Protein Fobut application in regulating pathogenicity of banana fusarium wilt pathogen

Abstract

The application discloses application of protein Fobut in regulation of pathogenicity of banana fusarium wilt fungus. The application obtains a deletion mutant and a back-supplementation mutant of Fobut by constructing a knock-out plasmid and a back-supplementation plasmid of a gene (Fobut) coding protein Fobut of banana fusarium wilt fungus and transforming corresponding protoplasts respectively. It is found through observation of the phenotype of the deletion mutant and the back-supplementation mutant and pathogenicity analysis that the knock-out gene Fobut can obviously reduce the pathogenicity of banana fusarium wilt fungus, and the pathogenicity is restored after back-supplementation, which indicates that the protein Fobut is related to the pathogenicity of banana fusarium wilt fungus and can be used as a target for prevention and treatment of banana fusarium wilt. The application enriches a protein database related to the pathogenicity of banana fusarium wilt fungus and is beneficial to prevention and treatment of banana fusarium wilt.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology. More specifically, it relates to the application of the protein Fobut in regulating the pathogenicity of Fusarium wilt pathogen in bananas. Background Technology

[0002] Banana wilt (Fusarium wilt) is a soil-borne vascular fungal disease caused by Fusarium oxysporum f.sp.cubense (Foc). This disease can lead to reduced banana yields or even total crop failure, severely hindering the development of the banana industry. Currently, there is no effective method to control banana wilt.

[0003] Research on the pathogenic factors and mechanisms of Fusarium wilt in bananas, and the identification of potential targets for its control, is crucial for the effective prevention and control of Fusarium wilt. Therefore, it is necessary to continuously explore genes or proteins related to the pathogenicity of Fusarium wilt in order to achieve effective control of the disease. Summary of the Invention

[0004] This invention provides the application of protein Fobut in regulating the pathogenicity of Fusarium wilt of banana, enriches the protein database related to the pathogenicity of Fusarium wilt of banana, and helps in the prevention and control of Fusarium wilt of banana.

[0005] The first objective of this invention is to provide the application of the protein Fobut in regulating the pathogenicity of Fusarium wilt in bananas.

[0006] A second objective of this invention is to provide the application of formulations that inhibit the expression of the protein Fobut or formulations for knocking out the gene encoding the protein Fobut in the prevention and control of banana wilt disease.

[0007] A third objective of this invention is to provide the use of formulations that inhibit the expression of the protein Fobut or for knocking out the gene encoding the protein Fobut in the preparation of products for the prevention and control of banana wilt disease.

[0008] A fourth objective of this invention is to provide the application of formulations that inhibit the expression of the protein Fobut or formulations for knocking out the gene encoding the protein Fobut in reducing the pathogenicity of Fusarium wilt in bananas.

[0009] A fifth object of the present invention is to provide the use of formulations that inhibit the expression of the protein Fobut or for knocking out the gene encoding the protein Fobut in the preparation of products that reduce the pathogenicity of Fusarium wilt of banana.

[0010] The sixth objective of this invention is to provide a method for reducing the pathogenicity of Fusarium wilt in bananas.

[0011] The seventh objective of this invention is to provide a method for preventing and controlling banana wilt disease.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution:

[0013] In the process of studying the proteomics of Foc4 secretion, this invention discovered an uncharacterized protein with high evolutionary conservation, named FoBut. By constructing a knockout plasmid encoding the FoBut gene and transforming it into *Fusarium wilt* protoplasts, the phenotype of the obtained FoBut knockout mutants and pathogenicity analysis revealed that knocking out the FoBut gene significantly reduced the pathogenicity of *Fusarium wilt*. However, after FoBut was reintroduced, the pathogenicity of the reintroduced mutants was restored, indicating that FoBut is a pathogenicity-related protein of *Fusarium wilt*, regulating the pathogenicity of the fungus. Therefore, this invention seeks protection for the following applications:

[0014] This invention seeks to protect the use of the protein Fobut in regulating the pathogenicity of Fusarium wilt in bananas.

[0015] Specifically, the amino acid sequence of the protein Fobut described in this invention is shown in SEQ ID NO.2.

[0016] Specifically, the application is the use of protein Fobut in reducing the pathogenicity of banana wilt pathogen.

