AU2021101598A4 - HarpinF PROTEIN AND USE THEREOF IN INDUCTION OF RESISTANCE OF POPULUS × CANADENSIS MOENCH POPLAR TO BACTERIAL CANKER - Google Patents

Abstract

The present disclosure provides a harpinF protein and use thereof in induction of resistance of Populus x canadensis Moench poplar (Populus x canadensis Moench.) to bacterial canker. The present disclosure provides an hrpF gene sequence of a pathogen of the bacterial canker of Populus x canadensis Moench poplar, Lonsdalea quercina subsp. populi strain, and a protein encoded thereby; His is used for fusion expression of harpin protein by a gene engineering method, and the expression thereof in Escherichia coli is significantly upregulated. A target protein is purified by a Ni column, and the pure protein concentration is about 1 mg/mL. To spray the harpinF obtained by the present disclosure to branches of the Populus x canadensis Moench poplar can significantly improve resistance of the Populus x canadensis Moench poplar to bacterial canker. The above conclusion proves that the harpinF has application value in developing biogenic pesticides.

Description

HarpinF PROTEIN AND USE THEREOF IN INDUCTION OF RESISTANCE OF POPUL US x CANADENSIS MOENCH POPLAR TO BACTERIAL CANKER

TECHNICAL FIELD The present disclosure belongs to the technical field of biological genes, and particularly relates to a harpinF protein and use thereof in induction of resistance of Populus x canadensis Moench to bacterial canker.

BACKGROUND The information disclosed in this Background section is only for enhancement of understanding of the general background of the present disclosure and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art. Poplar is one of the tree species widely distributed in China, which is the main afforestation tree species in the construction of ecological shelter forest or timber forest. Populus x canadensis Moench poplar is the fourth generation of poplar species in China, which is bred by crossing Populus nigra L. with Populus deltoides MarshallPopulus x canadensis Moench poplar is an excellent tree species with a wide range of uses, rapid growth, and high ecological and economic value. In recent years, massive afforestation with Populus x canadensis Moench poplar in North China has effectively improved the ecological environment of these areas and played a significant role in alleviating the contradiction between wood supply and demand. However, long-term and large-scale cultivation of pure breeds can leads to serious plant diseases and insect pests, ecological environment destruction, decline in biodiversity, and serious economic losses. Bacterial canker of Populus x canadensisMoench poplar (Lonsdalea quercinasubsp. populi) was first discovered in a plurality of forestlands of Populus x canadensis Moench poplar in Puyang, Henan Province in 2006, and subsequently found in Heze and Yanzhou, Shandong Province, Tianjin Municipality, and other cities, becoming an important disease threatening to the growth of Populus x canadensisMoench poplar. The disease mainly occurs in the branches and trunks of trees. The disease can occur in seedlings, young forests and mature forests between 2 and 20 years old. Diseased trees have swelling and canker on the branches and trunks, and rotten barks, resulting in weak tree vigor, deformed trunks, and lower value in wood use; when the canker is severe, the trunk can be broken from the diseased part due to the wind, resulting in the death of the entire tree and causing significant economic losses. The pathogen of the bacterial canker of Populus x canadensis Moench poplar invades and harms the branches and stems of plants mainly through stab wounds and cracks. At the early stage of the disease, cracks are formed in the trunk cortex and milky white to orange mucus flows out. As time goes by and the temperature rises, the disease progresses to the middle stage. The affected area expands from the epidermis to the phloem, the mucus turns brown, and cankers are formed at the wound and orifice. After that, the cortex rots and produces milky white slurry in the xylem, which gradually turns tawny. The mucus flows to the ground along the trunk, and diseased parts rot and ferment, emitting a rancid smell that attracts a variety of adnascent insects to multiply on the rotten parts, and accelerating the spread of the disease in the forest. At present, there are two main methods for the prevention and control of bacterial canker of Populus x canadensis Moench poplar. One method is chemical control, that is, prevention and control by applying chemical pesticides. When the disease breaks out, the spread of the disease can be effectively controlled, and the broad spectrum of chemical pesticides can target a plurality of diseases and is the main method of disease control; however, long-term chemical control has produced many disadvantages. High pesticide residues endanger human and animal health, and long term application leads to increasing resistance of pathogens to pesticides and causes irreversible damage to ecological environment. The other is prevention and control by applying antagonistic bacteria. XU Fangyuan isolated and screened strains from rhizosphere soil by dilution separation method, and screened out five antagonistic bacteria strains that had inhibitory effects on the pathogen of bacterial canker of Populus x canadensis Moench poplar by using plate confrontation method, which were identified as Bacillus subtilis and Streptomyces venezuelae. After root watering treatment, the control effect thereof reached 51%. However, when antagonistic bacteria are screened out by the plate confrontation method, only one pathogen is usually used as a target bacterium. However, due to complex field conditions and generally mixed infection of a plurality of pathogens, the disease control is limited.

