A folgpl gene targeting agent and its application in inhibiting plant pathogenic bacteria

Abstract

The application discloses a kind of FolGP1 Gene targeting agent and its application in inhibiting plant pathogenic fungi belong to the technical field of plant genetic engineering.The gene targeting agent disclosed in the application FolGP1 The gene targeting agent includes compound D264-0813 and / or compound STL496578, which can interact and combine with Q112, E128, E194 and Q197 sites of FolGP1 protein.The application is verified by experiment, FolGP1 The gene targeting agent can significantly inhibit various pathogenic fungi, including Fusarium oxysporum, Botrytis cinerea, Magnaporthe oryzae and Liriomyza trifolii, by affecting FolGP1 The normal function of FolGP1 protein encoded by the gene, so as to achieve the purpose of preventing and treating various plant diseases.Furthermore, FolGP1 The gene targeting agent is simple to use, has no effect on the plant itself, is safe and reliable, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a... FolGP1 Gene targeting agents and their application in inhibiting plant pathogens. Background Technology

[0002] Fusarium wilt of tomato is a serious disease that causes significant damage in tomato cultivation. It occurs extensively in my country, with an incidence rate of approximately 20%, especially in greenhouse cultivation. Early symptoms include stunted growth and weakened vegetative state, with leaves initially turning yellow and losing their green color, which gradually spreads to the entire plant. As the disease progresses, the vascular bundles are destroyed by the pathogen, hindering water and nutrient transport, and in severe cases, the entire plant wilts and dies from dehydration. In the early stages, the external stem may show no obvious abnormalities, but the internal vascular bundles will develop characteristic reddish-brown lesions. Under high humidity, a pinkish mold layer will appear on the stem surface, consisting of conidiophores and conidia of the pathogenic fungus. Because this disease spreads horizontally through soil and irrigation water in the field, it spreads rapidly and can easily cause large-scale outbreaks, leading to a significant drop in tomato yield and severe economic losses.

[0003] *Fusarium oxysporum*, a common soil-borne pathogenic fungus, is widely found in soil and plant debris. It is one of the world's top ten plant pathogenic fungi, infecting over 200 food and economic crops, including soybeans, tomatoes, melons, watermelons, and flowers, causing diseases such as wilt, stem rot, and root rot. Given the significant differences in host infection range among different *Fusarium oxysporum* strains, and the fact that some strains exhibit host specificity, these strains are classified into different specialized types (…). formae speciales, f. sp. Furthermore, different strains exhibit varying environmental adaptability, leading to differences in the geographical distribution of different specialized Fusarium oxysporum species. In addition to producing small conidia, this strain can also form large conidia and chlamydospores: small conidia are mostly elliptical and colorless; large conidia are often sickle-shaped or spindle-shaped and septate; chlamydospores have a smooth surface and are capable of resisting adverse environments.

[0004] Because *Fusarium oxysporum* is a soil-borne pathogenic fungus that produces chlamydospores to cope with various adverse environments, the diseases it causes are extremely difficult to control in production, and resistance easily develops. Currently, tomato cultivation is generally characterized by large planting areas, limited variety, and relatively enclosed, high-humidity greenhouse environments, which facilitate the growth and spread of *Fusarium oxysporum*, leading to a worsening of tomato wilt disease. Given the infection mode and characteristics of wilt, there are currently no highly effective control measures for this disease in production, and chemical control remains the primary method. However, the extensive use of chemical pesticides has led to a continuous increase in the pathogen's resistance, making the control of tomato wilt even more challenging. Therefore, finding new compounds or fungicides for pathogen control is of great significance to the development of the tomato industry. Summary of the Invention

[0005] Based on the above technical requirements, the purpose of this invention is to provide a FolGP1 Gene targeting agents and their application in inhibiting plant pathogens. The present invention describes... FolGP1 Genes are crucial for Fusarium oxysporum, being essential genes for pathogenicity. This invention utilizes... FolGP1 Gene-targeted screening yielded compounds D264-0813 and STL496578, which were then used as... FolGP1 Gene targeting agents: Through experiments, this invention has demonstrated that the compounds D264-0813 and STL496578 have broad-spectrum antibacterial activity against a variety of plant pathogens.

