Application of rice transcription factor gene OsNAC15 or its encoded protein in regulating disease resistance of rice

Abstract

This application belongs to the field of biotechnology and provides a rice transcription factor gene. OsNAC15 Or the application of its encoded protein in regulating rice disease resistance, said rice transcription factor gene OsNAC15 The nucleotide sequence is shown in SEQ ID NO:1; the amino acid sequence of the encoded protein is shown in SEQ ID NO:2. This invention confirms the transgenic function through verification. OsNAC15 It is a positive regulator of rice blast resistance, and overexpression OsNAC15 It can significantly enhance rice's resistance to rice blast (leaf blast), while knocking out this gene leads to a decrease in rice resistance. Moreover, the regulation of this gene does not affect agronomic traits such as plant height, number of panicles, number of grains per panicle, seed setting rate, thousand-grain weight, or rice quality. It provides an ideal gene resource for breeding disease-resistant, high-quality, and high-yield rice varieties.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a rice transcription factor gene. OsNAC15 Or the application of its encoded proteins in regulating disease resistance in rice. Background Technology

[0002] Rice blast is caused by Ascomycetes ( Magnaporthe grisea Rice blast is a global disease caused by fungi, one of the top ten fungal diseases of plants, commonly known as "rice cancer." It occurs in all rice-producing regions worldwide, with severe outbreaks in major rice-growing areas such as Asia and Africa. In my country's main rice-producing areas, it is the leading cause of disease outbreaks, generally more severe in mountainous regions than in plains, and more severe in japonica rice than indica rice. In epidemic years, rice blast can cause yield reductions of 10% to 20% in mild cases, and up to 40% to 50% in severe cases, or even complete crop failure. It also degrades rice quality.

[0003] Rice blast resistance mechanisms are complex, involving the synergistic regulation of multiple signaling pathways and a large number of genes. Currently, the number of resistance genes that can be directly applied in production is far from sufficient, and most genes have narrow resistance spectra or limited resistance persistence. Therefore, continuously exploring superior resistance gene resources with broad and persistent resistance spectra and systematically analyzing their resistance mechanisms is of great significance for breeding new rice varieties with broad-spectrum and durable rice blast resistance and ensuring food security.

[0004] The NAC transcription factor family is a class of transcription factors unique to plants, which are widely involved in various physiological processes such as plant growth and development and responses to abiotic stress. OsNAC15 As a member of the NAC family, its function in regulating rice blast resistance has not been reported. This invention systematically elucidates its role through plant molecular biology, genetics, and physiological and biochemical techniques. OsNAC15 To investigate the molecular characteristics and biological functions of [the gene], and to elucidate its molecular mechanism for regulating rice blast resistance, this study will provide new gene resources and theoretical basis for rice blast resistance breeding. Summary of the Invention

[0005] This invention aims to solve the problems in the prior art and provide a rice transcription factor gene. OsNAC15 Or the application of its encoded proteins in regulating disease resistance in rice.

[0006] To achieve the above objectives, the first aspect of this application provides a rice transcription factor gene. OsNAC15 Or the application of its encoded protein in regulating rice disease resistance, said rice transcription factor gene OsNAC15 The nucleotide sequence is shown in SEQ ID NO: 1; the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2.

[0007] The preferred application described above is through enhancing rice transcription factor genes. OsNAC15 The expression or activity of [a substance] can enhance the resistance of rice to rice blast.

[0008] Of the above applications, PCR amplification is preferred. OsNAC15 The CDS sequence was used to construct the 35S promoter-driven overexpression vector 35S-P1300- OsNAC15 -GFP; This was transferred into rice using Agrobacterium-mediated genetic transformation to obtain the rice transcription factor gene. OsNAC15 Transgenic lines overexpressing [the gene].

[0009] A second aspect of this application provides a rice transcription factor. OsNAC15 The application of proteins or their encoding genes as targets for regulating disease resistance in rice, said rice transcription factors OsNAC15 The amino acid sequence of the protein is as shown in SEQ ID NO: 2.

[0010] A third aspect of this application provides a recombinant vector comprising the above-described nucleotide sequence.

[0011] Preferably, the recombinant vector described above is an expression vector or a gene editing vector.

[0012] The fourth aspect of this application provides a method for enhancing the resistance of rice to rice blast, comprising the step of introducing the above-mentioned nucleotide sequence or the above-mentioned recombinant vector into a rice receptor.

[0013] The fourth aspect of this application provides a method for detecting or amplifying the aforementioned rice transcription factor gene. OsNAC15 The primer pairs are selected from any one of the following groups:

[0014] (a) Used for amplification OsNAC15 The primer pairs for the gene CDS sequence, with the nucleotide sequences of the forward and reverse primers shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; (b) Used for detection OsNAC15 The nucleotide sequences of the forward and reverse primers for qRT-PCR at gene expression levels are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. (c) Used for detection OsNAC1 5. Primer pairs for identifying gene editing status, the nucleotide sequences of the forward and reverse primers are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0015] The fifth aspect of this application provides a method for analysis OsNAC15Gene function kit, containing the primer pairs mentioned above.

[0016] The sixth aspect of this application provides a composition for synergistically regulating rice resistance to rice blast, comprising the aforementioned rice transcription factor gene. OsNAC15 The encoded protein, and the proteins that interact with it, wherein the interacting proteins are OsAPIP5, ONAC122, or OsOTUB1.1.

[0017] Compared with the prior art, this application has the following beneficial effects: (1) This invention confirms the transgenic function through verification. OsNAC15 It is a positive regulator of rice blast resistance, and overexpression OsNAC15 It can significantly enhance rice's resistance to rice blast (leaf blast), while knocking out this gene leads to a decrease in rice resistance. Moreover, the regulation of this gene does not affect agronomic traits such as plant height, number of panicles, number of grains per panicle, seed setting rate, thousand-grain weight, or rice quality. It provides an ideal gene resource for breeding disease-resistant, high-quality, and high-yield rice varieties.

[0018] (2) This invention analyzes OsNAC15 The molecular mechanism of resistance to rice blast disease is mainly mediated by the interaction with OsAPIP5, ONAC122 and OsOTUB1.1 proteins, which synergistically regulate rice blast resistance, providing a new perspective for a deeper understanding of the molecular network of rice blast resistance.

[0019] (3) The multiple sets of specific amplification primers provided by this invention can be used efficiently for OsNAC15 The gene expression detection, vector construction, and editing line identification are characterized by high specificity and high amplification efficiency, providing a convenient tool for further research and application of this gene. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 shows the result obtained in Example 1. OsNAC15 The spatiotemporal representation pattern results; Figure 2 shows the result obtained in Example 1. OsNAC15 Subcellular localization results of the protein: A is OsNAC15 In the subcellular localization of rice protoplasts, B is... OsNAC15 Subcellular localization within tobacco cells; Figure 3 shows the identification results of positive strains of ProOsNAC15-GUS material obtained in Example 1; Figure 4 shows the tissue-specific GUS staining results of the OsNAC15 gene obtained in Example 1: 1. Leaf during grain filling stage; 2. Leaf during tillering stage; 3. Leaf sheath during heading stage; 4. Stem base during tillering stage; 5. Root during seedling stage; 6. Leaf during tillering stage; 7. Leaf during heading stage; 8. Leaf sheath during heading stage; 9. Stem base during heading stage; 10. Spikelet during heading stage. Figure 5 shows the hygromycin identification (A) and qRT-PCR analysis results (B) of the OsNAC15 overexpression material obtained in Example 2; Figure 6 shows the sequencing identification results of the OsNAC15 gene knockout mutant obtained in Example 2: A and T deletions ( osnac15-1 ); B, T insertion ( osnac15-2 ); C, A Insert ( osnac15-3 D, GAGAACAAAGC permutation ( osnac15-4 E, ATCCG substitution ( osnac15-5 ); F, G insertion, CAT permutation, and A insertion ( osnac15- 6 ); Figure 7 shows the blast resistance phenotypes (A) and lesion length statistics (B) of the OsNAC15 overexpressing lines, knockout lines and wild-type rice obtained in Example 2. Figure 8 shows the statistical results of the main agronomic traits of the OsNAC15 overexpressing lines, knockout lines, and wild type obtained in Example 2: A, plant height; B, number of panicles per plant; C, number of panicles per mu (667 square meters); D, panicle length; E, number of grains per panicle; F, thousand-grain weight; G, seed setting rate; H, yield per plant; I, yield per mu (667 square meters); J, harvest index. Figure 9 shows the quality test results of the OsNAC15 genetic material and wild-type rice obtained in Example 2: (OE1, OsNAC15-OE1 OE2, OsNAC15-OE2 ;WT, NIP; MT1, osnac15-1 MT2, osnac15-2 ); Figure 10 shows the OsNAC15 candidate binding motif results obtained by reverse yeast one-hybrid (TF-centered Y1H) screening in Example 3: A, sequencing alignment results; B, candidate motif; C, candidate motif verification; (+) pGAD53m+pHIS2-p53 is the positive control; (-) pGADT7-Os07g0684800+pHIS2-p53 is the negative control; Figure 11 shows the yeast single hybrid (H1Y) verification results of OsNAC15 and candidate motif obtained in Example 3: pGADT7-Os07g0684800+pHIS2-p53 is the negative control; pGAD53m+pHIS2-p53 is the positive control. Os07g0684800 For genes OsNAC15 Gene ID; Figure 12 shows the yeast two-hybrid (H2Y) verification results of OsNAC15 and candidate interacting proteins obtained in Example 3. pGBKT7-p53+pGADT7-largeT is the positive control; pGBKT7-laminC+pGADT7-largeT is the negative control. Figure 13 shows the bimolecular fluorescence complementation (BIFC) verification results of OsNAC15 and the candidate interacting protein obtained in Example 3. Detailed Implementation

[0022] To facilitate understanding of this application, the following description will be more comprehensive and detailed in conjunction with the accompanying drawings and preferred embodiments, but the scope of protection of this application is not limited to the following specific embodiments.

[0023] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of this application.

[0024] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0025] Example 1: OsNAC15 Molecular characterization of genes A rice transcription factor gene OsNAC15 Its nucleotide sequence is shown in SEQ ID NO: 1. The amino acid sequence it encodes is shown in SEQ ID NO: 2.

