CN117721136A

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

Info

Publication number: CN117721136A
Application number: CN202311667882.2A
Authority: CN
Inventors: 李云锋; 周晓姝; 聂燕芳; 李华平
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2023-12-06
Filing date: 2023-12-06
Publication date: 2024-03-19

Abstract

The invention discloses application of protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt. The invention obtains the deletion mutant and the anaplerotic mutant of the Fobut by constructing knockout plasmid and anaplerotic plasmid of the gene (Fobut) encoding the Fobut of the banana fusarium wilt protein and respectively converting corresponding protoplast. Through observing the phenotypes of the deletion mutant and the anaplerotic mutant and carrying out pathogenicity analysis, the knock-out gene Fobut can obviously reduce the pathogenicity of banana fusarium wilt, and the pathogenicity of the anaplerotic gene Fobut is recovered after anaplerotic, which shows that the protein Fobut is related to the pathogenicity of the banana fusarium wilt and can be used as a target point for preventing and treating the banana fusarium wilt. The invention enriches protein databases related to pathogenicity of banana fusarium wilt and is beneficial to preventing and treating banana fusarium wilt.

Description

Application of protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt

Technical Field

The invention belongs to the technical field of plant genetic engineering. More particularly relates to the application of the protein Fobut in regulating and controlling the pathogenicity of banana fusarium wilt.

Background

Banana wilt (Banana Fusarium wilt) is a soil-borne vascular bundle fungal disease caused by banana wilt bacteria (Fusarium oxysporum f.sp.cube, foc) which can lead to banana yield reduction and even failure, severely restricting the development of banana industry. At present, no method for effectively preventing and treating banana vascular wilt exists.

Research on banana fusarium wilt pathogens and pathogenic mechanisms thereof is carried out, and searching for the banana fusarium wilt pathogens which can be used as a target point for preventing and controlling banana fusarium wilt is a key for effectively preventing and controlling banana fusarium wilt. Therefore, genes or proteins related to the pathogenicity of banana fusarium wilt bacteria are required to be continuously excavated, so that the banana fusarium wilt disease can be effectively prevented and treated.

Disclosure of Invention

The invention provides the application of the protein Fobut in regulating and controlling the pathogenicity of banana fusarium wilt, enriches a protein database related to the pathogenicity of banana fusarium wilt, and is beneficial to preventing and controlling banana fusarium wilt.

The first object of the invention is to provide an application of the protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt.

A second object of the present invention is to provide the use of an agent for inhibiting the expression of protein Fobut or an agent for knocking out the gene encoding protein Fobut for controlling banana vascular wilt.

A third object of the present invention is to provide the use of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out the gene encoding protein Fobut for the preparation of a product for controlling banana vascular wilt.

A fourth object of the present invention is to provide the use of an agent for inhibiting the expression of protein Fobut or an agent for knocking out the gene encoding protein Fobut for reducing the pathogenicity of banana vascular wilt.

A fifth object of the present invention is to provide the use of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out the gene encoding protein Fobut for the preparation of a product for reducing the pathogenicity of banana vascular wilt.

A sixth object of the invention is to provide a method for reducing the pathogenicity of banana vascular wilt.

The seventh object of the invention is to provide a method for preventing and treating banana vascular wilt.

The above object of the present invention is achieved by the following technical scheme:

in the course of research on Foc secretion proteomics, an unknown protein (uncharacterized protein) with high conservation in evolution is found and is named as protein FoBut. The phenotype of the obtained FoBut knockout mutant is observed and pathogenicity analysis is carried out by constructing a knockout plasmid of a gene (FoBut) for encoding the protein FoBut and transferring the FoBut into a banana fusarium wilt protoplast, and the obtained FoBut knockout gene FoBut obviously reduces the pathogenicity of the banana fusarium wilt, and after the FoBut is restored, the pathogenicity of the obtained restoring mutant is restored, so that the protein FoBut is a protein related to the pathogenicity of the banana fusarium wilt and regulates and controls the pathogenicity of the banana fusarium wilt. Accordingly, the present invention claims the following applications:

the invention discloses application of a protein Fobut in regulating and controlling pathogenicity of banana fusarium wilt.

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

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

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

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

In view of the fact that banana vascular wilt is a disease caused by banana vascular wilt, reducing pathogenicity of banana vascular wilt can prevent occurrence of banana vascular wilt and reduce harm of banana vascular wilt. The expression of the banana fusarium wilt gene Fobut or the inhibition gene Fobut can be knocked out to influence the expression of the protein Fobut, so that the pathogenicity of the banana fusarium wilt is reduced. Therefore, the invention also claims the use of a preparation for inhibiting the expression of protein Fobut or a preparation for knocking out the gene encoding protein Fobut for controlling banana vascular wilt.

