Application of HrpNlqp protein in improvement of forest disease resistance

Abstract

The invention relates to the technical field of gene engineering, in particular to application of HrpNlqp protein to improvement of forest disease resistance. The invention provides an application of HrpNlqp protein in improvement of forest tree disease resistance, the amino acid sequence of the HrpNlqp protein is shown as SEQ ID NO.1, and the sequence of the coding gene hrpNlqp is shown as SEQ ID NO.2. The invention also provides an application of the HrpNlqp protein in improvement of forest tree disease resistance. The screened HrpNlqp protein has immune induction activity, can obviously improve the disease resistance of forest plants (such as poplar) after being applied, has application value of being developed into biological source pesticides, can be used for preventing and treating poplar (especially poplar branches and trunks) diseases, and has wide 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 HrpN lqp The application of protein in improving the disease resistance of forest trees. Background Art

[0002] Poplar ( Populus spp.) as the main tree species for afforestation, timber forest, shelterbelt and greening, it plays an important role in forestry production and ecological construction. However, under natural conditions, the growth and development of poplars will inevitably be affected by various external stresses, among which the interference of adverse factors such as plant diseases and insect gnawing will cause large-scale destruction of poplar plantations. At the same time, due to the large-scale afforestation of a single asexual line of poplars, the ability of plantations to resist the invasion of pathogens has been weakened, making diseases a major problem in the development of forestry.

[0003] The main method of disease prevention and control is to apply chemical pesticides. Applying chemical pesticides can effectively control the spread of diseases when they break out, and the broad spectrum of chemical pesticides can target a variety of diseases. However, long-term chemical control also brings many disadvantages, such as high pesticide residues that harm human and livestock health, long-term application of pesticides leading to increasing drug resistance of pathogens, and irreversible damage to the ecological environment.

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

[0005] In order to solve the above technical problems, the present invention provides a HrpN lqp The application of protein in improving the disease resistance of forest trees.

[0006] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides HrpN lqp Application of protein in improving forest disease resistance, the HrpN lqp The amino acid sequence of the protein is as follows (a1) or (a2); (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO.1; (a2) A protein derived from (a1) with the same function as (a1) after one or more amino acid residues are substituted and / or deleted and / or added.

[0007] Preferably, the HrpN lqp The protein encoding gene is as follows (b1), (b2) or (b3); (b1) the nucleotide sequence shown in SEQ ID NO.2; (b2) a nucleotide sequence complementary to (b1); (b3) A nucleotide sequence that has ≥90% identity with the nucleotide sequence shown in (b1) or (b2) and encodes a protein with the same function.

[0008] Preferably, the HrpN lqp The 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 HrpN into the basic expression vector. lqp Protein coding genes obtained.

[0009] Preferably, the basic expression vector is pET-28a.

[0010] Preferably, the trees are poplars.

[0011] Preferably, the present invention converts HrpN lqp Protein is used to improve resistance to poplar rot.

[0012] Preferably, the present invention converts HrpN lqp Protein is used to improve resistance to anthracnose in poplars.

[0013] Preferably, the present invention converts HrpN lqp Protein is used to improve resistance to bacterial canker in poplars.

[0014] In a second aspect, the present invention provides a drug for improving the disease resistance of poplar, wherein the active ingredient is HrpN lqp Protein, the HrpN lqp The amino acid sequence of the protein is as follows (a1) or (a2); (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO.1; (a2) A protein derived from (a1) with the same function as (a1) after one or more amino acid residues are substituted and / or deleted and / or added.

[0015] Preferably, the drug of the present invention can promote plant growth and induce the expression of defense response PR genes in trees, and the defense response PR genes in trees include PR1, PR3, and PR4.

[0016] Beneficial effects: The present invention provides a HrpN lqp The application of protein in improving the disease resistance of forest trees, the amino acid sequence of which is shown in SEQ ID NO.1, and the encoding gene hrpN lqp The sequence is shown in SEQ ID NO. 2. lqpThe protein has immune-inducing activity and can significantly improve the disease resistance of forest plants (such as poplars) after application. It has application value in being developed into a bio-source pesticide and can be used for the prevention and control of poplar (especially poplar branches) diseases. It has broad application prospects and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0018] Figure 1 is HrpN in the embodiment of the present invention lqp The expression and purification results of HrpN lqp Protein 2 is purified HrpN lqp protein.

