Plant immunity inducing protein CcHE1 and its application

Abstract

The present invention discloses a plant immune inducing protein CcHE1 and its application, belonging to the field of biotechnology. The plant immune inducing protein CcHE1 secreted by plant pathogenic fungi disclosed in the present invention has an amino acid sequence as shown in SEQ ID NO.2, and a nucleotide sequence of the CcHE1 encoding gene is shown in SEQ ID NO.1. The present invention provides a new plant immune inducing protein CcHE1, which can induce plant resistance and immune defense response, and can significantly improve the disease resistance of plants. It has a low concentration and a significant disease resistance effect. CcHE1 activates the plant's own immune system, provides a new way to improve plant resistance, and has broad application prospects in green prevention and control of forest diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a plant immunity inducing protein CcHE1 and an application thereof. Background Art

[0002] Poplar is an important tree species for artificial forests in my country. It has the characteristics of strong adaptability, easy growth, and resistance to barrenness. It can show good rapid growth and stress resistance in mountainous and hilly areas, and has become the main tree species in my country's "Three Norths" shelterbelt system. With the large-scale promotion and planting of poplars and poor site environmental conditions, poplar diseases are becoming more and more serious. Poplar rot caused by golden shell cysts is one of the most serious diseases. The branches of affected poplars wither and rot, and even cause tree death and forest destruction. Poplar rot is a host-dominated disease, and the strength of host resistance affects the severity of the disease. At present, the prevention and control of this disease mainly adopts passive chemical control and physical control, and the input-output effect is poor. With the country's major strategic needs for green prevention and control of plant diseases, it is urgently needed to develop new green products that can be used for forest disease prevention and control.

[0003] When pests harm host plants, they secrete a large number of effector molecules, some of which can induce immune responses in plants, called elicitors. After being recognized by receptor molecules on the surface of the host plant cell membrane or in the cell, the elicitors stimulate the plant's defense system through a series of signal transduction pathways, induce the plant's defense response, and ultimately produce systemic resistance in the plant to reduce the harm of pests. Developing potential elicitors into plant immune inducers for use in green prevention and control of plant diseases will help improve my country's long-standing problems of ecosystem destruction, environmental pollution, and agricultural and forestry food safety, and meet the major strategic needs of the goal of zero growth in the use of chemical pesticides. However, there are no reports in the prior art on the plant immune induction protein CcHE1 secreted by the poplar rot pathogen (Cytospora chrysosperma (Pers.) Fr.). Summary of the invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the first object of the present invention is to provide a plant immunity inducing protein CcHE1 secreted by plant pathogenic fungi, the amino acid sequence of which is shown in SEQ ID NO.2.

[0005] The second object of the present invention is to provide a gene encoding the plant immunity inducing protein CcHE1, whose nucleotide sequence is shown in SEQ ID NO.1.

[0006] The third object of the present invention is to provide a recombinant vector, expression cassette or recombinant bacteria comprising the above encoding gene.

[0007] The recombinant vector can be a prokaryotic expression vector obtained by inserting a CcHE1 coding gene without a signal peptide, and specifically can be a prokaryotic expression vector obtained by inserting a CcHE1 coding gene without a signal peptide into pET-28a. In the specific implementation process, the recombinant vector can be used for Escherichia coli BL21 (DE3) expression to obtain a fusion expression protein (His-CcHE1) with a molecular weight of about 13.5 kDa, which can induce resistance reactions in plants such as poplar, tobacco and chestnut, improve plant immunity, and reduce the degree of damage caused by gray mold, poplar rot, gloeosporium anthracnose, Botrytis cinerea and chestnut blight.

[0008] The fourth object of the present invention is to provide the use of the above-mentioned plant immunity inducing protein CcHE1, encoding gene, recombinant vector, expression cassette or recombinant bacteria in inducing plant defense response or improving plant disease resistance.

[0009] Further, the plant is poplar, tobacco or chestnut.

[0010] Furthermore, the disease resistance is resistance to diseases caused by Botrytis cinerea, poplar rot, Colletotrichum gloeosporioides, Botrytis cinerea and chestnut blight.

