CN117721136A - Application of protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt - Google Patents

Abstract

The invention discloses the application of protein Fobut in regulating the pathogenicity of banana Fusarium wilt. The present invention obtains a deletion mutant and a complementation mutant of Fobut by constructing a knockout plasmid and a complementation plasmid encoding the gene encoding the banana Fusarium wilt protein Fobut (Fobut) and transforming the corresponding protoplasts respectively. By observing the phenotypes of deletion mutants and complementation mutants and conducting pathogenicity analysis, it was found that knocking out the gene Fobut can significantly reduce the pathogenicity of banana Fusarium wilt, while its pathogenicity was restored after complementation, indicating that the protein Fobut It is related to the pathogenicity of banana Fusarium wilt and can be used as a target for the control of banana Fusarium wilt. The invention enriches the protein database related to the pathogenicity of banana Fusarium wilt and is beneficial to the prevention and treatment of banana Fusarium wilt.

Description

Application of protein Fobut in regulating the pathogenicity of banana Fusarium wilt

Technical field

The invention belongs to the technical field of plant genetic engineering. More specifically, it relates to the application of the protein Fobut in regulating the pathogenicity of banana Fusarium wilt.

Background technique

Banana Fusarium wilt is a soil-borne vascular fungal disease caused by Fusarium oxysporum f.sp.cubense (Foc). The disease can lead to reduced banana yields or even no harvest, seriously restricting the development of the banana industry. There is currently no effective method to control banana fusarium wilt.

Carrying out research on the pathogenic factors and pathogenic mechanisms of banana Fusarium wilt and finding targets that can be used as targets for banana Fusarium wilt control is the key to effectively preventing and controlling banana Fusarium wilt. Therefore, it is necessary to continuously explore genes or proteins related to the pathogenicity of banana Fusarium wilt in order to achieve effective control of banana Fusarium wilt.

Contents of the invention

The invention provides the application of the protein Fobut in regulating the pathogenicity of banana Fusarium wilt, enriches the protein database related to the pathogenicity of banana Fusarium wilt, and contributes to the prevention and control of banana Fusarium wilt.

The first object of the present invention is to provide the application of protein Fobut in regulating the pathogenicity of banana Fusarium wilt.

The second object of the present invention is to provide the application of a preparation that inhibits the expression of the protein Fobut or a preparation that is used to knock out the gene encoding the protein Fobut in preventing and treating banana fusarium wilt.

The third object of the present invention is to provide the application of a preparation that inhibits the expression of the protein Fobut or a preparation that is used to knock out the gene encoding the protein Fobut in the preparation of products for preventing and treating banana fusarium wilt.

The fourth object of the present invention is to provide the application of a preparation that inhibits the expression of the protein Fobut or a preparation that is used to knock out the gene encoding the protein Fobut in reducing the pathogenicity of banana Fusarium wilt.

The fifth object of the present invention is to provide the application of a preparation that inhibits the expression of the protein Fobut or a preparation that is used to knock out the gene encoding the protein Fobut in the preparation of products that reduce the pathogenicity of banana Fusarium wilt.

The sixth object of the present invention is to provide a method for reducing the pathogenicity of banana Fusarium wilt.

The seventh object of the present invention is to provide a method for preventing and controlling banana fusarium wilt.

The above objects of the present invention are achieved through the following technical solutions:

In the process of studying Foc4 secretion proteomics, the present invention discovered an unknown protein (uncharacterized protein) that is highly conserved in evolution, named protein FoBut. By constructing a knockout plasmid of the gene encoding the protein FoBut (FoBut) and transferring it into banana Fusarium wilt protoplasts, we observed the phenotype of the obtained FoBut knockout mutant and analyzed the pathogenicity. It was found that the knockout gene FoBut significantly reduced the banana pathogenicity. Fusarium wilt pathogenicity, and after complementing FoBut, the pathogenicity of the resulting mutant was restored, indicating that the protein FoBut is a protein related to the pathogenicity of Fusarium wilt in banana and regulates the pathogenicity of Fusarium wilt in banana. Therefore, the present invention claims protection for the following applications:

The present invention claims the application of the protected protein Fobut in regulating the pathogenicity of banana Fusarium wilt.

Specifically, the amino acid sequence of the protein Fobut of the present invention is shown in SEQ ID NO. 2.

