CN113201054B - Application of protein FoUPE1 in regulating the pathogenicity of Fusarium oxysporum in banana - Google Patents

Abstract

The invention discloses the application of a protein FoUPE1 in regulating the pathogenicity of Fusarium wilt of banana, and belongs to the field of plant genetic engineering. In the present invention, a gene knockout vector is constructed, and it is introduced into the protoplast of Fusarium oxysporum, and the homologous recombination method is used to knock out the gene from Fusarium oxysporum, to obtain a knockout mutant △Foupe1; △Foupe1 colony growth rate decreased and spore production decreased significantly, and the tolerance to oxidative stress decreased; pathogenicity test showed that the deletion of FoUPE1 significantly reduced the pathogenicity of Foc4; after the gene was complemented, its pathogenicity decreased get restored. The present invention confirms that FoUPE1 is necessary for Fusarium wilt conidium production, response to oxidative stress and pathogenicity. Our research helps to elucidate the pathogenic molecular mechanism of Fusarium wilt and provides target genes for the development of effective fungicides.

Description

Application of protein FoUPE1 in regulating the pathogenicity of Fusarium oxysporum in banana

technical field

The invention belongs to the field of plant genetic engineering, and particularly relates to the application of an unknown protein (Uncharacterized protein) FoUPE1 of Fusarium wilt of banana in regulating the pathogenicity of Fusarium wilt of banana.

Background technique

Banana fusarium wilt is a soil-borne fungal disease caused by Fusarium oxysporum f.sp.cubense (Foc), which occurs in almost all major banana producing areas in my country , becoming one of the most important factors restricting the development of my country's banana industry. Foc can be divided into 3 races, of which race 4 (Foc4) is the most serious, and can harm almost all the current cultivars. Fully excavating the pathogenic genes of Fusarium wilt and carrying out functional research will help to fully understand the pathogenic molecular mechanism of Fusarium wilt and provide a theoretical basis for the prevention and control of Fusarium wilt.

FoUPE1 (Uncharacterized protein) is an unknown protein that does not contain a known domain and is highly conserved in Fusarium. The specific function of FoUPE1 in Fusarium oxysporum is unclear.

SUMMARY OF THE INVENTION

In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide the application of a protein FoUPE1 in regulating the pathogenicity of Fusarium wilt of banana.

The purpose of the present invention is to disclose a new function of a banana Fusarium wilt gene FoUPE1 and its encoded protein FoUPE1. The gene FoUPE1 is the nucleotide sequence shown in position 1 to position 2733 in SEQ ID NO: 1, and the protein FoUPE1 encoded by it is the protein shown in SEQ ID NO: 2. In the present invention, a gene knockout vector is constructed and introduced into the protoplast of Fusarium wilt of banana; the homologous recombination method is used to knock out the gene from Fusarium wilt of banana to obtain a knockout mutant △Foupe1; It was introduced into the △Foupe1 protoplast; the gene was backfilled into the knockout mutant by random insertion to obtain the complemented mutant △Foupe1-com. Knockout mutants of this gene were defective in conidia production and response to oxidative stress, and decreased colony diameter. Pathogenicity assays showed that the knockout mutant △Foupe1 significantly reduced the pathogenicity; the apoplectic mutant △Foupe1-com restored the pathogenicity to the Foc4 level. The above test proves that Fusarium oxysporum FoUPE1 is a pathogenic related gene of Fusarium oxysporum.

The object of the present invention is achieved through the following technical solutions:

The invention provides the application of a protein FoUPE1 in regulating the pathogenicity of Fusarium wilt of banana.

Further, the application of the protein FoUPE1 in regulating the growth and development of Fusarium oxysporum.

Further, the application of the protein FoUPE1 in regulating the sporulation of Fusarium oxysporum.

Further, the application of the protein FoUPE1 in regulating the stress resistance of Fusarium oxysporum.

Preferably, the stress is oxidative stress.

The present invention provides the application of a protein FoUPE1 in preventing and treating Fusarium wilt of banana caused by Fusarium oxysporum, the control is realized by blocking or inhibiting the expression of the gene encoding the protein FoUPE1.

The present invention provides the application of a protein FoUPE1 as a target of a drug for preventing and treating plant diseases, wherein the plant disease is banana fusarium wilt caused by Fusarium oxysporum.

The present invention further provides a method for treating Fusarium wilt of banana caused by Fusarium wilt, comprising blocking or inhibiting the expression of the gene encoding protein FoUPE1 in Fusarium wilt (for example, using antisense RNA or siRNA of the gene, etc.).

Use of a medicament for blocking or inhibiting the expression of the gene encoding protein FoUPE1 in Fusarium wilt (for example, using antisense RNA or siRNA of the gene, etc.) in the preparation of a medicament for controlling the banana caused by Fusarium wilt Fusarium wilt.

Wherein, the described protein FoUPE1, its amino acid sequence is as shown in SEQ ID NO:2, or the amino acid sequence shown in SEQ ID NO:2 is obtained by one or more amino acid replacement, insertion, deletion and still has control banana analogs of the virulence function of Fusarium wilt;

The gene encoding protein FoUPE1, its nucleotide sequence is one of the following A, B, C:

A. The DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 2;

B. DNA sequence as shown in SEQ ID NO: 1;

C, the above A and B obtained by base insertion, deletion or replacement and still have the analog that controls the virulence function of Fusarium oxysporum sp.;

Further, the banana Fusarium wilt is No. 4 physiological race (Foc4) of Fusarium oxysporum.

