Application of oszfp350 gene or its coded protein in regulating rice sheath blight resistance

Abstract

This invention belongs to the field of plant biotechnology, specifically involving OsZFP350 Application of genes or their encoded proteins in regulating resistance to rice sheath blight. This invention discovers the encoding of the amino acid sequence shown in SEQ ID NO:2. OsZFP350 Genes are associated with resistance to rice sheath blight. OsZFP350 Overexpression of the gene can significantly improve the resistance of rice to sheath blight, while OsZFP350 Loss of function reduces rice's resistance to sheath blight. Overexpression OsZFP350 Genes enable rice to form an effective defense response in the early stages of pathogen infection, thereby inhibiting the spread of lesions. Overexpression of these genes... OsZFP350 Genetically modified rice showed a significant reduction in lesion length after infection with Sheath blight pathogen. Furthermore, the disease resistance phenotype exhibited good reproducibility in different lines and batches of experiments, demonstrating stable disease resistance that did not depend on complex multi-gene combinations. This provides a new technical pathway for molecular breeding of rice resistant to Sheath blight.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically involving OsZFP350 Application of genes or their encoded proteins in regulating resistance to rice sheath blight. Background Technology

[0002] Plants are susceptible to various pathogens during their growth. Plant pathogens are diverse, including viruses, bacteria, fungi, and nematodes. Pathogen invasion of plants leads to two outcomes: (1) the pathogen successfully multiplies within the host plant, causing related symptoms; (2) the host plant develops a resistance response, killing the pathogen or inhibiting its growth. Utilizing resistance gene resources to improve plant disease resistance is a fundamental solution for preventing diseases while simultaneously protecting the environment.

[0003] Rice is a vital food crop worldwide, but sheath blight often leads to a decline in both yield and quality. Currently, there is a lack of rice varieties with high resistance to sheath blight in agricultural production. Furthermore, because most field-causing sheath blight pathogens possess binucleate or multinucleate genomes, genetic transformation is difficult, resulting in relatively slow progress in research on their pathogenic mechanisms. This poses a significant challenge to the control of this disease. Disease-resistant breeding is an efficient and environmentally friendly disease control strategy, and discovering new broad-spectrum, highly resistant genes is a crucial foundation for successful breeding. These aspects cannot be achieved using conventional plant breeding and improvement techniques. However, research on rice sheath blight resistance genes is limited; therefore, discovering new rice sheath blight resistance genes and obtaining resistant plants through cloning of these genes is particularly important in this field. Summary of the Invention

[0004] The purpose of this invention is to provide OsZFP350 The application of genes or their encoded proteins in regulating rice sheath blight resistance can enrich the types of sheath blight resistance genes and improve rice's resistance to sheath blight.

[0005] This invention provides OsZFP350 The application of genes or their encoded proteins in regulating resistance to rice sheath blight, the aforementioned OsZFP350 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0006] Preferably, the regulation is: overexpression OsZFP350 Genes or their encoded proteins enhance resistance to rice sheath blight.

[0007] This invention provides overexpression OsZFP350 The application of genes or their encoded proteins in rice breeding, wherein the rice breeding includes the development of rice varieties resistant to sheath blight; OsZFP350 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0008] Preferably, the OsZFP350 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0009] Preferably, the sheath blight is caused by infection with the blight fungus.

[0010] Preferably, the Rhizoctonia solani includes Rhizoctonia solani Y-36.

[0011] This invention provides a biomaterial for improving resistance to rice sheath blight, the biomaterial comprising a recombinant vector and / or recombinant bacteria.

[0012] The recombinant vector includes a base vector and a component inserted into the base vector. OsZFP350 Gene; The recombinant bacteria include a basic strain and the basic strain into which the recombinant bacteria are introduced. OsZFP350 Gene or the recombinant vector; The OsZFP350 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0013] This invention provides a method for improving resistance to rice sheath blight, comprising the following steps: overexpressing in rice receptors OsZFP350 Genes, the ones mentioned OsZFP350 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0014] Preferably, the rice receptor comprises rice callus tissue.