Specifically, the application is the application of protein Fobut in reducing the pathogenicity of banana Fusarium wilt.

More specifically, the application is achieved by inhibiting the expression of the protein Fobut or knocking out the gene encoding the protein Fobut.

As one of the options, the nucleotide sequence of the gene encoding the banana Fusarium wilt protein Fobut is shown in SEQ ID NO.1.

Given that banana Fusarium wilt is a disease caused by Fusarium wilt of banana, reducing the pathogenicity of banana Fusarium wilt can prevent the occurrence of banana Fusarium wilt and reduce the harm of banana Fusarium wilt. Knocking out the banana Fusarium wilt gene FoBut or inhibiting the expression of the gene FoBut can affect the expression of the protein Fobut, thereby reducing the pathogenicity of banana Fusarium wilt. Therefore, the present invention also claims the application of a preparation that inhibits the expression of the protein Fobut or a preparation that is used to knock out the gene encoding the protein Fobut in preventing and treating banana fusarium wilt.

Optionally, the agent that inhibits the expression of the protein Fobut is siRNA targeting the gene FoBut.

The present invention also claims the use of a preparation that inhibits the expression of the protein Fobut or a preparation that is used to knock out the gene encoding the protein Fobut in the preparation of products for preventing and treating banana fusarium wilt.

The present invention also claims the application of a preparation that inhibits the expression of the protein Fobut or a preparation that is used to knock out the gene encoding the protein Fobut in reducing the pathogenicity of banana Fusarium wilt.

The present invention also claims the application of a preparation that inhibits the expression of the protein Fobut or a preparation that is used to knock out the gene encoding the protein Fobut in the preparation of products that reduce the pathogenicity of banana Fusarium wilt.

Specifically, the amino acid sequence of the protein Fobut is shown in SEQ ID NO. 2.

Specifically, the preparation for knocking out the gene encoding the protein Fobut includes its knockout plasmid, which is constructed based on the filamentous fungal expression vector pCT74.

The invention also provides a method for reducing the pathogenicity of banana Fusarium wilt, which method is: blocking or inhibiting the expression of banana Fusarium wilt protein Fobut.

The invention also provides a method for preventing and treating banana Fusarium wilt, which method is: blocking or inhibiting the expression of banana Fusarium wilt protein Fobut.

Specifically, the banana Fusarium wilt disease is caused by Fusarium wilt of banana. By blocking or inhibiting the expression of Fusarium wilt protein Fobut and reducing the pathogenicity of Fusarium wilt, banana Fusarium wilt can be prevented and treated.

More specifically, the banana Fusarium wilt fungus is Fusarium wilt banana race No. 4.

The invention has the following beneficial effects:

The present invention obtains a deletion mutant and a complementation mutant of Fobut by constructing a knockout plasmid and a complementation plasmid encoding the gene encoding the banana Fusarium wilt protein Fobut (Fobut) and transforming the corresponding protoplasts respectively. By observing the phenotypes of deletion mutants and complementation mutants and conducting pathogenicity analysis, it was found that knocking out the gene Fobut can significantly reduce the pathogenicity of banana Fusarium wilt, and its pathogenicity was restored after complementation, indicating that the protein Fobut It is related to the pathogenicity of banana Fusarium wilt and can be used as a target for the control of banana Fusarium wilt. The invention enriches the protein database related to the pathogenicity of banana Fusarium wilt and is beneficial to the prevention and treatment of banana Fusarium wilt.

Description of the drawings

Figure 1 is a schematic diagram of the principle of knocking out the FoBut gene of banana Fusarium wilt.

Figure 2 is a schematic diagram of the banana Fusarium wilt gene FoBut anaplerotic plasmid.

Figure 3 shows the PCR verification analysis results of the hph gene in some hygromycin-positive transformants.

Figure 4 shows the PCR verification analysis results of the gene FoBut in some hygromycin-resistant transformants.

Figure 5 shows the PCR verification analysis results of the gene FoBut in the bleomycin-positive transformants.

Figure 6 shows the growth conditions and statistical analysis results of FoBut knockout mutants under different stress conditions; A in the figure shows the growth conditions of FoBut knockout mutants under different stress conditions; B in the figure shows the corresponding statistics Analyze the results.