The knockout vector containing the above-mentioned gene encoding the protein FoUPE1 and the application of the recombinant bacteria in the above-mentioned aspects also belong to the protection scope of the present invention.

Compared with the prior art, the present invention has the following advantages and effects:

The present invention provides a new function of an uncharacterized protein FoUPE1 (Uncharacterized protein) of Fusarium wilt No. 4 (Foc4) and its encoding gene FoUPE1. The gene FoUPE1 is the nucleotide sequence shown in positions 1 to 2733 in SEQ ID NO: 1, and the protein FoUPE1 encoded by it is the protein shown in SEQ ID NO: 2; the FoUPE1 protein does not contain a known structural domain , Uniprot analysis of its subcellular localization is unknown, its biological function in Fusarium oxysporum is not clear. The hygromycin phosphotransferase gene (hph) and the fluorescent protein gene (SGFP) were replaced by the FoUPE1-encoding gene FoUPE1 to obtain the Foc4 knockout mutant △Foupe1; the experiment proved that compared with Foc4, the colony growth rate of △Foupe1 decreased and the spores were produced. The amount of Foc4 was significantly reduced, and the tolerance to oxidative stress was reduced; the pathogenicity test showed that the deletion of FoUPE1 significantly reduced the pathogenicity of Foc4; after the gene was replenished, its pathogenicity was restored. The present invention confirms that FoUPE1 is necessary for Fusarium wilt conidium production, response to oxidative stress and pathogenicity. Our research helps to elucidate the pathogenic molecular mechanism of Fusarium wilt and provides target genes for the development of effective fungicides.

Description of drawings

Figure 1 is a schematic diagram of the construction of a Fusarium wilt gene FoUPE1 knockout vector.

Figure 2 is the agarose gel electrophoresis image of the PCR amplification products of some candidate knockout transformants hph gene; wherein, M: 2000 DNA Marker; lane 1: pCT74 plasmid; lane 2: sterile water; lane 3-6: transformation Subs 71, 76, 113, 117.

Figure 3 is the agarose gel electrophoresis image of the PCR amplification products of the target gene FoUPE1 of some candidate knockout transformants; wherein, M: 5000 DNA Marker; Swimming lane 1: Foc4; Swimming lane 2: sterile water; Swimming lanes 3-6: transformation Subs 71, 76, 113, 117.

Figure 4 is a Southern blot analysis of Foc4 knockout transformants using the FoUPE1 fragment as a probe; wherein, lane 1: Foc4; lane 2-4: transformants 71, 76, and 113.

Figure 5 is a Southern blot analysis of Foc4 knockout transformants using hph fragment as a probe; wherein, lane 1: Foc4; lane 2-4: transformants 71, 76, and 113.

Fig. 6 is a schematic diagram of the complementation vector of Fusarium oxysporum gene FoUPE1.

Figure 7 is the agarose gel electrophoresis image of the amplification products of the FoUPE1 gene of some candidate aplastic transformants; M: 5000 DNAMarker; Swimming lane 1: Foc4; Swimming lane 2: sterile water; 10, 21.

Fig. 8 is the analysis of the knockout mutant ΔFoupe1 and the complementation mutant ΔFoupe1-com under different stress conditions; A: colony diameter; B: colony growth inhibition rate.

Fig. 9 is the pathogenicity analysis of the knockout mutant ΔFoupe1 and the complement mutant ΔFoupe1-com.

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

The test methods that do not specify specific experimental conditions in the following examples are usually in accordance with conventional experimental conditions or in accordance with experimental conditions suggested by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are the reagents and materials obtained from commercial sources.

Example 1

1. Experimental materials

1.1 Test strains and plants

The tested strain was F. fusarium wilt No. 4 physiological race (Foc4), and the tested plant was Brazil banana (Cavendish, AAA) with 4-5 leaves.

1.2 Host bacteria and plasmid vector

The host strain is Escherichia coli DH5α strain. The cloning vector is pMD18-Tvector, the gene knockout vector is the filamentous fungal expression vector pCT74, and the gene complementing vector is pCTZN (which was transformed from the pCT74 plasmid in our laboratory, that is, the SGFP and hph genes on pCT74 were replaced with Bola Zeocin gene).

2. Experimental method

2.1 Amplification of upstream and downstream homologous fragments of Fusarium oxysporum FoUPE1 gene

The construction of the Fusarium oxysporum FoUPE1 gene knockout vector is shown in Figure 1. The upstream and downstream sequences of the FoUPE1 gene with a length of about 1000 bp (named as homology arm A fragment and homology arm B fragment respectively) were selected, and primers were designed (Table 1).

Table 1 Amplification primers for the A and B fragments of the homology arm of the FoUPE1 gene

primer name Primer sequence 5'-3' Restriction sites FoUPE1-AF GG<u>GGTACC</u>GGTGCGACGGGTCCATT KpnI FoUPE1-AR CCG<u>CTCGAG</u>ATGTCTCCGTCTGTTAGCAAGT XhoI FoUPE1-BF TCCC<u>CCCGGG</u>CTGATGACGATTCTCGGGCT XmaI FoUPE1-BR GC<u>TCTAGA</u>ACCTGATCGCCAACTATTCTTTAC XbaI

Foc4 genomic DNA was extracted according to the instructions of OMEGA's column-type fungal DNA extraction kit (OMEGA Fungal DNA Kit); the Foc4 genomic DNA was used as a template, and primers FoUPE1-AF and FoUPE1-AR were used for PCR amplification to obtain the homologue of the FoUPE1 gene. Source arm A fragment (FoUPE1-A); PCR amplification was performed with primers FoUPE1-BF and FoUPE1-BR to obtain the homology arm B fragment (FoUPE1-B) of the FoUPE1 gene.

