Pythium ultimum purwp1 gene and its application as a molecular target in the prevention of pythium ultimum disease

Abstract

The application belongs to the field of genetic engineering, and discloses a final pythium PuRWP1 gene and application of the gene as a molecular target in disease prevention caused by the final pythium. The application provides a gene PuRWP1 from the final pythium for controlling a sexual development stage and oospore formation. The gene is composed of 1184 nucleotides. A protein coded by the PuRWP1 gene is composed of 627 amino acids. Knockout of the PuRWP1 gene can cause a significant decrease in sporulation capacity and a decrease in spore activity of a mutant. The gene can be used as a molecular target in disease prevention of oomycetes. The application verifies the function of the final pythium PuRWP1 gene, and provides a new direction for further regulating physiological functions of the final pythium and other oomycetes and preventing oomycete diseases by using the gene.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the PuRWP1 gene of Pythium tertii and its application as a molecular target in the prevention and control of diseases caused by Pythium tertii. Background Technology

[0002] Soybean root rot, caused by various soil-borne pathogens such as fungi and oomycetes, is a devastating soil-borne disease that severely impacts soybean yield and quality. Among them, *Pythium terrestris* (…) is particularly prevalent. Pythium ultimum *Pythium oxysporum* is one of the major pathogenic fungi, capable of surviving in the soil as oospores for extended periods. It infects soybean roots and hypocotyls, causing root rot, stem rot, and even plant death, resulting in significant ecological and economic losses to soybean production. *Pythium oxysporum* is one of the most pathogenic Pythium fungi, capable of causing damping-off and root rot on over 300 different hosts, including soybeans, corn, wheat, and various ornamental plants.

[0003] Ultimate Pythium ( Pythium ultimum The life cycle of *Pythium cerevisiae* mainly includes the following stages: (1) *Pythium cerevisiae* overwinters in the soil or in diseased plant debris as oospores or hyphae. Oospores have thick walls and strong resistance, and can survive in the soil for many years, making them the main source of primary infection for this pathogen; (2) Under suitable environmental conditions, oospores germinate to form germ tubes, which directly penetrate the soybean root epidermis or root hairs and enter the root tissue to complete the primary infection; (3) Germ tubes develop into vegetative hyphae, which spread in the cortex and vascular bundles of soybean roots, causing tissue necrosis and leading to root rot, stem rot, and other diseases; (4) In the host tissue, vegetative hyphae undergo sexual development to form oothecia and anthers, with the vast majority being homologous; male and female gametophytes complete fertilization to form new oospores; (5) Newly formed oospores are deposited in diseased tissues or released into the soil and enter a dormant state, serving as the main inoculum source for the next infection cycle. Therefore, the formation and survival ability of oospores are considered the core basis for the survival and pathogenicity of oomycete pathogens.

[0004] With the development of molecular biology and functional genomics, the reproductive development of oomycetes is regulated by multiple transcription factors, especially the RKD subfamily within the RWP-RK family, which has attracted attention due to its crucial role in sexual development. However, current research on the functions of RWP-RK family members in reproductive processes in *Pythium terrestris* remains relatively scarce, particularly lacking in-depth analysis of the key factors specifically regulating oospore formation. Summary of the Invention

[0005] To address the aforementioned technical challenges, this invention, for the first time, identified and cloned the PuRWP1 gene from *Pythium truncatum*. Functional verification revealed that this gene plays a crucial role in the sexual development stage; its deletion significantly inhibits oospore formation and affects spore viability. The discovery of PuRWP1 not only provides new clues for a deeper understanding of the molecular mechanisms of oomycete oospore formation but also lays an important foundation for targeted regulation of oomycete reproductive development and innovative control strategies.

[0006] In a first aspect, the present invention provides the PuRWP1 gene of *Pythium oxyphylla*, the nucleotide sequence of which is shown in SEQ ID NO.1, or can hybridize under stringent conditions with the complementary sequence of the nucleotide sequence shown in SEQ ID NO.1 and encode a protein having the same function as the protein encoded by the nucleotide sequence shown in SEQ ID NO.2.

[0007] In a second aspect, the present invention also provides the ultimate Pythium PuRWP1 protein, which is encoded by the PuRWP1 gene described in the first aspect.

[0008] Furthermore, the amino acid sequence of the PuRWP1 protein is as shown in SEQ ID NO.2, or an amino acid sequence with more than 95% identity obtained by substituting, deleting and / or adding one or more amino acids and / or terminal modification of any one or more amino acids in the amino acid sequence shown in SEQ ID NO.2.

