物玉米为材料，以活性氧为免疫指标，分析了100nM融合蛋白His-MP7的免疫激活能力。具体实验操作如下：
取二周龄的玉米叶片，取中间部分剪成5cm长度浸入1μg/mL生长素6BA溶液中，加入终浓度为100nM的融合蛋白His-MP7，以生长素6BA的水溶液为空白对照。浸泡48小时，用1mg/mL的DAB溶液染色12小时，洗脱液(乙醇：乙酸：甘油＝3:1:1)洗脱12h，放置30分钟观察。
实验结果如图4所示：100nM融合蛋白His-MP7能够诱导植物产生活性氧爆发免疫反应。
上述三个实验证明，较低浓度的融合蛋白His-MP7(100nM)即可高效激活植物免疫，且同一浓度下的融合蛋白His-MP7的免疫激活能力优于单一的PAMP分子多肽。
实施例4 检测七肽融合蛋白His-MP7提高植物的抗病能力
一、 融合蛋白His-MP7能增强植物对DC3000病原菌的抗性
实验过程：
(1)取丁香假单胞菌DC3000菌种接入20mL SOC+str(链霉素)液体培养基，28℃过夜培养14-16小时，测定OD600值，梯度稀释至OD600＝0.00005，得到菌液；
(2)将融合蛋白His-MP7加入菌液至终浓度100nM，以加入水的菌液为阳性对照。同时注射四周龄健康的拟南芥叶片，一株4片(T0)，5株每株两片(T3)，分别于第0天和第三天采样，每片叶子用打孔器打孔，取打孔的小圆片一片，T0天加入500μL 10mM MgCl 2，在1.5mL  ep管中磨碎，同一处理的四个样，各取50μL点在同一个SOC+str(链霉素)固体平板上(平板必须吹干才能保持点样形状)，28℃培养16-24小时，拍照，观察统计菌落生长情况；
(3)T3天5株每株的两片叶子作为一个样本，同样打孔取小圆片，加入250μL 10mM MgCl 2，在1.5mL ep管中磨碎并用MgCl 2逐级稀释至1×10 -5。同一处理的五个样，包括稀释的每个样品(一个处理30个样品)各取10μL点在同一个SOC+str(链霉素)固体平板上(用方皿，平板必须吹干才能保持点样形状)，28℃培养16-24小时，拍照，观察统计菌落生长情况。
实验结果如图5的柱状图所示：对照组的细菌生长指数为5.38，添加100nM融合蛋白His-MP7处理的拟南芥的细菌生长指数为4.05，其丁香假单胞菌DC3000的生长量减少了10以上，His-MP7能够有效增强植物对病原菌的免疫能力。
二、 融合蛋白His-MP7能增强植物对禾谷镰刀菌的抗性
实验过程：
(1)将禾谷镰刀菌菌丝从平板接入CMC液体培养基，25℃避光培养3-7天，用纱布过滤，10000rpm/10min离心收集孢子，血球计数板统计孢子数目，调整浓度至2×10 5，放4℃保存(一个月内使用)；
(2)取两周龄玉米叶片，取中间部分剪成5cm长度浸入1μg/mL生长素6BA溶液中，每个处理取12-13片叶子，加入终浓度为100nM的融合蛋白His-MP7蛋白，以生长素6BA的水溶液为空白对照；
(3)分别将禾谷镰刀菌孢子液体均匀点在叶片上，28℃，12小时光照，12小时黑暗条件下培养3-4天，观察发病情况，通过imageJ软件统计病斑面积百分比。
实验结果如图6中a显示玉米叶片生长实测对比，b显示的柱状图所示：未加融合蛋白His-MP7处理的叶片病斑面积百分比为12.3％，加融合蛋白His-MP7处理的叶片病斑面积百分比为3.6％。说明融合蛋白His-MP7显著提高了玉米对禾谷镰刀菌的抗性，使禾谷镰刀菌的侵染率降低70.8％。
三、 融合蛋白His-MP7能增强植物对稻瘟病的抗性
实验过程：
(1)稻瘟病菌接种在CM固体培养基上，28℃正置培养13-15天，用枪头将菌丝全部刮下，取少量5-10mL无菌水冲洗，用纱布过滤置50mL离心管，10000rpm/10min离心收集孢子，血球计数板统计孢子数目，调整浓度至1×10 6，放常温保存(一周内使用)；
(2)取四周龄的水稻叶片，取中间部分剪成5cm长度浸入1μg/mL生长素6BA溶液中，每个处理取12-13片叶子，加入终浓度为100nM的融合蛋白His-MP7，以生长素6BA的水溶液为空白对照；
