Activator protein foemg1 and its use in plant immunity disease resistance

Abstract

The present application relates to the field of biotechnology, in particular to the application of the elicitor FoEmg1 in plant immunity and disease resistance. The FoEmg1 gene is cloned from Fusarium oxysporum, and a prokaryotic expression vector is constructed to obtain the elicitor FoEmg1 by expression in E. coli. Experiments prove that the elicitor FoEmg1 can significantly improve the root activity of Rehmannia glutinosa, induce plant defense response, enhance the resistance of the plant to brown root rot and root rot, and increase the content of secondary metabolites in the plant. The elicitor FoEmg1 has good thermal stability and pH stability, and has important application prospects in plant immunity and disease resistance and novel biological pesticides.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the activator protein FoEmg1 and its application in plant immune disease resistance. Background Technology

[0002] Rehmannia glutinosa is a perennial herb, with its dried or processed tuberous roots used medicinally. It is a major traditional Chinese medicine with extremely high medicinal value. During its cultivation, Rehmannia glutinosa is frequently susceptible to pests and diseases, including important ones such as ring spot, root rot, leaf spot, and yellow spot, which severely affect its quality and yield, causing significant losses in Rehmannia glutinosa production. Fusarium is one of the main pathogens causing root rot in Rehmannia glutinosa. This pathogen can infect Rehmannia glutinosa throughout its entire growth period, leading to root rot or plant wilting, causing serious damage to its cultivation and production, often resulting in significant yield reduction. Currently, the application of chemical fungicides is the main measure for controlling Rehmannia glutinosa diseases. However, long-term irrational and widespread use of chemical pesticides may lead to drug resistance in pathogens and environmental pollution. Therefore, there is an urgent need to research and develop new plant disease control technologies.

[0003] Activating proteins are a novel class of proteins derived from various fungi (including Fusarium, Alternaria, Trichoderma, and blast fungus). After isolation and purification, they have been confirmed to induce broad-spectrum disease resistance in plants and enhance plant immunity. Studies have shown that activating proteins can induce a series of defensive responses in plants by mimicking pathogen infection or stimulating plant immune responses, such as reactive oxygen species (ROS) bursts, lignin synthesis, accumulation of secondary metabolites, and expression of defense-related genes, thereby enhancing the plant's resistance to pathogens. Compared with traditional fungicides, activating proteins have the following significant advantages: they do not directly kill pathogenic microorganisms, activate the plant's own immune system, induce broad-spectrum disease resistance, have control effects on multiple diseases, do not induce drug resistance in pathogens, and are environmentally friendly, causing no environmental pollution. Various activating proteins have been isolated from multiple microorganisms. For example, PeaT1, isolated from Alternaria microphylla, can improve the drought resistance of rice, and VDAL, obtained from Verticillium dahliae, can effectively improve the resistance of potato plants to Verticillium wilt and late blight. However, there is currently little research on activating proteins that can induce disease resistance in Rehmannia glutinosa. Therefore, there is an urgent need to develop activating proteins that can induce disease resistance in Rehmannia glutinosa. Summary of the Invention

[0004] In view of the problems and shortcomings of the existing technology, the purpose of this invention is to provide the activator protein FoEmg1 and its application in plant immune disease resistance.

[0005] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0006] This invention provides an activating protein FoEmg1, which is the protein shown in A1), A2), A3), or A4) below:

[0007] A1) A protein with the amino acid sequence shown in SEQ ID NO.1;

[0008] A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.1;

[0009] A3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1;

[0010] A4) Proteins that have 85% or more similarity to the amino acid sequence shown in SEQ ID NO.1 and have the same function.

[0011] The amino acid sequence shown in SEQ ID NO.1 is as follows:

[0012] MSSPERRTAGVKRPRTQSLPPPSLPQLVAEQSTPIPPTDKDSQRLIVVLSNASLETYKASHGGTSRNREDKYSLLNSDEHIGVMRKMNRDISDARPDITHQCLLTLLDSPINKAGKLQIYIHTAKGVLIE VSPSVRIPRTFKRFAGLMVQLLHRLSIRSTNSNEKLLRVIQNPITDHLPPNCRKVTLSFDAPLVKVREYVETVDSKDSICVFVGAMAKGEDNFADALVDEKISISNYSLSASVACSKFCHAAEDVWDIM.

[0013] The second aspect provides biological materials related to the aforementioned activating protein FoEmg1, which are any of the following:

[0014] B1) A nucleic acid molecule encoding the activating protein FoEmg1 as described in claim 1;

[0015] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0016] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0017] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

[0018] The nucleotide sequence of the nucleic acid molecule described in B1) is shown in SEQ ID NO.2.

[0019] The nucleotide sequence shown in SEQ ID NO.2 is as follows:

[0020] .

[0021] The third aspect provides for the use of the activating protein FoEmg1 or the aforementioned biological material in any of the following:

[0022] C1) Improve plant root vitality;

[0023] C2) Prepare products that enhance plant root vitality;

[0024] C3) Improve plant disease resistance;

[0025] C4) Prepare products that enhance plant disease resistance;

[0026] C5) Cultivate plants with improved disease resistance;

[0027] C6) Prepare products from plants with enhanced disease resistance;

[0028] C7) induces plant defense responses;

[0029] C8) Enhances the induction of plant immune responses;

[0030] C9) increases the content of secondary metabolites in plants.