[0009] Thirdly, the present invention also provides biological materials containing the genes described in the first aspect, wherein the biological material is any one of the following:

[0010] 1) An expression cassette containing a substance that reduces the expression level of the PuRWP1 gene described in the first aspect;

[0011] 2) An expression vector containing a substance that reduces the expression level of the PuRWP1 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, the present invention also provides the application of substances capable of reducing the expression level of the PuRWP1 gene and / or substances capable of reducing the activity of the PuRWP1 protein in regulating the formation of terminal Pythium oospores.

[0016] Furthermore, reducing the expression level of the PuRWP1 gene and / or reducing the activity of the PuRWP1 protein can lead to a significant reduction in the yield and activity of Pythium oxyspores, or effectively prevent soybean root rot.

[0017] Fifthly, the present invention also provides a method for reducing the yield and activity of *Pythium oxysporum* oospores, the method comprising the steps of reducing the expression level of the *PuRWP1* gene and / or reducing the activity of the *PuRWP1* protein.

[0018] In a sixth aspect, the present invention also provides a method for preventing and controlling soybean root rot, the method comprising the steps of reducing the expression level of the PuRWP1 gene and / or reducing the activity of the PuRWP1 protein.

[0019] Further, the step includes introducing a gene-editing vector into *Pythium oxysporum*, the gene-editing vector being capable of reducing the expression level of the PuRWP1 gene and / or reducing the activity of the PuRWP1 protein.

[0020] Compared with the prior art, this invention provides the PuRWP1 gene from *Pythium terrestris*, which controls the sexual development stage and oospore formation. This gene consists of 1184 nucleotides, and its DNA sequence is shown in the figure. The protein encoded by the PuRWP1 gene consists of 627 amino acids. Knockout of the PuRWP1 gene leads to a significant decrease in sporulation capacity and spore activity in the mutant. This invention verifies the function of the *PuRWP1* gene from *Pythium terrestris*, providing a new direction for further utilizing this gene to regulate the physiological functions of *Pythium terrestris* and other oomycetes, and to control oomycete diseases. Furthermore, this invention, through functional verification, provides a theoretical basis and potential targets for targeting this gene to regulate the reproductive development of oomycetes (including *Pythium terrestris*), and for developing novel oomycete disease control strategies based on this. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the PuRWP1 gene knockout.

[0022] Figure 2 Electrophoresis diagrams for identifying PuRWP1 gene knockout mutants; lane 1 is WT, the ultimate wild-type Pythium strain; lane 2 is EV, the control strain whose protoplast transformation failed; lanes 3 and 4 are ΔPuRWP1 transformants 1 and 2; lane 5 is the control strain whose transformation failed during complementation transformation of ΔPuRWP1 transformants (complementation failure control strain); lanes 6 and 7 are complementation transformants C1 and C2.

[0023] Figure 3 The cell production of mutant and wild-type strains in solid culture medium.

[0024] Figure 4 The results of pathogenicity testing of soybean hypocotyls of mutant and wild-type strains are presented.

[0025] Figure 5 The results show the oospore yield of the mutant and wild-type strains.

[0026] Figure 6 The results show the activity of oospores in mutant and wild-type strains. Detailed Implementation

[0027] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0028] The formulations of some of the reagents and culture media used in the examples are as follows.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] WA medium: Water agar medium. Weigh 3g of agar powder and add pure water to make up to 200mL.

[0033] 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.

[0034] 0.5M CaCl2 solution: Weigh 7.3505g CaCl2·2H2O and add pure water to make up to 100mL.

[0035] 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.

[0036] 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.

[0037] 0.8M Mantiol solution: Weigh 145.736g of Mantiol powder, add ddH2O to make up to 1L, and autoclave at 121℃ for 20min.

[0038] 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 powder and then sterilize by filtration.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Example 1

[0044] Based on previous transcriptome data analysis of the *Pythium terrestris* RWP-RK transcription factor family, the *PuRWP1* gene was found to be highly expressed at multiple developmental stages in this species, indicating potential value for functional studies. Therefore, the *PuRWP1* gene was screened and knocked out for subsequent functional validation. The nucleotide sequence of the *PuRWP1* gene is shown in SEQ ID NO.1, and its encoded amino acid sequence is shown in SEQ ID NO.2. The full-length *PuRWP1* gene is 1884 bp.

[0045] Example 2

[0046] (1) Construction of the knockout vector

[0047] The gene knockout was performed using homologous chromosome recombination. A constructed vector was used to replace the coding sequence of the PuRWP1 gene in the wild-type strain PU18-6 with a fragment of the hygromycin phosphotransferase gene. The knockout pattern is described in [link to knockout diagram]. Figure 1 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 to construct the recombinant plasmid pPuRWP1 for gene knockout.

[0048] The primers used to construct the vector are shown in Table 1.