[0019] Figure 2 is HrpN in the embodiment of the present invention lqp The results of the immune response induced by protein on tobacco. Among them, A is HrpN lqp Tobacco allergic necrosis reaction caused by tobacco injection, B is HrpN lqp The reactive oxygen burst caused by tobacco, C is HrpN lqp Tobacco callus deposition caused by.

[0020] Figure 3 The recombinant protein HrpN in the embodiment of the present invention lqp Results of inducing immune response in poplar leaves. A is HrpN lqp The reactive oxygen burst caused by treating poplar leaves, B is HrpN lqp Callose deposition caused by treatment of poplar leaves, C is HrpN lqp Expression of genes related to poplar defense responses after treatment of poplar leaves.

[0021] Figure 4 The recombinant protein HrpN in the embodiment of the present invention lqp The results of the poplar resistance test to rot pathogens after treatment. lqp Treated, without HrpN lqp Disease status of poplar leaves after treatment with harpin protein Hpa1: (a) Disease symptoms of poplar leaves (CK is the culture medium inoculation point, 1-3 is the pathogen inoculation point); (b) Statistics of lesion diameter. lqp Treated, without HrpN lqp Disease status of poplar branches after treatment with HAPR1 and treatment with Harpin protein Hpa1: (a) disease symptoms of poplar branches, (b) statistics of lesion diameter.

[0022] Figure 5 The recombinant protein HrpN in the embodiment of the present invention lqp The results of the resistance test of poplar leaves to Colletotrichum gloeosporioides after treatment. A is the disease symptoms of poplar leaves (CK is the culture medium inoculation point, 1-3 is the pathogen inoculation point); B is the statistics of lesion diameter.

[0023] Figure 6 The recombinant protein HrpN in the embodiment of the present invention lqp The results of the resistance test of poplar branches to bacterial canker after treatment. A is the disease symptoms of poplar branches, and B is the average virulence level of the pathogen. DETAILED DESCRIPTION

[0024] 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), do not contain cysteine ​​(Cys), are heat-stable, and are sensitive to proteases. Harpins can act as effectors to trigger a hypersensitive response (HR), allowing plants to establish systemic acquired resistance (SAR) to respond to external danger signals. When harpin proteins are constitutively expressed in plant cells or applied exogenously to plants, they help plants produce immune defense responses to various plant pathogens without causing HR cell death. Erwinia amylovora HrpN was reported in 1992 as an elicitor that can regulate the PR1 gene through the SAR pathway to induce resistance in Arabidopsis. Erwinia amylovora The research on the harpinEa protein of strain Ea321 and the development of efficient and safe biopesticides have attracted widespread attention due to their significant disease prevention, insect resistance and yield increase effects. In addition to HrpN, other harpin proteins such as Hpa1, PopA1 and PopW also have the function of enhancing plant disease resistance. Xanthomonas axonopodis pv. glycine s Hpa1-Xag and Xanthomonas oryzae pv. oryzae Hpa1 XooIt can induce HR response in tobacco leaves. Under the induction of pathogens, PopA1 protein expression is activated in PopA1 transgenic tobacco, which produces resistance to parasitic oomycetes through a salicylic acid-dependent pathway. Exogenous application of PopW protein can improve the resistance of plants to mosaic virus (Tobacco mosaic virus, TMV). However, the current use of harpins to confer plant resistance and improve plant quality and yield is mainly concentrated in the agricultural field, while the application and development in the forestry field is relatively small, and the effect is not ideal. The present invention screened and identified a harpin protein (HrpN lqp ), which has good application effects in the forestry field, especially significantly improving the disease resistance of poplar, and has wide application value.

[0025] In a typical embodiment of the present invention, there is provided a HrpN lqp The application of a protein named HrpN in inducing disease resistance in poplar lqp The protein is derived from the pathogen of bacterial canker of Populus occidentalis Lonsdalea populi The strain has an amino acid sequence consisting of 328 amino acid residues, as specifically shown in SEQ ID NO.1.

