OsRbohB gene, OsRbohB gene mutant and use thereof

Abstract

The present invention relates to the technical field of plant genetic engineering, and in particular to an OsRbohB gene, an OsRbohB gene mutant and an application thereof. The nucleotide sequence of the OsRbohB gene provided by the present invention is shown in SEQ ID No.1, and the OsRbohB gene mutant is obtained by performing ubiquitination modification or phosphorylation modification site mutation on the OsRbohB gene, and its nucleotide sequence is shown in SEQ ID No.3 and SEQ ID No.5. The specific embodiment of the present invention confirms that overexpression of the OsRbohB gene and its mutants will improve the resistance of rice to rice blast, and the mutants obtained by the ubiquitination modification and phosphorylation modification site base mutation of the OsRbohB gene show a higher resistance level. The OsRbohB gene and its mutants can be introduced into other rice varieties as target genes to improve the disease resistance of rice, so as to improve rice varieties.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, in particular to an OsRbohB gene, an OsRbohB gene mutant and applications thereof. Background Art

[0002] Crops are affected by a variety of pathogens during the production process, which will eventually lead to crop yield reduction, seriously threatening food security. Since crop varieties with broad-spectrum resistance (BSR) are resistant to multiple pathogen families or multiple pathogen species, they are more capable of resisting pathogen infection.

[0003] Reactive oxygen species (ROS) play a key role in cellular signaling during plant-microbe interactions. In plants, NADPH oxidases belong to the respiratory burst oxidase homolog (RBOH) family and are responsible for ROS production. ROS production is a conserved signaling mechanism during plant growth, development, and immunity. Pathogen-triggered immunity (PTI) and effector-triggered immunity (ETI) are initiated through different mechanisms and involve different early signaling cascades. Recent studies have shown that Rbohs play a key regulatory role in both PTI and ETI. Knockdown or knockout of Rbohs in plants has been found to suppress ROS burst and immune responses, indicating that Rbohs actively regulate ROS production and plant immunity. However, it is still unclear whether upregulation of RBOH can enhance ROS production and plant immunity.

[0004] Previous studies have shown that phosphorylation plays an important role in regulating the activation mechanism of RBOHDs such as EFR and FLS2, and that the phosphorylated RLCK (receptor-like cytoplasmic kinase) BIK1 directly interacts with and phosphorylates specific residues of RBOHD, which is essential for RBOHD activation. 2+The kinase-dependent protein kinase and MAP4 kinase SIK1 also contribute to the full activation of RBOHD through phosphorylation. Plant RBOHD can also be regulated by CYSTEINE-RICH RLK2 (CRK2) to control ROS production. Maintaining ROS homeostasis is essential for plant cells. Rbohs are tightly controlled by multiple regulatory mechanisms. For example, through the vacuolar degradation pathway, AvrPphB-sensitive 1-like 13 (PBL13) and PBL13-interacting RING domain E3 ubiquitin ligase (PIRE) negatively regulate RBOHD abundance. In rice, OsRLCK118 / 176 phosphorylates OsRbohB, an ortholog of Arabidopsis RBOHD, to stimulate ROS production. The E3 ligase OsATL32 inhibits pathogen-induced ROS accumulation by mediating ubiquitination and degradation of the ROS-producing OsRac5-OsRbohB module. However, the precise regulatory mechanisms controlling Rbohs-mediated ROS production in rice, especially the down-regulation mechanism of Rbohs, remain unclear.

[0005] Phosphorylation and ubiquitination are two important modifications of proteins after translation. In recent years, several molecular and genetic studies have shown that there is a crosstalk between the two in signal transduction during plant immunity. This interaction between phosphorylation and ubiquitination occurs at different levels. Overall, phosphorylation and ubiquitination play a key role in rapidly reprogramming cells to obtain defense signals and regulate responses, thereby achieving cellular homeostasis at all levels of plant immune responses. However, there are few studies on the NADPH oxidase gene in rice and its post-translational modifications. At the same time, there are no reports on the biological functions of the NADPH oxidase gene in rice and NADPH ubiquitination and phosphorylation modifications on pathogenic fungi. Summary of the invention

[0006] The purpose of the present invention is to provide an OsRbohB gene, an OsRbohB gene mutant and their application to solve the problems existing in the above-mentioned prior art. The resistance of rice to rice blast can be significantly improved by overexpressing the OsRbohB gene and the OsRbohB gene mutant provided by the present invention.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention is the first to discover that the OsRbohB gene (NADPH oxidase gene OsRbohB) is related to rice blast resistance in rice. The results of the specific embodiments of the present invention confirm that overexpression of the OsRbohB gene improves rice resistance to rice blast fungi. Therefore, the gene can be used as a new disease resistance gene tool to be introduced into other rice varieties for variety improvement to improve rice blast resistance. In summary, the gene can be used to improve rice blast resistance, cultivate rice varieties with high resistance to rice blast, improve rice varieties, and rice breeding, providing a new approach for rice breeding.