[0031] The disease resistance refers to resistance to ring spot disease and / or root rot.

[0032] The plant in question is Rehmannia glutinosa, and its secondary metabolites are catalpol, verbascoside, rehmannia glycoside D, rehmannia glycoside A, and / or leonurin.

[0033] The present invention also provides a plant treatment agent, the active ingredient of which is activating protein FoEmg1.

[0034] Preferably, the concentration of the activating protein in the plant treatment agent is 1-300 μg / mL. More preferably, the concentration of the activating protein in the plant treatment agent is 1-100 μg / mL.

[0035] The present invention provides a method for improving plant disease resistance, the method comprising applying the plant treatment agent to the plant, preferably by spraying.

[0036] This invention provides a method for improving plant root vitality, the method comprising treating plant roots with the plant treatment agent, preferably by root irrigation.

[0037] Compared with the prior art, the positive and beneficial effects of the present invention are as follows:

[0038] 1. This invention is the first to clone and express a novel protein, FoEmg1, from Fusarium oxysporum, which has an immune-activating function, providing a new protein resource for the biological control of plant diseases.

[0039] 2. This invention is the first to discover and confirm that the activating protein FoEmg1 can effectively induce a defensive response in Rehmannia glutinosa. Experiments showed that after treatment with FoEmg1, the resistance of Rehmannia glutinosa to ring rot and root rot was significantly enhanced, the lesion area on leaves and tubers was significantly reduced, and the disease index was greatly decreased, with the highest induction resistance effects reaching 73.68% and 61.54%, respectively. Simultaneously, the relative biomass of pathogens (WY3 and B12) within the plant was significantly reduced. This indicates that FoEmg1 can effectively inhibit the colonization of pathogens and the development of diseases by stimulating the plant's own immune system.

[0040] 3. The activating protein FoEmg1 of this invention can activate the PTI and ETI immune pathways in plants. Specifically, it upregulates the expression of key immune-related genes (such as RgFLS2, RgMPK, RgBIK1, and RgCERK1), genes related to the salicylic acid signaling pathway (such as RgPBS3, RgEPS1, and RgTGA), and genes related to the jasmonic acid signaling pathway (such as RgAOC and RgJAZ). This indicates that FoEmg1 systematically activates the plant's comprehensive defense response by coordinating multiple immune signaling pathways.

[0041] 4. The activating protein FoEmg1 of this invention not only has immune-inducing activity, but also significantly improves the root vigor of plants and promotes the accumulation of various medicinal active ingredients (secondary metabolites), such as catalpol, leonurin, rehmannia glycoside A and rehmannia glycoside D, which has a promoting effect on improving the yield and quality of medicinal plants. Attached Figure Description

[0042] Figure 1 The image shows the bacterial culture PCR electrophoresis of the pET-28a-FoEmg1 plasmid (Note: M is the DNA molecular weight standard, lane 1 is the negative control, lanes 2-7 are the FoEmg1 gene, and lane 8 is the positive control).

[0043] Figure 2 Polyacrylamide gel electrophoresis image of activated protein FoEmg1 (Note: M is the molecular weight standard of protein; lanes 1 and 2 are the uninduced and induced bacterial cultures, respectively; lanes 3 and 4 are the supernatant and precipitate after sonication, respectively; lanes 5-11 are the imidazole gradient elution buffers of 50, 100, 200, 300, 400, 500 ①, and 500 ②, respectively; 500 mM was eluted twice, corresponding to two lanes).

[0044] Figure 3 Results of thermostability assay for activator protein FoEmg1;

[0045] Figure 4 The results of pH stability assay for the activating protein FoEmg1;

[0046] Figure 5The results of the detection of the effect of the activating protein FoEmg1 on the root vigor of Rehmannia glutinosa;

[0047] Figure 6 The results of the relative conductivity test of Rehmannia glutinosa leaves;

[0048] Figure 7 The effect of activating protein FoEmg1 on the phenotype of Rehmannia glutinosa leaves inoculated with WY3 (Note: n=9, the treatment groups from left to right are CK, P1, P2, P3).

[0049] Figure 8 The statistical results of leaf lesion area and disease index after inoculating Rehmannia glutinosa with WY3 are shown in the figure. The left figure shows the leaf lesion area and the right figure shows the disease index.

[0050] Figure 9 For WY3 relative biomass detection;

[0051] Figure 10 The effect of activating protein FoEmg1 on the phenotype of Rehmannia glutinosa tubers inoculated with B12 (Note: n=9, the treatment groups from left to right are CK, P1, P2, P3).

[0052] Figure 11 Statistical results of lesion area and disease index of tuberous roots after inoculation with B12 for Rehmannia glutinosa; the left figure shows the lesion area of ​​tuberous roots, and the right figure shows the disease index.

