HrpN lqp Application of protein in improving disease resistance of forest trees

Abstract

The present invention relates to the field of genetic engineering technology, and particularly relates to an application of HrpN lqp protein in improving the disease resistance of forest trees. The present invention provides an application of HrpN lqp protein in improving the disease resistance of forest trees, and its amino acid sequence is shown in SEQ ID NO.1, and its coding gene hrpN lqp sequence is shown in SEQ ID NO.2. The HrpN lqp protein screened by the present invention has immune induction activity. After application, it can significantly improve the disease resistance of forest trees (such as poplar trees), has the application value of being developed into a biogenic pesticide, can be used for the prevention and control of poplar tree (especially poplar tree branches and trunks) diseases, and has broad application prospects and economic benefits.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to an application of HrpN lqp protein in improving the disease resistance of forest trees. Background Art

[0002] Poplar ( Populus spp.) is a major tree species for afforestation, timber forest, shelter forest and greening, and plays an important role in forestry production and ecological construction. However, under natural conditions, the growth and development of poplar will inevitably be affected by various external stresses. Among them, the interference of adverse factors such as plant diseases and insect grazing will cause large-scale damage to poplar plantations. At the same time, due to the large-scale afforestation of a single poplar clone, the ability of the plantation to resist pathogen invasion is weakened, which makes diseases a major problem in the development of forestry.

[0003] The main method for controlling diseases is to apply chemical pesticides. Applying chemical pesticides can effectively control the spread of diseases during the outbreak of diseases, and at the same time, the broad-spectrum nature of chemical pesticides can target multiple diseases. However, long-term chemical control has also brought many disadvantages, such as the high residue of pesticides endangering the health of humans and livestock, the continuous increase in the drug resistance of pathogens caused by long-term application of pesticides, and irreversible damage to the ecological environment.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an application of HrpN lqp protein in improving the disease resistance of forest trees.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides an application of HrpN lqp protein in improving the disease resistance of forest trees, and the amino acid sequence of the HrpN lqp protein is as follows (a1) or (a2);

[0008] (a1) A protein composed of the amino acid sequence shown in SEQ ID NO.1;

[0009] (a2) A protein derived from (a1) through substitution and / or deletion and / or addition of one or several amino acid residues and having the same function.

[0010] Preferably, the coding gene of the HrpN lqp protein is as follows (b1), (b2) or (b3);

[0011] (b1) The nucleotide sequence shown in SEQ ID NO.2;

[0012] (b2) a nucleotide sequence complementary to (b1);

[0013] (b3) a nucleotide sequence having ≥ 90% identity with the nucleotide sequence shown in (b1) or (b2) and encoding the same functional protein.

[0014] Preferably, the HrpN lqp protein is expressed by a recombinant prokaryotic expression vector or a recombinant cell containing the recombinant prokaryotic expression vector; the recombinant prokaryotic expression vector is obtained by inserting the HrpN lqp protein coding gene into a basic expression vector.

[0015] Preferably, the basic expression vector is pET-28a.

[0016] Preferably, the forest tree is a poplar tree.

[0017] Preferably, the present invention uses the HrpN lqp protein to improve the resistance to poplar canker.

[0018] Preferably, the present invention uses the HrpN lqp protein to improve the resistance to poplar anthracnose.

[0019] Preferably, the present invention uses the HrpN lqp protein to improve the resistance to poplar bacterial canker.

[0020] In a second aspect, the present invention provides a drug for improving the disease resistance of poplar trees, the active ingredient of which is the HrpN lqp protein, and the amino acid sequence of the HrpN lqp protein is as follows (a1) or (a2);

[0021] (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO.1;

[0022] (a2) a protein derived therefrom by substitution and / or deletion and / or addition of one or several amino acid residues and having the same function.

[0023] Preferably, the drug of the present invention can promote plant growth and induce the expression of defense response PR genes in forest trees, and the defense response PR genes in forest trees include PR1, PR3, and PR4.