[0049] Table 1 Primer sequences used for constructing the knockout vector

[0050]

[0051] (2) Transformation of Pythium ultimatum and the acquisition of knockout mutants

[0052] PEG-mediated protoplast transformation

[0053] 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.);

[0054] 2) Activate the *Pythium guiyangense* strain on 1.5% KPYG2 solid medium and culture in the dark at 25°C. After 2 days of culture, cut mycelial blocks of about 1 mm*1 mm from the edge and culture them in KPYG2 liquid medium in 6 dishes. Incubate in the dark at 25°C for 48 h.

[0055] 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.

[0056] 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. It is best to achieve a protoplast concentration of 106 cells / mL.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 9) Add 1 mL of M Mg solution resuspended protoplast solution to each centrifuge tube containing plasmid. After adding, gently tap the tube wall to mix the plasmid and protoplast thoroughly, and incubate on ice for 6 min.

[0062] 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.

[0063] 11) Add ampicillin antibiotic (final concentration 50 μg / mL) (50 mg / mL ampicillin) to KPYG2 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.

[0064] 12) Add 8 mL of KPYG2 liquid medium to each centrifuge tube, gently tap the tube wall to mix thoroughly, and incubate on ice for 2 min.

[0065] 13) Invert and mix well. Pour the liquid from the centrifuge tube into a centrifuge tube containing 10 mL of KPYG2 culture medium. Incubate in the dark at 25°C for overnight regeneration for 14-16 hours.

[0066] 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.

[0067] 15) Cool 1% KPYG2 solid 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.

[0068] 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.

[0069] 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.

[0070] 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).

[0071] 19) After screening for transformants, verify the transformants, including DNA verification and qPCR verification.

[0072] (3) Detection of knockout mutants

[0073] ① Transformants were cultured in V8 medium with G416 resistance selection in 60mm dishes for 3-4 days. After the bacteria reached confluence, the genomic DNA of Pythium cerevisiae was extracted by CTAB method.

[0074] ② Using the extracted genomic DNA as a template, the transformants were amplified twice with the designed primers to verify their transformation.

[0075] 1) The amplified sequence was verified by amplification using primers PuRWP1-BY-F1 and HPH-R. The amplified sequence was a part of the fusion of the upper arm and the hygromycin phosphotransferase gene.

[0076] 2) Amplification and verification were performed using primers PuRWP1-BY-F1 and PuRWP1-MD-R, and the amplified sequence was found to be inside the target gene;

[0077] 3) Amplify and verify the gene using primers PuRWP1-BY-F1 and PuRWP1-BY-R1. The amplified sequence is the full-length gene. The PCR products of the pre-selected gene selected in the second screening are sent to Shanghai Bio-Biotech Co., Ltd. for sequencing. The correct transformants are screened out and transformed at least three times until the G418 resistance disappears. They are named △PuRWP1-1 and △PuRWP1-2.

[0078] The primer sequences used for mutant screening are shown in Table 2, and the screening results are as follows: Figure 2 As shown.

[0079] Table 2 Primer sequences used for mutant screening

[0080]

[0081] (4) Purification and identification of single mycelia

[0082] 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℃.

[0083] 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 the ultimate Pythium strain.

[0084] Example 3

[0085] 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.

[0086] Example 4

[0087] The hypocotyl of soybean variety Hefeng 47 was used for pathogenicity testing. Sixty Hefeng 47 soybeans were sown, covered with black plastic bags to maintain a dark environment, and grown in a 25℃ incubator for 4 days. On day 3 of growth, wild-type and mutant strains were inoculated onto 70mm plates for activation. After 1 day of growth, mycelial cakes (5mm in diameter) were punched from the edge of the colony, and the mycelial-containing side was placed onto the soybean hypocotyl. The plates were then kept moist and incubated at 25℃ for 1 day, after which disease development was observed. Results are as follows: Figure 4 The results showed that the mutant strain did not differ significantly from the wild-type strain in pathogenicity, and quantitative analysis also showed no significant difference in biomass.

[0088] Example 5

[0089] This experiment used microscopic observation to compare the sporulation of wild-type and mutant strains.

[0090] 1) Solid culture

[0091] wild type Pythium ultimum Strains PU18-6 and the target gene knockout mutant ΔPuRWP1 were inoculated into 70mm V8 plates (12.5mL per plate) and incubated at 25℃ 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 matured, 5mm 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 hyphal growth.

[0092] 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.

[0093] 2) Liquid culture

[0094] 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.

[0095] Experimental results are as follows Figure 5 The results showed that, under solid-state culture conditions, the oospore production of strain △PuRWP1 was significantly lower than that of the wild-type strain. Similarly, under liquid culture conditions, the oospore production of strain △PuRWP1 was also significantly lower than that of the wild-type strain, indicating... PuRWP1 Genes are involved in regulating the process of oospore formation.