[0053] Figure 12 The results are for the relative biomass of B12.

[0054] Figure 13 The results show the expression levels of immune-related genes in Rehmannia glutinosa.

[0055] Figure 14 The results show the expression levels of genes related to salicylic acid in Rehmannia glutinosa.

[0056] Figure 15 The results show the expression levels of genes related to rehmannia jasmonic acid.

[0057] Figure 16 The results show the content of secondary metabolites in Rehmannia glutinosa leaves. Detailed Implementation

[0058] The technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0059] Unless otherwise specified, the experimental methods used in the examples are all conventional methods, and the experimental materials used are all commercially available conventional materials.

[0060] The experimental materials, reagents, and culture media used are as follows:

[0061] 1. Experimental materials

[0062] Fusarium oxysporum: This invention was previously isolated from Rehmannia glutinosa by our research group and preserved in an ultra-low temperature freezer at -80℃ using the glycerol preservation method.

[0063] Fusarium solani WY3: This fungus was previously isolated from a plantlet infected with ring spot disease of Rehmannia glutinosa in our laboratory.

[0064] Fusarium vanettenii B12: This bacterium was previously isolated from Rehmannia glutinosa root rot diseased plants in our laboratory.

[0065] 2. Reagents

[0066] Trizol, 2xEs Taq MasterMix (Dye), and deoxyribonuclease I (DNase I) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; Escherichia coli DH-5α and BL21 (DE3) were purchased from Beijing TransGen Biotech Co., Ltd.; pUCm-T ligation vector was purchased from Shanghai Sangon Biotech Co., Ltd.; kanamycin (Kan) was purchased from Maclean's Reagent Co., Ltd.; and standards for ziziphus jujuba var. spinosa, rehmannia glycoside D, rehmannia glycoside A, and leonuridine were all purchased from Yuanye Biotechnology Co., Ltd.

[0067] PDA medium (1 L): 200 g potato, 20 g sucrose, 20 g agar.

[0068] LB medium (100 mL): 1 g sodium chloride, 0.5 g yeast extract, 1 g tryptone.

[0069] Dialysis solution A: 20 mM Tris-HCl, 0.2 M NaCl. Dialysis solution B: 20 ​​mM Tris-HCl, 0.1 M NaCl.

[0070] Example 1: Cloning, expression, and purification of the gene encoding the plant immune-activating protein FoEmg1

[0071] The applicant's previous research found that *Fusarium oxysporum*, as an endophytic fungus, significantly increases the content of secondary metabolites during its symbiotic relationship with *Rehmannia glutinosa*. Combined with *Rehmannia glutinosa* transcriptome data, it was found that the expression level of the FoEmg1 gene (as shown in SEQ ID NO.2) significantly increased before and after *Fusarium oxysporum* infection, with the expression level in the later stage of infection being 1.68 times that in the earlier stage, suggesting that the FoEmg1 gene responds to *Fusarium oxysporum* infection. To further investigate the effect of FoEmg1 on *Rehmannia glutinosa*, this invention first clones the FoEmg1 gene from *Fusarium oxysporum* using molecular cloning technology, analyzes its physicochemical properties through bioinformatics analysis, constructs a prokaryotic expression vector, and obtains the activating protein through expression in *Escherichia coli*, and then detects its activity and function.

[0072] 1. Extraction and reverse transcription of Fusarium oxysporum RNA

[0073] Total RNA was extracted from *Fusarium oxysporum* using the Trizol Total RNA Extraction Kit from Kangwei Century. The extracted RNA was analyzed using a multi-functional microplate reader to determine its concentration. RNA integrity was assessed using 1% agarose gel electrophoresis, followed by BeyoRT analysis. TM The III cDNA First Strand Synthesis Kit reverse transcribes intact RNA to obtain cDNA.

[0074] 2. PCR amplification of the target gene fragment

[0075] Primers FoEmg1-F (5'-ATGTCGGTTGTCACAAAGAACCCT-3') and FoEmg1-R (5'-CTAGTTGCCGCCGAGGCT-3') were designed based on the coding region sequence of FoEmg1. PCR amplification was performed using the cDNA obtained in step 1 as a template with primers FoEmg1-F and FoEmg1-R.

[0076] The PCR amplification reaction system was as follows: 2 μL cDNA template, 1 μL each of upstream and downstream primers, 10 μL 2xEs TaqMasterMix (Dye), and ddH2O to a final volume of 20 μL.

[0077] PCR reaction program: 95℃ incubation for 3 min; 95℃ for 30 s, 55.7℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ for 10 min.

[0078] The PCR amplification product was detected by agarose gel electrophoresis, and a specific band of 780 bp was successfully cloned. The band was recovered, and the FoEmg1 gene fragment was obtained.