[0026] MIQISIKTTPGADLGSQASGVEGYRSLSGGTGTQGSSGASADQLSSIIDKLTDLLTALMFANNGQSGTTQSDSQYSGAGTKGVQSPVNTQDSDAIDKLFSQALENLLGSDTVNALSQSGSNGGSLLSDSQATQGNQDAYSQGVKDALTLLGNNTSGSMGTPPSLSL GGNGLQGLSSPSDFSQLGSAVGSSVGKSAGLLALNNIDTHRDGVDRNFVNKEDRGTAKVIGQLMDQYPEIFGKPEYQKDNASSPVQDDKSWAQALSKPDDDGMTRESMDKFKQAVGMVKSALGGDSGNSNLNLRGAGGSLLGIDAAVAGEKIANLSLSKLAA (SEQ ID NO.1).

[0027] HrpN provided by the present invention lqp The protein can trigger allergic necrosis in tobacco, causing reactive oxygen burst and callose deposition; it can trigger reactive oxygen burst in poplar leaves within 20 minutes; it can trigger callose deposition in poplar leaves; it can activate the expression of a series of defense response-related genes in plants, giving poplars disease resistance. lqpAfter treatment, the incidence of poplar rot, anthracnose and bacterial canker was significantly reduced, the lesion diameter was significantly reduced, and the lesion expansion area was significantly reduced, which was better than the control group treated with Hpa1 (a very representative harpin protein used for plant disease resistance in the agricultural field).

[0028] Based on the above HrpN lqp The protein has significant advantages in improving the disease resistance of forestry plants. The protein composed of the amino acid sequence shown in SEQ ID NO.1 is obtained by replacing and / or deleting and / or adding one or several amino acid residues, and the protein derived therefrom has the same function. Its application in improving the disease resistance of forestry plants also falls within the scope of protection of the present invention.

[0029] The present invention provides a method for encoding the HrpN lqp Protein HkDJ lqp The gene has a nucleotide sequence consisting of 987 nucleotides, wherein the 1st to 984th nucleotides are coding sequences, and the 985th to 987th nucleotides are transcribed as stop codons to terminate peptide chain synthesis, as specifically shown in SEQ ID NO.2.

[0030] ATGATTCAAATCTCGATCAAGACCACGCCCGGCGCTGATCTCGGATCTCAAGCCTCGGGTGTTGAAGGCTACCGGTCTCTGTCCGGTGGAACGGGAACGCAGGGCAGCAGCGGTGCCAGCGCCGATCAGCTGAGTAGCATCATCGATAAACTGACCGACCTGCTGACAGCCCTGATGTTTGCCAACAACGGCCAGTCAGGCACGACGCAGAGCGATTCACAGTACAGCGGCGCAGGCACGAAAGGCGTGCAATCTCCCGTTAATACTCAGGATTCGGATGCCATCGATAAGCTGTTTTCCCAGGCGCTGGAGAATCTGCTCGGTAGCGATACGGTCAACGCCCTCAGCCAGTCCGGCAGCAATGGTGGATCGCTGCTGAGCGACAGTCAGGCGACACAGGGCAATCAGGATGCCTATAGCCAAGGGGTTAAAGATGCGCTGACGCTGCTGGGGAATAACACAAGCGGCAGCATGGGCACGCCGCCATCGCTTTCTCTGGGAGGCAATGGTCTTCAGGGGCTGAGCAGTCCTTCGGACTTCAGCCAGTTGGGGAGCGCCGTCGGTTCGAGCGTTGGCAAAAGTGCCGGTTTGCTGGCGCTGAATAATATCGATACCCACCGGGATGGCGTGGATCGCAACTTCGTCAACAAAGAGGATCGCGGCACGGCCAAGGTGATCGGGCAACTGATGGACCAGTATCCTGAGATTTTTGGCAAGCCGGAGTACCAGAAGGATAACGCGTCATCGCCGGTACAAGACGACAAAAGCTGGGCGCAGGCGTTGAGCAAACCGGACGACGACGGGATGACCCGTGAAAGTATGGATAAATTTAAACAGGCCGTCGGCATGGTGAAAAGTGCACTGGGGGGCGATAGCGGCAACAGCAATCTGAACCTGCGCGGCGCGGGCGGTTCCCTGCTGGGCATCGATGCAGCGGTCGCCGGAGAGAAGATCGCAAATCTCTCACTGTCGAAGCTGGCCGCCTAA(SEQ ID NO.2)。