[0024] Beneficial effects:

[0025] The present invention provides an application of an HrpN lqp protein in improving the disease resistance of forest trees, the amino acid sequence of which is shown in SEQ ID NO.1, and its coding genehrpN lqp The sequence is shown as SEQ ID NO.2. The HrpN lqp protein screened in the present invention has immune induction activity. After application, it can significantly improve the disease resistance of forest plants (such as poplar trees), has the application value of being developed into a biogenic pesticide, can be used for the prevention and control of diseases of poplar trees (especially poplar tree branches and trunks), and has broad application prospects and economic benefits. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will describe the drawings required for use in the examples or the description of the prior art.

[0027] Figure 1 is the expression and purification result of HrpN lqp in the embodiment of the present invention. Among them, 1 is the diluted and concentrated HrpN lqp protein, and 2 is the purified HrpN lqp protein.

[0028] Figure 2 is the immune response result stimulated by HrpN lqp protein on tobacco in the embodiment of the present invention. Among them, A is the hypersensitive necrosis reaction of tobacco caused by injecting HrpN lqp into tobacco, B is the burst of reactive oxygen species on tobacco caused by HrpN lqp , and C is the deposition of callose on tobacco caused by HrpN lqp .

[0029] Figure 3 is the immune response result of recombinant protein HrpN lqp induced in poplar leaves in the embodiment of the present invention. Among them, A is the burst of reactive oxygen species caused by treating poplar leaves with HrpN lqp , B is the deposition of callose caused by treating poplar leaves with HrpN lqp , and C is the expression of genes related to the defense response of poplar after treating poplar leaves with HrpN lqp .

[0030] Figure 4 is the detection result of the resistance of poplar to the rot pathogen after treatment with recombinant protein HrpN lqp in the embodiment of the present invention. Among them, A is the disease incidence of poplar leaves after treatment with HrpN lqp , without treatment with HrpN lqp , and treatment with the Harpin protein Hpa1: (a) is the disease symptom of poplar leaves (CK is the medium inoculation point, 1 - 3 are the pathogen inoculation points); (b) is the statistics of the lesion diameter. B is the disease incidence of poplar leaves after treatment with HrpN lqp , without treatment with HrpN lqpDisease conditions of poplar branches after treatment with and without Harpin protein Hpa1: (a) shows the disease symptoms of poplar branches, and (b) shows the statistics of lesion diameters.

[0031] Figure 5 is the recombinant protein HrpN in the embodiments of the present invention lqp Detection results of the resistance of poplar leaves to Colletotrichum gloeosporioides after treatment. Among them, A shows the disease symptoms of poplar leaves (CK is the medium inoculation point, and 1 - 3 are the pathogen inoculation points); B shows the statistics of lesion diameters.

[0032] Figure 6 is the recombinant protein HrpN in the embodiments of the present invention lqp Detection results of the resistance of poplar branches to bacterial canker pathogens after treatment. Among them, A shows the disease symptoms of poplar branches, and B shows the average virulence level of the pathogens. Detailed implementation manners

[0033] Harpin proteins are proteins encoded by members of the hrp gene cluster of plant Gram - negative pathogens and secreted through the type III secretion system (T3SS). They are rich in glycine (Gly), contain no cysteine (Cys), are heat - stable, and are sensitive to proteases. Harpins can act as effectors to trigger the hypersensitive response (HR) and enable plants to establish systemic acquired resistance (SAR) to cope with external danger signals. When harpin proteins are constitutively expressed in plant cells or externally applied to plants, they help plants generate immune defense responses against various plant pathogens without causing HR cell death. Erwinia amylovora HrpN, reported in 1992, is an elicitor that can regulate the PR1 gene through the SAR pathway to confer induced resistance to Arabidopsis thaliana. Through the Erwinia amylovora study of the harpinEa protein of strain Ea321, a highly efficient and safe biological pesticide has been developed, which has attracted wide attention due to its significant effects on disease prevention, insect resistance, and yield increase. In addition to HrpN, other harpin proteins such as Hpa1, PopA1, and PopW also have the effect of enhancing plant disease resistance. Derived from Xanthomonas axonopodis pv. glycine s of Hpa1 - Xag and Xanthomonas oryzae pv. oryzae of Hpa1 XooIt can cause the HR response in tobacco leaves. Under the induction of pathogens, the expression of PopA1 protein is initiated in PopA1 transgenic tobacco, and it generates resistance to parasitic oomycetes through the salicylic acid-dependent pathway. Exogenous application of PopW protein can enhance the resistance of plants to Tobacco mosaic virus (TMV). However, at present, the use of harpins to endow plants with resistance, improve plant quality and yield mainly focuses on the agricultural scope, while there is less application development in the forestry scope and the effect is not ideal. The present invention screens and determines a harpin protein (HrpN lqp ), which has good application effects in the forestry field, especially significantly improves the disease resistance performance of poplar trees and has wide application value.

