Fusion Protein, Amino Acid Sequence Thereof, Coding Nucleotide Sequence Thereof, Preparation Method Thereof and Use Thereof

Abstract

A fusion protein, an amino acid sequence thereof, a coding nucleotide sequence thereof, a preparation method thereof and a use thereof are in the technical field of agricultural biotechnology. The fusion protein contains or consists of at least three, four, five, six, seven, or eight same and/or different PAMP (Pathogen-Associated Molecular Pattern) polypeptides. Optionally, there is at least one linker or no linker between two adjacent PAMP polypeptides. A plurality of PAMP polypeptides are assembled into the fusion protein having multiple immune epitopes. The fusion protein may induce defense immune responses of plants, weaken infestation ability of pathogenic microorganisms and substantially improve the disease resistance of plants. The method for preparing the fusion protein combines technologies of PTI (PAMP-Triggered Immunity) mechanism and gene engineering to obtain the fusion protein having multiple immune epitopes can be used in preparation of plant immune PAMP polypeptides.

Claims

1. A fusion protein, wherein the fusion protein comprises or consists of at least three, four, five, six, seven, or eight same and/or different PAMP (Pathogen-Associated Molecular Pattern) polypeptides; and optionally, there is at least one linker or no linker between two adjacent PAMP polypeptides.

2. The fusion protein according to claim 1, wherein the PAMP polypeptides comprise a first polypeptide for activating an immune receptor FLS2, a second polypeptide for activating an immune receptor RLP23, a third polypeptide for activating an immune receptor EFR, a fourth polypeptide for activating an immune receptor RLK7, a fifth polypeptide for activating an immune receptor PEPR1, a sixth polypeptide for activating an immune receptor CORE1, a seventh polypeptide for activating an immune receptor FLS3, an eighth polypeptide for activating an immune receptor FER, a ninth polypeptide pep13 for activating immune responses of plants, a tenth polypeptide hrp24 and an eleventh polypeptides sys18.

3. The fusion protein according to claim 2, wherein the first polypeptides is flg15 and homoeotic mutants thereof, or flg22 and homoeotic mutants thereof; the second polypeptides is nlp20 and homoeotic mutants thereof; the third polypeptides is elf18 and homoeotic mutants thereof; the fourth polypeptides is pip1 and homoeotic mutants thereof; the fifth polypeptides is pep1 and homoeotic mutants thereof; the sixth polypeptides is csp15 and homoeotic mutants thereof, or csp22 and homoeotic mutants thereof; the seventh polypeptides is flgII-28 and homoeotic mutants thereof; the eighth polypeptides is ralf17 and homoeotic mutants thereof; the ninth polypeptides is pep13 and homoeotic mutants thereof; the tenth polypeptides is hrp15 and homoeotic mutants thereof, or hrp24 and homoeotic mutants thereof; and the eleventh polypeptides is sys18 and homoeotic mutants thereof.

4. The fusion protein according to claim 3, wherein 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.

5. The fusion protein according to claim 1, wherein the fusion protein consists of three same and/or different PAMP polypeptides, and optionally, there is at least one linker or no linker between two adjacent PAMP polypeptides; preferably, the fusion protein consists of four same and/or different PAMP polypeptides, and optionally, there is at least one linker or no linker between two adjacent PAMP polypeptides; preferably, the fusion protein consists of five same and/or different PAMP polypeptides, and optionally, there is at least one linker or no linker between two adjacent PAMP polypeptides; and preferably, the fusion protein consists of six same and/or different PAMP polypeptides, and optionally, there is at least one linker or no linker between two adjacent PAMP polypeptides.

6. The fusion protein according to claim 1, wherein the fusion protein comprises or consists of seven different PAMP polypeptides, and optionally, there is at least one linker or no linker between two adjacent PAMP polypeptides; and preferably, the seven different PAMP polypeptides are selected from any combination of seven of flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, ralf17, pep13, hrp24 or sys18.

7. The fusion protein according to claim 6, wherein the fusion protein comprises or consists of the following amino acid sequences: (1) an amino acid sequence shown as SEQ ID NO: 15; or (2) a functional homologous sequence having at least 80% sequence identity to the amino acid sequence shown as SEQ ID NO: 15.

8. A nucleotide sequence coding the fusion protein according to claim 1.

9. The nucleotide sequence coding the fusion protein according to claim 8, wherein the nucleotide sequence comprises or consists of the following nucleotide sequences: (1) a nucleotide sequence shown as SEQ ID NO: 16, or (2) a complementary sequence, a degenerate sequence or a homologous sequence of the nucleotide sequence shown as SEQ ID NO: 16, or (3) a nucleotide sequence hybridizing, under stringent conditions, to the nucleotide sequence shown as SEQ ID NO: 16 and capable of coding the fusion protein; and preferably, the homologous sequence is a polynucleotide sequence having at least 85% sequence identity to the nucleotide sequence shown as SEQ ID NO: 16 and coding the fusion protein.

10. A vector, into which the nucleotide sequence according to claim 8 is introduced.

11. A microorganism or cell, into which the nucleotide sequence according to claim 8 is introduced.

12. The microorganism or cell according to claim 11, wherein the microorganism or cell comprises one or more of Escherichia coli, Agrobacterium, Lactobacillus, a yeast or Bacillus subtilis; and preferably, Escherichia coli.

