Rice OsNPR3 gene and application thereof in resistance to rice blast

Abstract

The invention provides a rice OsNPR3 gene and application of the rice OsNPR3 gene in resisting rice blast, aiming at the production problem of how to improve the resistance of rice to the rice blast. Overexpression of the OsNPR3 gene can improve the rice blast resistance, and knockout or mutation of the OsNPR3 gene can improve the rice blast sensitivity. The nucleotide sequence of the OsNPR3 gene is as shown in SEQ ID NO. 1. According to the invention, rice blast resistance function identification is carried out on the OsNPR3 gene, and the positive regulation effect of the OsNPR3 gene in rice blast resistance is found and proved for the first time; experiments prove that the resistance of the rice to the rice blast is weakened after the OsNPR3 gene is knocked out, and the resistance of the rice to the rice blast can be enhanced by up-regulated expression of the OsNPR3 gene, so that the OsNPR3 gene positively regulates the resistance of the rice to the rice blast. The results show that the OsNPR3 gene is an excellent candidate gene for creating transgenic rice with enhanced rice blast resistance and molecular crop design, and has important theoretical value and wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of rice transgenic technology and disease-resistant breeding, and in particular to the application of rice OsNPR3 gene in rice resistance to rice blast. Background Art

[0002] Rice is one of the three major staple foods in the world. According to statistics, more than 3.5 billion people in the world rely on rice as their staple food, and more than 60% of the population in Asia relies on rice as their main food source. However, rice production is often affected by various diseases, such as bacterial blight, rice blast, sheath blight and other diseases. Plant diseases seriously threaten the safety of rice production, and the large-scale use of pesticides will pollute the environment and cause food safety hazards. Therefore, using the natural immune system of plants, exploring and using rice's own disease-resistant genes to select and breed resistant varieties is currently the most economical, effective and environmentally friendly measure to prevent and control diseases.

[0003] Rice blast is a rice disease caused by the fungus Magnaporthe oryzae. It is widely distributed in rice-growing areas around the world and can cause a 10%-50% reduction in rice yield, or even a total crop failure in severe cases. The disease can occur throughout the entire growth period of rice and can be divided into seedling blast, leaf blast, node blast, ear neck blast and grain blast according to the site of disease. Among them, leaf blast and ear neck blast have the greatest impact on yield. Leaf blast is manifested as spindle-shaped or fusiform lesions on the leaves, while ear neck blast causes browning of the ear, white ears or barren grains. The occurrence of rice blast is closely related to the disease resistance of the variety, climatic conditions (such as high temperature and high humidity) and fertilizer and water management. Prevention and control measures include the selection of disease-resistant varieties, reasonable fertilization, scientific irrigation and the use of chemical agents (such as tricyclazole and rice blast). In recent years, with the development of molecular biology technology, the exploration of disease-resistant genes and the analysis of disease-resistant mechanisms have become research hotspots, providing a new direction for the breeding of broad-spectrum disease-resistant varieties.

[0004] Therefore, it is of great significance to explore new molecular mechanisms of rice resistance to rice blast and rice blast resistance genes and apply them to rice breeding, which will open up broader prospects for the breeding of disease-resistant rice varieties in the future. Summary of the invention

[0005] The present invention aims at the production problem of how to improve the resistance of rice to rice blast, and provides the application of the rice blast resistance-related gene OsNPR3 in regulating rice disease resistance. Up-regulating the expression of the OsNPR3 gene can enhance the resistance of rice to rice blast, while mutating OsNPR3 will increase the susceptibility of rice to rice blast. Therefore, the OsNPR3 gene can be used for genetic improvement to improve the resistance of rice crops.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide the application of the OsNPR3 gene (LOC_Os03g46440) in regulating rice disease resistance. Overexpression of the OsNPR3 gene can improve the blast resistance of rice, and knockout or mutation of the OsNPR3 gene can improve the blast susceptibility of rice.

[0008]

[0009] The CDS sequence of the OsNPR3 gene is shown in SEQ ID NO.2:

