CN116693639A - Plant immunity activated egg CfCE61 secreted by fruit anthracis and application thereof - Google Patents

Abstract

The invention belongs to the field of biotechnology, and provides a plant immune activation egg CfCE61 secreted by fruit anthracnose and application thereof, wherein the plant immune activation egg CfCE61 can be identified by a plant immune system to generate immune response, and a new way is provided for improving plant resistance; the CfCE61 protein can induce plant defense reaction and provide new materials for synthesizing biopesticide; the invention can be applied to the aspects of improving the disease resistance of crop breeding, biopesticide and the like, is hopeful to improve the disease resistance of plants to epidemic diseases, thereby achieving the purposes of increasing yield and reducing medicine, and has wide application prospect in agricultural production.

Description

Plant immunity activated egg CfCE61 secreted by fruit anthracis and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a plant immunity activated egg CfCE61 secreted by fruit anthracis and application thereof.

Background

Anthrax species are important plant pathogens and can infect many plant hosts, and the resulting anthrax often poses serious damage to crops.

The pear anthracnose is an important fungal disease on pear trees, wherein the pear anthracnose caused by fruit anthracnose (Colletotrichum fructicola) is one of important diseases in main pear producing areas in China, and can occur in the growing period and the fruit ripening period of the pear trees, so that fruit rot and premature leaf drop are caused to cause weakening of tree vigor, the yield and quality of the pear trees are seriously influenced, and serious economic loss is caused to Chinese agricultural production.

The pear anthracnose is also called bitter rot and late rot, mainly caused by fruit, and can also infect branches, leaves and the like.

The pear anthracnose occurs to different degrees in the pear producing area of the yellow river hometown road of China, and has obvious harm to Dangshan pear in particular; in the year 2008, pear anthracnose is outbreak in Dangshan areas, the disease fruit rate reaches more than 70 percent, and the direct economic loss exceeds 7 hundred million yuan; at present, pear anthracnose is still one of the major hidden hazards threatening the safety of the human pear industry, and the development of the pear industry is greatly limited.

The effector is a kind of secreted protein which is transported to plant cells by fruit anthracnose, has important functions of regulating host physiology and immune response, and is a key for analyzing disease occurrence mechanism and plant disease resistance.

During infection of plants by pathogenic bacteria, the plants recognize effectors secreted by the pathogenic bacteria by means of pattern recognition receptors (Pattern Recognition Receptors, PRRs) on the cell surface, triggering the basic immune response of the plants. These factors that activate the plant immune response are commonly referred to as plant immune elicitors (Plant Immunity Inducer, PII), an important class of which is the plant immune activator protein.

At present, the identification of novel plant immunity induced antigen proteins secreted by pathogenic bacteria is the focus of attention of researchers at home and abroad.

Reference to the literature

1. Wu Liangqing, zhu Liwu, heng Wei, etc. Dangshan pear anthracnose pathogen identification and antibacterial agent screening [ J ]. Chinese agricultural science, 2010,43 (18): 3750-3758.

2.Zhang P F,Zhai L F,Zhang X K,et al.Characterization of Colletotrichum fructicola,a new causal agent of leaf black spot disease of sandy pear(Pyrus pyrifolia)[J].European Journal of Plant Pathology,2015,143(4):651-662.

3.Li H N,Jiang J J,Hong N,et al.First report of Colletotrichum fructicola causing bitter rot of pear(Pyrus bretschneideri)in China[J].Plant Disease,2013,97(7):1000-1000.

4.Shang S,Wang B,Zhang S,et al.A novel effector CfEC92 of Colletotrichum fructicola contributes to glomerella leaf spot virulence by suppressing plant defences at the early infection phase[J].Molecular plant pathology,2020,21(7):936-950.

5.Jones J D G,Dangl J L.The plant immune system[J].nature,2006,444(7117):323-329.

Disclosure of Invention

The invention aims to provide an effector molecule CfCE61 of fruit anthracnose, which has the activity of activating plant immunity.

Another object of the present invention is to provide the gene sequence of the effector molecule CfCE61 of the fruit-borne anthrax.

It is still another object of the present invention to provide a recombinant expression vector containing a gene encoding plant immune activator protein CfCE61.

It is still another object of the present invention to provide the use of the above-mentioned protein CfCE61 and its coding gene for activating plant immune response.