The PCR reaction system is:

2×TSINGKE Master Mix 12.5μL template DNA 0.5μL FoUPE1-AF/BF(10μmol/L) 0.5μL FoUPE1-AR/BR(10μmol/L) 0.5μL ddH₂O 11.0μL Total 25.0μL

PCR reaction conditions were: 94°C for 5 min; 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, 30 cycles in total; 72°C for 10 min. Use the OMEGA Cycle Pure Kit for clean recovery of PCR amplification products.

2.2 Construction of FoUPE1 gene knockout vector

Referring to the instructions of the pMD 18-T Vector Cloning Kit (TakaRa Company), FoUPE1-A and FoUPE1-B were respectively ligated with T vector to obtain recombinant plasmids pMD18T-FoUPE1-A and pMD18T-FoUPE1-B. Specifically: take 1 μL of pMD18-T vector, add 4 μL of the above PCR recovery product (homologous arm A fragment or homology arm B fragment) and 5 μL solution I respectively, and connect at 16°C for 3-4 h. Add 10 μL of the ligation product to 100 μL of E. coli DH5α competent cells, and place on ice for 30 min; heat shock in a water bath for 90 s at 42°C, and cool on ice for 5 min; add 800 μL of LB liquid medium, and incubate at 150 rpm for 1 h at 37°C; Centrifuge at 4000 rpm for 5 min, discard the supernatant, leave 100 μL of bacterial solution and mix with the precipitate, spread on LB solid medium (containing 50 μg/mL Amp), and culture at 37°C for 8 to 12 h.

The positive transformants with Amp resistance were picked, and the recombinant plasmid DNA was extracted and identified by sequencing. The pMD18T-FoUPE1-A and pCT74 vectors were double digested with KpnI and XhoI, respectively, and the A fragment and the pCT74 vector were recovered. The A fragment was ligated with pCT74 with T ₄ DNA ligase and transformed into E. coli DH5α competent cells; the recombinant plasmid pCT74-FoUPE1-A was obtained. According to the same procedure, pMD18T-FoUPE1-B and recombinant plasmid pCT74-FoUPE1-A were double digested with XmaI and XbaI, respectively, and the B fragment and the recombinant plasmid were recovered. The B fragment was ligated with pCT74-FoUPE1-A with T ₄ DNA ligase, and transformed into E. coli DH5α competent cells; the gene knockout vector pCT74-FoUPE1-KO was obtained by restriction enzyme digestion.

2.3 Amplification of the complement fragment of FoUPE1

The construction of the Fusarium oxysporum FoUPE1 gene complementing vector is shown in Figure 6. A promoter sequence with an upstream length of 1500 bp and a downstream terminator sequence with a length of 500 bp were selected for the FoUPE1 gene, and primers were designed (Table 2).

Table 2 Amplification primers for the complemented fragment of the FoUPE1 gene

primer name Primer sequence 5'-3' Restriction sites FoUPE1-com-F G<u>ACTAGT</u>TGCATAGTGATGAGAAAGTCCAA SpeI FoUPE1-com-R ATAAGAAT<u>GCGGCCGC</u>AAGTCAAAGAGTGGTGAGCACAT NotI

The Foc4 genomic DNA was extracted with a fungal DNA extraction kit (OMEGA Fungal DNA Kit); the genomic DNA was used as a template to perform PCR amplification with primers FoUPE1-com-F and FoUPE1-com-R to obtain a complement fragment of the FoUPE1 gene (FoUPE1-com).

The specific PCR reaction system is:

template DNA 1.0μL FoUPE1-com-F(10μmol/L) 1.0μL FoUPE1-com-R(10μmol/L) 1.0μL 10×Taq Buffer(Mg²⁺plus) 5.0μL dNTPs(2.5mmol/L) 4.0μL ExTaq(5U/μL) 0.5μL ddH₂O 37.5μL Total 50.0μL

PCR reaction conditions were: 94°C for 5 min; 94°C for 1 min, 60°C for 1 min, 72°C for 4 min, a total of 30 cycles; 72°C for 10 min. The PCR amplification products were cleaned and recovered using the OMEGA Cycle Pure Kit.

2.4 Construction of FoUPE1 gene complement vector

The FoUPE1-com and pCTZN vectors were double digested with SpeI and NotI, respectively, and the FoUPE1-com fragment and the pCTZN vector were recovered. The FoUPE1-com fragment was ligated with _pCTZN with T4 DNA ligase, and transformed into E. coli DH5α competent cells; the recombinant plasmid pCTZN-FoUPE1-com was obtained. The gene complementing vector pCTZN-FoUPE1-com was obtained by restriction enzyme digestion.