[0011] The fifth object of the present invention is to provide a method for preventing and controlling plant diseases, wherein the plants are treated with the plant immunity inducing protein CcHE1.

[0012] Furthermore, the specific treatment method is: spraying or applying the plant immunity inducing protein CcHE1 on the plant surface or injecting it into the plant leaves.

[0013] Further, the plant is poplar, tobacco or chestnut.

[0014] Furthermore, the plant diseases are diseases caused by Botrytis cinerea, poplar rot, Colletotrichum gloeosporioides, Botrytis cinerea and chestnut blight.

[0015] The present invention has the following beneficial effects:

[0016] The present invention provides a new plant immune inducing protein CcHE1, which can induce plant resistance and immune defense response, significantly improve plant disease resistance, and can be used as a plant immune inducing factor in the prevention and control of plant diseases. In addition, the protein is used at a low concentration and will not cause plant necrosis but can induce plant immune defense response, thereby improving the plant's disease resistance to gray mold, poplar rot pathogen, gloeosporium anthracnose, botrytis cinerea, and chestnut blight pathogen. CcHE1 activates the plant's own immune system, provides a new way to improve plant resistance, and has broad application prospects in green prevention and control of forest diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a Western blot detection diagram of the plant immune-inducing protein CcHE1 expressed and purified by a prokaryotic expression system. The antibody used is anti-His. The left side is Coomassie Brilliant Blue staining, and the right side is protein immunoblotting.

[0018] Figure 2 These are the results of trypan blue staining, reactive oxygen species burst, and callose deposition after Nicotiana benthamiana was treated with 10 μM, 8 μM, and 5 μM of the plant immune-inducing protein CcHE1.

[0019] Figure 3 This is the result of inducing the expression of tobacco disease resistance-related genes after injecting 5 μM plant immunity-inducing protein CcHE1 into tobacco leaves.

[0020] Figure 4 This is a picture of the disease symptoms of Nicotiana benthamiana after being treated with 5 μM plant immunity-inducing protein CcHE1 and then inoculated with Botrytis cinerea.

[0021] Figure 5 This is the result of reactive oxygen burst and callose deposition after treating poplar leaves with 5 μM plant immune-inducing protein CcHE1.

[0022] Figure 6 The results show that the expression of disease resistance-related genes in poplar trees was induced after the poplar leaves were treated with 5 μM plant immunity-inducing protein CcHE1.

[0023] Figure 7 This is a picture of the disease symptoms of poplar leaves treated with 5 μM plant immune-inducing protein CcHE1 and then inoculated with Colletotrichum gloeosporioides.

[0024] Figure 8 This is a picture of the symptoms of poplar branches treated with 5μM plant immune-inducing protein CcHE1 and then inoculated with poplar rot pathogen.

[0025] Fig. 9 This is a picture of the disease symptoms of poplar branches inoculated with Botrytis cinerea after being treated with 5 μM plant immune-inducing protein CcHE1.

[0026] Fig.10 This is a picture of the symptoms of chestnut branches treated with 5 μM plant immune-inducing protein CcHE1 and then inoculated with chestnut blight pathogen.

[0027] Fig.11 The results showed that 5 μM plant immune-inducing protein CcHE1 had no growth inhibitory effect on Botrytis cinerea, poplar rot pathogen, Colletotrichum gloeosporioides, Botrytis cinerea and chestnut blight pathogen. DETAILED DESCRIPTION

[0028] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0029] Example 1: Cloning of the gene encoding the plant immune-inducing protein CcHE1

[0030] The specific process of cloning the gene encoding plant immune-inducing protein CcHE1 is as follows:

[0031] (1) Total RNA extraction: Liquid cultured poplar rot mycelium was used as the material. Total RNA was extracted using the RNA extraction kit of TIANGEN Company according to the instructions, and the RNA content and quality were detected using Nanodrop.

[0032] (2) Reverse transcription to generate the first strand: Take 2 μg RNA as template and synthesize cDNA according to the instructions of Hifair reverse transcriptase reagent from YEASEN Company, and adjust the volume to 40 μL. Take an appropriate amount of reverse transcription product for subsequent gene cloning PCR.