The aim of the invention can be achieved by the following technical scheme: a plant immune activation protein CfCE61 secreted by fruit anthracis is as follows (a) or (b):

(a) Has the sequence shown in SEQ ID NO:2, a protein having an amino acid sequence shown in seq id no;

(b) Setting SEQ ID NO:2 by substitution and/or deletion and/or addition of one or several amino acid residues and associated with activating the plant immune response function, consisting of the amino acid sequence of SEQ ID NO: 2.

A gene encoding the plant immune activating protein CfCE 61; the gene can induce plant resistance and has a nucleotide sequence shown as SEQ ID NO. 1; the amino acid sequence of the plant immune activator protein CfCE61 is utilized to facilitate the expression in plants.

Recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the coding genes of the genes CfCE61.

The expression vector is obtained by inserting the gene into a plant expression vector pSuper vector and is obtained by double enzyme cutting of restriction enzymes XbaI and KpnI, and the recombinant expression vector is obtained by inserting the gene into a plant expression vector pET-28a and is obtained by double enzyme cutting of restriction enzymes BamHI and NdeI.

The method for improving the disease resistance of plants, improving the defensive power of the plants and improving the resistance of the plants to pathogenic bacteria is realized by a mode of secreting a plant immune activating protein CfCE61 of fruit anthracnose; the method specifically comprises the following steps:

s1: connecting the coding gene of the plant immunity activating protein CfCE61 secreted by the fruit anthracis to a plant reaching carrier, and then converting escherichia coli to obtain a recombinant plasmid;

s2: the recombinant plasmid was transferred into agrobacterium and transiently expressed on plants.

Preferably, the plant expression vector described in S1 is preferably a pSuper vector.

Preferably, the E.coli described in S1 is E.coli DH 5. Alpha.

Preferably, the agrobacterium described in S2 is preferably agrobacterium GV3101.

The application of the plant immune activation protein CfCE61 secreted by the fruit anthracnose in the aspect of inducing up-regulation of the expression of the plant defense related genes can be realized by expressing the plant immune activation protein CfCE61 secreted by the fruit anthracnose.

A method of inducing a plant resistance response comprising injecting into a plant leaf the plant immune activating protein CfCE61 described above.

Compared with the prior art, the invention has the following beneficial effects: the plant immune activating protein CfCE61 can be identified by a plant immune system to generate immune response, a new way is provided for improving plant resistance, the CfCE61 protein can induce plant defense response, and a new material is provided for synthesizing biological pesticides; the invention can be applied to the aspects of improving the disease resistance of crop breeding, biopesticide and the like, is hopeful to improve the disease resistance of plants to epidemic diseases, thereby achieving the purposes of increasing yield and reducing medicine, and has wide application prospect in agricultural production.

Drawings

FIG. 1 is a schematic diagram of a plant expression vector pSuper:: cfCE 61;

FIG. 2 is a Western blot detection chart of tobacco leaf allergic reaction detection and CfCE61 protein expression induced by injecting and expressing CfCE61 on tobacco leaves by using a plant expression vector, wherein the antibody is an anti-HA monoclonal antibody;

FIG. 3 shows SDS-PAGE and Coomassie brilliant blue staining of purified CfCE61 protein obtained by prokaryotic expression;

FIG. 4 is a diagram showing induction of expression of plant defense-related genes after injection of CfCE61 on tobacco leaves using a plant expression vector;

FIG. 5 is a graph showing the effect of plant immune activating protein CfCE61 on inducing tobacco to resist phytophthora capsici and a statistical graph of disease occurrence.

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.

Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer.

The reagents and starting materials used in the present invention are commercially available unless otherwise specified.

The primers related to the embodiment of the invention are synthesized by Nanjing qingke biotechnology Co.

Embodiment one: cloning of coding genes

(1) Total RNA extraction: the method uses the fruit anthracnose mycelium as an experimental material, extracts total RNA by adopting an RNA extraction kit of Vazyme company, and detects the RNA content and quality by using a spectroscope according to the illustrated operation.

(2) Reverse transcription generates the first strand: 1. Mu.g of RNA was used as a template for cDNA synthesis according to the instructions of the Vazyme HiScript II reverse transcription kit.

Appropriate amounts of the reverse transcription product were taken for subsequent gene cloning PCR.