(3)分别将稻瘟菌孢子液体均匀点在叶片上，28℃，12小时光照，12小时黑暗条件下培养3-4天，观察发病情况，过imageJ软件统计病斑面积百分比。
实验结果如图7中a显示水稻叶片生长实测对比，b显示的柱状图所示：未加融合蛋白His-MP7处理的叶片病斑面积百分比为11.6％，加融合蛋白His-MP7处理的叶片病斑面积百分比为1.9％。说明融合蛋白His-MP7显著提高了水稻对稻瘟病的抗性，使稻瘟病菌的侵染率降低83.6％，减少水稻稻瘟病对水稻的危害。
四、 融合蛋白His-MP7能增强烟草对烟草花叶病毒(TMV)的抗性
实验设实验组与对照组，每组20株烟草，实验组为注射重组蛋白后接种病毒，对照组为注射无菌水后接种病毒，接种方法如下：
(1)将新鲜的TMV病叶加入少量灭菌后的磷酸缓冲液(1∶200)，并在研钵中研磨，用灭菌纱布滤去病叶残体，取出新鲜汁液制成接种物，调节TMV病毒接种物的浓度，得到TMV病毒水溶液；
(2)当烟草苗处于4-5片真叶期时，选择充分展开的真叶，在叶面上均匀撒上适量的石英砂，用脱脂棉球蘸取TMV病毒水溶液轻轻摩擦1-2遍，然后立即用水冲洗叶面；
(3)接种21d后，观察烟草植株发病情况。以株为单位，分级标准如下：
0级：无病；
1级：心叶基部沿叶脉出现少量褪绿黄斑，不卷曲；
3级：新生叶出现与叶脉平行的黄绿相间条纹，轻微卷曲；
5级：新生叶出现大量与叶脉平行的失绿条纹，叶片卷曲、细弱；
7级：植株矮化，叶片出现黄白色条纹卷起，新生叶扭曲下垂，不能正常开张；
9级：植株严重矮化、失绿或死亡。
计算病情指数和防治效果，病情指数及防治效果计算方法：
病情指数＝[Σ(各级病株数×相对级数值)/(调查总株数×9)]×100
防治效果(％)＝[(对照病情指数-处理病情指数)/对照病情指数]×100。
结果如表1和表2所示：
表1 烟草病株数与病情分级
单位(株) 0级 1级 3级 5级 7级 9级
H 2O 0 0 2 2 9 7
His-MP7 1 11 6 2 0 0
表2 烟草病情指数与防治效果
Figure PCTCN2020135367-appb-000001
实验结果表明，经融合蛋白His-MP7处理后的实验组病情指数下降至21.7，防治效果达到72.5％，试验证明了融合蛋白His-MP7可增强烟草对TMV的抗性。
实施例5 多种三肽融合蛋白的分子设计、表达与纯化
一、多种三肽融合蛋白的分子设计
(1)在11种不同的PAMP分子多肽：flg22、nlp20、elf18、pip1、pep1、csp22、flgII-28、elf18、pep13、ralf17、hrp15、sys18及其同源突变体中，随机选取三种多肽组成多种融合蛋白，连接子为AKG，得到多种设计方案；
(2)使用在线分析平台ExPASy对上述蛋白质序列进行分子质量、氨基酸组成、和疏水性等基本性质分析；使用Phyre2在线平台对上述融合蛋白结构进行建模；
(3)结合分析结果，筛选出20种设计方案，分别命名为TP1，TP2，TP3，TP4，TP5，TP6，TP7，TP8，TP9，TP10，TP11，TP12，TP13，TP14，TP15，TP16，TP17，TP18，TP19，TP20。多种三肽融合蛋白的组成设计方案如表3所示：
表3 三肽融合蛋白设计方案
名称 序列(连接子AKG) 名称 序列(连接子AKG)
TP1 flg22-csp22-pep13 TP11 flg22 m2-flg22 m2-flg22 m2
TP2 flg22-elf18-pep1 TP12 flg22 m2-nlp20 m1-hrp15
TP3 flg22-elf18-pip1 TP13 flg22-nlp20-csp22
TP4 flg22-flg22-flg22 TP14 flg22-nlp20 m1-pep1
TP5 flg22-flgII-28-csp22 TP15 flg22-nlp20 m2-csp22 m1
TP6 flg22-flgII-28-nlp20 TP16 flg22-pep1-pip1
TP7 flg22-hrp15-sys18 TP17 flg22-ralf17 m1-hrp15
