Application of a rice protein kinase OsAFC1 and its encoding gene in disease resistance breeding

Abstract

The present invention discloses a rice protein kinase, OsAFC1, and its encoding gene for use in disease-resistant breeding, belonging to the field of molecular biotechnology. The present invention identifies a protein kinase, named OsAFC1. Experiments have demonstrated that the OsAFC1 protein has the function of enhancing rice disease resistance. By overexpressing the OsAFC1 gene from Zhonghua 11 (ZH11) in ZH11, the transgenic plants obtained exhibit a phenotype that is more resistant to rice blast than ZH11. This indicates that OsAFC1 positively regulates plant disease resistance and that the OsAFC1 protein and its encoding gene are of great significance for breeding disease-resistant rice varieties.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, in particular to a rice protein kinase OsAFC1 and an encoding gene thereof and application thereof in disease-resistant breeding. Background Art

[0002] Rice is one of the most important food crops in the world. However, during its growth, rice is affected by a variety of diseases, such as rice blast. These diseases not only lead to reduced yields, but also seriously affect the quality and safety of rice. Therefore, improving the disease resistance of rice is an important issue that needs to be urgently addressed in agricultural production. The existing technology has reported a variety of genes related to plant disease resistance, including effector molecule genes and regulatory genes. These genes are derived from crops such as rice, wheat, corn, soybeans, and model plants such as Arabidopsis. Some of them have been used as target genes in crop disease resistance genetic engineering, and disease-resistant rice, wheat, corn, and soybeans have been successfully cultivated. However, due to differences in genetic background, the application of some genes is hindered, so new related genes are still needed to meet the needs of breeding.

[0003] Protein kinases are important signal transduction molecules that regulate a variety of biological processes, including cell division, differentiation, metabolism, and stress responses, through phosphorylation. The rice protein kinase AFC1 (Activation of Fungal Cell Wall Synthesis 1) belongs to the mitogen-activated protein kinase (MAPK) family. Its amino acid sequence possesses a typical kinase domain, including an ATP-binding site and a substrate-binding site. Existing studies have shown that AFC1 plays a key role in plant heat stress tolerance. Therefore, previous research on rice OsAFC1 has focused on its regulation of heat stress tolerance. However, there are no reports on OsAFC1 regulating rice resistance to fungal diseases. Summary of the Invention

[0004] The present invention aims to provide a rice protein kinase, OsAFC1, and its encoding gene for use in disease resistance breeding to address the aforementioned problems of the prior art. By overexpressing the OsAFC1 gene from Zhonghua 11 (ZH11) in ZH11, the resulting transgenic plants exhibited a phenotype superior to that of ZH11 in resistance to rice blast. This indicates that OsAFC1 positively regulates plant disease resistance and that the OsAFC1 protein and its encoding gene are of great significance for breeding disease-resistant rice varieties.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a use of rice protein kinase OsAFC1 and / or related biomaterials in any one of the following A1)-A3):

[0007] A1) Regulate plant disease resistance;

[0008] A2) preparing products for improving plant disease resistance;

[0009] A3) Cultivating highly disease-resistant plants;

[0010] The amino acid sequence of the rice protein kinase OsAFC1 is shown in SEQ ID NO. 4, or has more than 80% identity with the amino acid sequence shown in SEQ ID NO. 4 and has the same function.

[0011] Optionally, the CDS sequence encoding the rice protein kinase OsAFC1 is shown as SEQ ID NO.3.

[0012] Optionally, the relevant biological material includes any one of the following B1)-B3):

[0013] B1) a nucleic acid molecule encoding the rice protein kinase OsAFC1;

[0014] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0015] B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0016] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3);

[0017] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0018] B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0019] B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0020] B8) a nucleic acid molecule that increases or promotes the expression of the rice protein kinase OsAFC1;

[0021] B9) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

[0022] Optionally, the regulating plant disease resistance is to increase the expression of the rice protein kinase OsAFC1 to enhance the disease resistance of the plant, or to decrease the expression of the rice protein kinase OsAFC1 to weaken the disease resistance of the plant.

[0023] Further optionally, the disease resistance is resistance to plant fungal diseases.

[0024] Further optionally, the plant fungal disease includes rice blast.

[0025] Optionally, the plant comprises rice.

