Rice brassinosteroid signal key transcription factor osbri gene and its cloning and application

Abstract

This invention discloses a key transcription factor for brassinolide signaling in rice. OsBZR1 Genes, their cloning, and applications. OsBZR1 Composed of 897 bases, it encodes a 31.9 kDa protein located in the nucleus and cytoplasm, namely the transcription factor OsBZR1. OsBZR1 is involved in rice autophagy and brassinolide signaling and responds to the biotic stress response to invasion by Southern Rice Black-Streaked Dwarf Virus. Knockout OsBZR1 The mutant rice of the gene is not significantly different from the wild-type rice, but the knockout gene... OsBZR1 The mutant rice strain exhibited enhanced resistance to Southern Rice Black-Streaked Dwarf Virus (SMRV), and the symptoms of infected plants were reduced, indicating that... OsBZR1 The gene is regulating the infection of rice with Southern Rice Black-Streaked Dwarf Virus. Therefore, gene editing technology can be used to knock out this gene to improve the resistance of rice to Southern Rice Black-Streaked Dwarf Virus.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and disease-resistant breeding, and particularly to a key transcription factor for brassinolide signaling in rice. OsBZR1 Genes, their cloning, and applications. Background Technology

[0002] In eukaryotes, autophagy is a highly conserved intracellular degradation and recycling pathway in biological evolution. Autophagy-related genes... ATGs Under the regulation of autophagosomes, abnormal intracellular substances (such as organelles, proteins, and lipids) are transported to lysosomes (in animal cells) or vacuoles (in plant cells) for degradation, and the degradation products are recycled, providing support for intracellular environmental stability and energy homeostasis. Autophagy is an important branch of plant antiviral immunity. Viral infection can induce autophagy activation, and the activated autophagy pathway can target and degrade viral coat proteins, replicases, or viral RNA, thereby limiting viral accumulation and spread. Meanwhile, some viruses have also evolved mechanisms to inhibit or escape autophagy. In-depth analysis of the regulatory network of autophagy in plant antiviral defense has significant theoretical value and application prospects for discovering new disease-resistant resources and cultivating broad-spectrum, durable disease-resistant varieties.

[0003] Southern rice black-streaked dwarf virus (SRBSDV) is one of the most serious viruses threatening rice production in Asia in recent years. It has frequently broken out in major rice-producing areas such as Vietnam, Thailand, China, and Japan, causing consecutive years of yield reductions. This virus relies on the white-backed planthopper (Syntaxelus chinensis). Sogatella furcifera As a vector, SRBSDV spreads in the field through a persistent, proliferative process. Typical symptoms caused by SRBSDV infection in rice include extremely stunted growth, dark green leaves, curling of the base or tip of new leaves, waxy white nodular protrusions of about 1-2 mm in size on the stem that later turn dark brown, high-node tillering, underdeveloped root system, swollen or absent panicles, and failure to produce grains. In severe cases, it can even lead to complete crop failure, posing a significant challenge to regional food security. Currently, control measures against SRBSDV remain scarce, and there are no commercially available highly resistant varieties for production. Practice has proven that breeding and promoting resistant varieties is the most economical and effective way to control this disease. Therefore, systematically identifying SRBSDV-resistant genetic resources in rice and deeply analyzing its resistance molecular mechanisms has significant theoretical value and application prospects for accelerating the breeding of persistently resistant varieties and ensuring stable and high rice yields.

[0004] OsBZR1 is a core transcription factor in the brassinolide signaling pathway in rice. By binding to BRRE elements and E-box motifs in the promoters of downstream target genes, it positively or negatively regulates the transcription of these genes, thus participating extensively in biological processes such as plant architecture, grain development, and stress resistance in rice. Determining whether OsBZR1 participates in rice's defense response to SRBSDV through a transcriptional regulatory network has significant theoretical and applied value. Therefore, cloning and analyzing OsBZR1 is crucial. OsBZR1 The functional mechanism of the gene not only helps to deepen the theoretical understanding of the cross-regulation of rice hormone signaling and antiviral immunity, but also provides a direct target for antiviral molecular breeding. By precisely editing or regulating the expression of this gene, it is expected to synergistically improve rice resistance to SRBSDV and key agronomic traits, and cultivate new germplasm that is disease-resistant, high-yielding, and stable-yielding. Summary of the Invention

[0005] The purpose of this invention is to address the critical problem of the scarcity of rice germplasm resources resistant to Southern Rice Black-Streaked Dwarf Virus (SMRV) by providing a key transcription factor for the disease-susceptibility gene—rice brassinolide signaling. OsBZR1 Genes, their cloning, and applications.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a key transcription factor for brassinolide signaling in rice. OsBZR1 The gene, whose base sequence is shown in SEQ ID NO: 1.

[0007] Secondly, the present invention provides a key transcription factor for brassinolide signaling in rice as described in the first aspect above. OsBZR1 The transcription factor OsBZR1 encoded by the gene has the amino acid sequence shown in SEQ ID NO:2, and it participates in the rice autophagy process and positively regulates the infection of rice with Southern Rice Black-Streaked Dwarf Virus.