[0034] In a typical embodiment of the present invention, there is provided an application of a protein named HrpN lqp in inducing disease resistance in poplar trees. The protein named HrpN lqp is derived from the pathogen of poplar bacterial canker Lonsdalea populi strain, and its amino acid sequence consists of 328 amino acid residues, specifically as shown in SEQ ID NO.1.

[0035] MIQISIKTTPGADLGSQASGVEGYRSLSGGTGTQGSSGASADQLSSIIDKLTDLLTALMFANNGQSGTTQSDSQYSGAGTKGVQSPVNTQDSDAIDKLFSQALENLLGSDTVNALSQSGSNGGSLLSDSQATQGNQDAYSQGVKDALTLLGNNTSGSMGTPPSLSLGGNGLQGLSSPSDFSQLGSAVGSSVGKSAGLLALNNIDTHRDGVDRNFVNKEDRGTAKVIGQLMDQYPEIFGKPEYQKDNASSPVQDDKSWAQALSKPDDDGMTRESMDKFKQAVGMVKSALGGDSGNSNLNLRGAGGSLLGIDAAVAGEKIANLSLSKLAA (SEQ ID NO.1).

[0036] The HrpN lqp protein provided by the present invention can trigger the hypersensitive necrosis response on tobacco, causing the burst of reactive oxygen species and the deposition of callose; it can trigger the burst of reactive oxygen species in poplar leaves within 20 min; it can trigger the deposition of callose in poplar leaves; it can activate the expression of a series of defense response-related genes in plants and endow poplar trees with disease resistance. It has been verified that HrpN lqpThe incidence degrees of poplar canker, anthrax and bacterial canker after treatment are significantly reduced, the diameters of lesions are significantly decreased, and the expanded areas of lesions are significantly reduced, which are superior to the control group treated with Hpa1 (a very representative harpin protein used for plant disease resistance in the agricultural field).

[0037] Based on the above HrpN lqp Due to the significant advantages of the protein in enhancing the disease resistance of forestry plants, the application of the protein derived from the protein composed of the amino acid sequence shown in SEQ ID NO.1, which is obtained by substitution and / or deletion and / or addition of one or several amino acid residues and has the same function, in enhancing the disease resistance of forestry plants also falls within the scope of protection of the present invention.

[0038] The present invention provides the lqp gene encoding the above HrpN HrpN lqp protein, the nucleotide sequence of which consists of 987 nucleotides, wherein the 1st to 984th nucleotides are the coding sequence, and the 985th to 987th nucleotides are transcribed into stop codons to terminate the synthesis of the peptide chain, specifically as shown in SEQ ID NO.2.