[0033] (3) Using the synthesized cDNA as a template and CcHE1-5F and CcHE1-3F as primers, the full length of the CcHE1 coding gene was amplified by conventional PCR:

[0034] The PCR primer sequences are as follows:

[0035] CcHE1-5F: 5'-ATGCAGATCACCACTACCAT-3' (SEQ ID NO.3);

[0036] CcHE1-3F: 5'-GTACCCCGGGACAGCATCAT-3' (SEQ ID NO. 4).

[0037] The 50μL reaction system was: 25μL Sangon Biotech 2×SanTaq PCR Mix enzyme, 1μL template cDNA, 2μL CcHE1-5F, 2μL CcHE1-3F, and water was added to 50μL; the PCR amplification program was 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 15s, 35 cycles, and 72℃ extension for 10min.

[0038] (4) Using agarose gel electrophoresis to detect whether the obtained band is the target band, and further cutting the gel to recover the PCR product of the CcHE1 encoding gene, that is, obtaining the plant immune inducing protein CcHE1 encoding gene.

[0039] The full-length nucleotide sequence of the CcHE1 coding gene of the poplar rot pathogen (Cytospora chrysosperma (Pers.) Fr.) is 396 bp long, and the specific sequence is as follows:

[0040] ATGCAGATCACCACTACCATCCTCGCGTTCGCCGCTGCTGCCATG

[0041] GCAGCTCCCTATGAGTGCACCTTCGGGCAGTACGTCTGCTCCAAGGAC

[0042] GGCCTGTCCATCTCCCAAATGTGACATCAGCGGCCACTGGGTTGAGATC

[0043] GGGCCCTGCCCTGATGGCAGCAAGTGCTCCAACATCGGAGACATTCCT

[0044] TACTGCCAGCCCGTCACCCAGAAGCGAGGCTCTCCTCCTTACTGCTCC

[0045] ACCCCCGGCACTTACACCTGCACTGGTGATTACGAGGGGATCAATGTC

[0046] TGCAATTCCCAGAACCAGCTGGTGTTCAACGGTGCCTGCCCTGAGAAG

[0047] ACCCACTGTGGGTATCTCAATGGCATTCCATTCTGTGTTGATGATGCT

[0048] GTCCCGGGGTACTGA(SEQ ID NO.1);

[0049] The full-length protein sequence of CcHE1 of Cytospora chrysosperma (Pers.) Fr. is 131 aa long, and the specific sequence is as follows:

[0050] MQITTTILAFAAAAMAAPYECTFGQYVCSKDGLSISQCDISGHWVEI GPCPDGSKCSNIGDIPYCQAVTQKRGSPPYCSTPGTYTCTGDYEGINVCNS QNQLVFNGACPEKTHCGYLNGIPFCVDDAVPGY (SEQ ID NO. 2).

[0051] Example 2: Prokaryotic expression and purification of plant immunity inducing protein CcHE1

[0052] The prokaryotic expression and purification process of plant immune-inducing protein CcHE1 is as follows:

[0053] (1) Construction of prokaryotic expression vector

[0054] Design specific primers for the gene encoding plant immune-inducing protein CcHE1, the primer sequences are:

[0055] CcHE1-P5F: 5'-CATGCCATGGATGGCTCCCTATGAGTGCACCTT-3' (SEQ ID NO. 5);

[0056] CcHE1-P3F: 5'-CCGCTCGAGGTACCCCGGGACAGCATCAT-3' (SEQ ID NO. 6).

[0057] PCR amplification was performed using the above primer sequences. The PCR reaction system was: 25 μL of Sangon Biotech 2×SanTaqPCR Mix enzyme, 1 μL of template CcHE1, 2 μL of CcHE1-P5F, 2 μL of CcHE1-P3F, and water was added to 50 μL. The PCR amplification program was 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 15 s, 35 cycles, and extension at 72°C for 10 min.

[0058] Agarose gel electrophoresis was used to detect whether the obtained band was the target band, and the gel was further cut to recover the PCR product of the CcHE1 coding gene carrying NcoI and XhoI (ABclonal) restriction sites.