[0008] Thirdly, the present invention provides a key transcription factor for rice brassinolide signaling as described in the first aspect above. OsBZR1 The gene cloning method involves extracting total RNA from rice leaves and synthesizing a gene band using a pair of gene sequences targeting the OsBZR1 protein. Eco RI and Bam Using primers upstream and downstream of the HI restriction site and the homologous arm sequence of the pGAD T7 vector, an 897 bp key transcription factor for rice brassinolide signaling was cloned from total RNA in rice leaves by RT-PCR. OsBZR1 Gene; and the sequences of the upstream and downstream primers are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0009] As a preferred embodiment of the third aspect above, the total RNA from the rice leaves is extracted using TRIzol reagent.

[0010] As a preferred option in the third aspect mentioned above, the gene cloned by RT-PCR needs to be cloned into the pGAD T7 vector through in-fusion seamless ligation, followed by first-generation nucleic acid sequencing, and the obtained nucleic acid sequence is compared with BLAST to verify the correctness of cloning.

[0011] Fourthly, this invention provides a method for improving the resistance of rice to Southern Rice Black-Streaked Dwarf Virus, which involves knocking out key transcription factors of brassinolide signaling in rice as described in the first aspect using gene editing technology. OsBZR1 Gene.

[0012] Fifthly, the present invention provides a key transcription factor for rice brassinolide signaling as described in the first aspect. OsBZR1 The application of the gene involves using gene editing technology to knock out this gene in rice to improve the rice's resistance to Southern Rice Black-Streaked Dwarf Virus.

[0013] Sixthly, the present invention provides a key transcription factor for rice brassinolide signaling as described in the first aspect. OsBZR1 The application of genes is to obtain resistant germplasm resources by knocking out the gene through gene editing technology in disease resistance breeding.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a simple and efficient RT-PCR method for cloning the brassinoazole tolerance factor gene from rice. OsBZR1 An effective cloning method; moreover, this invention utilizes gene editing and transgenic technologies to respectively clone rice... OsBZR1 Gene knockout and overexpression were used to analyze the gene's function, demonstrating... OsBZR1 The gene has the ability to respond to the biological stress of Southern Rice Black-Streaked Dwarf Virus, that is... OsBZR1 The gene positively regulates the infection of rice with Southern Rice Black-Streaked Dwarf Virus. Therefore, the results of this invention indicate that this virus can be knocked out using gene editing technology. OsBZR1 Genes were used to improve rice resistance to Southern Rice Black-Streaked Dwarf Virus. Attached Figure Description

[0015] Figure 1This is a subcellular localization analysis of the OsBZR1 protein. A: Subcellular localization of the OsBZR1-GFP fusion protein in *Nicotiana benthamiana* leaf cells observed using laser confocal microscopy. Scale bar: 20 μm. B: Localization of the OsBZR1-GFP fusion protein in rice protoplasts observed using laser confocal microscopy. Scale bar: 20 μm.

[0016] Figure 2 yes OsBZR1 Transcriptional function analysis of genes. A: Illustration of the construction of effector and reporter vectors for dual-luciferase reporter assay. pBD and pBD-VP16 vectors served as negative and positive controls, respectively. B: Fluorescence intensity of each combination was observed using a plant in vivo imaging system. C: LUC / REN ratio was measured using a microplate reader. Data are expressed as mean ± variance of three biological replicates. Data are presented using Student's... t Statistical difference analysis was performed using the test. ** indicates... P <0.01.

[0017] Figure 3 OsBZR1 acts as a transcriptional repressor for 14 autophagy-related genes. OsATGs and BR biosynthesis genes OsCPD1 Transcriptional effects analysis. A: PlantPAN 4.0 software analysis of 14 autophagy-related genes. OsATGs and BR biosynthesis genes OsCPD1 Position of the BRRE element binding to OsBZR1 in the promoter sequence. Numbers represent the distance from the transcription start site. B: Illustration of effector and reporter vector construction for the dual-luciferase reporter system. The sequence containing the binding motif 3000 bp upstream of the start codon (ATG) of each gene was amplified and cloned into the dual-luciferase reporter vector. C: Fluorescence intensity of each combination observed using a plant in vivo imaging system. D: LUC / REN ratio measured using a microplate reader. Data are expressed as mean ± variance of three biological replicates. Data are presented using Student's... t Statistical difference analysis was performed using the test. * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001, **** indicates P <0.0001.