[0026] SEQ ID NO: 1 ATGTCGGAGTCGGAGGTGTCGGTGATCAACCAGCTGGAGGAGGAGGAGACTCGGCTGGAGCTGCCGCCGGGGTTCCGGTTCCACCCCACCGACGAGGAGGTGGTGACCCACTACCTCACCCGCAAGGCCCAGGACCGGAGCTTCTCCTGCGTCGTCATCGCCGACGTGAACCTCAACAACTGCGAGCCATGGGACCTCCCAAGCAAGGCGAAGATGGGGGAGAAGGAGTGGTTCTTCTTCTGCCACAAGGACCGCAAGTACCCGACGGGGATGAGGACGAACCGGGCGACGGCCAGCGGGTACTGGAAGGCCACCGGGAAGGACAAGGAGATCTTCCGCGGCCGCGGCCTCCTCGTCGGCATGAAGAAGACGCTCGTCTTCTACATGGGCCGCGCCCCCCGCGGCGAGAAGACCCCCTGGGTCATGCACGAATACCGCCTCGACGGCAAGCTTCCCCCCAACCTCCCCCGCTCCGCAAAGGAGGAATGGGCGGTTTGCCGGGTGTTCAACAAAGACTTGGCGGCAAAGATTGCCCAAATGCCGCCGCCTCCCTTCCCGCGCAACGACTCCTTCGACCTCGACCTCGACGACTTCCTCCACCTCGACGCCGACCTGCCGCCGCTCATCGACGACCCCTTCGCCTCCACCTCCACGCTCAAGACGGAGCCACCTCCGCCGGCCAACCTGATGCACAACCACTACGGCTACTTCTCCCTCCCGGCGTCAGCGACCAATTATAACCACAGCTCCGGCGCCATGGCGGACCAGGCGATTCGGAGGTTCTGCAAGGCGGAGGCGTCGACGGCGTGCTTCTCCGGCGCCGACGCTGACGTGGATCCGGTGGTGGACGAGCTGCTCTCCTTCCCGGACTCCATCACGGACTACTCCTACATATGGAAGGCCTGA SEQ ID NO:2 MSESEVSVINQLEEEETRLELPPGFRFHPTDEEVVTHYLTRKAQDRSFSCVVIADVNLNNCEPWDLPSKAKMGEKEWFFFCHKDRKYPTGMRTNRATASGYWKATGKDKEIFRGRGLLVGMKKTLVFYMGRAPRGEKTPWVMHEYRLDGK LPPNLPRSAKEEWAVCRVFNKDLAAKIAQMPPPPFPRNDSFDLDLDDFLHLDADLPPLIDDPFASTSTLKTEPPPPANLMHNHYGYFSLPASATNYNHSSGAMADQAIRRFCKAEASTACFSGADADVDPVVDELLSFPDSITDYSYIWKA

[0027] Should NAC Transcription factor genes OsNAC15 Through transcriptome analysis, gene overexpression technology, CRISPRE The CAS9 technique, along with plant molecular genetics and physiological biochemical methods, was discovered in rice. Studies have shown that... OsNAC15 It is a positive regulator of rice blast resistance, and overexpression OsNAC15 It can significantly enhance rice's resistance to rice blast (leaf blast), while knocking out this gene leads to a weakening of rice resistance, providing an ideal gene resource for breeding disease-resistant rice varieties.

[0028] 1. OsNAC15 Tissue expression patterns of genes and subcellular localization of proteins (1) In order to obtain OsNAC15 To investigate gene expression patterns, RNA was extracted from various tissues (roots, stem base, stem, nodes, leaf sheaths, leaves, and panicles) during the seedling, tillering, heading, and grain-filling stages of rice (Nipponbare). Each sample was tested in triplicate. Total RNA was extracted using TRIzol, and first-strand cDNA was synthesized using the Hiscript II QRT SuperMix kit (Vazyme). Real-time quantitative PCR analysis was performed on a StepOnePlus instrument using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) premix, with qRT primers as the amplification primers. OsNAC15 Its forward and reverse primers are 5' and 5' respectively. TGCCACAAGGACCGCAAGTA 3' (as shown in SEQ ID NO: 7) and 5' AACCGCCCATTCCTCCTTT 3' (as shown in SEQ ID NO: 8). Using Action as the internal reference gene, the amplification primers were A:: Action Its forward and reverse primers are 5' and 5' respectively. GACTCTGGTGATGGTGTCAGC 3' (as shown in SEQ ID NO:13) and 5' GCTGGAAGAGGACCTCAGG 3' (as shown in SEQ ID NO: 14).

[0029] The specific steps are as follows: Total RNA extraction: After tissue sampling, the sample was flash-frozen in liquid nitrogen and ground into powder. 0.1 g of the sample was added to a 1.5 mL centrifuge tube, 1 mL of TRIzol was added, and the mixture was quickly mixed and allowed to stand at room temperature for 5 min. 200 μL of chloroform was added to the centrifuge tube, and the mixture was shaken vigorously for 30 s and allowed to stand at room temperature (25℃) for 10 min. The mixture was then centrifuged at 12000 rpm for 10 min at 4℃. 500 μL of the supernatant was carefully transferred to a new 1.5 mL centrifuge tube. An equal volume of isopropanol was added, and the mixture was gently inverted to mix. The mixture was allowed to stand at room temperature (25℃) for 10 min. The mixture was then centrifuged at 12000 rpm for 10 min at 4℃ and the supernatant was discarded. 1 mL of 75% ethanol was added, and the precipitate was gently inverted to suspend it. The mixture was then centrifuged at 8000 rpm for 5 min at 4℃ and the supernatant was discarded. The 75% ethanol washing step was repeated. The RNA precipitate was blown into a semi-transparent gel in a clean bench and 30-40 μL of DEPC water was added to dissolve the RNA.

[0030] RNA quality testing and concentration determination: RNA integrity was detected by 1% agarose gel rapid electrophoresis. The 18S and 28S bands were observed. The brightness of the 28S band was about twice that of the 18S band, indicating good RNA integrity. The purity and concentration of RNA were detected by Nano Drop nucleic acid analyzer. RNA samples with OD260 / 280 between 1.8 and 2.0 met the purity standards and could be used for subsequent experiments.

[0031] First-strand cDNA synthesis: Total RNA was synthesized using the Hiscript II QRT SuperMix reverse transcription kit. The total cDNA volume for each sample was 1 μg. The reverse transcription system was as follows: Total RNA (1 μg), 4×gDNA wiper Mix 4 μL, Hiscript II QRT Super Mix II 4 μL, and RNA-free water to a final volume of 20 μL. Reaction conditions: Incubation at 42℃ for 15 min, followed by inactivation at 85℃ for 5 s. After the reaction, an equal volume of RNA-free water was added to dilute the cDNA by half, and the mixture was thoroughly mixed and stored at -20℃ for later use.

[0032] Real-time quantitative PCR: Using the first strand of cDNA synthesized by reverse transcription as a template, quantification was performed in two steps on a Roche Light 96 real-time quantitative PCR instrument, with three technical replicates per sample for each gene. The reaction system was as follows: 1 μL cDNA, qRT:: OsNAC15 Forward primer (10 μM) 0.5 μL, qRT:: OsNAC15 0.5 μL of reverse primer (10 μM), 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix, and 8 μL of ddH2O. Reaction conditions: (1) Pre-denaturation stage: 95℃, 10 min; (2) Amplification stage: 95℃, 10 s, 60℃, 30 s, for a total of 40 cycles; (3) Melting curve stage: 96℃, 15 s, 60℃, 60 s, 95℃, 15 s.

[0033] The results are shown in Figure 1. OsNAC15 The expression was detected in all rice tissues tested. The highest expression was found in leaves during the seedling stage, followed by high expression levels in leaves and leaf sheaths during the tillering stage, and the lowest expression level at the stem base. During the heading stage, the highest expression level was found in leaves, followed by stems, nodes, spikelets, and roots, with the lowest expression level at the stem base. During the grain-filling stage, the highest expression level was found in leaves, followed by panicle stalks, spikelets, leaf sheaths, nodes, roots, and stem base. Throughout the entire growth period, the highest expression level was observed in leaves during the heading stage. Picture 1 ).

[0034] (2) For research OsNAC15 Subcellular localization of the protein was obtained by PCR amplification. OsNAC15 The CDS sequence (excluding the terminator) is used, and the amplification primers are OE:: OsNAC15 Its forward and reverse primers are 5' and 5' respectively. cgggggactctagaggatccATGTCGGAGTCGGAGGTGTCGGTGA 3' (as shown in SEQ ID NO: 3) and 5' tcggaggaggccatactagtGGCCTTCCATATGTAGGAGTAGTC 3' (as shown in SEQ ID NO: 4), the amplified fragment was cloned into the pCAMBIA1300-GFP vector to construct the pCAMBIA1300-GFP-OsNAC15 fusion expression vector. Rice protoplasts were transformed using a PEG-mediated method, and the GFP signal was observed using a laser confocal microscope.

[0035] The specific steps are as follows: Vector construction: Using rice Nipponbare cDNA as a template, OE:: OsNAC15 PCR amplification was performed using primers. The reaction mixture consisted of 1 μL template cDNA, 15 μL Max Master Mix, 1.5 μL forward primer, 1.5 μL reverse primer, and 11 μL ddH2O. The reaction conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, and 72℃ extension for 1 min, for 40 cycles; followed by a final extension at 72℃ for 10 min. The amplified products were recovered after electrophoresis, double-digested with the pCAMBIA1300-GFP vector, ligated, and transformed into DH5α competent cells. Positive clones were screened and sequenced for verification.

[0036] Rice protoplast preparation: Rice seedlings of Nipponbare variety were cultured in 1 / 2 MS medium and grown in the dark for 14 days. Fifteen seedlings with good growth were selected and cut into thin strips with a blade. The strips were placed in a petri dish containing 10 mL of enzymatic hydrolysate, vacuumed for 5 min, and then enzymatically hydrolyzed in a shaker in the dark for 5 h (28℃, 45 rpm). The hydrolysate was filtered through a 150-mesh sieve into a 10 mL round-bottom centrifuge tube. The tube was centrifuged at 200 g for 1 min at 4℃, the supernatant was discarded, and 1 mL of W5 was gently added along the wall to gently resuspend the protoplasts until there was no precipitate at the bottom. The tubes were incubated on ice for 40 min. The supernatant was discarded, and 2 mL of W5 was added and gently inverted to mix. The tubes were counted under a microscope and then incubated on ice for later use.

