CN116715679A - FolIws1 gene targeting agent and application thereof - Google Patents

Abstract

The invention discloses a FolIws1 gene targeting agent and its application. The FolIws1 gene targeting agent includes at least one of the compounds F2058-0186 and STOCK2S-90312. These two compounds can form hydrogen bonds with R288 of the FolIws1 protein, have a π-π stacking effect with F292 of the FolIws1 protein, and interact with FolIws1 R295 of the protein generates van der Waals forces. The present invention has also been experimentally verified that the FolIws1 gene targeting agent can significantly inhibit a variety of pathogenic bacteria - Fusarium oxysporum, Botrytis cinerea, Verticillium dahliae and Magnaporthe oryzae by affecting the normal function of the FolIws1 protein encoded by the FolIws1 gene. It can ultimately achieve the purpose of preventing and curing various plant diseases, such as tomato fusarium wilt. It is simple to use, has no impact on the plant itself, and is safe and reliable.

Description

A FolIws1 gene targeting agent and its application

Technical field

The invention belongs to the field of plant genetic engineering, and specifically relates to a FolIws1 gene targeting agent and its application.

Background technique

Tomato wilt caused by Fusarium oxysporum f.sp.lycopersici is a serious disease in tomato cultivation. It occurs in a large area in my country, with an incidence rate of about 20%. In greenhouses The incidence of disease is relatively high. Tomato wilt can cause plant stunting, yellowing, chlorosis and other symptoms on the leaves. In severe cases, the entire plant will lose water and cause death. In the early stage, there are no obvious changes on the outside of the stems of the diseased plants, but reddish-brown color changes will appear in the vascular bundles of the diseased plants. When the humidity is high, an obvious pink mold layer will appear, which is the conidiophore and conidiophore of the pathogenic bacteria. Conidia. Tomato fusarium wilt can easily cause horizontal expansion in the field, leading to large-scale disease occurrence and serious losses.

Fusarium oxysporum is a soil-borne pathogenic fungus that is distributed worldwide and is a facultative parasitic fungus that can survive in the soil for a long time. So far, no sexual form of this pathogenic fungus has been found, so it is classified as a fungus in the phylum Deuteromycota. Fusarium oxysporum can produce three types of spores, namely large conidia, small conidia and chlamydospores. Fusarium oxysporum has a wide host range and can infect more than 200 species of plants, covering some cash crops and food crops. According to the different host plants that pathogenic bacteria infect, they can be divided into different specialized types. There is a certain specificity between specialized strains. The main reason for this difference is the adaptive selection for different living environments.

Because Fusarium oxysporum can exist in the soil and can produce chlamydospores to cope with different adverse environments. In addition, the pathogen is prone to mutation, so it is difficult to control in production and it is easy to develop some resistance. Currently, there are only one variety of tomatoes grown in production, with large cutting areas. Some planting environments are relatively closed and have high humidity, which can easily lead to the occurrence of diseases and have extremely adverse effects on prevention and control. In view of the characteristics of the disease, there is currently no very efficient prevention and control method in production, but chemical control is still the main method. Due to the widespread use of chemical pesticides, the level of pesticide resistance continues to increase, which brings great difficulties to the prevention and control of plant diseases. Therefore, finding new compounds or fungicides for pathogen control is of great significance for the future development of agriculture.

Contents of the invention

The invention provides a FolIws1 gene targeting agent and its application. The FolIws1 gene is crucial to Fusarium oxysporum and is an essential gene for growth. The present invention obtains two compounds by screening with the FolIws1 gene as a target, and uses them as FolIws1 gene targeting agents. Experimental verification shows that these two compounds have A variety of plant pathogenic bacteria have broad-spectrum bacteriostatic properties.

In order to achieve the above objects of the invention, the present invention is implemented through the following solutions:

The invention provides a FolIws1 gene targeting agent, which includes at least one of compounds F2058-0186 and STOCK2S-90312.

Further, the chemical structural formula of the compound F2058-0186 is:

Further, the chemical structural formula of the compound STOCK2S-90312 is:

Further, the nucleotide sequence of the FolIws1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the FolIws1 protein encoded by it is shown in SEQ ID NO.2.

