Mutant ospbl8 gene, preparation method thereof and application of mutant ospbl8 gene in enhancing disease resistance of rice

Abstract

The application relates to the technical field of plant genetic engineering, and particularly relates to a mutant type OsPBL8 gene, a preparation method thereof and application of the mutant type OsPBL8 gene in enhancing the disease resistance of rice. The mutant type OsPBL8 gene comprises a full-length mutant type promoter and a full-length coding sequence of an OsPBL8 gene, and the nucleotide sequence of the mutant type OsPBL8 gene is shown as SEQ ID No: 5. The mutant type OsPBL8 gene sequence is loaded on a pZ469 (pZCB11-1300-3xMyc-GFP) vector which is single-enzyme cut by BamHI, and then is transferred into agrobacterium, and the gene is transfected into initial rice plants by a method of agrobacterium-mediated transformation to form transgenic rice. The promoter of the OsPBL8 gene is modified, the disease resistance of the rice can be significantly improved, and the mutant type OsPBL8 gene overexpression plant still has excellent rice blast resistance in an environment with a large diurnal temperature difference.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a mutant OsPBL8 gene, its preparation method, and its application in enhancing the disease resistance of rice. Background Technology

[0002] Rice blast, often referred to as "rice cancer," severely restricts rice yield and quality. Existing resistant varieties exhibit numerous limitations in combating rice blast, such as limited resistance types, the easy loss of resistance as the pathogen evolves, and some resistant varieties possessing undesirable agronomic traits. Furthermore, traditional disease-resistant breeding methods suffer from drawbacks such as lengthy cycles and low efficiency, and lack precise molecular markers closely linked to desirable agronomic traits. This makes precise, targeted improvement during the breeding process difficult, becoming a key bottleneck restricting breakthroughs in rice disease-resistant breeding technology and industrial development.

[0003] To effectively reduce the negative impact of rice blast on rice yield and quality, researchers have explored the application of biotechnology in rice variety improvement. For example, patent application number CN202610050298.X discloses the application of the OsPBL8 gene in enhancing rice resistance to rice blast fungus. This technology uses transgenic methods to regulate key nodes in the plant's immune signal transduction, providing more durable broad-spectrum resistance. However, this transgenic rice still has limitations in its resistance to rice blast fungus. More importantly, the expression regulation of the OsPBL8 gene is significantly affected by environmental factors, especially under conditions of large diurnal temperature variations, where its susceptibility and resistance show significant fluctuations, leading to a decrease in rice disease resistance. This severely restricts the stable application effect of this gene in complex and variable natural environments. Summary of the Invention

[0004] The purpose of this invention is to provide a mutant OsPBL8 gene, which improves the expression level of the OsPBL8 gene under conditions of large diurnal temperature differences by modifying the promoter of the OsPBL8 gene.

[0005] The present invention also provides the application of the above-mentioned mutant OsPBL8 gene in enhancing the disease resistance of rice. By overexpressing the OsPBL8 gene, the disease resistance of rice under conditions of large diurnal temperature difference is significantly improved.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a mutant OsPBL8 gene, the mutant OsPBL8 gene including the full length of the mutant OsPBL8 gene promoter and the full length of the coding sequence, the nucleotide sequence of the mutant OsPBL8 gene being shown in SEQ ID No:5.

[0007] Furthermore, the full length of the encoded sequence does not include untranslated regions (UTRs).

[0008] The method for preparing the mutant OsPBL8 gene described above includes:

[0009] Total DNA and total RNA were obtained from japonica rice seedlings, and cDNA was obtained by reverse transcription using the RNA as a template.

[0010] Using the DNA as a template and OsPBL8-Pro-F and OsPBL8-Pro-M2-R as primers, amplification was performed to obtain a fragment including the first half of the OsPBL8 gene promoter, the nucleotide sequence of which is shown in SEQ ID No:28.

[0011] Using the DNA as a template and OsPBL8-Pro-CDS-R and OsPBL8-Pro-M2-F as primers, amplification was performed to obtain a fragment including the latter half of the OsPBL8 gene promoter and the first half of the coding sequence, the nucleotide sequence of which is shown in SEQ ID No:29.

[0012] Using OsPBL8-Pro-F and OsPBL8-Pro-CDS-R as primers, the obtained first half of the OsPBL8 gene promoter fragment and the second half of the OsPBL8 gene promoter and the first half of the coding sequence fragment were used as templates for amplification to obtain the full length of the OsPBL8 gene mutant promoter and the first half of the coding sequence, the nucleotide sequence of which is shown in SEQ ID No:30;

[0013] Using the cDNA as a template and OsPBL8-Pro-CDS-F and OsPBL8-R as primers, amplification was performed to obtain a fragment including the latter half of the OsPBL8 gene promoter and the full length of the coding sequence, the nucleotide sequence of which is shown in SEQ ID No:27.