[0037] PEG-mediated protoplast transformation: MMG was added based on the protoplast concentration to achieve a final concentration of approximately 2 × 10⁻⁶. 5 Protoplasts / mL; prepare the reaction solution in a 1.5 mL round-bottom centrifuge tube: 150 μL protoplasts, 15 μg OsNAC15- GFP plasmid (1.0 μg / μL) was mixed evenly; 165 μL of PEG transformation solution was added, and the mixture was gently inverted several times and allowed to stand at room temperature for 5 min; 1 mL of W5 was added to stop the reaction; the mixture was centrifuged at 200 g for 5 min, the supernatant was discarded, and the mixture was resuspended in 200 μL of W5 and cultured overnight at 22℃ in the dark; the GFP signal was observed under a laser confocal microscope.

[0038] Agrobacterium transformation: Take 50 μL of Agrobacterium GV3101 competent cells, thaw them on ice, add 2 μg of recombinant plasmid (OsNAC15-GFP), mix gently, and incubate on ice for 30 min; flash freeze in liquid nitrogen for 5 min, heat shock in a water bath at 37℃ for 5 min, and incubate on ice for 2 min; add 500 μL of antibiotic-free LB medium, and culture at 28℃ and 200 rpm for 2 h with shaking; take 200 μL and spread it onto LB plates containing double antibiotics, and incubate upside down at 28℃ for 48 h.

[0039] Agrobacterium activation: Pick single colonies and inoculate them into 5 mL of LB medium containing antibiotics and viable antibiotics. Incubate at 28°C and 200 rpm for 16 h with shaking. Transfer the culture to 50 mL of fresh LB medium containing antibiotics and viable antibiotics at a 1:100 ratio and continue culturing until OD (Occurrence Limit). 600 = 0.6-0.8 (approximately 6-8 h). Centrifuge the activated Agrobacterium culture at 5000 rpm for 10 min, discard the supernatant; resuspend the precipitate in infection buffer and adjust the OD. 600 = 0.6-0.8, add acetylsuccinone (final concentration 200 μM), and let stand at room temperature for 3 h (protected from light).

[0040] Instantaneous transformation of tobacco leaves: Greenhouse conventional culture (16 h light / 8 h darkness, 25℃, 60-70% humidity) *Nicotiana benthamiana* ( Nicotiana benthamiana 4-6 weeks. Select healthy, disease-free functional leaves (3rd-5th true leaves), disinfect the leaf surface by wiping with 75% ethanol, and air dry. Using a 1 mL needleless syringe, draw up the Agrobacterium infection solution and gently inject it from the underside of the leaf, ensuring even penetration into the leaf tissue (avoid puncturing the leaf; inject 2-3 areas per leaf, approximately 0.5 cm² each). Place the injected tobacco leaves in a greenhouse and incubate in darkness for 12 h, then resume normal light (16 h / 8 h), continuing incubation for another 36 h. Take leaves 36 h post-injection, cut out the infected area (approximately 0.3 cm × 0.3 cm) with a blade, place it on a glass slide, add 1 drop of PBS buffer, and gently flatten the lower epidermis of the leaf. Excite with GFP at 488 nm and emit at 500-530 nm, adjusting the focus and laser intensity to capture the fluorescence signal distribution.

[0041] The results are shown in Figure 2. OsNAC15 The protein is located in the cell nucleus.

[0042] 2. OsNAC15 Tissue-specific expression analysis To investigate the promoter activity and expression pattern of the OsNAC15 gene, the plant expression vector DX2181-GUS was constructed. Based on data from the rice database... OsNAC15 The 2.0 kb sequence preceding the start codon ATG was used to design specific primers Pro:: OsNAC15 Its forward and reverse primers are as shown in SEQ ID NO: 15 (5'). cgacggccagtgccaagcttGCTCTAGAAGCTTGCATGCCT 3') and SEQ ID NO: 16 (5') gactgaccacccggggatccCGGATCCGGTACCTCTAGAGT As shown in 3'), PCR amplification was performed using rice Nipponbare genomic DNA as a template to obtain... OsNAC15 The promoter sequence was cloned into the DX2181-GUS vector, transformed into Agrobacterium EHA105, and then transformed into rice Nipponbare using Agrobacterium-mediated transformation.

[0043] The specific steps are as follows: Promoter amplification: Using rice Nipponbare genomic DNA as a template, Pro:: OsNAC15 PCR amplification was performed using primers. The reaction mixture consisted of 1 μL template DNA, 15 μL Max Master Mix, 1.5 μL forward primer, 1.5 μL reverse primer, and 11 μL ddH₂O. The reaction conditions were: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 58 °C annealing for 30 s, and 72 °C extension for 2 min, for 40 cycles; followed by a final extension at 72 °C for 10 min. The amplified products were recovered after electrophoresis.

[0044] Vector construction: The DX2181-GUS vector and promoter amplification fragment were double-digested with the corresponding restriction enzymes, recovered, and ligated using the II Recombinant Cloning Kit (Vazyme). The vector was then transformed into DH5α competent cells. Kanamycin was used for screening plates. Positive clones were picked and subjected to colony PCR detection and sequencing. After identification, the plasmid was extracted and named ProOsNAC15-DX2181-GUS.

[0045] Agrobacterium transformation: The recombinant plasmid was transformed into Agrobacterium EHA105, and the antibiotics were kanamycin and rifampin. After the bacterial culture was confirmed to be positive, it was used for genetic transformation of mature embryo callus tissue of wild-type rice.

[0046] Rice genetic transformation: (1) Rice mature embryo callus culture: Select healthy Nipponbare grains, remove the husks, sterilize the surface with 75% ethanol for 3 min, rinse twice with sterile water; disinfect with sodium hypochlorite stock solution for 30 min, shake gently every few minutes to ensure thorough disinfection, rinse 7 times with sterile water; transfer the seeds to sterile filter paper to dry, inoculate 20-25 seeds into NBM induction medium; culture in the dark at 25-26℃ for 10 days to induce callus; pick dry, dense, light yellow callus tissue, remove the grains and buds, transfer to subculture medium, and culture in the dark at 25-26℃ for 7 days.

[0047] (2) Co-culture of Agrobacterium and callus: Pick the callus and place it on sterile filter paper to air dry until the surface turns white; transfer it into the bacterial solution with the OD value adjusted, soak for 30 min, and shake it gently every 5 min; pour off the bacterial solution, wash it 4 times with sterile water, and air dry it again; take out the NBM (containing As) solid culture medium, cover the surface with sterile filter paper, transfer the callus into it, and incubate in the dark at 25~26℃ for 3 days.

[0048] (3) Screening culture: The co-cultured callus tissue was rinsed 7 times with sterile water and then soaked in sterile water containing 500 mg / L cefotaxime and 400 mg / L carbenicillin for 30 min; after drying, it was transferred to screening medium (J3 + 500 mg / L cefotaxime + 400 mg / L carbenicillin + 50 mg / L hygromycin) and cultured in the dark at 25~26℃; after 15 days, the active callus tissue was picked and transferred to a new screening medium for a second screening.

[0049] (4) Predifferentiation, differentiation, and rooting culture: The resistant callus was transferred to the predifferentiation medium (Y + 500 mg / L cefotaxime + 400 mg / L carbenicillin) and cultured at 25-26℃ for 14 h under light for 5 days; the green callus was transferred to the differentiation medium (DL + 500 mg / L cefotaxime + 400 mg / L carbenicillin) and cultured under light for 14 h, with the medium being changed every 15 days; the differentiated green seedlings (about 4-6 cm tall) were transferred to the rooting medium (R) to root.

[0050] (5) Hardening off and transplanting: After the roots have grown for about 15 days, open the culture bottle cap and add sterile water to harden the seedlings for 10 days; rinse the culture medium from the roots and transplant them into the soil. After they survive, they are transplanted into the experimental field.

[0051] (6) Identification of transgenic plants: DNA was extracted from transgenic plants, and specific primers (HYG::OsNAC15) were designed based on the hygromycin gene sequence. The forward primer is shown in SEQ ID NO: 5 (5'). CTGCCCGCTGTTCTACAACCGG As shown in SEQ ID NO: 6 (5'), the reverse primer is as shown in SEQ ID NO: 6 (5'). GGAGCATATACGCCCGGAGTC PCR identification was performed as shown in 3'), and transgenic positive seedlings were obtained.

[0052] The genetically transformed lines were cultured throughout their entire growth period, and the hygromycin identification results were as follows: Picture 3 As shown, a total of 16 positive plants were obtained.

[0053] After identification, tissues from positive plants at various stages were added to GUS staining solution and stained in a 37℃ incubator in the dark for about 10 hours. The tissues were then destained with 30%, 50%, and 70% alcohol sequentially until chlorophyll was completely removed, and observed and photographed under a microscope. The results are shown in Figure 4. GUS activity was detected in leaves and roots during the seedling stage; leaves, leaf sheaths, and stem base during the tillering stage; leaves, leaf sheaths, stem base, and spikelets during the heading stage; and leaves during the grain-filling stage, consistent with the spatiotemporal expression pattern, with expression mainly observed in leaves during the heading stage.

[0054] Example 2: OsNAC15 Biological function analysis of genes 1. The present invention OsNAC15 Obtaining Gene Overexpression Materials To explore OsNAC15 The biological functions are obtained through PCR amplification. OsNAC15 The CDS sequence (excluding the terminator) is used, and the amplification primers are OE:: OsNAC15 Its forward and reverse primers are 5' and 5' respectively. cgggggactctagaggatccATGTCGGAGTCGGAGGTGTCGGTGA 3' (as shown in SEQ ID NO: 3) and 5' tcggaggaggccatactagtGGCCTTCCATATGTAGGAGTAGTC 3' (as shown in SEQ ID NO: 4), the amplified fragment was cloned into the pCAMBIA1300-GFP vector to construct the pCAMBIA1300-GFP-OsNAC15 fusion expression vector, which was transformed into Agrobacterium tumefaciens GV3101. The positive strain was used for genetic transformation of wild-type rice Nipponbare to obtain... OsNAC15Overexpression of transgenic plants. Transgenic lines were identified using PCR amplification of the hygromycin gene, with the primer HYG:: OsNAC15 Its forward and reverse primers are 5' and 5' respectively. CTGCCCGCTGTTCTACAACCGG 3' (as shown in SEQ ID NO: 5) and 5' GGAGCATATACGCCCGGAGTC 3' (as shown in SEQ ID NO: 6). Forty-five positive lines were obtained through hygromycin identification. Quantitative detection of transgenic lines was performed using qRT-PCR. OsNAC15 The expression level was detected using the qRT:: primer. OsNAC15 Its forward and reverse primers are 5' and 5' respectively. TGCCACAAGGACCGCAAGTA 3' (as shown in SEQ ID NO: 7) and 5' AACCGCCCATTCCTCCTTT 3' (as shown in SEQ ID NO: 8), the internal reference primer is A:: Action Its forward and reverse primers are 5' and 5' respectively. GACTCTGGTGATGGTGTCAGC 3' (as shown in SEQ ID NO: 13), and 5' GCTGGAAGAGGACCTCAGG 3' (as shown in SEQ ID NO: 14). Analysis yielded 16 [items / items]. OsNAC15 Overexpression lines were used, and seeds of T3 generation pure lines were obtained by further generations (as shown in Figure 5). Two positive overexpression lines, 24-14 and 9-1, were selected and named as follows: OsNAC15-OE1 and OsNAC15-OE2 For use in subsequent research.