Furthermore, the FolIws1 gene targeting agent can bind to the FolIws1 protein, thereby targeting the FolIws1 gene.

Furthermore, the FolIws1 gene targeting agent forms a hydrogen bond with R288 of the FolIws1 protein, has a π-π stacking effect with F292 of the FolIws1 protein, and generates a van der Waals force with R295 of the FolIws1 protein.

The present invention also provides the application of the FolIws1 gene targeting agent in inhibiting plant pathogenic bacteria.

Further, the method of using the FolIws1 gene targeting agent is: when the plant grows leaves, spray the FolIws1 gene targeting agent to the entire plant.

Further, the concentration of the FolIws1 gene targeting agent is 8 μM to 100 μM.

Further, the pathogenic bacteria include Fusarium oxysporum, Botrytis cinerea, Verticillium dahliae and Magnaporthe oryzae.

Further, the plants include tomatoes, mung beans, cotton and rice.

Compared with the existing technology, the advantages and technical effects of the present invention are:

1. By constructing the 3D structure of the FolIws1 protein, the present invention directionally screened two small molecule compounds: F2058-0186 and STOCK2S-90312. Both of them can form hydrogen bonds with R288 of the FolIws1 protein, and have π-π stacking effects with F292. R295 generates van der Waals force, thereby achieving the purpose of targeting the FolIws1 gene.

2. The present invention has been experimentally verified that F2058-0186 and STOCK2S-90312 can significantly inhibit various plant pathogenic bacteria such as Fusarium oxysporum, Botrytis cinerea, Verticillium dahliae and Magnaporthe oryzae by affecting the function of FolIws1 protein. Finally, It can achieve the purpose of preventing and treating various plant diseases, such as tomato fusarium wilt, and the method of use is simple, has no impact on the plant itself, and is safe and reliable. Therefore, these two small molecule compounds have a good control effect on a variety of plant pathogenic bacteria. have a broad vision of application.

Description of the drawings

Figure 1 is a diagram of the binding patterns of (A) F2058-0186 and (B) STOCK2S-90312 with FolIws1 protein;

Figure 2 shows that F2058-0186 and STOCK2S-90312 significantly inhibited the sporulation of Fusarium oxysporum at a concentration of 8 μM, where Mock is a negative control without any treatment, and the sporulation amount was measured in 1/10 YEPD liquid medium;

Figure 3 shows that F2058-0186 and STOCK2S-90312 significantly inhibited the pathogenicity of Fusarium oxysporum at a concentration of 8 μM, where Mock is a negative control without any treatment, and the pathogenicity of Fusarium oxysporum was measured using the root dipping method, ( A) Photos taken 20 days after vaccination, (B) Disease index;

Figure 4 shows that F2058-0186 and STOCK2S-90312 significantly inhibited the sporulation of (A) Botrytis cinerea, (B) Verticillium dahliae and (C) Magnaporthe oryzae at a concentration of 8 μM, in which Mock was not treated in any way. negative control;

Figure 5 shows that F2058-0186 and STOCK2S-90312 significantly inhibited the sporulation of (A) Botrytis cinerea, (B) Verticillium dahliae and (C) Magnaporthe oryzae at a concentration of 8 μM, in which Mock was not treated in any way. Negative control, the right side is the disease index or lesion statistics;

Figure 6 shows that F2058-0186 and STOCK2S-90312 have no obvious effect on the growth of host plants at a concentration of 100 μM; tomatoes, mung beans, cotton and rice are sprayed with 100 μM compounds, where Mock is a negative control without any treatment; the left side is the host Photo of the plant after two weeks of continued growth. On the right is the measurement and statistics of the plant height.

Detailed ways

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

The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials, medicines, instruments, etc. used in the following examples are all commercially available products unless otherwise specified. The quantitative statistics in the following examples are all repeated experiments three times, and the average values are taken.

Example 1: Screening of effective agents for Fusarium oxysporum using FolIws1 as a target

The nucleotide sequence of the FolIws1 gene is shown in SEQ ID NO.1; the sequence of the FolIws1 protein encoded by it is shown in SEQ ID NO.2.