[0059] The DNA and pET-28a plasmid recovered from the gel were double-digested, and the reaction system of 50 μL was: template DNA / pET-28a 15 μL, 10×K Buffer 5 μL, BSA 5 μL, NcoI 1 μL, XhoI 1 μL, water was added to 50 μL, placed in a 37°C incubator for overnight digestion, and recovered using a common DNA product purification kit (TIANGEN). Subsequently, T4 DNA Ligase (ABclonal) was used for ligation, and the reaction system of 10 μL was: T4 DNA Ligase 1 μL, 10×T4 DNA LigaseReaction Buffer 2 μL, pET-28a recovered by digestion 3 μL, CcHE1 recovered by digestion 4 μL, placed in a 16°C metal bath for overnight ligation. The ligation product was transformed into Escherichia coli competent cells BL21 (DE3), coated with LB (containing Kanamycin) plates, and incubated at 37°C for 12 h before colony PCR verification. Three positive clones were picked and shaken, and the plasmid was extracted according to the plasmid extraction kit (TIANGEN). The plasmid was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the sequences were aligned on the MUSCLE multiple sequence alignment website, and the vector construction was completed.

[0060] (2) Prokaryotic expression of CcHE1

[0061] Pick a single clone of the recombinant plasmid into 50 mL of liquid LB medium containing 100 μg / mL Kanamycin, and culture it overnight at 37°C, 220 rpm for 16 h; inoculate the overnight cultured E. coli into 250 mL of liquid LB medium containing 100 μg / mL Kanamycin at a concentration of 1:50, and culture it at 37°C, 220 rpm until the OD 600 =0.04; incubate at 37°C for 2-3h to make OD 600=0.5-0.6; add IPTG (isopropyl-β-D-thiogalactopyranoside) to the cultured Escherichia coli culture to make the final concentration of 0.2mM, 20℃, 160rpm, and culture for 18h to induce protein expression; centrifuge the induced expression Escherichia coli culture at 4℃, 4000rpm for 15min, and discard the supernatant; use Beyotime's His tag protein purification kit (denaturation-resistant formulation) to lyse the bacteria, and before lysing the bacteria, add an appropriate amount of protease inhibitor to the lysate For fresh precipitates, add 4 mL of non-denaturing lysis buffer per gram of wet weight of precipitates to fully resuspend the bacteria; add lysozyme to a final concentration of 1 mg / mL and mix well, and place on ice for 30 minutes; ultrasonically lyse the bacteria on ice, with an ultrasonic power of 200-300 W, each ultrasonic treatment for 10 seconds, each interval of 10 seconds, and a total ultrasonic treatment of 20-30 minutes; centrifuge at 4°C, 12000 rpm for 30 minutes, collect the supernatant of the bacterial lysate and place on ice; take 1 mL of the well-mixed 50% BeyoGold TM Add His-tag Purification Resin (denaturation-resistant formulation) to a 2mL EP tube, centrifuge at 4℃, 3000rpm for 30s, and discard the supernatant; add 0.5mL non-denaturing lysis buffer to the gel and mix well to balance the gel, centrifuge at 4℃, 3000rpm for 30s, and discard the liquid. Repeat the balance 3 times, discard the liquid, and transfer to a 50mL centrifuge tube; add about 4mL of bacterial lysate supernatant, and shake slowly on a side-swinging shaker or horizontal shaker at 4℃ for 2h; mix the lysate and BeyoGold TM The mixture of His-tag Purification Resin (denaturation-resistant formulation) was loaded into the empty column tube of the affinity chromatography column provided by the kit, the lid at the bottom of the purification column was opened, and the liquid in the column was allowed to flow out under the action of gravity. The column was washed with 1 mL of 10 mM and 20 mM imidazole buffers in sequence, and then 500 μL of 500 mM imidazole buffer was added to elute the target protein, and the column was transferred to a 2 mL EP tube, and 500 μL of 500 mM imidazole buffer was added to elute the target protein, and then the column was transferred to the same EP tube again; gel electrophoresis was performed using the precast gel of Changzhou Boyi Biotechnology Co., Ltd. to test the protein expression in the above eluate, and subsequent protein purification was performed.