Figure 7 shows the analysis results of the pathogenicity of FoBut knockout mutants to Brazilian banana; A in the figure shows the symptoms of Brazilian banana plants and bulbs; B in the figure shows the statistical analysis results of disease index.

Different letters in the figure indicate significant differences, p<0.05.

Detailed ways

The invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

Unless otherwise stated, the reagents and materials used in the following examples were all commercially available.

Example 1 Construction of banana Fusarium wilt FoBut gene knockout mutants and complementation mutants

1. Experimental materials

(1) Test strains and plants

The Fusarium wilt fungus strain used in the embodiments of the present invention is Fusarium wilt of banana race 4 (Foc4), and the test plant is Cavendish (AAA).

(2) Host bacteria and plasmid vectors

The cloning vector used in the embodiment of the present invention is pMD18-T, the gene knockout vector used is pCT74, and the gene complement vector used is pCTZN. Wherein, the complementation vector pCTZN is modified based on the vector pCT74 by replacing the fluorescent protein gene (SGFP) and hygromycin phosphotransferase gene (hph) on pCT74 with the bleomycin gene.

2. Experimental methods

The schematic diagram of the principle of knocking out the FoBut gene of banana Fusarium wilt in the present invention is shown in Figure 1. That is, the FoBut gene in Fusarium wilt of banana was replaced with hph and SGFP through homologous recombination, thereby achieving the knockout of the FoBut gene.

(1) Amplification of the upstream and downstream homology arms of the FoBut gene of Fusarium wilt in banana

The nucleotide sequence of the Fusarium wilt banana FoBut gene is shown in SEQ ID NO. 1, which is a complete gene sequence, and the amino acid sequence of the FoBut protein encoded by it is shown in SEQ ID NO. 2. The present invention finds the upstream and downstream homologous fragments of the FoBut gene through comparison in the NCBI database, and selects sequences of about 1200 bp in length upstream and downstream of the FoBut gene as homology arms, which are respectively named homology arm A fragment and Homology arm B fragment, design corresponding amplification primers and introduce appropriate enzyme cutting sites. The nucleotide sequences of the designed primers are shown in Table 1.

Table 1 Amplification primers for fragment A and fragment B of FoBut gene homology arm

Note: The underlined position is the enzyme cutting site.

The genomic DNA of Foc4 was extracted using a fungal DNA extraction kit (OMEGA Fungal DNA Kit); using the extracted genomic DNA as a template, PCR amplification was performed with primers FoBut-AF and FoBut-AR to obtain the homology arm A fragment of the FoBut gene ( FoBut-A); use primers FoBut-BF and FoBut-BR to perform PCR amplification to obtain the homology arm B fragment of the FoBut gene (FoBut-B).

The PCR reaction system used to amplify homology arm fragments is shown in Table 2.

Table 2 PCR reaction system used to amplify homology arm fragments

PCR reaction conditions were: 94°C for 5 min; 98°C for 10 sec, 58°C for 30 sec, 72°C for 1.2 min, a total of 30 cycles; 72°C for 10 min.

After the PCR reaction is completed, the PCR amplification product is purified and recovered using the OMEGA Cycle Pure Kit.

(2)Construction of FoBut gene knockout vector

Referring to the instructions of the pMD18-T Vector Cloning Kit (TakaRa Company), connect FoBut-A and FoBut-B to the pMD18-T vector respectively to obtain the recombinant vectors pMD18-T-FoBut-A and pMD18-T-FoBut-B. Specifically: take 1 μL pMD18-T vector, add 4 μL of the above PCR recovery product (homology arm A fragment or homology arm B fragment) and 5 μL solution I respectively, mix evenly and ligate at 16°C overnight; take 10 μL of the ligation product and add to 100 μL Escherichia coli DH5α competent cells were placed on ice for 30 minutes, heat shocked in a 42°C water bath for 90 seconds, and cooled on ice for 5 minutes; add 800 μL of LB liquid culture medium, and culture with shaking at 37°C and 150 rpm for 1 hour; centrifuge at 4000 rpm for 5 minutes, discard the supernatant, and leave 100 μL Mix the bacterial solution and sediment, spread on LB solid medium (containing 50 μg/mL Amp); culture in a 37°C incubator overnight.