The 3D structure of FolIws1 was constructed using MODELLER 9.11 software, and the ChemDiv database was screened using the 3D structure. The results showed that there are two compounds, compound F2058-0186 and STOCK2S-90312, that can bind to the FolIws1 protein.

Among them, the specific chemical structure of compound F2058-0186 is as follows:

The specific chemical structure of compound STOCK2S-90312 is as follows:

The specific results are shown in Figure 1. The compounds F2058-0186 and STOCK2S-90312 form hydrogen bonds with R288 of the FolIws1 protein, have π-π stacking effects with F292 of the FolIws1 protein, and generate van der Waals forces with R295 of the FolIws1 protein.

Example 2: Inhibition test of F2058-0186 and STOCK2S-90312 on Fusarium oxysporum sporulation

Take the bacterial cake of wild-type Fusarium oxysporum Fol cultured on PDA medium for 4 days, place it in 100 mL 1/10 YEPD liquid medium, and shake it. Add F2058-0186 and STOCK2S- at a final concentration of 8 μM to the medium. 90312 and a 1:1 mixture of the two, while adding the same volume of DMSO as a negative control. Incubate for 48 h at 25°C and 180 rpm, collect conidia and count them under a microscope.

The results are shown in Figure 2. Compared with the control group, 8 μM F2058-0186 and STOCK2S-90312 significantly inhibited the spore production of Fusarium oxysporum.

Example 3: F2058-0186 and STOCK2S-90312 inhibit the pathogenicity of Fusarium oxysporum

Take the bacterial cake of wild-type Fusarium oxysporum Fol cultured on PDA medium for 4 days, add it to PDB medium and culture for 48 hours, collect the conidia, and adjust the concentration to 5.0×10 ⁶ /mL. Compounds F2058-0186 and STOCK2S-90312 were dissolved in DMSO respectively, and added to the spore solution to adjust the final concentration to 8 μM, using DMSO as a control. Clean the roots of 3-week-old tomato seedlings and cause micro-wounds. The roots of the treated tomato seedlings were soaked in the spore solution, and 20 minutes later they were planted back into vermiculite to continue culturing. 20 days later, photos were taken and the disease index was calculated.

The results are shown in Figure 3. Compared with the control group, 8 μM of F2058-0186 and STOCK2S-90312 significantly inhibited the pathogenicity of Fusarium oxysporum.

Example 4: F2058-0186 and STOCK2S-90312 inhibit sporulation test of Botrytis cinerea, Verticillium dahliae and Magnaporthe oryzae

1. Inoculate Botrytis cinerea into PDA medium supplemented with 8 μM F2058-0186 and STOCK2S-90312 compounds. DMSO is used as a blank control. After 7 days of culture, conidia on the PDA medium are collected and counted.

2. Inoculate Verticillium dahliae cultured on PDA into liquid Czapek Dox medium, and add F2058-0186 and STOCK2S-90312 compounds to a final concentration of 8 μM. Incubate for 24 hours at 25°C and 180 rpm, collect conidia and count them.

3. Inoculate Magnaporthe oryzae into oatmeal tomato medium (OTA) supplemented with 8 μM F2058-0186 and STOCK2S-90312 compounds. DMSO is used as a blank control. After 10 days of culture, conidia on the OTA medium are collected and counted.

The results are shown in Figure 4. Compared with the control group, 8 μM F2058-0186 and STOCK2S-90312 significantly inhibited the sporulation of Botrytis cinerea, Verticillium dahliae and Magnaporthe oryzae.

Example 5: Pathogenicity test of F2058-0186 and STOCK2S-90312 on inhibiting Botrytis cinerea, Verticillium dahliae and Magnaporthe oryzae

1. Collect conidia of wild-type B05.10 that have been cultured on PDA medium for about 7 days, and use infection buffer (6.7mM KH ₂ PO ₄ , 6.7mM glucose) to adjust the spore concentration to 5.0×10 ⁶ /mL . Compounds F2058-0186 and STOCK2S-90312 were added to the spore solution, and the final concentration was adjusted to 8 μM. DMSO was used as a negative control. The spore liquid was dropped on the center of the mung bean leaves, and after 3 days of moist culture, photos were taken and the diameter of the lesions was measured.