13. A plant immune inducer comprising the fusion protein according to claim 1 preferably, the plant immune inducer further comprises one or more of an agronomically acceptable vector, an excipient, a diluent or a solvent; and preferably, the plant immune inducer is in a form selected from the group consisting of a powder, a soluble powder, a wettable powder, a granule, an aqueous solution, a microemulsion, suspension and a water dispersible granule.

14. A method for preparing the fusion protein according to claim 1, the method comprises the following steps of: (a) synthesizing the nucleotide sequence, and preferably, before the synthesizing, analyzing, designing and assembling the nucleotide sequence coding the fusion protein; (b) transforming the synthesized nucleotide sequence (preferably, through the vector) into the microorganism or cell, and cultivating the microorganism or cell to express the fusion protein; and (c) optionally, collecting and purifying the expressed fusion protein.

15. A use of the fusion protein according to claim 1, inducing defense responses and/or resistance against pathogenic microorganisms of plants; preferably, the plants comprise Arabidopsis, corns, wheat, rice, tomatoes and tobaccos; and preferably, the pathogenic microorganisms comprise Pseudomonas syringae, Fusarium graminearum, Magaporthe grisea and a tobacco mosaic virus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0123] FIG. 1 is an SDS-PAGE electrophoretogram showing a heptapeptide fusion protein His-MP7.

[0124] FIG. 2 is a diagram showing accumulation of callose in Arabidopsis plants treated with different concentrations of fusion protein His-MP7 and a PAMP polypeptide flg22.

[0125] FIG. 3 is a diagram showing accumulation of callose in Arabidopsis plants treated with 100 nM fusion protein His-MP7 and different PAMP polypeptides.

[0126] FIG. 4 is a diagram showing production of reactive oxygen species (ROS) induced by a fusion protein His-MP7 in a corn plant.

[0127] FIG. 5 is a diagram showing an experimental result that a fusion protein His-MP7 enhances the resistance of an Arabidopsis plant to DC3000 pathogenic bacteria.

[0128] FIG. 6 is a diagram showing an experimental result that a fusion protein His-MP7 enhances the resistance of a corn plant to Fusarium graminearum, wherein “a” shows actual growth comparison on inhibition of Fusarium graminearum infection in corn by the fusion protein His-MP7; and “b” is an experimental result histogram of inhibiting Fusarium graminearum infection in corn by the fusion protein His-MP7.

[0129] FIG. 7 is a diagram showing an experimental result that a fusion protein His-MP7 enhances the resistance of a rice plant to Magaporthe grisea, wherein “a” shows actual growth comparison on inhibition of Magaporthe grisea infection in rice by the fusion protein His-MP7; and “b” is an experimental result histogram of inhibiting Magaporthe grisea infection in rice by the fusion protein His-MP7.

DETAILED DESCRIPTION OF THE INVENTION

[0130] In the present disclosure, the term “PAMP polypeptide” refers to a relatively conserved polypeptide fragment, having the immune activation ability, in a sequence of a protein molecule, which is found through contrastive analysis on homologous sequences in intrinsic proteins of various pathogenic fungi or bacteria or plants and usually serves as a signaling molecule polypeptide for perceiving infection of pathogenic bacteria.

[0131] In the present disclosure, the term “linker” refers to having at least one amino acid residue, preferably, at least two consecutive amino acid residues.

[0132] In the present disclosure, the term “plant immune inducer” refers to an exogenous organism or molecule capable of inducing or activating immune responses of the plants and improving the resistance of the plants to certain pathogenic microorganisms.

[0133] In the present disclosure, the term “PTI mechanism” is named as pathogen-associated molecular pattern-triggered immunity (PAMP-Triggered Immunity) mechanism, referring to a mechanism that the immune responses of the plants are activated after the PAMP signaling molecule is recognized by a plant cell receptor.

[0134] In the present disclosure, “protein tag” refers to a kind of polypeptide subjected to fusion expression with a target protein together by using DNA recombination in vitro for convenience in expression, detection and purification of the target protein.

[0135] The technical solutions in the examples of the present disclosure are described clearly and completely in the following with reference to accompanying figures in the examples of the present disclosure. Apparently, the described examples are only part rather than all of the examples of the present disclosure. Based on the examples of the present disclosure, all the other examples obtained by those of ordinary skill in the art without inventive effort are within the scope of the present disclosure.

[0136] In the following examples, there are introductions for sources of a part of plant materials, strains and viruses.

[0137] Plant materials: rice (Oryza sativa L.) selected is Nipponbare (NPB) which belongs to short-grain japonica rice (NPB is an international general variety which has been subjected to whole genome sequencing), corns selected are Jundan 20, and the rice and the corns are both purchased from the market; and cultivated tomatoes (Solanum lycopersicum) and a wild-type Arabidopsis col-0 from Columbia and N89 tobacco lines are all from a research group of Professor Yi Cai from College of Life Science in Sichuan Agricultural University.

[0138] Strains and viruses: Pseudomonas syringae DC3000, Magaporthe grisea race ZB15, a tobacco mosaic virus and Fusarium graminearum are all from the research group of Professor Yi Cai from College of Life Science in Sichuan Agricultural University.

[0139] In addition, materials, reagents, consumables and the like, sources of which are not mentioned in the examples, may all be purchased from the market.

[0140] An His tag protein purification kit is purchased from ComWin Biotech Co., Ltd with a catalog number of CW0894; and a BCA protein assay kit is purchased from Solarbio Company with a catalog number of PC0020-500 micropores (50T).