[0062] (3) Purification of CcHE1 protein

[0063] The above eluate was placed in a 15 mL ultrafiltration centrifuge tube and washed three times with 10 mL 1× PBS at 4°C, 4500 rpm, 30 min. When the total protein solution was concentrated to 1 mL, it was the purified His-tagged CcHE1 protein sample. The protein concentration was then detected using a BCA kit (Solarbio), and electrophoresis, Coomassie brilliant blue staining, and Western blot were performed to detect the purified target protein. The results are shown in Figure 1 .

[0064] Depend on Figure 1 It can be seen that after electrophoresis detection, a CcHE1 recombinant protein with a molecular weight of about 13.5 kDa containing a His tag at the C-terminus was obtained.

[0065] Example 3: Defense response induced by plant immune induction protein CcHE1 in tobacco

[0066] The plant immunity inducing protein CcHE1 obtained in Example 2 was applied to tobacco to induce a defense response. The specific process is as follows:

[0067] (1) 5 μM CcHE1 did not cause tobacco necrosis but induced reactive oxygen species burst and callose deposition in tobacco

[0068] After the concentration of CcHE1 protein was measured, its concentration was adjusted to 10μM, 8μM and 5μM. The middle leaves of tobacco grown to the 5-leaf stage were selected, and a needle was used to poke a needle hole at different positions of the tobacco leaves. Then, a 1mL syringe without a needle was used to inject different concentrations of CcHE1 protein into the leaves from the back of the leaves. At the same time, PBS was used as a control. After 48 hours of injection, the samples were collected. Tobacco dead cells were stained with trypan blue (Solarbio). The tobacco was immersed in a fixing liquid (acetic acid: ethanol = 3:1) for 48 hours, and then the fixing liquid was discarded. The leaves were washed twice with distilled water, and the leaves were immersed in a staining solution (0.67g trypan blue, 100mL phenol, 100mL lactic acid, 100mL glycerol, 100mL distilled water, 600mL ethanol) at room temperature for 12 hours. The leaves were taken out of the staining solution and immersed in a decolorizing solution (2g / mL chloral hydrate). The necrosis of tobacco was observed 48 hours after injection; for active oxygen detection, the tobacco leaves injected 48 hours after injection were immersed in 3,3'-diaminobenzidine (DAB) solution (Solarbio) for 12 hours, and then decolorized with 95% ethanol for observation; for callose observation, the tobacco leaves injected 48 hours after injection were immersed in decolorizing solution (acetic acid: ethanol = 1:6) for 12 hours, and after complete decolorization, they were stained with aniline blue solution for 12 hours, and the deposition of callose was observed using a fluorescent universal microscope (BX61). The above experiments were repeated three times, and the results are shown in Figure 2 .

[0069] Depend on Figure 2It can be seen that 5μM CcHE1 does not cause tobacco necrosis but can induce tobacco reactive oxygen burst and callose deposition.

[0070] (2) 5 μM CcHE1 induced a significant increase in the expression level of tobacco resistance-related genes

[0071] PBS was used as a control, and samples were collected 12 hours after tobacco injection to detect the expression level of resistance-related genes. RNA was extracted using the RNA extraction kit of TIANGEN Company according to the instructions, and the RNA content and quality were detected using Nanodrop.

[0072] Reverse transcription to generate the first strand: Take 2 μg RNA as template, and synthesize cDNA according to the instructions of Hifair reverse transcriptase reagent of YEASEN company, and adjust the volume to 40 μL. The reverse transcription product is diluted 4 times for real-time quantitative PCR reaction to detect the expression level of induced resistance-related genes.

[0073] The primers used for real-time fluorescence quantitative PCR reaction are as follows:

[0074] NbActin upstream primer NbActin-5F:

[0075] 5'-TGGTCGTACCACCGGTATTGTGTT-3' (SEQ ID NO. 7);

[0076] NbActin downstream primer NbActin-3F:

[0077] 5'-TCACTTGCCCATCAGGAAGCTCAT-3' (SEQ ID NO. 8);

[0078] NbHSR203J upstream primer NbHSR203J-5F:

[0079] 5'-ACGCAGATTTCAACCGAGTAT-3' (SEQ ID NO.9);

[0080] NbHSR203J downstream primer NbHSR203J-3F:

[0081] 5'-GCCAGTCGCATTGGAGATAA-3' (SEQ ID NO. 10);