[0039] ATGATTCAAATCTCGATCAAGACCACGCCCGGCGCTGATCTCGGATCTCAAGCCTCGGGTGTTGAAGGCTACCGGTCTCTGTCCGGTGGAACGGGAACGCAGGGCAGCAGCGGTGCCAGCGCCGATCAGCTGAGTAGCATCATCGATAAACTGACCGACCTGCTGACAGCCCTGATGTTTGCCAACAACGGCCAGTCAGGCACGACGCAGAGCGATTCACAGTACAGCGGCGCAGGCACGAAAGGCGTGCAATCTCCCGTTAATACTCAGGATTCGGATGCCATCGATAAGCTGTTTTCCCAGGCGCTGGAGAATCTGCTCGGTAGCGATACGGTCAACGCCCTCAGCCAGTCCGGCAGCAATGGTGGATCGCTGCTGAGCGACAGTCAGGCGACACAGGGCAATCAGGATGCCTATAGCCAAGGGGTTAAAGATGCGCTGACGCTGCTGGGGAATAACACAAGCGGCAGCATGGGCACGCCGCCATCGCTTTCTCTGGGAGGCAATGGTCTTCAGGGGCTGAGCAGTCCTTCGGACTTCAGCCAGTTGGGGAGCGCCGTCGGTTCGAGCGTTGGCAAAAGTGCCGGTTTGCTGGCGCTGAATAATATCGATACCCACCGGGATGGCGTGGATCGCAACTTCGTCAACAAAGAGGATCGCGGCACGGCCAAGGTGATCGGGCAACTGATGGACCAGTATCCTGAGATTTTTGGCAAGCCGGAGTACCAGAAGGATAACGCGTCATCGCCGGTACAAGACGACAAAAGCTGGGCGCAGGCGTTGAGCAAACCGGACGACGACGGGATGACCCGTGAAAGTATGGATAAATTTAAACAGGCCGTCGGCATGGTGAAAAGTGCACTGGGGGGCGATAGCGGCAACAGCAATCTGAACCTGCGCGGCGCGGGCGGTTCCCTGCTGGGCATCGATGCAGCGGTCGCCGGAGAGAAGATCGCAAATCTCTCACTGTCGAAGCTGGCCGCCTAA (SEQ ID NO.2).

[0040] Based on the significant advantages of the above-mentioned HrpN lqp protein in enhancing the disease resistance of forestry plants, the gene for expressing the above-mentioned HrpN lqp protein, or a gene complementary to the gene shown in SEQ ID NO.2, or a gene having ≥90% identity with the gene shown in SEQ ID NO.2 and encoding the same functional protein, or a biological material (such as a recombinant expression vector, recombinant cell, engineered bacterium, etc.) having the function of expressing the same protein containing the said gene, and a biological preparation (HrpN lqp protein + excipients) containing the said HrpN lqp protein applied to enhance the disease resistance of forestry plants also fall within the scope of protection of the present invention.

[0041] Preferably, the recombinant expression vector is a recombinant prokaryotic expression vector, and the recombinant prokaryotic expression vector is obtained by inserting the above-mentioned coding gene into the expression vector pET-28a.

[0042] In a more specific embodiment provided by the present invention, the recombinant expression vector is pET-28a- hrpN lqp recombinant vector, which is constructed by the following method:

[0043] The strain N-5-1 of Populus euramericana bacterial canker is cultured on an LB solid medium containing rifampicin resistance at 30 °C for 48 hours and then transferred to an LB liquid medium containing rifampicin resistance, and after continuous shaking culture for 48 h, fresh bacterial liquid is collected for extracting genomic DNA. According to hrpN lqp the sequence, primers for the complete open reading frame (ORF) are designed:

[0044] hrpN lqp -P1: ATGATTCAAATCTCGATCAAG (SEQ ID NO.3);

[0045] hrpN lqp -P2: GGCGGCCAGCTTCGACAGTG (SEQ ID NO.4).

[0046] Using the genomic DNA of strain N-5-1 as a template for PCR amplification, the reaction conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, with 30 cycles, and final extension at 72°C for 5 min. The PCR product was ligated to the pMD19-T vector and transformed into Escherichia coli DH5α. Positive transformants were obtained through plasmid PCR verification, and the recombinant plasmid was extracted and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing.