2.5 Preparation of Foc4 protoplasts

Foc4 was inoculated into Char's medium (FeSO ₄ 7H ₂ O 0.018g, KCl 0.5g, K ₂ HPO ₄ 3H ₂ O 1g, MgSO ₄ 7H ₂ O 0.5g, NaNO ₃ 3g, sucrose 30g, distilled water to volume to 1 L), cultured at 150 rpm at 28°C for 3 days, filtered through a 200-mesh cell sieve to obtain conidia liquid, centrifuged at 10,000 × g for 10 min at 4°C, discarded the supernatant to obtain concentrated conidia liquid, added CM medium (glucose 10.0g, peptone 2.0g, hydrolyzed casein 1.0g, yeast extract 1.0g, 20× nitrate 50mL, 1000×vitamin 1mL, 1000×trace element 1mL, dilute to 1L, adjust pH to 6.5 , wherein the components of 20× nitrate, 1000× vitamins, and 1000× trace elements are disclosed in “201710903818.8, a medium for Fusarium wilt of banana and its application”), so that the final concentration of the conidia solution is 1×10 ⁶ /mL; incubate at 120rpm for 11-12h at 28°C, filter with a 100-mesh cell sieve, and rinse with 0.8mol/L NaCl solution (osmotic pressure stabilizer) for 3-5 times to obtain fresh mycelium. According to the ratio of enzyme solution to mycelium (volume-to-mass ratio 10:1), an appropriate amount of 15g/L crash enzyme solution was added, and the enzyme solution was enzymatically hydrolyzed at 30°C and 120rpm for 3h to obtain a protoplast enzymolysis solution. Centrifuge at 4000 × g for 10 min at 4°C and discard the supernatant. Add 1 mL of pre-cooled STC solution (containing 10 mmol/L Tris-HCl (pH 7.5), 1.2 mol/L sorbitol, 50 mmol/L CaCl ₂ ) to resuspend the pellet; centrifuge, and discard the supernatant. Then 10-20 mL of pre-cooled STC was added to resuspend the precipitate to obtain a Foc4 protoplast suspension, so that the final concentration of protoplasts was about 1×10 ⁷ /mL.

Fusarium wilt of banana knockout mutant protoplast is prepared by referring to the above-mentioned preparation steps of Fusarium wilt of banana protoplast.

2.6 Transformation of Foc4 knockout mutant protoplasts

The knockout vector pCT74-FoUPE1-KO was digested with NotI to obtain a linearized fragment of the knockout vector. Thaw 200 μL of Foc4 protoplasts on ice, add about 5 μg of the linearized fragment of the knockout vector, flick and mix evenly, and let stand on ice for 20 min; alternatively, combine pCTZN-FoUPE1-com plasmid with 200 μL of Fusarium oxysporum to knock out mutants The protoplasts were mixed evenly; 1 mL of PTC (40% PEG-4000, 1.2 mol/L sorbitol, 50 mmol/L CaCl ₂ , 10 mmol/LTris-HCl, pH 7.5) was added dropwise, and after mixing, placed on ice for 15 min; Add 15mL of pre-cooled STC, mix well; centrifuge at 4000rpm for 15min at 4°C; remove the supernatant, leave 5mL of mixed solution, add 3mL of PSB regeneration medium (potato 200.0g, sucrose 273.6g, distilled water to dilute to 1L) The pellet was suspended and incubated at 28°C with shaking at 100rpm for 16h. Centrifuge at 4000 rpm for 15 min at 4°C, remove 5 mL of supernatant, add 12 mL of PSA regeneration medium (add 1.5% agar powder, 150 μg/mL hygromycin or 200 μg/mL bleomycin to PSB regeneration medium), mix and pour plate, cultured at 28°C in the dark for 2-3 days; pick hygromycin (or bleomycin) resistant transformants and transfer to PDA containing 150 μg/mL hygromycin (or 200 μg/mL bleomycin) for culture base (containing 200.0 g of potato, 20.0 g of anhydrous glucose, 15.0 g of agar, and distilled water to 1 L), cultured in the dark at 28°C for 2-3 days, and picked a single colony for identification.

2.7 PCR validation analysis of Foc4 knockout mutants

According to the instructions of the fungal DNA extraction kit (OMEGA Fungal DNA Kit), the genomic DNA of the above-mentioned hygromycin-positive transformants was extracted and analyzed by PCR for verification. The PCR amplification of the hph gene fragment was performed with primers hph-F/hph-R respectively; the PCR amplification analysis of the FoUPE1 gene fragment was performed with primers FoUPE1-F/FoUPE1-R.

hph-F: 5′-TGCTGCTCCATACAAGCCAA-3′,

hph-R: 5′-GACATTGGGGAGTTCAGCGA-3′;

FoUPE1-F: 5′-AGACCAACAACTAGGCAATCCTTT-3′,

FoUPE1-R: 5′-GTCTTGGGCCGGTACTGGTT-3′,

The PCR reaction system is as follows:

template DNA 0.5μL FoUPE1-F/hph-F(10μmol/L) 0.5μL FoUPE1-R/hph-R(10μmol/L) 0.5μL 10×Taq Buffer(Mg²⁺plus) 2.5μL dNTPs(2.5mmol/L) 2.0μL Taq(5U/μL) 0.25μL ddH₂O 18.75μL Total 25.0μL

PCR reaction conditions were: 94°C for 5 min; 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, a total of 30 cycles; 72°C for 10 min to obtain the amplified product.

2.8 PCR verification analysis of FoUPE1 aplasia mutants

According to the instructions of the fungal DNA extraction kit (OMEGAFungal DNA Kit), the genomic DNA of the above-mentioned bleomycin-positive transformants was extracted, and PCR analysis was carried out. PCR amplification of the gene fragment FoUPE1 was performed with primers FoUPE1-F/FoUPE1-R.