[0014] Using OsPBL8-Pro-F and OsPBL8-R as primers, the full-length OsPBL8 gene mutant promoter and the first half of its coding sequence, as well as the second half of its coding sequence and the full-length OsPBL8 gene mutant promoter, were amplified to obtain the full-length OsPBL8 gene mutant promoter and its full-length coding sequence, the nucleotide sequence of which is shown in SEQ ID NO:5.

[0015] The nucleotide sequence of OsPBL8-Pro-F is SEQ ID No:7, the nucleotide sequence of OsPBL8-Pro-CDS-R is SEQ ID No:8, the nucleotide sequence of OsPBL8-Pro-M2-R is SEQ ID No:17, the nucleotide sequence of OsPBL8-Pro-M2-F is SEQ ID No:16, the nucleotide sequence of OsPBL8-Pro-CDS-F is SEQ ID No:9, and the nucleotide sequence of OsPBL8-R is SEQ ID No:10.

[0016] This application also provides the application of the above-mentioned mutant OsPBL8 gene in enhancing rice resistance to rice blast.

[0017] Furthermore, the mutant OsPBL8 gene sequence was transferred into the initial rice plants to form transgenic rice with OsPBL8 gene overexpression.

[0018] Furthermore, the mutant OsPBL8 gene sequence was loaded onto the pZ469 (pZCB11-1300-3xMyc-GFP) vector digested with BamHI and then transformed into Agrobacterium, and the initial rice plants were transfected by Agrobacterium-mediated transformation to form the transgenic rice.

[0019] Furthermore, the initial rice variety is japonica rice.

[0020] Furthermore, the genetically modified rice maintains its resistance under diurnal temperature variations of 5°C to 15°C.

[0021] The beneficial effects of this invention are as follows: The mutant OsPBL8 gene provided in this application, by modifying the promoter of the OsPBL8 gene, can significantly improve the disease resistance of rice, so that the mutant OsPBL8 gene overexpression plants still have excellent resistance to rice blast in environments with large diurnal temperature differences, thereby significantly improving the adaptability of rice in environments with large temperature differences, which is helpful for agricultural production under different climatic conditions.

[0022] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the construction of binary plant overexpression vectors in Embodiments 1 and 2 of the present invention;

[0024] Figure 2This is an agarose gel electrophoresis image of DNA from multiple rice plants in Embodiment 1 of the present invention;

[0025] Figure 3 This is an agarose gel electrophoresis image of DNA from multiple rice plants in Example 2 of the present invention;

[0026] Figure 4 This is a graph showing the expression level analysis of the OsPBL8 gene in various rice plants as described in this invention.

[0027] Figure 5 Phenotypes of various rice plant leaves inoculated with rice blast spores and cultured at a constant temperature of 28°C for 7 days, as shown in this invention.

[0028] Figure 6 Phenotypic results of various rice plant leaves inoculated with rice blast spores and cultured for 7 days under a day-night temperature difference of 10°C;

[0029] Figure 7 This is a graph showing the relative biomass analysis of the leaves of various rice plants after inoculation with rice blast spores, as shown in this invention.

[0030] Figure 8 This is an analysis diagram of the release of reactive oxygen species after treating the base of various rice plants with chitin, as shown in this invention.

[0031] Figure 9 This is a diagram showing the expression of resistance genes in the leaves of various rice plants as described in this invention. Detailed Implementation

[0032] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] A preferred embodiment of this application illustrates a mutant OsPBL8 gene, which includes the full-length mutant OsPBL8 gene promoter and the full-length coding sequence (CDS). The nucleotide sequence of this mutant OsPBL8 gene is shown in SEQ ID No:5.

[0034] The promoter is a DNA sequence upstream of a gene, serving as the regulatory region for gene transcription and responsible for initiating transcription. The promoter region itself does not encode proteins; instead, it regulates gene expression by binding to transcription factors. Plant disease resistance is primarily regulated by the expression of resistance genes (R genes) or defense-related genes. Currently, most transgenic or gene-editing strategies focus on modifying coding sequence (CDS) regions, such as enhancing protein activity and stability, while neglecting the optimization of the promoter region at the transcriptional regulatory level. Natural promoters often suffer from insufficient expression intensity, limited spatiotemporal specificity, and delayed response to pathogen signals, leading to slow initiation of disease resistance responses, inadequate local expression, or excessive energy consumption, failing to rapidly establish an efficient defense barrier in the early stages of pathogen infection. The promoter is the region where RNA polymerase recognizes and binds, determining the timing, location, and intensity of gene expression. By directionally modifying promoters, such as inserting cis-acting elements, optimizing core promoter sequences, and fusing strongly inducible elements, precise regulation of the expression of target disease resistance genes can be achieved, enabling rapid, high-intensity, and localized expression during pathogen infection, thereby seizing the initiative in defense.

[0035] In this and other embodiments, the full-length coding sequence does not include the UTR (untranslated region). By removing the full-length UTR region of the coding sequence, it is possible to avoid interference from repressive regulatory elements (such as miRNA binding sites, uORF, etc.) in the UTR region from interfering with the efficient expression of the target protein, and it is also possible to reduce the vector size and improve cloning and viral packaging efficiency.