Example 1 Molecular Design and Nucleotide Sequence Obtaining of Heptapeptide Fusion Protein His-MP7

[0141] (1) 7 different PAMP polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22 and flgII-28 were selected from 11 different PAMP polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15 and sys18 and mutants thereof and were assembled into a fusion protein with linkers of AGA and GAG.

[0142] (2) Analysis (ProtScale) on molecular weight, amino acid composition (ProtParam) and basic properties of hydrophobicity and the like was conducted on the sequence of the protein by using an online analysis platform ExPASy (https://www.expasy.org); and a structure of the protein was modeled by using a Phyre2 online platform (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index).

[0143] (3) In combination with an analysis result, one design solution was screened out, and the fusion protein was named as MP7, having an amino acid sequence shown as SEQ ID NO: 15.

[0144] (4) A nucleotide sequence coding the fusion protein MP7 was designed by using the bioinformatics software Geneious R9 and an online analysis platform Jcat (http://www.jcat.de) to obtain a nucleotide sequence shown as SEQ ID NO: 16. The nucleotide sequence was artificially synthesized.

Example 2 Expression and Purification of Heptapeptide Fusion Protein His-MP7

[0145] (1) A nucleotide sequence shown as SEQ ID NO: 16 was cloned into a pET-28b(+) expression vector (Novagen) at sites HindIII and XhoII. The pET-28b(+) expression vector was heat shocked and transformed into Escherichia coli DH5a. A positive clone colony was selected, shaken, and extracted for plasmids. After being verified through enzyme digestion and sequencing, it was then heat shocked and transformed into Escherichia coli BL21(DE3) to obtain Escherichia coli containing a recombinant plasmid pET-28b-MP7, named as BL21(DE3)/pET-28b-MP7.

[0146] (2) BL21(DE3)/pET-28b-MP7 was subjected to induced expression through the following steps. An expression strain was inoculated to an LB liquid medium, and cultured overnight at 37° C. and 200 rpm/min shaking to obtain a first bacterial solution, which was then transferred to an LB liquid medium containing 100 μg/mL kanamycin based on a volume ratio of 1 to 100, and was cultured under shaking continuously at 37° C. and 200 rpm/min, until a concentration OD.sub.600 nm of the bacterial solution was 0.6. After that, it was added with IPTG with a final concentration of 0.5 mmol/L, cultured under shaking for 12 h at 28° C. and 200 rpm/min to obtain a second bacterial solution. The second bacterial solution was centrifuged at 12000 rpm/min to collect cell pellets, which were then added into a PBS buffer, ultrasonically broken and then centrifuged at 4° C. and 12000 rpm/min. A supernate was then collected.

[0147] (3) The supernate was purified with a His tag protein purification kit (soluble protein) through the following specific operation. 5 mL of Ni-Agarose was filled in an empty affinity column. The supernate was slowly flowed through the affinity column. It was then washed by using 6 column volumes of PBS buffer containing 10 mM imidazole to remove impurities. Finally, it was then eluted by using 5 column volumes of PBS buffer containing 500 mM imidazole, and the eluate passing the column was collected. Such eluate was the fusion protein His-MP7 solution.

[0148] (4) The concentration of the fusion protein His-MP7 was detected as 0.2 mg/mL by using a BCA protein assay kit. Detected by SDS-PAGE, as shown in FIG. 1, the fusion protein His-MP7 was purified to obtain an expressed protein (His-MP7) containing His tag and having a molecular weight of about 23 kDa.

Example 3 Study on Titer of Immune Activation of Heptapeptide Fusion Protein His-MP7

[0149] In immune responses of plant cells, callose might accumulate. The callose might enhance the mechanical strength of plant cell walls and block channels through which pathogens spread between the cells, thereby limiting invasion of pathogenic microorganisms. With a model plant Arabidopsis as a material and callose accumulation as immune index, the immune activation abilities of different concentrations of fusion proteins His-MP7 were analyzed and were compared to that of a single PAMP polypeptide. A specific experimental operation was as follows:

[0150] I. Comparison in Callose Accumulation, Caused By Different Concentrations of Fusion Protein His-MP7, of Arabidopsis

[0151] Final concentrations of the fusion protein His-MP7 and a polypeptide flg22 were adjusted to be 1 μM, 100 nM and 10 nM respectively, with water being served as a control. Four-week old Arabidopsis leaves were infiltrated with an injection syringe permeation method for 12 h, the treated leaves were collected and put in a 6-well plate, and a suitable amount of washing buffer was added for 4 h incubation. The washing buffer was substituted by an aniline blue staining solution with a final concentration of 0.1 mg/mL for staining at a room temperature in the dark for 1 h, and the callose accumulation was observed using a 10 times objective lens of a fluorescence microscope. Each treatment contained three biological replicates with three repeated experiments.

[0152] Experimental results were shown in FIG. 2. With 100 nM fusion protein His-MP7, callose accumulation of Arabidopsis could be obviously induced and the intensity was equivalent to that induced by 1 μM PAMP polypeptide flg22.

[0153] II. Comparison in Immune Activation Ability Between Fusion Protein His-MP7 and Different Polypeptides Under Same Concentration

[0154] A final concentration of the fusion protein His-MP7 and each of polypeptides flg22, nlp20, elf18, pip1, pep1, csp22 and flgII-28 was adjusted to be 100 nM, with csp22 and flgII-28 being served as negative controls. The four-week old Arabidopsis leaves were infiltrated with an injection syringe permeation method; and a sample treatment method was the same as mentioned above.