The PCR reaction system is as follows:

template DNA 0.5μL FoUPE1-F(10μmol/L) 0.5μL FoUPE1-R(10μmol/L) 0.5μL 10×Taq Buffer(Mg²⁺plus) 2.5μL dNTPs(2.5mmol/L) 2.0μL Taq(5U/μL) 0.25μL ddH₂O 18.75μL Total 25.0μL

PCR reaction conditions were: 94°C for 5 min; 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, a total of 30 cycles; 72°C for 10 min to obtain the amplified product.

2.9 Southern blot analysis of Foc4 knockout mutants

Southern blot hybridization was performed according to the instructions of DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Company). Use primers FoUPE1 probe-F/FoUPE1 probe-R to amplify the target gene probe, and use hph-F/hph-R to amplify the hph gene probe.

FoUPE1 probe-F: 5′-CCACCCTAGAAATCGCTCGT-3′,

FoUPE1 probe-R: 5′-GATCTTAGCGGTCTCAGCGT-3′,

hph-F: 5′-TGCTGCTCCATACAAGCCAA-3′,

hph-R: 5′-GACATTGGGGAGTTCAGCGA-3′;

The PCR amplification system of the DNA probe is as follows:

template DNA 1.0μL FoUPE1 probe-F/hph-F(20μmol/L) 1.0μL FoUPE1 probe-R/hph-R(20μmol/L) 1.0μL 10×Ex Taq Buffer(Mg²⁺plus) 5.0μL dNTPs(2.5mmol/L) 4.0μL Ex Taq (5U/μL) 0.5μL ddH₂O 37.5μL Total 50.0μL

PCR reaction conditions were: 94°C for 5 min; 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, a total of 30 cycles; 72°C for 10 min to obtain the amplified product.

2.10 Phenotypic observation of the Foc4 knockout mutant △Foupe1 and the apoplastic mutant △Foupe1-com

(1) Observation of colony morphology and determination of growth rate. Foc4, ΔFoupe1 and ΔFoupe1-com were respectively inoculated on PDA medium and cultured at 28°C in the dark. On the 5th day, the colony diameter was measured by the cross method, and the colony morphology was observed. 3 replicates were set up for each treatment.

(2) The acquisition of conidia. The banana Fusarium wilt was inoculated into Cha's medium, cultured at 28°C at 120 rpm, and the spore production was counted after 3 days.

2.11 Stress resistance analysis of knockout mutant △Foupe1 and apoplectic mutant △Foupe1-com

Foc4, △Foupe1 and △Foupe1-com were inoculated into different stress media (containing 1 mol/L NaCl, 1 mol/L sorbitol, 50 mmol/L H ₂ O ₂ , 0.1% SDS, 100 μg/mL fluorescent whitening agent (CFW, respectively). , calcium fluorescent white) and 100 μg/mL Congo red), cultured at 28 °C for 5 d, with PDA medium as blank control, the colony diameter was measured by the cross method, and the colony growth inhibition rate under different stress conditions was calculated. Processing sets 3 replicates.

2.12 Pathogenicity analysis of knockout mutant △Foupe1 and apoplectic mutant △Foupe1-com

Brazil bananas at the 4-leaf stage were taken, and the conidia (2 × 10 ⁵ /mL) suspensions of Foc4, △Foupe1 and △Foupe1-com were used for root immersion for 30 min, and then transplanted into nutrient soil; placed in 25 The plants were cultivated indoors at ±1°C, alternately cultivated in light/dark for 12h/12h, and the incidence of leaves and bulbs of banana seedlings was observed after 28d. Foc4 wild strain and sterile water were used as positive and negative controls, respectively.

3 Results and Analysis

3.1 Construction of Fusarium oxysporum wilt FoUPE1 gene knockout vector

The PCR amplification method was used to clone the homology arm A fragment and homology arm B fragment of the FoUPE1 gene with the Foc4 genomic DNA as the template. After sequencing and identification, recombinant plasmids pMD18T-FoUPE1-A and pMD18T-FoUPE1-B were obtained. The recombinant plasmid pCT74-FoUPE1-A was obtained by ligating pMD18T-FoUPE1-A with the pCT74 plasmid; it was double digested with pMD18T-FoUPE1-B, and the gene knockout vector was obtained by DNA ligation, E. coli transformation, and enzyme digestion identification. pCT74-FoUPE1-KO (Figure 1).

3.2 Screening of knockout mutant △Foupe1

3.2.1 PCR verification of gene fragment hph

Using homologous recombination, the gene knockout vector pCT74-FoUPE1-KO was transformed into Fusarium oxysporum protoplasts, and 117 hygromycin-resistant transformants were obtained. After DNA extraction, 117 hygromycin-positive transformants were analyzed by PCR using hph gene-specific primers. The results showed that the hph gene was amplified in the above 117 transformants. The verification results of transformants 71, 76, 113 and 117 are shown in Figure 2.

3.2.2 PCR verification of the gene fragment FoUPE1

Further, using FoUPE1 gene-specific primers, 117 positive transformants amplified by PCR to hph gene were subjected to PCR verification analysis of FoUPE1. The results showed that among the 117 transformants, 4 transformants (transformants 71, 76, 113, 117) did not amplify the FoUPE1 gene, further indicating that these 4 transformants were positive transformants (Figure 3).

3.2.3 Southern blot validation of knockout mutant △Foupe1

Southern blot analysis was performed on the 3 positive transformants (transformants 71, 76, 113) that amplified hph gene but not FoUPE1 gene. The results showed that when the target gene was used as a probe for hybridization, none of the three transformants had hybridization bands (Fig. 4). Hybridization was carried out with hph as a probe, and all three transformants had single-copy bands (Fig. 5). The above experiments further proved that these three transformants were positive transformants.