[0031] Based on the above HrpN lqp The protein has significant advantages in improving the disease resistance of forestry plants and will be used to express the above HrpN lqp A gene encoding a 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 a protein with the same function, or a biological material (such as an artificially constructed recombinant expression vector, recombinant cell, engineered bacteria, etc.) containing the gene and having the same protein function, and a gene containing the HrpN lqp Protein biological preparations (HrpN lqp Protein + auxiliary materials) is used to enhance the disease resistance of forestry plants, which also falls within the scope of protection of the present invention.

[0032] Preferably, the recombinant expression vector is a recombinant prokaryotic expression vector, and the recombinant prokaryotic expression vector is obtained by inserting the above-mentioned encoding gene into the expression vector pET-28a.

[0033] In a more specific embodiment provided by the present invention, the recombinant expression vector is pET-28a- hrpN lqp The recombinant vector is constructed by the following method: The Populus euphorbia N-5-1 strain was cultured on a solid LB medium containing rifampicin resistance at 30°C for 48 hours and then transferred to a liquid LB medium containing rifampicin resistance. After shaking culture for 48 hours, fresh bacterial culture was collected for genomic DNA extraction. hrpN lqp Sequence, design primers for complete open reading frame (ORF): hrpN lqp -P1:ATGATTCAAATCTCGATCAAG (SEQ ID NO.3); hrpN lqp -P2: GGCGGCCAGCTTCGACAGTG (SEQ ID NO. 4).

[0034] PCR amplification was performed using the genomic DNA of the N-5-1 strain as a template. 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 connected to the 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 Ruiboxingke Biotechnology Co., Ltd. for sequencing.

[0035] The positive recombinant plasmid pMD19-T- hrpN lqp As template, design primers hrpN lqp -P3 and hrpN lqp -P4 was amplified by PCR, and the amplified product was recovered and connected to the expression vector pET-28a by homologous recombination, and cultured at 37℃ for 12 hours. Single colonies were picked for PCR identification, and positive clone colonies were inoculated into LB liquid culture medium and cultured at 37℃ for 12 hours. Plasmid was extracted for sequencing. If the sequencing was correct, it was the recombinant expression vector pET-28- hrpN lqp .

[0036] hrpN lqp -P3: AGCAAATGGGTCGCGGATCCATGATTCAAATCTCGATCAAG (SEQ ID NO. 5).

[0037] hrpN lqp -P4: TGTCGACGGAGCTCGAATTCGGCGGCCAGCTTCGACAGTG (SEQ ID NO. 6).

[0038] Preferably, the recombinant cell is a prokaryotic cell; more preferably, a bacterium, further selected from Escherichia coli; further, the recombinant cell is BL21 containing the above-mentioned gene and / or recombinant expression vector.

[0039] In a more specific embodiment provided by the present invention, the recombinant cell is a recombinant Escherichia coli BL21 / pET-28a- hrpN lqp , which is constructed by the following method: The above recombinant plasmid pET-28a- hrpN lqp Transformed into E. coli BL21, recombinant E. coli BL21 / pET-28a- hrpN lqp .

[0040] In a more specific embodiment of the present invention, the coding gene, recombinant expression vector or recombinant cell for preparing protein HrpN is also provided. lqp The protein HrpN prepared by the method provided by the present invention lqp Therefore, the coding gene, recombinant expression vector or recombinant cell is applied to protein HrpNlqp The preparation and further application of the method to improve the disease resistance of forestry plants are also within the scope of invention protection.

[0041] The present invention provides a primer pair for amplifying the above coding gene, and the nucleotide sequences thereof are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0042] The present invention provides a primer pair for amplifying the above recombinant plasmid, and the nucleotide sequences thereof are shown as SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0043] In another specific embodiment of the present invention, the protein HrpN lqp The application as a protein inducer should also be within the scope of protection of the present invention. Specifically, the application at least includes: (1) inducing plant disease resistance; (2) promoting plant growth. Preferably, the plant is poplar.