[0018] Figure 4 yes OsBZR1 Gene knockout rice affects 14 autophagy-related genes OsATGs and BR biosynthesis genes OsCPD1 Analysis of transcriptional effects. A: OsBZR1 Identification results of gene knockout rice lines. B: RT-qPCR analysis OsBZR1Gene knockout and wild-type diseased rice plants OsATG1b , OsATG1c , OsATG3a , OsATG3b , OsATG6b , OsATG7 , OsATG8b , OsATG8c , OsATG9a , OsATG12 , OsATG13a , OsATG18b , OsATG18c , OsATG18e and OsCPD1 Gene expression levels, OsUBQ5 As an internal reference gene, this data is expressed as mean ± variance of three biological replicates and uses Student's... t Statistical difference analysis was performed using the test. * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001, **** indicates P <0.0001.

[0019] Figure 5 yes OsBZR1 Analysis of resistance of gene knockout rice to Southern Rice Black-Streaked Dwarf Virus. A: OsBZR1 Symptoms of disease 30 days after inoculation of gene knockout and wild-type rice plants with Southern Rice Black-Streaked Dwarf Virus. B: Statistical analysis of disease symptoms 30 days after virus inoculation. OsBZR1 Plant height of gene knockout and wild-type diseased rice plants. This data is expressed as mean ± variance of three biological replicates and uses Student's data. t Statistical difference analysis was performed using the test, ** indicates P <0.01. C: RT-qPCR analysis of the virus 30 days after inoculation. OsBZR1 SRBSDV in gene knockout and wild-type diseased rice plants P10 Gene expression levels, OsUBQ5 As an internal reference gene, this data is expressed as mean ± variance of three biological replicates and uses Student's... t Statistical difference analysis was performed using the test, and **** indicates... P <0.0001. D: Western blot analysis of SRBSDV vaccination 30 days later. OsBZR1 The accumulation level of SRBSDV P10 in gene knockout and wild-type rice plants. Detailed Implementation

[0020] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0021] This invention provides a key transcription factor for brassinolide signaling in rice. OsBZR1 The specific process of gene cloning is as follows: 1) Based on the gene sequence of gene number Os07g0580500 and transcription factor OsBZR1 protein in the RAP-DB (The Rice Annotation Project Database, http: / / rice.plantbiology.msu.edu / index.shtml) database, a pair of genes were designed and synthesized. Eco RI and Bam The upstream and downstream primers for the HI restriction site and the homologous arm sequence of the pGAD T7 vector are shown in SEQ ID NO: 3, specifically 5'-GCCATGGAGGCCAGTGAATTCATGACGTCCGGGGCGGCG-3', and the downstream primer T2 is shown in SEQ ID NO: 4, specifically 5'-CAGCTCGAGCTCGATGGATCCTCATTTCGCGCCGACGCC-3'. 2) Extract total RNA from rice leaves using TRIzol reagent according to the product instructions; 3) Using the primers described above, an 897 bp gene was cloned from total RNA in rice leaves by RT-PCR. This gene was cloned into the pGAD T7 vector and subjected to first-generation nucleic acid sequencing. The nucleic acid sequence was submitted to GenBank for BLAST alignment, which confirmed that the cloned gene is a key transcription factor for brassinolide signaling in rice. OsBZR1 Gene.

[0022] The above-mentioned key transcription factor for brassinolide signaling in rice cloned in this invention OsBZR1 The gene contains 897 bases, and its DNA sequence is shown in SEQ ID NO: 1. A key transcription factor for brassinolide signaling in rice. OsBZR1 The gene-encoded transcription factor OsBZR1, OsBZR1The protein encoded by the gene has the amino acid sequence shown in SEQ ID NO:2, and this protein participates in rice autophagy and brassinolide signal transduction, positively regulating the infection of rice with Southern Rice Black-Streaked Dwarf Virus. Therefore, in practical applications, it can be knocked out using gene editing technology. OsBZR1 Genes can be used to improve the resistance of rice to viruses such as Southern Rice Black-Streaked Dwarf Virus, providing resistant germplasm resources for disease-resistant breeding.