Positive transformants with Amp resistance were selected, the recombinant plasmid DNA was extracted for sequencing identification, and the recombinant plasmid DNA with correct sequencing results was selected to construct the FoBut gene knockout vector. Specifically, the pMD18T-FoBut-A and pCT74 vectors were digested with Kpn I and Apa I respectively, the homology arm A fragment and the linearized pCT74 vector were recovered, and T4 DNA ligase was used to connect the A fragment to pCT74, and then transformed into the large intestine. Bacillus DH5α; the recombinant plasmid pCT74-FoBut-A was obtained. According to the same method, use EcoR I and Spe I to digest pMD18-T-FoBut-B and recombinant plasmid pCT74-FoBut-A, recover B fragment and recombinant plasmid pCT74-FoBut-A, and use T4 DNA ligase to combine B fragment with pCT74 -FoBut-A ligation, transformed into E. coli DH5α; after enzyme digestion and identification, the gene knockout vector pCT74-FoBut-KO was obtained.

(3) Amplification of the complementary fragment of the FoBut gene

The complementation fragment (FoBut-com) of the FoBut gene selected in the present invention includes a promoter sequence with a length of 1932 bp selected upstream of the FoBut gene, and a terminator sequence with a length of 475 bp selected downstream of the FoBut gene. PCR amplification primers are designed and introduced. Suitable enzyme cutting sites. The designed amplification primers for the FoBut gene complement fragment are shown in Table 3.

Table 3 Amplification primers for the complemented fragment of FoBut gene

The genomic DNA of Foc4 was extracted using a fungal DNA extraction kit (OMEGA Fungal DNA Kit); using the extracted genomic DNA as a template, PCR amplification was performed using primers FoBut-comF and FoBut-comR to obtain the complemented fragment of the FoBut gene (FoBut- com).

The PCR reaction system used to amplify the complemented fragment of the FoBut gene is shown in Table 4.

Table 4 PCR reaction system used to amplify FoBut gene complementation fragment

PCR reaction conditions were: 94°C for 5 min; 94°C for 1 min, 55°C for 1 min, 72°C for 3.2 min, a total of 30 cycles; 72°C for 10 min.

After the PCR reaction, use the OMEGA Cycle Pure Kit to purify and recover the PCR amplification products.

(4)Construction of FoBut gene complementation vector

The schematic diagram of the banana Fusarium wilt FoBut gene complementation vector constructed in the present invention is shown in Figure 2. The construction process is as follows: use EcoR I and Spe I to digest the FoBut-com and pCTZN vectors respectively, recover the FoBut-com fragment and the linearized pCTZN vector; use T4 DNA ligase to combine the FoBut-com fragment and the pCTZN vector at 16 The ligation was carried out overnight at ℃, and the ligation product was transformed into E. coli DH5α. Positive transformants were selected, and their recombinant plasmids were extracted and identified by enzyme digestion. After enzyme digestion and identification, the gene complement vector pCTZN-FoBut-com was successfully obtained.

(5) Preparation of Foc4 protoplasts

Foc4 was inoculated into Chad's medium (FeSO ₄ .7H ₂ O 0.018g, KCl 0.5g, K ₂ HPO ₄ .3H ₂ O1g, MgSO ₄ .7H ₂ O 0.5g, NaNO ₃ 3g, sucrose 30g, and measured with distilled water (to 1L), incubate for 3 days with shaking at 28°C and 150rpm; filter the culture medium with a cell sieve, and centrifuge the filtrate at 5000rpm for 10 minutes at 4°C, discard the supernatant; use CM liquid medium (2g tryptone, 1g yeast extract) for precipitation. , casein hydrolyzate 1g, 20× nitrate 50mL, 1000× vitamins 1mL, 1000× trace elements 1mL, dilute to 800mL, pH 6.5; glucose 10g, dilute to 200mL), resuspend and dilute to prepare conidia suspension liquid; inoculate the conidia suspension into CM liquid culture medium, and culture with shaking at 120 rpm for 11 to 13 hours at 28°C; filter the mycelium with a cell sieve, and rinse it with 0.8 mol/L NaCl solution 3 to 5 times to obtain fresh mycelium. Protoplast enzymatic hydrolyzate was obtained by mixing the enzymatic hydrolyzate and mycelium at a volume/mass ratio of 10:1 at 120 rpm for 3 hours to obtain the protoplast enzymatic hydrolyzate; centrifuge at 2000 rpm for 10 min at 4°C, discard the supernatant, and add pre-cooled STC. The solution (containing 100mmol/L Tris-HCl pH 7.5, 1.2mol/L sorbitol, 50mmol/L CaCl ₂ ) resuspends the pellet; centrifuge, discard the supernatant, then add pre-cooled STC to resuspend the pellet to obtain Foc4 protoplasts suspension.