[0155] Experimental results were shown in FIG. 3. The immune activity effect of 100 nM fusion protein His-MP7 was superior to those of all the PAMP polypeptides.

[0156] III. Induction of Oxidative Burst Immune Response in Plants with Fusion Protein His-MP7

[0157] Oxidative burst was considered as one of earliest responses of plants to pathogenic microorganisms and played an important role in defense responses of the plants. Researches had proved that reactive oxygen species might directly serve as an antimicrobial agent in the plants to result in direct toxicity to the pathogenic microorganisms and inhibition in growth of the pathogenic microorganisms. After the plants were infected by the pathogenic microorganisms, the reactive oxygen species could be produced and accumulate, and oxidative burst could be caused. With plant corns as a material and the reactive oxygen species as an immune index, the immune activation ability of 100 nM fusion protein His-MP7 was analyzed. A specific experimental operation was as follows:

[0158] Two-week old corn leaves were taken, the intermediate portion of each leaf was taken, shorn with a length of 5 cm and immersed into 1 μg/mL auxin 6BA solution. It was added with the fusion protein His-MP7 with a final concentration of 100 nM, with an aqueous solution of auxin 6BA being served as a blank control. The intermediate portions were soaked for 48 h, stained for 12 h using 1 mg/mL DAB solution, eluted for 12 h by a washing buffer (ethyl alcohol:acetic acid:glycerin=3:1:1) and observed after being placed for 30 min.

[0159] Experimental results were shown in FIG. 4. The 100 nM fusion protein His-MP7 could induce the oxidative burst immune response in the plants.

[0160] The above three experiments had proved that plant immune could be efficiently activated with a relatively low concentration of fusion protein His-MP7 (100 nM); and under the same concentration, the immune activation ability of the fusion protein His-MP7 was superior to that of a single PAMP polypeptide.

Example 4 Detection in Improvement of Disease Resistance of Plants By Heptapeptide Fusion Protein His-MP7

[0161] I. A Fusion Protein his-MP7 could Enhance the Resistance of Plants to DC3000 Pathogenic Bacteria.

[0162] Experimental Process:

[0163] (1) A strain DC3000 of Pseudomonas syringae was inoculated to 20 mL of SOC+str (streptomycin) liquid medium for overnight culture for 14-16 h at 28° C., OD.sub.600 was determined, and a product was subjected to gradient dilution until OD.sub.600 was 0.00005 to obtain a bacterial solution.

[0164] (2) The fusion protein His-MP7 was added in the bacterial solution until a final concentration was 100 nM, and a bacterial solution, to which water was added, was served as a control. Meanwhile, four-week old healthy Arabidopsis leaves were injected with the bacterial solution and were sampled at Day 0 (four leaves from one plant) and Day 3 (five plants, two leaves from each plant) respectively. Each leaf was punched by a puncher. The punched small discs were taken; 500 μL of 10 mM MgCl.sub.2 was added at Day 0; the small discs were ground in a 1.5 mL EP tube; and 50 μL of each of four samples in the same treatment was spotted on one SOC+str (streptomycin) solid plate (the plate required to be blow-dried, and then the shape of each spotted sample could be kept) and cultured for 16-24 h at 28° C. Samples were photographed and observed for statistics of colony growth.

[0165] (3) At Day 3, the two leaves on each plant of the five plants were punched to take small discs similarly; 250 μL of 10 mM MgCl.sub.2 was added; and the small discs were ground in the 1.5 mL EP tube and diluted by MgCl.sub.2 stepwise until 1×10.sup.−5. The five samples were subjected to the same treatment: 10 μL of each diluted sample (for treating 30 samples) was spotted on one SOC+str (streptomycin) solid plate (the plate selected was a square vessel and required to be blow-dried, and then the shape of each spotted sample could be kept) for 16-24 h culture at 28° C., photographed and observed for statistics of colony growth.

[0166] Experimental results were shown as a histogram in FIG. 5. In a control group, a bacterium growth index was 5.38; for Arabidopsis treated by adding 100 nM fusion protein His-MP7, a bacterium growth index was 4.05, and a growth amount of Pseudomonas syringae DC3000 was reduced by 10 or above. Therefore, the His-MP7 could effectively enhance the immunity of the plants to the pathogenic bacteria.

[0167] II. A Fusion Protein his-MP7 could Enhance the Resistance of Plants to Fusarium graminearum

[0168] Experimental Process:

[0169] (1) Mycelia of Fusarium graminearum were inoculated to a CMC liquid medium from the plate for culturing in dark for 3-7 days at 25° C. The resulting product was filtered with gauze, and centrifugated at 10000 rpm/10 min to collect spores. The number of the spores was counted by a blood counting chamber; a concentration of the spores was adjusted to 2×10.sup.5; and the spores were preserved at 4° C. (used within one month).

[0170] (2) Two-week old corn leaves were taken; the intermediate portion of each leaf was taken, shorn with a length of 5 cm and immersed into 1 μg/mL auxin 6BA solution; 12-13 leaves were taken in each treatment; the fusion protein His-MP7 with a final concentration of 100 nM was added; and an aqueous solution of auxin 6BA was served as a control.