[0015] This invention provides a rice material, the rice material comprising exogenously introduced... OsZFP350 Genes, the ones mentioned OsZFP350 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0016] Beneficial effects: This invention discovers that encodes the amino acid sequence shown in SEQ ID NO:2 OsZFP350 Genes are associated with resistance to rice sheath blight. OsZFP350 Overexpression of the gene can significantly improve the resistance of rice to sheath blight, while OsZFP350 Loss of function reduces rice's resistance to sheath blight. Overexpression OsZFP350 Genes enable rice to form an effective defense response in the early stages of pathogen infection, thereby inhibiting the spread of lesions. Overexpression of these genes... OsZFP350 Genetically modified rice showed a significant reduction in lesion length after infection with Sheath blight pathogen. Furthermore, the disease resistance phenotype exhibited good reproducibility in different lines and batches of experiments, demonstrating stable disease resistance that did not depend on complex multi-gene combinations. This provides a new technical pathway for molecular breeding of rice resistant to Sheath blight. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 For wild-type rice and overexpression OsZFP350 rice OsZFP350 Relative gene expression levels; Figure 2 Wild-type rice and OsZFP350 Mutant rice OsZFP350 Gene sequencing results; Figure 3 Wild-type rice, overexpression OsZFP350 rice and OsZFP350 Length of lesions in mutant rice infected with Sheath blight pathogen; Figure 4 Wild-type rice, overexpression OsZFP350 rice and OsZFP350 Phenotypic diagram of the mutant rice leaf sheath blight in living organisms. Detailed Implementation

[0019] This invention provides OsZFP350 The application of genes or their encoded proteins in regulating resistance to rice sheath blight, the aforementioned OsZFP350 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0020] As one implementation method, the regulation described in this invention is: overexpression OsZFP350 Genes or their encoded proteins enhance resistance to rice sheath blight.

[0021] This invention provides overexpression OsZFP350 The application of genes or their encoded proteins in rice breeding, wherein the rice breeding includes the development of rice varieties resistant to sheath blight; OsZFP350 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0022] As one implementation method, the present invention described OsZFP350The nucleotide sequence of the gene is shown in SEQ ID NO:1. In one embodiment, the sheath blight described in this invention is sheath blight caused by infection with *Rhizoctonia solani*. In one embodiment, the *Rhizoctonia solani* described in this invention includes *Rhizoctonia solani* Y-36. *Rhizoctonia solani* Y-36 used in the embodiments of this invention is disclosed in *Comparative transcriptome analysis of rice cultivars resistant and susceptible to *Rhizoctonia solani* AG1-IA*.

[0023] This invention provides a biomaterial for improving resistance to rice sheath blight, the biomaterial comprising a recombinant vector and / or recombinant bacteria, the recombinant vector comprising a base vector and a bacterium inserted into the base vector. OsZFP350 Gene; the recombinant bacteria include a basic bacterium and a gene into which the basic bacterium is introduced. OsZFP350 The gene or the recombinant vector; the OsZFP350 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0024] This invention provides a method for improving resistance to rice sheath blight, comprising the following steps: overexpressing in rice receptors OsZFP350 Genes, the ones mentioned OsZFP350 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2.

[0025] In one embodiment, the rice receptor of the present invention comprises rice callus tissue. The present invention addresses the overexpression of [the receptor] in rice. OsZFP350 There are no strict requirements regarding the gene delivery method; any method conventional in the field can be used. For example, Agrobacterium-mediated gene delivery can be used. OsZFP350 Genes are introduced into rice receptors.

[0026] This invention provides a rice material, the rice material comprising exogenously introduced... OsZFP350 Genes, the ones mentioned OsZFP350 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO:2. In this invention, the rice material is exogenously introduced. OsZFP350 After genes, OsZFP350 Non-stable genetic materials with significantly upregulated gene expression levels are different from new rice varieties. They lack uniformity and stability, meaning that there is phenotypic segregation in other phenotypic traits.

[0027] SEQ ID NO:1:ATGGATCCAGCAAGGTACTGGATCTTGGCCAGGAGGAAGCTGGGAGATCAGAAGGCGCCTCTCTTCCCCACCCCTCACATCACCATCGGCGCCGGCGGCAGCTCCACCTCCTACTACGAGTCATGGGAGGAACGTGCCTTCGCGGAAGACTCCGCAGGGCATCTCGGGGGCTGCATCTGGCCGCCGAGGTCCTACTCCTGCAGCTTCTGCGGCCGTGAGTTCCGGTCGGCGCAGGCGCTGGGCGGGCACATGAACGTCCACCGGAGGGACCGGGCAAGGCTCAAGCTCTCTGGGGTTGTGGAGGACGGCACCGGCGGCGAAAGCCACGGCATGCCGCCAAACCAAAACTACATGATACAACCATGCCCTCCCCAAATTGGCACCCTGCAGCACGCCTACAGCCCAAACCCTAGAAGTGGCAGTATTCTCGCTGCTGACACTAACCCTAATTCCATATGTGATGTTGTCGCGTACCCTGCTAGATCCTTGCTTCAAGTTGCAGCTGCTAGAACTGCCTTGGGCAAACAAGTCCTGAACGCCCCTCTTGTCTCATCGAAGTCGCCGTCGGCTGGTAGAGAACATGGTAAGAGGGAGACGTTGTTTCTTGGTGCTGTTCGATTGGCACAGGATCATGACACGGTGAGTTCTAATCTTGACCTGCGTGTTGGGAAAAATGAATTGAAGATAACTACTGTTTTGGGGTGCCGATCAAGAAGGGACTTTATGTACGATAACGACCGCGCGGATGATGAAAAGACTGTTGAAGCAAGTCACAAGAAAAGAAGAATCGATTTGGAGGTAAACCCCCTGATTCTAACTTCTTCAACCAGTAATCATCAACAGCAAGATGGTGGTGATGACCAACATCATGAAAAGATACTAAAACTTTATCGTAGCTCCTCGGTTGAAGAACTAGATCTTGAGCTTAGACTCGGGGAAGCCCCAAAAGAACAGTAG。

