Application of FoErv25 gene in regulating endophytic fungus Sclerospora oryzae to promote rice growth

Abstract

The application discloses FoErv25 The application discloses a gene in the regulation of endophytic fungus Pyricularia oryzae promoting rice growth and belongs to the technical field of genetic engineering. FoErv25 The nucleotide sequence of the gene is shown as SEQ ID NO. 1. FoErv25 The application discloses a gene in the regulation of endophytic fungus Pyricularia oryzae promoting rice growth and belongs to the technical field of genetic engineering. FoErv25 The application discloses a gene in the regulation of endophytic fungus Pyricularia oryzae promoting rice growth and belongs to the technical field of genetic engineering. FoErv25 The application discloses a gene in the regulation of endophytic fungus Pyricularia oryzae promoting rice growth and belongs to the technical field of genetic engineering. FoErv25 FoErv25 FoErv25 FoErv25 FoErv25 FoErv25 FoErv25 FoE The application discloses a gene in the regulation of endophytic fungus Pyricularia oryzae promoting rice growth and belongs to the technical field of genetic engineering. This research result provides a research idea for promoting rice plant growth, improving rice yield and reducing the use of chemical fertilizers.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to FoErv25 Application of genes in regulating the endophytic fungus Fusarium oryzae in promoting rice growth. Background Technology

[0002] In recent years, the development and utilization of agricultural microbial resources have received increasing attention, among which endophytic fungi that can coexist in plants for a long time have shown good application potential. These fungi usually do not cause diseases, but can promote root development and improve nutrient absorption efficiency by secreting plant hormone analogs or regulating host metabolic pathways, thereby improving plant growth and increasing yield.

[0003] Existing research and technical practices have shown that some rice-derived endophytic fungi can significantly enhance seedling vigor and field yield after establishing a stable symbiotic relationship with the host. For example, the rice endophytic fungus Fusarium oryzae reported in patent document CN113388526A... Falciphora oryzae The FO-R20 strain demonstrated its ability to promote rice growth. Compared to traditional breeding or transgenic improvement methods, the functional enhancement strategy based on beneficial endophytic microorganisms provides a new technical approach for improving crop quality and efficiency.

[0004] However, systematic research on genes regulating plant growth in endophytic fungi remains relatively scarce. In-depth analysis of the molecular mechanisms of fungal-plant interactions, screening and functional validation of key regulatory factors, and optimization of dominant traits using molecular biology techniques to construct efficient and stable microbial formulation systems are of significant practical importance for promoting their large-scale application in agricultural production.

[0005] Plant growth and development are regulated by a variety of organelles, among which protein sorting, transport, and secretion pathways play a crucial role in maintaining cellular homeostasis and signal transduction. The endoplasmic reticulum-Golgi apparatus secretion pathway is not only involved in the transport of cell wall components, enzymes, and signaling molecules, but is also closely related to plant-microbe interactions (Brandizzi F, Barlowe C. Organization of the ER-Golgi interface for membrane traffic control). Nat Rev Mol Cell Biol(Belden WJ, Barlowe C. Erv25p, a Component of COPII-coated Vesicles, Forms a Complex with Emp24p That Is Required for Efficient Endoplasmic Reticulum to Golgi Transport). In eukaryotes, Erv25 is a key component of the COPII-coated Vesicle complex, primarily located at the endoplasmic reticulum exit site, participating in the selective loading of cargo proteins and vesicle budding, thereby regulating the efficiency of protein transport from the endoplasmic reticulum to the Golgi apparatus (Belden WJ, Barlowe C. Erv25p, a Component of COPII-coated Vesicles, Forms a Complex with Emp24p That Is Required for Efficient Endoplasmic Reticulum to Golgi Transport). Journal of Biological Chemistry (Barlowe C. COPII: A membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum). This protein, through synergistic action with factors such as Sec23 / 24, ensures the correct sorting and transport of specific transmembrane or secretory proteins, which is of great significance for maintaining the function of the cellular secretory system (Barlowe C. COPII: A membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum). Cell ,1994, 77: 895-907.).