(3) Using cDNA as PCR template, amplifying the mature gene sequence of CfCE61 gene:

PCR primer amplification sequence:

an upstream primer: SEQ ID NO.3

TACACCAAATCGACTCTAGAATGGTCAACTCATTCGTC

A downstream primer: SEQ ID NO.4

GGAAATTCGAGCTCGGTACCCTACGAGGAAGTACACTTG

50. Mu.L of reaction system: 2X Phanta Max Mix Buffer. Mu.L, 10mM dNTPs 1. Mu.L, vazyme DNA Polymerase. Mu.L, template cDNA 2. Mu.L, and water to 50. Mu.L; the PCR amplification procedure comprises pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 1min, circulation for 35 times, and extension at 72 ℃ for 5min; the PCR reaction products were subjected to fragment size verification by DNA agarose gel electrophoresis.

Adding nucleic acid dye into 1 Xelectrophoresis buffer solution according to the ratio of 1:1000, performing electrophoresis separation, recording the result, and cutting gel to recover PCR product.

The electrophoresis band is recovered by FastPure Gel DNA Extraction Mini Kit (Vazyme) (the PCR product band is the plant immune activating protein CfCE61 gene, and is sent to Nanjing qinghao company for sequencing, the sequence is shown as SEQ ID NO.1, and the coded amino acid sequence of the plant immune activating protein CfCE61 is shown as SEQ ID NO. 2).

The PCR product recovered from the cut gel was ligated according to the procedure described for XbaI and KpnI on a pSuper vector containing pSuper:: cfCE61 ligation product according to CloneExpress II One Step Cloning Kit (Vazyme).

Wherein the pSuper vector contains XbaI and KpnI cleavage sites.

The ligation product was transformed into E.coli competent cells DH5 a, plated with LB (containing 50. Mu.g/mL) plates, incubated at 37℃for 12h, and then subjected to colony PCR validation to pick up two positive clones, which were subjected to plasmid extraction according to plasmid extraction kit procedure (Vazyme) to extract plasmids, and sent to Nanjing engine company for sequencing.

The plasmid with correct sequence was transformed into Agrobacterium GV3101, and LB plates (calicheamicin 50. Mu.g/mL, rifampicin 50. Mu.g/mL) containing the corresponding antibiotics were applied, and after 48h incubation at 28℃colony PCR confirmed that the correct clone was picked for subsequent experiments.

Results: the plant expression vector pSuper is CfCE61, and the pattern diagram is shown in figure 1

Embodiment two: transient expression of CfCE61 in tobacco induces generation of necrosis response

(1) Agrobacterium culture

Agrobacterium GV3101 single colonies containing the relevant vector were picked from the plates, inoculated into 2mL LB liquid medium (containing 50. Mu.g/mL of kananamycin, 50. Mu.g/mL of rifampin) and incubated overnight at 200rpm on a constant temperature shaker at 28℃to an OD600 of 0.6. The cultured overnight GV3101 Agrobacterium solution was centrifuged at 6000g for 3min to collect the cells.

The bacterial cells were collected by centrifugation after suspending the bacterial cells in a buffer (composition: 10mM2- [ N-morpholino ] ethanesulfonic acid,10mM MgCl2,0.1mM acetosyringone pH5.6). After 2 times of repeated washing, the bacterial solutions were diluted with buffer solutions, and the final concentrations were 0.5 each.

(2) Transient expression of CfCE61 in tobacco leaves

The prepared Agrobacterium was injected into tobacco leaves using a 1mL syringe with the needle removed, and the tobacco was cultured in a greenhouse (22-25 ℃ C., 16h light/8 h dark) after injection.

(3) Detection of necrosis

After the agrobacterium is transiently expressed for 5 days, the necrosis of the leaves is observed, after obvious phenotype appears, the leaves are collected, and the leaves are photographed by a camera and stored.

(4) Western blot detection of CfCE61 protein accumulation

Taking a Bentonite sample injected by agrobacterium after 2d injection, putting the sample into liquid nitrogen for quick freezing, grinding the sample into powder by a grinder, and carefully loading the powder into a precooled 1.5mL centrifuge tube; adding protein lysis buffer (50mM Tris,pH7.5, 150mM NaCl,1mM EDTA,1%TritonX-100,1% sodium deoxycholate, 1mM PMSF,1%proteinase inhibitor cocktail (Sigma)), and placing on ice for lysis for 40min, and vortexing once every 10 min; the sample was put into a centrifuge, centrifuged at 13000rpm at 4℃for 10min, the supernatant was added to an equal volume of 2 XSDS-PAGE loading buffer (120 mM Tris, pH6.8, 20% glycerol, 4% SDS,0.04% bromophenol blue, 10% mercaptoethanol), boiled for 10min, and 10. Mu.L of the sample was separated by electrophoresis on SDS-PAGE gel for 1.5h at 120V.