[0028] SEQ ID NO:2:MDPARYWILARRKLGDQKAPLFPTPHITIGAGGSSTSYYESWEERAFAEDSAGHLGGCIWPPRSYSCSFCGREFRSAQALGGHMNVHRRDRARLKLSGVVEDGTGGESHGMPPNQNYMIQPCPPQIGTLQHAYSPNPRSGSILAADTNPNSICDVVA YPARSLLQVAAARTALGKQVLNAPLVSSKSPSAGREHGKRETLFLGAVRLAQDHDTVSSNLDLRVGKNELKITTVLGCRRSRRDFMYDNDRADDEKTVEASHKKRRIDLEVNPLITSSTSNHQQQDGGDDQHHEKILKLYRSSSVEELDLELRLGEAPKEQ.

[0029] To further illustrate the present invention, the following description, in conjunction with the accompanying drawings and embodiments, explains the invention. OsZFP350 The application of genes or their encoded proteins in regulating resistance to rice sheath blight is described in detail, but should not be construed as limiting the scope of protection of this invention.

[0030] Example 1 1. OsZFP350 Construction of overexpression vectors Total mRNA was extracted from wild-type rice ZH11 and reverse transcribed into cDNA; design OsZFP350 upstream and downstream primers, thereby amplifying OsZFP350 The CDS fragment, with its nucleotide sequence shown in SEQ ID NO:1, was inserted into the vector pEXT06 / g via homologous recombination. Hind III and Kpn Between the I sites, positive vectors were screened to obtain... OsZFP350 Overexpression vector.

[0031] 2. OsZFP350 Obtaining rice through overexpression The result obtained in step 1 OsZFP350 An overexpression vector was introduced into Agrobacterium strain EHA105. The Agrobacterium carrying the vector was used to infect rice callus tissue for co-culture. The callus tissue was then transferred to a selection medium containing the corresponding antibiotic, inducing differentiation and regeneration of resistant callus tissue into complete plants, thus obtaining... OsZFP350 Overexpression in rice.

[0032] 3. OsZFP qRT-PCR identification of overexpressing rice plants (1) Select rice wild-type plants (WT) with uniform growth and​ Overexpressing rice plants (OE) were used, and functional leaves were collected at the tillering stage.

[0033] (2) RNA extraction and reverse transcription Take 100-200 mg of fresh leaves and extract total RNA using the BayBiopure Magnetic Bead Total RNA Extraction Kit (Bayzol), controlling the RNA concentration at 200-1000 ng / μL; take 1-2 μg of RNA for reverse transcription to obtain cDNA.

[0034] (3) qRT-PCR identification Using cDNA obtained from reverse transcription as a template, PCR amplification was performed using ChamQ universal SYBR qPCR Master Mix (Vazyme, China) to obtain the amplification products. The total PCR amplification volume was 20 μL, and the final primer concentration was 0.2–0.4 μM. ​ The primers used as internal reference genes are as follows: ​ -F (SEQ ID NO:3):TACCCTGCTAGATCCTTGCTT; ​ -R (SEQ ID NO:4):CCATGTTCTCTACCAGCCGAC; ​ -F (SEQ ID NO:5):GAGTATGATGAGTCGGGTCCAG; ​ -R (SEQ ID NO:6): ACACCAACAATCCCAAACAGAG.