[0017] More specifically, the application is achieved by inhibiting the expression of the protein Fobut or knocking out the gene encoding the protein Fobut.

[0018] As one alternative, the nucleotide sequence of the gene encoding the banana wilt pathogen protein Fobut is shown in SEQ ID NO.1.

[0019] Since banana wilt is a disease caused by *Fusarium wiltii*, reducing its pathogenicity can prevent its occurrence and mitigate its damage. Knocking out or inhibiting the expression of the *Fusarium wiltii* gene *FoBut* can affect the expression of the *FoBut* protein, thereby reducing the pathogenicity of the fungus. Therefore, this invention also claims protection for the use of preparations that inhibit *FoBut* protein expression or preparations for knocking out the gene encoding *FoBut* in the control of banana wilt.

[0020] Optionally, the reagent for inhibiting the expression of the Fobut protein is siRNA targeting the FoBut gene.

[0021] The present invention also claims protection for the use of formulations that inhibit the expression of the Fobut protein or for the use of formulations that knock out the gene encoding the Fobut protein in the preparation of products for the prevention and control of banana wilt disease.

[0022] The present invention also claims protection for the use of preparations that inhibit the expression of the protein Fobut or preparations for knocking out the gene encoding the protein Fobut in reducing the pathogenicity of Fusarium wilt of banana.

[0023] The present invention also claims protection for the use of formulations that inhibit the expression of the protein Fobut or for the use of formulations that knock out the gene encoding the protein Fobut in the preparation of products that reduce the pathogenicity of Fusarium wilt of banana.

[0024] Specifically, the amino acid sequence of the protein Fobut is shown in SEQ ID NO.2.

[0025] Specifically, the formulation for knocking out the gene encoding the protein Fobut includes a knockout plasmid, which is constructed based on the filamentous fungal expression vector pCT74.

[0026] The present invention also provides a method for reducing the pathogenicity of Fusarium wilt in bananas, wherein the method comprises: blocking or inhibiting the expression of the protein Fobut of Fusarium wilt in bananas.

[0027] The present invention also provides a method for preventing and controlling banana wilt disease, wherein the method comprises: blocking or inhibiting the expression of the banana wilt pathogen protein Fobut.

[0028] Specifically, the banana wilt disease is caused by the fungus *Fusarium wiltii*. The disease is controlled by blocking or inhibiting the expression of the *Fusarium wiltii* protein Fobut, thereby reducing the pathogenicity of the fungus.

[0029] More specifically, the fungus causing banana wilt is race 4 of the fungus causing banana wilt.

[0030] The present invention has the following beneficial effects:

[0031] This invention constructs knockout and complement plasmids of the gene encoding the protein Fobut, the causal agent of banana wilt, and transforms them into corresponding protoplasts, obtaining Fobut deletion and complement mutants. Phenotypic observation and pathogenicity analysis of the deletion and complement mutants revealed that knocking out the Fobut gene significantly reduces the pathogenicity of banana wilt, while complementation restores pathogenicity. This indicates that the Fobut protein is associated with the pathogenicity of banana wilt and can be used as a target for the control of banana wilt. This invention enriches the database of proteins related to the pathogenicity of banana wilt, which is beneficial for the control of banana wilt. Attached Figure Description

[0032] Figure 1 A schematic diagram illustrating the principle of knocking out the FoBut gene in the banana wilt pathogen.

[0033] Figure 2 This is a schematic diagram of the FoBut gene complementation plasmid for the banana wilt pathogen.

[0034] Figure 3 The results are PCR validation analysis of the hph gene in some hygromycin-positive transformants.

[0035] Figure 4 The results are PCR validation analysis of the FoBut gene in some hygromycin-resistant transformants.

[0036] Figure 5 The results of PCR validation analysis of the FoBut gene in bleomycin-positive transformants.

[0037] Figure 6 Figure A shows the growth of the FoBut knockout mutant under different stress conditions and the statistical analysis results; Figure B shows the corresponding statistical analysis results.

[0038] Figure 7 The results of pathogenicity analysis of FoBut knockout mutants on Brazilian bananas are shown; A in the figure represents the symptoms of Brazilian banana plants and bulbs; B in the figure represents the statistical analysis results of the disease index.

[0039] Different letters in the figure represent significant differences, p < 0.05. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present 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.

