Application of OsMYB23 gene in improving resistance of rice to rice blast

Abstract

The application discloses application of an OsMYB23 gene in improving resistance of rice to rice smut fungus and belongs to the technical field of genetic engineering. The nucleotide sequence of the CDS region of the OsMYB23 gene is shown in SEQ ID NO. 2. The application finds that the OsMYB23 gene is related to the resistance of rice to rice smut, overexpression of the OsMYB23 gene significantly reduces the disease resistance of rice to the rice smut fungus, mutation of the OsMYB23 gene improves the defense capacity of rice to the rice smut, and the mutation of the OsMYB23 gene does not affect the normal growth of rice, so the rice OsMYB23 gene can be used to improve the resistance of rice to the rice smut fungus, and provides a theoretical basis for prevention and control of the rice smut fungus and the disease caused by the rice smut fungus.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the OsMYB23 gene in improving the resistance of rice to rice false smut. Background Technology

[0002] As one of the world's most important food crops, rice holds a pivotal position in global food production due to its excellent nutritional quality and wide ecological adaptability. However, the persistent infection by various pathogens seriously threatens its yield and quality. In recent years, rice false smut, caused by *Ustilaginoidea virens*, has shown a continuously worsening trend, characterized by specific parasitism on the panicle, and has become a significant fungal disease threatening rice yield and quality safety. The most prominent pathological feature of this disease is the formation of dark green spore balls (rice false spore balls) at the rice spikelet, accompanied by the production of various mycotoxins such as arbusculin and arbusculin. These toxic metabolites not only significantly inhibit normal grain development, leading to an increase in empty grains and a decrease in thousand-grain weight, causing severe yield losses, but also reduce commercial quality due to their residues in rice, posing a potential toxic risk to human and animal health, and becoming a significant biological stressor restricting modern green and safe rice production.

[0003] In actual production, spraying chemical agents remains a routine measure for controlling rice false smut in the field, but its inherent defects are becoming increasingly prominent: the application window period is difficult to precisely synchronize with the peak infection period of the pathogen, resulting in unstable control efficacy in the field; long-term use of single pesticides not only disrupts the ecological balance of farmland but also forces the pathogen population to undergo adaptive evolution, forming pesticide-resistant populations. In contrast, using disease-resistant genes to breed resistant new varieties through modern molecular design is a more ideal green control approach. This eco-friendly control strategy can ensure stable and high-quality yields and aligns with the macro-guidance of green agricultural transformation. In recent years, systematic research on the interaction mechanism between rice and rice false smut has made continuous breakthroughs, opening up new perspectives for analyzing the pathogenic process at the molecular level and laying important theoretical basis and practical guidance for more precise design of resistance improvement programs.

[0004] Some genes in rice may influence resistance to rice false smut. For example, invention publication CN116590311A discloses the application of the rice gene OsBCAT2 or its encoded protein, a method for improving rice resistance to rice false smut, a recombinant plasmid vector and its application, and a method for constructing rice varieties resistant to rice false smut. This invention, by silencing or knocking out the OsBCAT2 gene in the rice genome, can result in the OsBCAT2 gene being unexpressed or poorly expressed, significantly improving rice resistance to rice false smut, thereby enhancing the rice's ability to resist infection by the fungus, promoting healthy rice growth, and ultimately benefiting rice production. Therefore, the OsBCAT2 gene or its encoded protein can be used to breed rice varieties resistant to rice false smut. However, currently, there are relatively few genes known to resist rice false smut, and there is an urgent need to discover new genes resistant to the fungus. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the OsMYB23 gene in improving the resistance of rice to rice false smut, so as to solve the problems existing in the prior art. By silencing or knocking out the OsMYB23 gene, the resistance of rice to rice false smut can be significantly improved, providing a theoretical basis for the prevention and control of rice false smut and the diseases it causes.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of the OsMYB23 gene in the following (1) or (2): (1) Application in improving rice resistance to rice false smut; (2) Application in the cultivation of rice resistant to rice false smut; The nucleotide sequence of the CDS region of the OsMYB23 gene is shown in SEQ ID NO.2.

[0007] The present invention also provides the use of the protein encoded by the OsMYB23 gene in the following (1) or (2): (1) Application in improving rice resistance to rice false smut; (2) Application in the cultivation of rice resistant to rice false smut; The nucleotide sequence of the CDS region of the OsMYB23 gene is shown in SEQ ID NO.2.

