Gene porwp2 for improving production of pythium oligandrum oospores and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and specifically discloses a gene PoRWP2 for improving the production of Pythium oligandrum oospores and an application thereof. The application analyzes and transforms the Pythium oligandrum PoRWP2 gene, which is composed of 837 nucleotides, and the protein coded by the gene contains 278 amino acids, and can regulate the sexual development and oospore formation process of Pythium oligandrum. The application verifies the function of the PoRWP2 gene, provides a new direction for regulating the physiological function of Pythium oligandrum by using the gene and improving the sporulation capacity of the biocontrol bacteria, and provides a theoretical basis and technical support for developing biocontrol agents based on Pythium oligandrum and carrying out biological control of oomycete diseases.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering technology, specifically relating to the gene PoRWP2, which increases the yield of oospores of the biocontrol fungus Pythium oligomaleum, and its application. Background Technology

[0002] The control of plant diseases in agricultural production has long relied on chemical pesticides. While these pesticides can quickly control diseases, they easily lead to pesticide residues, ecological pollution, and the development of pesticide resistance in pathogens, severely hindering the development of green agriculture. Against this backdrop, biological control technologies, with the application of biocontrol bacteria at their core, have become a key direction for replacing chemical control due to their environmentally friendly and sustainable advantages.

[0003] Pythium oligandrum is a biocontrol fungus with great application potential. It can effectively inhibit a variety of plant pathogenic fungi and oomycetes through mechanisms such as niche competition, production of antagonistic metabolites, and hyperparasitism, making it a high-quality strain for developing biocontrol agents. However, commercially available Pythium oligandrum biocontrol agents generally suffer from problems such as low sporulation, weak field colonization, and poor control stability. The core reason is the insufficient production of oospores by Pythium oligandrum. Ooospores, as its dormant reproductive bodies, germinate the following year and directly determine the strain's resistance, colonization ability, and the persistence of its biocontrol effect.

[0004] Therefore, increasing oospore production is the core requirement for overcoming the bottleneck in the application of Pythium oligosporum preparations. It has important theoretical significance and practical application value for solving the application limitations of Pythium oligosporum preparations and promoting the industrialization of biological control technology. Summary of the Invention

[0005] To address the aforementioned technical challenges, this application, while researching the RWP-RK gene family, discovered that the PoRWP2 gene in *Pythium oligoandrogenes* plays a crucial regulatory role in oospore formation: knocking out this gene significantly increases the number of oospores in *Pythium oligoandrogenes*, while overexpression of the gene results in the inability to produce oospores. Therefore, this application aims to regulate its expression using gene knockout technology to increase the oospore yield of *Pythium oligoandrogenes*, thereby enhancing its biocontrol capacity and application value.

[0006] In a first aspect, this application provides the PoRWP2 gene of Pythium oligostere, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Secondly, this application provides the PoRWP2 protein of Pythium oligosterolum, which is encoded by the PoRWP2 gene described in the first aspect.

[0008] Furthermore, the amino acid sequence of the PoRWP2 protein is shown in SEQ ID NO.2.

[0009] Thirdly, this application provides biological material containing the PoRWP2 gene described in the first aspect, wherein the biological material is any one of the following:

[0010] 1) An expression cassette containing the PoRWP2 gene described in the first aspect;

[0011] 2) An expression vector containing the PoRWP2 gene described in the first aspect;

[0012] 3) An expression carrier containing the expression box described in 1);

[0013] 4) Recombinant microorganisms containing the expression vector described in 2);

[0014] 5) Recombinant microorganisms containing the expression vector described in 3).

[0015] Fourthly, this application provides the use of substances capable of reducing the expression level of the PoRWP2 gene described in the first aspect and / or substances capable of reducing the activity of the PoRWP2 protein described in the second aspect in regulating the formation of Oospores of Pythium oligoandrogenes.

[0016] Furthermore, the substance is any of the following:

[0017] A1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the gene PoRWP2 that encodes the protein;

[0018] A2) An expression cassette containing the nucleic acid molecules described in A1);

[0019] A3) A recombinant expression vector containing the nucleic acid molecule described in A1) or the expression cassette described in A2);

[0020] A4) Recombinant microorganisms containing the recombinant expression vector described in A3).