[0006] Erv25 was first observed in model yeast. Saccharomyces cerevisiae It was identified that the absence of Erv29 leads to impaired transport of some membrane proteins and enhanced endoplasmic reticulum stress response. Subsequent studies have shown that Erv25 homologs are ubiquitous in various eukaryotes and participate in regulating the maturation of secretory proteins, the transport of cell wall synthesis-related enzymes, and environmental adaptive responses (Qian B, et al. MoErv29 promotes apoplastic effector secretion contributing to virulence of the rice blast fungus Magnaporthe oryzae). New Phytologist (2022, 233:1289-1302.). However, systematic research on the biological functions of Erv25 in plant endophytic fungi, especially its mechanism of action in promoting host plant growth, is still lacking. Summary of the Invention

[0007] This invention aims to identify key functional genes derived from rice endophytic fungi, elucidate their regulatory roles in promoting host growth, and clarify their biological significance through molecular cloning and functional identification. Systematic research on these genes can provide new molecular targets for optimizing the interaction between endophytic fungi and rice, while also providing important theoretical support and genetic resources for constructing efficient and stable symbiotic systems, enhancing rice yield potential, and achieving sustainable yield increases.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention identifies *Fusarium oryzae*, a plant endophytic fungus, through genome annotation and bioinformatics analysis. FoErv25 Genes, in *Fusarium graminearum* Falciphora oryzae In the symbiotic system of FOR20 and rice, the expression of this gene was significantly upregulated.

[0009] Specifically, FoErv25 The full-length gene is 976 bp, and its nucleotide sequence is shown in SEQ ID NO.1; the CDS length of this gene is 762 bp, and its nucleotide sequence is shown in SEQ ID NO.2. The DNA sequence has a high GC content, which is consistent with the characteristics of fungal genes.

[0010] FoErv25 The sequence encodes a protein with 253 amino acid residues, as shown in SEQ ID NO. 3. The amino acid sequence contains a hydrophobic signal peptide at the N-terminus and a hydrophobic core in the middle, exhibiting an overall α / β folded structure. Hydrophilic regions on the surface may be involved in interactions. Structural prediction suggests that this protein may be a secretory protein with a stable folded core and potential functional domains.

[0011] Based on the functional prediction results, FoErv25 Genes may influence the efficiency of fungal-host interaction by regulating the transmembrane transport of secreted proteins or signaling molecules, thereby participating in plant growth promotion processes.

[0012] This invention utilizes homologous recombination technology to target endophytic fungi in rice. Falciphora oryzae In the FOR20 genome FoErv25 Gene knockout yields mutant Δ FoErv25 Compared to the wild-type strain, this mutant exhibits a weakened growth-promoting effect on rice. During the rice seedling stage, root length is significantly reduced by 22.95%, stem length by 31.1%, above-ground fresh weight by 59.6%, and underground fresh weight by 55.1%. Total root length is significantly reduced by 36.0%, total root surface area by 40.4%, average root diameter by 55%, and root volume by 47.5%.

[0013] This invention utilizes overexpression technology to FoErv25The gene was overexpressed in the rice endophytic fungus Fusarium oryzae to obtain an overexpressing strain. Compared with the wild-type strain, the overexpressing strain had a stronger growth-promoting effect on rice when symbiotic with rice. The root length of rice seedlings increased significantly by 22.96%, the stem length increased significantly by 28.4%, the aboveground fresh weight increased significantly by 33.2%, the underground fresh weight increased significantly by 45.9%, the total root length increased significantly by 11.8%, the total root surface area increased significantly by 26.5%, the average root diameter increased significantly by 37.6%, and the root volume increased significantly by 26.6%.

[0014] The above results indicate that FoErv25 Genes regulate the growth of rice endophytic fungus Fusarium graminearum (Fusarium graminearum). Falciphora oryzae (Key genes that promote rice growth)

[0015] Therefore, the present invention provides FoErv25 The application of the gene in regulating the endophytic fungus Fusarium oryzae in promoting rice growth, the aforementioned FoErv25 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0016] In this invention, the *Fusarium graminearum* is an endophytic fungus that can coexist with rice and promotes rice growth; however, it is not limited to... Falciphora oryzae FO-R20 strain.

[0017] The indicators for rice growth include at least one of the following: root length, stem length, above-ground (stem and leaf) fresh weight, underground (root) fresh weight, total root length, total root surface area, average root diameter, and root volume.