[0008] The present invention also provides the use of the OsMYB23 gene knockout vector in the following (1) or (2): (1) Application in improving rice resistance to rice false smut; (2) Application in the cultivation of rice resistant to rice false smut; The knockout vector targets the OsMYB23 gene and is designed with a CRISPR / Cas9-based sgRNA sequence. The DNA fragment containing the sgRNA sequence is ligated into a vector carrying CRISPR / Cas. The nucleotide sequence of the CDS region of the OsMYB23 gene is shown in SEQ ID NO.2, and the sgRNA sequence is CTGCAATCAGCAAACAGGCG.

[0009] The present invention also provides the use of recombinant engineered bacteria containing the knockout vector in the following (1) or (2): (1) Application in improving rice resistance to rice false smut; (2) Application in the cultivation of rice resistant to rice false smut.

[0010] Preferably, the resistance of rice plants to rice false smut is improved by reducing the expression level of the OsMYB23 gene in rice plants.

[0011] The present invention also provides a method for improving the resistance of rice to rice false smut, comprising silencing or knocking out the OsMYB23 gene in rice to improve the resistance of said rice to rice false smut; wherein the nucleotide sequence of the CDS region of said OsMYB23 gene is shown in SEQ ID NO.2.

[0012] Preferably, the silencing or knockout method includes: using the OsMYB23 gene as a target, designing a CRISPR / Cas9-based sgRNA sequence, ligating a DNA fragment containing the encoding the sgRNA sequence into a CRISPR / Cas vector to construct a knockout vector, and then introducing the knockout vector into rice cells through callus transformation to cultivate the rice cells into plants.

[0013] The present invention also provides a method for breeding rice resistant to rice false smut, comprising silencing or knocking out the OsMYB23 gene in rice, reducing or inhibiting the expression of the rice OsMYB23 gene, and improving the resistance of the rice to rice false smut to obtain rice resistant to rice false smut; wherein the nucleotide sequence of the CDS region of the OsMYB23 gene is shown in SEQ ID NO.2.

[0014] Preferably, the silencing or knockout method includes: using the OsMYB23 gene as a target, designing a CRISPR / Cas9-based sgRNA sequence, ligating a DNA fragment containing the encoding the sgRNA sequence into a CRISPR / Cas vector to construct a knockout vector, and then introducing the knockout vector into rice cells through callus transformation to cultivate the rice cells into plants.

[0015] The present invention discloses the following beneficial effects: This invention reveals that the OsMYB23 gene is associated with rice false smut resistance. Mutations in the OsMYB23 gene enhance rice's defense against false smut, while overexpression of the OsMYB23 gene significantly reduces rice's resistance to the pathogen. Therefore, the rice OsMYB23 gene, as a negative regulator of plant immunity, is an ideal susceptibility gene that can be used to improve crop resistance. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The figures show the sequence and transcriptional levels of osmyb23 knockout positive plants and OE-OsMYB23 overexpressing positive plants; where A: target site location and sequence information in the construction of the OsMYB23 knockout vector; B: sequence alignment results between knockout positive plants and wild type; C: changes in OsMYB23 transcriptional levels in OE-OsMYB23 overexpressing plants. Figure 2 Figure 1 shows the results of rice false smut resistance identification in osmyb23 mutant and OE-OsMYB23 plants; where A: phenotypic diagram of osmyb23 mutant and OE-OsMYB23 plants after inoculation with rice false smut fungus; B: statistical results of susceptible spikelets in Figure A. Figure 3 Phenotypic diagrams of osmyb23 mutant and OE-OsMYB23 plants. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1 The amino acid sequence of the rice pathogenic factor OsMYB23 protein is shown in SEQ ID NO.1; the nucleotide sequence of the CDS region of the OsMYB23 gene is shown in SEQ ID NO.2; and the genomic DNA sequence of rice OsMYB23 is shown in SEQ ID NO.3, which includes non-coding regions such as introns.

