TGL protein as drug target for screening and preventing rice false smut and application of TGL protein

Abstract

The invention discloses a TGL protein as a drug target for screening and preventing rice false smut and application of the TGL protein. The invention provides a drug target for screening and preventing rice false smut. The target is triglyceride lipase, namely TGL protein. The invention also provides application of the triglyceride lipase, namely the TGL protein, as a drug target in screening drugs for preventing and treating rice false smut. The invention provides a bactericide taking TGL protein as a target. The invention aims to solve the problems that the activity of TGL protein is increased, the phospholipid synthesis of a cell membrane is possibly changed, and the flowability and permeability of the cell membrane are influenced. The TGL protein serves as a drug target of a potential Ustilaginoidea virens bactericide for the first time, and it is found that the plant source substance anisaldehyde and the Ustilaginoidea virens TGL protein have excellent binding performance through experiments such as electron microscope observation, propidium iodide staining, multiomics joint analysis, molecular simulation docking and molecular dynamics; the TGL protein can become a potential bactericide target for preventing and controlling ustilaginoidea virens.

Description

Technical Field

[0001] The invention relates to a TGL protein used as a drug target for screening and preventing and treating rice false smut and an application thereof, belonging to the technical field of plant disease prevention and treatment. Background Art

[0002] Rice false smut has become one of the three most devastating new rice diseases in my country. It is a fungal disease caused by the smut fungus Ustilaginoidea virens (asexual form, Ustilaginoidea virens; sexual form, Villosiclava virens) infecting the rice panicle. In recent years, with the widespread cultivation of susceptible high-yield japonica and hybrid rice, excessive fertilizer use, and changes in climate and cultivation practices, rice false smut has evolved from a historically minor disease to one of the most destructive panicle diseases in rice production today. Chemical pesticides remain the primary method for controlling rice false smut, and rice quality and safety continue to face the dual threats of pesticide residues and mycotoxins.

[0003] The plant-derived compound anisaldehyde exhibits significant bioinhibitory activity against a variety of pathogens. Anisaldehyde, scientifically known as anisaldehyde, also known as p-anisaldehyde and anisaldehyde, is widely found in plants such as the genus Anise and Acacia. In recent years, anisaldehyde has been shown to exhibit antioxidant activity, activating the antioxidant defense system by enhancing the antioxidant potential of the ascorbic acid-reduced glutathione cycle, alleviating the accumulation of reactive oxygen species and thus slowing the oxidative decay of fruit. Studies have shown that anisaldehyde exhibits significant antifungal activity, making it a hot topic in antifungal drug research. This suggests that anisaldehyde is a promising plant-derived active substance. Summary of the Invention

[0004] Based on the above, the present invention provides a TGL protein as a drug target for screening and preventing and treating rice false smut and its application.

[0005] The technical solution of the present invention is:

[0006] In a first aspect, the present invention provides a target for screening drugs for preventing and treating rice false smut, wherein the target is triglyceride lipase, ie, TGL protein.

[0007] In a second aspect, the present invention provides a use of triglyceride lipase, namely TGL protein, as a drug target in screening drugs for preventing and treating rice false smut.

[0008] In a third aspect, the present invention provides a drug for preventing and controlling rice false smut by using triglyceride lipase, i.e., TGL protein, as a target for screening. The drug is anisaldehyde, a plant-derived active substance.

[0009] The technical principle of the present application: fatty acids are the key raw materials for synthesizing fungal cell membrane phospholipids. The increase of triglyceride lipase activity increases the production of fatty acids, which may change the synthesis of cell membrane phospholipids, affect the fluidity and permeability of the cell membrane, and the function and distribution of proteins on the membrane. If the structure and function of the cell membrane are seriously damaged, it will lead to the leakage of intracellular substances, affecting the normal physiological function of the fungal cell.

[0010] The beneficial effects of the present application: the present application aims at the increase of TGL protein activity, which may change the synthesis of cell membrane phospholipids and affect the fluidity and permeability of the cell membrane. For the first time, TGL protein is used as a potential fungicide target for Ustilaginoidea virens. Through electron microscopy observation, iodinated propylene dyeing, multi-omics joint analysis, molecular docking, molecular dynamics and other experiments, it is found that anethole has excellent binding performance with TGL protein of Ustilaginoidea virens, and TGL protein can become a potential fungicide target for preventing and controlling Ustilaginoidea virens. In addition, the drug anethole screened by the present application has good biological activity on Ustilaginoidea virens. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Inhibition effect of anethole on mycelium of Ustilaginoidea virens;

[0012] Figure 2 Mycelial morphology of Ustilaginoidea virens after anethole treatment;

[0013] Figure 3 Ultrastructure of Ustilaginoidea virens cells after anethole treatment;