[0021] Furthermore, the nucleic acid molecule is an sgRNA molecule that targets the PoRWP2 gene encoded in the first aspect, and the target sequence of the sgRNA molecule is SEQ ID NO.3 or SEQ ID NO.4.

[0022] Fifthly, this application also provides a method for increasing the yield and activity of *Pythium oligoandrogenes* oospores, the method comprising the steps of reducing the expression level of the PoRWP2 gene as described in the first aspect and / or reducing the activity of the PoRWP2 protein as described in the second aspect.

[0023] Further, the step includes introducing the substance into the oligomale pythium via a CRISPR / Cas9 system.

[0024] Compared with existing technologies, this application discovers the PoRWP2 gene in Pythium oligandrum and verifies its function. By regulating the expression of this gene through genetic engineering, the yield of oospores can be increased. This has important theoretical significance and practical application value for solving the application limitations of Pythium oligandrum preparations and promoting the industrialization of biological control technology. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the PoRWP2 gene knockout.

[0026] Figure 2 Electrophoresis diagrams for identifying PoRWP2 gene knockout mutants; lane 1 is WT, wild-type Pythium oligoandrogenes; lane 2 is EV, control strain with failed protoplast transformation; lanes 3, 4, and 5 are ΔPoRWP2 transformants 1, 2, and 3.

[0027] Figure 3 The cell production and oospore yield of mutant and wild-type strains in solid culture medium are shown.

[0028] Figure 4 The biocontrol effects of mutant and wild-type strains on oomycete and fungal diseases are shown; where CK represents Hefeng 47 without pathogen inoculation, RS represents Rhizoctonia solani, and Fg represents Fusarium graminearum.

[0029] Figure 5 The biocontrol effects of mutant and wild-type strains against oomycete and fungal diseases were evaluated: CK represents Hefeng 47 without pathogen inoculation, RS represents Rhizoctonia solani, and Fg represents Fusarium graminearum. Plant height and root length were measured for five plants of each treatment. Detailed Implementation

[0030] The following embodiments are provided to facilitate a better understanding of this application, but do not limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0031] The formulations of some of the reagents and culture media used in the examples are as follows.

[0032] LB liquid medium: Weigh 10g peptone, 5g yeast extract, and 10g sodium chloride, add distilled water, mix well with a magnetic stirrer, and bring the volume to 1L. Adjust the pH to 7.0 and autoclave at 121℃ for 20min.

[0033] 10% V8 medium: Add 1g of calcium carbonate to 100mL of V8 juice, centrifuge at 2500rpm for 5min or mix well and filter through double-layer gauze. Dilute the supernatant after centrifugation or filtration with water at a ratio of 1:9, and autoclave at 121℃ for 20min. To prepare solid medium, add 1.5% agar powder by volume.

[0034] 0.6M KPYG2 medium: Weigh 1.5g glucose, 1g yeast extract, 1g peptone, 0.1g CaCl2·2H2O, 0.02g cholesterol, 1g corn oil, and 109.302g Manitol powder. Add pure water to a final volume of 200mL and autoclave at 121℃ for 20min. For solid medium, use protoplast transformation agar powder. 1.5% fixation medium is used to activate the strain, and 1% fixation medium is used for covering the strain the following day.

[0035] WA medium: Water agar medium. Weigh 3g of agar powder and add pure water to make up to 200mL.

[0036] 0.5M MES solution: Weigh 4.88g MES or 5.33g hydrated MES and add ultrapure water to 45mL. Adjust the pH to 5.7 with KOH and bring the volume to 50mL. After complete dissolution, sterilize by filtration.

[0037] 0.5M CaCl2 solution: Weigh 7.3505g CaCl2·2H2O and add pure water to make up to 100mL.

[0038] MMg solution: Weigh 18.22g Manitol powder, 4.6g MgCl2·6H2O, 2mL MES solution, add ddH2O to make up to 250mL, and autoclave at 121℃ for 20min.

[0039] W5 solution: Weigh 0.093g KCl, 2.25g NaCl, 4.5g CaCl2·2H2O, and 7.97g Glucose, add ddH2O to make up to 250mL, and autoclave at 121℃ for 20min.

[0040] 0.8M Mantiol solution: Weigh 145.736g of Mantiol powder, add ddH2O to make up to 1L, and autoclave at 121℃ for 20min.