3.3 Screening of apoplastic mutants

The FoUPE1 gene complement fragment was cloned and obtained by PCR amplification; it was ligated with the pCTZN vector, and the recombinant plasmid pCTZN-FoUPE1-com was obtained through E. coli transformation, Amp resistance screening, plasmid extraction and sequencing identification (Figure 6).

Using the method of random insertion, the gene complement vector pCTZN-FoUPE1-com was transformed into the knockout mutant △Foupe1 (△Foupe1-71) protoplasts, and 21 bleomycin-resistant transformants were obtained. The above transformants were verified by PCR using the FoUPE1 gene-specific primers through the extraction of the genomic DNA of Fusarium wilt of banana ( FIG. 7 ). The results showed that 4 transformants could amplify the target gene, which further indicated that these 4 transformants were positive transformants.

3.4 Determination of the colony morphology and growth rate of the knockout mutant △Foupe1 and the complementing mutant △Foupe1-com

Foc4, knockout mutant △Foupe1 (△Foupe1-71) and apoplectic mutant △Foupe1-com (△Foupe1-71-com-2) were inoculated into PDA medium respectively, and the colony morphology was carried out 5 days after inoculation. Observation and colony diameter determination. The results showed that, compared with Foc4, the growth rate of ΔFoupe1 decreased significantly, while the growth rate of the apoplectic mutant recovered to the level of wild-type Foc4.

3.5 Analysis of spore production of knockout mutant △Foupe1 and apoplectic mutant △Foupe1-com

Foc4, knockout mutant △Foupe1 (△Foupe1-71), and apoplectic mutant △Foupe1-com (△Foupe1-71-com-2) were inoculated in Chase medium respectively, and the spore production was analyzed after culturing for 3 days. . The results showed that the sporulation yield of the knockout mutant △Foupe1 was significantly lower than that of its Foc4, while the sporulation yield of the apoplectic mutant △Foupe1-com recovered to the level of wild-type Foc4.

3.6 Analysis of knockout mutant △Foupe1 and apoplectic mutant △Foupe1-com under different stress conditions

Foc4, △Foupe1 (△Foupe1-71, △Foupe1-113) and △Foupe1-com (△Foupe1-71-com-2) were inoculated in 1 mol/L NaCl, 1 mol/L sorbitol, 0.1% SDS, respectively. , 50mmol/LH ₂ O ₂ , 100μg/mL fluorescent whitening agent (CFW) and 100μg/mL Congo red PDA medium, the colony diameter was measured after culturing at 28 ℃ for 5 days. The results showed that compared with Foc4, △Foupe1 was more sensitive to 50 mmol/L H ₂ O ₂ and to 1 mol/L NaCl, 1 mol/L sorbitol, 0.1% SDS, 100 μg/mL fluorescent whitening agent (CFW) and 100 μg/mL There was no significant difference in the sensitivity of Congo red, indicating that FoUPE1 plays an important role in the response of Foc4 to oxidative stress (Fig. 8).

3.7 Pathogenicity analysis of knockout mutant △Foupe1 and apoplectic mutant △Foupe1-com

Using the wounded root inoculation method, the conidia of Foc4, △Foupe1 (△Foupe1-71) and △Foupe1-com (△Foupe1-71-com-2) were inoculated in Brazil bananas, and observed after 28 days. The results showed that 28 days after inoculation of Foc4 and △Foupe1-com spore liquid, the lower leaves of Brazil banana seedlings appeared yellow, and the yellowed area of leaves accounted for about 50-60% of the leaf area. Longitudinal dissection of the bulbs of the diseased Brazil banana seedlings showed dark brown lesions, and the browning area was close to 50% of the area of the bulb; however, after 28 days of inoculation with △Foupe1-71 spore fluid, the yellowed area of the lower leaves of Brazil banana accounted for about 30% of the leaf area. . Longitudinal dissection of the diseased Brazil banana seedlings showed dark brown lesions, and the browning area accounted for about 25% of the bulb area. This indicated that after the FoUPE1 gene was knocked out, the virulence of Fusarium oxysporum was significantly decreased (Fig. 9).

Therefore, the gene provided by the present invention can be used for the control of plant diseases, especially the banana fusarium wilt caused by Fusarium wilt. In addition, the gene provided by the present invention can be used as a target of a drug for plant disease control. Those skilled in the art can follow the teaching and inspiration of this specification to develop medicines for controlling plant diseases, especially banana wilt.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

sequence listing

<110> South China Agricultural University

Application of <120> protein FoUPE1 in regulating the pathogenicity of Fusarium wilt of banana

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2733

<212> DNA

<213> Artificial Sequence

<220>

<223> Base sequence of FoUPE1 gene

<220>

<222> (1)..(1041)

<223> Non-coding region 1

<220>

<222> (1042)..(1099)

<223> Exon 1

<220>

<222> (1100)..(1155)

<223> Intron 1

<220>

<222> (1156)..(1996)

<223> Exon 2

<220>

<222> (1997)..(2051)

<223> Intron 2

<220>

<222> (2052)..(2349)

<223> Exon 3

<220>

<222> (2350)..(2733)