TP8 flg22 m1-flgII-28 m1-flg22 m2 TP18 flg22-ralf17-hrp24
TP9 flg22 m1-flg22 m1-flg22 m1 TP19 flg22-ralf17-pip1
TP10 flg22 m1-ralf17 m1-csp22 m1 TP20 flg22-ralf17-sys18
(4)编码上述20种三肽融合蛋白的核苷酸序列获取方法同实施例1相同；
二、多种三肽融合蛋白的表达与纯化
(1)将获取的编码上述20种三肽融合蛋白的核苷酸序列分别克隆到pEGX-4T-1表达载体的BamHI和XholI位点，热激转化到大肠杆菌DH5α中，挑取阳性克隆，摇菌并提取质粒，酶切并测序验证正确后，热激转化到大肠杆菌BL21(DE3)中，得到含有重组质粒pEGX-4T-1-TP的大肠杆菌命名为BL21(DE3)/pEGX-4T-1-TP；
(2)对上述大肠杆菌进行诱导表达，包括以下步骤：表达菌株接种于LB液体培养基中，37℃，200rpm/min振荡培养过夜，得到第一菌液；将过夜的菌液以1:100的体积比转接至含100μg/mL氨苄青霉素的LB液体培养基中，37℃、200rpm/min继续振荡，培养至菌液的浓度OD600nm值为0.6，然后加入终浓度为0.3mmol/L IPTG，于25℃、200rpm/min振荡培养12h，得到第二菌液；将第二菌液12000rpm/min离心，收集菌体加入PBS缓冲液，经超声破碎菌体后，4℃，12000rpm/min离心，收集上清液；
(3)上清液采用GST标签蛋白纯化试剂盒纯化(可溶性蛋白)，得到GST-TP融合蛋白溶液。 采用BCA法蛋白质浓度测定试剂盒对蛋白溶液进行定量。
实施例6 多种四肽融合蛋白的分子设计、表达与纯化
一、多种四肽融合蛋白的分子设计
在11种不同的PAMP分子多肽：flg22、nlp20、elf18、pip1、pep1、csp22、flgII-28、elf18、pep13、ralf17、hrp15、sys18及其同源突变体中，随机选取四种多肽组成融合蛋白，连接子为AKG，设计方法同实施例5。筛选得到20种设计方案，分别命名为FP1，FP2，FP3，FP4，FP5，FP6，FP7，FP8，FP9，FP10，FP11，FP12，FP13，FP14，FP15，FP16，FP17，FP18，FP19，FP20。多种四肽融合蛋白的组成设计方案如表4所示：
表4 四肽融合蛋白设计方案
名称 序列(连接子AKG) 名称 序列(连接子AKG)
FP1 flg22-csp22-pep13-ralf17 FP11 flg22 m1-elf18 m1-nlp20 m2-csp22 m1
FP2 flg22-elf18-cap22-nlp20 FP12 flg22 m1-flg22 m1-flg22 m1-elf18
FP3 flg22-elf18-pep1-pip1 FP13 flg22 m1-nlp20 m2-csp22 m1-pip1 m1
FP4 flg22-elf18-pip1-pep1 FP14 flg22 m1-ralf17 m1-csp22-ralf17
FP5 flg22-elf18-nlp20 m2-csp22 m1 FP15 flg22 m2-flg22 m2-flg22 m2-pip1 m1
FP6 flg22-flg22-flg22-flg22 FP16 flg22 m2-nlp20 m1-csp22-hrp15
FP7 flg22-flgII-28-csp22-hrp24 FP17 flg22-ralf17 m1-hrp15-flg22
FP8 flg22-flgII-28-nlp20-hrp24 FP18 flg22-ralf17-pep1-csp22
FP9 flg22-flgII-28-nlp20 m1-csp22 m1 FP19 flg22-ralf17-pip1-pep1
FP10 flg22-hrp15-sys18-sys18 m1 FP20 flg22-ralf17-sys18-hrp15
编码上述20种四肽融合蛋白的核苷酸序列获取方法同实施例1相同。