[0023] Example 1: Key Transcription Factor for Brassinolide Signaling in Rice OsBZR1 Cloning of genes 1) Design a pair of genes based on the gene sequence (gene number: Os07g0580500) of the 31.9 kDa protein in the transcription factor OsBZR1 from the RAP-DB database. Eco RI and Bam The primers for the HI restriction site and the homologous arm sequence of the pGAD T7 vector, namely the upstream primer T1 being 5'-GCCATGGAGGCCAGTGAATTCATGACGTCCGGGGCGGCG-3' (as shown in SEQ ID NO: 3) and the downstream primer T2 being 5'-CAGCTCGAGCTCGATGGATCCTCATTTCGCGCCGACGCC-3' (as shown in SEQ ID NO: 4), were synthesized with the assistance of other companies. 2) Total RNA was extracted from rice leaves using Takara's TRIzol reagent according to the product instructions; 3) RNA was reverse transcribed into cDNA using the Takara Reverse Transcriptase M-MLV (RNase H-) kit. The reverse transcription process is as follows: Using the extracted total RNA as template RNA, a template RNA / primer mixture was first prepared, comprising 1 µL template RNA, 1 µL gene-specific primer, 0.5 µL dNTP mixture, and 3.5 µL water. The prepared template RNA / primer mixture was reacted at 65°C for 5 minutes, then cooled on ice for 2 minutes, and subsequently used as an RNA template / primer denaturation solution for reverse transcription. The reverse transcription system consisted of 6 μL of the above RNA template / primer denaturation solution, 2 μL of 5 × Reverse Transcriptase M-MLV Buffer, 0.25 μL of RNase Inhibitor (40 U / μl), 0.25 μL of Reverse Transcriptase M-MLV (RNase H-) (200 U / μl), and 1.5 µL of RNase-free deionized water. The mixture prepared according to this reverse transcription system was reverse transcribed at 42°C for 1 hour, and then inactivated at 70°C for 15 minutes to obtain the reverse transcription product cDNA. 4) PCR amplification was performed using TOYOBO's high-fidelity enzyme TOROBlue Flash KOD Dye Mix with the reverse transcription product cDNA as a template. The PCR reaction system included 25 µL of 2 × TOROBlue Flash KOD Dye Mix, 1 µL of template cDNA, 1.5 µL each of the upstream primer T1 and the downstream primer T2, and 21 µL of deionized water. The PCR reaction conditions were as follows: 98℃ pre-denaturation for 2 minutes, 98℃ for 10 seconds, 60℃ for 5 seconds, 68℃ for 20 seconds, 30 cycles, and a final filler at 68℃ for 5 minutes. The PCR product was run on a 1% agarose gel, and the specific band was excised. The PCR-specific product was recovered by excising the gel using Vazyem's FastPure Gel DNA Extraction MiniKit according to its product instructions.

[0024] 5) Using Thermo Fisher Scientific Eco RI and Bam The pGADT7 vector was digested with HI restriction endonuclease. The digestion reaction system included 7 µL of plasmid and 2 µL of 10 × FastDigest buffer. Eco RI and Bam 1 µL each of HI restriction endonuclease and 13 µL of sterile deionized water were added, and the enzyme digestion reaction was carried out at 37°C for 30 minutes. The digestion products were then cleaned and recovered using the Vazyme Gel DNA Extraction Mini Kit according to the product instructions. Then, the digestion products were processed using TransGen... pEASY ® The Pro Seamless Cloning and Assembly Kit seamlessly ligates recovered PCR-specific product genes into the enzyme-digested pGAD T7 vector. The seamless ligation reaction system includes 3 µL of gel-extracted PCR-specific product and gene, 2 µL of enzyme-digested pGAD T7 vector, and 5 µL of 2× pEASY ® Pro Assembly Mix, 50℃ in-fusion for 15 minutes for seamless bonding to obtain bonding products.

[0025] 6) Transform the ligation product into *E. coli* DH5α competent cells. Add 10 µL of the ligation product to 100 µL of competent cells, place on ice for 30 minutes, heat shock at 42°C for 90 seconds, then immediately place on ice for 5 minutes. Add 700 µL of preheated LB broth at 37°C and incubate at 37°C with shaking for 1 hour. Centrifuge the bacterial culture at 12000 rpm for 1 minute, resuspend the pellet, and spread it onto LB agar plates containing ampicillin. Use 2 × Rapid Taq Master Mix PCR to screen for positive colonies. The colony PCR reaction system includes 10 µL of 2 × Rapid Taq Master Mix, 1 µL each of the upstream primer T1 and downstream primer T2, 8 µL of sterile deionized water, and a single colony. The PCR reaction conditions are as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, and extension at 72°C. PCR was performed for 1 minute, with 27 cycles in total, followed by a final 5-minute time at 72°C. The PCR products were run on 1% agarose gel, and colonies exhibiting specific bands were selected and cultured with shaking on LB broth containing ampicillin. The resulting bacterial culture was sent to Beijing Qingke Biotechnology Co., Ltd. for gene sequencing, and the sequences were submitted to GenBank for BLAST alignment. The results showed that the cloned gene contained 897 bases. OsBZR1 The gene, whose gene sequence is shown in SEQ ID NO: 1, includes a complete open reading frame (ORF) that encodes a protein of 298 amino acids as shown in SEQ ID NO: 2.