[0026] The present invention also provides a method for improving plant disease resistance, comprising the step of overexpressing rice protein kinase OsAFC1 in the plant; the amino acid sequence of the rice protein kinase OsAFC1 is shown in SEQ ID NO. 4, or has more than 80% identity with the amino acid sequence shown in SEQ ID NO. 4 and has the same function.

[0027] Optionally, the disease resistance is resistance to plant fungal diseases; the plant fungal diseases include rice blast.

[0028] Optionally, the plant comprises rice.

[0029] The present invention discloses the following technical effects:

[0030] The present invention identified a protein kinase, named OsAFC1. Experimental studies have shown that the OsAFC1 protein enhances rice disease resistance. By overexpressing the OsAFC1 gene from Zhonghua 11 (ZH11) in ZH11, the transgenic plants obtained exhibited a phenotype superior to that of ZH11 in resistance to rice blast. This suggests that OsAFC1 positively regulates plant disease resistance, and that the OsAFC1 protein and its encoding gene are of great significance for the development of disease-resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0032] Figure 1 Schematic diagram of the protein structure of OsAFC1 and the sgRNA designed for the knockout target site;

[0033] Figure 2 Sequencing and characterization of genetically stable OsAFC1 knockout lines OsAFC1-KO1 and OsAFC1-KO2 in the ZH11 background;

[0034] Figure 3 To identify the expression levels of OsAFC1 overexpression lines OsAFC1-OE1 and OsAFC1-OE2 in the ZH11 background and genetically stable;

[0035] Figure 4 Figure 2 is the puncture wound inoculation phenotype of the control and OsAFC1 knockout plants; A is a representative picture of lesions on leaves of three-week-old Zhonghua 11 (ZH11), OsAFC1-KO1, and OsAFC1-KO2 plants 5 days after inoculation with rice blast fungus (physiological subspecies zhong10-8-14); B is the statistical result of lesion length on leaves of three-week-old Zhonghua 11 (ZH11), OsAFC1-KO1, and OsAFC1-KO2 plants 5 days after inoculation with rice blast fungus (physiological subspecies zhong10-8-14);

[0036] Figure 5 Figure 2 is the stab wound inoculation phenotype of the control and OsAFC1 overexpressing plants; A is a representative picture of lesions on the leaves of three-week-old Zhonghua 11 (ZH11), OsAFC1-OE1 and OsAFC1-OE2 plants 7 days after inoculation with rice blast fungus (physiological subspecies zhong10-8-14); B is the statistical result of the lesion length on the leaves of three-week-old Zhonghua 11 (ZH11), OsAFC1-OE1 and OsAFC1-OE2 plants 7 days after inoculation with rice blast fungus (physiological subspecies zhong10-8-14). DETAILED DESCRIPTION

[0037] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] The technical problem to be solved by the present invention is how to improve the disease resistance of rice or how to cultivate rice with high disease resistance.

[0043] To solve the above technical problems, the present invention first provides a protein. The protein is OsAFC1. The OsAFC1 can be the following protein A1), A2), or A3):

[0044] A1) a protein with an amino acid sequence as shown in SEQ ID NO. 4;

[0045] A2) a protein derived from A1) or having more than 80% identity with the protein of A1) and having the same function, obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO. 4;

[0046] A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0047] In the above protein, the amino acid sequence shown in SEQ ID NO. 4 consists of 370 amino acid residues.

[0048] The above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0049] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a GFP tag, a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and / or a SUMO tag, etc.

[0050] In the above-mentioned proteins, identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by searching in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, all filters to OFF, BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively. The identity value (%) can then be obtained.

[0051] In the above proteins, the above 80% identity or more may be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0052] In order to solve the above technical problems, the present invention further provides a biomaterial related to the above protein, which may be any one of the following B1) to B9):

[0053] B1) a nucleic acid molecule encoding the above protein;

[0054] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0055] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B1);

[0056] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0057] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1) or a transgenic plant cell line containing the expression cassette described in B2);

[0058] B6) transgenic plant tissue containing the nucleic acid molecule described in B1) or transgenic plant tissue containing the expression cassette described in B2);

[0059] B7) a transgenic plant organ containing the nucleic acid molecule described in B1) or a transgenic plant organ containing the expression cassette described in B2);

[0060] B8) Nucleic acid molecules that increase or promote the expression of the above-mentioned proteins;

[0061] B9) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

[0062] In the biological material described above, the nucleic acid molecule in B1) may be a gene encoding the protein shown in b1), b2) or b3) below:

[0063] b1) a cDNA molecule or a DNA molecule whose coding sequence is the nucleotide sequence of SEQ ID NO. 3;

[0064] b2) the nucleotide sequence is a DNA molecule of SEQ ID NO. 3,

[0065] b3) A cDNA molecule or DNA molecule that hybridizes with the cDNA or DNA molecule defined in b2) and encodes a protein having the same function.