[0079] 3. Ligation and transformation of PCR products with pUCm-T vector

[0080] The purified FoEmg1 gene fragment was ligated to the pUCm-T vector using the pUCm-T kit (Shanghai Sangon Biotech), and incubated at 16℃ for 12 h. 5 μL of the ligation product was transformed into 50 μL of DH-5α competent cells and cultured overnight at 37℃. Positive clones were selected using blue-white screening for colony PCR verification. The colony PCR results showed that the negative control showed no amplification band, and the bacterial PCR bands were single and of the correct size (e.g., ...). Figure 1 (As shown). The verified positive clones were sent to Shanghai Sangon Biotech for sequencing analysis. The sequencing results were consistent with SEQ ID NO.2, proving that the expression vector pUCm-T-FoEmg1 was successfully constructed and can be used for subsequent experiments.

[0081] 4. Construction of pET-28a-FoEmg1 plasmid

[0082] (1) Amplification of the target gene fragment with restriction enzyme sites

[0083] Plasmids were extracted from the bacterial culture of the correctly sequenced positive clones from step 3 using the Tiangen Plasmid Mini-Extraction Kit. Based on the open reading frame sequence of FoEmg1, the restriction enzyme sites in its base sequence were analyzed. Combining this with the restriction enzyme sites on the pET-28a multiple cloning site, EcoRI and HindIII restriction sites were selected, and expression primers FoEmg1-EcoRI IF and FoEmg1-HindIII-R containing these restriction sites were designed accordingly. Using the plasmid as a template, PCR amplification was performed using expression primers FoEmg1-EcoRI IF and FoEmg1-HindIII-R (PCR amplification reaction system and conditions were the same as in step 2), yielding the target gene fragment.

[0084] (2) Construction of pET-28a-FoEmg1 plasmid

[0085] The vector pET-28a and the purified product of the target gene fragment obtained by amplification in step 1) were simultaneously digested using Takara restriction endonuclease.

[0086] The enzyme digestion system was as follows: the concentration of the purified product of plasmid pET-28a / FoEmg1 was less than or equal to 2.5 μg; 1 μL each of EcoRI and HindIII; 5 μL of 10× M buffer; and ddH2O to a final volume of 50 μL. The digestion conditions were incubation at 37℃ for 3.5 h. After digestion, electrophoresis was performed for detection and fragment recovery. The digested target gene fragment and pET-28a vector were ligated overnight using T4 DNA ligase and transformed into DH-5α competent cells. Single colonies were then selected and subjected to colony PCR detection before being sent to Shanghai Sangon Biotech for sequencing. The results showed that the pET-28a-FoEmg1 plasmid was successfully constructed.

[0087] 5. Induction of expression of the activating protein FoEmg1

[0088] The obtained pET-28a-FoEmg1 plasmid was transformed into BL21(DE3) competent cells. Single colonies were selected and subjected to colony PCR detection. Positive colonies were then selected for induction of expression. Positive colonies were cultured to OD28a. 600The concentration of the culture medium was 0.6-0.8, and IPTG was added to a final concentration of 0.4 mM for induction of expression. The culture was then incubated at 16℃ and 150 rpm with shaking for 12 h. The bacterial cells were then collected by centrifugation and resuspended in phosphate buffer at a volume ratio of 1:5. The resuspended bacterial solution was then sonicated, and the supernatant was collected for SDS-PAGE analysis.

[0089] Test results ( Figure 2 The results showed that, compared with the uninduced supernatant, the induced supernatant showed a specific expression band, the size of which was consistent with the theoretical molecular weight of FoEmg1 (28.8 kDa), indicating that the activating protein FoEmg1 is mainly expressed in a soluble form.

[0090] 6. Purification of the activating protein FoEmg1

[0091] The supernatant was purified using a His-tagged protein purification kit (Kangwei Century) to remove contaminating proteins. A Millipore centrifuge was used to remove salt and imidazole, purifying the activating protein FoEmg1. The protein solution after elution was collected and added to the centrifuge. The mixture was centrifuged at 4°C and 9000 rpm. When the total protein solution was concentrated to approximately 1 mL, the waste liquid was discarded. Dialysis buffer A was added to the tube, and the protein solution was concentrated again to approximately 1 mL. Dialysis buffer B was added to the tube, and this process was repeated until the purified activating protein FoEmg1 was obtained. The concentration of the activating protein FoEmg1 was determined using the Coomassie Brilliant Blue assay.

[0092] The amino acid sequence of the activator protein FoEmg1 is shown in SEQ ID NO.1.

[0093] 7. Physicochemical properties of the activating protein FoEmg

[0094] Using the Expasy website to predict the physicochemical properties of the protein, it was found that the molecular weight of FoEmg1 protein is 28.8 kDa and the isoelectric point is 8.94.

[0095] Example 2: Stability detection of activating protein FoEmg1

[0096] 1. Thermal stability

[0097] Take 1 mL of the activating protein FoEmg1 solution prepared in Example 1, and incubate at 4, 20, 40, 60, 80, and 100 °C for 10 min each. After cooling to room temperature, filter. Use a syringe to inject 50 μL of Tris-HCl and the treated protein solution into Rehmannia glutinosa leaves. Detect the Rehmannia glutinosa leaves 48 h after injection. The results are as follows: Figure 3 As shown. By Figure 3It can be seen that the activating protein FoEmg1 can induce cell death in Rehmannia glutinosa leaves under all temperature treatments, indicating that the activating protein has good thermal stability in the range of 4-100℃.