[0018] Furthermore, the application includes: utilizing biological techniques to... FoErv25 The gene was upregulated in the rice endophytic fungus Fusarium oryzae, enhancing the growth-promoting effect of Fusarium oryzae on rice.

[0019] This invention utilizes techniques known in the art to upregulate the expression of the rice endophytic fungus Fusarium graminearum. FoErv25 Genes. Compared to wild-type strains, FoErv25 The gene-overexpressing strains significantly enhanced the growth-promoting ability of rice. The biological techniques used may include, but are not limited to, genetic engineering techniques.

[0020] Furthermore, the application includes: transferring the... FoErv25 Gene fragments were cloned into overexpression vectors to construct recombinant plasmids. The target gene fragments were then introduced into *Fusarium oxysporum* using Agrobacterium-mediated transformation, and the resulting plasmids were screened for optimal expression. FoErv25 Transgenic Fusarium graminearum with overexpressed genes was then co-cultured with rice to colonize the rice root tissue.

[0021] The present invention can optimize the coding sequence of FoErv25 based on the codon preference of Fusarium graminearum, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.3.

[0022] Furthermore, the *Fusarium oxysporum* species is identified by accession number CCTCC NO: M 2021505. Falciphora oryzae FO-R20.

[0023] Furthermore, the overexpression vector is pKD5-GFP.

[0024] Furthermore, the Agrobacterium used is Agrobacterium AGL1.

[0025] Furthermore, before co-culturing, the transgenic strain was inoculated into PDA medium for activation culture and cultured in the dark at 22-25℃ for 7-10 days.

[0026] Furthermore, the co-culture includes: inoculating germinating rice seeds and transgenic strain blocks onto sterile 1 / 2 MS medium for co-culture. The co-culture conditions are: 20-25℃ for 20-25 days, with 16 hours of light and 8 hours of dark culture per day.

[0027] Another co-cultivation method involves inoculating the transgenic strain into sterilized barley grains to obtain solid fermented microbial fertilizer, which is then mixed into the rice planting substrate to promote rice growth.

[0028] The present invention also provides a method for promoting rice growth, comprising: using nucleotide sequences as shown in SEQ ID NO.1 or SEQ ID NO.2. FoErv25 Gene fragments were introduced into Fusarium oryzae to obtain overexpressing strains, and then the overexpressing strains were co-cultured with rice to obtain an overexpressing strain-rice symbiotic system.

[0029] The beneficial effects of this invention are as follows: This invention discloses for the first time genes in the rice endophytic fungus Fusarium graminearum that regulate rice growth. FoErv25 Through the FoErv25 The gene was cloned and analyzed, and its function was verified using gene knockout and gene overexpression techniques. The knockout technique was then used to identify key gene mutations. FoErv25 The gene caused a significant reduction in the growth-promoting effect of the strain on rice, and overexpression of it... FoErv25 The gene significantly enhances the growth-promoting effect of the strain on rice. This research helps to reveal the molecular basis of endophytic fungi promoting plant growth, and provides theoretical support for the molecular improvement and agricultural application of functional strains. It also provides research ideas for promoting rice plant growth, increasing rice yield, and reducing fertilizer use. Attached Figure Description

[0030] Figure 1 Wild type of endophytic fungus Falciphora oryzae FO-R20, FoErv25 mutant strain (Δ FoErv25 ), FoErv25 complement strains (Δ FoErv25 C) and FoErv25 overexpression strains ( FoErv25 A comparison of colony morphology (OE), where: A represents wild-type strain; B represents... FoErv25 mutant strain; C is FoErv25 complement strain; D is FoErv25 Overexpression strains.

[0031] Figure 2 Wild type FoErv25 mutant strains, FoErv25 complement strains, FoErv25 Colony of the overexpressing strain in rice roots.

[0032] Figure 3 This image shows the growth of rice in a tissue culture bottle experiment with endophytic fungi in symbiosis. A shows a picture of the rice plant inside the tissue culture bottle; B shows a picture of the specific morphology of the rice plant.

[0033] Figure 4 This presents the statistical results of growth indicators of endophytic fungi symbiotic rice in tissue culture bottles, where: A represents the statistical results of rice root length; B represents the statistical results of rice stem length; C represents the statistical results of rice aboveground fresh weight (stems and leaves); and D represents the statistical results of rice underground (roots) fresh weight. Significance: * indicates P <0.05; ** indicates P <0.01; *** indicates P <0.001; **** indicates P <0.0001; ns indicates no significant difference.