Optionally, the agent that inhibits protein Fobut expression is an siRNA that targets the gene Fobut.

The invention also claims the application of the preparation for inhibiting the expression of the protein Fobut or the preparation for knocking out the gene encoding the protein Fobut in the preparation of the product for preventing and treating banana vascular wilt.

The invention also claims the application of the preparation for inhibiting the expression of the protein Fobut or the preparation for knocking out the gene encoding the protein Fobut in reducing the pathogenicity of banana fusarium wilt.

The invention also claims the application of the preparation for inhibiting the expression of the protein Fobut or the preparation for knocking out the gene encoding the protein Fobut in the preparation of products for reducing the pathogenicity of banana fusarium wilt.

Specifically, the preparation for knocking out the gene encoding the protein Fobut comprises a knocking-out plasmid thereof, wherein the knocking-out plasmid is constructed based on a filamentous fungus expression vector pCT 74.

The invention also provides a method for reducing the pathogenicity of banana fusarium wilt, which comprises the following steps: blocking or inhibiting the expression of banana fusarium wilt protein Fobut.

The invention also provides a method for preventing and treating banana vascular wilt, which comprises the following steps: blocking or inhibiting the expression of banana fusarium wilt protein Fobut.

Specifically, the banana fusarium wilt is caused by banana fusarium wilt, and the banana fusarium wilt is prevented and controlled by blocking or inhibiting the expression of banana fusarium wilt protein Fobut and reducing the pathogenicity of banana fusarium wilt.

More specifically, the banana fusarium wilt is banana fusarium wilt No. 4 race.

The invention has the following beneficial effects:

the invention obtains the deletion mutant and the anaplerotic mutant of the Fobut by constructing knockout plasmid and anaplerotic plasmid of the gene (Fobut) encoding the Fobut of the banana fusarium wilt protein and respectively converting corresponding protoplast. Through observing the phenotypes of the deletion mutant and the anaplerotic mutant and carrying out pathogenicity analysis, the knock-out gene Fobut can obviously reduce the pathogenicity of banana fusarium wilt, and the pathogenicity of the anaplerotic gene Fobut is recovered after anaplerotic, which shows that the protein Fobut is related to the pathogenicity of the banana fusarium wilt and can be used as a target point for preventing and treating the banana fusarium wilt. The invention enriches protein databases related to pathogenicity of banana fusarium wilt and is beneficial to preventing and treating banana fusarium wilt.

Drawings

FIG. 1 is a schematic diagram of the principle of knocking out FoBut gene of banana vascular wilt.

FIG. 2 is a schematic diagram of the FoBut anaplerotic plasmid of the banana vascular wilt gene.

FIG. 3 shows the result of PCR-validated analysis of gene hph in a portion of hygromycin-positive transformants.

FIG. 4 shows the results of PCR-validated analysis of the FoBut gene in a portion of hygromycin resistant transformants.

FIG. 5 shows the results of PCR-validated analysis of the gene FoBut in bleomycin positive transformants.

FIG. 6 shows growth and statistical analysis results of FoBut knockout mutants under different stress conditions; a in the graph is the growth condition of FoBut knockout mutants under different stress conditions; b in the figure is the corresponding statistical analysis result.

FIG. 7 is a result of a pathogenicity analysis of FoBut knockout mutants on Brazilian banana; a in the figure is the brazil banana plant and bulb symptoms; b in the figure is the result of statistical analysis of the disease index.

The different letters in the graph represent significant differences, p < 0.05.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 construction of FoBut Gene knockout mutant and anaplerotic mutant of Banana fusarium wilt

1. Experimental materials

(1) Test strain and plant

The banana fusarium wilt bacterial strain used in the embodiment of the invention is banana fusarium wilt bacterial No. 4 race (Foc 4), and the plant to be tested is Brazil banana (Cavendsh, AAA).

(2) Host bacterium and plasmid vector

The cloning vector used in the embodiment of the invention is pMD18-T, the gene knockout vector used is pCT74, and the gene complement vector used is pCTZN. Wherein, the repayment vector pCTZN is obtained by replacing a fluorescent protein gene (SGFP) and a hygromycin phosphotransferase gene (hph) on pCT74 with bleomycin genes on the basis of a vector pCT 74.