[0098] 2. pH stability

[0099] The activated protein solution from Example 1 was dialyzed into Tris-HCl solutions with pH values ​​of 2, 4, 6, 8, 11, and 13, respectively. 50 μL of the protein solution and corresponding pH Tris-HCl solution at each pH were then injected into Rehmannia glutinosa leaves using a syringe. The leaves were examined 48 hours after injection, and the results are as follows: Figure 4 As shown.

[0100] Depend on Figure 4 It was found that protein solutions with pH values ​​of 2, 4, 6, 8, and 11 could induce cell death in Rehmannia glutinosa leaves. This indicates that the activating protein FoEmg1 has good stability within the pH range of 2-13.

[0101] Example 3: Effect of activator protein FoEmg1 on the root vigor of Rehmannia glutinosa

[0102] The root activity of Rehmannia glutinosa was determined by the triphenyltetrazolium chloride (TTC) method.

[0103] Specific operating method: Select Rehmannia glutinosa tissue culture seedlings with uniform growth, and apply 1 mL of activating protein FoE mg1 solution with concentrations of 1, 10, and 100 μg / mL to their roots (denoted as P1, P2, and P3, respectively). The control group is treated with 1 mL of 20 mM Tris-HCl (denoted as CK group). After 3 days of treatment, weigh 0.3 g of Rehmannia glutinosa roots into a 10 mL centrifuge tube, add 3 mL of 0.4% TTC and 3 mL of phosphate buffer, and treat in the dark at 37℃ for 2 h. Then, add 2 mL of 1 M sulfuric acid solution to terminate the reaction. Remove the roots, wipe them dry, add 6 mL of methanol, and decolorize overnight at 37℃ in the dark until the roots turn white. Measure the absorbance at 485 nm and calculate the root activity.

[0104] Test results as follows Figure 5 As shown. By Figure 5 It can be seen that the root activity of Rehmannia glutinosa in groups P1, P2 and P3 was 1.53, 1.54 and 1.03 times that of group CK, respectively, indicating that low concentration of activating protein FoEmg1 can significantly improve the root activity of Rehmannia glutinosa.

[0105] Example 4: Effect of activating protein FoEmg1 on the relative conductivity of Rehmannia glutinosa carrying pathogens

[0106] The activating protein FoEmg1 solution prepared in Example 1 was diluted to 1, 10, and 100 μg / mL, respectively. Potted Rehmannia glutinosa seedlings with uniform growth were selected. The treatment groups were sprayed with the activating protein FoEmg1 solution at concentrations of 1, 10, and 100 μg / mL (referred to as groups P1, P2, and P3, respectively), while the control group was sprayed with 20 mM Tris-HCl (referred to as group CK). Three days after treatment, all seedlings in both the treatment and control groups were uniformly sprayed with WY3 spore solution. Fresh leaves from the potted Rehmannia glutinosa seedlings were collected 12 h and 72 h after spraying to determine their electrical conductivity.

[0107] Conductivity test results as follows Figure 6 As shown, different concentrations of the activating protein FoEmg1 can reduce the relative electrical conductivity of leaves. 12 h after spraying with WY3 spore solution, the relative electrical conductivity of Rehmannia glutinosa leaves in groups P1, P2, and P3 were 0.58, 0.55, and 0.38 times that of the control group (CK), respectively. 72 h after spraying with WY3 spore solution, the relative electrical conductivity of leaves in groups P1, P2, and P3 were 0.59, 0.40, and 0.51 times that of the control group (CK), respectively. A higher relative electrical conductivity indicates a greater degree of damage to plant tissue. The relative electrical conductivity of leaves in the FoEmg1-treated groups was lower than that in the control group, indicating that FoEmg1 can effectively reduce the damage to leaves caused by pathogenic fungal spores, thereby improving the disease resistance of Rehmannia glutinosa.

[0108] Example 5: Activating protein FoEmg1 enhances the resistance of Rehmannia glutinosa to ring spot disease.

[0109] The activating protein FoEmg1 prepared in Example 1 was diluted to concentrations of 1, 10, and 100 μg / mL. Potted Rehmannia glutinosa seedlings with uniform growth were selected. The treatment groups were sprayed with 1, 10, and 100 μg / mL FoEmg1 solutions (referred to as P1, P2, and P3 groups, respectively), while the control group was sprayed with 20 mM Tris-HCl (referred to as CK group). Three days after treatment, the leaves of the potted Rehmannia glutinosa seedlings were punctured using a sterile syringe needle via a puncture inoculation method, and WY3 mycelial cakes were inoculated. When obvious lesions appeared in the CK group, images of the lesions were taken, and the lesion area was statistically analyzed using Adobe Photoshop (2021) software, expressed in pixels. The disease grade was determined by the percentage of lesion area to the total leaf area (the grading standards for Rehmannia glutinosa ring rot disease are shown in Table 1 below), and the disease index was calculated accordingly.