[0044] In another specific embodiment of the present invention, a protein inducer is provided, wherein the protein inducer comprises at least the above-mentioned protein HrpN lqp , and also includes an adjuvant acceptable in pesticides or microbial agents. Preferably, the adjuvant is selected from one or more of a surfactant, a penetrant and a solvent. The present invention has no special restrictions on the source of the adjuvant, and generally commercially available products can be used. Preferably, the surfactant is Silwet-L77. Preferably, the penetrant is sodium di-sec-octyl maleate sulfonate. Preferably, the solvent is deionized water.

[0045] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and 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, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0047] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0048] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0049] Abbreviations used in the present invention: LB: bacterial culture medium, yeast powder 5 g / L + tryptone 10 g / L + NaCl 10 g / L; binding buffer (1 L): 20mM Tris + 150mM NaCl + 1mM PMSF; IPTG: isopropylthio-β-galactosidase; PMSF: protease inhibitor phenylmethylsulfonyl fluoride; EVP: protein expressed and purified by BL21 / pET-28a; Hpa1: derived from Xanthomonas oryzae pv. oryzicola The amino acid sequence of the Harpin protein Hpa1 is: MNSLNTQFGGSASNFQVDQSQNAQSDSSQGSNGSQGISEKQLDQLLCQLIQALLQPNKNAEEGKGQQGGENGGGQGGNQQAGKENGASPLTQMLMNIVGEILQAQNGGGAGGAGGSSGGDFGGSFASSFSNDSGSMQ* (SEQ ID NO. 7).

[0050] Example 1

[0051] Populus euphorbia bacterial canker strain N-5-1 hrpN lqp Gene cloning and sequencing.

[0052] Populus euphorbia N-5-1 strain (stored in 309, Science Building, Beijing Forestry University) was cultured on LB solid medium containing rifampicin resistance at 30°C for 48 hours and then transferred to LB liquid medium containing rifampicin resistance. After shaking culture for 48 hours, fresh bacterial culture was collected for genomic DNA extraction. hrpN lqp Sequence, design primers for complete open reading frame (ORF): hrpN lqp -P1:ATGATTCAAATCTCGATCAAG (SEQ ID NO. 3).

[0053] hrpN lqp -P2: GGCGGCCAGCTTCGACAGTG (SEQ ID NO. 4).

[0054] PCR amplification was performed using the genomic DNA of the N-5-1 strain as a template. 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 connected to the 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 Ruiboxingke Biotechnology Co., Ltd. for sequencing.

[0055] Example 2

[0056] HkDJ lqp Protein expression and purification.

[0057] (one) hrpN lqp Construction of genetically engineered expression strains.

[0058] The positive recombinant plasmid pMD19-T- hrpN lqp As template, design primers rPb lqp -P3 and hrpN lqp -P4 was amplified by PCR, and the amplified product was recovered and connected to the expression vector pET-28a by homologous recombination, and transformed into the expression host bacteria BL21 (DE3), and the positive recombinant strain was obtained by plasmid PCR verification and screening.

[0059] hrpN lqp -P3: AGCAAATGGGTCGCGGATCCATGATTCAAATCTCGATCAAG (SEQ ID NO. 5).

[0060] hrpN lqp -P4: TGTCGACGGAGCTCGAATTCGGCGGCCAGCTTCGACAGTG (SEQ ID NO. 6).

[0061] (2) HrpN lqp Protein expression

[0062] (1) Activated stock: recombinant strain BL21 / pET-28a- hrpN lqp Inoculate into LB liquid medium containing 50 mg / ml kanamycin and culture with shaking at 37℃ (200 rpm / min) overnight. The next day, transfer to LB liquid containing 50 mg / ml kanamycin at a ratio of 2%, culture with shaking at 37℃ for 2 h, add IPTG with a final concentration of 1 mM, and culture with shaking at 25℃.

[0063] (2) Cell disruption: Collect the induced bacterial solution at 4000 rpm / min, centrifuge at 4°C for 10 min to collect the cells, wash once with binding buffer, redissolve in 20 ml binding buffer, and disrupt by ultrasonication. Centrifuge at 8000 rpm / min for 40 min, and collect the supernatant, which is the soluble HrpN. lqp The crude protein was purified by Histrap column to obtain HrpN lqp Pure protein.