二、多种四肽融合蛋白的表达与纯化，与实施例5相同。
实施例7 多种五肽融合蛋白的分子设计、表达与纯化
一、多种四肽融合蛋白的分子设计
在11种不同的PAMP分子多肽：flg22、nlp20、elf18、pip1、pep1、csp22、flgII-28、elf18、pep13、ralf17、hrp15、sys18及其突变体中，随机选取五种多肽组成融合蛋白，连接子为AKG，设计方法同实施例5。筛选得到20种设计方案，分别命名为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。多种五肽融合蛋白的组成设计方案如表5所示：
表5 五肽融合蛋白设计方案
名称 序列(连接子AKG)
MP5-1 elf18-flg22-csp22-flgII-28-nlp20
MP5-2 elf18-flg22-flgII-28-pep13-nlp20
MP5-3 elf18-flg22-flgII-28-pip1-pep1
MP5-4 elf18-flg22-flgII-28-ralf17-hrp15
MP5-5 elf18-flg22-flgII-28-ralf17-sys18
MP5-6 flg22-flg22-flg22-flg22-flg22
MP5-7 flg22-flgII-28-flg22-flgII-28-flg22
MP5-8 flg22-flgII-28-nlp20-hrp24-sys18
MP5-9 flg22-flgII-28-nlp20 m1-csp22 m1-pep13
MP5-10 flg22-hrp15-sys18-pep13-ralf17
MP5-11 flg22-flgII-28-nlp20-csp22-pep13
MP5-12 nlp20-flg22 m1-flg22 m1-flg22 m1-elf18
MP5-13 nlp20-flg22 m1-nlp20 m2-csp22 m1-pip1
MP5-14 nlp20 m1-flg22 m1-ralf17 m1-csp22-ralf17
MP5-15 nlp20 m2-flg22 m2-flg22 m2-flg22 m2-pip1
MP5-16 nlp20 m1-flg22 m2-nlp20 m1-csp22-hrp15
MP5-17 flg22-ralf17 m1-hrp15-flg22-csp22
MP5-18 flgII-28-flg22-ralf17-pep1-csp22
MP5-19 flgII-28-flg22-ralf17-pip1-pep1
MP5-20 flgII-28-flg22-ralf17-sys18-hrp15
编码上述20种五肽融合蛋白的核苷酸序列获取方法同实施例1相同。
二、多种五 肽融合蛋白的表达与纯化，与实施例5相同。
实施例8 多种六肽及七肽融合蛋白的分子设计
在11种不同的PAMP分子多肽：flg22、nlp20、elf18、pip1、pep1、csp22、flgII-28、elf18、pep13、ralf17、hrp15、sys18及其突变体中，随机选取六种组成多种融合蛋白，连接子为AKG，设计方法同实施例5。筛选得到20种设计方案，分别命名为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。多种六肽融合蛋白的组成设计方案如表6所示：
表6 六肽融合蛋白设计方案
名称 序列(连接子AKG)
MP6-1 elf18-flg22-csp22-flgII-28-nlp20-pep1
MP6-2 elf18-flg22-flgII-28-csp22-pip1-pep1
MP6-3 elf18-flg22-flgII-28-nlp20-ralf17-hrp15
MP6-4 elf18-flg22-flgII-28-pep13-nlp20-pip1
MP6-5 elf18-flg22-flgII-28-ralf17-sys18-hrp15
MP6-6 flg22-flg22-flg22-flg22-flg22-flg22
MP6-7 flg22-flgII-28-flg22-flgII-28-flg22-pep13