The protoplasts of banana Fusarium wilt fungus knockout mutants were prepared by referring to the above preparation steps of Foc4 protoplasts.

(6) Transformation of protoplasts

Take protoplast transformation of banana Fusarium wilt FoBut gene knockout plasmid as an example. The gene knockout vector pCT74-FoBut-KO was digested with Kpn I and Spe I to obtain the A-hph-SGFP-B fragment; the A-hph-SGFP-B fragment was mixed with the prepared protoplasts of Fusarium wilt of banana. Evenly, keep on ice for 20 minutes; add freshly prepared PTC conversion buffer (40% PEG4000, 1.2mol/L sorbitol, 50mmol/LCaCl ₂ , 10mmol/L Tris-HCl, pH7.5), mix well and let stand on ice. 7min; add the freshly prepared PTC solution again, mix well, let stand on ice for 7min, add pre-cooled STC, mix well, let stand at room temperature for 7min; centrifuge at 4000rpm for 15min at 4℃; add 3mL PDA liquid regeneration medium (Potato 200.0g, sucrose 273.6g, distilled water adjusted to 1L) Resuspend the pellet and culture with shaking at 28°C and 100rpm for 13 to 16h for cell wall regeneration; centrifuge the above solution at 4000rpm for 15min, remove the supernatant and add PDA solid for regeneration. Base (add 1.5% agar powder and 150 μg/mL hygromycin to PDA liquid regeneration medium), mix well, pour the plate, and culture in the dark at 28°C for 2 to 3 days; pick hygromycin-resistant transformants and transfer them to a medium containing 150 μg/mL hygromycin. mL of hygromycin PDA medium (containing 200.0g of potato, 20.0g of anhydrous glucose, 15.0g of agar, and distilled water to 1L), culture it in the dark at 28°C for 3 to 4 days, and pick single colonies for identification.

The transformation of banana Fusarium wilt FoBut gene anaplerotic plasmid is the same as above. The protoplast used for transformation is the protoplast of banana Fusarium wilt knockout mutant. The antibiotic used to screen positive transformants is bleomycin, and the bleomycin concentration is 200 μg/mL.

(7) PCR verification analysis of FoBut knockout mutant

According to the instructions of the Fungal DNA Extraction Kit (OMEGA Fungal DNA Kit), the genomic DNA of the above hygromycin-positive transformants was extracted and PCR verification analysis was performed. First use the primers hph-F/hph-R to perform PCR amplification of the hph gene fragment, and then use the primers FoBut-F/FoBut-R to perform PCR amplification analysis of the FoBut gene fragment; if the gene fragment hph can be amplified but the amplification cannot If the gene fragment FoBut is found, it indicates that the detected transformant is a FoBut knockout mutant.

hph-F: 5′-TGCTGCTCCATACAAGCCAA-3′;

hph-R: 5′-GACATTGGGGAGTTCAGCGA-3′.

FoBut-F: 5′-CAGGTCATCCCAGTTGGTGAA-3′;

FoBut-R: 5′-GTCTTCATTCCAGAGCCACCT-3′.

The PCR reaction system used for PCR verification analysis of FoBut knockout mutants is shown in Table 5.

Table 5 PCR reaction system used for PCR verification analysis of FoBut knockout mutants

The PCR reaction conditions were: 94°C for 5 min; 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min, a total of 30 cycles; 72°C for 10 min to obtain the amplification product.