[0047] Using the correctly sequenced positive recombinant plasmid pMD19-T- hrpN lqp as a template, primers hrpN lqp -P3 and hrpN lqp -P4 were designed for PCR amplification. After the amplified product was recovered, it was ligated to the expression vector pET-28a through homologous recombination. The culture was incubated at 37°C for 12 h, and single colonies were picked for PCR identification. The positive clone colonies were inoculated into LB liquid medium and cultured at 37°C for 12 h, and then the plasmid was extracted for sequencing. Correct sequencing indicated the recombinant expression vector pET-28- hrpN lqp .

[0048] hrpN lqp -P3:

[0049] AGCAAATGGGTCGCGGATCCATGATTCAAATCTCGATCAAG (SEQ ID NO.5).

[0050] hrpN lqp -P4:

[0051] TGTCGACGGAGCTCGAATTCGGCGGCCAGCTTCGACAGTG (SEQ ID NO.6).

[0052] Preferably, the recombinant cell is a prokaryotic cell; more preferably a bacterium, further selected from Escherichia coli; furthermore, the recombinant cell is BL21 containing the above gene and / or recombinant expression vector.

[0053] In a more specific embodiment provided by the present invention, the recombinant cell is recombinant Escherichia coli BL21 / pET-28a- hrpN lqp , which is constructed by the following method:

[0054] The above recombinant plasmid pET-28a- hrpN lqpTransforming into Escherichia coli BL21, the recombinant Escherichia coli BL21 / pET-28a- hrpN lqp .

[0055] In a more specific embodiment of the present invention, there is also provided a method for preparing the protein HrpN using the said coding gene, recombinant expression vector or recombinant cell. lqp The protein HrpN prepared by the method provided by the present invention lqp significantly enhances the disease resistance of forestry plants. Therefore, the application of the said coding gene, recombinant expression vector or recombinant cell in the preparation of the protein HrpN lqp and further in the scheme for improving the disease resistance of forestry plants is also within the scope of protection of the present invention.

[0056] The present invention provides a primer pair for amplifying the above coding gene, and its nucleotide sequences are respectively shown as SEQ ID NO.3 and SEQ ID NO.4.

[0057] The present invention provides a primer pair for amplifying the above recombinant plasmid, and its nucleotide sequences are respectively shown as SEQ ID NO.5 and SEQ ID NO.6.

[0058] In yet another specific embodiment of the present invention, the application of the protein HrpN lqp as a protein inducer for disease resistance should also be within the scope of protection of the present invention. Specifically, the said application at least includes: (1) inducing disease resistance in plants; (2) promoting plant growth. Preferably, the plant is a poplar tree.

[0059] In yet another specific embodiment of the present invention, there is provided a protein inducer for disease resistance, which at least includes the above protein HrpN lqp , and also includes excipients acceptable in pesticide or microbial agent. Preferably, the excipients are selected from one or more of surfactants, penetrants and solvents. The present invention has no special restrictions on the sources of the said excipients, and generally commercially available products can be used. Preferably, the surfactant is Silwet-L77. Preferably, the penetrant is sodium di-sec-octyl sulfosuccinate. Preferably, the solvent is deionized water.

[0060] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the scope of protection of the present invention.

[0061] The endpoints and any values within the ranges disclosed in this specification are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0062] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "specific implementation manners", or "some specific implementation manners" etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the embodiments of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0063] In the embodiments provided in this specification, for those without specifying specific techniques or conditions, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For the reagents or instruments without specifying the manufacturer, they are all conventional products that can be obtained through regular channels.