[0014] Figure 4 Effect of anethole on cell membrane permeability of Ustilaginoidea virens;

[0015] Figure 5 KEGG enrichment pathway diagram of differential genes and differential metabolites of Ustilaginoidea virens after anethole treatment;

[0016] Figure 6 Correlation network diagram of differential genes and differential metabolites of Ustilaginoidea virens after anethole treatment;

[0017] Figure 7 Binding ability prediction of anethole and target protein of Ustilaginoidea virens;

[0018] Figure 8 Molecular docking diagram of anethole and TGL protein of Ustilaginoidea virens;

[0019] Figure 9 Molecular dynamics analysis diagram of anethole and TGL protein of Ustilaginoidea virens. DETAILED DESCRIPTION

[0020] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0021] (1) Determination of the antibacterial activity of anisaldehyde against rice smut pathogen

[0022] Anisaldehyde was dissolved in N,N-dimethylformamide (DMF), diluted with Tween-80 and sterile water (1:1000 v / v), and mixed with quantitative PSA at 40-45°C to obtain five anisaldehyde concentrations (5, 10, 20, 40, and 80 μg / mL). After the culture medium solidified, a mycelial block (6 mm in diameter) cut from the edge of a U.S. smut colony was placed in the center of the PSA. The culture dish was sealed with sealing film and incubated upside down at 28°C in the dark. The diameter (cm) of each colony was measured, and the inhibition rate was calculated using a bioassay data processing system.

[0023] As the concentration of anisaldehyde treatment increased, the colony diameter of the rice smut pathogen decreased; when the concentration of anisaldehyde was 80 μg / mL, the mycelial growth of the rice smut pathogen was severely inhibited, indicating that anisaldehyde treatment had a significant inhibitory effect on the growth of the rice smut pathogen ( Figure 1 ), the effective inhibitory concentration of anisaldehyde on rice smut pathogen EC 50 The value was 11.08 μg / mL. It can be seen that the natural product anisaldehyde has good antibacterial activity against rice smut pathogen.

[0024] (2) Observation of the effects of anisaldehyde on the mycelial morphology and cell ultrastructure of U.S. smut pathogen

[0025] Scanning electron microscopy (SEM) was used to observe the changes in mycelial morphology of U. oryzae var. oryzae after anisaldehyde treatment. Figure 2 The results showed that untreated mycelial morphology of the rice smut pathogen was naturally plump, with a smooth surface, well-extended, and complete morphology. However, 48 hours after treatment with 28 μg / mL anisaldehyde, the mycelial morphology showed severe shrinkage, distortion, and deformity, with a rough surface and abnormal structure. The mycelial morphology of the treated group differed from that of the control group, with slightly twisted mycelia. After treatment with 65 μg / mL anisaldehyde, the mycelia became more disorganized and aggregated than in the control group, with severe twisting and entanglement, and a small number of mycelia were broken. Therefore, anisaldehyde treatment of mycelial morphology caused abnormal mycelial growth and severely inhibited normal mycelial growth.

[0026] Transmission electron microscopy (TEM) was used to observe the changes in the ultrastructure of the cells of U. oryzae var. oryzae after anisaldehyde treatment. Figure 3It can be seen that anisaldehyde treatment of U.S. oryzae caused severe damage to the cellular ultrastructure. In the control group, the mycelial cell membrane, nuclear membrane, and cellular tissue were clearly visible, the organelles were intact, and the cell wall thickness was uniform. However, after treatment with 28 μg / mL and 65 μg / mL anisaldehyde, the mycelial organelles of U.S. oryzae spp. swelled, the cell membrane and organelle membranes became blurred, the cytoplasm dissolved, the intracellular lipid droplets increased, the cell membrane permeability increased, the intracellular substances effluxed, and vacuolation occurred inside the cells. Anisaldehyde treatment of U.S. oryzae spp. resulted in abnormal cell structure and morphology, which prevented the mycelium from growing and developing normally.

[0027] (3) Determination of cell membrane permeability of U.S. smut pathogen

[0028] Propidium iodide (PI) dye cannot pass through normal cell membranes. However, when cells undergo apoptosis or necrosis, the integrity of the cell membrane is damaged. PI can pass through the damaged cell membrane and enter the cell to bind to DNA, releasing strong red fluorescence. Figure 4 As shown in the results, the mycelia of the U.S. smut fungus in the control group showed no red fluorescence; after treatment with 28 μg / mL anisaldehyde, weak fluorescence was observed in the mycelia; after treatment with 65 μg / mL anisaldehyde, strong red fluorescence was produced in the mycelia. This suggests that the natural product anisaldehyde may cause cell death in U.S. smut fungus by destroying its cell membrane structure.