[0035] (4) Data Analysis Use 2 -ΔΔCt Method Calculation ​ Relative gene expression levels were measured, with three biological replicates per sample. Results are as follows: ​ As shown, compared to the wild type, ​ Overexpression in rice plants ​ The transcriptional level was significantly increased, with expression levels approximately 15–25 times higher than that of the wild type, and the difference was statistically significant. P <0.05 indicates successful acquisition. ​ Overexpression in rice plants.

[0036] Example 2 1. ​ Construction of knockout vector The sgRNA with the nucleotide sequence shown in SEQ ID NO:7 (TGGGCTGTAGGCGTGCTGCAGGG) was inserted into the backbone vector BGK03. ​ I site, screen for positive vectors, obtain ​ Knock out the carrier.

[0037] 2. ​ Obtaining mutant rice Referring to existing technology (Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed), this method utilizes Agrobacterium-mediated transformation of rice callus tissue obtained in step 1. ​ Knockout vector introduced into rice callus tissue to obtain ​ Mutant rice.

[0038] 3. ​ qRT-PCR identification of mutant rice plants (1) Select rice wild-type plants (WT) with uniform growth and ​ Functional leaves of mutant rice plants were collected at the tillering stage.

[0039] (2) Genomic DNA extraction Take 100-200 mg of fresh leaves and use the BayBiopure Magnetic Bead Bacterial Genomic DNA Extraction Kit (Bayzol) to perform the operation according to the kit instructions, controlling the final DNA concentration to be 50-200 ng / μL.

[0040] (3) PCR amplification Using extracted genomic DNA as a template, covering ​ PCR amplification was performed using primers specific to the coding region. The PCR product was purified and then sent for Sanger sequencing. The total PCR amplification volume was 50 μL, and the final primer concentration was 10 μM. The primers used were as follows: ​ -CDS-F (SEQ ID NO:8):GCAGCTCCACCTCCTACTAC; ​ -CDS-R (SEQ ID NO:9): CGGCGACTTCGATGAGACAA.

[0041] Sanger sequencing results are as follows ​ As shown, compared to the wild type, OsZFP350 ​The insertion of one A base in the coding region caused a frame shift in the reading frame, verifying the molecular level. ​ The authenticity of mutant rice plants.

[0042] Example 3 ​ Identification of resistance to sheath blight in overexpressing and mutant rice (leaf sheath inoculation method) 1. Experimental rice materials Wild-type rice plants (WT), obtained in Example 1 ​ Overexpression in rice plants ( ​ OE) and the results obtained in Example 2 ​ mutant rice plants ( ​ Each treatment had 8 plants.

[0043] 2. Test Methods (1) Culture of *Rhizoctonia solani* Activated Rhizoctonia solani Y-36 was inoculated in the center of PDA medium, and wood bark was placed radially around the mycelium. The medium was then cultured in the dark at 25-28°C until the mycelium covered the entire culture dish.

[0044] (2) Leaf sheath inoculation Take a piece of bark at the same distance from the center of the mushroom cake and place it inside the leaf sheath of the experimental rice material. Spray it with distilled water twice and wrap it with plastic wrap to maintain a high humidity environment.

[0045] (3) Investigation of the disease Seven days after inoculation, observe the lesions on the rice leaf sheaths, measure the length of the lesions, and calculate the average lesion length. The results are as follows: ​ As shown in Table 1.

[0046] Table 1. Length of lesions (cm) in rice after inoculation with Sheath blight pathogen.

[0047] according to ​ As shown in Table 1, wild-type rice develops obvious lesions after infection with Sheath blight pathogen. ​ The lesion length of the mutant plants was significantly longer than that of the wild-type control. ​ The lesion length of the overexpressing plants was significantly shorter than that of the wild type. ​ Overexpression of this substance can significantly improve the resistance of rice to sheath blight, while ​ Functional loss reduces rice's resistance to sheath blight.

[0048] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. OsZFP350 The application of genes in regulating rice sheath blight resistance, the aforementioned OsZFP350 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:2; The regulation is: overexpression OsZFP350 Genes enhance resistance to rice sheath blight.

2. Overexpression OsZFP350 The application of genes in rice breeding, wherein rice breeding includes the development of rice varieties resistant to sheath blight; OsZFP350 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

3. The application according to claim 1 or 2, characterized in that, The OsZFP350 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

4. The application according to claim 1 or 2, characterized in that, The aforementioned sheath blight is caused by infection with Rhizoctonia solani.

5. A method for improving resistance to rice sheath blight, characterized in that, The steps include: overexpression in rice receptors OsZFP350 Genes, the ones mentioned OsZFP350 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

6. The method according to claim 5, characterized in that, The rice receptors include rice callus tissue.

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