[0041] 40% PEG solution: Weigh 6g of PEG4000 powder, 3.75mL of 0.8M Manitol solution, 3mL of 0.5M CaCl2 solution, and 3mL of ddH2O. Dissolve all the powders and then sterilize by filtration.

[0042] Enzyme lysis buffer for protoplast transformation: Weigh 0.5g of domestic enzyme powder, 10mL of 0.8M Manitol solution, 400μL of 0.5M CaCl2 solution, 800μL of 0.5M KCl solution, 800μL of MES solution, and 8mL of ddH2O. Dissolve all the contents and then sterilize by filtration.

[0043] Piper's medium: Weigh 0.5g KH2PO4, 0.5g Yeast Extract, 0.25g MgSO4·7H2O, 0.001g VB1, 25g Glucose, 1g Asparagines, and 15g agar powder, add water, mix well, and bring the volume to 1L. Autoclave at 121℃ for 20min.

[0044] MTT thiazolyl blue reagent: Dissolve 0.5g of MTT powder in 100mL of 1xPBS solution to a final concentration of 5mg / mL. Filter through a 22μm filter membrane and store at 4℃ protected from light.

[0045] Cotton blue lactophenol oil staining solution: 10 mL lactic acid, 10 mL glycerol, 10 g phenol, and 10 mg trypan blue are dissolved in 10 mL sterile water.

[0046] Example 1

[0047] Based on transcriptomic data analysis of the RWP-RK transcription factor family of *Pythium oligoandrogenesis*, the PoRWP2 gene was found to be highly expressed at multiple developmental stages, indicating potential value for functional studies. The PoRWP2 gene was screened and knocked out for subsequent functional validation. The full-length PoRWP2 gene is 837 bp, and its nucleotide sequence is shown in SEQ ID NO. 1. The full-length amino acid sequence it encodes is 278 amino acids, and its amino acid sequence is shown in SEQ ID NO. 2.

[0048] Example 2: Knockout Analysis of the PoRWP2 Gene

[0049] (1) Construction of knockout vector

[0050] Knockout target 1: 5'-ACTGGCGCGAGGAGAGGACACGG-3' (SEQ ID NO.3) and knockout target 2: 5'-CGCCGCGCTGGCCTCTCTGTCGG-3' (SEQ ID NO.4) were selected, involving sgRNA1 and sgRNA2. Amplification was performed using primers PoRWP2-sgRNA1-F1 / R1 and PoRWP2-sgRNA2-F2 / R2, and the sgRNA1 and sgRNA2 fragments were recovered from the products. Then, the pYF515 empty vector was digested with Nhe I and Bsa I (digestion system: total volume 50 μL, CUTSMART(10X) 5 μL, NHEI 1 μL, BSAI 1 μL, vector pyf515 1 μg / recovered sgRNA1 fragment 20 μL, ddH2O to make up the volume). The digested vector was ligated with the recovered sgRNA fragment using T4 ligase, and then transformed into E. coli and validated by colony PCR. Positive colonies were selected for testing to obtain the vector pYF515.

[0051] The gene knockout was achieved using homologous chromosome recombination. A constructed vector was used to replace the coding sequence of the PoRWP2 gene in the wild-type strain Pyo 34-3 with a fragment of the hygromycin phosphotransferase gene. The upper and lower arm fragments were digested with enzymes (ECoRV) and then ligated to both sides of the knockout vector pCE-ZERO hygromycin phosphotransferase gene, thus constructing the recombinant plasmid pPoRWP2 for gene knockout.

[0052] Experimental principle as follows Figure 1 As shown in Table 1, the primers used to construct the vector are listed below.

[0053] Table 1 Primer sequences used for constructing the knockout vector

[0054]

[0055] (2) Transformation of Pythium oligandrum and the acquisition of knockout mutants

[0056] PEG-mediated protoplast transformation

[0057] 1) Large-scale extraction of recombinant plasmids (cultured in test tubes containing 2 mL LB liquid medium at 37 ℃ and 200 rpm for 8 h with shaking, followed by expansion culture in Erlenmeyer flasks at a 1:100 ratio for 8-10 h. Plasmids were extracted using a non-toxic plasmid extraction kit provided by Tiangen Biotech Co., Ltd.