[0009] The present invention provides the use of the OsRbohB gene or the OsRbohB protein encoded by it in any of the following items:

[0010] (1) Application in improving rice blast resistance;

[0011] (2) Application in breeding rice varieties with high resistance to rice blast;

[0012] (3) Application in breeding improved rice varieties with high resistance to rice blast;

[0013] (4) Application in rice breeding;

[0014] The nucleotide sequence of the OsRbohB gene is shown in SEQ ID No.1; the amino acid sequence of the OsRbohB protein is shown in SEQ ID No.2.

[0015] Preferably, the improvement of rice blast resistance is achieved by overexpressing the OsRbohB gene in rice.

[0016] Further preferably, the present invention also provides the use of a vector containing the OsRbohB gene in any of the following:

[0017] (1) Application in improving rice blast resistance;

[0018] (2) Application in breeding rice varieties with high resistance to rice blast;

[0019] (3) Application in breeding improved rice varieties with high resistance to rice blast;

[0020] (4) Application in rice breeding;

[0021] The nucleotide sequence of the OsRbohB gene is shown in SEQ ID No.1.

[0022] Preferably, the improvement of rice blast resistance is achieved by overexpressing the OsRbohB gene in rice.

[0023] The present invention provides a method for improving the resistance of rice to rice blast, comprising the steps of overexpressing the OsRbohB gene in rice to improve the resistance of the rice to rice blast;

[0024] The nucleotide sequence of the OsRbohB gene is shown in SEQ ID No.1.

[0025] The present invention provides a method for cultivating transgenic rice with high resistance to rice blast, comprising the steps of overexpressing the OsRbohB gene in rice to obtain the transgenic rice;

[0026] The nucleotide sequence of the OsRbohB gene is shown in SEQ ID No.1.

[0027] The present invention provides an OsRbohB gene mutant (named as OsRbohB K266R and OsRbohB S2A ), the nucleotide sequence of the OsRbohB gene mutant is as shown in SEQ ID No.3 (OsRbohB K266R ) or SEQ ID No.5 (OsRbohB S2A ) as shown.

[0028] Further preferably, the OsRbohB K266R It is obtained by mutagenesis of the ubiquitination modification site of the OsRbohB gene (site-directed mutagenesis), that is, the 797th base of the nucleotide sequence shown in SEQ ID No. 1 is mutated from A to G;

[0029] The OsRbohB S2A The phosphorylation modification site mutation of the OsRbohB gene was obtained by mutating the nucleotides at positions 964-965 and 976-977 of the nucleotide sequence shown in SEQ ID No. 1 from AG to GC, respectively.

[0030] The experiment verified that overexpression of the OsRbohB gene mutant can significantly improve rice resistance to rice blast. It can be seen that the OsRbohB gene mutant can also be used to improve rice resistance to rice blast, cultivate rice varieties with high resistance to rice blast, improve rice varieties and rice breeding, providing a new approach for rice breeding.

[0031] The present invention provides a protein encoded by the above-mentioned OsRbohB gene mutant, the amino acid sequence of the protein is shown as SEQ ID No.4 or SEQ ID No.6.

[0032] The present invention provides the use of the above-mentioned OsRbohB gene mutant or the above-mentioned protein in any of the following items:

[0033] (1) Application in improving rice blast resistance;

[0034] (2) Application in breeding rice varieties with high resistance to rice blast;

[0035] (3) Application in breeding improved rice varieties with high resistance to rice blast;

[0036] (4) Application in rice breeding.

[0037] Preferably, the improvement of rice blast resistance is achieved by overexpressing an OsRbohB gene mutant in rice.

[0038] Further preferably, the present invention also provides use of a vector containing the OsRbohB gene mutant in any of the following:

[0039] (1) Application in improving rice blast resistance;

[0040] (2) Application in breeding rice varieties with high resistance to rice blast;

[0041] (3) Application in breeding improved rice varieties with high resistance to rice blast;

[0042] (4) Application in rice breeding;

[0043] The nucleotide sequence of the OsRbohB gene mutant is shown in SEQ ID No.3 or SEQ ID No.5.

[0044] Preferably, the improvement of rice blast resistance is achieved by overexpressing an OsRbohB gene mutant in rice.

[0045] The present invention provides a method for improving the resistance of rice to rice blast, comprising the steps of overexpressing an OsRbohB gene mutant in rice to improve the resistance of the rice to rice blast;

[0046] The nucleotide sequence of the OsRbohB gene mutant is shown in SEQ ID No.3 or SEQ ID No.5.

[0047] The present invention provides a method for cultivating transgenic rice with high resistance to rice blast, comprising the steps of overexpressing an OsRbohB gene mutant in rice to obtain the transgenic rice;

[0048] The nucleotide sequence of the OsRbohB gene mutant is shown in SEQ ID No.3 or SEQ ID No.5.