SUMMARY In view of the shortcomings of the prior art, the present disclosure provides a harpinF protein and use thereof in induction of resistance of Populus x canadensisMoench poplar to bacterial canker. The present disclosure obtains a harpinF protein through research. The protein can serve as a safe and efficient protein elicitor that induces plants to produce broad-spectrum resistance and can be used to prevent and treat bacterial canker of Populus x canadensis Moench poplar. Therefore, the protein has excellent practical application value. To achieve the above objectives, the present disclosure relates to the following technical solutions: In a first aspect of the present disclosure, a protein named harpinF is provided, and the protein named harpinF is derived from a pathogen of bacterial canker of Populus x canadensis Moench poplar, Lonsdalea quercina subsp. populi strain. The harpinF is the following (al) or (a2): (al) a protein composed of an amino acid sequence shown in SEQ ID No. 1;

(a2) a protein derived from (al) with the same function after substitution and/or deletion and/or addition of one or more amino acid residues. In a second aspect of the present disclosure, a gene encoding the harpinF is provided. Herein, the gene may have any one of nucleotide sequences of (bl) to (b3): (bI) a nucleotide sequence shown in SEQ ID No. 2; (b2) a nucleotide sequence complementary to (bI); and (b3) a nucleotide sequence that has >90% identity (for example, 90%, 91%, 92%, 93%, 94%, %, 96%, 97%, 98%, 99%, or 100% (complete) sequence) with the nucleotide sequence shown in (bI) or (b2) and encodes the same functional protein. Herein, SEQ ID No. 2 consists of 228 nucleotides, where nucleotides 1 to 225 are the coding sequences, and nucleotides 226 to 228 are transcribed as a stop codon to terminate peptide chain synthesis. In a third aspect of the present disclosure, a recombinant expression vector or recombinant cell containing the gene further falls within the protection scope of the present disclosure. The recombinant expression vector may be a recombinant prokaryotic expression vector, and the recombinant prokaryotic expression vector may be obtained by inserting the foregoing encoding gene into an expression vector pET30a. The recombinant cell may be a prokaryotic cell and preferably a bacterium, and be further selected from Escherichia coli and Bacillus sp.; further, the recombinant cell may be BL21 containing the foregoing gene and/or recombinant expression vector. In a fourth aspect of the present disclosure, use of the coding gene, recombinant expression vector, or recombinant cell in the preparation of the harpinF protein further falls within the protection scope of the present disclosure. In a fifth aspect of the present disclosure, a primer pair for amplifying the foregoing encoding gene is provided, where nucleotide sequences thereof are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively. In a sixth aspect of the present disclosure, use of the harpinF protein as a protein elicitor should further fall within the protection scope of the present disclosure. Specifically, the use may at least include: 1) prevention and/or treatment of bacterial canker of Populus x canadensis Moench poplar; 2) induction of broad-spectrum resistance of plant products; and 3) promotion of plant growth. The plant may be a poplar, and more preferably Populus x canadensis Moench poplar. In a seventh aspect of the present disclosure, a protein elicitor is provided, where the protein elicitor at least includes the foregoing harpinF protein. The protein elicitor may further include a pesticidally acceptable excipient.