The present invention utilizes the homologous recombination method and transforms the gene knockout vector into banana Fusarium wilt protoplasts, thereby obtaining 26 hygromycin-positive transformants. Genomic DNA of 26 hygromycin-positive transformants was extracted and amplified using hph gene-specific primers. It was found that the gene hph was amplified in all 26 transformants, and the gene hph of some hygromycin-positive transformants was amplified. The PCR verification analysis results are shown in Figure 3. On this basis, we continued to use FoBut gene-specific primers to conduct PCR verification analysis of the FoBut gene on the 26 positive transformants amplified by the above PCR to the gene hph. The results of PCR verification analysis of the gene FoBut in some hygromycin-resistant transformants are shown in Figure 4. It can be seen from the figure that among the 26 transformants, 8 transformants did not amplify the FoBut gene fragment, indicating that these 8 transformants were FoBut knockout mutants.

(9) PCR verification analysis of FoBut complement mutant

According to the instructions of the fungal DNA extraction kit (OMEGA Fungal DNA Kit), the genomic DNA of the above-mentioned bleomycin-positive transformants was extracted and PCR verification analysis was performed. Use primers FoBut-F/FoBut-R to perform PCR amplification of the gene fragment FoBut.

The PCR reaction system used for PCR verification analysis of FoBut complementation mutants is shown in Table 6.

Table 6 PCR reaction system used in PCR verification analysis of FoBut complement mutants

The PCR reaction conditions were: 94°C for 5 min; 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min, a total of 30 cycles; 72°C for 10 min to obtain the amplification product.

The present invention uses a random insertion method to transform the gene complement plasmid pCTZN-FoBut-com into banana Fusarium wilt bacteria ΔFoBut protoplasts, and obtains four bleomycin-positive transformants. After extracting the genomic DNA of the bleomycin-positive transformants, PCR verification analysis of these bleomycin-positive transformants was performed using FoBut gene-specific primers. The results of PCR verification analysis of the gene FoBut in the bleomycin-positive transformants are shown in Figure 5. As shown in Figure 5, there are two positive transformants that can amplify the target gene fragment, indicating that these two transformants contain the FoBut gene, that is, these two transformants are FoBut complement mutants (ΔFoBut-com).

Example 2 Phenotypic observation and stress resistance analysis of FoBut knockout mutants

1. Phenotypic observation of FoBut knockout mutant (ΔFoBut)

(1) Observation of colony morphology and measurement of growth rate

The Foc4 wild type, knockout mutant ΔFoBut and complementation mutant ΔFoBut-com were inoculated on PDA medium and cultured in the dark at 28°C. The colony diameter was measured on the 5th day and the colony morphology was observed. The results showed that there was no significant difference in the colony morphology and growth rate of ΔFoBut compared with the banana Fusarium wilt wild type.

(2) Observation of conidia production and germination

The Foc4 wild type, knockout mutant ΔFoBut, and complement mutant ΔFoBut-com were inoculated into Chad's medium respectively, and cultured at 28°C and 120 rpm for 3 days with shaking, and then the sporulation amount was counted. In addition, the conidia suspension of Foc4 wild type, knockout mutant ΔFoBut and complement mutant ΔFoBut-com were inoculated into CM medium, cultured at 28°C and 120 rpm with shaking, and samples were taken at 7 h to observe the germination of conidia. situation and record. The results showed that the spore production of the mutant ΔFoBut was not significantly different from that of the wild type, and its conidial germination was also the same as that of the wild type, indicating that deleting FoBut did not affect the production and germination of conidia of Fusarium wilt in banana. .

2. Stress resistance analysis of FoBut knockout mutants

Foc4 wild type, knockout mutant ΔFoBut and complementation mutant ΔFoBut-com were respectively inoculated into cells containing 1mol/L NaCl, 1mol/L Sorbitol (Sorbitol), 300mmol/LH ₂ O ₂ , 0.05% SDS, 100 μg/mL On the PDA medium containing Congo red (CR) and 50 μg/mL fluorescent whitening agent (CFW), the growth conditions were observed and statistically analyzed after incubation at 28°C for 5 days.

The growth conditions and statistical analysis results of the FoBut knockout mutant under different stress conditions are shown in Figure 6; A in Figure 6 shows the growth of the FoBut knockout mutant under different stress conditions; B in Figure 6 is Corresponding statistical analysis results. It can be seen from Figure 6: ① In the PDA medium containing NaCl and sorbitol, ΔFoBut has no significant difference from the wild type, indicating that FoBut has no effect on the ability of Foc4 to resist high osmotic pressure; ② Under H ₂ O ₂ stress conditions, compared with the wild type Compared with the wild type, there is no significant difference between the colonies of the ΔFoBut mutant and the wild type, indicating that FoBut has no effect on Foc4's ability to resist oxidative stress; ③ In PDA medium containing 100 μg/mL CR, the growth of the mutant ΔFoBut is different from that of the wild type. The difference is significant (different letters in the figure indicate significant differences, p<0.05), indicating that knocking out FoBut affects the cell wall integrity of Foc4.