2. Experimental method

The principle schematic diagram of the invention for knocking out FoBut gene of banana fusarium wilt is shown in figure 1. Namely, foBut genes in banana fusarium wilt are replaced by hph and SGFP through homologous recombination, so that the knockout of the FoBut genes is realized.

(1) Amplification of upstream and downstream homology arms of FoBut gene of banana fusarium wilt

The nucleotide sequence of the FoBut gene of banana fusarium wilt is shown as SEQ ID NO.1, and is a complete gene sequence, and the amino acid sequence of the encoded FoBut protein is shown as SEQ ID NO. 2. The invention finds the upstream and downstream homologous fragments of the FoBut gene by comparison in NCBI database, sequences with the length of about 1200bp are respectively selected as homology arms at the upstream and downstream of the FoBut gene and named as a homology arm A fragment and a homology arm B fragment respectively, corresponding amplification primers are designed, proper enzyme digestion sites are introduced, and the nucleotide sequences of the primers are designed as shown in the table 1.

TABLE 1 amplification primers for FoBut Gene homology arm A fragment and B fragment

Note that: the underlined position is the cleavage site.

Extracting Foc4 genomic DNA with a fungal DNA extraction kit (OMEGA Fungal DNA Kit); PCR amplification is carried out by using the extracted genome DNA as a template and using primers FoBut-AF and FoBut-AR to obtain a homologous arm A fragment (FoBut-A) of the FoBut gene; PCR amplification was performed using the primers FoBut-BF and FoBut-BR to obtain a homology arm B fragment (FoBut-B) of the FoBut gene.

The PCR reaction system used for amplifying the homology arm fragment is shown in Table 2.

TABLE 2 PCR reaction System for amplifying homology arm fragments

The PCR reaction conditions were: reacting at 94 ℃ for 5min; reacting at 98 ℃ for 10sec,58 ℃ for 30sec, and 72 ℃ for 1.2min for 30 cycles; the reaction was carried out at 72℃for 10min.

After the PCR reaction was completed, the PCR amplified product was purified and recovered by using OMEGA Cycle Pure Kit kit.

(2) Construction of FoBut Gene knockout vector

Referring to the specification of the pMD18-T Vector Cloning Kit (TakaRa) kit, foBut-A and FoBut-B were ligated to pMD18-T vectors, respectively, to obtain recombinant vectors pMD18-T-FoBut-A and pMD18-T-FoBut-B. The method comprises the following steps: taking 1 mu L of pMD18-T vector, adding 4 mu L of the PCR recovery product (homologous arm A fragment or homologous arm B fragment) and 5 mu L of solution I respectively, uniformly mixing, and connecting at 16 ℃ overnight; adding 10 μl of the ligation product into 100 μl of Escherichia coli DH5 α competent cells, standing on ice for 30min, performing water bath heat shock at 42deg.C for 90s, and cooling on ice for 5min; adding 800 mu L of LB liquid medium, and culturing at 37 ℃ for 1h by shaking at 150 rpm; centrifuging at 4000rpm for 5min, discarding supernatant, mixing 100 μl of bacterial liquid with the precipitate, and coating on LB solid medium (containing 50 μg/mL Amp); the culture was carried out in an incubator at 37℃overnight.

And selecting positive transformants with Amp resistance, extracting recombinant plasmid DNA, carrying out sequencing identification, and selecting recombinant plasmid DNA with correct sequencing results to construct a FoBut gene knockout vector. Specifically, the pMD18T-FoBut-A and pCT74 vectors are respectively subjected to enzyme digestion by Kpn I and Apa I, a homology arm A fragment and a linearized pCT74 vector are recovered, and the A fragment is connected with pCT74 by using T4 DNA ligase to transform escherichia coli DH5 alpha; the recombinant plasmid pCT74-FoBut-A was obtained. The pMD18-T-FoBut-B and the recombinant plasmid pCT74-FoBut-A are digested with EcoR I and SpeI in the same manner, the B fragment and the recombinant plasmid pCT74-FoBut-A are recovered, and the B fragment is ligated with pCT74-FoBut-A by using T4 DNA ligase to transform E.coli DH 5. Alpha; and obtaining the gene knockout vector pCT74-FoBut-KO through enzyme digestion identification.

(3) Amplification of the anaplerotic fragment of the FoBut Gene

The patch segment (FoBut-com) of the FoBut gene selected by the invention comprises the steps of selecting a promoter sequence with the length of 1932bp at the upstream of the FoBut gene and selecting a terminator sequence with the length of 475bp at the downstream of the FoBut gene, designing PCR amplification primers and introducing proper enzyme cutting sites. The amplification primers for the designed FoBut gene make-up fragments are shown in Table 3.