[0034] Figure 5 This is a scan of the roots of rice in a tissue culture flask experiment with endophytic fungi. Scale bar: 1 cm.

[0035] Figure 6 This presents the statistical results of rice root parameters in a tissue culture flask experiment involving endophytic fungi. A represents the statistical results of rice root surface area; B represents the statistical results of average rice root diameter; C represents the statistical results of total rice root length; and D represents the statistical results of rice root volume. Significance: * indicates significance. P <0.05; ** indicates P <0.01; *** indicates P <0.001; **** indicates P <0.0001; ns indicates no significant difference.

[0036] Figure 7 This image shows the growth of rice plants in a pot experiment with endophytic fungi in symbiosis. A shows a picture of the potted rice plant; B shows a picture of the specific morphology of the rice plant.

[0037] Figure 8 This presents the statistical results of growth indicators of rice symbiotic with endophytic fungi in a pot experiment, where: A represents the statistical results of rice root length; B represents the statistical results of rice stem length; C represents the statistical results of rice above-ground fresh weight; and D represents the statistical results of rice below-ground fresh weight. Significance: * indicates P <0.05; ** indicates P <0.01; *** indicates P <0.001; **** indicates P <0.0001; ns indicates no significant difference. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0040] This invention builds upon our previous work, which isolated a novel endophytic fungus from the roots of wild rice species *Oryza sativa* in Yunnan. Falciphora oryzae FOR20 is the material, and it is obtained by knocking out FoErv25 Gene or overexpression FoErv25 Genetic studies on the symbiotic interaction between the endophytic fungus FOR20 and rice are of great significance for elucidating the molecular mechanism by which the endophytic fungus FOR20 promotes rice growth.

[0041] Example 1: FoErv25 Gene analysis Endophytic fungi Falciphora oryzae FO-R20 was isolated from the root system of wild rice, and deposited on May 8, 2021, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2021505. See Chinese patent document with publication number CN113388526A.

[0042] Previous studies have shown that endophytic fungi Falciphora oryzae When FOR20 colonizes the root tissues of rice plants, it can significantly promote rice growth. This mutualistic relationship provides a basis for studying the molecular mechanisms of rice growth.

[0043] We Falciphora oryzae Proteomics analysis of FOR20 co-cultured with rice revealed a significantly upregulated expression of the Erv25 protein. We selected this gene as a candidate gene and named it... FoErv25 Genes, exploring their role Falciphora oryzae The role of FOR20 in promoting host growth.

[0044] Specifically, FoErv25 The full-length gene is 976 bp, and its nucleotide sequence is shown in SEQ ID NO.1; the CDS length of this gene is 762 bp, and its nucleotide sequence is shown in SEQ ID NO.2. The DNA sequence has a high GC content, which is consistent with the characteristics of fungal genes.

[0045] FoErv25 The sequence encodes a protein with 253 amino acid residues, as shown in SEQ ID NO. 3. The amino acid sequence contains a hydrophobic signal peptide at the N-terminus and a hydrophobic core in the middle, exhibiting an overall α / β folded structure. Hydrophilic regions on the surface may be involved in interactions. Structural prediction suggests that this protein may be a secretory protein with a stable folded core and potential functional domains.

[0046] Example 2: Plate co-culture verification FoErv25 Gene function Tested endophytic fungi: Falciphora oryzae FO-R20; Test plant: Rice Oryza sativa L., a common variety, CO39.

[0047] 1. Strains culture The FO-R20 strain preserved on PDA slant was inoculated onto potato dextrose agar (PDA) solid medium for activation culture and cultured in the dark at 25°C for 7 days for later use.

[0048] PDA medium: Each liter contains 20 g glucose, 200 g potato, and 15 g agar. Weigh the required amount of potato according to the volume of the medium to be prepared, boil it in water, mash and dissolve it, filter it, add glucose and agar, and autoclave at 121°C for 20 min.