MP6-8 flg22-flgII-28-flg22-ralf17-sys18-hrp15
MP6-9 flg22-flgII-28-nlp20-csp22-pep13-pep13
MP6-10 flg22-flgII-28-nlp20-hrp24-sys18-csp22
MP6-11 flg22-flgII-28-nlp20 m1-csp22 m1-pep13-elf18
MP6-12 flg22-hrp15-sys18-pep13-ralf17-csp22
MP6-13 flg22 m1-flgII-28-flg22-ralf17-pep1-csp22
MP6-14 flg22 m2-flgII-28-flg22-ralf17-pip1-pep1
MP6-15 nlp20-flg22 m1-flg22 m2-elf18-pep1-nlp20
MP6-16 nlp20-flg22 m1-nlp20 m2-csp22 m1-pip1-pep1
MP6-17 nlp20 m1-flg22 m2-nlp20 m1-csp22-hrp24-ralf17
MP6-18 nlp20 m1-flg22 m1-ralf17 m1-csp22-ralf17-hrp15
MP6-19 nlp20 m2-flg22 m2-flg22 m2-flg22 m2-pep13-pep1
MP6-20 ralf17 m1-hrp15-flg22-csp22-pip1-pep13
编码上述20种六肽融合蛋白的核苷酸序列获取方法同实施例1相同。
二、多种六 肽融合蛋白的表达与纯化，与实施例5相同。
实施例9 多种七肽融合蛋白的分子设计、表达与纯化
在11种不同的PAMP分子多肽：flg22、nlp20、elf18、pip1、pep1、csp22、flgII-28、elf18、pep13、ralf17、hrp15、sys18及其突变体中，随机选取七种多肽组成融合蛋白，连接子为AKG，设计方法同实施例5。筛选得到20种设计方案，分别命名为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。多种七肽融合蛋白的组成设计方案如表7所示：
表7 七肽融合蛋白设计方案
名称 序列(连接子AKG)
MP7-1 elf18-flg22-csp22-flgII-28-nlp20-pep1-pip1
MP7-2 elf18-flg22-flgII-28-csp22-nlp20-pep1-pip1
MP7-3 elf18-flg22-flgII-28-nlp20-csp22-ralf17-hrp15
MP7-4 elf18-flg22-flgII-28-nlp20-csp22-hrp24-sys18
MP7-5 flg22-flgII-28-nlp20-csp22-ralf17-sys18-hrp15
MP7-6 flg22-flgII-28-nlp20-csp22-pep13-ralf17-hrp15
MP7-7 flg22-flgII-28-nlp20-csp22-pep13-ralf17-sys18
MP7-8 flg22-flgII-28-nlp20-csp22-pep13-ralf17-hrp24
MP7-9 flg22-flgII-28-nlp20-csp22-pep13-ralf17-nlp20
MP7-10 flg22-flg22 m1-flg22 m2-flg22 m1-flg22 m2-flg22 m1-flg22 m2
名称 序列(连接子KRK)
MP7-11 elf18-flg22-csp22-flgII-28-nlp20-pep1-pip1
MP7-12 elf18-flg22-flgII-28-csp22-nlp20-pep1-pip1
MP7-13 elf18-flg22-flgII-28-nlp20-csp22-ralf17-hrp15
MP7-14 elf18-flg22-flgII-28-nlp20-csp22-hrp24-sys18
MP7-15 flg22-flgII-28-nlp20-csp22-ralf17-sys18-hrp15