[0041] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0042] Example 1: Construction of FoBut gene knockout mutant and complement mutant of Fusarium wilt of banana

[0043] 1. Experimental materials

[0044] (1) Test strains and plants

[0045] The strain of Fusarium wilt used in this embodiment of the invention is race 4 of Fusarium wilt (Foc4), and the test plant is Cavendish (AAA).

[0046] (2) Host bacteria and plasmid vector

[0047] The cloning vector used in this embodiment of the invention is pMD18-T, the gene knockout vector is pCT74, and the gene complementation vector is pCTZN. Specifically, the complementation vector pCTZN is obtained by modifying the pCT74 vector by replacing the fluorescent protein gene (SGFP) and hygromycin phosphotransferase gene (hph) on pCT74 with the bleomycin gene.

[0048] 2. Experimental Methods

[0049] A schematic diagram illustrating the principle of knocking out the FoBut gene in the banana wilt pathogen of this invention is shown below. Figure 1 As shown, the FoBut gene in the banana wilt pathogen was knocked out by replacing it with hph and SGFP through homologous recombination.

[0050] (1) Amplification of upstream and downstream homologous arms of the FoBut gene of Fusarium wilt of banana

[0051] The nucleotide sequence of the FoBut gene from the banana wilt pathogen is shown in SEQ ID NO.1, which is a complete gene sequence. The amino acid sequence of the FoBut protein it encodes is shown in SEQ ID NO.2. In this invention, homologous fragments upstream and downstream of the FoBut gene were identified by comparison in the NCBI database. Sequences of approximately 1200 bp in length were selected upstream and downstream of the FoBut gene as homologous arms, named homologous arm A and homologous arm B, respectively. Corresponding amplification primers were designed and suitable restriction enzyme sites were introduced. The nucleotide sequences of the designed primers are shown in Table 1.

[0052] Table 1 Primers for amplification of FoBut gene homologous arms A and B fragments.

[0053]

[0054] Note: The underlined positions are enzyme cleavage sites.

[0055] Genomic DNA of Foc4 was extracted using the 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 homologous arm A fragment of the FoBut gene (FoBut-A). PCR amplification was performed with primers FoBut-BF and FoBut-BR to obtain the homologous arm B fragment of the FoBut gene (FoBut-B).

[0056] The PCR reaction system used to amplify homologous arm fragments is shown in Table 2.

[0057] Table 2. PCR reaction system used for amplifying homologous arm fragments.

[0058]

[0059] The PCR reaction conditions were as follows: 94℃ for 5 min; 98℃ for 10 sec; 58℃ for 30 sec; 72℃ for 1.2 min, for a total of 30 cycles; 72℃ for 10 min.

[0060] After the PCR reaction was completed, the PCR amplification products were purified and recovered using the OMEGA Cycle Pure Kit.

[0061] (2) Construction of FoBut gene knockout vector

[0062] Referring to the instructions of the pMD18-T Vector Cloning Kit (TakaRa), FoBut-A and FoBut-B were ligated to the pMD18-T vector to obtain recombinant vectors pMD18-T-FoBut-A and pMD18-T-FoBut-B, respectively. Specifically: Take 1 μL of pMD18-T vector, add 4 μL of the above-mentioned PCR recovered product (homologous arm A fragment or homologous arm B fragment) and 5 μL of solution I, mix well and ligate overnight at 16℃; take 10 μL of ligation product and add it to 100 μL of E. coli DH5α competent cells, place on ice for 30 min, then heat shock at 42℃ for 90 s, cool on ice for 5 min; add 800 μL of LB liquid medium, and culture at 37℃ and 150 rpm for 1 h with shaking; centrifuge at 4000 rpm for 5 min, discard the supernatant, keep 100 μL of bacterial solution and mix it with the precipitate, spread it on LB solid medium (containing 50 μg / mL Amp); incubate overnight at 37℃.