[0006] To achieve the above-mentioned objectives, the present invention is implemented through the following solution: This invention provides FolGP1 Gene targeting agents, the FolGP1 Gene targeting agents include compound D264-0813 and / or compound STL496578; the chemical structural formula of compound D264-0813 is as follows: ; The chemical structural formula of the compound STL496578 is as follows: .

[0007] Furthermore, the aforementioned FolGP1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] Furthermore, the aforementioned FolGP1 The amino acid sequence of the FolGP1 protein encoded by the gene is shown in SEQ ID NO.2.

[0009] Furthermore, the aforementioned FolGP1Gene targeting agents can interact and bind to the Q112, E128, E194, and Q197 sites of the FolGP1 protein.

[0010] The present invention also provides the aforementioned FolGP1 Application of gene-targeting agents in inhibiting plant pathogens.

[0011] Furthermore, the plant pathogens include at least one of Fusarium oxysporum, Botrytis cinerea, rice blast fungus, and Verticillium dahliae.

[0012] Furthermore, the aforementioned FolGP1 The concentration of gene-targeting agents used is 8 µM-100 µM.

[0013] Furthermore, the aforementioned FolGP1 The method of using gene-targeting agents is as follows: FolGP1 Gene-targeting agents are prepared to the appropriate concentration and applied to the plants via root irrigation or spraying.

[0014] Furthermore, the plant includes at least one of tomato, mung bean, rice, and cotton.

[0015] Furthermore, the aforementioned FolGP1 Gene-targeting agents can prevent and control plant diseases by significantly inhibiting the pathogenicity of plant pathogens.

[0016] Compared with the prior art, the advantages and technical effects of the present invention are as follows: 1. This invention, through the construction of the 3D structure of the FolGP1 protein, targeted screening yielded two small molecule compounds: D264-0813 and STL496578. Both compounds can interact with the Q112, E128, E194, and Q197 sites of the FolGP1 protein, thereby achieving targeted targeting. FolGP1 The purpose of genes.

[0017] 2. Experimental verification of this invention shows that compounds D264-0813 and STL496578 can significantly inhibit various plant pathogenic fungi, such as Fusarium oxysporum, Botrytis cinerea, Rice blast fungus, and Verticillium dahliae, by affecting the biological function of the FolGP1 protein, ultimately achieving the purpose of preventing and controlling various plant diseases, such as tomato wilt. Furthermore, the application method is simple, has no impact on the plant itself, and is safe and reliable. Therefore, the two small molecule compounds D264-0813 and STL496578 provided by this invention have excellent control effects against various plant pathogens and have broad application prospects. Attached Figure Description

[0018] Figure 1The diagram shows the binding patterns of two compounds to the FolGP1 protein, where A is compound D264-0813 and B is compound STL496578.

[0019] Figure 2 Compounds D264-0813 and STL496578 significantly inhibited the pathogenicity of Fusarium oxysporum at a concentration of 8 µM. Mock was the negative control group without any treatment. A is a photograph taken 20 days after inoculation, and B is the disease index.

[0020] Figure 3 The images show the significant inhibition of plant pathogens by compounds D264-0813 and STL496578 at a concentration of 8 µM. A represents Botrytis cinerea, B represents blast fungus, C represents Verticillium dahliae, and Mock represents the negative control group without any treatment. The left-hand images show the appearance of plants with the corresponding pathogens inhibited, while the right-hand images show the size or biomass of the corresponding pathogen lesions.

[0021] Figure 4 Compounds D264-0813 and STL496578 had no significant effect on the growth of host plants at a concentration of 100 µM; where A was tomato, B was mung bean, C was rice, D was cotton, and the Mock group was the negative control group without any treatment. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials, reagents, and instruments used in the following examples are all commercially available products. The quantitative statistics in the following examples are all based on three replicate experiments, with the average value taken.