[0024] (1) In order to elucidate the biological function of the OsMYB23 gene in rice growth and development and rice false smut resistance, this invention constructed a rice OsMYB23 gene knockout mutant. The knockout mutant was constructed according to the following method: First, according to the gene number of OsMYB23 (LOC_Os02g17190), the reference sequence of the OsMYB23 gene in Nipponbare was downloaded from the MSU website. Then, the CRISPR-P website was used to screen for suitable knockout target sites of the OsMYB23 gene, and the target site detection primer OsMYB23-check-F / R was designed. The genomic DNA of japonica rice TP309 (Taipei 309) was used as a template for PCR amplification. Finally, the amplified target band was recovered, purified and sequenced to confirm that the target site sequence was not changed in TP309. Subsequently, the target gene was knocked out using CRISPR / Cas9 gene editing technology. A specific sgRNA target site (5'-CTGCAATCAGCAAACAGGCG-3') was designed targeting the coding region of the OsMYB23 gene, and an OsMYB23 gene knockout vector was constructed by transfecting it into the pYLCRISPR / Cas9-MH vector. Finally, the osmyb23 knockout mutant was obtained by transforming it into TP309 plants.

[0025] The primer sequences are as follows: OsMYB23-check-F: 5'-CCCTAGTTGTGGCAATAAAT-3' (SEQ ID NO.4); OsMYB23-check-R: 5'-AAGTGTGTTAGATGCATGCTG-3' (SEQ ID NO.5); MH-OsMYB23-F: 5'-ggcaCTGCAATCAGCAAACAGGCG-3' (SEQ ID NO.6); MH-OsMYB23-R: 5'-aaacCGCCTGTTTTGCTGATTGCAG-3' (SEQ ID NO. 7).

[0026] Detailed steps for constructing the OsMYB23 gene knockout mutant: ① Target adapter preparation: Annealing of 1 μM MH-OsMYB23-F / R primers by incubation at 90℃ for 30 s.

[0027] ② Perform gRNA expression cassette ligation reaction: The reaction system is shown in Table 1.

[0028] Table 1 Connection Reaction System ③ First round of amplification: gRNA expression cassette amplification was performed using UF / gRNA-R primers.

[0029] The primer sequences are as follows: UF: 5'-CTCCGTTTTACCTGTGGAATCG-3' (SEQ ID NO.8); gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3' (SEQ ID NO. 9).

[0030] The system is shown in Table 2.

[0031] Table 2 First-round amplification system The PCR reaction conditions were as follows: 95℃ pre-denaturation for 1 min; 95℃ denaturation for 10 s, 60℃ annealing for 15 s, 68℃ extension for 20 s, for 10 cycles; 95℃ denaturation for 10 s, 60℃ annealing for 15 s, 68℃ extension for 30 s, for 20 cycles.

[0032] ④ Second round of amplification: Amplify gRNA expression cassettes at specific locations using B1' and BL primers.

[0033] The primer sequences are as follows: B1': 5'-TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG-3' (SEQ ID NO. 10); BL: 5'-AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC-3' (SEQ ID NO. 11).

[0034] The system is shown in Table 3.

[0035] Table 3 Second-round amplification system The PCR reaction conditions were as follows: 95℃ pre-denaturation for 1 min; 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 68℃ extension for 30 s, for a total of 25 cycles; and 68℃ extension for 5 min.

[0036] ⑤ Simultaneous digestion and ligation: Mix the second-round amplification product from step ④ with an equal volume of the pYLCRISPR / Cas9-MH vector, and then perform simultaneous digestion and ligation using T4 ligase. The reaction system is shown in Table 4, taking one target site as an example: Table 4 Enzyme digestion system After reacting at 37℃ for 10 min, add 1.6 μL of 10× DNA ligase buffer and 1.6 μL of T4 DNA ligase.

[0037] The final reaction program was: 37℃ for 5 min, 10℃ for 5 min, 20℃ for 5 min, for a total of 15 cycles.

[0038] ⑥ E. coli DH5α transformation and sequencing verification: After the recombinant vector was verified to be correct by sequencing, Wuhan Boyuan Biotechnology Co., Ltd. was commissioned to carry out rice genetic transformation. The CRISPR / Cas9 editing vector was introduced into the callus tissue of TP309 variety through Agrobacterium-mediated transformation. After infection, co-culture and antibiotic resistance screening, the osmyb23 gene knockout mutant was finally obtained.

[0039] (2) Construction of OE-OsMYB23 overexpression plants: In order to verify the biological significance of OsMYB23 in rice development and pathogenicity, the OsMYB23 gene was overexpressed in TP309 background material.