[0055] 2. The present invention OsNAC15 Obtaining gene knockout materials To further verify the present invention OsNAC15 The role of rice in rice blast resistance was investigated using Nipponbare (NIP) as a background model. OsNAC15 CRISPR / Cas9 knockout vector for the gene: The pYLCRISPR / Cas9-MH basic vector containing the CaMV 35S promoter driving SpCas9, the dual U6-26 promoter, and the hygromycin resistance marker was selected. The target sequence 5' was knocked out using the Golden Gate one-step method. CTACCTCACCCGCAAGGCCCAGG 3' (as shown in SEQ ID NO: 9, located at positions 42-64 of the CDS in exon 1 of the OsNAC15 gene, corresponding to the core binding region of the NAC domain) and 5' CCGACGTGAACCTCAACAACTGC 3' (as shown in SEQ ID NO: 10, located at positions 285-307 of the CDS in exon 2, corresponding to the region connecting the transcriptional activation region) was inserted into the vector (simultaneous enzyme digestion and ligation at 55℃ for 15 min, verified by E. coli DH5α resistance selection and Sanger sequencing). The recombinant vector was transformed into Agrobacterium tumefaciens GV3101 using the freeze-thaw method, and after double-antibiotic selection for positive strains, it was transformed into wild-type rice Nipponbare using Agrobacterium-mediated callus infection. After induction, selection, differentiation, and hardening, the resulting seedlings were obtained. OsNAC15 Gene-edited transgenic lines were developed. The selection of the two target sequences strictly followed CRISPR / Cas9 target design principles. Both sequences contained NGG-type PAM sequences recognized by SpCas9 at their 3' ends, and no highly homologous sequences were found across the entire genome after comparison with the RGAP database, thus avoiding off-target effects. Both sequences targeted the critical N-terminal NAC domain of the OsNAC15 gene, and the GC content (56.5%, 52.2%) was within the optimal range of 40%-60%, ensuring sgRNA binding stability and gene function disruption efficiency. Transgenic lines were identified using PCR amplification (20 μL system, 94℃ pre-denaturation for 5 min followed by 35 cycles of amplification, annealing at 58℃). The identification primers were Cas9:: OsNAC15 Its forward and reverse primers are 5' and 5' respectively. TGATCAACCAGCTGGAGGAG 3' (as shown in SEQ ID NO: 11), and 5' GACGATGAGGAAGCTAGGC 3' (as shown in SEQ ID NO: 12), positive lines were detected by 1.5% agarose gel electrophoresis, and then identified by Sanger sequencing as 6 different types of mutant lines: T deletion ( osnac15-1 ), T insertion ( osnac15-2 A Insert ( osnac15-3 ), GAGAACAAAGC substitution ( osnac15-4 ), ATCCG replacement ( osnac15-5 ) and G insertion, as well as CAT permutation and A insertion ( osnac15-6 ), and then further generation to obtain T3 generation pure materials (as shown in Figure 6), select osnac15-1 and osnac15-2 Further functional analysis will be conducted.

[0056] 3. The present invention OsNAC15The influence of genes on rice blast resistance Rice blast resistance identification: using Nipponbare (NIP) and genetically stable varieties with NIP background. OsNAC15 Overexpression materials OsNAC15-OE1 and OsNAC15-OE2 With NIP background and genetically stable OsNAC15 Knockout strains osnac15-1 and osnac15-2 Using these materials, rice blast seedlings were treated with puncture wound inoculation to identify the pathogen. OsNAC15 The impact on rice blast resistance.

[0057] The specific steps are as follows: Rice blast fungus cultivation: 1. Culture medium preparation: Potato dextrose agar (PDA, for strain activation / preservation): 200 g potato, 20 g glucose, 15-20 g agar, 1000 mL distilled water, pH 5.6-6.0. Peel and chop the potato, boil for 30 min, filter through 4 layers of gauze and collect the filtrate; add glucose and agar, stir to dissolve, and bring the volume to 1000 mL; dispense into Erlenmeyer flasks / petries, autoclave at 121℃ for 20 min, cool to about 50℃ and pour into plates (about 20 mL per plate). Oatmeal agar (OMA, for sporulation): 40 g oatmeal, 15-20 g agar, 1000 mL distilled water, pH 5.6-6.0. Boil oatmeal with distilled water for 30 min, filter through 4 layers of gauze; add agar to dissolve and bring the volume to a final level, sterilize, and pour into plates. 2. Activation, purification, and sporulation of *Magnaporthe oryzae* strains: A small amount of *Magnaporthe oryzae* strain was selected and inoculated onto the center of a PDA plate using a sterile inoculation needle within a clean bench. The plate was then sealed with Parafilm. The plate was incubated in the dark at 25°C until the mycelium covered the entire plate. Using a sterile punch (6 mm), holes were made along the edge of the mycelium on the PDA plate, and mycelial discs (with mycelium) were collected. 3-4 discs were inoculated onto each OMA plate and evenly distributed. After incubation in the dark at 25°C for 7 days until the mycelium covered the plate, the plate was placed in a 25°C artificial climate chamber and cultured under alternating 12-hour light / 12-hour dark conditions for 5 days to induce sporulation, while maintaining a slightly moist environment within the petri dish. After light induction, the plate surface was washed with sterile water, and the sporulation was observed under a microscope. The sporulation density was adjusted to 1×10⁻⁶. 6 / mL.

[0058] Rice blast inoculation: Select healthy, plump, and uniform seeds, disinfect them in 75% alcohol for 5 min, then sterilize them in 2% sodium hypochlorite solution for 15 min, and finally rinse them 5 times with pure water. Place the test materials in petri dishes (9 cm in diameter) lined with double-layered filter paper and soak for 48 h. Select seeds with sprouting white leaves and sow them in pots containing nutrient soil and vermiculite (3:1 ratio). Incubate at 26℃ and approximately 75% relative humidity under 16 h light / 8 h dark conditions. At the 5-leaf stage, cut 5-6 cm long fully extended terminal leaves of rice, place them on filter paper in a petri dish, and add 0.001% 6-benzylaminopurine solution. Then, use a sterile pipette tip to create a mechanical lesion point at the center of the upper surface of the leaf. Place 10 μL of conidial suspension (1×10⁻⁶) at intervals on the leaf surface. 6 / mL). Inoculated leaves were cultured in darkness under moist conditions for 24 hours, then cultured normally. Lesion size was measured 7 days post-inoculation.

[0059] The results are as follows Picture 7 As shown, OsNAC15 The lesion length on the leaves of the overexpression material was significantly smaller than that of the wild-type control, while OsNAC15 The lesion length of the mutant material was significantly larger than that of the wild-type control, confirming overexpression. OsNAC15 It can enhance the resistance of rice to rice blast (leaf blast). OsNAC15 It is a positive regulator of rice blast resistance. The values ​​are the mean ± standard deviation of five replicate experiments. p The value represents the statistical test of significance for differences. (Student's t) test analysis p <0.05, p <0.01.

[0060] 4. The present invention OsNAC15 The Influence of Genes on Agronomic Traits Related to Rice Yield and Quality Agronomic traits and quality analysis: Nipponbare (NIP) and NIP-based and genetically stable varieties. OsNAC15 Overexpression materials OsNAC15-OE1 and OsNAC15-OE2 With NIP background and genetically stable OsNAC15 Knockout strains osnac15-1 and osnac15-2 Using these materials, field plot experiments were conducted to analyze... OsNAC15 The effects on rice yield and quality traits.

[0061] The specific steps are as follows: Field experiments were conducted under natural conditions at the Henan Modern Agricultural Development Base in Xinxiang City, Henan Province. The test soil was paddy soil. The main nutrient characteristics of the top 20 cm layer of the paddy soil were as follows: pH value (1:1 soil-to-water ratio) 5.66, organic matter 34.57 g / kg. -1 Total nitrogen 1.72 g kg -1 Total phosphorus 2.85 g kg -1 Total potassium 12.60 g kg -1 Available phosphorus 58.62 mg kg -1 Available potassium 112.38 mg / kg -1 Alkaline nitrogen uptake: 156.26 mg / kg -1 Seedlings were raised in mid-April and transplanted in mid-May. Strong seedlings with uniform growth were selected, one seedling per hill, with a planting density of 25 cm × 25 cm. Plot sizes were set at 3 m × 4 m, with three replicates per material, arranged in a randomized block design. A 1 m protective row was set around the perimeter of each plot to avoid marginal effects. A 40 cm wide ditch was left between each plot for irrigation and drainage. Conventional field fertilization was applied. Precise management was implemented throughout the growth period, with pesticides and herbicides applied according to different growth stages and actual needs to prevent pests, diseases, and weeds. Bird netting was erected before heading to prevent damage from birds and animals that could cause yield losses. At maturity, 15 marked rice plants from each plot were harvested close to the base of the stem and allowed to dry naturally in an outdoor greenhouse for agronomical trait assessment. Two weeks later, five individual plants were selected and weighed every three days until the weight no longer changed. The total weight of each plant, the straw weight of each plant, and the panicle weight of each plant were recorded. The grains were threshed manually. A winnowing machine was used to separate the grains into full and empty grains. After air-drying, the weight of the full grains was measured, which is the yield per plant. The harvest index was calculated based on the yield and total weight per plant. Full and empty grains were manually counted to calculate the number of grains per plant and the seed setting rate per plant. 1000 full grains were counted using a seed analyzer, and the thousand-grain weight was calculated, repeated three times. After weighing the above-ground parts, the weight of the straw per plant, the weight of the ear per plant, the thousand-grain weight, grain length, grain width, the number of grains per plant, the seed setting rate, and the harvest index were measured. The rice was milled using a rice milling machine, and the shape characteristics of each material were analyzed using a rice appearance quality analyzer, a rice grain taste analyzer, and a cooked rice taste analyzer.