[0110] Table 1. Grading criteria for Rehmannia glutinosa ringworm

[0111]

[0112] The formula for calculating the disease index is: Disease Index (%) =

[0113] Test results as follows Figure 7 , Figure 8 As shown. By Figure 7 It can be seen that after inoculation with WY3 mycelium, obvious lesions appeared on the leaves of Rehmannia glutinosa. Compared with the CK group, the leaf lesion area in groups P1, P2, and P3 was significantly smaller. Figure 8 The leaf lesion areas of groups P1, P2, and P3 were 0.90, 0.59, and 0.10 times that of the control group (CK), respectively. Compared with the control group, the disease index of groups P1, P2, and P3 decreased by 11.11%, 35.55%, and 62.22%, respectively, and the induction effects of P1, P2, and P3 were 13.16%, 42.10%, and 73.68%, respectively. These results indicate that treatment with the activating protein FoEmg1 can effectively reduce the diseased area and improve the resistance of Rehmannia glutinosa leaves to leaf spot.

[0114] In addition, the relative biomass of the pathogenic fungus (WY3) in the leaves of *Rehmannia glutinosa* in groups P1, P2, P3, and CK was statistically analyzed. The method for detecting the relative biomass of the pathogenic fungus in *Rehmannia glutinosa* leaves is as follows: DNA was extracted from the lesion sites using the Ezup column-based super plant genomic DNA extraction kit (Shanghai Sangon Biotech Co., Ltd.). Using the extracted DNA as a template, q-PCR was performed using WY3-specific amplification primers (these primers only amplify WY3; the upstream primer is 3'-ATTCCACATCGAATTCCCTCCCT-5', and the downstream primer is 3'-GAACCACGTGATGATGCGCG-5'). The relative biomass of the pathogenic fungus WY3 in the leaves of *Rehmannia glutinosa* was detected, and the results are as follows: Figure 9 As shown.

[0115] The internal reference gene used in q-PCR was RgTIP4.

[0116] Upstream primer: 5'-TGGCTCAGAGTTGATGGAGTGCT-3';

[0117] Downstream primer: 5'-CTCTCCAGCAGCTTTCTCGGAGA-3'.

[0118] The q-PCR reaction system is shown in Table 2, and the reaction conditions are shown in Table 3.

[0119] Table 2 q-PCR reaction system

[0120]

[0121] Table 3 q-PCR reaction conditions

[0122]

[0123] Depend on Figure 9It was found that, compared with the CK group, the relative biomass of WY3 in Rehmannia glutinosa leaves in groups P1, P2, and P3 decreased by 31.02%, 52.61%, and 90.45%, respectively, and the trend of WY3 relative biomass change was consistent with the statistical analysis of the ring spot phenotype. These results indicate that treatment with the activating protein FoEmg1 can reduce the occurrence of the disease by decreasing the colonization of WY3 in Rehmannia glutinosa leaves, and the disease resistance effect of the activating protein on Rehmannia glutinosa against ring spot is positively correlated with its concentration.

[0124] Example 6: Activating protein FoEmg1 enhances the resistance of Rehmannia glutinosa to root rot.

[0125] The FoEmg1 activating protein solution prepared in Example 1 was diluted to obtain FoEmg1 activating protein solutions with concentrations of 1, 10, and 100 μg / mL. Uniformly sized Rehmannia glutinosa tubers were selected, rinsed clean, soaked in 75% alcohol for 30 seconds, rinsed three times with sterile water, dried, and placed in germination boxes lined with filter paper. Each tuber in the treatment groups was poured with 5 mL of FoEmg1 activating protein solution at concentrations of 1, 10, and 100 μg / mL (referred to as groups P1, P2, and P3, respectively), while the control group tubers were poured with 5 mL of 20 mM Tris-HCl (referred to as group CK). The tubers were placed in an incubator and incubated in the dark at 28°C for 3 days. B12 mycelial cakes were inoculated using the puncture inoculation method. When obvious lesions appeared in the CK group, images of the lesions were taken, and the lesion area was calculated using Adobe Photoshop (2021) software, expressed in pixels. At the same time, the disease severity level is determined by the percentage of the lesion area to the total cross-sectional area of ​​the tuber, and the disease index is calculated accordingly (see Example 5 for the grading standards and disease index calculation formula).

[0126] Test results as follows Figure 10 , Figure 11 As shown. By Figure 10 It was found that after inoculation with B12 mycelium, obvious rotting symptoms appeared in the inoculated area, and the lesion area in groups P1, P2, and P3 was significantly smaller than that in the CK group. The lesion area was measured and statistically analyzed. Figure 11 The lesion areas in groups P1, P2, and P3 were 0.69, 0.51, and 0.27 times larger than those in the control group (CK). Compared with the CK group, the disease index of groups P1, P2, and P3 decreased by 18.33%, 23.33%, and 40%, respectively. The induction effects of groups P1, P2, and P3 were 28.2%, 35.89%, and 61.54% of those in the CK group, respectively. These results indicate that treatment with the activating protein FoEmg1 can reduce the diseased area and improve the resistance of Rehmannia glutinosa tubers to root rot.