After the reaction, the protein samples were transferred to PVDF membranes and the membranes were incubated with 5% pbst skim milk. After adding HA primary antibody (Sigma) diluted 1:2000 and incubating for 3h, the membrane was washed three times with PBST for 5min, and PVDF membrane was immersed in ECL luminescence solution (Cytiva) and then scanned for imaging.

Results: cfCE61 was able to induce tobacco leaf cell necrosis as shown in figure 2.

Example 3: production of a defense response by prokaryotic expression of CfCE61 and purification of the protein

(1) Construction of prokaryotic expression vectors

The cDNA library of the interaction of the fruit anthracnose and the pear is taken as a template, a specific primer is designed according to the enzyme cutting site of the carrier,

an upstream primer: SEQ ID NO.5

GTGCCGCGCGGCAGCCATATGGGGATCCTCTCCCCGCGC

A downstream primer: SEQ ID NO.6

ACGGAGCTCGAATTCGGATCCCTACGAGGAAGTACACTTG

The gene of interest was cloned using Phusion High-Fidelity DNA Polymerase (New England Biolabs). The cloning fragment was then ligated into the plant expression vector pET-28a using the ClonExp one-step cloning kit (Vazyme), the vector was transformed into E.coli DH 5. Alpha. Competent by heat shock, positive clones were picked up, shaken and plasmids were extracted, and verified by sequencing.

(2) Inducible expression and crude protein extraction

The correct plasmid obtained in the step (1) was transformed into E.coli BL21 (DE 3) competent by heat shock, and after kanamycin selection, single clones were picked up to 5mL LB,37℃and cultured at 200rpm for 10h. Expanded culture was performed in 100mL of LB at a ratio (1:100) until OD600 = 0.6, induced expression was performed by adding 0.3M IPTG, culturing at 16℃for 24h at 200rpm, collecting the cells by centrifugation for 10min, and washing twice with PBS buffer (pH 7.4). The bacterial cells were resuspended in lysis buffer (20mM Na2HPO4, 300mM NaCl,pH7.4,1mg/mL lysozyme,1mM PMSF,1.98mM mercaptoethanol), sonicated for 15min, centrifuged at 10000g for 10min, and the supernatant was aspirated to obtain CfCE61 crude protein.

(3) Protein purification

The column was packed with an appropriate amount of Ni-NTA (Thermo Scientific) resin.

The storage buffer is allowed to drain from the resin by gravity flow. Samples were prepared by mixing the protein extract with an equal volume of equilibration buffer (20 mM sodium phosphate, 300mM sodium chloride and 10mM imidazole; pH 7.4).

The column was equilibrated with twice the volume of the resin bed of equilibration buffer and the buffer was allowed to flow out of the column. The prepared protein extract was added to the resin and the flow-through was collected in a tube.

The flow-through is reapplied once if necessary to maximize adsorption. The resin was washed with twice the resin bed volume of wash buffer (25 mM imidazole in PBS; pH 7.4) and the effluent was collected.

This procedure was repeated using a new collection tube. His-tagged proteins were eluted from the resin with twice the resin bed volume of elution buffer (250 mM imidazole in PBS; pH 7.4). This procedure was repeated twice and each fraction was collected in a separate tube.

Dialysis was performed with PBS (20 mM sodium phosphate, 300mM sodium chloride; pH 7.4) buffer.

Results: the purified CfCE61 protein (His-CfCE 61) obtained was subjected to SDS-PAGE, and a distinct band of interest (about 23 kDa) was seen after Coomassie blue staining, as shown in FIG. 3.

Example 4: cfCE61 purified protein induces up-regulated expression of defense tobacco related genes

(1) Tobacco leaf injection CfCE61 purified protein

The His-CfCE61 purified protein obtained in example 3 was subjected to concentration measurement and diluted to 1. Mu.M. The diluted purified protein was injected into tobacco leaves using a 1mL syringe with the needle removed, while PBS Buffer was injected as a control (Buffer), and after completion of the injection, the purified protein was incubated in a greenhouse (22-25 ℃ C., 16h light/8 h dark).