[0062] The results are as follows Picture 8 As shown in Figure 9, OsNAC15 The overexpression and knockout mutant materials showed no significant differences from the wild type in terms of plant height, number of spikes, number of grains per spike, seed setting rate, and thousand-grain weight (Figure 8), and there were no significant differences in quality among the materials (Figure 9), indicating that... OsNAC15 This result proves that it does not affect other agronomic traits of rice. OsNAC15The mediated enhancement of disease resistance was not negatively correlated with yield and quality traits. Values ​​are the mean ± standard deviation of 10 replicates. p The value represents the statistical test of significance for differences. (Student's t) test analysis p <0.05, p <0.01.

[0063] Example 3: OsNAC15 Research on the interaction and regulatory relationship with target genes 1. OsNAC15 Filtering and Validation of Candidate Motifs For screening OsNAC15 We constructed a collection of motifs that could be combined. OsNAC15 The pGADT7 bait vector was obtained by PCR amplification. OsNAC15 The CDS amplification primers are TF:: OsNAC15 Its forward and reverse primers are 5' and 5' respectively. CCCGAATTCATGTCGGAGTCGGAGGTGTCGGTGA 3' (as shown in SEQ ID NO: 17), and 5' CGGGGATCCGGCCTTCCATATGTAGGAGTAGTC 3' (as shown in SEQ ID NO: 18). The fragment was digested using EcoRI and BamHI restriction enzymes, and the amplified fragment was cloned into the pGADT7 vector, producing pGADT7- OsNAC15 Bait carrier.

[0064] The specific steps are as follows: design OsNAC15Specific primers for CDS were used, with EcoRI and BamHI restriction sites added. Using cDNA from young rice leaves at the seedling stage as a template, PCR amplification was performed using Phanta Max high-fidelity polymerase (Vazyme). The reaction system was as follows: 25 μL 2×PhantaMax Buffer, 1 μL dNTP Mix (10 mM each), 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 0.5 μL Phanta Max enzyme (2 U / μL), 1 μL rice cDNA template, and 18.5 μL ddH2O (enzyme-free and DNA-free). The components were added sequentially, gently mixed, and briefly centrifuged. The PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 60 s, 35 cycles; and 72℃ extension for 5 min. OsNAC15 PCR amplification products of the CDS sequence were detected by electrophoresis and then recovered using a gel extraction kit. The pGBKT7 bait vector and pGADT7 prey vector plasmid were double-digested with EcoRI and BamHI. The digestion system (30 μL) consisted of approximately 1 μg of PCR gel-recovered product, approximately 0.5 μg of pGBKT7 / pGADT7 empty vector, 3 μL of 10×CutSmart Buffer, 1 μL of EcoRI (10 U / μL), 1 μL of BamHI (10 U / μL), and ddH2O to a final volume of 30 μL. The mixture was incubated at 37 ℃ for 3 h. The digested products were subjected to 1% agarose gel electrophoresis, purified using a gel extraction kit, and 5 μL was taken for concentration determination. The large fragment was then detected by gel electrophoresis and recovered. The recovered double-digested large fragment and the small CDS sequence fragment were recombined using the Vazyme recombinant cloning kit. The reaction system is as follows: 200 ng of the large fragment of pGADT7 plasmid, 80 ng of the fragment recovered from PCR amplification, 4 μL of 5X CE II Buffer, 4 μL of Exnase II, and ddH2O to a final volume of 20 μL. Add the above components sequentially, gently mix with a pipette, and collect the reaction solution at the bottom of the tube by brief centrifugation. Incubate at 37℃ for 30 min, and immediately cool on ice after the reaction. Transform the recombinant plasmid into competent DH5α E. coli cells. Ampicillin was used as the antibiotic for selection plates. Positive clones were picked for colony PCR detection and sequencing. After successful sequencing identification, the bacterial culture was expanded and the plasmid was extracted and named pGADT7- OsNAC15 Competent cells were prepared using yeast culture containing motifs as recipient bacteria, and the correctly sequenced pGADT7- OsNAC15The bait plasmid was transferred into the plate and plated on an SD-TLH selection plate containing 100 mM 3AT, and incubated at 30°C for 5 days. Initial positive transformants grown on the SD-TLH+100 mM 3AT selection plate were streaked onto an SD-TLH+100 mM 3AT selection plate and incubated at 30°C for 3 days. The resulting positive clones were amplified from yeast cells and sequenced for DNA comparison. Simultaneously, using pGAD53m+pHIS2-p53 as a positive control and pGADT7-OsNAC15+pHIS2-p53 as a negative control, the positive clones grown on the SD-TLH+100 mM 3AT plate were diluted with sterile water and spotted onto SD-TLH, SD-TLH, and SD-TLH+100 mM 3AT plates, and incubated at 30°C for 4 days. Colony growth was observed.

[0065] The results showed that 25 different motif sequences were obtained through screening, and all of them could grow on SD-TL, SD-TLH, and SD-TLH+100 mM 3AT plates. This indicates... OsNAC15 It can be combined with the 25 motifs obtained through screening (Figure 10).

[0066] The 488 reported genes involved in rice blast regulation were compared and screened with the obtained candidate genes. A total of 71 candidate genes (mainly involving ubiquitin ligases, MYB / NAC / WRKY transcription factors, auxin responses, and ethylene responses) were obtained from 10 motifs (CCCGTCG, CCCTACC, CCGACGT, CCGGCCC, CGCAGTG, CGCCAAT, CGGAAAG, GGGCCGG, TAGCGGG, and TCCGCGG) (Figure 10). After further analysis, four candidate motifs—CCCTACC, CCGACGT, CGCAGTG, and CGGAAAG—were selected for further validation.

[0067] To further verify OsNAC15 To determine whether it can combine with the four candidate motifs CCCTACC, CCGACGT, CGCAGTG, and CGGAAAG, we constructed a motif carrying... OsNAC15 The pGADT7 prey vector and the pHIS2 bait vectors carrying CCCTACC, CCGACGT, CGCAGTG, and CGGAAAG, respectively, were obtained by PCR amplification. OsNAC15 The CDS and three co-directional tandem positive and reverse single-stranded DNA sequences (CCCTACC, CCGACGT, CGCAGTG, and CGGAAAG) synthesized by Sangon Biotech were used for amplification. The amplification primers were TF:: OsNAC15The forward and reverse primers are 5' each. CCCGAATTCATGTCGGAGTCGGAGGTGTCGGTGA 3' (as shown in SEQ ID NO: 17), and 5' CGGGGATCCGGCCTTCCATATGTAGGAGTAGTC 3' (as shown in SEQ ID NO: 18). The positive and reverse single-stranded DNA sequences of pHIS2-CCCTACC are 5'-GGAGAATTCCCCTACCTCCCTACCTCCCTACCGAGCTCGGC-3' (as shown in SEQ ID NO: 19) and 5'-GCCGAGCTCGGTAGGGAGGTAGGGAGGTAGGGGAATTCTCC-3' (as shown in SEQ ID NO: 20), respectively; the positive and reverse single-stranded DNA sequences of pHIS2-CCGACGT are 5'-GGAGAATTCCCCTACCTCC-3' (as shown in SEQ ID NO: 19) and 5'-GCCGAGCTCGGTAGGGAGGTAGGGAGGTAGGGGAATTCTCC-3' (as shown in SEQ ID NO: 20), respectively; GGAGAATTCCCGACGTTCCGACGTTCCGACGTGAGCTCGGC 3' (as shown in SEQ ID NO: 21) and 5' GCCGAGCTCACGTCGGAAGTCGGAAGTCGGGAATTCTCC 3' (as shown in SEQ ID NO: 22); the positive and reverse single-stranded DNA sequences of pHIS2-CGCAGTG are 5'-GGAGAATTCCGCAGTGTCCGCAGTGTCCGCAGTGGAGCTCGGC-3' (as shown in SEQ ID NO: 23) and 5'-GCCGAGCTCCACTGCGGAACACTGCGGAACACTGCGGAATTCTCC-3' (as shown in SEQ ID NO: 24), respectively; the positive and reverse single-stranded DNA sequences of pHIS2-CGGAAAG are 5'-GGAGAATTCCGGAAAGTCCGGAAAGTCCGGAAAGGAGCTCGGC-3' (as shown in SEQ ID NO: 25) and 5'-GCCGAGCTCCTTTCCGGAACTTTCCGGAACTTTCCGGAATTCTCC-3' (as shown in SEQ ID NO: 26), respectively; EcoRI and BamH were used. The pGADT7 prey vector fragment was digested by an I restriction enzyme, and the amplified fragments were cloned into the pGADT7 prey vector, producing pGADT7- OsNAC15Prey carriers. EcoRI and SacI were used to cleave the pHIS2 bait carrier fragments, and the synthesized CCCTACC, CCGACGT, CGCAGTG and CGGAAAG double-stranded fragments were cloned into the pHIS2 bait carriers, producing pHIS2-CCGACG, pHIS2-CCGACGT, pHIS2-CGCAGTG and pHIS2-CGGAAAG bait carriers.