[0171] (3) Spore liquid of Fusarium graminearum was evenly spotted on each leaf; the leaves were cultured for 3-4 days under the condition of 12-h light and 12-h darkness and at 28° C.; the morbidity was observed; and the scab area percentage was counted by an imageJ software.

[0172] Experimental results were shown as growth measurement comparison of infections in a corn leaf, shown as in a, and a histogram shown as b in FIG. 6. For the leaves treated without the fusion protein His-MP7, the scab area percentage was 12.3%; and for the leaves treated with the fusion protein His-MP7, the scab area percentage was 3.6%. It indicated that the fusion protein His-MP7 had significantly enhanced the resistance of the corns to Fusarium graminearum, so that the infection rate of Fusarium graminearum was lowered by 70.8%.

[0173] III. A Fusion Protein his-MP7 could Enhance the Resistance of Plants to Magaporthe grisea

[0174] Experimental Process:

[0175] (1) Magaporthe grisea was inoculated to a CM solid medium for upright standing culture for 13-15 days at 28° C., mycelia were all scraped off by a pipette tip, washed by a small amount of 5-10 mL of sterile water and filtered by a gauze into a 50 mL centrifuge tube; spores were collected through centrifugation at 10000 rpm/10 min; the number of the spores was counted by a blood counting chamber; a concentration of the spores was adjusted to 1×10.sup.6; and the spores were preserved at a normal temperature (used within one week).

[0176] (2) Four-week old rice leaves were taken; the intermediate portion of each leaf was taken, shorn with a length of 5 cm and immersed into 1 μg/mL auxin 6BA solution; 12-13 leaves were taken in each treatment; the fusion protein His-MP7 with a final concentration of 100 nM was added; and an aqueous solution of auxin 6BA was served as a control.

[0177] (3) Spore liquid of Magaporthe grisea was evenly spotted on each leaf; the leaves were cultured for 3-4 days under the condition of 12-h light and 12-h darkness and at 28° C.; the morbidity was observed; and the scab area percentage was counted by an imageJ software.

[0178] Experimental results of pathogen growth comparison on rice leaves were shown as “a”, and a histogram was shown as “b” in FIG. 7. For the leaves treated without the fusion protein His-MP7, the scab area percentage was 11.6%; and for the leaves treated with the fusion protein His-MP7, the scab area percentage was 1.9%. It indicated that the fusion protein His-MP7 had significantly enhanced the resistance of the rice to Magaporthe grisea; so that the infection rate of Magaporthe grisea had been Lowered by 83.6%, and Harms of Rice Blasts to Rice had been Reduced.

[0179] IV. A fusion protein His-MP7 could enhance the resistance of plants to Tobacco mosaic virus (TMV)

[0180] The experiment was divided into an experimental group and a control group (with 20 lines of tobacco plants in each group). After the experimental group was injected with a recombinant protein, viruses were inoculated to the experimental group; and after the control group was injected with the sterile water, the viruses were inoculated to the control group. It was inoculated through the following steps:

[0181] (1) Fresh leaves infected with TMV were added to a small amount of sterilized phosphate buffer (1:200) and were ground in a mortar and filtered with sterilized gauze to remove the residues of the infected leaves. The above fresh juice was prepared as an inoculum and a concentration of the inoculum of the TMV was adjusted to obtain an aqueous solution of TMV.

[0182] (2) When tobacco seedlings were in the 4-5 true leaves period, fully expanded true leaves were selected; a suitable amount of quartz sands were uniformly scattered on the leaf surfaces; and the leaf surfaces were slightly rubbed by an absorbent ball dipped with the aqueous solution of TMV for 1-2 times and then immediately washed with water.

[0183] (3) After inoculation for 21 days, the morbidity of tobacco plants was observed. With each plant as a unit for assessment, the grading standard was as follows:

[0184] Grade 0: freedom from disease

[0185] Grade 1: the base of each interior leaf had a small amount of fading yellow spots along a leaf vein without curling;

[0186] Grade 3: newborn leaf had yellow-green stripes parallel to the leaf vein with slightly curling;

[0187] Grade 5: newborn leaf had a great amount of chlorotic strips parallel to the leaf vein with a curled, thin and weak leaves;

[0188] Grade 7: plant was dwarfed, leaf had yellowish-white strips with curling, and newborn leaves were twisted and prolapsed and could not be normally expanded; and

[0189] Grade 9: plant was dwarfed seriously and subjected to chlorina or died.

[0190] A disease index and the control effect were computed with the following methods:

Disease index=[Σ(number of infected plants in various grades×relative grade value)/(total number of plants investigated×9)]×100;

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

[0191] Results are shown in Table 1 and Table 2:

TABLE-US-00001 TABLE 1 Number of Infect Tobacco Plants and Disease Grading Unit (plant) 0 grade 1 grade 3 grade 5 grade 7 grade 9 grade H.sub.2O 0 0 2 2 9 7 His-MP7 1 11 6 2 0 0

TABLE-US-00002 TABLE 2 Disease Index and Control Effect for Tobaccos Treatment Disease index Control effect H.sub.2O 78.9 0 His-MP7 21.7 72.5%

[0192] Experimental results showed that, for the experiment group treated with the fusion protein His-MP7, the disease index was lowered to 21.7, and the control effect reached 72.5%. The test proved that the fusion protein His-MP7 might enhance the resistance of the tobaccos to TMV.