[0040] The primer sequences are as follows: OE-OsMYB23-F:5'-tacttctgcactaggtaccATGATTAACATATATGGGCATAACAACTG-3' (SEQ ID NO. 12); OE-OsMYB23-R:5'-tcttagaattcccggggatccTCATATGAAATCACCATCGTATGGG-3' (SEQ ID NO. 13).

[0041] The CDS sequence of OsMYB23 was amplified using primers OE-OsMYB23-F / R and cloned into the pCAMBIA1390 vector (KpnI / BamHI double restriction sites) using homologous recombination ligase. After successful sequencing, the sample was sent to Wuhan Boyuan Biotechnology Co., Ltd. for rice genetic transformation. The overexpression vector was introduced into the callus tissue of TP309 cultivar using Agrobacterium-mediated transformation. After infection, co-culture, and antibiotic resistance screening, transgenic plants with high expression of the OsMYB23 gene were obtained.

[0042] Example 2: Identification of OsMYB23 gene knockout mutants and overexpression plants The detection primer OsMYB23-check-F / R was used to target the site in wild-type TP309 and transgenic knockout plants, respectively. Figure 1 PCR amplification was performed on the mutants (as shown in Figure A). After agarose gel electrophoresis confirmed that the band size met expectations, sequencing and sequence alignment analysis were conducted to obtain different types of knockout mutants. The results are as follows: Figure 1 In section B, osmyb23-3 and osmyb23-7 mutant plants were selected for further research.

[0043] Subsequently, quantitative primers qOsMYB23-F / R were selected using NCBI BLAST and analyzed by real-time quantitative PCR. Primer qOsUBQ-F / R was used to amplify the rice internal reference gene Ubiquitin, and the results are as follows: Figure 1 In the middle C, OE-OsMYB23-6 and OE-OsMYB23-9 overexpressing plants were selected for further research.

[0044] qOsMYB23-F:AACTGGGAAACTGTTGCACG (SEQ ID NO. 14); qOsMYB23-R: CCTGTTTGCTGATTGCAGGG (SEQ ID NO. 15); qOsUBQ-F: AAGAAGCTGAAGCATCCAGC (SEQ ID NO. 16); qOsUBQ-R: CCAGGACAAGATGATCTGCC (SEQ ID NO. 17).

[0045] Example 3: Effect of OsMYB23 on the pathogenicity of rice false smut. The artificial inoculation method for *Strombus oryzae* is as follows: After activating *Strombus oryzae* strain JS60-2, incubate it for 7 days at 28℃ and 180 rpm using PS liquid potato medium (200 g of potatoes cut into small pieces, boiled in distilled water until softened, filtered through gauze, and the supernatant is reserved; add 20 g of sucrose). Remove the cultured *Strombus oryzae* inoculum and blend it in a small blender. Adjust the number of conidia in the mixture of *Strombus oryzae* mycelia and spores to 1×10⁻⁶ using fresh PS liquid potato medium. 6 Prepare a solution of mycelium / mL for later use. Select plants from the field 5-7 days before the rice panicles break open, attach tags, and carefully dig them up by the roots and transplant them into the greenhouse. Inject the mycelium and spore solution into the rice panicles using a syringe until the spore solution overflows. After injection, attach a label with the name and date to the flag leaf of the panicle for easy recording and observation. Shade and moisture are required for the first 2 days after inoculation. After inoculation, cover the greenhouse roof with the shade curtain to maintain a room temperature of 22℃, and spray water for about 20 minutes every 3 hours. After 2 days, open the shade curtain and adjust the room temperature to 28℃, spraying water for about 15 minutes every 4 hours, with 12 hours of light and 12 hours of darkness during this period. After 25-30 days of inoculation, investigate the disease status of the rice panicles by counting the number of rice blasts.

[0046] The results are as follows Figure 2As shown, the number of rice false smut balls was counted 30 days after inoculation. The results showed that compared with the wild type, the number of rice false smut balls formed by the osmyb23 mutant plants was reduced, and the defense ability against rice false smut fungus was significantly increased. On the other hand, the OE-OsMYB23 overexpression plants formed more rice false smut balls than the wild type, indicating that OsMYB23 negatively regulates the resistance of rice to rice false smut.