[0068] The specific steps are as follows: design OsNAC15Specific primers were used, and EcoRI and BamHI restriction enzyme sites were added. Using cDNA from young rice leaves at the seedling stage as a template, PCR amplification was performed using Phanta Max high-fidelity polymerase (Vazyme). The reaction system was as follows: 25 μL 2×PhantaMax Buffer, 1 μL dNTP Mix (10 mM each), 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 0.5 μL Phanta Max enzyme (2 U / μL), 1 μL rice cDNA template, and 18.5 μL ddH2O (enzyme-free and DNA-free). The components were added sequentially, gently mixed, and briefly centrifuged. The PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 60 s, 35 cycles; and 72℃ extension for 5 min. The PCR amplification product of the OsNAC15 CDS sequence was detected by electrophoresis and then recovered using a gel extraction kit. The pGADT7 bait vector was double-digested with EcoRI and BamHI. The digestion system (30 μL) consisted of: 1 μg of PCR gel-recovered product, 0.5 μg of pGADT7 empty vector, 3 μL of 10×CutSmart Buffer, 1 μL of EcoRI (10 U / μL), 1 μL of BamHI (10 U / μL), and ddH2O to a final volume of 30 μL. The system was incubated at 37°C for 3 h. The digested product was subjected to 1% agarose gel electrophoresis, purified using a gel extraction kit, and 5 μL was taken for concentration determination. The large fragment was then recovered by gel electrophoresis. The recovered double-digested large fragment and the CDS sequence small fragment were recombined using the Vazyme recombinant cloning kit. The reaction system was as follows: 200 ng of the pGADT7 plasmid fragment, 80 ng of the recovered PCR amplification product fragment, 4 μL of 5X CE II Buffer, 4 μL of Exnase II, and ddH2O to a final volume of 20 μL. The components were added sequentially, gently pipetted to mix, and the reaction mixture was collected at the bottom of the tube after a brief centrifugation. The reaction was carried out at 37 °C for 30 min, and immediately cooled on ice after completion. The recombinant plasmid was transformed into competent DH5α E. coli cells. Ampicillin was used as the antibiotic for selection plates. Positive clones were selected for colony PCR detection and sequencing. After successful sequencing, the bacterial culture was expanded, and the plasmid was extracted and named pGADT7-OsNAC15. Three tandemly linked positive and reverse single-stranded DNA sequences, CCCTACC, CCGACGT, CGCAGTG, and CGGAAAG, containing EcoRI and SacI restriction sites, were synthesized.Annealing was performed using 2× annealing buffer. The reaction system was as follows: 5 μL each of positive and reverse strand single-stranded DNA (100 μM), 50 μL of 2× annealing buffer, and ddH2O to a final volume of 100 μL. The components were added sequentially, gently swirled to mix, and then briefly centrifuged. Annealing was performed at 95℃ for 5 min, followed by natural cooling to room temperature. The annealed product was ready for subsequent use. The pGADT7 bait vector was digested with EcoRI and SacI. The digestion system (50 μL) was: 0.5 μg of empty pGADT7 vector, 5 μL of 10×CutSmart Buffer, 2 μL of EcoRI (10 U / μL), 2 μL of BamHI (10 U / μL), and ddH2O to a final volume of 50 μL. The mixture was incubated at 37℃ for 4 h. The enzyme digestion products were subjected to 1% agarose gel electrophoresis, purified using a gel extraction kit, and 5 μL was taken to determine the concentration before gel electrophoresis to detect and recover the large fragment. The linear pHIS2 vector and the annealed Motif double-stranded fragment were recombined using T4 DNA ligase (Vazyme). The reaction system was as follows: 1 μL linear pHIS2 vector, 5 μL annealed Motif double-stranded fragment, 1 μL 10×T4 Ligase Buffer, 0.5 μL T4 DNA ligase (350 U / μL), and ddH2O (enzyme-free) to a final volume of 10 μL. The above components were added sequentially, gently pipetted to mix, and the reaction mixture was collected at the bottom of the tube by brief centrifugation. The mixture was incubated overnight at 16°C in a metal bath. The recombinant plasmid was transformed into competent DH5α Escherichia coli cells. Ampicillin was used as the antibiotic for screening plates. Positive clones were selected for PCR detection and then sent for sequencing. After the sequencing confirmed that the bacterial culture was correct, the bacterial culture was expanded and the plasmid was extracted. The plasmids were named pHIS2-CCGACG, pHIS2-CCGACGT, pHIS2-CGCAGTG, and pHIS2-CGGAAAG, respectively. The experimental groups pHIS2-CCGACG+pGADT7-OsNAC15, pHIS2-CCGACGT+pGADT7-OsNAC15, pHIS2-CGCAGTG+pGADT7-OsNAC15, and pHIS2-CGGAAAG+pGADT7-OsNAC15, the control group pGADT7-OsNAC15+pHIS2-p53, and the positive control pGAD53m+pHIS2-p53 were co-transformed into yeast Y187. Single colonies were randomly selected from the yeast transformants grown from the co-transformed Y187 for PCR detection. Three colonies from each group that tested correctly by PCR were randomly selected, resuspended in 2 ml of ddH2O water, and OD was adjusted. 600 =0.002, concentration set to three gradients of 10 0 10 -1 10-2 Take 10 μL for a spotting experiment, spotting it onto SD-TL, SD-TLH, and SD-TLH+100 mM 3AT medium, three spots on each plate, and incubate at 30℃ for 4 days to observe yeast growth.

[0069] The results are as follows Picture 11 As shown, the positive control grew normally on SD-TL, SD-TLH, and SD-TLH+100 mM 3AT plates. The control group grew normally on SD-TL and SD-TLH plates, but not on SD-TLH+100 mM 3AT plates. The experimental group grew normally on SD-TL, SD-TLH, and SD-TLH+100 mM 3AT plates, indicating that OsNAC15 interacts with CCCTACC, CCGACGT, CGCAGTG, and CGGAAAG (Figure 11).

[0070] 2. OsNAC15 Screening and validation of candidate interacting proteins The results of yeast single-hybrid validation confirmed that OsNAC15 It can bind to CCCTACC, CCGACGT, CGCAGTG, and CGGAAAG, with the four motifs containing 32, 8, 8, and 7 candidate genes, respectively. CCCTACC contains the most candidate genes, among which... OsAPIP5 Belonging to the bZIP transcription factor family, it negatively regulates the rice immune response by participating in reactive oxygen species and salicylic acid metabolism. (Inhibition) OsAPIP5 The expression of [certain substances] promotes the production of reactive oxygen species, while simultaneously targeting [certain substances] directly. OsMYBS1 This weakens the degradation of reactive oxygen species, thereby enhancing resistance to rice blast (Wu et al. 2024). ONAC122 It is an NCA transcription factor in rice, whose expression is induced by infection with rice blast fungus, and can be silenced or reduced. ONAC122 The expression of OsOTUB1.1 leads to reduced resistance to rice blast (Sun et al. 2013). OsOTUB1.1 is an Otubain-like protease with deubiquitinating enzyme activity. OsOTUB1.1 and OsSnRK1a Interaction, overexpression OsOTUB1.1 weaken OsSnRK1a Polyubiquitination linked to K63 leads to increased SnRK1 activity, thereby enhancing rice blast resistance (Liu et al. 2023). In summary, OsAPIP5 , ONAC122 and OsOTUB1.1 It plays an important role in regulating rice blast resistance.

[0071] Therefore, OsAPIP5, ONAC122, and OsOTUB1.1, which play important roles in the regulation of rice blast resistance, were selected as potential interacting proteins of OsNAC15. To verify whether OsNAC15 interacts with OsAPIP5, ONAC122, and OsOTUB1.1, we constructed a protein carrying... OsNAC15 pGBKT7 decoy carriers and respectively carrying OsAPIP5 , ONAC122 and OsOTUB1.1 The pGADT7 prey vector was obtained by PCR amplification. OsNAC15 , OsAPIP5 , ONAC122 and OsOTUB1.1 The CDS amplification primers are H2Y:: OsNAC15 The forward and reverse primers are 5' and 5' respectively. CCCCATATGATGTCGGAGTCGGAGGTGTCGGTGA 3' (as shown in SEQ ID NO: 27) and 5' CGGGGATCCGGCCTTCCATATGTAGGAGTAGTC 3' (as shown in SEQ ID NO: 28); H2Y:: OsAPIP5 The forward and reverse primers are 5' and 5' respectively. CCCGAATTCATGGAGCAGCAGAACGTCGT 3' (as shown in SEQ ID NO: 29) and 5' CGGGGATCCGCTGCTGTTGATGATCTGCT 3' (as shown in SEQ ID NO: 30); H2Y:: ONAC122 The forward and reverse primers are 5' and 5' respectively. CCCGAATTCATGTCGTCGATGCTGCTGCC 3' (as shown in SEQ ID NO: 31) and 5' CGGGGATCCGATGATGCTGTTGCTGCTGA 3' (as shown in SEQ ID NO: 32); H2Y:: OsOTUB1.1 The forward and reverse primers are 5' and 5' respectively. CCCGAATTCATGGCGAACGTCGAGCAGCT 3' (as shown in SEQ ID NO: 33) and 5' CGGGGATCCGCTGTTGATGATGCTGCTGC 3' (as shown in SEQ ID NO: 34); the fragment was digested using HindIII and BamHI restriction enzymes, and the amplified fragment was cloned into the pGBKT7 bait vector and the pGADT7 prey vector, respectively, to produce pGBKT7- OsNAC15 bait carriers and pGADT7- OsAPIP5 pGADT7- ONAC122 pGADT7- OsOTUB1.1 Carrier of prey.