[0063] Positive transformants resistant to Amp were selected, and recombinant plasmid DNA was extracted and sequenced for identification. Recombinant plasmid DNA with correct sequencing results was selected to construct the FoBut gene knockout vector. Specifically, pMD18T-FoBut-A and pCT74 vectors were digested with Kpn I and Apa I, respectively. The homologous arm A fragment and the linearized pCT74 vector were recovered. The A fragment was ligated to pCT74 using T4 DNA ligase, and the resulting vector was transformed into *E. coli* DH5α to obtain the recombinant plasmid pCT74-FoBut-A. Following the same method, pMD18-T-FoBut-B and recombinant plasmid pCT74-FoBut-A were digested with EcoR I and Spe I. The B fragment and recombinant plasmid pCT74-FoBut-A were recovered. The B fragment was ligated to pCT74-FoBut-A using T4 DNA ligase, and the resulting vector was transformed into *E. coli* DH5α to obtain the gene knockout vector pCT74-FoBut-KO.

[0064] (3) Amplification of the complement fragment of the FoBut gene

[0065] The FoBut gene complement fragment (FoBut-com) selected in this invention includes a 1932 bp promoter sequence upstream of the FoBut gene and a 475 bp terminator sequence downstream of it. PCR amplification primers were designed and suitable restriction enzyme sites were introduced. The amplification primers for the designed FoBut gene complement fragment are shown in Table 3.

[0066] Table 3 Primers for amplifying the FoBut gene complement fragment

[0067]

[0068] Genomic DNA of Foc4 was extracted using the OMEGA Fungal DNA Kit. Using the extracted genomic DNA as a template, PCR amplification was performed with primers FoBut-comF and FoBut-comR to obtain the complement fragment of the FoBut gene (FoBut-com).

[0069] The PCR reaction system used to amplify the FoBut gene complement fragment is shown in Table 4.

[0070] Table 4. PCR reaction system used for amplifying the FoBut gene complementation fragment.

[0071]

[0072] The PCR reaction conditions were: 94℃ for 5 min; 94℃ for 1 min, 55℃ for 1 min, 72℃ for 3.2 min, for a total of 30 cycles; and 72℃ for 10 min.

[0073] After the PCR reaction was completed, the PCR amplification products were purified and recovered using the OMEGA Cycle Pure Kit.

[0074] (4) Construction of FoBut gene complementation vector

[0075] A schematic diagram of the FoBut gene complementation vector constructed by the present invention for the banana wilt pathogen is shown below. Figure 2As shown in the figure, the construction process was as follows: The FoBut-com and pCTZN vectors were digested with EcoRI and SpeI, respectively, and the FoBut-com fragment and the linearized pCTZN vector were recovered. The FoBut-com fragment and the pCTZN vector were ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into *E. coli* DH5α, positive transformants were selected, and their recombinant plasmids were extracted and identified by restriction enzyme digestion. Restriction enzyme digestion confirmed the successful acquisition of the gene complementation vector pCTZN-FoBut-com.

[0076] (5) Preparation of Foc4 protoplasts

[0077] Foc4 cells were inoculated into Czapek's medium (FeSO4·7H2O 0.018g, KCl 0.5g, K2HPO4·3H2O 1g, MgSO4·7H2O 0.5g, NaNO3 3g, sucrose 30g, and diluted to 1L with distilled water) and cultured at 28℃ with shaking at 150rpm for 3 days. The culture medium was filtered through a cell sieve, and the filtrate was centrifuged at 5000rpm for 10min at 4℃, and the supernatant was discarded. The precipitate was diluted to 800mL with CM liquid medium (tryptone 2g, yeast extract 1g, casein hydrolysate 1g, 20× nitrate 50mL, 1000× vitamin 1mL, 1000× trace element 1mL, pH 1000). 6.5; glucose 10g, adjusted to 200mL) resuspended and diluted to prepare a conidia suspension; the conidia suspension was inoculated into CM liquid medium and cultured at 28℃ with shaking at 120rpm for 11-13h; the mycelium was filtered through a cell sieve and washed 3-5 times with 0.8mol / L NaCl solution to obtain fresh mycelium; the mycelium was mixed with the enzymatic hydrolysate at a volume ratio of 10:1 and enzymatically hydrolyzed at 120rpm for 3h to obtain protoplast hydrolysate; the mycelium was centrifuged at 2000rpm for 10min at 4℃, the supernatant was discarded, and the precipitate was resuspended in pre-cooled STC solution (containing 100mmol / L Tris-HCl pH 7.5, 1.2mol / L sorbitol, 50mmol / L CaCl2); the precipitate was centrifuged, the supernatant was discarded, and the precipitate was resuspended in pre-cooled STC solution to obtain Foc4 protoplast suspension.