[0127] In addition, the relative biomass of pathogenic fungi (B12) in the tuberous roots of *Rehmannia glutinosa* in groups P1, P2, P3, and CK was statistically analyzed. The specific method for detecting the relative biomass of pathogenic fungi in the tuberous roots of *Rehmannia glutinosa* is as follows: DNA was extracted from the diseased areas using the Ezup column-based super plant genomic DNA extraction kit (Shanghai Sangon Biotech Co., Ltd.). Using the extracted DNA as a template, q-PCR detection was performed using B12-specific amplification primers (these primers only amplify B12; the upstream primer is 3'-ATGCTTAGGGTCCGAGTGGA-5', and the downstream primer is 3'-TCCCTCCGAGCCTCTGATTG-5') (the method is the same as step 2 above). The relative biomass of pathogenic fungi WY3 in the leaves of *Rehmannia glutinosa* was then detected. The detection results are as follows: Figure 12 As shown.

[0128] Depend on Figure 12 It was found that, compared with the CK group, the relative biomass of B12 in groups P1, P2, and P3 was significantly reduced by 17.40%, 75.73%, and 93.34%, respectively. Moreover, the trend of B12 relative biomass change was consistent with the statistical analysis of root rot phenotype. This indicates that treatment with the activating protein FoEmg1 can reduce the occurrence of disease by decreasing the colonization of B12 in Rehmannia glutinosa tubers.

[0129] Example 7: FoEmg1 activator protein induces expression of Rehmannia glutinosa resistance-related genes.

[0130] This study investigated the effects of treatment with the activating protein FoEmg1 on the expression of immune-related genes, salicylic acid (SA), and jasmonic acid (JA) signaling pathways in Rehmannia glutinosa.

[0131] 1. Experimental Methods

[0132] The FoEmg1 activating protein solution prepared in Example 1 was diluted to obtain FoEmg1 activating protein solutions with concentrations of 1, 10, and 100 μg / mL. Potted Rehmannia glutinosa seedlings with uniform growth were selected, and foliar spraying with FoEmg1 activating protein solutions at concentrations of 1, 10, and 100 μg / mL was performed (referred to as groups P1, P2, and P3, respectively). The control group was sprayed with 20 mM Tris-HCl (referred to as group CK). Rehmannia glutinosa leaves were collected 72 h after spraying with the FoEmg1 activating protein solution, and genomic DNA was extracted from the leaves for detection of Rehmannia glutinosa resistance-related genes (including immune-related genes, salicylic acid-related genes, jasmonic acid-related genes, etc.).

[0133] 2. Experimental Results

[0134] (1) Effect of activating protein treatment on the expression levels of Rehmannia glutinosa immune-related genes

[0135] Results of Rehmannia glutinosa immune-related gene expression level detection: Figure 13 As shown, group P1 increased the relative expression levels of RgFLS2 and RgMPK by 1.76 and 46.78 times, respectively, compared to the CK group; group P2 increased the relative expression levels of RgMPK, RgBIK1, and RgCERK1 by 5.50, 1.89, and 1.66 times, respectively, compared to the CK group; and group P3 increased the relative expression levels of RgMPK and RgBIK1 by 32.95 and 1.93 times, respectively, compared to the CK group. This suggests that different immune genes respond differently to the concentration of the activating protein FoEmg1, but treatment with the activating protein can effectively initiate the PTI defense pathway of Rehmannia glutinosa.

[0136] (2) Effect of protein activation treatment on the expression levels of salicylic acid-related genes in Rehmannia glutinosa

[0137] The results of the detection of the effect of activation protein treatment on the expression level of rehmannia salicylic acid-related genes are as follows: Figure 14 As shown. By Figure 14 It was found that salicylic acid-related genes were increased to varying degrees under different concentrations of treatment. Group P1 significantly increased the relative expression levels of genes RgPBS3 and RgTGA, which were 1.48 times and 2.57 times that of the CK group, respectively; Group P2 significantly increased the relative expression levels of genes RgPBS3 and RgTGA, which were 2.11 times and 3.60 times that of the CK group, respectively; Group P3 significantly increased the relative expression level of gene RgTGA, which was 2.79 times that of the CK group.

[0138] Therefore, it is inferred that treatment with low concentration of activating protein FoEmg1 can activate the heterobranched acid synthesis pathway to synthesize SA by increasing the expression levels of genes RgPBS3 and RgEPS1. High concentration of activating protein FoEmg1 can activate salicylic acid signaling pathway transduction by increasing the expression level of gene RgTGA, enabling Rehmannia glutinosa to receive SA signals and make a series of defense responses. This indicates that FoEmg1 can activate the synthesis and signal transduction of SA.