[0049] The present invention discloses the following technical effects:

[0050] The present invention is the first to discover that the OsRbohB gene (NADPH oxidase gene OsRbohB) is related to rice blast resistance. The specific embodiment of the present invention verifies its function by creating a rice NADPH oxidase gene OsRbohB overexpressing transgenic plant. The experimental results show that overexpression of the NADPH oxidase gene OsRbohB can indeed significantly improve the resistance of rice to rice blast fungi. Therefore, the gene can be used as a new disease-resistant gene tool to be introduced into other rice varieties for variety improvement and to improve rice blast resistance. In summary, the gene can be used to improve rice blast resistance, cultivate rice varieties with high resistance to rice blast, and in rice breeding, providing a new approach for rice breeding. At the same time, the present invention provides a new application of the NADPH oxidase gene OsRbohB, which can bring inspiration to other crops.

[0051] The present invention also provides an OsRbohB gene mutant (OsRbohB K266R and OsRbohB S2A ) sequence and its application in improving rice blast resistance. In a specific embodiment of the present invention, the OsRbohB introduced into rice K266R It can protect its own proteins from degradation by inhibiting the degradation of rice 26S proteasome, effectively improving the defense ability of rice; OsRbohB transferred into rice S2A It can stabilize the NADPH oxidase gene OsRbohB by inhibiting the phosphorylation state of the NADPH oxidase gene OsRbohB; therefore, the OsRbohB gene mutant (OsRbohB K266R and OsRbohB S2A ) can also achieve the effect of improving rice blast disease, and overexpression of rice NADPH oxidase gene OsRbohB post-translational modification site mutants in monocotyledonous plants such as corn and wheat may also improve the disease resistance of the crop. It can be seen that the OsRbohB gene mutant can also be used to improve rice blast resistance, cultivate rice varieties with high resistance to rice blast disease, improve rice varieties and rice breeding, providing a new approach for rice breeding.

[0052] Furthermore, the specific examples of the present invention confirm that overexpression of the OsRbohB gene improves the resistance of rice to rice blast, and mutants obtained by mutating the bases at the ubiquitination and phosphorylation modification sites of the OsRbohB gene exhibit a higher level of resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0054] Figure 1 This is the map of the pRHV-cHA expression vector;

[0055] Figure 2 The relative expression level of OsRbohB and overexpression transgenic plants is shown in the figure; WT is the wild type, OV-OsRbohB, OV-OsRbohB S2A ,OV-OsRbohB S2D and OV-OsRbohB K266R For overexpressing transgenic plants;

[0056] Figure 3 OV-OsRbohB transgenic rice, OV-OsRbohB S2A Transgenic rice, OV-OsRbohB K266R The lesion phenotype of transgenic rice and WT rice after infection with rice blast fungus race RB22; WT is wild-type rice ZH11;

[0057] Figure 4 OV-OsRbohB transgenic rice, OV-OsRbohB S2A Transgenic rice, OV-OsRbohB K266R Statistical graph of fungal biomass in the lesion area of ​​transgenic rice and WT rice; WT is the wild-type rice ZH11. DETAILED DESCRIPTION

[0058] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0059] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0060] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0061] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0062] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0063] Example 1 Creation of rice overexpression transgenic plants

[0064] 1) Extraction of total RNA

[0065] The japonica rice variety Nipponbare was selected, and the rice seedlings were grown to about two weeks old. Some leaves were taken, frozen with liquid nitrogen, crushed with a mortar, and transferred into a 1.5 mL EP tube containing Trizol lysis solution. After sufficient shaking, total RNA was extracted and the quality of total RNA was identified by electrophoresis.

[0066] 2) Cloning of rice NADPH oxidase gene OsRbohB and its mutant and construction of plant expression vector

[0067] Design of primers at both ends: This experiment uses homologous recombination to construct vectors. The design of primers for forward amplification of inserted fragments is: 5'--homologous sequence at the end of upstream vector + restriction site + gene-specific forward amplification primer sequence--3'; the design of primers for reverse amplification of inserted fragments is: 5'--homologous sequence at the end of downstream vector + restriction site + gene-specific reverse amplification primer sequence--3';

[0068] Design of point mutation primers: Point mutation is achieved by bridging. The forward primer contains 12bp sequences before and after the mutation, and the reverse primer is the reverse complementary sequence of the forward primer. The specific primers are as follows:

[0069] P1: 5'-GGTGAGCTCGGTACCATGGCTGACCTGGAAGCAGG-3' (SEQ ID No. 7);

[0070] P2: 5'-AGCGCCGCACTAGTGAAGTTCTCCTTGTGGAAAT-3' (SEQ ID No. 8);

[0071] P3: 5'-GCATCAGCAACAGACTTTCCAAGATC-3' (SEQ ID No. 9);

[0072] P4: 5'-GATCTTGGAAAGTCTGTTTGCTGATGC-3' (SEQ ID No. 10);

[0073] P5: 5'-TCCAAACTTGCCAAAGCTCTTGCCATGAAGCTTGCG-3' (SEQ ID No. 11);

[0074] P6: 5'-CGCAAGCTTCATGGCAAGAGCTTTGGCAAGTTTGGA-3' (SEQ ID No. 12);

[0075] P7: 5'-TCCAAACTTGACAAAGCTCTTGACATGAAGCTTGCG-3' (SEQ ID No. 13);

[0076] P8: 5'-CGCAAGCTTCATGTCAAGAGCTTTGTCAAGTTTGGA-3' (SEQ ID No. 14).