MP7-16 flg22-flgII-28-nlp20-csp22-pep13-ralf17-hrp15
MP7-17 flg22-flgII-28-nlp20-csp22-pep13-ralf17-sys18
MP7-18 flg22-flgII-28-nlp20-csp22-pep13-ralf17-hrp24
MP7-19 flg22-flgII-28-nlp20-csp22-pep13-ralf17-nlp20
MP7-20 flg22-flg22 m1-flg22 m2-flg22 m1-flg22 m2-flg22 m1-flg22 m2
编码上述20种七肽融合蛋白的核苷酸序列获取方法同实施例1相同。
二、多种七肽融合蛋白的表达与纯化，与实施例5相同。
实施例10 检测不同融合蛋白的免疫反应
以模式植物拟南芥为材料，以胼胝质积累为免疫指标，并以水为空白对照，上述实施例5-9获得的所有融合蛋白调整溶液终浓度均为100nM，通过注射器渗透法侵染四周龄的拟南芥叶片，具体实验操作同实施例3。所得胼胝质积累荧光图像，通过图像处理软件ImageJ计算凭借荧光密度，量化融合蛋白对植物的免疫激活能力。计算方法如下：
测量IntDen＝integrated density＝综合光密度；
测量Area＝图片面积；
MD(mean optical density)＝IntDen/Area。
实验结果如表8所示：
表8：多种融合蛋白的免疫反应强度鉴定
Figure PCTCN2020135367-appb-000002
Figure PCTCN2020135367-appb-000003
Figure PCTCN2020135367-appb-000004
由表8的实验结果可知，与空白对照组相比，实施例5-9提供的多种三肽、多种四肽、多种五肽、多种六肽、多种七肽等不同的融合蛋白均具有免疫激活能力。

Claims (15)

  1. 一种融合蛋白，其特征在于，所述融合蛋白包含或由至少三个、四个、五个、六个、七个、或八个相同和/或不同的PAMP分子多肽组成，任选地，相邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子。
  2. 根据权利要求1所述的融合蛋白，其特征在于，所述PAMP分子多肽包括激活FLS2免疫受体的第一多肽，激活RLP23免疫受体的第二多肽，激活EFR免疫受体的第三多肽，激活RLK7免疫受体的第四多肽，激活PEPR1免疫受体的第五多肽，激活CORE1免疫受体的第六多肽，激活FLS3免疫受体的第七多肽，激活FER受体的第八多肽，激活植物免疫反应的第九多肽pep13，第十多肽hrp24，第十一多肽sys18。
  3. 根据权利要求2所述的融合蛋白，其特征在于，所述第一多肽为flg15及其同源突变体，或flg22及其同源突变体；
    所述第二多肽为nlp20及其同源突变体；
    所述第三多肽为elf18及其同源突变体；
    所述第四多肽为pip1及其同源突变体；
    所述第五多肽为pep1及其同源突变体；
    所述第六多肽为csp15及其同源突变体，或csp22及其同源突变体；
    所述第七多肽为flgII-28及其同源突变体；
    所述第八多肽为ralf17及其同源突变体；
    所述第九多肽为pep13及其同源突变体；
    所述第十多肽为hrp15及其同源突变体，或hrp24及其同源突变体；
    所述第十一多肽为sys18及其同源突变体。
  4. 根据权利要求3所述的融合蛋白，其特征在于，所述第一多肽为flg22，第二多肽为nlp20，第三多肽为elf18，第四多肽为pip1，第五多肽为pep1，第六多肽为csp22，第七多肽为flgII-28，第八多肽为ralf17，第九多肽为pep13，十多肽为hrp24，第十一多肽为sys18。
  5. 根据权利要求1-4任一项所述的融合蛋白，其特征在于，所述融合蛋白由三个相同和/或不同的PAMP分子多肽组成，任选地，相邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子；