[0139] (3) Effect of protein activation treatment on the expression levels of rehmannia jasmonic acid-related genes

[0140] The results of the detection of the effect of activating protein treatment on the expression level of rehmannia jasmonic acid-related genes are as follows: Figure 15 As shown. By Figure 15It was found that the relative expression levels of RgAOC and RgJAZ genes in group P1 were significantly increased, being 1.50-fold and 1.42-fold higher than those in group CK, respectively. High concentrations of the activating protein inhibited the relative expression of RgAOC, RgJAZ, and RgMYC2 genes. The relative expression levels of RgJAZ gene in groups P2 and P3 were 0.32-fold and 0.52-fold higher than those in group CK, respectively, while the relative expression levels of RgMYC2 gene were 0.32-fold and 0.55-fold higher than those in group CK, respectively. Therefore, it is speculated that low concentrations of the activating protein can promote the synthesis of jasmonic acid in Rehmannia glutinosa, while high concentrations can enhance the transduction capacity of jasmonic acid signaling in Rehmannia glutinosa.

[0141] Example 8: Activation of protein FoEmg1 promotes the synthesis of secondary metabolites of Rehmannia glutinosa.

[0142] This embodiment is used to study the effect of treatment with activating protein FoEmg1 on the content of the main active ingredients (secondary metabolites) in Rehmannia glutinosa leaves.

[0143] 1. Experimental Methods

[0144] The FoEmg1 activating protein solution prepared in Example 1 was diluted to obtain FoEmg1 activating protein solutions with concentrations of 1, 10, and 100 μg / mL. Potted Rehmannia glutinosa seedlings with uniform growth were selected, and the leaves of these seedlings were sprayed with FoEmg1 activating protein solutions at concentrations of 1, 10, and 100 μg / mL (referred to as groups P1, P2, and P3, respectively). The control group was sprayed with 20 mM Tris-HCl (referred to as group CK). 72 h after spraying with the FoEmg1 activating protein solution, the Rehmannia glutinosa leaves were dried at 55℃ and ground into powder. The contents of secondary metabolites (such as catalpol, verbascoside, rehmannia glycoside D, rehmannia glycoside A, and leonurin) were determined using high-performance liquid chromatography.

[0145] 2. Experimental Results

[0146] Iridoid glycosides are the most abundant and diverse group of components in Rehmannia glutinosa, with catalpol being a representative compound and one of its main active ingredients. The effect of activating protein FoEmg1 on the content of secondary metabolites in Rehmannia glutinosa leaves is as follows: Figure 16 As shown. By Figure 16The results showed that after treatment with the activating protein FoEmg1, the contents of leonurin, verbascoside, and catalpol in Rehmannia glutinosa in group P1 were significantly increased, reaching 1.27, 1.20, and 1.81 times that of the control group (CK), respectively; while the content of rehmannia glycoside A decreased slightly, reaching 0.87 times that of the CK group. In group P2, the content of leonurin in Rehmannia glutinosa showed an increasing trend, reaching 1.18 times that of the CK group, and the content of verbascoside was 0.95 times that of the CK group. In group P3, the contents of rehmannia glycoside A, rehmannia glycoside D, and leonurin were significantly increased, reaching 1.29, 1.10, and 1.23 times that of the CK group, respectively. These results indicate that high concentrations of the activating protein more effectively promote the accumulation of rehmannia glycoside A, rehmannia glycoside D, and leonurin in Rehmannia glutinosa leaves, while low concentrations of the activating protein more effectively promote the accumulation of verbascoside and catalpol in Rehmannia glutinosa leaves.

Claims

1. An activating protein FoEmg1, characterized in that, The proteins shown in A1), A2), A3), or A4) below: A1) The amino acid sequence is that of the protein shown in SEQ ID NO.1; A2) A tag is attached to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.1 to obtain a fusion protein; A3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1; A4) Proteins that have 85% or more similarity to the amino acid sequence shown in SEQ ID NO.1 and have the same function.

2. The biological material related to the activating protein FoEmg1 of claim 1 is any one of the following: B1) A nucleic acid molecule encoding the activating protein FoEmg1 as described in claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that, B1) The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

4. The use of the activating protein FoEmg1 of claim 1 or the biomaterial of claim 2 or 3 in any of the following: C1) Improve plant root vitality; C2) Prepare products that enhance plant root vitality; C3) Improve plant disease resistance; C4) Prepare products that enhance plant disease resistance; C5) Cultivate plants with improved disease resistance; C6) Prepare and cultivate plant products with enhanced disease resistance; C7) induces plant defense responses; C8) Enhances the induction of plant immune responses; C9) increases the content of secondary metabolites in plants.

5. The application according to claim 4, characterized in that, The disease resistance refers to resistance to ring spot and / or root rot.

6. The application according to claim 5, characterized in that, The plant is Rehmannia glutinosa, and the secondary metabolites are catalpol, verbascoside, rehmannia glycoside D, rehmannia glycoside A, or / and leonurin.

7. A plant treatment agent, characterized in that, The active ingredient in the plant treatment agent is the activating protein FoEmg1.

8. The plant treatment agent according to claim 7, characterized in that, The concentration of the activating protein in the plant treatment agent is 1-300 μg / mL.

9. A method for improving plant disease resistance, characterized in that, This includes applying the plant treatment agent as described in claim 7 or 8 to the plants.

10. A method for improving plant root vitality, characterized in that, This includes treating plant roots with the plant treatment agent as described in claim 7 or 8.

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