<223> Non-coding region 2

<400> 1

cgtccagtcc acactggagt ctggagtcca gctcgccctg gcatcgtctt gggccggtac 60

tggttccttt ttggtcttca gtgtctgagt gcccctggga taatgggccc cgaaactggt 120

gactgacctg tcggtgccct gcttttgtcc atgaacttag gttttgccat ccaactcgaa 180

cccaccctgg tagctccccc catgccccat gcaggcggcg ccgaattccc accgtgcact 240

catctcggtc gaccatcttt catctcctcc ctcccatctc ctcctctctt tttctctcgc 300

tccttcgaac ttcttctttc ctctccttgt cggtaatatt ctcgtccagt ttttctcttc 360

acgccactcg ttttcgtcag gctctactct gccagacttt atctagttct aggaatattc 420

ttaactgttc cgtgatatat ctcagtcgtt cgccgagaaa ctctcaagga tttgaagaat 480

caacaccatc caacaaaatc acaggatcct ttccccttac aaattaggta cgtacgaacg 540

ttcatcgctc ggtcgactgg ctcgatccat tgtgaggtgc ttgccgagac gcgcatttac 600

ttggcttgtg ctccttgatt ggtttcgacc gttccgtccc gtctttcttg ccgtcttgtt 660

ttctttcttc cttcccctgc ctcaaataat ctttttgtgt acactcttcc tcctccattc 720

actttgcgat ttggtctatt cttgcctacg attggaattg acgcatctcc tcatcccccc 780

ttcacgcatt ccttccaggt tggttgccat tgaacattcc ctttcctgcc aaagggggcg 840

ttgctatcga gcatcgacat ctccacgaca ttgtactgca gttgcttgct cgacaccact 900

ctacatcagc atcacttctt tcgaggcggt tctactttgg accgacattc atagggacca 960

ccgcgacaga ccagcccaca gttttcttca cgcactcgtg tataatcaac gttatctaat 1020

tgtttgttta tccaggccat aatgcattcc gttaaggtcc tctcggccat tgcggccctc 1080

agtgtctctg ccgtttctgg taggtgaaat cagatacacc gtctcggata tcctgctgac 1140

aacgaccttg tctagctgcc acttgtacca aggacgtcaa gatcaccgaa cctacacaag 1200

tcatcagctg cgatgttgtc gacgccgata tcatcattga caagtctgtt gctggtgccg 1260

ttgtcatcaa cggccccaag cagatcaagg gcaacttcca ggccaagaac gccggtgacc 1320

ttatctctat ctccagtacc tctattaatt ccatcactgg aaagttcgag ctcaacaacc 1380

tcgaggctct caacaacctc gacttctctt ctctcgagag ccttggcgag cttagcttca 1440

tcaagcttcc ccgactcggc gagctgaact tcggtaccga aggcgttact aagatcaaga 1500

gcattcgaat taccgacacc ttcatcagtg atcttagcgg tctcagcgtc gccagtgtcg 1560

agagcttcca gattgacaac aaccgaaaga tgaacgtctt caactccgac cttgttaatg 1620

ttactaccca gctcctgatc ttcgacaatg gcaacgacgt tatggaaatt accatgaaca 1680

agctcgagac tgctgccgag atccagattt ctagcgccaa gtccttcaag gtccctgctc 1740

tccaggaggt gaccaagagc ttgaagttga gcgccaaccc cgagctcaag tctttctccg 1800

cccccaacct gaccacgatc acggagaccc tgtcccttat cgacatgaac aagctcacca 1860

acgtttcctt ccccgccctc gaggagattg gcggcggttt caccatccag aacaacacca 1920

agcttgaggc tattgatgat ttccccaagc tcgagaaggt caccggtggt attgccctcc 1980

gaggtagctt cgagaagtga gtaaaacccc tctagcatat agcatttcca tatctaacga 2040

gcgatttcta gggtggaact tcccaagctc gaccaagtta gtggcagtgt cgttgtttcc 2100

tcgaccaccg atattaagga gttctgtgat tacttcgaca acctcaagaa ggacaagaag 2160

atcgatggtg aggagaagtg cacatccaac aacaaggcgg ccaatgaggg caaggacggt 2220

ggtgaggaga gcgacggctc ttccgagagc agcaacagtg acgatcagaa ggatgctgct 2280

ggcattgtca gcgtcaacat ggctgttctt gctcttgcag gtattgctgc cgttgcccag 2340

ctcttttaag catggagatg aagattgctg gcttttcttt taaaggcgtc attatcgtcg 2400

tcatcatgtt tcctgacttt tctctacgac atattgtgct tggtcaacat ggggcacaaa 2460

atgcttttca tgggtggggg taactgtgtg taaaggataa cctgttagag cttaataatc 2520

gattgtattt ggactttatt cttgagatca tgttcgccga gctctgaaaa ttgacatctc 2580

tcttaactca tcatataaag atgtttagtt tgtgataagc gcttcttatt tcttctgaac 2640

atcgagagtc atcaggaaac cttttcggtc tgcaactgat ataaaggatt gcctagttgt 2700

tggtctatta tgaatataag tatatctcca tcc 2733

<210> 2

<211> 398

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of FoUPE1 protein

<400> 2

Met His Ser Val Lys Val Leu Ser Ala Ile Ala Ala Leu Ser Val Ser

1 5 10 15

Ala Val Ser Ala Ala Thr Cys Thr Lys Asp Val Lys Ile Thr Glu Pro

20 25 30

Thr Gln Val Ile Ser Cys Asp Val Val Asp Ala Asp Ile Ile Ile Asp

35 40 45

Lys Ser Val Ala Gly Ala Val Val Ile Asn Gly Pro Lys Gln Ile Lys

50 55 60

Gly Asn Phe Gln Ala Lys Asn Ala Gly Asp Leu Ile Ser Ile Ser Ser

65 70 75 80

Thr Ser Ile Asn Ser Ile Thr Gly Lys Phe Glu Leu Asn Asn Leu Glu

85 90 95

Ala Leu Asn Asn Leu Asp Phe Ser Ser Leu Glu Ser Leu Gly Glu Leu

100 105 110

Ser Phe Ile Lys Leu Pro Arg Leu Gly Glu Leu Asn Phe Gly Thr Glu

115 120 125

Gly Val Thr Lys Ile Lys Ser Ile Arg Ile Thr Asp Thr Phe Ile Ser

130 135 140

Asp Leu Ser Gly Leu Ser Val Ala Ser Val Glu Ser Phe Gln Ile Asp