Example 3 Pathogenicity Analysis of Knockout Mutant ΔFoBut

Brazilian bananas at the 4-leaf stage with consistent growth were taken, and the roots were soaked in a suspension of conidia (1 × 10 ⁵ /mL) of the Foc4 wild type, knockout mutant ΔFoBut, and complement mutant ΔFoBut-com. The clean water treatment group was used as a control; after 40 minutes of treatment, it was transplanted into sterile nutrient soil, placed in a plant culture room at 28°C, and cultured in 12h/12h alternating light and dark conditions. After 28 days, the incidence of banana seedling leaves and bulbs was observed and calculated. Condition index, perform statistical analysis on the condition index. The disease classification standard refers to the method of Huang Yonghui (2016) (Table 7).

Table 7 Banana Fusarium Wilt Disease Grading Standards (Huang Yonghui, 2016)

The calculation method of disease index is: disease index = ∑ (disease level × number of plants affected by this disease level) / (highest level × total number of plants investigated) × 100

The results of the analysis of the pathogenicity of FoBut knockout mutants to Brazilian banana are shown in Figure 7; A in Figure 7 shows the symptoms of Brazilian banana plants and bulbs; B in Figure 7 shows the statistical analysis results of disease index. After 28 days of cultivation, it was observed that the leaves of the Brazilian banana seedlings treated with clean water did not show yellowing, and the bulbs did not change color. After Foc4 wild type inoculation, obvious yellowing appeared on the leaves of the entire banana plant from the lower part to the upper part, and more than 50% of the bulb area showed browning; after ΔFoBut inoculation, only the lower leaves of the banana plant showed yellowing, and the bulb The discolored area does not exceed 20%; after inoculation of the complement mutant ΔFoBut-com, the upper and lower leaves of the banana plant also showed large-scale yellowing, and more than 50% of the bulb area was browned (A in Figure 7). The statistical analysis results of disease index showed that the disease index of ΔFoBut-com and Foc4 wild type was similar, indicating that the pathogenicity of ΔFoBut-com returned to the wild type level. At the same time, the disease index of ΔFoBut was significantly lower than that of the wild type and the complement mutant, indicating that the pathogenicity of Fusarium wilt in banana was significantly reduced after knocking out the FoBut gene. The above results show that knocking out FoBut reduces the pathogenicity of Fusarium wilt in banana.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims (10)

1. The application of the protein Fobut in regulating and controlling the pathogenicity of banana fusarium wilt is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.

2. The use according to claim 1, characterized in that the use is of the protein Fobut for reducing the pathogenicity of banana vascular wilt.

3. The use according to claim 2, wherein the use is effected by inhibiting the expression of the protein Fobut or by knocking out the gene encoding the protein Fobut.

4. The application of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out a gene encoding the protein Fobut in preventing and treating banana vascular wilt is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.

5. The application of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out a gene encoding the protein Fobut in the preparation of a product for preventing and treating banana vascular wilt is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.

6. The application of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out a gene encoding the protein Fobut in reducing the pathogenicity of banana fusarium wilt is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.

7. The application of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out a gene encoding the protein Fobut in the preparation of a product for reducing the pathogenicity of banana fusarium wilt is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2.

8. A method for reducing pathogenicity of banana fusarium wilt is characterized by blocking or inhibiting expression of banana fusarium wilt protein Fobut; the amino acid sequence of the protein is shown as SEQ ID NO. 2.

9. A method for preventing and treating banana vascular wilt is characterized by blocking or inhibiting the expression of banana vascular wilt protein Fobut.

10. The method according to claim 9, wherein the banana vascular wilt is caused by banana vascular wilt and the banana vascular wilt is controlled by blocking or inhibiting expression of banana vascular wilt protein Fobut and reducing pathogenicity of banana vascular wilt.