[0066] In the aforementioned biological material, the expression cassette containing the nucleic acid molecule described in B2) refers to DNA capable of expressing the protein described in the aforementioned application in a host cell. This DNA may include not only a promoter for initiating transcription of the protein-encoding gene, but also a terminator for terminating transcription of the protein-encoding gene. Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0067] Available existing plant expression vector construction contains the recombinant expression vector of described protein encoding gene expression cassette.Described plant expression vector comprises binary agrobacterium vector and can be used for the carrier etc. of plant microprojectile bombardment.As pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA2300, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company) etc.Described plant expression vector can also comprise the 3 ' end non-translated region of foreign gene, promptly comprise polyadenylic acid signal and any other DNA fragmentation that participates in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor. For example, the untranslated region transcribed at the 3' end of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the nopaline synthase gene Nos) and plant genes (such as the rice starch synthase gene) all have similar functions. When using the genes of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence.

[0068] In the above-mentioned biological materials, the recombinant microorganisms can specifically be yeast, bacteria, algae and fungi.

[0069] Any of the following applications of the above-mentioned protein and / or the above-mentioned biomaterial also falls within the scope of protection of the present invention:

[0070] Q1. Application of the protein and / or the biomaterial in regulating plant disease resistance;

[0071] Q2. Use of the protein and / or the biomaterial in preparing a product for improving plant disease resistance;

[0072] Q3. Use of the protein and / or the biomaterial in cultivating highly disease-resistant plants;

[0073] Q4. Use of the protein and / or the biological material in plant breeding.

[0074] 1) Connected to various plant-expressed promoters to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the temporal and spatial requirements of expression and also depends on the target species; for example, a tissue- or organ-specific expression promoter, depending on the stage of development at which the receptor is required; although many promoters derived from dicots have been shown to function in monocots and vice versa, ideally, dicot promoters are selected for expression in dicots and monocot promoters are selected for expression in monocots;

[0075] 2) Linking to a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml from CaMV, E9 from rbcS; any available terminator known to function in plants can be linked to the gene of the present invention;

[0076] 3) Introducing enhancer sequences, such as intron sequences (e.g., from Adhl and bronze) and viral leader sequences (e.g., from TMV, MCMV, and AMV).

[0077] In the above method, the plant resistant to fungal disease stress may be a transgenic plant.

[0078] In the above methods, the transgenic plants are understood to include not only first- and second-generation transgenic plants, but also their progeny. Transgenic plants can be propagated within their species or transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. Transgenic plants include seeds, callus, whole plants, and cells.

[0079] The plant mentioned above may be any of the following:

[0080] D1) Dicotyledons;

[0081] D1) Monocots;

[0082] D3) Gramineae;

[0083] D4) Grasses;

[0084] D5) Oryza plants;

[0085] D6) Rice.

[0086] The disease resistance mentioned above is resistance to fungal diseases.

[0087] The technical solution of the present invention is now described in detail with reference to specific embodiments.

[0088] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0089] The rice variety Zhonghua 11 (O. Sativa L. spp. japonica, var. zhonghua11, AA genome, ZH11) belongs to the japonica subspecies and is described in the non-patent document "Ni Yuchong, A New Rice Variety Cultivated by Flower Culture—Zhonghua 11," Agricultural Science and Technology Communications, July 35, 1989; provided by Professor Chen Xuewei of Sichuan Agricultural University.

[0090] OsAFC1 knockout and overexpression transgenic rice were produced by Boyuan Biotechnology Company.

[0091] The plant binary expression vector pTCRISPR was provided by Associate Professor Tang Yongyan from the State Key Laboratory of Sichuan Agricultural University.