TABLE 3 amplification primers for FoBut Gene make-up fragments

Extracting Foc4 genomic DNA with a fungal DNA extraction kit (OMEGA Fungal DNA Kit); and (3) taking the extracted genome DNA as a template, and carrying out PCR amplification by using the primers FoBut-comF and FoBut-comR to obtain a patch fragment (FoBut-com) of the FoBut gene.

The PCR reaction system used for amplifying the FoBut gene anaplerotic fragment is shown in Table 4.

TABLE 4 PCR reaction System for amplifying FoBut Gene anaplerotic fragments

The PCR reaction conditions were: reacting at 94 ℃ for 5min;94℃for 1min,55℃for 1min and 72℃for 3.2min for 30 cycles; the reaction was carried out at 72℃for 10min.

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

(4) Construction of FoBut Gene repair vector

A schematic diagram of the banana fusarium wilt bacteria FoBut gene anaplerosis vector constructed by the invention is shown in figure 2. The construction process is as follows: digestion of the FoBut-com and pCTZN vectors with EcoR I and SpeI, respectively, to recover the FoBut-com fragment and linearized pCTZN vector; the FoBut-com fragment was ligated with pCTZN vector overnight at 16℃with T4 DNA ligase, the ligation product was transformed into E.coli DH 5. Alpha. And positive transformants were picked up, recombinant plasmids thereof were extracted and subjected to restriction enzyme identification. And successfully obtaining the gene compensation vector pCTZN-FoBut-com through enzyme digestion identification.

(5) Preparation of Foc protoplasts

Foc 4A culture 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, 30g of sucrose, and distilled water to 1L), and culturing at 28 ℃ for 3d at 150 rpm; filtering the culture solution with a cell sieve, centrifuging the filtrate at 5000rpm at 4deg.C for 10min, and discarding the supernatant; the precipitate was resuspended in CM broth (tryptone 2g, yeast extract 1g, casein hydrolysate 1g,20 Xnitrate 50mL,1000 Xvitamin 1mL,1000 Xtrace element 1mL, constant volume to 800mL, pH 6.5; glucose 10g, constant volume to 200 mL) and diluted to give a conidium suspension; inoculating the conidium suspension to a CM liquid culture medium, and shake culturing at 120rpm for 11-13 h at 28 ℃; filtering mycelium by using a cell sieve, and flushing 3-5 times by using 0.8mol/L NaCl solution to obtain fresh mycelium; mixing the enzymolysis liquid and mycelium in a volume-mass ratio of 10:1, and carrying out enzymolysis for 3 hours at 120rpm to obtain protoplast enzymolysis liquid; centrifuging at 2000rpm at 4deg.C for 10min, discarding supernatant, adding pre-chilled STC solution (containing 100mmol/L Tris-HCl pH7.5, 1.2mol/L sorbitol, 50mmol/L CaCl) ₂ ) Re-suspending and precipitating; centrifuging, discarding the supernatant, adding pre-cooled STC, and re-suspending the precipitate to obtain Foc protoplast suspension.

Protoplast of the banana fusarium wilt knock-out mutant is prepared by referring to the preparation steps of Foc protoplast.

(6) Transformation of protoplasts

The transformation of protoplasts of the FoBut gene knockout plasmid of banana vascular wilt is exemplified. Cleavage of the knock-out vector pCT74-FoBut-KO with Kpn I and Spe IObtaining an A-hph-SGFP-B fragment; uniformly mixing the A-hph-SGFP-B fragment with the prepared protoplast of banana vascular wilt bacteria, and carrying out ice bath for 20min; fresh PTC conversion buffer (40% PEG4000,1.2mol/L sorbitol, 50 mmol/LCaCl) was added ₂ 10mmol/L Tris-HCl, pH 7.5), mixing, and standing on ice for 7min; adding the freshly prepared PTC solution again, mixing uniformly, standing on ice for 7min, adding pre-cooled STC, mixing uniformly, and standing at room temperature for 7min; centrifuging at 4000rpm at 4deg.C for 15min; adding 3mL of PDA liquid regeneration culture medium (200.0 g of potato, 273.6g of sucrose and distilled water to 1L) for re-suspending and precipitating, and performing shake culture at 28 ℃ and 100rpm for 13-16 h to regenerate cell walls; centrifuging the solution at 4000rpm for 15min, removing the supernatant, adding a PDA solid regeneration medium (1.5% agar powder and 150 mug/mL hygromycin are added into the PDA liquid regeneration medium), uniformly mixing, pouring into a plate, and culturing in darkness at 28 ℃ for 2-3 d; hygromycin resistant transformants were picked up and transferred to PDA medium (200.0 g potato, 20.0g anhydrous dextrose, 15.0g agar, distilled water to volume 1L) containing 150. Mu.g/mL hygromycin, and incubated in the dark at 28℃for 3-4 d, and single colonies were picked up for identification.