2. Collect the conidia of Magnaporthe oryzae on the OTA medium, and adjust the spore concentration to 1.0×10 ⁵ /mL with sterile water containing 0.1% Tween-20. F2058-0186 and STOCK2S-90312 compounds were added to the spore liquid at a final concentration of 8 μM, and DMSO was used as a negative control. Add the spore mixture dropwise to the rice leaves, keep them moisturized, take photos 4 days later, and measure the diameter of the lesions.

3. Inoculate Verticillium dahliae cultured on PDA into liquid Czapek Dox medium, collect conidia and adjust the concentration to 5.0×10 ⁶ /mL. Compounds F2058-0186 and STOCK2S-90312 were dissolved in DMSO respectively, and added to the spore solution to adjust the final concentration to 8 μM, using DMSO as a control. Soak the roots of the cotton seedlings in the spore solution for 20 minutes. Plant the cotton seedlings again in vermiculite to continue culturing. After 20 days, take photos and count the disease index.

The results are shown in Figure 5. Compared with the control group, 8 μM F2058-0186 and STOCK2S-90312 significantly inhibited the pathogenicity of Botrytis cinerea, Verticillium dahliae and Magnaporthe oryzae.

Example 6: F2058-0186 and STOCK2S-90312 have no obvious effect on the growth of host plants

The two compounds F2058-0186 and STOCK2S-90312 were dissolved in DMSO to 100mM respectively, and 1ml of the mother solution was added to 999mL of water to prepare a final concentration of 100μM F2058-0186 and 100μM STOCK2S-90312 for later use. The entire plants of tomatoes, mung beans, cotton and rice were sprayed with 100 μM of F2058-0186 or 100 μM of STOCK2S-90312 compound. Two weeks later, photos were taken and the height of the plants was measured.

The results are shown in Figure 6. Compared with the control group, the two compounds F2058-0186 and STOCK2S-90312 had no obvious effect on the growth of host plants at a concentration of 100 μM, indicating that these two compounds would not affect the normal growth of plants.

The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still make modifications to the foregoing embodiments. Modifications are made to the recorded technical solutions, or equivalent substitutions are made to some of the technical features; however, these 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 (10)

1. A FolIws1 gene targeting agent, characterized in that the FolIws1 gene targeting agent includes at least one of compounds F2058-0186 and STOCK2S-90312. 2. The FolIws1 gene targeting agent according to claim 1, characterized in that the chemical structural formula of the compound F2058-0186 is: 3. The FolIws1 gene targeting agent according to claim 1, characterized in that the chemical structural formula of the compound STOCK2S-90312 is: 4. The FolIws1 gene targeting agent according to claim 1, characterized in that the nucleotide sequence of the FolIws1 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the FolIws1 protein encoded by it is as SEQ ID NO. 2 shown. 5. The FolIws1 gene targeting agent according to claim 4, characterized in that the FolIws1 gene targeting agent can form a hydrogen bond with R288 of the FolIws1 protein, has a π-π stacking effect with F292 of the FolIws1 protein, and has a π-π stacking effect with FolIws1 protein. R295 of the protein generates van der Waals forces. 6. Application of the FolIws1 gene targeting agent according to any one of claims 1 to 5 in inhibiting plant pathogenic bacteria. 7. The application according to claim 6, characterized in that the method of using the FolIws1 gene targeting agent is: when the plant grows leaves, spray the FolIws1 gene targeting agent to the entire plant. 8. The application according to claim 7, wherein the FolIws1 gene targeting agent is used at a concentration of 8 μM to 100 μM. 9. The application according to claim 6, characterized in that the plant pathogenic bacteria include Fusarium oxysporum, Botrytis cinerea, Verticillium dahliae and Magnaporthe oryzae. 10. The application according to claim 6, characterized in that the plants include tomatoes, mung beans, cotton and rice.