[0092] The plant binary expression vector pCambia2300-35S:eGFP-nosT was provided by Professor Xuewei Chen from the State Key Laboratory of Sichuan Agricultural University.

[0093] Total RNA extraction kit: TRIzol purchased from Invitrogen, USA, catalog number 15596026.

[0094] Reverse transcription kit: HiScript III RT SuperMix for qPCR (+gDNA wiper) purchased from Vanzyme, China, catalog number R323-01.

[0095] Homologous recombination kit: ClonExpress II One Step Cloning Kit purchased from Vanzyme, China, with the catalog number C112-01.

[0096] Example 1 Rice protein kinase OsAFC1 and its cloning

[0097] A protein kinase, OsAFC1, was screened in rice. Primers were designed based on the reference gene sequence of the rice ZH11 genome:

[0098] OsAFC1-F: ATGGAGGCGCAGTGGCTCGC, SEQ ID NO.1;

[0099] OsAFC1-R: GTAACCACATCTTCTGTGGCAT, SEQ ID NO.2.

[0100] Using ZH11 total cDNA as a template, the aforementioned primers amplified a DNA band of approximately 1.3 kb. Sequencing confirmed that it was the OsAFC1 gene, which belongs to the protein kinase family. Alignment of the LOC_Os01g62080 (OsAFC1) sequence in the rice genome reference sequence with the cloned sequence amplified from ZH11 total cDNA revealed that the cloned sequence from ZH11 was identical to LOC_Os01g62080 (OsAFC1). The amino acid sequence of the OsAFC1 protein is shown in SEQ ID NO. 4. The CDS sequence of the coding strand of the gene encoding the OsAFC1 protein, OsAFC1, is shown in SEQ ID NO. 3.

[0101] SEQ ID NO.3:

[0102]

[0103] SEQ ID NO.4:

[0104] MEAQWLAEYPHQGADKRPRKRPRLAWDVAPPLFQPPKAIPMLYCGQELINGNFATAFLPPPPIYYTGPPRNLSPPWRPDDKDGHYVFAVGENLTPRYRILSKMGEGTF GQVLECWDLEHQETVAIKIVRSLQKYREAAMIEIDVLQRLGKHDFTGSRCVQIRNWFDYRNHICIVFERLGPSLYDFLRKNSYRAFPIDLVREFARQILESVAFMHDLR LIHTDLKPENILLVSSESIRVPDYKVTIRPPKDGSFFKNLPKSSAIKLIDFGSTTFEHQDHNYVVSTRHYRAPEVILGLGWNYSCDLWSVGCILVELCSGEALFQTHEN LEHLAMMERVLGPLPKHMIVRADRRAEKYFRRGLRLDWPEGAASRESLKAVWKLPRLQNLVMQHVDHSAGDLIDLLQGLLRYDPDARLKAREALQHPFFTRCHRRCGY.

[0105] Example 2 Construction of OsAFC1 plant knockout vector

[0106] Schematic diagram of the protein structure of OsAFC1 and the sgRNA designed at the knockout target site Figure 1 Using the genome of rice ZH11 as a template, the knockout target sequence was designed as: CTCCCTTCAGAAATATCGAG (SEQ ID NO. 5).

[0107] The following primers were synthesized:

[0108] F:TGTGCTCCCTTCAGAAATATCGAGG, SEQ ID NO.6;

[0109] R:AAAACCTCGATATTTCTGAAGGGAG, SEQ ID NO.7.

[0110] After denaturation at 95°C, anneal to form a double-stranded DNA. The pTCRISPR vector was digested with BsaI to recover the linearized vector. According to the system shown in Table 1, the above DNA double-stranded DNA and the linearized vector were mixed and ligated with T4 DNA ligase at room temperature for 30 minutes. The pTCRISPR-sgRNA was obtained. OsAFC1 Knockout vector.

[0111] Table 1 Construction of knockout vector ligation system

[0112]

[0113]

[0114] pTCRISPR-sgRNA OsAFC1 The knockout vector was transformed into DH5α competent medium. After selection with kanamycin, single clones were picked for colony PCR identification. Plasmids were extracted from positive colonies and sequenced to confirm the correct pTCRISPR-sgRNA. OsAFC1 OsAFC1 knockout transgenic rice was produced by Boyuan Biotechnology Company.