[0036] In one embodiment, the method for preparing the mutant OsPBL8 gene includes:

[0037] Total DNA and total RNA were obtained from japonica rice seedlings. Using RNA as a template, cDNA was obtained by reverse transcription.

[0038] Using DNA as a template and OsPBL8-Pro-F and OsPBL8-Pro-M2-R as primers, amplification was performed to obtain a fragment including the first half of the OsPBL8 gene promoter.

[0039] Using DNA as a template and OsPBL8-Pro-CDS-R and OsPBL8-Pro-M2-F as primers, amplification was performed to obtain a fragment including the latter half of the OsPBL8 gene promoter and the first half of the coding sequence.

[0040] Using OsPBL8-Pro-F and OsPBL8-Pro-CDS-R as primers, the first half of the OsPBL8 gene promoter fragment, the second half of the OsPBL8 gene promoter, and the first half of the coding sequence fragment were used as templates for amplification to obtain the full length of the OsPBL8 gene mutant promoter and the first half of the coding sequence fragment.

[0041] Using the cDNA as a template and OsPBL8-Pro-CDS-F and OsPBL8-R as primers, amplification was performed to obtain a fragment including the latter half of the OsPBL8 gene promoter and the full length of the coding sequence.

[0042] Using OsPBL8-Pro-F and OsPBL8-R as primers, the full-length OsPBL8 gene mutant promoter and the latter half of the OsPBL8 gene promoter and the full-length coding sequence were used as templates for amplification to obtain the full-length OsPBL8 gene mutant promoter and the full-length coding sequence.

[0043] The nucleotide sequence of OsPBL8-Pro-F is SEQ ID No:7, the nucleotide sequence of OsPBL8-Pro-M2-R is SEQ ID No:17, the nucleotide sequence of OsPBL8-Pro-CDS-R is SEQ ID No:8, the nucleotide sequence of OsPBL8-Pro-M2-F is SEQ ID No:16, the nucleotide sequence of OsPBL8-Pro-CDS-F is SEQ ID No:9, and the nucleotide sequence of OsPBL8-R is SEQ ID No:10.

[0044] This application also provides the application of the above-mentioned mutant OsPBL8 gene in enhancing rice resistance to rice blast.

[0045] In one embodiment, the mutant OsPBL8 gene sequence was transferred into the initial rice plant to form transgenic rice with OsPBL8 gene overexpression.

[0046] In this embodiment and other embodiments, the mutant OsPBL8 gene sequence was first loaded onto the pZ469 (pZCB11-1300-3xMyc-GFP) vector digested with BamHI, then transformed into Agrobacterium, and the initial rice plants were transfected by Agrobacterium-mediated transformation to form transgenic rice.

[0047] In some embodiments, the initial rice is japonica rice, such as the Kitaake variety.

[0048] In some embodiments, the genetically modified rice maintains its resistance under diurnal temperature variations of 5°C to 15°C.

[0049] Example 1

[0050] Transgenic rice was obtained by inserting the rice OsPBL8 gene into the genome of a primary rice variety, thereby overexpressing the OsPBL8 gene. In this example, the primary rice variety used was japonica rice, named Kitaake.

[0051] The OsPBL8 gene sequence and its promoter sequence were obtained from an online database. The nucleotide sequence of the OsPBL8 gene is shown in SEQ ID No:1, and its coding sequence (CDS) is shown in SEQ ID No:2. The nucleotide sequence of the OsPBL8 gene promoter is shown in SEQ ID No:3. The specific procedures for preparing transgenic rice are as follows:

[0052] Based on the nucleotide sequence of the OsPBL8 gene and the nucleotide sequence of the OsPBL8 gene promoter, primers for amplifying its complete open reading frame (ORF), primers for obtaining the full length of its promoter, and primers for obtaining the full length of the promoter plus the full length of the CDS (coding sequence) were designed. Specifically, these primers are: OsPBL8-Pro-F (SEQ ID NO:7), OsPBL8-Pro-CDS-R (SEQ ID NO:8), OsPBL8-Pro-CDS-F (SEQ ID NO:9), and OsPBL8-R (SEQ ID NO:10). These primers were prepared into solutions with concentrations ranging from 10 pmol / L to 15 pmol / L.

[0053] Total DNA and total RNA were obtained from Kitaake seedlings. Reverse transcription was performed using the total RNA as a template to obtain cDNA, which was then used as a template for subsequent amplification reactions. The polymerase required for amplification was a premixed DNA polymerase kit from PrimeSTAR, a brand under Takara Bio Inc. (Dalian). This kit includes 2×KOD buffer, a dNTP mixture, and KOD(Plus) DNA polymerase. Specific amplification systems and reaction conditions are shown in Tables 1 and 2.

[0054] Table 1. Amplification System

[0055]

[0056] Table 2. Reaction conditions for the amplification reaction

[0057]

[0058] The specific steps are as follows:

[0059] (1) Using DNA as a template and OsPBL8-Pro-F and OsPBL8-Pro-CDS-R as primers, the amplification system was prepared according to Table 1 using the premixed DNA polymerase kit described above, and PCR amplification was performed according to the conditions recorded in Table 2. The final product obtained was a fragment including the full length of the OsPBL8 gene promoter and the first half of the coding sequence, as shown in SEQ ID No:26, that is, extending from the promoter start site to the first 16 nucleotides of the coding sequence.