[0077] The total RNA obtained in step 1) was reverse transcribed to synthesize the first-chain cDNA, which was used as a template for PCR amplification using high-fidelity KOD enzyme, with primers P1+P2, and the PCR program is shown in Table 1.

[0078] Table 1 PCR amplification procedure

[0079]

[0080] The target fragment is recovered. The nucleotide sequence of the amplified fragment (OsRbohB) of the NADPH oxidase gene OsRbohB is shown in SEQ ID No.1, and the amino acid sequence of the encoded OsRbohB protein is shown in SEQ ID No.2.

[0081]

[0082] SEQ ID No.2:MADLEAGMVAAADTQGNSTRSQDDAATLIPNSGNLGSSNRSTKTARFK *。

[0083] The total RNA obtained in step 1) was reverse transcribed to synthesize the first strand of cDNA, which was used as a template for PCR amplification using high-fidelity KOD enzyme. The amplification steps were divided into two steps: the first step template was the same as above, primers P1+P4 amplified fragment 1, and primers P2+P3 amplified fragment 2; the second step template was fragment 1 and fragment 2 obtained in the first step, primers P1+P2, and the PCR program was shown in Table 2.

[0084] Table 2 PCR amplification program

[0085]

[0086] Recover the target fragment. The amplified fragment of the NADPH oxidase gene OsRbohB point mutation (OsRbohB K266R ) The nucleotide sequence is shown in SEQ ID No.3, and the amino acid sequence of the protein encoded by it is shown in SEQ ID No.4.

[0087] SEQ ID No.3:ATGGCTGACCTGGAAGCAGGCATGGTTGCTGCTGCCACAGACCA GGGCAATTCAACAAGGTCACAAGATGACGCAGCCACACTGATCCCGAACAGTGGCAATCTGGGCTCGAGCAACAGGAGCACCAAGACGGCCAGGTTCAAGGACGACGACGAGCTGGTCGAGATCACCCTCGACGTGCAGCGCGATTCGGTGGCAATCCAAGAAGTGAGAGGGGTGGATGAGGGTGGCTCCGGGCACGGTACCGGGTTCGACGGCCTGCCACTGGTGTCACCCTCGTCGAAGAGCGGAAAGCTGACGTCAAAGCTCAGGCAGGTGACCAATGGGCTCAAGATGAAGAGCTCCAGCAGGAAGGCGCCATCCCCGCAGGCGCAGCAGTCTGCGAAGAGGGTGAGGAAGAGGCTGGACAGGACCAAGAGCAGCGCCGCCGTGGCGCTCAAAGGATTGCAGTTTGTGACTGCAAAGGTTGGCAATGACGGCTGGGCCGCGGTGGAGAAGCGGTTCAATCAGCTGCAGGTGGATGGTGTGCTGCTCCGTTCAAGATTTGGGAAATGCATTGGAATGGATGGGTCCGACGAGTTTGCGGTGCAAATGTTCGATTCTCTGGCGAGGAAGAGAGGGATAGTGAAGCAGGTGCTCACTAAGGACGAGCTCAAAGATTTCTATGAGCAATTGACTGATCAGGGGTTTGACAATCGTCTTCGGACATTCTTTGACATGGTTGACAAGAACGCTGATGGAAGGCTCACAGCAGAAGAGGTTAAGGAGATTATTGCCCTTAGTGCATCAGCAAACA G