[0082] NbHIN1 upstream primer NbHIN1-5F:

[0083] 5'-CCAACTTGAACGGAGCCTATTA-3' (SEQ ID NO. 11);

[0084] NbHIN1 downstream primer NbHIN1-3F:

[0085] 5'-AGGCATCCAAAGAGACAACTAC-3' (SEQ ID NO. 12);

[0086] NbWRKY7 upstream primer NbWRKY7-5F:

[0087] 5'-CACAAGGGTACAAACAACACAG-3' (SEQ ID NO. 13);

[0088] NbWRKY7 downstream primer NbWRKY7-3F:

[0089] 5'-GGTTGCATTTGGTTCATGTAAG-3' (SEQ ID NO. 14);

[0090] NbWRKY8 upstream primer NbWRKY8-5F:

[0091] 5'-AACAATGGTGCCAATAATGC-3' (SEQ ID NO. 15);

[0092] NbWRKY8 downstream primer NbWRKY8-3F:

[0093] 5'-TGCATATCCTGAGAAACATT-3' (SEQ ID NO. 16);

[0094] NbAcre31 upstream primer NbAcre31-5F:

[0095] 5'-AATTCGGCCATCGTGATCTTGGTC-3' (SEQ ID NO.17); NbAcre31 downstream primer NbAcre31-3F:

[0096] 5'-GAGAAACTGGGATTGCCTGAAGGA-3' (SEQ ID NO. 18);

[0097] NbPR1 upstream primer NbPR1-5F:

[0098] 5'-CCGCCTTCCCTCAACTCAAC-3' (SEQ ID NO. 19);

[0099] NbPR1 downstream primer NbPR1-3F:

[0100] 5'-GCACAACCAAGACGTACTGAG-3' (SEQ ID NO. 20).

[0101] The PCR reaction system contained 0.5 μL of cDNA, 10 μL of qPCR SYBR Green Master Mix (Yeasen), 0.4 μL of upstream and downstream primers, and 8.7 μL of water. Reaction procedure: I: 95°C for 2 min, II: 95°C for 10 s, 60°C for 30 s, and 40 cycles of step II. The melting curve analysis procedure was: 95°C for 15 s, 60°C for min, and 95°C for 15 s. Data analysis was performed using 2 -ΔΔCT Method. Each sample was repeated three times, and the results are shown in Figure 3 .

[0102] Depend on Figure 3 It can be seen that after 12 hours of treating tobacco leaves with 5 μM CcHE1 protein, the expression levels of tobacco disease resistance-related genes were significantly increased.

[0103] Example 4: Plant immune induction protein CcHE1 enhances tobacco disease resistance

[0104] Using PBS as a control, 5 μM CcHE1 protein was injected into tobacco from the back of the leaves. 5 mm gray mold cake was inoculated on the leaves treated with 5 μM CcHE1 protein for 24 h, and the infection area was counted using image J 48 h after inoculation. Each sample was repeated three times, and the results are shown in Figure 4 .

[0105] Depend on Figure 4 It can be seen that compared with the negative control PBS treatment, the lesions infected by gray mold on tobacco leaves treated with 5 μM CcHE1 were significantly reduced.

[0106] Example 5: Plant immune-inducing protein CcHE1 induces immune response in poplar

[0107] (1) 5 μM CcHE1 protein induces reactive oxygen species burst and callose deposition in poplar

[0108] Reactive oxygen detection: PBS was used as a negative control, and tobacco treated with 5 μM CcHE1 was used as a positive control. Specifically, leaf discs were collected from poplar leaves and immersed in 5 μM CcHE1. The discs were placed in a vacuum pump and evacuated for 5-10 seconds, then allowed to stand for 15 minutes. The discs were then immersed in 3,3'-diaminobenzidine (DAB) solution (Solarbio) for 12 hours, and then decolorized with 95% ethanol for observation.

[0109] Callose deposition detection: Poplar leaves were treated with 5 μM CcHE1 according to the above method and then left to stand for 15 h. After complete decolorization, the poplar leaves were soaked in aniline blue for 12 h and callose deposition was observed using a fluorescence universal microscope (BX61). Each sample was repeated three times, and the results are shown in Figure 5.