[0024] Example 1: With FolGP1 Screening for effective agents against Fusarium oxysporum The present invention provides FolGP1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the sequence of the FolGP1 protein it encodes is shown in SEQ ID NO.2.

[0025] The 3D structure of FolGP1 was constructed using MODELLER 9.11 software. The ChemDiv database was then used to screen the 3D structure. The results showed that two compounds, D264-0813 and STL496578, could bind to the FolGP1 protein.

[0026] The specific chemical structure of compound D264-0813 is shown below: ; The specific chemical structure of compound STL496578 is shown below: .

[0027] Specific results are as follows Figure 1 As shown, compounds D264-0813 and STL496578 interact with the Q112, E128, E194, and Q197 sites of the FolGP1 protein.

[0028] Example 2: Inhibition of Fusarium oxysporum pathogenicity by compounds D264-0813 and STL496578 This embodiment uses the root-dipping method to determine the pathogenicity of Fusarium oxysporum. The specific operation includes the following steps: Wild-type Fusarium oxysporum cultured on PDA medium for 4 days was extracted. Fol The mycelial cake was added to PDB medium and cultured for 48 hours. Conidia were then collected and the concentration was adjusted to 5.0 × 10⁻⁶. 6 CFU / mL. Compounds D264-0813 and STL496578 were dissolved in DMSO and added to the spore solution to adjust the final concentration to 8 µM. The specific compound contents of the spore solution in the treatment group were: 8 µM of compound D264-0813, 8 µM of compound STL496578, and 8 µM of a mixture of D264-0813 and STL496578 (mass ratio 1:1). DMSO containing only spore solution was used as a negative control, and Mock was used as a negative control group without any treatment. The roots of 3-week-old tomato seedlings were washed to create micro-wounds. The roots of the treated tomato seedlings were immersed in the spore solution for 20 minutes, and then planted back in vermiculite for further cultivation. After 20 days, photos were taken and disease indices were recorded.

[0029] See results Figure 2 Compared with the control group, 8 µM D264-0813 and STL496578 significantly inhibited the pathogenicity of Fusarium oxysporum.

[0030] Example 3: Inhibition of pathogenicity of compounds D264-0813 and STL496578 against Botrytis cinerea, Verticillium dahliae, and blast fungus. This embodiment investigates the inhibitory effects of compounds D264-0813 and STL496578 on the pathogenicity of Botrytis cinerea, Verticillium dahliae, and Bacillus thuringiensis. The specific operation includes the following steps: 1. Collect conidia of *Botrytis cinerea* cultured on PDA medium for approximately 7 days, and adjust the spore concentration to 5.0 × 10⁻⁶ using an infection buffer (6.7 mM KH₂PO₄, 6.7 mM glucose). 6CFU / mL. Compounds D264-0813 and STL496578 were dissolved in DMSO and added to the spore suspension, adjusting the final concentration to 8 µM. The specific compound contents in the spore suspension of the treatment group were: 8 µM of compound D264-0813 and 8 µM of a mixture of D264-0813 and STL496578 (mass ratio 1:1). DMSO containing only spore suspension served as a negative control, and Mock served as a negative control group without any treatment. The spore suspension was dropped onto the center of mung bean leaves, and after 3 days of moist incubation, photographs were taken and the diameter of the lesions was measured.

[0031] 2. Collect conidia of *Magnapordica oryzae* from OTA medium and adjust the conidia concentration to 1.0 × 10⁻⁶ using sterile water containing 0.1% Tween-20. 5 CFU / mL. Compounds D264-0813 and STL496578 were added to the spore solution at a final concentration of 8 µM. The specific compound contents in the spore mixture of the treatment groups were: 8 µM of compound D264-0813, 8 µM of compound STL496578, and 8 µM of a mixture of D264-0813 and STL496578 (mass ratio 1:1). DMSO, containing only spore solution, served as a negative control, while Mock, a negative control group without any treatment, was used. The spore mixture was dropped onto rice leaves, kept moist, and photographed after 4 days, with the diameter of the lesions measured.