[0058] 2) Activate the Oligomaeus strain on 1.5% V8 solid medium and culture in the dark at 25°C. After 2 days of culture, cut mycelial blocks of about 1 mm*1 mm in size from the edge and culture them in V8 liquid medium in 6 dishes. Incubate in the dark at 25°C for 48 hours.

[0059] 3) Collect the mycelium using a 500 mL beaker wrapped with gauze, and use heated tweezers to place the mycelium into a petri dish containing 0.8 M Mannitol and rinse once for 2 minutes. Filter the mycelium using a beaker wrapped with gauze, and then transfer the rinsed mycelium into a clean sterile centrifuge tube. Add 0.8 M Mannitol to about 30 mL, and shake at 25°C and 70 rpm for 10 minutes.

[0060] 4) Collect mycelia by filtering in a 500mL beaker wrapped with gauze. Add the washed mycelia to a centrifuge tube containing enzyme solution and incubate at 25℃ and 70rpm for about 45 minutes. Examine the enzymatic digestion effect under a microscope. Do not exceed 50 minutes to achieve a protoplast concentration of 106 cells / mL.

[0061] 5) Use a 50 mL beaker with a layer of mira-cloth to filter the mycelium and collect the protoplasts. Then pour the collected protoplasts into a 50 mL Falcon centrifuge tube and centrifuge at 4°C and 1500 rpm for 4 min in a horizontal rotor centrifuge.

[0062] 6) Discard the supernatant, place the centrifuge tube on ice, add about 10 mL of W5 solution to gently resuspend the protoplasts, then add W5 solution to 35 mL, and centrifuge at 4℃ and 1500 rpm for 4 min.

[0063] 7) Discard the supernatant, add about 7 mL of W5 solution to gently resuspend the protoplasts, then place on ice for at least 30 min, and then centrifuge at 4℃ and 1500 rpm for 4 min.

[0064] 8) Discard the supernatant, slowly add an appropriate volume of M Mg solution along the sidewall into the protoplast, resuspend the protoplast, and let it stand at room temperature for 10 min.

[0065] 9) Add 1 mL of M Mg solution resuspended protoplast solution to each centrifuge tube containing the knockout plasmid. After adding, gently tap the tube wall to mix the plasmid and protoplast thoroughly, and incubate on ice for 6 min.

[0066] 10) Add 580 μL of 40% PEG 4000 solution to each centrifuge tube in three portions, rotating the centrifuge tube as you add the solution to allow the PEG to slowly flow into the protoplasts. Gently push the centrifuge tube to mix it thoroughly and incubate on ice for 20 min.

[0067] 11) Add ampicillin antibiotic (final concentration 50 μg / mL) (50 mg / mL ampicillin) to V8 liquid medium. After incubation, add 2 mL of KPYG2 liquid medium to each centrifuge tube, gently tap the tube wall to mix thoroughly, and incubate on ice for 2 min.

[0068] 12) Add 8 mL of V8 liquid culture medium to each centrifuge tube, gently tap the tube wall to mix thoroughly, and incubate on ice for 2 min.

[0069] 13) Invert and mix well. Pour the liquid from the centrifuge tube into a centrifuge tube containing 10 mL of V8 culture medium. Incubate in the dark at 25°C for overnight regeneration (14-16 h).

[0070] 14) After overnight regeneration, aspirate 5 μL to examine the regeneration under a microscope to see if hyphae have grown. Resuspend the cells in each tube and divide them into three tubes, each containing 7.5 mL. Prepare sterile 150 mm culture dishes and label them.

[0071] 15) Cool 1.5% V8 solid culture medium to 50°C, add G418 (final concentration 30 μg / mL) and ampicillin antibiotic (final concentration 50 μg / mL), shake well to mix, add to the resuspended protoplast fluid from the previous step, so that the total volume of each tube is 45 mL, invert and mix well, and pour into 3 labeled culture dishes, about 15 mL in each dish.

[0072] 16) After drying, seal the container and incubate it upside down in the dark in a 25℃ incubator. Observe whether the transformants have grown every day. The normal time range is about 24h-48h.