[0047] Example 4: Growth phenotype of OsMYB23 transgenic plants When the plants reached the yellow ripening stage in the field, representative wild-type TP309, osmyb23 mutant plants, and OE-OsMYB23 overexpressing plants were selected, and their growth phenotypes were photographed for observation. For example... Figure 3 As shown, the OsMYB23 transgenic plants showed no significant difference in growth compared to the wild type, indicating that knocking out OsMYB23 can improve the resistance of rice to rice false smut without affecting rice growth, further demonstrating that it can be applied to rice disease resistance breeding.

[0048] The amino acid and nucleotide sequences involved in this invention are as follows: SEQ ID NO.1: MINIYGHNNWETVARAIPGRNAQQCRVRWKFNLDPAISKQAWSREEELRLIHVQQIFGNKWCRMAEHFTGRTSAAIKEHWRGPMKRKLNSYLASGLLKKSPGLPENLSVSQSSDSNI LQQCDVSSDENKLLSDLRPSLKSKEGVSSKCDGSSSDNQLLSDLRASLKSKGASSKCDGSSGDNQLLSDLRASLKSKGASSKCDGSSGDNQLLSDLRASLKSKGASSKCDGSSDENKL LSDLRACLKSKQGASSKCDGSSGENQLLSDLQASLMSKQRTSSKYKQGASSKYKQGASSKSKQGTSSKSKQGFIDLCENTDTSEGESSELICTERPDPDSGEASQRIRDRLNWRKRA RKKLVFLSSPVELKVSAVAESERHLQESKEMSPEVNIVSPPAVLQGFSPEVPSVCEKIVEPPLADFNQAKNVCSLETSSDPCTLEQHLANVSDLLDMSYIDGLMIIPPSGCPYDGDFI .

[0049] SEQ ID NO.2:

[0050] SEQ ID NO.3:

[0051] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of silencing or knocking out the OsMYB23 gene in the following (1) or (2): (1) Application in improving rice resistance to rice false smut; (2) Application in the cultivation of rice resistant to rice false smut; in, The nucleotide sequence of the CDS region of the OsMYB23 gene is shown in SEQ ID NO.

2.

2. Application of OsMYB23 gene knockout vector in the following (1) or (2): (1) Application in improving rice resistance to rice false smut; (2) Application in the cultivation of rice resistant to rice false smut; in, The knockout vector targets the OsMYB23 gene and is designed with a CRISPR / Cas9-based sgRNA sequence. The DNA fragment containing the encoding the sgRNA sequence is ligated into a vector carrying CRISPR / Cas. The nucleotide sequence of the CDS region of the OsMYB23 gene is shown in SEQ ID NO.2, and the sgRNA sequence is CTGCAATCAGCAAACAGGCG.

3. The use of the recombinant engineered bacteria containing the knockout vector of claim 2 in either (1) or (2): (1) Application in improving rice resistance to rice false smut; (2) Application in the cultivation of rice resistant to rice false smut.

4. A method for improving the resistance of rice to rice false smut, characterized in that, This includes silencing or knocking out the OsMYB23 gene in rice to improve the rice's resistance to rice false smut; wherein the nucleotide sequence of the CDS region of the OsMYB23 gene is shown in SEQ ID NO.

2.

5. The method as described in claim 4, characterized in that, The silencing or knockout method includes: using the OsMYB23 gene as a target, designing a CRISPR / Cas9-based sgRNA sequence, ligating a DNA fragment encoding the sgRNA sequence into a CRISPR / Cas vector to construct a knockout vector, and then introducing the knockout vector into rice cells through callus transformation to cultivate the rice cells into plants; the sgRNA sequence is CTGCAATCAGCAAACAGGCG.

6. A method for cultivating rice resistant to rice false smut, characterized in that, The method includes silencing or knocking out the OsMYB23 gene in rice, reducing or inhibiting the expression of the OsMYB23 gene in rice, and improving the resistance of rice to rice false smut, so as to obtain rice resistant to rice false smut; wherein the nucleotide sequence of the CDS region of the OsMYB23 gene is shown in SEQ ID NO.

2.

7. The method as described in claim 6, characterized in that, The silencing or knockout method includes: using the OsMYB23 gene as a target, designing a CRISPR / Cas9-based sgRNA sequence, ligating a DNA fragment encoding the sgRNA sequence into a CRISPR / Cas vector to construct a knockout vector, and then introducing the knockout vector into rice cells through callus transformation to cultivate the rice cells into plants; the sgRNA sequence is CTGCAATCAGCAAACAGGCG.

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