[0026] Example 2 OsBZR1 Analysis and application of gene characteristics and functions 1. Subcellular localization of OsBZR1 protein According to the clone obtained in Example 1 OsBZR1 Design a pair of genes (as shown in SEQ ID NO: 1) BamThe primer pair for the HI restriction site and the homologous arm sequence of the pGD 35S-GFP vector is as follows: upstream primer T1 is 5'-TACAAAATCTATCTCTGGATCCATGACGTCCGGGGCGGCG-3', and downstream primer T2 is 5'-TCCAGAACCTCCATCGGATCCTTTCGCGCCGACGCCGAG-3'; the amplified... OsBZR1 Using the gene fragment as a template, the gene was amplified using the high-fidelity enzyme TOROBlue Flash KOD Dye Mix from TOYOBO, following the experimental system and method described in Example 1. OsBZR1 Genes and products were run on agarose gels and harvested. The pGD 35S-GFP vector plasmid was obtained using a Thermo Fisher Scientific product. Bam HI restriction endonuclease digestion was performed. The digestion reaction system consisted of 7 µL plasmid, 2 µL 10 × FastDigest buffer, 2 µL restriction endonuclease, and 13 µL sterile deionized water. Digestion was carried out at 37°C for 30 minutes. The reaction product was then gel-extracted and recovered using the Vazyme Gel DNA Extraction Mini Kit according to the product instructions. TransGen was used for further analysis. pEASY ® The Pro Seamless Cloning and Assembly Kit ligates the recovered gene fragments into the enzyme-digested plant expression vector pGD 35S-GFP, enabling... OsBZR1 The gene was fused to the N-terminus of GFP. The in-fusion seamless ligation reaction system included 5 µL of 2× pEASY ® Pro Assembly Mix, 2 µL pGD 35S-GFP vector and 3 µL OsBZR1 The gene-derived product was recovered from the gel and reacted at 50°C for 15 minutes. The ligation product was transformed into *E. coli* DH5α, and positive clones pGD35S-OsBZR1-GFP were obtained by colony PCR screening. Positive colonies were cultured with shaking, and plasmids were extracted using the Vazyme FastPure Plasmid Mini Kit-BOX and its instructions. The plasmids were then transformed into *Agrobacterium* strain EHA105 by electroporation. Positive transformed colonies were screened by PCR, and the positive *Agrobacterium* strains were cultured with shaking until OD200. 600The concentration of *Agrobacterium tumefaciens* was 0.6-0.8. Leaves of *N. benthamiana* at the 4-leaf stage were infiltrated with the bacteria. After 48 hours, the infiltrated leaves were collected and the expression of GFP was observed under a confocal microscope. The results showed that the OsBZR1-GFP protein was localized in the cytoplasm and nucleus, indicating that the OsBZR1 protein can be localized in the cytoplasm and nucleus of plant cells (e.g., ...). Figure 1 (As shown in Figure A). Furthermore, protoplast cells from wild-type rice leaves were prepared according to the literature reported by Zeng et al., and transformed with PEG vector (Zeng et al.). et al. Viral proteins suppress ricedefenses by boosting OsTSN1 RNA decay via phase separation and multimerization. Nat Commun (2025, 16, 7481) OsBZR1-GFP was transiently expressed in rice protoplast cells, and its expression and subcellular localization were observed under a confocal microscope. The results showed that OsBZR1-GFP protein was localized in the cytoplasm and nucleus of rice protoplast cells, further demonstrating that OsBZR1 protein can be localized in the cytoplasm and nucleus of plant cells (e.g., 2025, 16, 7481). Figure 1 (As shown in B).

[0027] 2. OsBZR1 Functional analysis and application of genes To analyze the transcriptional function of transcription factor OsBZR1, a dual-luciferase assay was performed according to the method reported by Zeng et al. (Zeng et al.) et al. Viral proteins suppress rice defenses by boosting OsTSN1 RNA decay via phase separation and multimerization. Nat Commun (2025, 16, 7481). According to the clone obtained in Example 1... OsBZR1 Design a pair of genes (as shown in SEQ ID NO: 1) Stu Primer pairs for the I restriction site and the homologous arm sequence of the effector pBD vector of the dual-luciferase reporter system: upstream primer T1 is 5'-GTATACGCCGACCGGTAGGCCTATGACGTCCGGGGCGGCG-3', downstream primer T2 is 5'-TGAAACCAGAGTTAAAGGCCTTCATTTCGCGCCGACGCC-3'; [The text abruptly ends here, likely due to an incomplete sentence or missing information.] OsBZR1Using the gene fragment as a template, the gene was amplified using the high-fidelity enzyme TOROBlue FlashKOD Dye Mix from TOYOBO, following the experimental system and method described in Example 1. OsBZR1 Genes and products were run on agarose gels and harvested. The effector pBD vector plasmid for the dual-luciferase reporter system was obtained using plasmids from Thermo Fisher Scientific. Stu I. Restriction endonuclease digestion: The digestion reaction system consisted of 7 µL plasmid, 2 µL 10 × FastDigest buffer, 2 µL restriction endonuclease, and 13 µL sterile deionized water. Digestion was performed at 37°C for 30 minutes. The reaction product was then recovered by gel extraction using the Vazyme Gel DNA Extraction Mini Kit according to its product instructions. TransGen... pEASY ® The Pro Seamless Cloning and Assembly Kit ligates recovered gene fragments into the enzyme-digested vector pBD. The in-fusion seamless ligation reaction system includes 5 µL 2× pEASY ® Pro Assembly Mix, 2 µL pBD vector and 3 µL OsBZR1 The gene-derived product was recovered from the gel and reacted at 50°C for 15 minutes. The ligation product was transformed into *E. coli* DH5α, and positive clone pBD-OsBZR1 was obtained by colony PCR screening. The positive colonies were cultured with shaking, and plasmids were extracted using the Vazyme FastPure Plasmid Mini Kit-BOX and its instructions. The plasmid was then transformed into *Agrobacterium* strain EHA105 using electroporation. Figure 2 As shown in Figure A, four-leaf stages of *Nicotiana benthamiana* leaves were co-infiltrated with *Agrobacterium* containing pBD-OsBZR1, pBD (negative control), and pBD-VP16 (positive control), respectively, along with the reporter vector *Agrobacterium*. After 48 hours, the infiltrated leaves were observed using a plant imaging system, and dual-luciferase activity was measured using a microplate reader. The results showed that the fluorescence signal intensity of the combination of the transient expression of the pBD-OsBZR1 effector and the reporter vector was the weakest (e.g., ...). Figure 2 As shown in Figure B), the luciferase activity of this combination was significantly lower than that of the negative control, indicating that OsBZR1 is a transcriptional repressor (as shown in Figure B). Figure 2 (As shown in C).