[0073] 17) After the transformants grow, perform a second covering. Add G418 (final concentration 60 μg / mL) and ampicillin antibiotic (final concentration 50 μg / mL) to V8 solid medium, shake well to mix, and ensure that the temperature is not too high. Pour into the culture dish of the grown transformants, cover, blow dry and seal, and incubate in the dark and upside down in a 25℃ incubator.

[0074] 18) After the transformants grow, screen them and try to select single colonies of transformants that are visible to the naked eye, avoiding picking multiple colonies at the same time. Transfer them to V8 solid medium containing G418 (final concentration 130 μg / mL) and ampicillin antibiotic (final concentration 50 μg / mL).

[0075] 19) After screening for transformants, verify the transformants, including DNA verification and qPCR verification.

[0076] (3) Detection of knockout mutants

[0077] ① Transformants were cultured in V8 medium with G416 resistance selection in 60mm dishes for 3-4 days. After the bacteria reached confluence, genomic DNA of Pythium oligoandrogenesis was extracted by CTAB method.

[0078] ② Using the extracted genomic DNA as a template, the transformants were amplified twice with the designed primers to verify their transformation.

[0079] 1) The amplified sequence was verified by primers HPH-F and PoRWP2-BY-R. The amplified sequence was a part of the fusion of the upper arm and the hygromycin phosphotransferase gene.

[0080] 2) The amplified sequence was verified using primers PoRWP2-BY-F and PoRWP2-MD-R, and the amplified sequence was internal to the target gene;

[0081] 3) The gene was amplified and verified using primers PoRWP2-BY-F and PoRWP2-BY-R. The amplified sequence was the full-length gene. The PCR product of the preselected gene selected in the second screening was sent to Shanghai Bio-Biotech Co., Ltd. for sequencing. The correct transformant was screened out and transformed at least three times until the G418 resistance disappeared. It was named △PoRWP2.

[0082] The primer sequences used for mutant screening are shown in Table 2, and the screening results are as follows: Figure 2 As shown.

[0083] Table 2 Primer sequences used for mutant screening

[0084]

[0085] (4) Purification and identification of single mycelia

[0086] The correct mutant strain was purified and identified using single-hyphae isolation and purification techniques. Three 0.5mm*0.5mm hyphal blocks were cut and placed in sterile BD tubes, and 15mL of ddH2O was added. A homogenate was then prepared using a sterile homogenizer to obtain a uniform oospore suspension. 100mL of the oospore suspension was added to a 90mm WA plate containing ampicillin and rifampin, spread evenly with a spreader, and incubated upside down in the dark at 25℃.

[0087] The next day, the microscopic examination was used to observe whether single hyphae had grown and marked them with a black pen. Then, the marked agar blocks were inoculated into 60 mm V8 plates (with antibiotics ampicillin and rifampin added) using a 1 mL needle. After incubation at 25°C for 2-3 days, genomic DNA was extracted using the CTAB method and ITS1 / 4 sequencing was used to verify whether it was an Oligandromyces strain.

[0088] Example 3

[0089] The study used solid V8 and Piper nigrum agar plate methods for observation. Wild-type and mutant strains were inoculated onto 70 mm plates for activation. After 1 day of growth, 5 mm diameter mycelial discs were punched from the edge of the colonies and re-inoculated onto 15 mL solid V8 and Piper nigrum agar plates in 90 mm plates. The plates were incubated at 25°C for 1 day, and then the cell growth was observed. Results are as follows: Figure 3 The results showed that the mutant strain did not differ significantly in growth from the wild-type strain.

[0090] Example 4

[0091] This experiment used microscopic observation to compare the sporulation of wild-type and mutant strains.

[0092] 1) Solid culture

[0093] Wild-type *Pythium oligandrum* strain Pyo 34-3 and the target gene knockout mutant ΔPoRWP2 were inoculated into 12.5 mL 70 mm V8 plates and incubated at 25 °C in the dark for 5–6 days. During this period, the growth of oospores of the wild-type strain was observed in real time under a microscope. When most of the wild-type oospores had matured, 5 mm diameter mycelial cakes were cut from the edge of the colony using a sterile punch. Subsequently, the mycelial cakes were treated with GDP sol to remove the agar substrate, retaining only the biofilm structure formed by mycelial growth.