[0072] The specific steps are as follows: design OsNAC15 , OsAPIP5 , ONAC122 and OsOTUB1.1 Specific primers for CDS were used, with EcoRI and BamHI restriction sites added. Using cDNA from young rice leaves at the seedling stage as a template, PCR amplification was performed using Phanta Max high-fidelity polymerase (Vazyme). The reaction system was as follows: 25 μL 2×PhantaMax Buffer, 1 μL dNTP Mix (10 mMeach), 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 0.5 μL Phanta Max enzyme (2 U / μL), 1 μL rice cDNA template, and 18.5 μL ddH2O (enzyme-free and DNA-free). The components were added sequentially, gently mixed, and briefly centrifuged. The PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1.5 min, 35 cycles; and 72℃ extension for 5 min. OsNAC15 , OsAPIP5 , ONAC122 and OsOTUB1.1The PCR amplification product of the CDS sequence was detected by electrophoresis and then recovered using a gel extraction kit. The pGBKT7 bait vector and pGADT7 prey vector plasmid were double-digested with EcoRI and BamHI. The digestion system (30 μL) consisted of approximately 1 μg of the PCR gel-recovered product, approximately 0.5 μg of the pGBKT7 / pGADT7 empty vector, 3 μL of 10×CutSmart Buffer, 1 μL of EcoRI (10 U / μL), 1 μL of BamHI (10 U / μL), and ddH2O to a final volume of 30 μL. The mixture was incubated at 37 ℃ for 3 h. The digested product was subjected to 1% agarose gel electrophoresis, purified using a gel extraction kit, and 5 μL was taken to determine the concentration before being detected by gel electrophoresis to recover the large fragment. The recovered double-digested large fragment and the CDS sequence small fragment were recombined using the Vazyme Recombinant Cloning Kit. The reaction system was as follows: 200 ng of the large fragment of pGBKT7 / pGADT7 plasmid, 80 ng of the fragment recovered from PCR amplification, 4 μL of 5X CE II Buffer, 4 μL of Exnase II, and ddH2O to a final volume of 20 μL. The above components were added sequentially, and the mixture was gently pipetted and centrifuged briefly to collect the reaction solution at the bottom of the tube. The reaction was incubated at 37℃ for 30 min, and immediately cooled on ice after completion. The recombinant plasmid was transformed into competent DH5α E. coli cells. Ampicillin was used as the antibiotic for screening plates. Positive clones were selected for colony PCR detection and sequencing. After successful sequencing, the bacterial culture was expanded, and the plasmids were extracted and named pGBKT7-OsNAC15, pGADT7-OsAPIP5, pGADT7-ONAC122, and pGADT7-OsOTUB1.1, respectively. The experimental groups pGBKT7-OsNAC15+pGADT7-OsAPIP5, pGBKT7-OsNAC15+pGADT7-ONAC122, pGBKT7-OsNAC15+pGADT7-OsOTUB1.1, the control groups pGBKT7+pGADT7-OsAPIP5, pGBKT7+pGADT7-ONAC122, pGBKT7+pGADT7-OsOTUB1.1, and pGBKT7-OsNAC15+pGADT7, the positive control pGBKT7-p53+pGADT7-largeT, and the negative control pGBKT7-laminC+pGADT7-largeT were co-transformed into hybrid yeast AH109. Eight single colonies were randomly selected from each of the yeast transformants grown from the co-transformed AH109 for PCR detection. Three colonies were randomly selected from each of the single colonies that tested correctly by PCR, and each colony was resuspended in 2 ml of sterile ddH2O. The OD values ​​were adjusted accordingly. 600 The concentration was set to 0.2, with three gradients of 10. 0 10-1 10 -2 Take 10 μl of the sample and perform a spotting experiment. Spot the sample onto defective plates containing SD-TL, SD-TLH+20 mM 3AT, SD-TLH+20 mM 3AT+X-α-gal, SD-TLHA+20 mM 3AT, and SD-TLHA+20 mM 3AT+X-α-gal, respectively. Incubate at 30℃ for 4 days and observe the yeast growth.

[0073] The results are as follows Picture 12 As shown, the positive control grew normally on SD-TL, SD-TLH+20 mM 3AT, SD-TLH+20 mM 3AT+X-α-gal, SD-TLHA+20 mM 3AT, and SD-TLHA+20 mM 3AT+X-α-gal defect plates, and showed a blue color on SD-TLH+20 mM 3AT+X-α-gal and SD-TLHA+20 mM 3AT+X-α-gal defect plates. The negative control grew normally on SD-TL plates, but could not grow on SD-TLH+20 mM 3AT, SD-TLH+20 mM 3AT+X-α-gal, SD-TLHA+20 mM 3AT, and SD-TLHA+20 mM 3AT+X-α-gal defect plates. The control group grew normally on SD-TL plates, but could not grow on SD-TLH+20 mM 3AT, SD-TLH+20 mM 3AT+X-α-gal, SD-TLHA+20 mM 3AT, and SD-TLHA+20 mM 3AT+X-α-gal defective plates. The experimental group grew normally on all SD-TL, SD-TLH+20 mM 3AT, SD-TLH+20 mM 3AT+X-α-gal, SD-TLHA+20 mM 3AT, and SD-TLHA+20 mM 3AT+X-α-gal defective plates, and showed a blue color on SD-TLH+20 mM 3AT+X-α-gal and SD-TLHA+20 mM 3AT+X-α-gal defective plates, indicating that OsNAC15 interacts with OsAPIP5, ONAC122, and OsOTUB1.1.

[0074] To further verify the interactions between OsNAC15 and OsAPIP5, ONAC122, and OsOTUB1.1, we constructed the pCAMBIA1300-nYFP vector carrying OsNAC15 and the pCAMBIA1300-cYFP vector carrying OsAPIP5, ONAC122, and OsOTUB1.1, respectively. CDS values ​​for OsNAC15, OsAPIP5, ONAC122, and OsOTUB1.1 were obtained by PCR amplification using BIFC:: OsNAC15 The forward and reverse primers are 5' and 5' respectively. CACAACGTCTATATCATGGCCGTCGACATGTCGGAGTCGGAGGTGTCGGTGA 3' (as shown in SEQ ID NO: 35) and 5' TCCATCATGGTCTTTGTAGTCGTCGACGGCCTTCCATATGTAGGAGTAGTC 3' (as shown in SEQ ID NO: 36); BIFC:: OsAPIP5 The forward and reverse primers are 5' and 5' respectively. ACACGGGGGACGAGCTCGGTACCATGGATGACGGGGACCTCGATTTC 3' (as shown in SEQ ID NO: 37) and 5' ATCGTATGGGTACTCCATGGTACCTTTCTTTTCAGAATTTGGCATACAGCC 3' (as shown in SEQ ID NO: 38); BIFC:: ONAC122 The forward and reverse primers are 5' and 5' respectively. ACACGGGGGACGAGCTCGGTACCATGGGCGGGGACTACTACCACTC 3' (as shown in SEQ ID NO: 39) and 5' ATCGTATGGGTACTCCATGGTACCCTTCGGGTAGAGAATGTCGTAGTG 3' (as shown in SEQ ID NO: 40); BIFC:: OsOTUB1.1 The forward and reverse primers are 5' and 5' respectively. ACACGGGGGACGAGCTCGGTACCATGCCGAGCAGCGGCGGCGCCATG 3' (as shown in SEQ ID NO: 41) and 5' ATCGTATGGGTACTCCATGGTACCCTGCATCTGCAGATGATTGTTCAG 3' (as shown in SEQ ID NO: 42); the fragments were digested using Sal1 or Kpn1 restriction enzymes, and the amplified fragments were cloned into the pCAMBIA1300-nYFP vector and the pCAMBIA1300-cYFP vector, respectively, to generate the pCAMBIA1300-nYFP-OsNAC15, pCAMBIA1300-cYFP-OsAPIP5, pCAMBIA1300-cYFP-ONAC122, and pCAMBIA1300-cYFP-OsOTUB1.1 vectors.

[0075] The specific steps are as follows: design OsNAC15 CDS-specific primers were used, and Sal1 restriction sites were added, along with the design of... OsAPIP5 , ONAC122 and OsOTUB1.1 Specific primers for CDS were added, along with a Kpn1 restriction site. Using cDNA from young rice leaves at the seedling stage as a template, PCR amplification was performed using Phanta Mix high-fidelity polymerase (Vazyme). The reaction mixture was as follows: 25 μL of 2×PhantaMax, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 1 μL of rice cDNA template, and 23 μL of ddH2O. The mixture was added sequentially, gently tapped to mix, and then briefly centrifuged. The PCR reaction conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 1.5 min, 30 cycles; and 72℃ extension for 10 min. OsNAC15 , OsAPIP5 , ONAC122 and OsOTUB1.1PCR amplification products of the CDS sequence were detected by electrophoresis and then recovered using a gel extraction kit (Novizan). The pCAMBIA1300-nYFP and pCAMBIA1300-cYFP vectors were digested with Sal1 and Kpn1, respectively. The digestion system (30 μL) consisted of approximately 1 μg of PCR gel-recovered product, approximately 0.5 μg of the empty pCAMBIA1300-nYFP / pCAMBIA1300-cYFP vector, 3 μL of 10×CutSmart Buffer, 1 μL of Sal1 / Kpn1 (10 U / μL), and ddH2O to a final volume of 30 μL. The mixture was incubated at 37 ℃ for 3 h. The digested products were subjected to 1% agarose gel electrophoresis, purified using a gel extraction kit, and 5 μL was taken for concentration determination. The large fragment was then detected by gel electrophoresis and recovered. The recovered double-digested large fragment and the CDS sequence small fragment were recombined using a recombinant cloning kit (Vazyme). The reaction system is as follows: 200 ng of the large fragment of pCAMBIA1300-nYFP / pCAMBIA1300-cYFP plasmid, 80 ng of the fragment recovered from PCR amplification, 4 μL of 5X CE II Buffer, 4 μL of Exnase II, and ddH2O to a final volume of 20 μL. Add the above components sequentially, gently pipette and mix, then briefly centrifuge to collect the reaction solution at the bottom of the tube. Incubate at 37℃ for 30 min, and immediately cool on ice after the reaction is complete.

[0076] The recombinant plasmid was transformed into competent DH5α Escherichia coli cells. Ampicillin was used as the antibiotic for screening plates. Positive clones were selected for PCR detection and then sequenced. After the sequencing confirmed that the bacterial culture was correct, the bacterial culture was expanded and the plasmids were extracted. The plasmids were named pCAMBIA1300-nYFP-OsNAC15, pCAMBIA1300-cYFP-OsAPIP5, pCAMBIA1300-cYFP-ONAC122, and pCAMBIA1300-cYFP-OsOTUB1.1, respectively. The experimental groups were pCAMBIA1300-nYFP-OsNAC15+pCAMBIA1300-cYFP-OsAPIP5, pCAMBIA1300-nYFP-OsNAC15+pCAMBIA1300-cYFP-ONAC122, pCAMBIA1300-nYFP-OsNAC15+pCAMBIA1300-cYFP-OsOTUB1.1, and the control group was pCAMBIA1300-nYFP-OsNAC15+pCAMBIA1300-cYFP, pCAMBIA13 The following gene sequences were co-transformed into rice protoplasts via PEG-mediated transformation: pCAMBIA1300-cYFP-OsAPIP5, pCAMBIA1300-nYFP-OsNAC15+pCAMBIA1300-cYFP, pCAMBIA1300-nYFP+pCAMBIA1300-cYFP-ONAC122, pCAMBIA1300-nYFP-OsNAC15+pCAMBIA1300-cYFP, and pCAMBIA1300-nYFP+pCAMBIA1300-cYFP-OsOTUB1.1. The GFP signal was observed using a laser confocal microscope.

[0077] The results are as follows Picture 13 As shown in Figure 13, GFP fluorescence signals of the fusion proteins pCAMBIA1300-nYFP-OsNAC15, pCAMBIA1300-cYFP-OsAPIP5, pCAMBIA1300-cYFP-ONAC122, and pCAMBIA1300-cYFP-OsOTUB1.1 were detected in the cell nuclei of each experimental group, and co-localized with the nuclear marker D53-mCherry. In contrast, only the nuclear marker D53-mCherry signal was detected in the control group, and no GFP fluorescence signal of the fusion protein was detected (Figure 13). This further confirms that OsNAC15 interacts with OsAPIP5, ONAC122, and OsOTUB1.1, synergistically regulating rice resistance to rice blast. Combined with the functional analysis results of Example 2, this indicates... OsNAC15It interacts with proteins such as OsAPIP5, ONAC122 and OsOTUB1.1 to synergistically regulate rice resistance to rice blast.