In an eighth aspect of the present disclosure, a method for inducing resistance to bacterial canker of Populus x canadensis Moench poplar is provided, where the method includes: applying the harpinF protein or the protein elicitor to the leaves, branches or rhizomes, and preferably leaves or branches, of Populus x canadensisMoench poplar. One or more of the above technical solutions have the following beneficial technical effects: 1. Disclosed is an hrpF gene sequence of a pathogen of the bacterial canker of Populus x canadensisMoench poplar, Lonsdalea quercina subsp. populi strain, and a protein encoded thereby; His is used for fusion expression of harpin protein by a gene engineering method, and the expression thereof in Escherichiacoli is significantly upregulated. A target protein is purified by a Ni column, and the pure protein concentration is about 1 mg/mL. 2. To spray the harpinF protein obtained by the above technical solutions to branches of the Populus x canadensis Moench poplar may significantly improve resistance of the Populus x canadensis Moench poplar to bacterial canker, thereby proving that the harpinF has application value in developing biogenic pesticides. BRIEF DESCRIPTION OF DRAWINGS The accompanying drawings of the specification constituting a part of the present disclosure are intended to provide a further understanding of the present disclosure; the exemplary examples and descriptions thereof are intended to explain the present disclosure, and do not constitute an improper limitation of the present disclosure. FIG. 1 illustrates the double digests of a vector fragment and the PCR verification of recombinant plasmid pET30a(+)-hrpF colony in Example 2 of the present disclosure: A is a double digested fragment, where M is a DNA marker DL2000; No. 1 is hrpF double digests; B is the PCR verification of recombinant plasmid pET30a(+)-hrpF colony, where 1 to 5 are transformants and 6 is a negative control. FIG. 2 illustrates the expression and purification of harpin F in Example 4 of the present disclosure: where 1 is a purified harpinF protein, and 2 is a crude harpin F protein. FIG. 3 illustrates the biological activity detection of harpinF in Example 4 of the present disclosure: where a is hrpF; b is an N-5-1 strain;c is a BL21/pET30a(+) empty vector control. DETAILED DESCRIPTION It should be noted that the following detailed descriptions are all illustrative and are intended to provide further descriptions of the present disclosure. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by a person of ordinary skill in the art to which the present disclosure belongs. It should be noted that the terms used herein are only intended to describe specific implementations and are not intended to limit the exemplary implementations of the present invention. As used herein, a singular form is intended to include a plural form unless otherwise indicated explicitly in the context. Furthermore, it should be further understood that the terms "includes" and/or "including" used in this specification specify the presence of features, steps, operations, devices, components and/or combinations thereof. It should be understood that the protection scope of the present disclosure is not limited to the following specific example; it should be further understood that the terms used in the examples of the present disclosure are intended to describe specific examples, not to limit the protection scope of the present disclosure. In an exemplary example of the present disclosure, a protein named harpinF is provided, and the protein named harpinF is derived from a pathogen of bacterial canker of Populus x canadensis Moench poplar, Lonsdalea quercina subsp. populi strain. In a further specific example of the present disclosure, the harpinF is the following (al) or (a2): (al) a protein composed of an amino acid sequence shown in SEQ ID No. 1; (a2) a protein derived from (al) with the same function after substitution and/or deletion and/or addition of one or more amino acid residues. Herein, SEQ ID No. 1 consists of 75 amino acid residues. In another specific example of the present disclosure, a gene encoding the harpinF is provided. Herein, the gene may have any one of nucleotide sequences of (bl) to (b3): (bI) a nucleotide sequence shown in SEQ ID No. 2; (b2) a nucleotide sequence complementary to (bI); and (b3) a nucleotide sequence that has >90% identity (for example, 90%, 91%, 92%, 93%, 94%, %, 96%, 97%, 98%, 99%, or 100% (complete) sequence) with the nucleotide sequence shown in (bI) or (b2) and encodes the same functional protein. Herein, SEQ ID No. 2 consists of 228 nucleotides, where nucleotides 1 to 225 are the coding sequences, and nucleotides 226 to 228 are transcribed as a stop codon to terminate peptide chain synthesis. In still another specific example of the present disclosure, a recombinant expression vector or recombinant cell containing the gene further falls within the protection scope of the present disclosure. The recombinant expression vector may be a recombinant prokaryotic expression vector, and the recombinant prokaryotic expression vector may be obtained by inserting the foregoing encoding gene into an expression vector pET30a. That is, the vector may be a pET30a(+)-hrpF recombinant vector and be constructed by the following method: (1) N-5-1 and pET-30a(+) vectors are streaked and inoculated on LB solid plates, and cultured at 37C for 24 h; N-5-I genomic DNA is extracted by a bacterial genomic DNA extraction kit. Using N-5-1gDNA as a template, and a target gene is amplified by using PrimeSTAR and hrpF specific primers: hrpF-F: 5'-GGGGTACCATGTCTTCATCGCTGGGATTACAG-3'(SEQ ID No.3); hrpF-R: 5'-CGGAATTCTCAGTTGAAGTCATTGATGATGGCC-3'(SEQ ID No.4); PCR system is 25 L, and reaction conditions are: 98°C for 5 min; 32 cycles of 98°C for 10 s, 62°C for 15 s, and 72°C for 10 s; 72°C for 5 min. PCR product is detected by 1% agarose gel electrophoresis, and a gel is cut to recover target bands. Fragments recovered from the cut gel are ligated to a T vector; a ligation product is transformed into DH5a chemically competent cells by heat shock; the cells are spread on an LB agar medium supplemented with Kn and cultured at 37°C for 12 h; a single colony is picked for PCR identification; positive clones are inoculated in Kn resistant LB broth and cultured at 37°C for 12 h to obtain a recombinant plasmid T-hrpF. (2) The T-hrpF and pET-30a(+) vectors are double digested with Kpn I and EcoRI, respectively, and a gel is cut to recover bands with hrpF and pET-30a(+). Recovered fragments are ligated with the vector with T4 ligase overnight at 16°C; a ligation product is transformed into DH5a chemically competent cells by heat shock; the cells are spread on a LB agar medium supplemented with Kn and cultured at 37°C for 12 h; a single colony is picked for PCR identification; positive clones are inoculated in Kn-resistant LB broth and cultured at 37°C for 12 h; plasmids are extracted and sequenced. The sequencing results are aligned with the target gene through SnapGene software, and correctly sequenced one is the recombinant expression vector pET30-hrpF. The recombinant cell may be a prokaryotic cell and preferably a bacterium, and be further selected from Escherichia coli and Bacillus sp.; further, the recombinant cell may be BL21 containing the foregoing gene and/or recombinant expression vector; that is, the recombinant cell is recombinant E. coli BL21/pET3Oa(+)-hrpF. A construction method thereof may include transformation of the foregoing recombinant plasmid pET30a(+)-hrpF into E. coli BL21 to obtain the recombinantE. coli BL21/pET30a(+)-hrpF. In yet another specific example of the present disclosure, use of the coding gene, recombinant expression vector, or recombinant cell in the preparation of the harpinF protein further falls within the protection scope of the present disclosure. In yet another specific example of the present disclosure, a primer pair for amplifying the foregoing encoding gene is provided, where nucleotide sequences thereof are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively. In yet another specific example of the present disclosure, use of the harpinF protein as a protein elicitor should further fall within the protection scope of the present disclosure. Specifically, the use may at least include: 1) prevention and/or treatment of bacterial canker of Populus x canadensis Moench poplar; 2) induction of broad-spectrum resistance of plant products; and 3) promotion of plant growth. The plant may be a poplar, and more preferably Populus x canadensisMoench poplar.