[0094] The above samples were placed on a glass slide and observed and imaged using an optical microscope. The number of oospores was statistically analyzed under multiple fields of view. The experimental results are as follows: Figure 3 The results showed that the ΔPoRWP2 strain had a significantly higher oospore production compared to the wild-type strain, indicating that the PoRWP2 gene may enhance the sporulation capacity of Pythium oligostere.

[0095] 2) Liquid culture

[0096] Using a 20mL syringe, 5mL of V8 liquid culture medium was added to each well of a six-well plate. Then, using a sterile punch, activated mycelial blocks from the V8 plate were placed into the wells, with three 5mm mycelial discs placed in each well. The plates were sealed and incubated statically at 25°C for a specified time to induce oospore formation. After 3 days of dark incubation at 25°C, the original mycelial blocks were removed with a sterile toothpick. Approximately 2mL of the mixture was aspirated using a sterile pipette tip and dropped onto a glass slide. After standing for a short time, the number of oospores was directly observed and counted under an optical microscope. Multiple fields of view were used for repeated statistical analysis for each sample to improve data representativeness.

[0097] Experimental results are as follows Figure 3 The results showed that, under liquid culture conditions, the oospore production of strain ΔPoRWP2 was significantly increased compared to wild-type strain Pyo 34-3, indicating that the PoRWP2 gene may enhance the sporulation capacity of Pythium oligostereum.

[0098] Example 5

[0099] To determine the biocontrol effect of Pythium oligandrum ΔPoRWP2 on fungal diseases, a pot inoculation experiment was conducted. Mycelial blocks of wild-type Pythium oligandrum Pyo 34-3, ΔPoRWP2, and representative fungi (Rhizoctonia solani and Fusarium graminearum) were mixed with sterilized vermiculite and placed in pots. Fifteen seeds of Hefeng 47 were sown. Controls were provided by adding only blank culture medium, only Pythium oligandrum, and only pathogens. After 16 days, the germination rate, fresh weight, plant height, and root length were recorded.

[0100] Experimental results are as follows Figure 4 The results showed that ΔPoRWP2 significantly reduced damage compared to the wild type, with a marked increase in germination rate and fresh weight. Meanwhile, as... Figure 5 The results showed that after 16 days, the plant height and root length of ΔPoRWP2 plants were increased compared to the wild type after the addition of Pythium oligandrum oospores. Therefore, Pythium oligandrum ΔPoRWP2 can inhibit fungal infection of soybean seeds, and its control effect is more significant.

[0101] The specific embodiments of this application have been described in detail above, but they are only examples, and this application is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this application are also within the scope of this application. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of this application should be covered within the scope of this application.

Claims

1. Eliminate Oligomalacia PoRWP2 The application of genetic material in increasing the yield of Oospores of Pythium oligoandrogenesis is characterized by, The Oligoma PoRWP2 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the substance is any of the following: A1) Knock out the aforementioned Oligopyrophyll PoRWP2 Nucleic acid molecules of genes; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant expression vector containing the nucleic acid molecule described in A1) or the expression cassette described in A2); A4) Recombinant microorganisms containing the recombinant expression vector described in A3); The nucleic acid molecule targets the oligoandrophyte. PoRWP2 The gene contains sgRNA1 and sgRNA2 molecules, wherein the target sequence of the sgRNA1 molecule is SEQ ID NO.3, and the amplification primer sequences are SEQ ID NO.5 and SEQ ID NO.6; the target sequence of the sgRNA2 molecule is SEQ ID NO.4, and the amplification primer sequences are SEQ ID NO.7 and SEQ ID NO.

8.

2. A method for increasing the yield of Oospores of Pythium oligoandrogenes, characterized in that, The method includes knocking out Oligomaeus. PoRWP2 Genetic steps, the oligoandrogenic fungus PoRWP2 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. The method according to claim 2, characterized in that, The knockout of Oligopyrophyll PoRWP2 The gene-targeting step is to target the oligomale pythium. PoRWP2 The sgRNA1 and sgRNA2 molecules of the gene were introduced into the *Pythium oligoandrogenes* via a CRISPR / Cas9 system. The target sequence of the sgRNA1 molecule is SEQ ID NO.3, and the amplification primer sequences are SEQ ID NO.5 and SEQ ID NO.

6. The target sequence of the sgRNA2 molecule is SEQ ID NO.4, and the amplification primer sequences are SEQ ID NO.7 and SEQ ID NO.8.

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