[0064] (III) HrpN lqp Protein purification

[0065] The crude protein was purified by Histrap column to obtain HrpN lqp Pure protein. The purified sample was ultrafiltered and concentrated ( Figure 1 The pure protein was stored at -80°C.

[0066] Example 3

[0067] HkDJ lqp Applications of protein.

[0068] 1. HrpN lqp Protein induces hypersensitive response in tobacco leaves.

[0069] Purified HrpN lqpProtein injection inoculated tobacco leaves, while Harpin protein Hpa1 (amino acid sequence as shown in SEQID NO.7), EVP and ddH2O were used as controls to observe the allergic necrosis reaction of tobacco, and DAB and aniline blue staining were used to observe the active oxygen burst and callose deposition of tobacco after inoculation. The results showed that HrpN lqp It can stimulate allergic necrosis reaction on tobacco, causing active oxygen burst and callus deposition ( Figure 2 ).

[0070] (2) HrpN lqp Protein triggers reactive oxygen burst and callose deposition in poplar leaves.

[0071] Poplar leaves of the same size and growth state were selected and purified HrpN lqp The leaves of poplars were treated with Silwet-L77 (0.03% w / v) diluted to 20 μg / ml, and DAB staining was performed to detect the release of reactive oxygen species at 15 min, 20 min, and 25 min. lqp In 20 min, the active oxygen burst of poplar leaves was induced ( Figure 3 (Figure A in the middle).

[0072] Select leaves of the same size and growth state and transfer the purified HrpN lqp Silwet-L77 (0.03% w / v) was added to treat poplar leaves after dilution to 20 μg / ml, and aniline blue staining was performed to detect callose deposition after 15 h, 24 h, and 48 h of incubation. lqp Can induce callus deposition in poplar leaves ( Figure 3 (Figure B).

[0073] (III) HrpN lqp Protein induces the expression of defense response-related genes in poplar.

[0074] Poplar leaves of the same size and growth state were selected and purified HrpN lqp The Silwet-L77 (0.03% w / v) was diluted to 20 μg / ml and treated with poplar leaves. ddH2O was used as a control. Leaf RNA was extracted at 24 h, 48 h, and 96 h, and the expression of defense response genes in poplar was detected by Real-time PCR. The results showed that HrpN lqp It can activate the expression of plant PR genes and confer disease resistance to poplars ( Figure 3 (Figure C).

[0075] (IV) HrpN lqp Protein induces resistance of poplar trees to poplar rot disease.

[0076] Poplar leaves of the same size and growth state were selected and purified HrpN lqp Silwet-L77 (0.03% w / v) was diluted to 30 μg / ml and sprayed on poplar leaves. ddH2O and Harpin protein Hpa1 (30 μg / ml) were used as controls. 24 hours later, poplar rot pathogens were inoculated on poplar leaves, the leaves were observed for disease occurrence, and the lesion diameter was measured. The results showed that the rot lesion diameter was 2.00 cm under ddH2O treatment, and after Harpin protein Hpa1 treatment, the rot lesion diameter decreased from 2.00 cm to 1.39 cm, while HrpN lqp After treatment, the diameter of the rot lesions was reduced to 0.99 cm, the area of ​​lesion expansion was significantly reduced, and the severity of the disease was significantly alleviated ( Figure 4 (Figure A in the middle).

[0077] Poplar branches with the same length, thickness and growth state were selected and purified HrpN lqp Silwet-L77 (0.03% w / v) and sodium di-sec-octyl maleate sulfonate (2% - 5%) were added to dilute to 30 μg / ml, and sprayed on poplar branches. ddH2O and Harpin protein Hpa1 (30 μg / ml) were used as controls. 24 hours later, poplar rot pathogens were inoculated on poplar branches, the branches were observed for disease, and the lesion diameter was measured. The results showed that the rot lesion diameter was 6.62 cm under ddH2O treatment, and after Harpin protein Hpa1 treatment, the rot lesion diameter decreased from 6.62 cm to 4.11 cm, while HrpN lqp After treatment, the diameter of the rot lesions dropped to 2.05 cm, the expansion speed slowed down, the expansion area was significantly reduced, and the severity of the disease was significantly reduced ( Figure 4 (Figure B).