[0110] Depend on Figure 5 It can be seen that 5μM CcHE1 can induce reactive oxygen burst and callose deposition in poplar leaves.

[0111] (2) 5 μM CcHE1 induced a significant increase in the expression level of resistance-related genes in poplar

[0112] Using PBS as a control, 5 μM CcHE1 was used to soak poplar leaves for 12 hours, and then samples were collected to detect the expression level of resistance-related genes in poplars. RNA was extracted using the RNA extraction kit of TIANGEN Company according to the instructions, and the RNA content and quality were detected using Nanodrop.

[0113] Reverse transcription to generate the first strand: 2 μg RNA was used as a template, and cDNA was synthesized according to the instructions for use of the Hifair reverse transcriptase reagent from YEASEN, and the volume was fixed to 40 μL. The reverse transcription product was diluted 4 times for real-time quantitative PCR reaction to detect the expression level of genes related to induced resistance in poplars.

[0114] The primers used for real-time fluorescence quantitative PCR reaction are as follows:

[0115] PtActin upstream primer PtActin-5F:

[0116] 5'-TCATCGGAATGGAAGCTGCTGGTA-3' (SEQ ID NO. 21);

[0117] PtActin downstream primer PtActin-3F:

[0118] 5'-TAGTGGAACCACCACTGAGCACAA-3' (SEQ ID NO. 22);

[0119] PtPR1 upstream primer PtPR1-5F:

[0120] 5'-TGGGTTGATGAGAAACCAAAGTATG-3' (SEQ ID NO. 23);

[0121] PtPR1 downstream primer PtPR1-3F:

[0122] 5'-GCTGCACCTTGCTTTAGCAC-3' (SEQ ID NO. 24);

[0123] PtPR4 upstream primer PtPR4-5F:

[0124] 5'-GACGGAGATGGATACGCAAAAGG-3' (SEQ ID NO. 25);

[0125] PtPR4 downstream primer PtPR4-3F:

[0126] 5'-TCGACGATAGAGAGCAGAGGTTTAG-3' (SEQ ID NO. 26).

[0127] The PCR reaction system contained 0.5 μL of cDNA, 10 μL of qPCR SYBR Green Master Mix (Yeasen), 0.4 μL of upstream and downstream primers, and 8.7 μL of water. Reaction procedure: I: 95°C for 2 min, II: 95°C for 10 s, 60°C for 30 s, and 40 cycles of step II. The melting curve analysis procedure was: 95°C for 15 s, 60°C for min, and 95°C for 15 s. Data analysis was performed using 2 -ΔΔCT Method. Each sample was repeated three times, and the results are shown in Figure 6 .

[0128] Depend on Figure 6 It can be seen that after 12 hours of treating poplar leaves with 5μM CcHE1 protein, the expression levels of poplar disease resistance-related genes were significantly increased.

[0129] Example 6: Plant immune induction protein CcHE1 enhances the resistance of poplar leaves to Colletotrichum gloeosporioides

[0130] Using PBS as a control, 5 μM CcHE1 protein was sprayed or applied on poplar leaves. 5 mm gloeosporioides cake was inoculated on leaves 24 h after protein treatment, and image J was used to count the infected area 5 days after inoculation. Each sample was repeated three times, and the results are shown in Figure 7 .

[0131] Depend on Figure 7 It can be seen that compared with the negative control PBS treatment, the lesions infected by Colletotrichum gloeosporioides on the poplar leaves treated with 5 μM CcHE1 were significantly reduced.

[0132] Example 7: Plant immune induction protein CcHE1 enhances the resistance of poplar branches to poplar rot pathogens

[0133] Poplar branches were disinfected with 75% alcohol and then scalded. CK was used as a negative control and wild-type WT was used as a positive control. 50 μL of 5 μM CcHE1 protein was dripped on the wound. After 24 hours of absorption, 5 mm poplar rot bacteria cake was inoculated on the wound and fixed with sealing film. The branches were placed in a ventilated and humid environment for 7 days and the results were observed. Image J was used to count the lesion area. 15 branches were selected for each treatment for the experiment. The results are shown in the figure. Figure 8 .