[0078] In summary, the rice gene of this invention OsNAC15 It is located in the cell nucleus. NAC This gene is a transcription factor that interacts with proteins such as OsAPIP5, ONAC122, and OsOTUB1.1. Overexpression of this gene significantly enhances rice resistance to rice blast, while knockout significantly reduces rice resistance to rice blast without affecting agronomic traits and quality. Its application in rice blast resistance breeding has shown significant effects, providing important genetic resources and technical support for developing new rice varieties with broad-spectrum and durable resistance.

[0079] The above are merely preferred embodiments of this application. It should be noted that this application is not limited to the above embodiments. For those skilled in the art, several improvements and modifications can be made without departing from the principles of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should also be considered within the scope of protection of this application.

[0080] sequence list SEQ ID NO: 1 ATGTCGGAGTCGGAGGTGTCGGTGATCAACCAGCTGGAGGAGGAGGAGACTCGGCTGGAGCTGCCGCCGGGGTTCCGGTTCCACCCCACCGACGAGGAGGTGGTGACCCACTACCTCACCCGCAAGGCCCAGGACCGGAGCTTCTCCTGCGTCGTCATCGCCGACGTGAACCTCAACAACTGCGAGCCATGGGACCTCCCAAGCAAGGCGAAGATGGGGGAGAAGGAGTGGTTCTTCTTCTGCCACAAGGACCGCAAGTACCCGACGGGGATGAGGACGAACCGGGCGACGGCCAGCGGGTACTGGAAGGCCACCGGGAAGGACAAGGAGATCTTCCGCGGCCGCGGCCTCCTCGTCGGCATGAAGAAGACGCTCGTCTTCTACATGGGCCGCGCCCCCCGCGGCGAGAAGACCCCCTGGGTCATGCACGAATACCGCCTCGACGGCAAGCTTCCCCCCAACCTCCCCCGCTCCGCAAAGGAGGAATGGGCGGTTTGCCGGGTGTTCAACAAAGACTTGGCGGCAAAGATTGCCCAAATGCCGCCGCCTCCCTTCCCGCGCAACGACTCCTTCGACCTCGACCTCGACGACTTCCTCCACCTCGACGCCGACCTGCCGCCGCTCATCGACGACCCCTTCGCCTCCACCTCCACGCTCAAGACGGAGCCACCTCCGCCGGCCAACCTGATGCACAACCACTACGGCTACTTCTCCCTCCCGGCGTCAGCGACCAATTATAACCACAGCTCCGGCGCCATGGCGGACCAGGCGATTCGGAGGTTCTGCAAGGCGGAGGCGTCGACGGCGTGCTTCTCCGGCGCCGACGCTGACGTGGATCCGGTGGTGGACGAGCTGCTCTCCTTCCCGGACTCCATCACGGACTACTCCTACATATGGAAGGCCTGA SEQ ID NO:2 MSESEVSVINQLEEEETRLELPPGFRFHPTDEEVVTHYLTRKAQDRSFSCVVIADVNLNNCEPWDLPSKAKMGEKEWFFFCHKDRKYPTGMRTNRATASGYWKATGKDKEIFRGRGLLVGMKKTLVFYMGRAPRGEKTPWVMHEYRLDGKLPPNLPRSAKEEWAVCRVFNKDLAAKIAQMPPPPFPRNDSFDLDLDDFLHLDADLPPLIDDPFASTSTLKTEPPPPANLMHNHYGYFSLPASATNYNHSSGAMADQAIRRFCKAEASTACFSGADADVDPVVDELLSFPDSITDYSYIWKA

[0081] SEQ ID NO:3 cgggggactctagaggatccATGTCGGAGTCGGAGGTGTCGGTGA SEQ ID NO:4 tcggaggaggccatactagtGGCCTTCCATATGTAGGAGTAGTC SEQ ID NO:5 CTGCCCGCTGTTCTACAACCGG SEQ ID NO:6 GGAGCATATACGCCCGGAGTC SEQ ID NO:7 TGCCACAAGGACCGCAAGTA SEQ ID NO:8 AACCGCCCATTCCTCCTTT SEQ ID NO:9 CTACCTCACCCGCAAGGCCCAGG SEQ ID NO:10 CCGACGTGAACCTCAACAACTGC SEQ ID NO:11 TGATCAACCAGCTGGAGGAG SEQ ID NO:12 GACGATGAGGAAGCTAGGC SEQ ID NO:13 GACTCTGGTGATGGTGTCAGC SEQ ID NO:14 GCTGGAAGAGGACCTCAGG SEQ ID NO:15 cgacggccagtgccaagcttGCTCTAGAAGCTTGCATGCCT SEQ ID NO:16 gactgaccacccggggatccCGGATCCGGTACCTCTAGAGT SEQ ID NO:17 CCCGAATTCATGTCGGAGTCGGAGGTGTCGGTGA SEQ ID NO:18 CGGGGATCCGGCCTTCCATATGTAGGAGTAGTC SEQ ID NO:19 GGAGAATTCCCCTACCTCCCTACCTCCCTACCGAGCTCGGC SEQ ID NO:20 GCCGAGCTCGGTAGGGAGGTAGGGAGGTAGGGGAATTCTCC SEQ ID NO:21 GGAGAATTCCCGACGTTCCGACGTTCCGACGTGAGCTCGGC SEQ ID NO:22 GCCGAGCTCACGTCGGAAGTCGGAAGTCGGGAATTCTCC SEQ ID NO:23 GGAGAATTCCGCAGTGTCCGCAGTGTCCGCAGTGGAGCTCGGC SEQ ID NO:24 GCCGAGCTCCACTGCGGAACACTGCGGAACACTGCGGAATTCTCC SEQ ID NO:25 GGAGAATTCCGGAAAGTCCGGAAAGTCCGGAAAGGAGCTCGGC SEQ ID NO:26 GCCGAGCTCCTTTCCGGAACTTTCCGGAACTTTCCGGAATTC SEQ ID NO:27 CCCCATATGATGTCGGAGTCGGAGGTGTCGGTGA SEQ ID NO:28 CGGGGATCCGGCCTTCCATATGTAGGAGTAGTC SEQ ID NO: 29 CCCGAATTCATGGAGCAGCAGAACGTCGT SEQ ID NO:30 CGGGGATCCGCTGCTGTTGATGATCTGCT SEQ ID NO:31 CCCGAATTCATGTCGTCGATGCTGCTGCC SEQ ID NO:32 CGGGGATCCGATGATGCTGTTGCTGCTGA SEQ ID NO:33 CCCGAATTCATGGCGAACGTCGAGCAGCT SEQ ID NO:34 CGGGGATCCGCTGTTGATGATGCTGCTGC SEQ ID NO:35 CACAACGTCTATATCATGGCCGTCGACATGTCGGAGTCGGAGGTGTCGGTGA SEQ ID NO:36 TCCATCATGGTCTTTGTAGTCGTCGACGGCCTTCCATATGTAGGAGTAGTC SEQ ID NO:37 ACACGGGGGACGAGCTCGGTACCATGGATGACGGGGACCTCGATTTC SEQ ID NO:38 ATCGTATGGGTACTCCATGGTACCTTTCTTTTCAGAATTTGGCATACAGCC SEQ ID NO:39 ACACGGGGGACGAGCTCGGTACCATGGGCGGGGACTACTACCACTC SEQ ID NO:40 ATCGTATGGGTACTCCATGGTACCCTTCGGGTAGAGAATGTCGTAGTG SEQ ID NO:41 ACACGGGGGACGAGCTCGGTACCATGCCGAGCAGCGGCGGCGCCATG SEQ ID NO:42 ATCGTATGGGTACTCCATGGTACCCTGCATCTGCAGATGATTGTTCAG

Claims

1. Rice transcription factor genes OsNAC15 Or the application of its encoded protein in regulating disease resistance in rice, characterized in that, The rice transcription factor gene OsNAC15 The nucleotide sequence is shown in SEQ ID NO: 1; the amino acid sequence of the encoded protein is shown in SEQ ID NO:

2.

2. The application as described in claim 1, characterized in that, The application involves enhancing rice transcription factor genes. OsNAC15 The expression or activity of [a substance] can enhance the resistance of rice to rice blast.

3. The application as described in claim 2, characterized in that, Amplification using PCR method OsNAC15 The CDS sequence was used to construct the 35S promoter-driven overexpression vector 35S-P1300- OsNAC15 -GFP; This was transferred into rice using Agrobacterium-mediated genetic transformation to obtain the rice transcription factor gene. OsNAC15 Transgenic lines overexpressing [the gene].

4. A rice transcription factor OsNAC15 The application of proteins or their encoding genes as targets for regulating disease resistance in rice is characterized by, The rice transcription factor OsNAC15 The amino acid sequence of the protein is as shown in SEQ ID NO:

2.

5. A recombinant vector, characterized in that, It comprises the nucleotide sequence described in claim 1.

6. The recombinant vector as described in claim 5, characterized in that, The recombinant vector is an expression vector or a gene editing vector.

7. A method for enhancing rice resistance to rice blast, characterized in that, The method includes the step of introducing the nucleotide sequence of claim 1 or the recombinant vector of claim 5 or 6 into a rice receptor.

8. A method for detecting or amplifying the rice transcription factor gene of claim 1. OsNAC15 The primer pair is characterized in that, The primer pairs are selected from any one of the following groups: (a) Used for amplification OsNAC15 The primer pairs for the gene CDS sequence, with the nucleotide sequences of the forward and reverse primers shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; (b) Used for detection OsNAC15 The nucleotide sequences of the forward and reverse primers for qRT-PCR at gene expression levels are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. (c) Used for detection OsNAC1 5. Primer pairs for identifying gene editing status, the nucleotide sequences of the forward and reverse primers are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

9. A method for analysis OsNAC15 A gene function kit, characterized in that, It includes the primer pair described in claim 12.

10. A composition for synergistically regulating rice resistance to rice blast, characterized in that, Contains the rice transcription factor gene as described in claim 1 OsNAC15 The encoded protein, and the proteins that interact with it, wherein the interacting proteins are OsAPIP5, ONAC122, or OsOTUB1.1.

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