    优选地，所述融合蛋白由四个相同和/或不同的PAMP分子多肽组成，任选地，相邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子；
    优选地，所述融合蛋白由五个相同和/或不同的PAMP分子多肽组成，任选地，相 邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子；
    优选地，所述融合蛋白由六个相同和/或不同的PAMP分子多肽组成，任选地，相邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子。
  6. 根据权利要求1-4任一项所述的融合蛋白，其特征在于，所述融合蛋白包含或由七个不同的PAMP分子多肽组成，任选地，相邻两个PAMP分子多肽之间具有至少一个连接子或者没有连接子；
    优选地，所述七个不同的PAMP分子多肽选自flg22、nlp20、elf18、pip1、pep1、csp22、flgII-28、ralf17、pep13、hrp24或sys18中的任意七种的组合。
  7. 根据权利要求6所述的融合蛋白，其特征在于，所述融合蛋白的氨基酸序列包含或由如下序列组成：
    (1)SEQ ID NO.15所示的氨基酸序列；或，
    (2)与SEQ ID NO.15所示的氨基酸序列具有至少80％序列同一性的功能性同源序列。
  8. 编码权利要求1-7任一项所述的融合蛋白的核苷酸序列。
  9. 根据权利要求8所述的编码融合蛋白的核苷酸序列，其特征在于，所述核苷酸序列包含或由如下序列组成：
    (1)SEQ ID NO.16所示的核苷酸序列；或，
    (2)SEQ ID NO.16所示的核苷酸序列的互补序列、简并序列或同源序列；或，
    (3)在严格条件下与SEQ ID NO.16的核苷酸序列杂交，且能够编码所述融合蛋白的核苷酸序列；
    优选地，所述同源序列为与SEQ ID NO.16所示的核苷酸序列具有至少85％或以上同一性且具有编码所述融合蛋白的多核苷酸序列。
  10. 导入了权利要求8或9所述核苷酸序列的载体。
  11. 导入了权利要求8或9所述核苷酸序列，和/或权利要求10所述载体的微生物或细胞。
  12. 如权利要求11所述的微生物或细胞，其特征在于，所述微生物或细胞包括大肠杆菌、农杆菌、乳酸菌、酵母菌或枯草芽孢杆菌中的一种或几种；优选大肠杆菌。
  13. 包含权利要求1-7任一项所述融合蛋白，或权利要求10所述的载体，或权利要求11或12所述的微生物或细胞的植物免疫诱抗剂；
    优选地，所述植物免疫诱抗剂还包括一种或多种农学上可接受的载体、赋形剂、稀释剂或溶剂；
    优选地，植物免疫诱抗剂的剂型选自由粉剂、可溶粉剂、可湿性粉剂、颗粒剂、水 剂、微乳剂、悬浮剂和水分散粒剂组成的组。
  14. 一种权利要求1-7任一项所述的融合蛋白的制备方法，其特征在于，包括培养包含权利要求11或12所述微生物或细胞的步骤，或包括人工合成权利要求1-7任一项所述融合蛋白的步骤；
    优选地，所述方法包括以下步骤：
    (a)合成权利要求8或9所述核苷酸，优选地在合成之前分析和设计拼接成编码权利要求1-7任一项所述融合蛋白的核苷酸序列；
    (b)将合成的核苷酸序列转化(优选通过载体转化)至微生物或细胞中，并培养所述微生物或细胞，以表达融合蛋白；和，
    (c)任选地，收集和纯化所表达的融合蛋白。
  15. 权利要求1-7任一项所述的融合蛋白，或权利要求13所述的植物免疫诱抗剂，或权利要求14所述的融合蛋白的制备方法制备得到的融合蛋白在提高植物抗病性、诱导植物防御反应和/或抵抗病原微生物中的应用；
    优选地，所述植物包括拟南芥、玉米、小麦、水稻、番茄、烟草；
    优选地，所述病原微生物包括丁香假单胞菌、禾谷镰刀菌、稻瘟病菌、烟草花叶病毒。
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