[0049] 2. Gene mutant strain Δ FoErv25 Δ FoErv25 C FoErv25 Obtaining OE Genomic DNA extraction: Use a toothpick to collect a 1×1 cm sample. 2Fresh mycelial cakes, aged 5-7 days, were mixed with an appropriate amount of quartz sand and 500 μL of Extraction Buffer (1 M KCl, 100 mM Tris-HCl (pH=7.5), 10 mM EDTA), and then shaken in an MP Fast Prep®-24 homogenizer (65 Hz, 120 s). The shaken liquid was centrifuged at 12000 rpm for 10 min. 300-350 μL of the supernatant was collected and mixed with an equal volume of isopropanol, shaken 7-8 times, and centrifuged at 12000 rpm for 10 min. The supernatant was discarded, and 800 μL of 70% ethanol was added. The mixture was inverted 7-8 times and centrifuged at 12000 rpm for 2 min. The supernatant was discarded, and the centrifuge tube was placed upside down on absorbent paper until the alcohol evaporated. 50 μL of dd H2O was added to dissolve the mixture at room temperature for 3 h, and then it was stored at -20°C.

[0050] Design upstream and downstream primers for the target gene, and amplify using genomic DNA of strain FOR-R20 as a template. FoErv25 Upstream and downstream segments of genes FoErv25 Gene fragments and G418 fragments. Specific primers are shown in Table 1.

[0051] Table 1

[0052] By restriction endonuclease XbaI and HindⅢ The pKO1B restriction vector was digested. Following the instructions of the ClonExpress II OneStep clone Kit (Vazyme, China), the G418 resistance gene fragment was ligated to the upstream and downstream fragments of the target gene, and then ligated to the digested pKO1B vector to construct a gene knockout vector. This vector was then transformed into competent DH5α cells of *E. coli*. Transformants were selected and placed in 1 mL of LB liquid medium (containing 50 μg / mL kanamycin), incubated at 37°C with shaking at 200 rpm for 4–6 h. 1–2 μL of the bacterial culture was then added to the validation system for PCR amplification and gel electrophoresis.

[0053] Plasmid extraction was performed according to the AxyPrep-96 (Axygen, US) operating instructions. The plasmid was then transformed into Agrobacterium competent cells AGL1, and transformed into wild-type cells using Agrobacterium-mediated transformation. Falciphora oryzae For FOR-R20, DNA was extracted from transformants for length and copy number verification. Verified transformants were then inoculated onto PDA medium for subsequent experiments and stored on PDA slants to obtain the mutant Δ. FoErv25 Mutant Δ FoErv25Growth on PDA medium as follows Figure 1 As shown in B.

[0054] In addition, FoErv25 The gene fragment was constructed on the overexpression vector pKD5-GFP via homologous recombination, transformed into E. coli DH5α, plasmid was extracted, and transformed into Agrobacterium tumefaciens AGL1 using the freeze-thaw method. Finally, it was transformed into AGL1 using Agrobacterium-mediated transformation. FoErv25 The knockout mutant was correctly identified through sequencing, and the replacement strain Δ was obtained. FoErv25 C. Replacement strain Δ FoErv25 The growth of C in PDA medium is as follows Figure 1 As shown in C.

[0055] The overexpression and complementation use the same vectors and steps, and will FoErv25 The gene fragment was constructed on pKD5-GFP via homologous recombination, transformed into E. coli DH5α, plasmid was extracted, and transformed into Agrobacterium AGL1 using the freeze-thaw method. ATMT transformation was then performed on wild-type strains, and sequencing confirmed the correct acquisition of an overexpressing strain. FoErv25 OE. Overexpression strain FoErv25 The growth of OE on PDA medium is as follows: Figure 1 As shown in D.

[0056] The results showed that the gene knockout mutant strain Δ FoErv25 , replenishment strain Δ FoErv25 C. Overexpression strains FoErv25 OE colony morphology is no different from wild-type strains. The hyphae are creeping, the aerial hyphae are not abundant, and the hyphae are dark brown.

[0057] 3. FOR20 wild-type strain and mutant strain Δ FoErv25 , replenishment strain Δ FoErv25 C. Overexpression strains FoErv25 OE co-culture with rice roots After hulling, rice seeds were placed in Erlenmeyer flasks and surface-sterilized with 1% NaClO for 10 min, then with 70% ethanol for 10 min, and rinsed three times with sterile water. The sterilized seeds were then evenly spread on 1 / 2 MS (Murashige and Skoog) medium, sealed with Parafilm, and placed in a 25°C plant culture incubator (16 h light / 8 h dark). After 3 days, when the seeds showed signs of sprouting, they were transferred to tissue culture flasks containing 200 mL of 1 / 2 MS medium, 5 seeds per flask. Five mycelial blocks (0.5 cm in diameter) were also inoculated simultaneously. The control group consisted of sterile PDA agar blocks. Five replicates were performed.