[0064] Abbreviations used in the present invention:

[0065] LB: Bacterial culture medium, yeast powder 5 g / L + tryptone 10 g / L + NaCl 10 g / L;

[0066] binding buffer (1 L): 20 mM Tris + 150 mM NaCl + 1 mM PMSF;

[0067] IPTG: Isopropyl β-D-thiogalactoside;

[0068] PMSF: Protease inhibitor phenylmethylsulfonyl fluoride;

[0069] EVP: Protein expressed and purified by BL21 / pET-28a;

[0070] Hpa1: Derived from Xanthomonas oryzae pv. oryzicola The Harpin protein Hpa1, and the amino acid sequence of the Hpa1 protein is:

[0071] MNSLNTQFGGSASNFQVDQSQNAQSDSSQGSNGSQGISEKQLDQLLCQLIQALLQPNKNAEEGKGQQGGENGGGQGGNQQAGKENGASPLTQMLMNIVGEILQAQNGGGAGGAGGSSGGDFGGSFASSFSNDSGSMQ* (SEQ ID NO.7).

[0072] Example 1

[0073] Bacterial canker pathogen N - 5 - 1 strain of Populus euramericana hrpN lqp Cloning and sequencing of the gene.

[0074] The bacterial canker pathogen N - 5 - 1 strain of Populus euramericana (preserved in Room 309, Research Building of Beijing Forestry University) was cultured on LB solid medium containing rifampicin resistance at 30 °C for 48 hours, then transferred to LB liquid medium containing rifampicin resistance and continuously cultured with shaking for 48 h. After that, fresh bacterial liquid was collected for genomic DNA extraction. According to hrpN lqp the sequence, primers for the complete open reading frame (ORF) were designed:

[0075] hrpN lqp -P1: ATGATTCAAATCTCGATCAAG (SEQ ID NO.3).

[0076] hrpN lqp -P2: GGCGGCCAGCTTCGACAGTG (SEQ ID NO.4).

[0077] Using the genomic DNA of strain N - 5 - 1 as a template for PCR amplification, the reaction conditions were: pre - denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, 30 cycles, and extension at 72 °C for 5 min; the PCR product was ligated with pMD19 - T vector and transformed into Escherichia coli DH5α. Positive transformants were obtained by plasmid PCR verification, and the recombinant plasmid was extracted and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing.

[0078] Example 2

[0079] HrpN lqp Expression and purification of the protein.

[0080] (I) hrpN lqp Construction of genetically engineered expression strains.

[0081] Using the correctly sequenced positive recombinant plasmid pMD19-T- hrpN lqp as a template, primers rpN lqp -P3 and hrpN lqp -P4 were designed for PCR amplification. After recovery of the amplification product, it was ligated to the expression vector pET-28a by homologous recombination and transformed into the expression host strain BL21(DE3). Positive recombinant strains were obtained by plasmid PCR verification and screening.

[0082] hrpN lqp -P3:

[0083] AGCAAATGGGTCGCGGATCCATGATTCAAATCTCGATCAAG (SEQ ID NO.5).

[0084] hrpN lqp -P4:

[0085] TGTCGACGGAGCTCGAATTCGGCGGCCAGCTTCGACAGTG (SEQ ID NO.6).

[0086] (II) Expression of HrpN lqp protein

[0087] (1) Activation of the original strain: The recombinant strain BL21 / pET-28a- hrpN lqp was inoculated into LB liquid medium containing 50 mg / ml kanamycin and cultured overnight at 37°C (200 rpm / min). The next day, it was transferred to LB liquid containing 50 mg / ml kanamycin at a ratio of 2% and cultured at 37°C for 2 h. IPTG with a final concentration of 1 mM was added, and the culture was continued at 25°C with shaking.

[0088] (2) Cell disruption: The induced bacterial liquid was collected and centrifuged at 4000 rpm / min at 4°C for 10 min to collect the bacterial cells. The cells were washed once with binding buffer and resuspended in 20 ml of binding buffer, then disrupted by sonication. After centrifugation at 8000 rpm / min for 40 min, the supernatant was collected as the soluble HrpN lqp crude protein. The crude protein was purified by Histrap column to obtain HrpN lqp pure protein.

[0089] (III) Purification of HrpN lqp protein

[0090] The crude protein was purified by Histrap column to obtain HrpN lqp pure protein. The purified sample was ultrafiltered and concentrated by an ultrafiltration column ( Figure 1 ), and the pure protein was stored at -80 °C.