In yet another specific example of the present disclosure, a protein elicitor is provided, where the protein elicitor at least includes the foregoing harpinF protein. The protein elicitor may further include a pesticidally acceptable excipient. In yet another specific example of the present disclosure, the excipient may be selected from one or more of dispersant, wetting agent, disintegrant, binder, defoamer, antifreeze, thickener, filler, and solvent. The present disclosure has no special restriction on the sources of the acceptable excipients of the bacterial agent, and generally, commercially available products may be used. Herein, the dispersant may be an anionic dispersant and/or a nonionic dispersant, and may be selected from one or more of sodium lignosulfonate, naphthalenesulfonate formaldehyde condensate, sodium methylenebisnaphthalene sulfonate, formaldehyde condensate sulfate, polycarboxylate, polyoxyethylene alkyphenol phosphorate, and polyoxyethylene fatty acid. The wetting agent may be selected from one or more of sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, saponin powder, soapberry powder, sasangua cake powder, and nekal BX. The disintegrant may be selected from one or more of bentonite, ammonium sulfate, aluminum chloride, urea, magnesium chloride, and glucose. The binder may be selected from one or more of starch, diatomaceous earth, cyclodextrin, rosin, carboxymethyl cellulose, carboxyethyl cellulose, and carboxymethyl cellulose salt. The defoamer may be selected from one or more of C8-C20 fatty alcohol compounds, C1O-C20 saturated fatty acid compounds, epoxidized soybean oil, ethanol, silicone compounds, and organic silicone oil. The antifreeze may be selected from one or more of sorbitol, ethylene glycol, polyethylene glycol, propanediol, glycerol, urea, and sodium chloride. The thickener may be selected from one or more of gelatin, xanthan gum, polyethylene glycol, and polyvinyl alcohol. The filler may be selected from one or more of light-weight calcium carbonate, diatomaceous earth, bentonite, attapulgite, and white carbon black. The solvent may be selected from water (preferably deionized water) or methyl oleate. In yet another specific example of the present disclosure, a method for inducing resistance to bacterial canker of Populus x canadensis Moench poplar is provided, where the method includes: applying the harpinF protein or the protein elicitor to the leaves, branches or rhizomes, and preferably leaves or branches, ofPopulus x canadensisMoench poplar. The present disclosure will be further described below with reference to the examples, but they do not constitute a limitation to the present disclosure. It should be understood that these examples are only intended to illustrate the present disclosure and not to limit the scope of the present disclosure. Abbreviations used in the present disclosure are as follows:

LB: Bacteria culture medium, yeast powder 5 g/L + tryptone 10 g/L + NaCl 10 g/L, PH 7.0; Kn50: 50 g/ml kanamycin; IPTG: isopropyl-p-D-thiogalactoside; PMSF: protease inhibitor phenylmethylsulfonyl fluoride. Example 1 Cloning of hrpF gene Lonsdalea quercina subsp. populi N-5-1 strain (deposited in A507, Microbiology Building, College of Plant Protection, Shandong Agricultural University on March 15, 2018) was cultured on an LB agar medium at 37°C. After the strain grew to the logarithmic phase, genomic DNA was extracted from the N-5-1 strain, specific primers were designed, and Kpn I and EcoR I restriction sites were introduced at the 5'end, respectively; HrpF-F: 5'-GGGGTACCATGTCTTCATCGCTGGGATTACAG-3'(SEQ ID No. 3); HrpF-R: 5'-CGGAATTCTCAGTTGAAGTCATTGATGATGGCC-3'(SEQ ID No. 4); PCR amplification was performed using the genomic DNA of the N-5-1 strain as a template. PCR system was 25 L. Reaction conditions were: initial denaturation at 98°C for 5 min; 32 cycles of denaturation at 98°C for 10 s, annealing at 62°C for 15 s, and extension at 72°C for 10 s; final extension at 72°C for 5 min; permanent holding at 4°C. PCR product was ligated with a pCE2-TA vector and transformed into E. coli DH5a, and a positive transformant was obtained by colony PCR verification. Recombinant plasmid DNA was extracted and sequenced. SnapGene software was used for sequence analysis, and homology comparison was performed by the Blast program in GenBank (http://www.ncbi.nlm.gov). Example 2 Expression and purification of harpinF protein A correctly sequenced positive recombinant plasmid pCE2-TA-hrpF was used as a template for PCR amplification. After an amplified product was recovered, the amplified product was double digested with Kpn I and EcoR I and ligated to an expression vector pET30a(+), so that hrpF gene was fused with polyhistidine tag His-tag on pET30a(+) to further obtain a recombinant plasmid pET30a(+)-hrpF. The recombinant plasmid and pET30a(+) were transformed into E. coli BL21 and a positive recombinant strain was identified by colony PCR. The recombinant strain BL21/pET30a(+)-hrpF was inoculated into an LB broth supplemented with Kn and cultured overnight at 37°C. The overnight bacterial suspension was added to the LB broth supplemented with Kn in a ratio of 1:100, and cultured at 37°C for 2 h, during which OD value was measured several times, preferably OD600 = 0.8; IPTG was added to a final concentration of 1 mmol/L, and a part of the bacterial solution without IPTG was taken as a control and incubated at 200 r/min and 37°C for 3 h. Bacterial cells were collected in a 50 mL centrifuge tube and centrifuged at 12,000 rpm and 4°C for 5 min (note that the bacterial cells should always be in a low temperature state). A supernatant was discarded; the bacterial cells were washed twice with sterile water, and centrifuged at 5,000 rpm and 4°C for 15 min. A supernatant was discarded, and pre-cooled PMSF (100 mM) was added in a ratio of 1:100; an appropriate amount of PBS buffer was added, about 3 mL per 50 mL of bacterial suspension. The bacterial cells were sonicated (worked for 4 s, and paused for 4 s); the bacterial cells were held on the ice-water mixture until the bacteria suspension was clear, and stored at -80°C. After sonication and centrifugation, the supernatant was collected to obtain crude protein. The expression of the target protein was detected by SDS-PAGE. HisTrap HP column was used to purify the tagged protein. For specific operations, refer to the operating instructions. The pure protein concentration was about 1 mg/mL. The purified harpinF obtained above might be used in promoting plant growth and inducing plant disease resistance. Example 3 Cloning and sequencing of the gene of Lonsdalea quercina subsp. populi N-5-1 strain Lonsdalea quercina subsp. populi N-5-1 strain (deposited in A507, Microbiology Building, College of Plant Protection, Shandong Agricultural University on March 15, 2018) was cultured on an LB agar medium at 37°C. After the strain grew to the logarithmic phase, genomic DNA was extracted from the N-5-1 strain, specific primers were designed, and Kpn I and EcoR I restriction sites were introduced at the 5'end, respectively; HrpF-F: 5'-GGGGTACCATGTCTTCATCGCTGGGATTACAG-3'(SEQ ID No. 3); HrpF-R: 5'-CGGAATTCTCAGTTGAAGTCATTGATGATGGCC-3'(SEQ ID No. 4); PCR amplification was performed using the genomic DNA of the N-5-1 strain as a template. PCR system was 25 L. Reaction conditions were: initial denaturation at 98°C for 5 min; 32 cycles of denaturation at 98°C for 10 s, annealing at 62°C for 15 s, and extension at 72°C for 10 s; final extension at 72°C for 5 min; permanent holding at 4°C. PCR product was ligated with a pCE2-TA vector and transformed into E. coli DH5a, and a positive transformant was obtained by colony PCR verification. Recombinant plasmid DNA was extracted and sent to Tsingke Biotechnology Co., Ltd. for sequencing. The sequencing results of the recombinant plasmid pCE2-TA-hrpF showed that the hrpF gene was 228 bp in size and encoded 76 amino acids; the encoded protein thereof contains no cysteine and was rich in glycine (6.7%). Example 4 Expression and purification of harpinF protein 1. Construction of genetically engineered hrpF strain A correctly sequenced positive recombinant plasmid pCE2-TA-hrpF was used as a template for PCR amplification. After an amplified product was recovered, the amplified product was double digested with Kpn I and EcoR I and ligated to an expression vector pET30a(+), so that hrpF gene was fused with polyhistidine tag His-tag on pET30a(+) to further obtain a recombinant plasmid pET30a(+)-hrpF. The recombinant plasmid and pET30a(+) were transformed into E. coli BL21 and a positive recombinant strain was identified by colony PCR. 2. Expression of harpinF protein (1) Stock activation: The recombinant strain BL21/pET30a(+)-hrpF was inoculated into an LB broth supplemented with Kn and cultured overnight at 37°C. The overnight bacterial suspension was added to the LB broth supplemented with Kn in a ratio of 1:100, and cultured at 37°C for 2 h, during which OD value was measured several times, preferably OD600 = 0.8; IPTG was added to a final concentration of 1 mmol/L, and a part of the bacterial solution without IPTG was taken as a control and incubated at 200 r/min and 37°C for 3 h. (2) Cell sonication: Bacterial cells were collected in a 50 mL centrifuge tube and centrifuged at 12,000 rpm and 4°C for 5 min (note that the bacterial cells should always be in a low temperature state). A supernatant was discarded; the bacterial cells were washed twice with PBS, and centrifuged at 5,000 rpm and 4°C for 15 min. A supernatant was discarded, and pre-cooled PMSF (100 mM) was added in a ratio of 1:100; an appropriate amount of PBS buffer was added, about 3 mL per 50 mL of bacterial suspension. The bacterial cells were sonicated (worked for 4 s, and paused for 4 s); the bacterial cells were held on the ice-water mixture until the bacteria suspension was clear, and stored at -80°C. After sonication and centrifugation, the supernatant was collected to obtain crude protein. As detected by SDS-PAGE, the size of the fusion protein was 8.84 kDa, the size of harpinF was 8.0 kDa, and the size of His-tag was 0.84 kDa (FIG. 2). 3. Purification of harpinF HisTrap HP was used to purify the tagged protein. The specific steps followed the method shown in the instructions. Before purification, crude harpinF protein was filtered through a 0.45 m filtration membrane to remove some cell debris or other impurities from the crude protein to avoid clogging cartridges. A sample was loaded by using a peristaltic pump during purification. The purified sample was ultrafiltered and concentrated by an ultrafiltration spin column, and the protein concentration was determined using the BCA Protein Quantification Kit. The pure protein was stored at -80°C. 4. Biological activity detection of harpinF protein The protein concentration was diluted to 10 mg/mL; 50 L each of hrpF, wild-type N-5-1 and BL21/pET30a(+) empty vector control was inoculated on the same tobacco leaf, and the HR response was observed every 12 h (FIG. 3). It should be noted that the above examples are only intended to illustrate the technical solutions of the present disclosure, but not to limit them. Although the present disclosure has been described in detail with reference to the given examples, those of ordinary skill in the art may modify or equivalently replace the technical solutions of the present disclosure as desired without departing from the spirit and scope of the technical solutions of the present disclosure.