[0078] The protoplasts of the banana wilt pathogen knockout mutant were prepared following the same steps as those for preparing the Foc4 protoplasts.

[0079] (6) Transformation of protoplasts

[0080] The transformation of protoplasts from the FoBut gene knockout plasmid of *Fusarium wilt* (the causal agent of banana wilt) is illustrated using this 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 *Fusarium wilt* protoplasts and incubated on ice for 20 min. Freshly prepared PTC transformation buffer (40% PEG4000, 1.2 mol / L sorbitol, 50 mmol / L CaCl2, 10 mmol / L Tris-HCl, pH 7.5) was added, mixed, and incubated on ice for 7 min. Freshly prepared PTC solution was added again, mixed, and incubated on ice for 7 min. Pre-chilled STC was added, mixed, and incubated at room temperature for 7 min. The mixture was centrifuged at 4000 rpm for 15 min at 4°C. 3 mL of the solution was added. The precipitate was resuspended in PDA liquid regeneration medium (200.0g potato, 273.6g sucrose, and distilled water to a final volume of 1L) and cultured at 28℃ with shaking at 100rpm for 13–16h to regenerate the cell wall. The solution was then centrifuged at 4000rpm for 15min, the supernatant was removed, and PDA solid regeneration medium (PDA liquid regeneration medium with 1.5% agar powder and 150μg / mL hygromycin) was added. The mixture was poured onto a plate and incubated in the dark at 28℃ for 2–3 days. Hygromycin-resistant transformants were picked and transferred to PDA medium containing 150μg / mL hygromycin (containing 200.0g potato, 20.0g anhydrous glucose, 15.0g agar, and distilled water to a final volume of 1L) and incubated in the dark at 28℃ for 3–4 days. Single colonies were then picked for identification.

[0081] The transformation of the FoBut gene complementation plasmid of Fusarium wilt of banana was carried out in the same manner as above. The protoplasts used for transformation were the protoplasts of the Fusarium wilt of banana knockout mutant. Bleomycin was used as the antibiotic for screening positive transformants, and the concentration of bleomycin was 200 μg / mL.

[0082] (7) PCR validation analysis of FoBut knockout mutant

[0083] Following the instructions of the OMEGA Fungal DNA Kit, genomic DNA was extracted from the hygromycin-positive transformants and subjected to PCR verification analysis. First, the hph gene fragment was amplified by PCR using primers hph-F / hph-R, followed by the FoBut gene fragment amplification by PCR using primers FoBut-F / FoBut-R. If the hph gene fragment was amplified but the FoBut gene fragment was not, it indicated that the detected transformant was a FoBut knockout mutant.

[0084] hph-F: 5′-TGCTGCTCCATACAAGCCAA-3′;

[0085] hph-R: 5′-GACATTGGGGAGTTCAGCGA-3′.

[0086] FoBut-F: 5′-CAGGTCATCCCAGTTGGTGAA-3′;

[0087] FoBut-R: 5′-GTCTTCATTCCAGAGCCACCT-3′.

[0088] The PCR reaction system used for PCR validation analysis of the FoBut knockout mutant is shown in Table 5.

[0089] Table 5. PCR reaction system used for PCR validation analysis of the FoBut knockout mutant.

[0090]

[0091] The PCR reaction conditions were as follows: 94℃ for 5 min; 94℃ for 1 min, 56℃ for 1 min, 72℃ for 1 min, for a total of 30 cycles; 72℃ for 10 min to obtain the amplification product.

[0092] This invention utilizes homologous recombination to obtain 26 hygromycin-positive transformants by transforming gene knockout vectors into protoplasts of *Fusarium wilt* fungus. Genomic DNA was extracted from these 26 transformants and amplified using hph gene-specific primers. The hph gene was found to be amplified in all 26 transformants. PCR verification analysis results for the hph gene in some hygromycin-positive transformants are shown below. Figure 3 As shown in the figure. Based on this, using FoBut gene-specific primers, the 26 positive transformants amplified to the hph gene by PCR were further analyzed for FoBut gene validation. The results of PCR validation analysis of the FoBut gene in some hygromycin-resistant transformants are shown below. Figure 4 As shown in the figure, among the 26 transformants, 8 transformants did not amplify the FoBut gene fragment, indicating that these 8 transformants are FoBut knockout mutants.