[0115] Example 3 Construction of OsAFC1 plant expression vector

[0116] The cDNA obtained by reverse transcription of total RNA from rice ZH11 was used as the template, and the cloning primers were:

[0117] F: TATCCAGATCCAGTGGGATCC ATGGAGGCGCAGTGGCTCGC, SEQ ID NO.8;

[0118] R: CGCACTAGTAAGCTTGGTACC GTAACCACATCTTCTGTGGCAT, SEQ ID NO.9.

[0119] The full-length OsAFC1 cDNA was amplified by PCR. The pCambia2300-35S:eGFP-nosT vector was digested with BamHI and KpnI to recover the linearized vector. The OsAFC1 full-length cDNA recovered by PCR and the linearized vector were mixed according to the system in Table 2 and homologous recombination was performed to obtain the pCambia2300-35S:OsAFC1 recombinant vector.

[0120] Table 2 Construction of recombinant vector ligation system

[0121] cDNA fragments 0.5-5μL Linearized vector 0.5-5μL 5×CEIIBuffer 4μL ExnaseII 2μL <![CDATA[ddH2O]]> Make up to 20 μL

[0122] The pCambia2300-35S:OsAFC1 recombinant vector was transformed into a DH5α competent cell. After selection with kanamycin, single colonies were selected and identified by colony PCR. Plasmids from positive colonies were extracted and sequenced to confirm the identity of pCambia2300-35S:OsAFC1 and to proceed with genetic transformation. Transgenic rice overexpressing OsAFC1 was produced by Boyuan Biotechnology.

[0123] Example 4 OsAFC1 is involved in regulating plant disease resistance

[0124] In plants, OsAFC1 has primarily been studied for its role in heat stress tolerance. The inventors discovered that OsAFC1 plays a key role in regulating plant disease resistance. Therefore, they tested the effect of OsAFC1 on rice disease resistance.

[0125] The test materials were three-week-old Zhonghua 11 (ZH11), the genetically stable OsAFC1 knockout lines OsAFC1-KO1 and OsAFC1-KO2 in the ZH11 background, and the genetically stable OsAFC1 overexpression lines OsAFC1-OE1 and OsAFC1-OE2 in the ZH11 background. Figure 2 The expression levels of OsAFC1 overexpression lines OsAFC1-OE1 and OsAFC1-OE2, which were genetically stable and based on the ZH11 background, were identified as shown in Figure 3 shown.

[0126] Seedling-stage wound inoculation: Select plump seeds and place them in a conical flask filled with tap water. Germinate in a dark incubator at 37°C, changing the water daily. After two days, select white seeds and place them in a 96-well seedling plate. Place the 96-well seedling plate on a float and grow in Hoagland nutrient solution. After 21 days, select the second-to-last rice leaf of uniform growth and size, wound it, and inoculate it with 5 μL of a 5×10 5 mL -1 The 10-8-14 rice blast fungus was used and the length of the lesions was observed and counted after 5-7 days.

[0127] Statistics showed that 5 days after inoculation, the lesion length of OsAFC1-KO1 and OsAFC1-KO2 plants under the ZH11 background was significantly increased and the disease resistance was weakened compared with ZH11 ( Figure 4 Seven days after inoculation, the lesion length of OsAFC1-OE1 and OsAFC1-OE2 plants under the ZH11 background was significantly reduced compared with that of ZH11, and the disease resistance of the plants was significantly enhanced ( Figure 5 This indicates that OsAFC1 positively regulates plant disease resistance and its encoding gene OsAFC1 can be used as a target gene for molecular breeding to improve plant disease resistance.

[0128] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A use of overexpressed rice protein kinase OsAFC1 in any one of the following A1)-A3): A1) Improve rice resistance to rice blast; A2) preparing a product for improving the resistance of rice to rice blast; A3) Cultivating rice with high resistance to rice blast; The amino acid sequence of the rice protein kinase OsAFC1 is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that The CDS sequence encoding the rice protein kinase OsAFC1 is shown in SEQ ID NO.

3.

3. A method for improving rice resistance to rice blast, characterized in that: The method comprises the step of overexpressing rice protein kinase OsAFC1 in the rice; the amino acid sequence of the rice protein kinase OsAFC1 is shown as SEQ ID NO.4.

Images