[0060] (2) Using cDNA as a template and the above-mentioned OsPBL8-Pro-CDS-F and OsPBL8-R as primers, PCR amplification was performed under the same conditions. The final product obtained included the latter half of the OsPBL8 gene promoter and the full-length coding sequence, as shown in SEQ ID No:27, that is, the fragment extending from the latter half of the promoter region to the stop codon of the coding region.

[0061] After amplification, agarose gel electrophoresis was performed and the gel was cut. The amplification products were recovered using an agarose gel recovery kit purchased from Omega Bio-Tek, yielding two fragments with high purity: the full-length OsPBL8 gene promoter and the first half of the coding sequence, and the second half of the OsPBL8 gene promoter and the full-length coding sequence.

[0062] Next, using OsPBL8-Pro-F and OsPBL8-R as primers, and the first half of the amplified full-length OsPBL8 gene promoter and coding sequence, as well as the second half of the OsPBL8 gene promoter and coding sequence, as templates, the amplification reaction was performed according to the conditions described in Table 2. The reaction system for this amplification reaction is shown in Table 3. After amplification, agarose gel electrophoresis was performed and the gel was cut. Subsequently, the amplification products were recovered using an agarose gel recovery kit to obtain the full-length OsPBL8 gene promoter and coding sequence (OsPBL8-Pro-CDS), the nucleotide sequence of which is shown in SEQ ID No:4. In the obtained full-length OsPBL8 gene promoter and coding sequence, the UTR region in the original coding sequence region was removed, which can avoid the repressive regulatory elements that may exist in the UTR region.

[0063] Table 3. Amplification System

[0064]

[0065] Based on the full-length promoter and coding sequence of the OsPBL8 gene obtained above, the binary plant overexpression vector pZ469(pZCB11-1300-3xMyc-GFP)-OsPBL8 was constructed. A schematic diagram of this pZ469(pZCB11-1300-3xMyc-GFP)-OsPBL8 vector is shown below. Figure 1 As shown in (A). The specific construction process is as follows:

[0066] The pZ469 (pZCB11-1300-3xMyc-GFP) vector, also purchased from Thermo Fisher Scientific, was digested with the restriction endonuclease BamHI (purchased from Thermo Fisher Scientific). The digestion system is shown in Table 4. After digestion at 37°C for 1 h, agarose gel electrophoresis was performed, and the gel was cut. The digested vector was recovered using an agarose gel recovery kit and kept for later use.

[0067] Table 4. Enzyme digestion system

[0068]

[0069] Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize various primer sequences. Specifically, an adapter sequence was added to the 5' end to obtain pZ469-OsPBL8-Pro-BamHI-F (SEQ ID NO:11); an adapter sequence was added to the 3' end to obtain pZ469-OsPBL8-BamHI-R (SEQ ID NO:12). Using pZ469-OsPBL8-Pro-BamHI-F and pZ469-OsPBL8-BamHI-R as primers, and using the full-length promoter and coding sequence obtained above as templates, amplification was performed under the amplification system shown in Table 5 and the amplification reaction conditions shown in Table 2. After amplification, agarose gel electrophoresis was performed and the gel was cut. The amplification product was recovered using an agarose gel recovery kit to obtain the PZ469-PBL8-Pro+CDS fragment.

[0070] Table 5. Amplification System

[0071]

[0072] The amplified PZ469-PBL8-Pro+CDS fragment was cloned into the pZ469 vector digested with BamHI using an INFUSION recombination kit purchased from Takara Corporation, Japan. The recombination reaction system is shown in Table 6.

[0073] Table 6. Recombination Reaction System

[0074]

[0075] After recombination, the mixture was briefly centrifuged and then placed in a 50°C water bath for 20 min. The recombinant product was transformed into *E. coli* DH5α competent cells (Beijing Tiangen) using a heat shock method. The transformed cells were plated on Luria-Betani (LB) solid medium containing 50 mg / L kanamycin and incubated at 37°C for 16 h. Single colonies were picked and sent to Nanjing Genscript Biotech Co., Ltd. for sequencing. The correctly sequenced plasmid was obtained and named pZ469-OsPBL8-Pro-CDS.