[0096] Example 6

[0097] Oospore viability was determined using the MTT thiazolyl blue staining method and microscopic observation. Using a 20 mL syringe, 5 mL of V8 liquid medium was added to each well of a six-well plate. Then, using a sterile punch, activated wild-type and mutant mycelial blocks from the V8 plate were placed into the wells, with three 5 mm mycelial cakes placed in each well. The plates were sealed and incubated statically at 25°C for a specified time until mature ospore formation (generally 15 days). The ospore suspension was collected and mixed with thiazolyl blue reagent at a 1:1 ratio. After incubation at 37°C for 24 h and 48 h, 2 mL of the liquid was aspirated and placed on a glass slide. Oospore viability was determined using microscopic observation.

[0098] The results are as follows Figure 6 The results showed that, compared with the wild type, the mortality rate of oospores in the △PuRWP1 strain was significantly increased, and their viability was significantly reduced, indicating that... PuRWP1 Genes are involved in regulating the process of oospore formation.

[0099] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A *Pythium terrestris* PuRWP1 gene that regulates the formation of *Pythium terrestris* oospores, characterized in that, The nucleotide sequence of the *Pythium spp.* PuRWP1 gene is shown in SEQ ID NO.

1.

2. A *Pythium terrestris* PuRWP1 protein that regulates the formation of *Pythium terrestris* oospores, characterized in that, The *Pythium oxyphylla* PuRWP1 protein is encoded by the *Pythium oxyphylla* PuRWP1 gene as described in claim 1, and the amino acid sequence of the *Pythium oxyphylla* PuRWP1 protein is shown in SEQ ID NO.

2.

3. A biomaterial containing the *Pythium cerevisiae* PuRWP1 gene as described in claim 1, characterized in that, The biomaterial is any of the following: 1) An expression cassette containing the PuRWP1 gene of Pythium oxyphylla as described in claim 1; 2) An expression vector containing the PuRWP1 gene of Pythium oxyphylla as described in claim 1; 3) An expression carrier containing the expression box described in 1); 4) Recombinant microorganisms containing the expression vector described in 2); 5) Recombinant microorganisms containing the expression vector described in 3).

4. The application of the *P. truncatella* PuRWP1 gene as described in claim 1 in regulating *P. truncatella* oospore formation or in the control of diseases caused by *P. truncatella*, characterized in that... Knocking out the PuRWP1 gene of Pythium oxyphylla as described in claim 1 results in a significant reduction in the yield and activity of Pythium oxyphylla oospores or effectively controls soybean root rot.

5. A method for reducing the yield and activity of *Pythium oxysporum* oospores, characterized in that, The method includes the steps of knocking out the PuRWP1 gene of Pythium oxyphylla as described in claim 1 and / or reducing the activity of the PuRWP1 protein of Pythium oxyphylla as described in claim 2, wherein the reduction of the activity of the PuRWP1 protein of Pythium oxyphylla as described in claim 2 is achieved by knocking out the PuRWP1 gene of Pythium oxyphylla as described in claim 1.

6. The method according to claim 5, characterized in that, The steps include introducing a gene-editing vector containing sgRNA and Cas9 into *Pythium oxyphylla*, wherein the gene-editing vector is capable of knocking out the *Pythium oxyphylla* PuRWP1 gene of claim 1 and / or reducing the activity of the *Pythium oxyphylla* PuRWP1 protein of claim 2, wherein the reduction of the *Pythium oxyphylla* PuRWP1 protein activity of claim 2 is achieved by knocking out the *Pythium oxyphylla* PuRWP1 gene of claim 1.

7. The method according to claim 6, characterized in that, The amplification primer sequences for the sgRNA are shown in SEQ ID NO. 3-4 or SEQ ID NO. 5-6.

8. A method for preventing soybean root rot caused by Pythium cerevisiae, characterized in that, The method includes the steps of knocking out the PuRWP1 gene of Pythium oxyphylla as described in claim 1 and / or reducing the activity of the PuRWP1 protein of Pythium oxyphylla as described in claim 2, wherein the reduction of the activity of the PuRWP1 protein of Pythium oxyphylla as described in claim 2 is achieved by knocking out the PuRWP1 gene of Pythium oxyphylla as described in claim 1.

9. The method according to claim 8, characterized in that, The steps include introducing a gene-editing vector containing sgRNA and Cas9 into *Pythium oxyphylla*, wherein the gene-editing vector is capable of knocking out the *Pythium oxyphylla* PuRWP1 gene of claim 1 and / or reducing the activity of the *Pythium oxyphylla* PuRWP1 protein of claim 2, wherein the reduction of the *Pythium oxyphylla* PuRWP1 protein activity of claim 2 is achieved by knocking out the *Pythium oxyphylla* PuRWP1 gene of claim 1.

10. The method according to claim 9, characterized in that, The amplification primer sequences for the sgRNA are shown in SEQ ID NO. 3-4 or SEQ ID NO. 5-6.

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