[0134] Depend on Figure 8 It can be seen that compared with the positive control WT treatment, the lesions infected with poplar rot pathogen on the poplar branches treated with 5 μM CcHE1 were significantly reduced.

[0135] Example 8: Plant immunity inducing protein CcHE1 enhances the resistance of poplar branches to Botrytis cinerea

[0136] Poplar branches were disinfected with 75% alcohol and then scalded. CK was used as a negative control and wild-type WT was used as a positive control. 50 μL of 5 μM CcHE1 protein was dripped on the wound. After 24 hours of absorption, 5 mm Botrytis ciliata cake was inoculated on the wound and fixed with sealing film. The branches were placed in a ventilated and humid environment for 10 days and the results were observed. Image J was used to count the lesion area. 15 branches were selected for each treatment for the experiment. The results are shown in the table. Fig. 9 .

[0137] Depend on Fig. 9 It can be seen that compared with the positive control WT treatment, the lesions infected by Botrytis cinerea on the poplar branches treated with 5 μM CcHE1 were significantly reduced.

[0138] Example 9: Plant immunity inducing protein CcHE1 enhances the resistance of chestnut branches to chestnut blight pathogen

[0139] The chestnut branches were disinfected with 75% alcohol and then scalded. CK was used as a negative control and wild-type WT was used as a positive control. 50 μL of 5 μM CcHE1 protein was dripped on the wound. After 24 hours of absorption, 5 mm chestnut blight bacteria cake was inoculated on the wound and fixed with sealing film. The branches were placed in a ventilated and humid environment for 12 days and the results were observed. Image J was used to count the lesion area. Seven branches were selected for each treatment for the experiment. The results are shown in the figure. Fig.10 .

[0140] Depend on Fig.10 It can be seen that compared with the positive control WT treatment, the lesions infected with chestnut blight fungus on chestnut branches treated with 5 μM CcHE1 were significantly reduced.

[0141] Example 10: Plant immunity inducing protein CcHE1 has no inhibitory effect on the growth of Botrytis cinerea, poplar rot pathogen, Colletotrichum gloeosporioides, Botrytis cinerea and chestnut blight pathogen

[0142] Inoculate activated Botrytis cinerea, poplar rot, Colletotrichum gloeosporioides, Botrytis cinerea, and Crysalis solani on a PDA plate. When the plate expands to 1 / 3, punch two holes on both sides of the plate at the midpoint of the plate with a 5 mm hole puncher. Use PBS as a control, add 50 μL of 5 μM CcHE1 protein to the holes, and place the plate in a 25°C incubator for culture. When the plate grows to the periphery of the hole, take a photo and record. Each sample was repeated three times. The results are shown in the table below. Fig.11 .

[0143] Depend on Fig.11 It can be seen that compared with the control PBS treatment, 5 μM CcHE1 had no inhibitory effect on Botrytis cinerea, Populus rot, Colletotrichum gloeosporioides, Botrytis cinerea and Crypterocarpium blight.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of plant immunity inducing protein CcHE1 or a gene encoding plant immunity inducing protein CcHE1 or a recombinant vector, expression cassette or recombinant bacteria containing a gene encoding plant immunity inducing protein CcHE1 in inducing plant defense response or improving plant disease resistance; in, The amino acid sequence of the plant immunity inducing protein CcHE1 is shown in SEQ ID NO.2; The nucleotide sequence of the gene encoding the plant immunity inducing protein CcHE1 is shown in SEQ ID NO.1; The plants are poplar, tobacco or chestnut; The plant is resistant to diseases caused by gray mold, poplar rot, Colletotrichum gloeosporioides, Botrytis cinerea and chestnut blight.

2. A method for preventing and controlling plant diseases, characterized in that: Treating plants with the plant immunity inducing protein CcHE1 according to claim 1; Among them, the plant is poplar, tobacco or chestnut; Plant diseases are caused by gray mold, poplar rot, Colletotrichum gloeosporioides, Botrytis cinerea and chestnut blight.

3. The method according to claim 2, characterized in that The specific treatment method is: spray or apply the plant immune inducing protein CcHE1 on the plant surface or inject it into the plant leaves.

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