[0076] The constructed expression vector was transformed into Agrobacterium strain EHA105 purchased from Intense Biotechnology, USA, using a liquid nitrogen freeze-thaw method. The specific procedure was as follows: EHA105 competent cells were thawed in an ice bath, at least 100 ng of the recovered and purified overexpression vector plasmid pZ469-OsPBL8-Pro-CDS was added, and the mixture was gently mixed. The cells were then incubated on ice for 5 min, flash-frozen in liquid nitrogen for 5 min, and heat-shocked at 37°C for 5 min. After these steps, the cells were immediately placed on ice and allowed to stand for 1–2 min. 800 μL of antibiotic-free LB medium was added to the system, and the cells were incubated at 200 rpm for 3.5 h at 28°C for recovery. The cells were then centrifuged at 4000 rpm for 3 min, the surface medium was removed with a pipette, and the remaining bacterial culture was mixed thoroughly. The bacterial culture was spread onto LB solid medium supplemented with 100 mg / mL kanamycin and 100 mg / mL rifampin, and incubated upside down at 28°C for 30-48 hours. Colonies were picked for colony PCR, and the obtained positive clones were named EHA105:pZ469-OsPBL8-Pro-CDS and stored at 4°C for later use.

[0077] Kitaake rice plants were transformed with pZ469-OsPBL8-Pro-CDS strain using the conventional Agrobacterium-mediated transformation method. Three transgenic plants with EHA105:pZ469-OsPBL8-Pro-CDS strain were obtained in the T0 generation.

[0078] Genomic DNA from the three single plants mentioned above was used as templates, and positive and negative samples were set up. Primers hyg283-F (SEQ ID NO:14) and hyg283-R (SEQ ID NO:15) were used to amplify a specific fragment in the transgenic genetic transformation marker gene (hygromycin phosphotransferase gene) for detection. The amplification system is shown in Table 7. The 2×Taq DNA Polymerase Mix was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0079] Table 7. Amplification System

[0080]

[0081] Amplification was performed using the Eppendorf Mastercycler PCR thermal cycler. The amplification reaction program was as follows: 95℃, 3 min; 95℃, 30 s; 58℃, 30 s; 72℃, 4 min, 30 cycles; 72℃, 5 min; 16℃, storage. After amplification, the amplified products were separated by agarose gel electrophoresis, and the results were photographed and recorded using a gel imaging system. The results are shown below. Figure 2 As shown.

[0082] Depend on Figure 2 It can be seen that the electrophoretic patterns of the DNA from the three transgenic plants of EHA105:pZ469-OsPBL8-Pro-CDS are similar to those of the positive control template, indicating that all three transgenic plants amplified the target product.

[0083] Example 2

[0084] The difference between this embodiment and Embodiment 1 is that: a mutant OsPBL8 gene fragment is inserted into the initial rice genome to obtain transgenic rice, thereby overexpressing the OsPBL8 gene. Specifically, the binding site of the OsPBL8 gene promoter is modified based on its nucleotide sequence and binding characteristics; then, the full-length mutant OsPBL8 gene promoter and its full-length coding sequence fragment are inserted into the initial rice genome to obtain transgenic rice, thereby overexpressing the OsPBL8 gene.

[0085] The specific process for modifying the promoter of the OsPBL8 gene is as follows:

[0086] Based on the nucleotide sequence of the OsPBL8 gene promoter, amplification primers were designed using software. Specifically, they are OsPBL8-Pro-M2-F (SEQ ID NO:16) and OsPBL8-Pro-M2-R (SEQ ID NO:17). These primers were prepared into solutions with concentrations ranging from 10 pmol / L to 15 pmol / L.

[0087] Similarly, total DNA and total RNA were obtained from Kitaake seedlings, and cDNA was obtained by reverse transcription using the obtained RNA as a template. This DNA or cDNA was then used as a template for amplification. Specific amplification systems and reaction conditions are shown in Tables 1 and 2 above.

[0088] (1) Using the DNA as a template, and the above-mentioned OsPBL8-Pro-F and OsPBL8-Pro-M2-R as primers, the above-mentioned premixed DNA polymerase kit was used to prepare the amplification system according to Table 1, and PCR amplification was performed according to the conditions recorded in Table 2. The final product obtained was a fragment including the first half of the OsPBL8 gene promoter, as shown in SEQ ID No:28.

[0089] (2) Using the DNA as a template and the above-mentioned OsPBL8-Pro-CDS-R and OsPBL8-Pro-M2-F as primers, PCR amplification was performed under the same conditions. The final product included fragments of the latter half of the OsPBL8 gene promoter and the first half of the coding sequence, as shown in SEQ ID No:29.

[0090] After amplification, agarose gel electrophoresis was performed and the gel was cut. The amplification products were then recovered using an agarose gel recovery kit to obtain two fragments with high purity: the first half of the OsPBL8 gene promoter fragment, the second half of the OsPBL8 gene promoter fragment, and the first half of the coding sequence fragment.

[0091] (3) Using OsPBL8-Pro-F and OsPBL8-Pro-CDS-R as primers, and the amplified first half of the OsPBL8 gene promoter fragment, the second half of the OsPBL8 gene promoter fragment, and the first half of the coding sequence fragment as templates, the amplification reaction was carried out according to the conditions described in Table 2. After amplification, agarose gel electrophoresis was performed and the gel was cut. Subsequently, the amplification products were recovered using an agarose gel recovery kit to obtain the full length of the mutant promoter of the OsPBL8 gene and the first half of the coding sequence, as shown in SEQ ID NO:30. In the full length of the mutant promoter of the OsPBL8 gene and the first half of the coding sequence fragment, the first half of the coding sequence includes a nucleotide fragment corresponding to approximately 16 amino acids.