[0028] Referring to the literature reported by Zhang et al., it is demonstrated that OsBZR1 can directly bind to the brassinosteroid response element (BRRE), namely the CGTGCG motif (Zhang et al. The dual effect of the brassinosteroid pathway on Rice black-streaked dwarf virus infection by modulating the peroxidase-mediated oxidative burst and plant defense. MolPlant Microbe In, 2019, 32, 685-696). To analyze whether OsBZR1, as a transcriptional repressor, can inhibit autophagy-related genes, the promoter sequences of all rice autophagy-related genes OsATGs and the BR biosynthesis gene OsCPD1 available in the RAP-DB database were analyzed using the plant promoter analysis website PlantPAN 4.0 (The Plant Promoter Analysis Navigator) to check whether the BRRE element bound by OsBZR1 was present. Analysis revealed that 14 rice OsATG genes (OsATG1b, OsATG1c, OsATG3a, OsATG3b, OsATG6b, OsATG7, OsATG8b, OsATG8c, OsATG9a, OsATG12, OsATG13a, OsATG18b, OsATG18c, and OsATG18e) and the rice BR biosynthesis gene OsCPD1 contain one or more BRRE elements (e.g., OsATG1b, OsATG1c, OsATG3a, OsATG3b, OsATG6b, OsATG7, OsATG8b, OsATG8c, OsATG9a, OsATG12, OsATG13a, OsATG18b, OsATG18c, and OsATG18e) within a 3000 bp upstream of the promoter, which may bind to OsBZR1. Figure 3(As shown in A). The inhibitory function of OsBZR1 on autophagy-related genes was analyzed by dual-luciferase assay according to the method reported by Zeng et al. (Zeng et al. Viral proteins suppress rice defenses by boosting OsTSN1 RNA decay via phase separation and multimerization. Nat Commun, 2025, 16, 7481). Based on the OsBZR1 gene sequence cloned in Example 1 (as shown in SEQ ID NO: 1), a pair of primers with Stu I restriction sites and homologous arm sequences of the effector vector containing dual-luciferase reporter system were designed. The upstream primer T1 is 5'-CTGCCCAAATTCGCGAGGCCTATGACGTCCGGGGCGGCG-3', and the downstream primer T2 is 5'-AACAGTAAATTCAAAAGGCCTTCATTTCGCGCCGACGCC-3'. Based on the 14 OsATGs genes containing BRRE elements and the full-length promoter sequence of 1 OsCPD1 gene downloaded from the RAP-DB database, 14 pairs of primers with Sma Primer pairs for the restriction enzyme sites and the homologous arm sequences of the reporter vector of the dual-luciferase reporter system were used. Using the amplified OsBZR1 gene fragment and the full-length promoter fragments of 14 OsATGs and 1 OsCPD1 gene as templates, the OsBZR1 gene and the full-length promoter sequences of the 14 OsATGs gene were amplified using the high-fidelity enzyme TOROBlue Flash KOD Dye Mix from TOYOBO. The products were run on agarose gels and then recovered by gel cutting. The effector and reporter vector plasmids of the dual-luciferase reporter system were digested with Stu I and Sma I restriction endonucleases from Thermo Fisher Scientific, respectively. The digestion reaction system included 7 µL of plasmid, 2 µL of 10 × FastDigest buffer, 2 µL of restriction endonuclease, and 13 µL of sterile deionized water. Digestion was performed at 37°C for 30 minutes. The reaction products were then recovered by gel cutting using the Vazyme Gel DNA Extraction Mini Kit according to its product instructions. Using TransGen's pEASY ® The ProSeamless Cloning and Assembly Kit ligates the recovered gene fragments into enzyme-digested effector and reporter vectors, respectively. The in-fusion seamless ligation reaction system includes 5 µL of 2×pEASY. ®Pro Assembly Mix, 2 µL of effector and reporter vectors, and 3 µL of the full-length promoter fragments of the OsBZR1 gene and 14 OsATGs genes were collected by gel extraction and incubated at 50°C for 15 minutes. The ligation products were transformed into *E. coli* DH5α, and positive clones, effector-OsBZR1 and reporter-OsATGs, were obtained by colony PCR screening. The positive colonies were cultured with shaking, and plasmids were extracted using the Vazyme FastPure Plasmid Mini Kit-BOX and its instructions. The plasmids were then transformed into *Agrobacterium* strain EHA105 by electroporation. Figure 3 As shown in Figure B, leaves of *Nicotiana benthamiana* at the 4-leaf stage were co-infiltrated with *Agrobacterium* containing the effector vector *effector-OsBZR1*, the reporter vector *reporter-OsATGs*, and the empty reporter vector *EV* (negative control). After 48 hours, the infiltrated leaves were observed using a plant imaging system, and dual-luciferase activity was measured using a microplate reader. The results showed that the fluorescence intensity of leaves co-infiltrated with the promoter sequences of the *OsATG*3a, *OsATG*3b, *OsATG*6b, *OsATG*7, *OsATG*8b, *OsATG*8c, *OsATG*9a, *OsATG*12, *OsATG*13a, *OsATG*18b, *OsATG*18c, *OsATG*18e, and *OsCPD* genes was significantly lower on the side co-infiltrated with the *OsBZR1* effector vector than on the side co-infiltrated with the empty effector vector *EV* (e.g., ...). Figure 3 As shown in Figure C), and the luciferase activity of these combinations was significantly lower than that of the negative control, indicating that OsBZR1 inhibits the transcriptional activity of the aforementioned autophagy-related genes and the OsCPD1 gene (e.g., Figure 3 (As shown in D). The fluorescence intensity of the reporter vector co-infiltrated with the promoter sequences of the OsATG1b and OsATG1c genes, the OsBZR1 effector vector, and the empty vector EV on both sides of the leaf was very weak, almost unobservable (e.g., Figure 3 (As shown in C), but the luciferase activity in the leaves of the reporter vector and the OsBZR1 effector vector co-infiltrated with the promoter sequences of the OsATG1b and OsATG1c genes was significantly lower than that in the negative control (e.g., ...). Figure 3 As shown in Figure D, this indicates that OsBZR1 also inhibits the transcriptional activity of the promoters of the OsATG1b and OsATG1c genes.