Example 5 Molecular Design, Expression and Purification of Various Tripeptide Fusion Proteins

[0193] I. Molecular Design of Various Tripeptide Fusion Proteins

[0194] (1) 3 PAMP polypeptides were randomly selected from 11 different PAMP polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15 and sys18 and homoeotic mutants thereof and were assembled into various fusion proteins with a linker of AKG, thereby obtaining various design solutions.

[0195] (2) Analysis on molecular weight, amino acid composition and basic properties of hydrophobicity and the like was conducted on a sequence of the protein by using an online analysis platform ExPASy; and a structure of the fusion protein was modeled by using a Phyre2 online platform.

[0196] (3) In combination with an analysis result, 20 design solutions were screened out and named as TP1, TP2, TP3, TP4, TP5, TP6, TP7, TP8, TP9, TP10, TP11, TP12, TP13, TP14, TP15, TP16, TP17, TP18, TP19 and TP20 respectively. The composition design solutions for various tripeptide fusion proteins were shown in Table 3:

TABLE-US-00003 TABLE 3 Design Solutions for Various Tripeptide Fusion Proteins Name Sequence (linker AKG) TP1 flg22-csp22-pep13 TP2 flg22-elf18-pep1 TP3 flg22-elf18-pip1 TP4 flg22-flg22-flg22 TP5 flg22-flgII-28-csp22 TP6 flg22-flgII-28-nlp20 TP7 flg22-hrp15-sys18 TP8 flg22.sub.m1-flgII-28.sub.m1-flg22.sub.m2 TP9 flg22.sub.m1-flg22.sub.m1-flg22.sub.m1 TP10 flg22.sub.m1-ralf17.sub.m1-csp22.sub.m1 TP11 flg22.sub.m2-flg22.sub.m2-flg22.sub.m2 TP12 flg22.sub.m2-nlp20.sub.m1-hrp15 TP13 flg22-nlp20-csp22 TP14 flg22-nlp20.sub.m1-pep1 TP15 flg22-nlp20.sub.m2-csp22.sub.m1 TP16 flg22-pep1-pip1 TP17 flg22-ralf17.sub.m1-hrp15 TP18 flg22-ralf17-hrp24 TP19 flg22-ralf17-pip1 TP20 flg22-ralf17-sys18

[0197] (4) An obtaining method for a nucleotide sequence coding the above 20 tripeptide fusion proteins was the same as that in Example 1.

[0198] II. Expression and Purification of Various Tripeptide Fusion Proteins

[0199] (1) The obtained nucleotide sequence for conding the above 20 tripeptide fusion proteins was cloned into a pEGX-4T-1 expression vector at sites BamHI and XhoII, the pEGX-4T-1 expression vector was transformed into Escherichia coli DH5a with thermal activation, a positive clone colony was selected for shaking, subjected to plasmid extraction and transformed into Escherichia coli BL21(DE3) with thermal activation after being verified to be correct by digestion and sequencing to obtain Escherichia coli containing a recombinant plasmid pEGX-4T-1-TP, named as BL21(DE3)/pEGX-4T-1-TP.

[0200] (2) The Escherichia coli was subjected to induced expression through the following steps. An expression strain was inoculated to an LB liquid medium and cultured overnight at 37° C. and 200 rpm/min shaking to obtain a first bacterial solution which was transferred to an LB liquid medium containing 100 μg/mL ampicillin based on a volume ratio of 1 to 100, and cultured under shaking continuously at 37° C. and 200 rpm/min, until a concentration OD.sub.600 nm of the bacterial solution was 0.6. After that, it was added with a final concentration of 0.3 mmol/L IPTG, and cultured under shaking for 12 h at 25° C. and 200 rpm/min to obtain a second bacterial solution. The second bacterial solution was centrifuged at 12000 rpm/min to collect cell pellets, which were then added, with a PBS buffer, ultrasonically broken, and centrifuged at 4° C. and 12000 rpm/min. A supernate was collected.

[0201] (3) The supernate was purified with a GST tag protein purification kit (soluble protein) to obtain a GST-TP fusion protein solution. The protein solution was quantified by the BCA protein assay kit.

Example 6 Molecular Design, Expression And Purification of Various Tetrapeptide Fusion Proteins

[0202] I. Molecular Design of Various Tetrapeptide Fusion Proteins

[0203] (1) 4 PAMP polypeptides were randomly selected from 11 different PAMP polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15 and sys18 and homoeotic mutants thereof and were assembled into various fusion proteins with a linker of AKG. The design methods were the same as those in Example 5. 20 design solutions were screened out and named as FP1, FP2, FP3, FP4, FP5, FP6, FP7, FP8, FP9, FP10, FP11, FP12, FP13, FP14, FP15, FP16, FP17, FP18, FP19 and FP20 respectively. The composition design solutions for various tetrapeptide fusion proteins were shown in Table 4:

TABLE-US-00004 TABLE 4 Design Solutions for Various Tetrapeptide Fusion Proteins Name Sequence (linker AKG) FP1 flg22-csp22-pep13-ralf17 FP2 flg22-elf18-cap22-nlp20 FP3 flg22-elf18-pep1-pip1 FP4 flg22-elf18-pip1-pep1 FP5 flg22-elf18-nlp20.sub.m2-csp22.sub.m1 FP6 flg22-flg22-flg22-flg22 FP7 flg22-flgII-28-csp22-hrp24 FP8 flg22-flgII-28-nlp20-hrp24 FP9 flg22-flgII-28-nlp20.sub.m1-csp22.sub.m1 FP10 flg22-hrp15-sys18-sys18.sub.m1 FP11 flg22.sub.m1-elf18.sub.m1-nlp20.sub.m2-csp22.sub.m1 FP12 flg22.sub.m1-flg22.sub.m1-flg22.sub.m1-elf18 FP13 flg22.sub.m1-nlp20.sub.m2-csp22.sub.m1-pip1.sub.m1 FP14 flg22.sub.m1-ralf17.sub.m1-csp22-ralf17 FP15 flg22.sub.m2-flg22.sub.m2-flg22.sub.m2-pip1.sub.m1 FP16 flg22.sub.m2-nlp20.sub.m1-csp22-hrp15 FP17 flg22-ralf17.sub.m1-hrp15-flg22 FP18 flg22-ralf17-pep1-csp22 FP19 flg22-ralf17-pip1-pep1 FP20 flg22-ralf17-sys18-hrp15

[0204] An obtaining method for a nucleotide sequence coding the above 20 tetrapeptide fusion proteins was the same as that in Example 1.

[0205] II. Expression and Purification of Various Tripeptide Fusion Proteins, Same as Those in Example 5

Example 7 Molecular Design, Expression and Purification of Various Pentapeptide Fusion Proteins

[0206] I. Molecular Design of Various Tetrapeptide Fusion Proteins

[0207] 5 PAMP polypeptides were randomly selected from 11 different PAMP polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15 and sys18 and mutants thereof and were assembled into various fusion proteins with a linker of AKG. The design methods were the same as those in Example 5. 20 design solutions were screened out and named as 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 and MP5-20 respectively. The composition design solutions for various pentapeptide fusion proteins were shown in Table 5:

TABLE-US-00005 TABLE 5 Design Solutions for Various Pentapeptide Fusion Proteins Name Sequence (linker 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.sub.m1-csp22.sub.m1-pep13 MP5-10 flg22-hrp15-sys18-pep13-ralf17 MP5-11 flg22-flgII-28-nlp20-csp22-pep13 MP5-12 nlp20-flg22.sub.m1-flg22.sub.m1-flg22.sub.m1-elf18 MP5-13 nlp20-flg22.sub.m1-nlp20.sub.m2-csp22.sub.m1-pip1 MP5-14 nlp20.sub.m1-flg22.sub.m1-ralf17.sub.m1-csp22-ralf17 MP5-15 nlp20.sub.m2-flg22.sub.m2-flg22.sub.m2-flg22.sub.m2-pip1 MP5-16 nlp20.sub.m1-flg22.sub.m2-nlp20.sub.m1-csp22-hrp15 MP5-17 flg22-ralf17.sub.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

[0208] An obtaining method for a nucleotide sequence coding the above 20 pentapeptide fusion proteins was the same as that in Example 1.

[0209] II. Expression and Purification of Various Pentapeptide Fusion Proteins, Same as Those in Example 5

Example 8 Molecular Design of Various Hexapeptide and Heptapeptide Fusion Proteins

[0210] 6 PAMP polypeptides were randomly selected from 11 different PAMP polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15 and sys18 and mutants thereof and were assembled into various fusion proteins with a linker of AKG. The design methods were the same as those in Example 5. 20 design solutions were screened out and named as 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 and MP6-20 respectively. The composition design solutions for various hexapeptide fusion proteins were shown in Table 6:

TABLE-US-00006 TABLE 6 Design Solutions for Various Hexapeptide Fusion Proteins Name Sequence (linker 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.sub.m1-csp22.sub.m1-pep13-elf18 MP6-12 flg22-hrp15-sys18-pep13-ralf17-csp22 MP6-13 flg22.sub.m1-flgII-28-flg22-ralf17-pep1-csp22 MP6-14 flg22.sub.m2-flgII-28-flg22-ralf17-pip1-pep1 MP6-15 nlp20-flg22.sub.m1-flg22.sub.m2-elf18-pep1-nlp20 MP6-16 nlp20-flg22.sub.m1-nlp20.sub.m2-csp22.sub.m1-pip1-pep1 MP6-17 nlp20.sub.m1-flg22.sub.m2-nlp20.sub.m1-csp22-hrp24-ralf17 MP6-18 nlp20.sub.m1-flg22.sub.m1-ralf17.sub.m1-csp22-ralf17-hrp15 MP6-19 nlp20.sub.m2-flg22.sub.m2-flg22.sub.m2-flg22.sub.m2-pep13-pep1 MP6-20 ralf17.sub.m1-hrp15-flg22-csp22-pip1-pep13

[0211] An obtaining method for a nucleotide sequence coding the above 20 hexapeptide fusion proteins was the same as that in Example 1.

[0212] II. Expression and Purification of Various Hexapeptide Fusion Proteins, Same as Those in Example 5

Example 9 Molecular Design, Expression And Purification of Various Heptapeptide Fusion Proteins

[0213] 7 PAMP polypeptides were randomly selected from 11 different PAMP polypeptides: flg22, nlp20, elf18, pip1, pep1, csp22, flgII-28, elf18, pep13, ralf17, hrp15 and sys18 and mutants thereof and were assembled into various fusion proteins with a linker of AKG. The design methods were the same as those in Example 5. 20 design solutions were screened out and named as 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 and M7-20 respectively. The composition design solutions for various heptapeptide fusion proteins were shown in Table 7:

TABLE-US-00007 TABLE 7 Design Solutions for Various Heptapeptide Fusion Proteins Name Sequence (linker 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.sub.m1-flg22.sub.m2-flg22.sub.m1-flg22.sub.m2- flg22.sub.m1-flg22.sub.m2 Name Sequence (linker 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.sub.m1-flg22.sub.m2-flg22.sub.m1-flg22.sub.m2- flg22.sub.m1-flg22.sub.m2

[0214] An obtaining method for a nucleotide sequence coding the above 20 heptapeptide fusion proteins was the same as that in Example 1.