[0092] (4) Using cDNA as a template and OsPBL8-Pro-CDS-F and OsPBL8-R as primers, amplification was performed to obtain a fragment including the latter half of the OsPBL8 gene promoter and the full length of the coding sequence, the nucleotide sequence of which is shown in SEQ ID No:27;

[0093] (5) Using OsPBL8-Pro-F and OsPBL8-R as primers, and the full-length OsPBL8 gene mutant promoter and the first half of its coding sequence, as well as the second half of the OsPBL8 gene mutant promoter and the full-length coding sequence, as templates, the amplification reaction was carried out according to the conditions described in Table 2. After amplification, agarose gel electrophoresis was performed and the gel was cut. Subsequently, the amplification products were recovered using an agarose gel recovery kit to obtain the full-length OsPBL8 gene mutant promoter and the full-length coding sequence (OsPBL8-Pro-M2-CDS), i.e., the mutant OsPBL8 gene, whose nucleotide sequence is shown in SEQ ID NO:5.

[0094] like Figure 1 As shown in (B), using the same method as in Example 1, the amplified full-length promoter and coding sequence fragments of the OsPBL8 gene mutant were transformed into the single-enzyme-digested pZ469 (pZCB11-1300-3xMyc-GFP) vector to prepare the pZ469-OsPBL8-Pro-M2-CDS plasmid. The pZ469-OsPBL8-Pro-M2-CDS plasmid was then transformed into Agrobacterium EHA105, and Kitaake rice plants were transformed with the pZ469-OsPBL8-Pro-M2-CDS strain. Five transgenic plants of EHA105:pZ469-OsPBL8-Pro-M2-CDS were obtained in the T0 generation.

[0095] Similarly, using genomic DNA from the above five individual plants as templates, and setting up positive and negative samples, primers hyg283-F and hyg283-R were used to amplify a specific fragment in the transgenic genetic transformation marker gene, namely the hygromycin phosphotransferase gene, for detection. The results are as follows: Figure 3 As shown.

[0096] Depend on Figure 3 It can be seen that the electrophoretic patterns of DNA from the five transgenic plants of EHA105:pZ469-OsPBL8-Pro-M2-CDS are similar to those of the positive control template, indicating that the target product was amplified in all of them.

[0097] Plants corresponding to DNA amplified with the target product in Example 1 and numbered 3 were randomly named the OsPBL8-Pro-CDS group. Similarly, plants corresponding to DNA amplified with the target product in Example 2 and numbered 2 were randomly named the OsPBL8-Pro-M2-CDS group. Untreated Kitaake rice plants were named the WT group.

[0098] Whole plants from the OsPBL8-Pro-CDS, OsPBL8-Pro-M2-CDS, and WT groups were collected, and RNA was extracted from each. The expression of the OsPBL8 gene in the tissue samples was detected by real-time quantitative polymerase chain reaction (qRT-PCR). For the reverse transcription in the qRT-PCR, the primers used were RT-OsPBL8-F (SEQ ID No:18) and RT-OsPBL8-R (SEQ ID No:19), respectively. The results are as follows: Figure 4 As shown.

[0099] Depend on Figure 4 It was found that the expression level of the OsPBL8 gene was significantly upregulated in both the OsPBL8-Pro-CDS and OsPBL8-Pro-M2-CDS groups compared to WT, indicating that the OsPBL8 gene was overexpressed in both groups. Furthermore, the expression level of the OsPBL8 gene in the OsPBL8-Pro-M2-CDS group was significantly higher than that in the OsPBL8-Pro-CDS group. Therefore, it can be concluded that the promoter modification in this application can increase the expression level of the target disease resistance gene at the infection site by 5–7 times, effectively shortening the defense response time and achieving rapid activation.

[0100] The OsPBL8-Pro-CDS, OsPBL8-Pro-M2-CDS, and WT groups were inoculated with rice blast fungus. Specifically, the rice blast fungus strain was inoculated onto complete (CM) medium for activation and cultured at 28°C for 12 days, with 12 hours of light and 12 hours of darkness every 24 hours. The medium was washed with 0.05% Tween 20 sterile water, and the spore solution was washed off with a glass rod. After filtration through a 40 μm filter membrane, the spore concentration was measured under a microscope, and the final concentration was adjusted to 1×10⁻⁶. 5 Spores / mL are used for foliar spraying inoculation of rice.

[0101] The spore suspension was evenly sprayed onto the surface of rice leaves corresponding to the OsPBL8-Pro-CDS, OsPBL8-Pro-M2-CDS, and WT groups. These groups were then placed in a 28℃ constant-temperature incubator for cultivation. After a 24-hour dark treatment, a 12-hour light treatment followed by a 12-hour dark treatment cycle was performed. Phenotypic results were observed after 7 days of cultivation. Figure 5As shown in the figure. The OsPBL8-Pro-CDS group, OsPBL8-Pro-M2-CDS group, and WT group in the replicate group were placed in an incubator with a day-night temperature difference of approximately 10℃. Specifically, after 24 hours of darkness, they were treated with light at 28℃ for 12 hours, followed by 12 hours of darkness at 18℃. This cycle was repeated for 7 days, and the phenotype was observed. The results are shown in the figure. Figure 6 As shown.