[0029] In order to obtain OsBZR1 Gene knockout rice plants were found to utilize CRISPR Cas9 technology specifically to knock out genes, according to Baige Gene Technology (Jiangsu) Co., Ltd. OsBZR1 Rice mutant seeds (gene) osbzr1 ) Specific knockout OsBZR1Rice mutant seeds (gene) osbzr1 ) cultivated into rice plants, such as Figure 4 As shown in Figure A, the specific knockout described above in this embodiment is illustrated. OsBZR1 The results of gene identification in rice plants. OsBZR1 In the sequencing results after gene knockout, OsBZR1 The gene's coding sequence contains a mutation that deletes 8 bases, which will lead to... OsBZR1 The gene's reading frame shifted, failing to encode the correct OsBZR1 protein. Following the experimental system and method in Example 2, RT-qPCR was used to... OsBZR1 Gene knockout rice and wild-type rice plants OsATG1b , OsATG1c , OsATG3a , OsATG3b , OsATG6b , OsATG7 , OsATG8b , OsATG8c , OsATG9a , OsATG12 , OsATG13a , OsATG18b , OsATG18c , OsATG18e and OsCPD1 The transcriptional level of genes, namely these 14 OsATGs Genes and OsCPD1 Primer pairs for genes and rice internal reference genes UBQ5 Primer pairs 5'-AGCAGAAGCACAAGCACAA-3' and 5'-AGCCTGCTGGTTGTAGACG-3' were used for RT-qPCR reactions using a Roche LightCycler® 480 instrument and a Vazyme ChamQ SYBR Color qPCR Master Mix kit. The reaction conditions were as follows: 95℃ for 30 s, followed by alternating cycles of 95℃ for 10 s and 60℃ for 30 s, for a total of 45 cycles. Relative quantification was performed using Roche's LightCycler® 480 Gene Scanning Software to analyze these 14 genes in rice. OsATGs Genes and OsCPD1 Gene expression levels. The results showed that, compared to WT rice, osbzr1 The above 14 in rice plants OsATGs as well as OsCPD1 The transcriptional levels of all genes were significantly reduced (e.g.) Figure 4 (As shown in Figure B). This further demonstrates that OsBZR1 can inhibit the transcription of autophagy-related genes and BR biosynthesis genes.