[0058] Determination of root colonization of rice strains: Wild-type strain FO-R20 and mutant strain Δ were collected separately. FoErv25 , replenishment strain Δ FoErv25 C. Overexpression strains FoErv25 The roots of wild-type rice were thoroughly rinsed with sterile water to remove surface impurities. To assess endophytic fungal colonization, the roots were sequentially treated with 75% ethanol for 30 seconds, then with 2% sodium hypochlorite for 2-3 minutes, followed by five rinses with sterile water for surface sterilization. The sterilized root tissue was flash-frozen in liquid nitrogen and ground into powder. Approximately 100 mg of root tissue was used to extract total DNA using a plant genomic DNA extraction kit according to the manufacturer's instructions. The DNA concentration and quality were determined using NanoDrop. The wild-type strain was used as the starting point for further analysis. Tubulin Design specific primers for the target gene in rice. Actin The gene was used as an internal reference gene for qPCR detection. Primer sequences are shown in Table 1. The total volume of the qPCR reaction system was 20 μL, including: 10 μL 2×SYBR Green Master Mix, 0.4 μL each of forward and reverse primers (10 μM), 1 μL DNA template, and 8.2 μL nuclease-free water. The amplification program was: 95°C pre-denaturation for 3 min; followed by 40 cycles: 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s. The relative colonization of endophytic fungi in rice roots was determined using a 22... -ΔCt The method is used to calculate ΔCt = Ct_Tubulin - Ct_Actin. Three biological replicates are set up for each treatment.

[0059] Stem length measurement: Rice was compared with wild-type strains and mutant strains Δ FoErv25 , replenishment strain Δ FoErv25 C. Overexpression strains FoErv25 After 20 days of OE co-culture, three plants were selected from each culture bottle to measure stem length, with three replicates each time, and each experiment was repeated three times.

[0060] Root system analysis was performed using a root scanner (Epson Expression 12000XL, America) to scan images, and the root data was analyzed using the WinRHIZO Pro root system analysis system (Canada).

[0061] Results of endophytic fungal colonization: Figure 2 As shown, relative to the wild-type strain, FoErv25 The mutant strain showed a significant 59.9% reduction in root colonization in rice. FoErv25 The overexpression strain significantly increased the root colonization of rice by 21.3%.

[0062] The results of the growth of tissue-cultured rice with endophytic fungi symbiosis are as follows: Figure 3 and Figure 4 As shown, compared with inoculating wild-type rice, inoculating Δ FoErv25 The rice growth weakened, with a significant decrease in root length (22.95%), stem length (31.1%), above-ground fresh weight (59.6%), and below-ground fresh weight (55.1%). The condition of the rice roots with endophytic fungi symbiosis was as follows: Figure 5 and Figure 6 As shown, the total root length decreased significantly by 36.0%, the total root surface area decreased significantly by 40.4%, the average root diameter decreased significantly by 55%, and the root volume decreased significantly by 47.5%.

[0063] Inoculation with replenishment strain Δ FoErv25 C's rice strain restored the rice's growth-promoting properties, consistent with the function of the wild-type strain.

[0064] Compared with inoculating wild-type rice, inoculating with the overexpressing strain FoErv25 OE rice root length increased by 22.96%, stem length increased significantly by 28.4%, aboveground fresh weight increased significantly by 33.2%, underground fresh weight increased significantly by 45.9%, total root length increased significantly by 11.8%, total root surface area increased significantly by 26.5%, average root diameter increased significantly by 37.6%, and root volume increased significantly by 26.6%.

[0065] These results indicate that FoErv25 The absence of Falciphora oryzae FOR20 has a reduced ability to promote rice growth. FoErv25 Overexpression can lead to Falciphora oryzae FOR20 enhances the growth-promoting ability of rice.