The transformation of the banana fusarium wilt FoBut gene anaplerotic plasmid is the same as that of the banana fusarium wilt knockout mutant, and the antibiotic used for screening positive transformants is bleomycin with the bleomycin concentration of 200 mug/mL.

(7) PCR validation analysis of FoBut knockout mutants

Genomic DNA of the hygromycin positive transformant was extracted and analyzed by PCR according to the instructions of the fungus DNA extraction kit (OMEGA Fungal DNA Kit). Firstly, carrying out PCR amplification on the hph gene fragment by using a primer hph-F/hph-R, and then carrying out PCR amplification analysis on the FoBut gene fragment by using a primer FoBut-F/FoBut-R; if the gene fragment hph can be amplified but the gene fragment FoBut cannot be amplified, the detected transformant is shown to be 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 validation analysis of FoBut knockout mutants is shown in Table 5.

TABLE 5 PCR reaction System for FoBut knockout mutant PCR validation analysis

The PCR reaction conditions were: reacting at 94 ℃ for 5min;94 ℃ for 1min,56 ℃ for 1min and 72 ℃ for 1min, 30 cycles; and (3) reacting for 10min at 72 ℃ to obtain an amplification product.

The invention utilizes a homologous recombination method to obtain 26 hygromycin positive transformants by transforming the gene knockout vector into banana fusarium wilt protoplast. Genomic DNA of 26 hygromycin positive transformants was extracted and amplified by using hph gene-specific primers, and it was found that gene hph was amplified in all of the 26 transformants, and the result of PCR verification analysis of gene hph of a part of hygromycin positive transformants is shown in FIG. 3. On this basis, the PCR verification analysis of the gene FoBut was performed on the 26 positive transformants amplified to the gene hph by the PCR using the FoBut gene-specific primers. The results of the PCR-validated analysis of the gene FoBut in a portion of hygromycin resistant transformants are shown in FIG. 4. As can be seen from the figure, of the 26 transformants, 8 transformants did not amplify the FoBut gene fragment, indicating that these 8 transformants were FoBut knockout mutants.

(9) PCR validation analysis of FoBut make-up mutants

Genomic DNA of the bleomycin positive transformant was extracted and analyzed by PCR as per the protocol of the fungal DNA extraction kit method (OMEGA Fungal DNA Kit). PCR amplification of the gene fragment FoBut was performed with the primer FoBut-F/FoBut-R.

The PCR reaction system used for the PCR validation analysis of the FoBut make-up mutant is shown in Table 6.

Table 6 PCR reaction System for FoBut make-up mutant PCR validation analysis

The invention uses random insertion method to transform gene compensation plasmid pCTZN-FoBut-com into banana fusarium wilt delta FoBut protoplast, to obtain 4 bleomycin positive transformants. After extracting genomic DNA of bleomycin positive transformants, PCR validation analysis was performed on these bleomycin positive transformants using FoBut gene-specific primers. The results of PCR-validated analysis of the gene FoBut in bleomycin positive transformants are shown in FIG. 5. As can be seen from FIG. 5, 2 positive transformants were amplified to the target gene fragment, indicating that these 2 transformants contained the FoBut gene, i.e., these 2 transformants were FoBut-make-up mutants (. DELTA.FoBut-com).

Example 2 phenotypic observations and stress resistance analysis of FoBut knockout mutants

1. Phenotypic observations of FoBut knockout mutant (. DELTA.FoBut)

(1) Colony morphology observation and growth rate measurement

Respectively inoculating Foc wild type, knockout mutant delta FoBut and anaplerosis mutant delta FoBut-com on PDA culture medium, and culturing at 28deg.C in dark condition; colony diameter was measured at 5d and colony morphology was observed. The results show that there is no significant difference in colony morphology and growth rate of ΔFoBut compared to wild type banana fusarium wilt.