[0093] (9) PCR validation analysis of FoBut complement mutant

[0094] Genomic DNA was extracted from the bleomycin-positive transformants according to the instructions of the OMEGA Fungal DNA Kit and then analyzed by PCR. The FoBut gene fragment was amplified by PCR using primers FoBut-F / FoBut-R.

[0095] The PCR reaction system used for PCR validation analysis of the FoBut complement mutant is shown in Table 6.

[0096] Table 6. PCR reaction system used for PCR validation analysis of the FoBut complementation mutant.

[0097]

[0098] The PCR reaction conditions were as follows: 94℃ for 5 min; 94℃ for 1 min, 56℃ for 1 min, 72℃ for 1 min, for a total of 30 cycles; 72℃ for 10 min to obtain the amplification product.

[0099] This invention utilizes a random insertion method to transform the gene complementation plasmid pCTZN-FoBut-com into the protoplast of *Fusarium wilt* ΔFoBut, obtaining four bleomycin-positive transformants. After extracting genomic DNA from these bleomycin-positive transformants, PCR verification analysis was performed using FoBut gene-specific primers. The PCR verification analysis results of the FoBut gene in the bleomycin-positive transformants are as follows: Figure 5 As shown. By Figure 5 It can be seen that two positive transformants can amplify to the target gene fragment, indicating that these two transformants contain the FoBut gene, that is, these two transformants are FoBut complement mutants (ΔFoBut-com).

[0100] Example 2: Phenotypic observation and stress resistance analysis of FoBut knockout mutants

[0101] 1. Phenotypic observation of FoBut knockout mutant (ΔFoBut)

[0102] (1) Observation of colony morphology and determination of growth rate

[0103] Wild-type Foc4, the knockout mutant ΔFoBut, and the complement mutant ΔFoBut-com were inoculated onto PDA medium and cultured at 28°C in the dark. Colony diameter and morphology were measured on day 5. The results showed that ΔFoBut exhibited no significant difference in colony morphology or growth rate compared to the wild-type banana wilt pathogen.

[0104] (2) Observation on conidium production and germination

[0105] Wild-type Foc4, the knockout mutant ΔFoBut, and the complement mutant ΔFoBut-com were inoculated onto Czapek's medium and cultured at 28°C with shaking at 120 rpm for 3 days. Conidial production was then recorded. Furthermore, conidial suspensions from wild-type Foc4, the knockout mutant ΔFoBut, and the complement mutant ΔFoBut-com were inoculated onto CM medium and cultured at 28°C with shaking at 120 rpm. Samples were taken after 7 hours to observe and record conidial germination. The results showed that the conidial production of the mutant ΔFoBut was not significantly different from that of the wild-type, and its conidial germination was also identical to that of the wild-type, indicating that knocking out FoBut did not affect the production and germination of conidia of the banana wilt pathogen.

[0106] 2. Stress resistance analysis of FoBut knockout mutants

[0107] Wild-type Foc4, the knockout mutant ΔFoBut, and the complement mutant ΔFoBut-com were inoculated onto PDA medium containing 1 mol / L NaCl, 1 mol / L sorbitol, 300 mmol / L H2O2, 0.05% SDS, 100 μg / mL Congo red (CR), and 50 μg / mL fluorescent whitening agent (CFW), respectively. After incubation at 28°C with the medium inverted for 5 days, the growth was observed and statistically analyzed.

[0108] The growth and statistical analysis results of FoBut knockout mutants under different stress conditions are as follows: Figure 6 As shown; Figure 6 In the figure, A represents the growth of the FoBut knockout mutant under different stress conditions; Figure 6 In the figure, B represents the corresponding statistical analysis result. Figure 6 It can be concluded that: ① In PDA medium containing NaCl and sorbitol, ΔFoBut showed no significant difference from the wild type, indicating that FoBut had no effect on the Foc4's resistance to high osmotic pressure; ② Under H2O2 stress, the colonies of the ΔFoBut mutant showed no significant difference from the wild type, indicating that FoBut had no effect on the Foc4's resistance to oxidative stress; ③ In PDA medium containing 100 μg / mL CR, the growth of the mutant ΔFoBut was significantly different from that of the wild type (different letters in the figure indicate significant differences, p < 0.05), indicating that knocking out FoBut affected the cell wall integrity of Foc4.