[0032] 3. The *Verticillium dahliae* cultured on the PDA was inoculated into liquid Czapek-Dox medium. After collecting conidia, the concentration was adjusted to 5.0 × 10⁻⁶. 6 CFU / mL. Compounds D264-0813 and STL496578 were dissolved in DMSO and added to the spore solution to adjust the final concentration to 8 µM. The specific compound contents in the spore solution of the treatment group were: 8 µM of compound D264-0813, 8 µM of compound STL496578, and 8 µM of a mixture of D264-0813 and STL496578 (mass ratio 1:1). DMSO containing only spore solution was used as a negative control, and Mock was used as a negative control group without any treatment. The roots of cotton seedlings were soaked in the spore solution for 20 minutes, and the cotton seedlings were then planted in vermiculite for further cultivation. After 20 days, photos were taken and the disease index was recorded.

[0033] See results Figure 3 Compared with the control group, compounds D264-0813 and STL496578 at 8 µM significantly inhibited the pathogenicity of Botrytis cinerea, Verticillium dahliae and blast fungus.

[0034] Example 4: Compounds D264-0813 and STL496578 had no significant effect on the growth of the host plant. This embodiment investigates whether compounds D264-0813 and STL496578 have an effect on the growth of host plants. The specific operation includes the following steps: Two compounds, D264-0813 and STL496578, were dissolved in DMSO to a final concentration of 100 mM. One ml of the stock solution was added to 999 mL of water to prepare 100 µM solutions of D264-0813 and STL496578, respectively. The entire plants of tomatoes, mung beans, cotton, and rice were sprayed with 100 µM of D264-0813, 100 µM of STL496578, and a 1:1 mixture of both compounds (by mass). Photos were taken two weeks later. DMSO alone served as the negative control group, while no treatment was used as the mock negative control group.

[0035] The results are as follows Figure 4 As shown, compared with the control group, the two compounds D264-0813 and STL496578 had no significant effect on the growth of the host plant at a concentration of 100 µM, indicating that these two compounds do not affect the normal growth of the plant.

[0036] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A kind FolGP1 Gene targeting agents, characterized in that, The FolGP1 Gene targeting agents include compound D264-0813 and / or compound STL496578; the chemical structural formula of compound D264-0813 is as follows: ； The chemical structural formula of the compound STL496578 is as follows: 。 2. As described in claim 1 FolGP1 Gene targeting agents, characterized in that, The FolGP1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. As described in claim 1 FolGP1 Gene targeting agents, characterized in that, The FolGP1 The amino acid sequence of the FolGP1 protein encoded by the gene is shown in SEQ ID NO.

2.

4. As described in claim 1 FolGP1 Gene targeting agents, characterized in that, The FolGP1 Gene targeting agents can interact and bind to the Q112, E128, E194, and Q197 sites of the FolGP1 protein.

5. The claim 1 to 4 FolGP1 Application of gene-targeting agents in inhibiting plant pathogens.

6. The application according to claim 5, characterized in that, The plant pathogens include at least one of Fusarium oxysporum, Botrytis cinerea, rice blast fungus, and Verticillium dahliae.

7. The application according to claim 5, characterized in that, The FolGP1 The concentration of gene-targeting agents used is 8 µM to 100 µM.

8. The application according to claim 5, characterized in that, The FolGP1 The method of using gene-targeting agents is as follows: FolGP1 Gene-targeting agents are prepared to the appropriate concentration and applied to the plants via root irrigation or spraying.

9. The application according to claim 5, characterized in that, The plant includes at least one of tomato, mung bean, rice and cotton.

10. The application according to claim 1, characterized in that, The FolGP1 Gene-targeting agents achieve the effect of preventing and controlling plant diseases by significantly inhibiting the pathogenicity of plant pathogens.

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