[0030] Will OsBZR1Gene knockout rice plants and control wild-type ZH11 rice (WT) were cultured together under the same conditions to observe their phenotypes. osbzr1 The rice phenotype is normal and indistinguishable from the wild type (e.g. Figure 5 (As shown in A). For further analysis OsBZR1 The function of the gene in SRBSDV infection of rice was investigated by inoculating rice with SRBSDV via feeding infected white-backed planthoppers. OsBZR1 Gene knockout rice and wild-type rice plants were observed daily after inoculation with SRBSDV. Infection was confirmed by RT-PCR, and the plant height of infected rice was recorded. Results showed that compared to wild-type rice, at 30 days after SRBSDV inoculation, osbzr1 The mutant rice showed milder disease symptoms than the wild-type control, and its plant height was significantly higher than the wild-type control at 30 days after SRBSDV infection (e.g., Figure 5 (As shown in A and B). osbzr1 The disease incidence rate in mutant rice was 35.7% (5 / 14), while that in wild-type rice was 66.7% (14 / 21). Following the experimental system and methods described in Example 2, RT-qPCR was used to detect the accumulation level of SRBSDV p10 mRNA in these plants, i.e., SRBSDV... P10 Primer pairs for the gene 5'-TGTCGTGAAGTTCCTGCTCAA-3' and 5'-GGTCGTAACCGCCATAGTGT-3' and the rice internal reference gene UBQ5 Primer pairs 5'-AGCAGAAGCACAAGCACAA-3' and 5'-AGCCTGCTGGTTGTAGACG-3' were used for RT-qPCR reactions using a Roche LightCycler® 480 instrument and a Vazyme ChamQ SYBR Color qPCR Master Mix kit. The reaction conditions were: 95℃ for 30 s, followed by alternating cycles of 95℃ for 10 s and 60℃ for 30 s, for a total of 45 cycles. Relative quantification was performed using Roche's LightCycler® 480 Gene Scanning Software to analyze the components in rice. P10 mRNA expression levels. Results showed... osbzr1 mutant rice P10 The mRNA accumulation level was significantly lower in rice than in wild-type rice (e.g., Figure 5 (As shown in C). Following the method reported by Zeng et al., Western blot analysis was performed to analyze the accumulation level of SRBSDV P10 protein in rice (Zeng et al.). et al.Viral proteins suppress ricedefenses by boosting OsTSN1 RNA decay via phase separation and multimerization. Nat Commun (2025, 16, 7481), using SRBSDV P10 antibody detection to determine the SRBSDV P10 content in rice from each treatment group 30 days after infection, it was found that in osbzr1 The viral load in mutant rice was significantly lower than that in wild-type rice control (e.g., Figure 5 As shown in Figure D, this indicates that the OsBZR1 protein positively regulates SRBSDV infection in rice. These results demonstrate that the cloned rice... OsBZR1 The gene has high application value, namely, by editing and knocking out this gene in rice, the resistance of rice plants to SRBSDV can be improved, providing resistant germplasm resources for disease-resistant breeding.

[0031] The embodiments described above are only some preferred embodiments of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A key transcription factor for brassinolide signaling in rice OsBZR1 Genes, characterized by, The base sequence of this gene is shown in SEQ ID NO:

1.

2. A key transcription factor for rice brassinolide signaling as described in claim 1 OsBZR1 The protein encoded by the gene has the amino acid sequence shown in SEQ ID NO:2, and it participates in rice autophagy and brassinolide signal transduction and positively regulates the infection of rice by Southern Rice Black-Streaked Dwarf Virus.

3. A key transcription factor for rice brassinolide signaling as described in claim 1 OsBZR1 A gene cloning method, characterized in that, Total RNA was extracted from rice leaves, and a banding agent was synthesized using a pair of gene sequences targeting the OsBZR1 protein. Eco RI and Bam Using primers upstream and downstream of the HI restriction site and the homologous arm sequence of the pGAD T7 vector, an 897 bp key transcription factor for rice brassinolide signaling was cloned from total RNA in rice leaves by RT-PCR. OsBZR1 Gene; and the sequences of the upstream and downstream primers are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

4. The cloning method as described in claim 3, characterized in that, Total RNA from the rice leaves was extracted using TRIzol reagent.

5. The cloning method as described in claim 3, characterized in that, Genes cloned using RT-PCR need to be cloned into the pGAD T7 vector using in-fusion seamless ligation, followed by first-generation nucleic acid sequencing. The obtained nucleic acid sequences are then compared using BLAST to verify the correctness of the cloning.

6. A method for improving the resistance of rice to Southern Rice Black-Streaked Dwarf Virus, characterized in that, By using gene editing technology to knock out the key transcription factor for brassinolide signaling in rice as described in claim 1. OsBZR1 Gene.

7. A key transcription factor for rice brassinolide signaling as described in claim 1 OsBZR1 The application of genes is characterized by This study aimed to improve rice's resistance to Southern Rice Black-Streaked Dwarf Virus by knocking out this gene in rice using gene editing technology.

8. A key transcription factor for rice brassinolide signaling as described in claim 1 OsBZR1 The application of genes is characterized by, In disease-resistant breeding, this gene is knocked out using gene editing technology to obtain resistant germplasm resources.

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