[0078] (V) HrpN lqp Protein induces resistance of poplar trees to poplar anthracnose.

[0079] Poplar leaves of the same size and growth state were selected and purified HrpN lqp Silwet-L77 (0.03% w / v) was diluted to 30 μg / ml and sprayed on poplar leaves. ddH2O and Harpin protein Hpa1 (30 μg / ml) were used as controls. 24 hours later, Colletotrichum gloeosporioides was inoculated on poplar leaves, the leaf disease was observed, and the lesion diameter was measured. The results showed that the diameter of the anthracnose lesion was 1.83 cm under ddH2O treatment, and the diameter of the rot lesion was reduced from 1.83 cm to 1.22 cm after Harpin protein Hpa1 treatment, while HrpNlqp After treatment, the diameter of the rot lesions was reduced to 0.95 cm, and the severity of anthracnose was significantly reduced ( Figure 5 ).

[0080] (VI) HrpN lqp Protein induces resistance of poplar trees to poplar bacterial canker.

[0081] Poplar branches with the same length, thickness and growth state were selected and purified HrpN lqp Silwet-L77 (0.03% w / v) and sodium di-sec-octyl maleate sulfonate (2% - 5%) were added to dilute to 30 μg / ml, and sprayed on poplar branches. ddH2O and Harpin protein Hpa1 (30 μg / ml) were used as controls. 24 hours later, poplar bacterial canker pathogens were inoculated on poplar branches, the disease of the branches was observed, the virulence level was statistically calculated, and the disease index was calculated. The results showed that the pathogenicity level of the pathogen under ddH2O treatment was 3.86, and the disease index was 77.14%. After Harpin protein Hpa1 treatment, the pathogenicity level of the pathogen dropped from 3.86 to 3.40, and the disease index dropped to 68%, while HrpN lqp After treatment, the pathogenicity level of the pathogen dropped to 1.70, and the disease index dropped to 56.67%, reaching a very significant level compared with the control, and the severity of the disease was significantly reduced ( Figure 6 ).

[0082] The above examples show that the HrpN cloned from the bacterial canker bacteria of Populus euphorbia is lqp Can stimulate allergic response on tobacco. lqp Treatment of poplars can induce immune responses in poplars and effectively improve their disease resistance, especially in the resistance to poplar trunk diseases. The above conclusions prove that HrpN lqp It has application value in developing into bio-source pesticide.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. HrpN lqp Application of protein in improving forest disease resistance, the HrpN lqp The amino acid sequence of the protein is as follows (a1) or (a2); (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO.1; (a2) A protein derived from (a1) with the same function as (a1) after one or more amino acid residues are substituted and / or deleted and / or added.

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

3. The use according to claim 1 or 2, characterized in that: HrpN lqp The 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 HrpN into the basic expression vector. lqp Protein coding genes obtained.

4. The use according to claim 3, characterized in that: The basic expression vector is pET-28a.

5. The use according to any one of claims 1 to 4, characterized in that: The trees described are poplars.

6. The use according to any one of claims 1 to 5, characterized in that: HrpN lqp Protein is used to improve resistance to poplar rot.

7. The use according to any one of claims 1 to 5, characterized in that: HrpN lqp Protein is used to improve resistance to anthracnose in poplars.

8. The use according to any one of claims 1 to 5, characterized in that: HrpN lqp Protein is used to improve resistance to bacterial canker in poplars.

9. A drug for improving the disease resistance of poplar, characterized in that: Its active ingredient is HrpN lqp Protein, the HrpN lqp The amino acid sequence of the protein is as follows (a1) or (a2); (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO.1; (a2) A protein derived from (a1) with the same function as (a1) after one or more amino acid residues are substituted and / or deleted and / or added.

10. The drug according to claim 9, characterized in that: The invention can promote plant growth and induce the expression of defense response PR genes in forest trees, wherein the defense response PR genes in forest trees include PR1, PR3 and PR4.

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