[0088] SEQ ID No.4: MADLEAGMVAAATDQGNSTRSQDDAATLIPNSGNLGSSNRSTKTARFKDDDELVEITLDVQRDSVAIQEVRGVDEGGSGHGTGFDGLPLVSPSSKSGKLTSKLRQVTNGLKMKSSSRKAPSPQAQQSAKRVRKRLDRTKSSAAVALKGLQFVTAKVGNDGWAAVEKRFNQLQVDGVLLRSRFGKCIGMDGSDEFAVQMFDSLARKRGIVKQVLTKDELKDFYEQLTDQGFDNRLRTFFDMVDKNADGRLTAEEVKEIIALSASAN R LSKIKERADEYTALIMEELDPTNLGYIEMEDLEALLLQSPSEAAARSTTTHSSKLSKALSMKLASNKEMSPVRHYWQQFMYFLEENWKRSWVMTLWISICIALFIWKFIQYRNRAVFGIMGYCVTTAKGAAETLKFNMALVLLPVCRNTITWIRSKTQVGAVVPFNDNINFHKVIAAGVAVGVALHAGAHLTCDFPRLLHASDAQYELMKPFFGEKRPPNYWWFVKGTEGWTGVVMVVLMAIAFTLAQPWFRRNKLKDSNPLKKMTGFNAFWFTHHLFVIVYTLLFVHGTCLYLSRKWYKKTTWMYLAVPVVLYVSERILRLFRSHDAVGIQKVAVYPGNVLALYMSKPPGFRYRSGQYIFIKCTAVSPYEWHPFSITSAPGDDYLSVHIRTRGDWTSRLRTVFSEACRPPTEGESGLLRADLSKGITDEKARFPKLLVDGPYGAPAQDYREYDVLLLIGLGIGATPLISIVKDVLNHIQGEGSVGTTEPESSSKAKKKPFMTKRAYFYWVTREEGSFEWFRGVMNEVSEKDKDGVIELHNHCSSVYQEGDARSALIVMLQELQHAKKGVDILSGTSVKTHFARPNWRSVFKKVAVSHENQRVGVFYCGEPVLVPQLRQLSADFTHKTNTRFDFHKENF*; Among them, the bold and underlined part is the mutation site.

[0089] The total RNA obtained in step 1) was reverse transcribed to synthesize the first strand of cDNA, which was used as a template for PCR amplification using high-fidelity KOD enzyme. The amplification steps were divided into two steps: the first step template was the same as above, primers P1+P6 amplified fragment 1, and primers P2+P5 amplified fragment 2; the second step template was fragment 1 and fragment 2 obtained in the first step, primers P1+P2, and the PCR program was shown in Table 3.

[0090] Table 3 PCR amplification program

[0091]

[0092] Recover the target fragment. The amplified fragment of the NADPH oxidase gene OsRbohB point mutation (OsRbohB S2A ) The nucleotide sequence is shown in SEQ ID No.5, and the amino acid sequence of the protein encoded by it is shown in SEQ ID No.6.

[0093] SEQ ID No.5:ATGGCTGACCTGGAAGCAGGCATGGTTGCTGCTGCCACAGACCAG GGCAATTCAACAAGGTCACAAGATGACGCAGCCACACTGATCCCGAACAGTGGCAATCTGGGCTCGAGCAACAGGAGCACCAAGACGGCCAGGTTCAAGGACGACGACGAGCTGGTCGAGATCACCCTCGACGTGCAGCGCGATTCGGTGGCAATCCAAGAAGTGAGAGGGGTGGATGAGGGTGGCTCCGGGCACGGTACCGGGTTCGACGGCCTGCCACTGGTGTCACCCTCGTCGAAGAGCGGAAAGCTGACGTCAAAGCTCAGGCAGGTGACCAATGGGCTCAAGATGAAGAGCTCCAGCAGGAAGGCGCCATCCCCGCAGGCGCAGCAGTCTGCGAAGAGGGTGAGGAAGAGGCTGGACAGGACCAAGAGCAGCGCCGCCGTGGCGCTCAAAGGATTGCAGTTTGTGACTGCAAAGGTTGGCAATGACGGCTGGGCCGCGGTGGAGAAGCGGTTCAATCAGCTGCAGGTGGATGGTGTGCTGCTCCGTTCAAGATTTGGGAAATGCATTGGAATGGATGGGTCCGACGAGTTTGCGGTGCAAATGTTCGATTCTCTGGCGAGGAAGAGAGGGATAGTGAAGCAGGTGCTCACTAAGGACGAGCTCAAAGATTTCTATGAGCAATTGACTGATCAGGGGTTTGACAATCGTCTTCGGACATTCTTTGACATGGTTGACAAGAACGCTGATGGAAGGCTCACAGCAGAAGAGGTTAAGGAGATTATTGCCCTTAGTGCATCAGCAAACAGACTTTCCAAGATCAAGGAGCGAGCTGATGAGTACACAGCACTCATTATGGAAGAGCTTGACCCTACAAACTTGGGATACATCGAGATGGAGGACTTGGAAGCACTATTGCTTCAGTCACCATCTGAAGCTGCTGCAAGATCAACAACGACGCACAGCTCCAAACTT GC CAAAGCTCTT GC