(2) Total RNA extraction

Tobacco samples treated for 4h with CfCE61 purified protein are used as experimental materials, total RNA is extracted by adopting an RNA extraction kit of Vazyme company according to the description operation, and the RNA content and quality of the total RNA are detected by using a spectroscope.

(3) Reverse transcription to generate the first strand

1. Mu.g of RNA was used as a template for cDNA synthesis according to the instructions of the Vazyme HiScript II reverse transcription kit. Appropriate amounts of reverse transcription product were taken for subsequent real-time quantitative PCR reactions.

(4) Real-time quantitative PCR reaction

NbPR1 quantitative pre-primer: SEQ ID NO.7

CCGCCTTCCCTCAACTCAAC

NbPR1 post-quantification primer: SEQ ID NO.8

GCACAACCAAGACGTACTGAG

NbPR4 quantitative pre-primer: SEQ ID NO.9

GGCCAAGATTCCTGTGGTAGAT

Quantitative post primer for NbPR 4: SEQ ID NO.10

CACTGTTGTTTGAGTTCCTGTTCCT

NbCYP71D20 quantitative pre-primer: SEQ ID NO.11

AAGGTCCACCGCACCATGTCCTTAGAG

NbCYP71D20 post-quantification primer: SEQ ID NO.12

AAGAATTCCTTGCCCCTTGAGTACTTGC

The PCR reaction system contained 1. Mu.L of cDNA, realStar Fast SYBR qPCR Mix. Mu.L of each of the front and rear primers, 1. Mu.L of water, and 7. Mu.L of water.

The reaction procedure: 95 degrees for 2 minutes, 95 degrees for 15 seconds, 60 degrees for 30 seconds, step II should be 40 cycles.

The dissolution profile analysis procedure was: 95 degrees 15 seconds, 60 degrees 1 minute, 95 degrees 15 seconds.

Results: the 1. Mu. MCfCE61 purified protein was able to induce a large up-regulated expression of the tobacco defense related genes compared to the control (Buffer), as shown in FIG. 4.

Example 5: the CfCE61 purified protein enhances tobacco resistance to Sclerotinia sclerotiorum and Phytophthora capsici

(1) Tobacco leaf injection CfCE61 purified protein

The concentration of the purified protein obtained in example 3 was measured and diluted to 1. Mu.M.

The diluted purified protein was injected into tobacco leaves using a 1mL syringe with the needle removed, while PBS Buffer was injected as a control (Buffer), and after completion of the injection, the purified protein was incubated in a greenhouse (22-25 ℃ C., 16h light/8 h dark).

(2) Respectively inoculating sclerotinia sclerotiorum and phytophthora capsici on tobacco leaves

Sclerotinia sclerotiorum is a pathogen causing sclerotinia rot of colza, belongs to fungi, has wide host range and is harmful to crops; phytophthora capsici is a pathogen causing induction of pepper epidemic disease, belongs to oomycetes, and has important harm to farming. After 12h of protein induction, the treated leaves are respectively inoculated with sclerotinia sclerotiorum and phytophthora capsici which are prepared in advance, and finally placed in a greenhouse (22-25 ℃ C., 16h light/8 h dark) for culture.

Observing and counting the disease condition of the sclerotinia sclerotiorum after 24 hours and photographing; and after 36 hours, observing, counting and photographing the disease condition of phytophthora capsici.

Results: compared to the control (Buffer), 1 μm CfCE61 purified protein significantly enhanced tobacco leaf resistance to sclerotinia and phytophthora capsici as shown in fig. 5.

While embodiments of the present invention have been shown and described above for purposes of illustration and description, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Sequence description:

full-length nucleotide sequence of CfCE61 gene: SEQ ID NO.1

ATGGTCAACTCATTCGTCAAGCTCTTCGCCTTCGCTGGCCTCGCCTCCGCCGGGATCCTCTCCCCGCGCCAGAACACGACGCCTGCTGTTGACATTACGGCGCTGACCAAGAACGTCACGGCCACTTCCGGAACCGGCGCCGTCAGCGCCGCGGGAACCTTGTCTCCCTTTGGAGGTATCGGAGTTGGTTGTGGCATCAACTGGGCTGAGGGCCAGTCTTTTGGAGGTGGTCTTCAATCCGGCTCGGACTCTTTCGGTCTCGGCGGCGGATTCACCATTACCAAGGACACCATGATCATTGGTCTTGGCATTGGAATCAACCCCATTAAGTTCAACTCCAGCGTGAACTATGAAGCTTCGACTAACGGCACCGTGACCATGACCTTCACCTCCACGACGGCGATCAAGTGCGAAGAGACCACCGTTGATGGCGTCAAGGGTGTCAAGTGTACTTCCTCGTAG