(2) Observation of yield and germination of conidia

Foc4 wild type, knockout mutant ΔFoBut and anaplerotic mutant ΔFoBut-com were inoculated into Charles medium respectively, and cultured with shaking at 28℃and 120rpm for 3d, and then the spore yield was counted. In addition, a suspension of the conidia of Foc wild type, knockout mutant ΔFoBut and anaplerotic mutant ΔFoBut-com was inoculated in CM medium, shake-cultured at 28℃and 120rpm, sampled at 7 hours, and the germination of the conidia was observed and recorded. The result shows that the spore yield of the mutant delta FoBut is not obviously different from that of the wild type, and the germination of the conidium is also not different from that of the wild type, so that the generation and the germination of the conidium of the banana fusarium wilt are not influenced after the FoBut is knocked out.

2. Stress resistance analysis of FoBut knockout mutants

Foc4 wild type, knockout mutant ΔFoBut and make-up mutant ΔFoBut-com were inoculated with 1mol/L NaCl, 1mol/L Sorbitol (Sorbitol), 300mmol/L H, respectively ₂ O ₂ Growth was observed and statistically analyzed on PDA medium, 0.05% SDS, 100. Mu.g/mL Congo Red (CR), 50. Mu.g/mL fluorescent whitening agent (CFW), after 5d incubation in an incubator at 28℃in an inverted position.

The growth conditions and statistical analysis results of the FoBut knockout mutants under different stress conditions are shown in FIG. 6; a in fig. 6 is the growth of the FoBut knockout mutant under different stress conditions; b in fig. 6 is the corresponding statistical analysis result. As can be seen from fig. 6: (1) in PDA medium containing NaCl and sorbitol, there is no obvious difference between ΔFoBut and wild type, indicating that FoBut has no effect on Foc ability of resisting high osmotic pressure; (2) at H ₂ O ₂ Under the stress condition, compared with the wild type, the bacterial colony of the delta FoBut mutant has no obvious difference with the wild type, which proves that the FoBut has no influence on the oxidation stress resistance of Foc 4; (3) the growth of mutant ΔFoBut was significantly different from wild type in PDA medium containing 100 μg/mL CR (different letters in the figure indicate significant differences, p < 0.05), indicating that knockout of FoBut affects the cell wall integrity of Foc.

Example 3 pathogenic analysis of knockout mutant ΔFoBut

Brazil banana with consistent growth vigor and 4 leaf stage is taken, and conidia (1×10) of Foc wild type, knockout mutant ΔFoBut and complementation mutant ΔFoBut-com are respectively used ⁵ individual/mL) suspension was root-dipped, and the fresh water treatment group was used as a control; transplanting the banana seedlings into sterile nutrient soil after 40min of treatment, placing the banana seedlings into a plant culture room at 28 ℃ for culture, alternately culturing the banana seedlings in 12h/12h of light and dark, observing the disease condition of the banana seedlings and the bulbs after 28d, calculating the disease condition index, and carrying out statistical analysis on the disease condition index. Disease grading criteria reference Huang Yonghui (2016) method (Table 7).

TABLE 7 Banana wilt disease grading Standard (Huang Yonghui, 2016)

The disease index calculating method comprises the following steps: disease index = Σ (disease grade x number of plants with disease grade)/(highest grade x total number of plants investigated) ×100

The pathogenicity analysis result of the FoBut knockout mutant on Brazilian banana is shown in FIG. 7; a in fig. 7 is the brazil banana plant and bulb symptoms; b in FIG. 7 is the result of statistical analysis of the disease index. After 28d of culture, the observation shows that no leaf yellowing phenomenon occurs in the Brazilian banana seedlings treated by clear water, and the bulb does not change color. After Foc wild type inoculation, the whole banana plant is obviously yellowing from the lower part to the upper part, and more than 50% of bulb areas are browned; after the delta FoBut inoculation, only the lower leaves of banana plants are yellowing, and the bulb discoloration area is not more than 20%; after inoculation of the anaplerotic mutant ΔFoBut-com, the upper and lower leaves of the banana plants also showed large area yellowing, and more than 50% of the bulb area was browned (A in FIG. 7). Statistical analysis of the disease index shows that ΔFoBut-com is similar to Foc wild type disease index, indicating that the pathogenicity of ΔFoBut-com is restored to wild type level. Meanwhile, the disease index of delta FoBut is obviously lower than that of wild type and anaplerotic mutants, which indicates that after FoBut gene is knocked out, the pathogenicity of banana fusarium wilt is obviously reduced. The result shows that the FoBut is knocked out to reduce the pathogenicity of banana fusarium wilt.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

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.