[0094] SEQ ID No.6: MADLEAGMVAAATDQGNSTRSQDDAATLIPNSGNLGSSNRSTKTARF KDDDELVEITLDVQRDSVAIQEVRGVDEGGSGHGTGFDGLPLVSPSSKSGKLTSKLRQVTNGLKMKSSSRKAPSPQAQQSAKRVRKRLDRTKSSAAVALKGLQFVTAKVGNDGWAAVEKRFNQLQVDGVLLRSRFGKCIGMDGSDEFAVQMFDSLARKRGIVKQVLTKDELKDFYEQLTDQGFDNRLRTFFDMVDKNADGRLTAEEVKEIIALSASANKLSKIKERADEYTALIMEELDPTNLGYIEMEDLEALLLQSPSEAAARSTTTHSSKL A KAL A MKLASNKEMSPVRHYWQQFMYFLEENWKRSWVMTLWISICIALFIWKFIQYRNRAVFGIMGYCVTTAKGAAETLKFNMALVLLPVCRNTITWIRSKTQVGAVVPFNDNINFHKVIAAGVAVGVALHAGAHLTCDFPRLLHASDAQYELMKPFFGEKRPPNYWWFVKGTEGWTGVVMVVLMAIAFTLAQPWFRRNKLKDSNPLKKMTGFNAFWFTHHLFVIVYTLLFVHGTCLYLSRKWYKKTTWMYLAVPVVLYVSERILRLFRSHDAVGIQKVAVYPGNVLALYMSKPPGFRYRSGQYIFIKCTAVSPYEWHPFSITSAPGDDYLSVHIRTRGDWTSRLRTVFSEACRPPTEGESGLLRADLSKGITDEKARFPKLLVDGPYGAPAQDYREYDVLLLIGLGIGATPLISIVKDVLNHIQGEGSVGTTEPESSSKAKKKPFMTKRAYFYWVTREEGSFEWFRGVMNEVSEKDKDGVIELHNHCSSVYQEGDARSALIVMLQELQHAKKGVDILSGTSVKTHFARPNWRSVFKKVAVSHENQRVGVFYCGEPVLVPQLRQLSADFTHKTNTRFDFHKENF*; Among them, the bold and underlined part is the mutation site.

[0095] The total RNA obtained in step 1) was reverse transcribed to synthesize the first strand of cDNA, which was used as a template for PCR amplification using high-fidelity KOD enzyme. The amplification steps were divided into two steps: the first step template was the same as above, primers P1+P8 amplified fragment 1, and primers P2+P7 amplified fragment 2; the second step template was fragment 1 and fragment 2 obtained in the first step, primers P1+P2, and the PCR program was shown in Table 4.

[0096] Table 4 PCR amplification program

[0097]

[0098] Recover the target fragment. The amplified fragment of the NADPH oxidase gene OsRbohB point mutation (OsRbohB S2D ) The nucleotide sequence is shown in SEQ ID No.15, and the amino acid sequence of the protein encoded by it is shown in SEQ ID No.16.

[0099] SEQ ID No.15:ATGGCTGACCTGGAAGCAGGCATGGTTGCTGCTGCCACAGACCAG GGCAATTCAACAAGGTCACAAGATGACGCAGCCACACTGATCCCGAACAGTGGCAATCTGGGCTCGAGCAACAGGAGCACCAAGACGGCCAGGTTCAAGGACGACGACGAGCTGGTCGAGATCACCCTCGACGTGCAGCGCGATTCGGTGGCAATCCAAGAAGTGAGAGGGGTGGATGAGGGTGGCTCCGGGCACGGTACCGGGTTCGACGGCCTGCCACTGGTGTCACCCTCGTCGAAGAGCGGAAAGCTGACGTCAAAGCTCAGGCAGGTGACCAATGGGCTCAAGATGAAGAGCTCCAGCAGGAAGGCGCCATCCCCGCAGGCGCAGCAGTCTGCGAAGAGGGTGAGGAAGAGGCTGGACAGGACCAAGAGCAGCGCCGCCGTGGCGCTCAAAGGATTGCAGTTTGTGACTGCAAAGGTTGGCAATGACGGCTGGGCCGCGGTGGAGAAGCGGTTCAATCAGCTGCAGGTGGATGGTGTGCTGCTCCGTTCAAGATTTGGGAAATGCATTGGAATGGATGGGTCCGACGAGTTTGCGGTGCAAATGTTCGATTCTCTGGCGAGGAAGAGAGGGATAGTGAAGCAGGTGCTCACTAAGGACGAGCTCAAAGATTTCTATGAGCAATTGACTGATCAGGGGTTTGACAATCGTCTTCGGACATTCTTTGACATGGTTGACAAGAACGCTGATGGAAGGCTCACAGCAGAAGAGGTTAAGGAGATTATTGCCCTTAGTGCATCAGCAAACAGACTTTCCAAGATCAAGGAGCGAGCTGATGAGTACACAGCACTCATTATGGAAGAGCTTGACCCTACAAACTTGGGATACATCGAGATGGAGGACTTGGAAGCACTATTGCTTCAGTCACCATCTGAAGCTGCTGCAAGATCAACAACGACGCACAGCTCCAAACTT GA CAAAGCTCTT GA