[0091] Example 3

[0092] HrpN lqp Application of the protein.

[0093] (I) HrpN lqp The protein induces the hypersensitive response in tobacco leaves.

[0094] The purified HrpN lqp protein was inoculated into tobacco leaves by injection. At the same time, Harpin protein Hpa1 (amino acid sequence shown in SEQ ID NO.7), EVP and ddH2O were used as controls. The hypersensitive necrosis reaction of tobacco was observed, and the burst of reactive oxygen species and the deposition of callose in tobacco after inoculation were observed by DAB and aniline blue staining. The results showed that HrpN lqp could trigger the hypersensitive necrosis reaction in tobacco, resulting in the burst of reactive oxygen species and the deposition of callose ( Figure 2 ).

[0095] (II) HrpN lqp The protein induces the burst of reactive oxygen species and the deposition of callose in poplar leaves.

[0096] Poplar leaves of the same size and growth state were selected. The purified HrpN lqp was diluted to 20 μg / ml and added with Silwet-L77 (0.03% w / v) to treat poplar leaves. The burst of reactive oxygen species was detected by DAB staining at 15 min, 20 min and 25 min respectively. The results showed that HrpN lqp induced the burst of reactive oxygen species in poplar leaves at 20 min ( Figure 3 Figure A in).

[0097] Leaves of the same size and growth state were selected. The purified HrpN lqp was diluted to 20 μg / ml and added with Silwet-L77 (0.03% w / v) to treat poplar leaves. The deposition of callose was detected by aniline blue staining after incubation for 15 h, 24 h and 48 h respectively. The results showed that HrpN lqp could induce the deposition of callose in poplar leaves ( Figure 3 Figure B in).

[0098] (III) HrpN lqp The protein induces the expression of defense response-related genes in poplar.

[0099] Select poplar leaves with consistent size and growth status, and dilute the purified HrpN lqp to 20 μg / ml and add Silwet-L77 (0.03% w / v) to treat poplar leaves. At the same time, use ddH2O as a control. Extract leaf RNA at 24 h, 48 h, and 96 h respectively, and detect the expression of defense response genes in poplar by Real-time PCR. The results show that HrpN lqp can activate the expression of plant PR genes and confer disease resistance to poplar ( Figure 3 Figure C in

[0100] (IV) HrpN lqp protein induces poplar resistance to poplar canker disease.

[0101] Select poplar leaves with consistent size and growth status, and dilute the purified HrpN lqp to 30 μg / ml and add Silwet-L77 (0.03% w / v), then spray and treat poplar leaves. At the same time, use ddH2O and Harpin protein Hpa1 (30 μg / ml) as controls. Inoculate poplar canker pathogen on poplar leaves 24 h later, observe the disease incidence of the leaves, and measure the lesion diameter. The results show that the lesion diameter of poplar canker under ddH2O treatment is 2.00 cm. After treatment with Harpin protein Hpa1, the lesion diameter of poplar canker decreases from 2.00 cm to 1.39 cm. However, after HrpN lqp treatment, the lesion diameter of poplar canker decreases to 0.99 cm, the lesion expansion area is significantly reduced, and the disease severity is significantly alleviated ( Figure 4 Figure A in

[0102] Select poplar branches with consistent length, thickness and growth status, and dilute the purified HrpN lqp to 30 μg / ml and add Silwet-L77 (0.03% w / v) and sodium di-sec-octyl sulfosuccinate (2% - 5%), then spray and treat poplar branches. At the same time, use ddH2O and Harpin protein Hpa1 (30 μg / ml) as controls. Inoculate poplar canker pathogen on poplar branches 24 h later, observe the disease incidence of the branches, and measure the lesion diameter. The results show that the lesion diameter of poplar canker under ddH2O treatment is 6.62 cm. After treatment with Harpin protein Hpa1, the lesion diameter of poplar canker decreases from 6.62 cm to 4.11 cm. However, after HrpN lqp treatment, the lesion diameter of poplar canker decreases to 2.05 cm, the expansion speed slows down, the expansion area is significantly reduced, and the disease severity is significantly alleviated ( Figure 4 Figure B in

[0103] (V) HrpN lqpProtein induces poplar resistance to poplar anthracnose.