145 150 155 160

Asn Asn Arg Lys Met Asn Val Phe Asn Ser Asp Leu Val Asn Val Thr

165 170 175

Thr Gln Leu Leu Ile Phe Asp Asn Gly Asn Asp Val Met Glu Ile Thr

180 185 190

Met Asn Lys Leu Glu Thr Ala Ala Glu Ile Gln Ile Ser Ser Ala Lys

195 200 205

Ser Phe Lys Val Pro Ala Leu Gln Glu Val Thr Lys Ser Leu Lys Leu

210 215 220

Ser Ala Asn Pro Glu Leu Lys Ser Phe Ser Ala Pro Asn Leu Thr Thr

225 230 235 240

Ile Thr Glu Thr Leu Ser Leu Ile Asp Met Asn Lys Leu Thr Asn Val

245 250 255

Ser Phe Pro Ala Leu Glu Glu Ile Gly Gly Gly Phe Thr Ile Gln Asn

260 265 270

Asn Thr Lys Leu Glu Ala Ile Asp Asp Phe Pro Lys Leu Glu Lys Val

275 280 285

Thr Gly Gly Ile Ala Leu Arg Gly Ser Phe Glu Lys Val Glu Leu Pro

290 295 300

Lys Leu Asp Gln Val Ser Gly Ser Val Val Val Ser Ser Thr Thr Asp

305 310 315 320

Ile Lys Glu Phe Cys Asp Tyr Phe Asp Asn Leu Lys Lys Asp Lys Lys

325 330 335

Ile Asp Gly Glu Glu Lys Cys Thr Ser Asn Asn Lys Ala Ala Asn Glu

340 345 350

Gly Lys Asp Gly Gly Glu Glu Ser Asp Gly Ser Ser Glu Ser Ser Asn

355 360 365

Ser Asp Asp Gln Lys Asp Ala Ala Gly Ile Val Ser Val Asn Met Ala

370 375 380

Val Leu Ala Leu Ala Gly Ile Ala Ala Val Ala Gln Leu Phe

385 390 395

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> FoUPE1-AF

<400> 3

ggggtaccgg tgcgacgggt ccatt 25

<210> 4

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> FoUPE1-AR

<400> 4

ccgctcgaga tgtctccgtc tgttagcaag t 31

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> FoUPE1-BF

<400> 5

tccccccggg ctgatgacga ttctcgggct 30

<210> 6

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> FoUPE1-BR

<400> 6

gctctagaac ctgatcgcca actattcttt ac 32

<210> 7

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> FoUPE1-com-F

<400> 7

gactagttgc atagtgatga gaaagtccaa 30

<210> 8

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> FoUPE1-com-R

<400> 8

ataagaatgc ggccgcaagt caaagagtgg tgagcacat 39

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223>hph-F

<400> 9

tgctgctcca tacaagccaa 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223>hph-R

<400> 10

gacattgggg agttcagcga 20

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> FoUPE1-F

<400> 11

agaccaacaa ctaggcaatc cttt 24

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> FoUPE1-R

<400> 12

gtcttgggcc ggtactggtt 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> FoUPE1 probe-F

<400> 13

ccaccctaga aatcgctcgt 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> FoUPE1 probe-R

<400> 14

gatcttagcg gtctcagcgt 20

Claims (7)

1. the application of protein FoUPE1 in reducing the virulence of Fusarium wilt of banana, is characterized in that: The amino acid sequence of the protein FoUPE1 is shown in SEQ ID NO: 2, and the application is realized by knocking out the gene encoding the protein FoUPE1. 2. application according to claim 1, is characterized in that: The application is the application of the protein FoUPE1 in inhibiting the growth and development of Fusarium oxysporum. 3. application according to claim 1, is characterized in that: The application is the application of the protein FoUPE1 in reducing the sporulation of Fusarium oxysporum. 4. application according to claim 1, is characterized in that: The application is the application of the protein FoUPE1 in reducing the antioxidant stress of Fusarium oxysporum. 5. The application according to any one of claims 1 to 4, characterized in that: The nucleotide sequence of the gene encoding the protein FoUPE1 is one of the following A and B: A. The DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 2; B. The DNA sequence shown in SEQ ID NO:1. 6. A method for reducing the pathogenicity of Fusarium oxysporum, characterized in that: the method is realized by knocking out the gene encoding the protein FoUPE1, and the amino acid sequence of the protein FoUPE1 is shown in SEQ ID NO: 2. 7. The method according to claim 6, wherein: The nucleotide sequence of the gene encoding the protein FoUPE1 is one of the following A and B: A. The DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 2; B. The DNA sequence shown in SEQ ID NO:1.

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