[0066] Example 3: The promoting effect of solid fermented microbial fertilizer on the growth of potted rice 1. Rice seed germination: Wash rice seeds in sequence with clean water, 1% NaClO (soak for 10 min), and sterile water. Place the washed seeds in a glass bowl containing moist absorbent paper and germinate in the dark at 37°C for 2 days.

[0067] 2. Preparation of microbial fertilizer: Wild-type... Falciphora oryzae FO-R20, mutant strain Δ obtained in Example 2 FoErv25, FoErv25 OE was cultured in PDA medium for 7 days, and the mycelial cake was placed in 150 mL PDB medium and cultured at 25℃ and 150 rpm for 3 days. Then the mycelial suspension was poured into barley grains that had been autoclaved (121℃ for 30 min) (150 mL of mycelial solution per 200 g of barley grains) and fermented at 25℃ for 20 days.

[0068] 3. Potted Planting: Allow the fermented microbial fertilizer to air dry naturally. Mix the dried microbial fertilizer with the rice planting substrate and fill the pots. Plant the germinated rice seeds (30 seeds and 40 g of fertilizer per pot) into the pots containing the fermented microbial fertilizer. Use sterile barley seed fertilizer as a control group.

[0069] 4. Phenotypic analysis of rice plants: After 24 days of rice cultivation, the rice seedlings were pulled out of the soil substrate by the roots, washed repeatedly with clean water, and the water on the rice plants was dried with absorbent paper. The height of the rice stems and leaves, root length, above-ground fresh weight and underground fresh weight were measured respectively.

[0070] 5. Results Analysis: For example... Figure 7 and Figure 8 As shown, in the pot experiment, compared with rice co-cultured with the wild-type strain, Δ FoErv25 The co-cultured rice showed a significant reduction in root length (32.3%), stem and leaf height (26.7%), aboveground fresh weight (30.2%), and underground fresh weight (49.9%). Compared with rice co-cultured with wild-type strains, FoErv25 The root length of rice co-cultured with OE increased significantly by 24.5%, the stem and leaf height increased significantly by 25.9%, the aboveground fresh weight increased significantly by 16.0%, and the underground fresh weight increased significantly by 37.9%.

[0071] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. FoErv25 The application of genes in regulating the endophytic fungus *Fusarium oryzae* to promote rice growth is characterized by, The application includes: utilizing biological techniques to... FoErv25 The gene was upregulated in the rice endophytic fungus *Fusarium oryzae*, enhancing the growth-promoting effect of *Fusarium oryzae* on rice; FoErv25 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The FoErv25 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

3.

3. The application as described in claim 1, characterized in that, The application includes: [the following] FoErv25 Gene fragments were cloned into overexpression vectors to construct recombinant plasmids. The target gene fragments were then introduced into *Fusarium oxysporum* using Agrobacterium-mediated transformation, and the resulting plasmids were screened for optimal expression. FoErv25 Transgenic Fusarium graminearum with overexpressed genes was then co-cultured with rice to colonize the rice root tissue.

4. The application as described in any one of claims 1-3, characterized in that, The *Fusarium graminearum* mentioned is the strain with accession number CCTCCNO: M 2021505. Falciphora oryzae FO-R20.

5. The application as described in claim 3, characterized in that, The overexpression vector is pKD5-GFP; the Agrobacterium is Agrobacterium AGL1.

6. The application as described in claim 3, characterized in that, Before co-culturing, the transgenic strain was inoculated into PDA medium for activation culture and cultured in the dark at 22-25℃ for 7-10 days.

7. The application as described in claim 3, characterized in that, The co-culture includes: inoculating germinating rice seeds and transgenic strain blocks onto sterile 1 / 2 MS medium for culture. The co-culture conditions are: culture at 22-25℃ for 20-25 days, with 16 hours of light and 8 hours of dark culture per day.

8. The application as described in claim 3, characterized in that, The co-cultivation includes: inoculating the transgenic strain into sterilized barley grains to obtain solid fermented microbial fertilizer, which is then mixed into rice planting substrate to promote rice growth.

9. A method for promoting rice growth, characterized in that, include: The nucleotide sequence is as shown in SEQ ID NO.1 or SEQ ID NO.

2. FoErv25 Gene fragments were introduced into Fusarium oryzae to obtain overexpressing strains, and then the overexpressing strains were co-cultured with rice to obtain an overexpressing strain-rice symbiotic system.

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