[0102] Depend on Figure 5 It can be seen that, under conditions of no diurnal temperature variation, the lesion area ratio after inoculation with rice blast spores in the OsPBL8-Pro-CDS group was 15%, while that in the OsPBL8-Pro-M2-CDS group was 8%. The lesion area ratio after inoculation with rice blast spores in the WT group was significantly larger. Figure 6 It was found that under conditions of large diurnal temperature variations, the lesion area ratio after inoculation with rice blast spores was 20% in the OsPBL8-Pro-CDS group, while it was 10% in the OsPBL8-Pro-M2-CDS group. The lesion area in the WT group was also larger after inoculation with rice blast spores. This indicates that under conditions of large diurnal temperature variations, inserting the mutant OsPBL8 gene helps to further improve the rice's resistance to rice blast.

[0103] Seven days after spraying with rice blast spores, DNA was extracted from leaves of the OsPBL8-Pro-CDS, OsPBL8-Pro-M2-CDS, and WT groups. The contents of the rice internal reference gene UBQ and the rice blast fungus internal reference gene MoPot in the DNA were detected by qRT-PCR, thus obtaining the relative biomass of rice blast fungus relative to leaf tissue in the OsPBL8-Pro-CDS, OsPBL8-Pro-M2-CDS, and WT groups. For qRT-PCR detection of the UBQ gene content, the primers used for reverse transcription were UBQ-RT-F (SEQ ID No: 20) and UBQ-RT-R (SEQ ID No: 21). For qRT-PCR detection of the MoPot gene content, the primers used for reverse transcription were MoPot-RT-F (SEQ ID No: 22) and MoPot-RT-R (SEQ ID No: 23). The experimental results are as follows: Figure 7 As shown.

[0104] Depend on Figure 7 It can be seen that in the OsPBL8-Pro-M2-CDS group lines with OsPBL8 gene overexpression, the relative biomass of rice blast fungus was significantly lower than that in the OsPBL8-Pro-CDS group, indicating that the development of rice blast was better suppressed.

[0105] Rice seeds obtained from the OsPBL8-Pro-CDS and OsPBL8-Pro-M2-CDS groups were shelled, sterilized, and spotted into Hoagland nutrient solution in glass tubes. After 7 days of aseptic culture, multiple rice seedlings were obtained. 150 μL of double-distilled water was added to each well of a black 96-well ELISA plate. Rice seedlings of uniform size and stem thickness were selected, and the base of the stems was cut to obtain segments approximately 1 mm in length. Five segments were placed in each well of the black ELISA plate. Care was taken to remove approximately 2 mm of white stem near the seed before cutting to obtain the stem base tissue. The black ELISA plate was incubated overnight at room temperature in the dark, and the measured room temperature was 25°C. The next day, a 2 mg / mL chitin stock solution was prepared, and 10 mL of reaction solution was obtained using this stock solution. The reaction solution was prepared by adding 20 μL of horseradish peroxidase (HRP) solution, 20 μL of chemiluminescent probe L-012 solution, and 20 μL of chitin stock solution to a test tube, and then adding double-distilled water to bring the volume to 10 mL. The horseradish peroxidase solution was a pre-prepared laboratory solution, prepared by adding 15 mg of horseradish peroxidase powder to 1 mL of sterile water and stored at -20°C. The chemiluminescent probe L-012 solution was also a pre-prepared laboratory solution, prepared by adding 15 mg of chemiluminescent probe L-012 powder to 1 mL of sterile water and stored at -20°C. Remove the black ELISA plate that has been left to stand overnight in the dark. Use a pipette to remove and discard the liquid from each well. Add 100 μL of reaction solution or double-distilled water to each well to obtain six groups of samples treated with the reaction solution or water. The samples obtained from the WT group after chitin treatment are labeled Group 1, the samples obtained from the OsPBL8-Pro-CDS group after chitin treatment are labeled Group 2, and the samples obtained from the OsPBL8-Pro-M2-CDS group after chitin treatment are labeled Group 3. Place the uncapped ELISA plate in the microplate reader and perform a test for approximately 30 ms per well. The microplate reader settings are shown in Table 8 below. The test results are as follows: Figure 8 As shown.

[0106] Table 8: Microplate reader settings

[0107]

[0108] Depend on Figure 8 It can be seen that chitin can trigger a burst of reactive oxygen species (ROS) in plants, and the insertion of the OsPBL8 gene or the mutant OsPBL8 gene does not affect this ROS burst. In addition, after chitin treatment, plants overexpressing the mutant OsPBL8 gene produced approximately twice the level of ROS compared to plants with the OsPBL8 gene.