[0100] SEQ ID No.16: MADLEAGMVAAATDQGNSTRSQDDAATLIPNSGNLGSSNRSTKTARFKDDDELVEITLDVQRDSVAIQEVRGVDEGGSGHGTGFDGLPLVSPSSKSGKLTSKLRQVTNGLKMKSSSRKAPSPQAQQSAKRVRKRLDRTKSSAAVALKGLQFVTAKVGNDGWAAVEKRFNQLQVDGVLLRSRFGKCIGMDGSDEFAVQMFDSLARKRGIVKQVLTKDELKDFYEQLTDQGFDNRLRTFFDMVDKNADGRLTAEEVKEIIALSASANKLSKIKERADEYTALIMEELDPTNLGYIEMEDLEALLLQSPSEAAARSTTTHSSKL D KAL D MKLASNKEMSPVRHYWQQFMYFLEENWKRSWVMTLWISICIALFIWKFIQYRNRAVFGIMGYCVTTAKGAAETLKFNMALVLLPVCRNTITWIRSKTQVGAVVPFNDNINFHKVIAAGVAVGVALHAGAHLTCDFPRLLHASDAQYELMKPFFGEKRPPNYWWFVKGTEGWTGVVMVVLMAIAFTLAQPWFRRNKLKDSNPLKKMTGFNAFWFTHHLFVIVYTLLFVHGTCLYLSRKWYKKTTWMYLAVPVVLYVSERILRLFRSHDAVGIQKVAVYPGNVLALYMSKPPGFRYRSGQYIFIKCTAVSPYEWHPFSITSAPGDDYLSVHIRTRGDWTSRLRTVFSEACRPPTEGESGLLRADLSKGITDEKARFPKLLVDGPYGAPAQDYREYDVLLLIGLGIGATPLISIVKDVLNHIQGEGSVGTTEPESSSKAKKKPFMTKRAYFYWVTREEGSFEWFRGVMNEVSEKDKDGVIELHNHCSSVYQEGDARSALIVMLQELQHAKKGVDILSGTSVKTHFARPNWRSVFKKVAVSHENQRVGVFYCGEPVLVPQLRQLSADFTHKTNTRFDFHKENF*; Among them, the bold and underlined part is the mutation site.

[0101] At the same time, the pRHVcHA vector was double-digested with restriction endonucleases KpnI and SpeI. The map of the pRHVcHA vector is shown in Figure 1 As shown, the vector fragment after enzyme digestion was recovered, and OsRbohB and point mutation OsRbohB were further cloned. S2A , OsRbohB S2D and OsRbohB K266R The expression vectors pRHVcHA and pRHVcHA-OsRbohB were cloned into the overexpression vector pRHVcHA, and sequencing was performed to ensure that the reading frame of the coding region in the expression vector was correct. S2A , pRHVcHA-OsRbohB S2D and pRHVcHA-OsRbohB K266R .

[0102] 3) Obtaining transgenic plants

[0103] The expression vectors pRHVcHA-OsRbohB and pRHVcHA-OsRbohB obtained in step 2) were K266R , pRHVcHA-OsRbohB S2D and pRHVcHA-OsRbohB K266R Transferred to Agrobacterium strain EHA105, coated with kanamycin and rifampicin double-resistance plates, cultured at 28℃ for 2 days, picked single clones in kanamycin and rifampicin double-resistance medium, cultured at 28℃ and 200 rpm for 1 day, aspirated 2 μL for colony PCR, and confirmed whether the single clone was correct or not according to the size of the product band. Aspirated 100 μL of bacterial solution with the same size as the target band was added to 10 mL of culture medium containing kanamycin and rifampicin double-resistance and shaken vigorously, cultured at 28℃ and 200 rpm for 12-14 h, and then further transferred to rice japonica rice variety ZH11 to obtain transgenic rice.

[0104] Young leaves of ZH11 and transgenic rice plants were taken, RNA was extracted and reverse transcribed, and the expression levels of OsRbohB-related transgenic plants were detected by qRT-PCR using quantitative primers qF (GCTACTTCTCATCGGGCTGG, SEQ ID No.17) and qR (TCATGACGCCTCTGAACCAC, SEQ ID No.18); the specific system was as follows: 1 μL of primers qF and qR, 10 μL of 2×SYBR qPCR Mix, and 8 μL of diluted cDNA; the specific procedure was as follows: 95℃ 30 s, 95℃ 10 s, 60℃ 30 s, 40 cycles, 95℃ 15 s, 60℃ 60 s, and 95℃ 15 s to generate the melting curve. After testing, the rice NADPH oxidase gene OsRbohB and point mutation overexpression transgenic plants were obtained, which were respectively recorded as OV-OsRbohB transgenic plants (abbreviated as OV-OsRbohB), OV-OsRbohB S2A Transgenic plants (abbreviated as OV-OsRbohB S2A )、OV-OsRbohB S2D Genetically modified rice (abbreviated as OV-OsRbohB S2D ) and OV-OsRbohB K266R Transgenic plants (abbreviated as OV-OsRbohB K266R )( Figure 2 ).