Claims (5)

1. l1

   What claimed is: 1. A protein named harpinF, wherein the harpinF protein is the following (al) or (a2): (al) a protein comprising an amino acid sequence shown in SEQ ID No. 1; (a2) a protein derived from (al) with the same function after substitution and/or deletion and/or addition of one or more amino acid residues.
2. 2. A gene encoding the harpinF protein according to claim 1; wherein the gene has any one of nucleotide sequences of (bl) to (b3): (bI) a nucleotide sequence shown in SEQ ID No. 2; (b2) a nucleotide sequence complementary to (bI); and (b3) a nucleotide sequence that has >90% identity with the nucleotide sequence shown in (bI) or (b2) and encodes the same functional protein.
3. 3. A recombinant expression vector comprising the gene according to claim 2; wherein the recombinant expression vector is a recombinant prokaryotic expression vector, and the recombinant prokaryotic expression vector is obtained by inserting the foregoing encoding gene into an expression vector pET30a.
4. 4. The primer pair for amplifying the gene according to claim 2, wherein a nucleotide sequence thereof is as shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.
5. 5. A protein elicitor, wherein the protein elicitor comprises the harpinF protein according to claim 1; preferably, the protein elicitor further comprises a pesticidally acceptable excipient.

   FIGURES

   FIG. 1 B A 1/2

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   FIG. 3 FIG. 2