[0109] Leaves from the OsPBL8-Pro-CDS, OsPBL8-Pro-M2-CDS, and WT groups, seven days after application of rice blast spores, were infected. Total RNA was extracted from these leaves, and the expression of phenylalanine ammonia-lyase (PAL) and WRKY transcription factor 45 (WRKY45) genes in the tissue samples was detected by qRT-PCR. For PAL expression detection, the primers used for reverse transcription in qRT-PCR were RT-OsPAL-F (SEQ ID No: 24) and RT-OsPAL-R (SEQ ID No: 25). For WRKY transcription factor 45 expression detection, the primers used for reverse transcription in qRT-PCR were RT-OsWRKY-F (SEQ ID No: 6) and RT-OsWRKY-R (SEQ ID No: 13). The results are shown below. Figure 9 As shown in (A) and (B).

[0110] Depend on Figure 9 As shown in (A) and (B), plants overexpressing the OsPBL8 gene have a significantly stronger resistance to rice blast than plants without the OsPBL8 gene. Furthermore, plants with the OsPBL8-Pro-M2-CDS gene inserted have a significantly stronger resistance to rice blast than plants with the OsPBL8-Pro-CDS gene inserted.

[0111] In summary, this application demonstrates that by modifying the promoter and inserting the full-length modified promoter and its coding sequence into the plant, the disease resistance of transgenic plants can be significantly improved. Furthermore, under conditions of large temperature differences, the transgenic plants can effectively resist rice blast. In addition, the modified promoter can drive the synergistic expression of multiple disease resistance-related genes, forming a "multi-target defense network," reducing the risk of pathogens escaping through mutations and improving the durability of resistance. By precisely controlling the promoter activity range, overexpression of disease resistance genes in vegetative tissues can be avoided, ensuring that plant type, yield, and quality are not affected, thus meeting the needs of agricultural applications.

[0112] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0113] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A mutant OsPBL8 gene, characterized in that, The mutant OsPBL8 gene includes the full-length mutant promoter and the full-length coding sequence of the OsPBL8 gene, and the nucleotide sequence of the mutant OsPBL8 gene is shown in SEQ ID No:

5.

2. The mutant OsPBL8 gene as described in claim 1, characterized in that, The full length of the encoded sequence does not include non-translated regions.

3. The method for preparing the mutant OsPBL8 gene as described in claim 1 or 2, characterized in that, include: Total DNA and total RNA were obtained from japonica rice seedlings, and cDNA was obtained by reverse transcription using the RNA as a template. Using the DNA as a template and OsPBL8-Pro-F and OsPBL8-Pro-M2-R as primers, amplification was performed to obtain a fragment including the first half of the OsPBL8 gene promoter, the nucleotide sequence of which is shown in SEQ ID No:

28. Using the DNA as a template and OsPBL8-Pro-CDS-R and OsPBL8-Pro-M2-F as primers, amplification was performed to obtain a fragment including the latter half of the OsPBL8 gene promoter and the first half of the coding sequence, the nucleotide sequence of which is shown in SEQ ID No:

29. Using OsPBL8-Pro-F and OsPBL8-Pro-CDS-R as primers, the obtained first half of the OsPBL8 gene promoter fragment and the second half of the OsPBL8 gene promoter and the first half of the coding sequence fragment were used as templates for amplification to obtain the full length of the OsPBL8 gene mutant promoter and the first half of the coding sequence, the nucleotide sequence of which is shown in SEQ ID No:30; Using the cDNA as a template and OsPBL8-Pro-CDS-F and OsPBL8-R as primers, amplification was performed to obtain a fragment including the latter half of the OsPBL8 gene promoter and the full length of the coding sequence, the nucleotide sequence of which is shown in SEQ ID No:

27. Using OsPBL8-Pro-F and OsPBL8-R as primers, the full-length OsPBL8 gene mutant promoter and the first half of its coding sequence, as well as the second half of its coding sequence and the full-length OsPBL8 gene mutant promoter, were amplified to obtain the full-length OsPBL8 gene mutant promoter and its full-length coding sequence, the nucleotide sequence of which is shown in SEQ ID NO:

5. The nucleotide sequence of OsPBL8-Pro-F is SEQ ID No:7, the nucleotide sequence of OsPBL8-Pro-CDS-R is SEQ ID No:8, the nucleotide sequence of OsPBL8-Pro-M2-R is SEQ ID No:17, the nucleotide sequence of OsPBL8-Pro-M2-F is SEQ ID No:16, the nucleotide sequence of OsPBL8-Pro-CDS-F is SEQ ID No:9, and the nucleotide sequence of OsPBL8-R is SEQ ID No:

10.

4. The application of the mutant OsPBL8 gene as described in claim 1 or 2 in enhancing rice resistance to rice blast, characterized in that, The mutant OsPBL8 gene sequence was transferred into the initial rice plants to overexpress the OsPBL8 gene, thus forming transgenic rice. The transgenic rice maintained its resistance under diurnal temperature ranges of 5℃-15℃.

5. The application as described in claim 4, characterized in that, The initial rice variety was japonica rice.

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