[0029] (4) Transcriptome and metabolome association analysis

[0030] KEGG enrichment analysis was performed on the differential genes and metabolites of U.S. smut pathogen after treatment with 65 μg / mL anisaldehyde for 24 h and 48 h, as follows Figure 5 As shown: The differential genes and differential metabolites of U.S. smut fungus are particularly enriched in the Glycerophospholipid metabolism pathway. It can be seen that after the U.S. smut fungus is treated with anisaldehyde, the differential genes and differential metabolites are mostly concentrated in the membrane lipid synthesis-related pathway of the cell membrane. The correlation network diagram can be used to represent the correlation between metabolites and genes. Metabolites are marked in red, genes are marked in green, solid lines represent positive correlations, and dotted lines represent negative correlations. After the U.S. smut fungus was treated with anisaldehyde for 48 hours, the differential genes and differential metabolites with a correlation greater than 0.5 in the glycerol phospholipid metabolism pathway were selected for mapping. The differential genes are as follows Figure 6 As shown, UV8b_03588, UV8b_03378, UV8b_00401, UV8b_02648, UV8b_07228, UV8b_04396, UV8b_08160 and UV8b_06725.

[0031] (5) Molecular simulation docking

[0032] The binding ability of U.S. smut pathogen proteins UV8b_03588 (TGL), UV8b_03378, UV8b_00401, UV8b_02648, UV8b_07228, UV8b_04396, UV8b_08160 and UV8b_06725 with anisaldehyde was determined by molecular simulation docking technology. It was found that TGL had the strongest binding ability with anisaldehyde ( Figure 7 The grey dashed line represents the hydrophobic interaction, and the orange dashed line represents the π-π stacking force ( Figure 8 ). This indicates that anisaldehyde has a high degree of binding to TGL protein, which is likely to affect the structure, function and biological activity of TGL protein.

[0033] Molecular simulation dynamics: Molecular simulation dynamics analysis of TGL protein and anisaldehyde was performed to predict the binding ability of the two. Figure 9 A) It can be seen that the RMSD curve of the complex of TGL protein and anisaldehyde fluctuates within 1 nm throughout the whole process, and reaches a stable range of about 0.3 nm after 40 ns of fluctuation. Figure 9 The fluctuations of B, ) are all within 1nm, without large fluctuations, indicating that the addition of anisaldehyde has little effect on the stability of the amino acid residues in TGL protein, and the formed complex is very stable. Figure 9 As shown in Figure C), the curve fluctuation is stable in the range of 2.5-2.6nm and is stable throughout the process without large fluctuations, indicating that TGL protein and anisaldehyde form a tight and stable complex, and the addition of anisaldehyde does not cause significant changes in the overall structure of the protein. Hydrogen bond analysis is as follows Figure 9 D, the number of bonds between TGL protein and anisaldehyde shows that no stable hydrogen bonds are formed in the time of 0-40ns, while 2-3 hydrogen bonds are formed in the time of 40-100ns, and the curve fluctuates smoothly, indicating that a good hydrogen bond interaction is formed between TGL protein and anisaldehyde after 40ns of movement, and the complex is highly stable. Figure 9 E), the fluctuation is stable throughout the whole process, without large fluctuations, and the fluctuation range is 260nm 2 This indicates that the complex of TGL protein and anisaldehyde is very stable.

[0034] Free energy landscape ( Figure 9 F) shows that the free energy distribution diagram of the complex of TGL protein and anisaldehyde forms a single minimum energy cluster, and the energy cluster distribution is concentrated, indicating that the complex formed between TGL protein and anisaldehyde has good stability. Average binding free energy ( Figure 9G) It can be seen that the average binding free energy between TGL protein and anisaldehyde is -14.6 kcal·mol -1 , indicating that the binding between TGL protein and anisaldehyde is strong. By comparing the molecular dynamics simulation of the complex conformation at five moments ( Figure 9 At five moments (H), 0, 25, 50, 75, and 100 ns, anisaldehyde and TGL protein all bind to the same position without significant changes, indicating that the binding stability between TGL protein and anisaldehyde is very good. Figure 9 I) It can be seen that anisaldehyde forms a good binding with the amino acid residues LEU-412 and TRP-463 in the TGL protein, with binding energies of -1.69 and -1.12 kcal / mol, respectively, indicating that LEU-412 and TRP-463 play a major role in the binding of anisaldehyde to TGL protein.

Claims

1. A TGL protein as a drug target for screening and controlling rice false smut, characterized in that: The target is triglyceride lipase, or TGL protein.

2. The application of triglyceride lipase, namely TGL protein, as a drug target in screening drugs for preventing and controlling rice false smut.

3. A drug for preventing and controlling rice false smut by using triglyceride lipase (TGL protein) as a target for screening, characterized in that: The drug is a botanical fungicide anisaldehyde.

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