[0105] Example 2 Identification of disease resistance of rice OsRbohB and point mutation overexpressing transgenic plants

[0106] Punch inoculation: wild-type rice ZH11, OV-OsRbohB transgenic rice, OV-OsRbohB S2A Transgenic rice, OV-OsRbohB S2D Transgenic rice and OV-OsRbohB K266R When the transgenic rice was 6-8 weeks old, the second leaf was selected for inoculation with rice blast fungus (Pyricularia oryzae RB22). 0.5‰ Tween water was used to elute the spores of Pyricularia oryzae RB22 and the concentration was adjusted to 2×10 5 spores / mL, stick tape on the middle of the leaf, make a non-penetrating hole with a mouse ear clip, add 10 μL of spore suspension, fix the drop with tape, and wrap it tightly.

[0107] The rice materials (wild-type rice ZH11, OV-OsRbohB transgenic rice, OV-OsRbohB S2A Transgenic rice, OV-OsRbohB S2D Transgenic rice and OV-OsRbohBK266R The transgenic rice plants were placed in a plant growth chamber with a temperature of 25°C, a humidity of 75%, and a photoperiod of 12 h light / 12 h dark. After 14 days, the expansion of the lesions was observed, the disease was investigated, and the fungal biomass was counted. The transgenic rice plants with obvious resistance compared with the wild-type ZH11 were the obtained rice blast-resistant transgenic plants. The investigation results are shown in Tables 5 and Figure 3-Figure 4 shown.

[0108] Table 5 Relative biomass of diseased leaves of transgenic lines and wild type

[0109]

[0110] From Table 5 and Figure 3-Figure 4 It can be seen from the records that compared with wild-type rice ZH11, OV-OsRbohB transgenic rice, OV-OsRbohB S2A Transgenic rice and OV-OsRbohB K266R The transgenic plants all showed enhanced resistance to rice blast fungus race RB22, OV-OsRbohB S2A and OV-OsRbohB K266R The resistance of transgenic plants was significantly improved, while OV-OsRbohB S2D Transgenic rice has reduced resistance to rice blast race RB22 ( Figure 3 At the same time, the relative fungal biomass on the diseased leaves of transgenic rice lines and wild-type rice ZH11 lines was counted (Table 5). S2A Transgenic rice and OV-OsRbohB K266R After the transgenic plants were inoculated with rice blast race RB22, the fungal biomass in the lesion area decreased by about 50% of that in wild-type rice ZH11. S2D After inoculation with rice blast race RB22, transgenic rice increased the fungal biomass in the lesion area ( Figure 4 ).

[0111] In summary, the present invention creates rice overexpression transgenic plants (OV-OsRbohB transgenic rice, OV-OsRbohB S2A Transgenic rice, OV-OsRbohB S2D Transgenic rice and OV-OsRbohB K266R Transgenic plants) verified the transgenic plants overexpressing the OsRbohB gene - OV-OsRbohB transgenic rice, OV-OsRbohBS2A Transgenic rice and OV-OsRbohB K266R Transgenic plants can achieve the effect of improving rice blast resistance. Therefore, the OsRbohB gene and OsRbohB gene mutant provided by the present invention can be used as new disease resistance gene tools to be introduced into other rice varieties to improve varieties and improve rice blast resistance. It can be seen that the OsRbohB gene and OsRbohB gene mutant provided by the present invention can be used to improve rice blast resistance, cultivate rice varieties with high resistance to rice blast, improve rice varieties and rice breeding, and provide a new approach for rice breeding.

[0112] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Ob A gene mutant, characterized in that Said Ob The nucleotide sequence of the gene mutant is shown as SEQ ID No.3 or SEQ ID No.

5.

2. The method according to claim 1 Ob The protein encoded by the gene mutant is characterized in that The amino acid sequence of the protein is shown in SEQ ID No.4 or SEQ ID No.

6.

3. The method according to claim 1 Ob Use of the gene mutant or the protein according to claim 2 in any of the following: (1) Application in improving rice blast resistance; (2) Application in breeding rice varieties with high resistance to rice blast; (3) Application in breeding improved rice varieties with high resistance to rice blast; The method for improving rice blast resistance is carried out by overexpressing the Ob Gene mutants reached.

4. A method for improving rice blast resistance, characterized in that: Including overexpression in rice Ob Gene mutants, a step of improving the resistance of the rice to rice blast; Said Ob The nucleotide sequence of the gene mutant is shown as SEQ ID No.3 or SEQ ID No.

5.

5. A method for cultivating transgenic rice with high resistance to rice blast, characterized in that: Including overexpression in rice Ob Gene mutants, a step of obtaining the transgenic rice; Said Ob The nucleotide sequence of the gene mutant is shown as SEQ ID No.3 or SEQ ID No.5.

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