[0215] II. Expression and Purification of Various Heptapeptide Fusion Proteins, Same as Those in Example 5

Example 10 Detection on Immune Responses of Different Fusion Proteins

[0216] With a model plant Arabidopsis as a material, callose accumulation as immune index and water as a blank control, all the fusion proteins obtained in Examples 5-9 were adjusted until final concentrations of solutions were all 100 nmol; and four-week old Arabidopsis leaves were infected with an injection syringe permeation method. The specific experimental operation was the same as that in Example 3. Computation was conducted through image processing software ImageJ according to obtained callose accumulation fluorescence images, and the immune activation ability of the fusion proteins to the plants was quantified by virtue of the fluorescence intensity. Computing methods were as follows:

[0217] Measured IntDen=Integrated Density=Integrated Optical Density;

[0218] Measured Area=Image Area;

[0219] MD(mean optical density)=IntDen/Area.

[0220] Experimental results are shown in Table 8:

TABLE-US-00008 TABLE 8 Identification on Intensity of Immune Responses of Various Fusion Proteins Tripeptide Tetrapeptide Pentapeptide Immune Immune Immune Name MD activation Name MD activation Name MD activation H.sub.2O 0.002 No H.sub.2O 0.003 No H.sub.2O 0.002 No TP1 0.058 Yes FP1 0.035 Yes MP5-1 0.386 Yes TP2 0.292 Yes FP2 0.061 Yes MP5-2 0.235 Yes TP3 0.061 Yes FP3 0.111 Yes MP5-3 0.111 Yes TP4 0.089 Yes FP4 0.356 Yes MP5-4 0.139 Yes TP5 0.135 Yes FP5 0.171 Yes MP5-5 0.097 Yes TP6 0.034 Yes FP6 0.348 Yes MP5-6 0.166 Yes TP7 0.358 Yes FP7 0.051 Yes MP5-7 0.018 Yes TP8 0.388 Yes FP8 0.236 Yes MP5-8 0.295 Yes TP9 0.242 Yes FP9 0.373 Yes MP5-9 0.037 Yes TP10 0.378 Yes FP10 0.156 Yes MP5-10 0.115 Yes TP11 0.148 Yes FP11 0.228 Yes MP5-11 0.278 Yes TP12 0.042 Yes FP12 0.279 Yes MP5-12 0.224 Yes TP13 0.193 Yes FP13 0.223 Yes MP5-13 0.127 Yes TP14 0.087 Yes FP14 0.011 Yes MP5-14 0.195 Yes TP15 0.113 Yes FP15 0.217 Yes MP5-15 0.367 Yes TP16 0.146 Yes FP16 0.163 Yes MP5-16 0.091 Yes TP17 0.077 Yes FP17 0.094 Yes MP5-17 0.098 Yes TP18 0.096 Yes FP18 0.141 Yes MP5-18 0.048 Yes TP19 0.160 Yes FP19 0.044 Yes MP5-19 0.299 Yes TP20 0.170 Yes FP20 0.037 Yes MP5-20 0.301 Yes Hexapeptide Heptapeptide Immune Immune Name MD activation Name MD activation H.sub.2O 0.003 No H.sub.2O 0.002 No MP6-1 0.396 Yes MP7-1 0.386 Yes MP6-2 0.359 Yes MP7-2 0.324 Yes MP6-3 0.114 Yes MP7-3 0.019 Yes MP6-4 0.065 Yes MP7-4 0.115 Yes MP6-5 0.266 Yes MP7-5 0.237 Yes MP6-6 0.382 Yes MP7-6 0.253 Yes MP6-7 0.317 Yes MP7-7 0.391 Yes MP6-8 0.250 Yes MP7-8 0.290 Yes MP6-9 0.051 Yes MP7-9 0.168 Yes MP6-10 0.116 Yes MP7-10 0.125 Yes MP6-11 0.071 Yes MP7-11 0.328 Yes MP6-12 0.386 Yes MP7-12 0.220 Yes MP6-13 0.288 Yes MP7-13 0.330 Yes MP6-14 0.221 Yes MP7-14 0.295 Yes MP6-15 0.348 Yes MP7-15 0.323 Yes MP6-16 0.123 Yes MP7-16 0.065 Yes MP6-17 0.314 Yes MP7-17 0.047 Yes MP6-18 0.023 Yes MP7-18 0.030 Yes MP6-19 0.090 Yes MP7-19 0.392 Yes MP6-20 0.102 Yes MP7-20 0.022 Yes

[0221] From the experimental results in Table 8, compared with the blank control group, the various tripeptide fusion proteins, the various tetrapeptide fusion proteins, the various pentapeptide fusion proteins, the various hexapeptide fusion proteins, the various heptapeptide fusion proteins and other different fusion proteins provided in Examples 5-9 all had the immune activation ability.