CfCE61 mature protein sequence: SEQ ID NO.2

MGILSPRQNTTPAVDITALTKNVTATSGTGAVSAAGTLSPFGGIGVGCGINWAEGQSFGGGLQSGSDSFGLGGGFTITKDTMIIGLGIGINPIKFNSSVNYEASTNGTVTMTFTSTTAIKCEETTVDGVKGVKCTSS

PCR primer amplification sequences in example 1:

an upstream primer: SEQ ID NO.3

TACACCAAATCGACTCTAGAATGGTCAACTCATTCGTC

A downstream primer: SEQ ID NO.4

GGAAATTCGAGCTCGGTACCCTACGAGGAAGTACACTTG

PCR primer amplification sequences in example 3:

an upstream primer: SEQ ID NO.5

GTGCCGCGCGGCAGCCATATGGGGATCCTCTCCCCGCGC

A downstream primer: SEQ ID NO.6

ACGGAGCTCGAATTCGGATCCCTACGAGGAAGTACACTTG

Real-time quantitative PCR reaction primers in example 4

NbPR1 quantitative pre-primer: SEQ ID NO.7

CCGCCTTCCCTCAACTCAAC

NbPR1 post-quantification primer: SEQ ID NO.8

GCACAACCAAGACGTACTGAG

NbPR4 quantitative pre-primer: SEQ ID NO.9

GGCCAAGATTCCTGTGGTAGAT

Quantitative post primer for NbPR 4: SEQ ID NO.10

CACTGTTGTTTGAGTTCCTGTTCCT

NbCYP71D20 quantitative pre-primer: SEQ ID NO.11

AAGGTCCACCGCACCATGTCCTTAGAG

NbCYP71D20 post-quantification primer: SEQ ID NO.12

AAGAATTCCTTGCCCCTTGAGTACTTGC。

Claims (10)

1. A plant immune activation egg CfCE61 secreted by fruit anthracis is either (a) or (b) as follows:

(a) Has the sequence shown in SEQ ID NO:2, a protein having an amino acid sequence shown in seq id no;

(b) Setting SEQ ID NO:2 by substitution and/or deletion and/or addition of one or several amino acid residues and associated with activating the plant immune response function, consisting of the amino acid sequence of SEQ ID NO: 2.

2. A gene encoding the plant immune activator protein CfCE61 of claim 1, which is capable of inducing plant resistance, having a nucleotide sequence as shown in SEQ ID No. 1.

3. A recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium comprising the gene encoding the gene CfCE61 of claim 2.

4. The expression vector of claim 2, wherein the gene is inserted into a plant expression vector pSuper vector, the gene is obtained by double digestion of restriction enzymes XbaI and KpnI, and the recombinant expression vector is obtained by double digestion of restriction enzymes BamHI and NdeI selected for inserting the gene into a plant expression vector pET-28 a.

5. The method for improving plant disease resistance, improving plant defense capacity and improving plant resistance to pathogenic bacteria according to claim 1 by means of plant immune activating protein CfCE61 secreted by fruit anthracnose; the method specifically comprises the following steps:

s1: connecting the coding gene of the plant immunity activating protein CfCE61 secreted by the fruit anthracis to a plant reaching carrier, and then converting escherichia coli to obtain a recombinant plasmid;

s2: the recombinant plasmid was transferred into agrobacterium and transiently expressed on plants.

6. The plant-immune-activating egg CfCE61 secreted by anthrax fruit as in claim 5, wherein: the plant expression vector is preferably a pSuper vector.

7. The plant-immune-activating egg CfCE61 secreted by anthrax fruit as in claim 5, wherein: the escherichia coli is preferably escherichia coli DH5 alpha.

8. The plant-immune-activating egg CfCE61 secreted by anthrax fruit as in claim 5, wherein: the agrobacterium is preferably agrobacterium GV3101.

9. The use of plant immune activating egg CfCE61 according to claim 1 for inducing up-regulation of expression of plant defense-related genes by expressing plant immune activating protein CfCE61 secreted by anthrax fruit.

10. A method of inducing a plant resistance response comprising injecting into a plant leaf the plant immune activating protein CfCE61 described above.