[0109] Example 3: Pathogenicity analysis of the knockout mutant ΔFoBut

[0110] Take Brazilian bananas with uniform growth at the 4-leaf stage, and use conidia (1×10⁻⁶) from wild-type Foc4, knockout mutant ΔFoBut, and replenishment mutant ΔFoBut-com, respectively.5 The roots were treated with a suspension of (number of plants / mL), with a water treatment group serving as a control. After 40 minutes of treatment, the seedlings were transplanted into sterile nutrient soil and cultured in a plant culture room at 28℃. The plants were then cultured under alternating light and dark conditions for 12 hours / 12 hours. After 28 days, the disease incidence on the leaves and bulbs of the banana seedlings was observed, and the disease index was calculated. Statistical analysis was performed on the disease index. The disease grading criteria were based on the method of Huang Yonghui (2016) (Table 7).

[0111] Table 7 Grading Standards for Banana Fusarium Wilt Disease (Huang Yonghui, 2016)

[0112]

[0113] The disease index is calculated as follows: Disease Index = ∑(Disease Grade × Number of Plants with Disease at That Grade) / (Highest Grade × Total Number of Plants Surveyed) × 100

[0114] The results of the pathogenicity analysis of the FoBut knockout mutant against the Brazilian banana are as follows: Figure 7 As shown; Figure 7 In the diagram, A represents the symptoms of the Brazilian banana plant and its bulb; Figure 7 B in the table represents the statistical analysis results of the disease index. After 28 days of cultivation, observation revealed that the Brazilian banana seedlings treated with water showed no leaf yellowing, and the corms did not change color. After inoculation with the Foc4 wild-type, the entire banana plant showed obvious yellowing on the leaves from bottom to top, and more than 50% of the corm area showed browning; after inoculation with ΔFoBut, only the lower leaves of the banana plant showed yellowing, and the discolored area of ​​the corm did not exceed 20%; after inoculation with the replacement mutant ΔFoBut-com, the upper and lower leaves of the banana plant also showed large-scale yellowing, and more than 50% of the corm area showed browning. Figure 7 (A) Disease index statistical analysis results showed that the disease index of ΔFoBut-com was similar to that of the wild-type Foc4, indicating that the pathogenicity of ΔFoBut-com recovered to the wild-type level. Meanwhile, the disease index of ΔFoBut was significantly lower than that of the wild-type and the complementation mutant, indicating that knocking out the FoBut gene significantly reduced the pathogenicity of Fusarium wilt of banana. These results indicate that knocking out FoBut reduces the pathogenicity of Fusarium wilt of banana.

[0115] 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 changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of protein Fobut in reducing the pathogenicity of Fusarium wilt pathogen in bananas, characterized in that, The application is achieved by knocking out the gene encoding the protein Fobut; the amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. The application of an agent used to knock out the gene encoding the protein Fobut in the control of banana wilt disease, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. The application of a formulation for knocking out the gene encoding the protein Fobut in the preparation of products for controlling banana wilt disease, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

4. The application of a preparation for knocking out the gene encoding the protein Fobut in reducing the pathogenicity of Fusarium wilt in bananas, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

5. The application of a formulation for knocking out the gene encoding the protein Fobut in the preparation of products that reduce the pathogenicity of Fusarium wilt in bananas, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

6. A method for reducing the pathogenicity of Fusarium wilt in bananas, characterized in that, The method is achieved by knocking out the gene encoding the protein Fobut; the amino acid sequence of the protein is shown in SEQ ID NO.

2.

7. A method for controlling banana wilt disease, characterized in that, The method is achieved by knocking out the gene encoding the protein Fobut; the amino acid sequence of the protein is shown in SEQ ID NO.

2.

8. The method according to claim 7, characterized in that, The aforementioned banana wilt disease is caused by the fungus *Fusarium wiltum*. Controlling banana wilt disease involves reducing the pathogenicity of the fungus by blocking or inhibiting the expression of the *Fusarium wiltum* protein, Fobut.

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