[0104] Select poplar leaves of the same size and growth status, dilute the purified HrpN lqp to 30 μg / ml and add Silwet-L77 (0.03% w / v), spray and treat the poplar leaves. At the same time, use ddH2O and Harpin protein Hpa1 (30 μg / ml) as controls. After 24 h, inoculate Colletotrichum gloeosporioides on the poplar leaves, observe the disease incidence of the leaves, and measure the diameter of the lesions. The results show that the diameter of the anthracnose lesions under the ddH2O treatment is 1.83 cm. After treatment with Harpin protein Hpa1, the diameter of the rot lesions decreased from 1.83 cm to 1.22 cm, while after treatment with HrpN lqp the diameter of the rot lesions decreased to 0.95 cm, and the incidence of anthracnose was significantly reduced ( Figure 5 ).

[0105] (VI) HrpN lqp Protein induces poplar resistance to poplar bacterial canker.

[0106] Select poplar branches of the same length, thickness and growth status, dilute the purified HrpN lqp to 30 μg / ml and add Silwet-L77 (0.03% w / v) and sodium di-sec-octyl sulfosuccinate (2% - 5%), spray and treat the poplar branches. At the same time, use ddH2O and Harpin protein Hpa1 (30 μg / ml) as controls. After 24 h, inoculate poplar bacterial canker pathogen on the poplar branches, observe the disease incidence of the branches, count the virulence level, and calculate the disease index. The results show that the pathogenic level of the pathogen under the ddH2O treatment is 3.86, and the disease index is 77.14%. After treatment with Harpin protein Hpa1, the pathogenic level of the pathogen decreased from 3.86 to 3.40, and the disease index decreased to 68%. While after treatment with HrpN lqp the pathogenic level of the pathogen decreased to 1.70, and the disease index decreased to 56.67%, reaching a highly significant level compared with the control, and the disease incidence was significantly reduced ( Figure 6 ).

[0107] The above examples show that the HrpN cloned from the poplar bacterial canker pathogen of Populus euramericana in the present invention lqp can elicit hypersensitive response on tobacco. The HrpN of the present invention lqp treats poplar, can induce the immune response of poplar, effectively improve the disease resistance of poplar, and has a significant effect on resisting poplar trunk diseases. The above conclusions prove that HrpN lqp has the application value of being developed into a biogenic pesticide.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. HrpN lqp Application of the protein in improving disease resistance of forest trees, wherein the lqp amino acid sequence of the protein is a protein composed of the amino acid sequence shown in SEQ ID NO. 1; The forest tree is a poplar tree; The disease resistance refers to the resistance to poplar canker, poplar anthracnose, and poplar bacterial canker.

2. The application according to claim 1, wherein The HrpN lqp protein has a coding gene as follows: (b1), (b2), or (b3); (b1) The nucleotide sequence shown in SEQ ID NO.2; (b2) The nucleotide sequence complementary to (b1); (b3) The nucleotide sequence having ≥90% identity with the nucleotide sequence shown in (b1) or (b2) and encoding the same functional protein.

3. The application according to claim 1 or 2, characterized in that The HrpN lqp protein is expressed by a recombinant prokaryotic expression vector or a recombinant cell containing the recombinant prokaryotic expression vector; the recombinant prokaryotic expression vector is obtained by inserting the HrpN lqp protein coding gene into a basic expression vector.

4. The application according to claim 3, characterized in that The basic expression vector is pET-28a. 5.HrpN lqp Application of protein in inducing expression of PR gene for defense response in poplar, characterized in that The defense response PR genes in the poplar tree include PR1, PR3, and PR4; The HrpN lqp protein has an amino acid sequence which is a protein composed of the amino acid sequence shown in SEQ ID NO.1.

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