[0010] ATGGAGACGTCCACCATAAGCTTCTCCTCCTCGTCGCCGCCGTCCCCTCCGCCGCCGCAG

[0011] CCGGCTCCGGGCGACATCGACGCCGTCAGCCTCGGCCGCCTCAGCAGGAACCTCGAGA

[0012] ACCTCCTCGACCCCGCCTTTCTCAACTGCGCCGACGCCGAGATCGTCCTCGCCTCCGGAG

[0013] GCGGCGACCCCGGCGGCGGCGCCGTCGTGGGCGTCCACCGCTGCATCCTCGCCGCCAGG

[0014] AGCCGCTTCTTCTACGACCACTTCTCCTCCGCCCCCGCCCCCGCCCCCGCCACCGCCGGC

[0015] GACAAGCCGCAGCTGGACCTCGACGGGCTGGTCCCCGGCGGGCGCCACATCGGCCGAG

[0016] ACGCCCTCGTCGCCGTTCTCAGCTACCTGTACACCGGCCGCCTCAGGTCGGCGCCCCCC

[0017] GAGGCCGCCGCCTGCCTGGACGACGGCTGCAGCCACGACGCGTGCCGCCCGGCGATCG

[0018] ACTTCGTCGTCGAGTCCACGTACGCCGCCTCCGGCTTCCAGATCTCCGAGCTCGTCTCCC

[0019] TCTTCCAGCGCCGATTATCTGATTTTGTGAACAAAGCTTTGGCTGAGGACATACTGCCAA

[0020] TTCTTGTGGTTGCCTCCACCTGCCATCTTCCAGAGCTGCTAAATCAATGTATCCAGAGGG

[0021] TTGCCAACTCGAACCTGGACAATCGTTACCTCGAGAAGCGGCTTCCGGATGATCTGTAC

[0022] GCCAAGCTGAAGGAGTTTCGCGTGCCTGATGAACCACACAGTGGCATTCTTGACCCTGA

[0023] GCATGAGAAGAGGGTCAGAAACATCCACAAGGCCTTGGATTCCGATGATGTCGATCTTG

[0024] TTGGCATGCTTCTGAAGGAGTCCCCGGTCACCTTGGATGATGCATTCGCCATACACTAC

[0025] GCTGCGGCCTACTGTGAGCCAAAAGTGTTAGCAGAATTGCTGAAACTGGAATCTGCAAA

[0026] TGTGAACCTGAAGAACTCAAGTGGATACACGCCGCTCCACATGGCTTGCATGAGGCGAG

[0027] AACCGGATATCATTGTTTCGCTTATAGAAAAGGGGGCCTCTGTTCTGGAAAGGACACAG

[0028] GATGGACGTGATGCTCTTACCATCTGCAAGAGATTAACAAGGGAGAAAGACCGCAACG

[0029] AGAAATCAGAAAAATGCAAGGAGAGAAGCAAGGCTTACTTGTGCATTGGCGTCTTACA

[0030] GCAAGAAATAAAGAGGAGACCACAAATTTTGGAGGACCAGATGTCTGCAGAGGAGTCA

[0031] ATTGCTACACCTCTGCTGGTTGATAATTTTCACATGAGGCTACTAAACTTGGAGAATAGA

[0032] GTTGCCTTTGCAAGAATATTTTTCCCTTCGGAAGCCAAACTTGTGATGCGCATAGCACAA

[0033] GCTGACTCAACTCAAGAGTTTGCTGGTCTCACATCTGCTAATTTCAGTAAACTTAAGGA

[0034] GGTTGACCTAAATGAGACCCCCACAATGCAAAACAGGAGGTTGCGAGAACGCCTTGAT

[0035] GCTTTGACAAAAACAGTTGAACTCGGACGACGATACTTCCCACATTGTTCAGAAGTTCT

[0036] CGACAAGTTCCTGAATGAAGAATCCACCGATTTGATCTTGCTTGAAAGTGGCACAGCAG

[0037] AGGACCAGCAAACCAAGAGGATGCGCTTTTCTGAACTCAGAGAGGATGTACGGAAGGC

[0038] CTTTACCAAAGATAAGGCAGCCGGCGCTGCAATATCTTCCTCAACATCTGCGTCTTCATC

[0039] ACCAAGGTATGAGACAAAGTTAAGACCTGGCAACAAGAAAGGGAAGCTATCACGGTAA. The full length of the CDS is 1770 bp.

[0040] The second object of the present invention is to provide the application of the protein encoded by the OsNPR3 gene in regulating rice blast resistance. Upregulating the expression level of the OsNPR3 protein can improve rice blast resistance, and downregulating the expression level of the OsNPR3 protein can improve rice blast susceptibility. The amino acid sequence of the said protein is shown as SEQ ID NO.3:

[0041] METSTISFSSSSPPSPPPPQPAPGDIDAVSLGRLSRNLENLLDPAFLNCADAEIVLASGGGD

[0042] PGGGAVVGVHRCILAARSRFFYDHFSSAPAPAPATAGDKPQLDLDGLVPGGRHIGRDALVA

[0043] VLSYLYTGRLRSAPPEAAACLDDGCSHDACRPAIDFVVESTYAASGFQISELVSLFQRRLSDF

[0044] VNKALAEDILPILVVASTCHLPELLNQCIQRVANSNLDNRYLEKRLPDDLYAKLKEFRVPDE

[0045] PHSGILDPEHEKRVRNIHKALDSDDVDLVGMLLKESPVTLDDAFAIHYAAAYCEPKVLAEL

[0046] LKLESANVNLKNSSGYTPLHMACMRREPDIIVSLIEKGASVLERTQDGRDALTICKRLTREK

[0047] DRNEKSEKCKERSKAYLCIGVLQQEIKRRPQILEDQMSAEESIATPLLVDNFHMRLLNLENR

[0048] VAFARIFFPSEAKLVMRIAQADSTQEFAGLTSANFSKLKEVDLNETPTMQNRRLRERLDALT

[0049] KTVELGRRYFPHCSEVLDKFLNEESTDLILLESGTAEDQQTKRMRFSELREDVRKAFTKDKAAGAAISSSTSASSSPRYETKLRPGNKKGKLSR, with a total length of 589 aa.

[0050] The third object of the present invention is to provide the application of the recombinant vector containing the OsNPR3 gene in improving the blast resistance of rice.

[0051] Furthermore, the basic vector of the recombinant vector containing the OsNPR3 gene is pCAMBIA1305 - 3xMYC, and the OsNPR3 gene is inserted between the SpeI and KpnI restriction enzyme sites to construct the recombinant vector.

[0052] The fourth object of the present invention is to provide the application of the recombinant engineering bacteria containing the aforementioned recombinant vector in improving the blast resistance of rice.

[0053] The fifth object of the present invention is to provide the application of the primer for amplifying the aforementioned OsNPR3 gene in improving the blast resistance of rice, and the primer is:

[0054] OsNPR3 - F: 5’-GGGGTACCGATGTCCATCACTACTTGTACA-3’, SEQ ID NO.4;

[0055] OsNPR3 - R: 5’-GACTAGTCCGTGATAGCTTCCCTTTCTTG-3’, SEQ ID NO.5.

[0056] The positive regulation of the rice disease resistance by the OsNPR3 gene includes the positive regulation of the rice blast resistance by the OsNPR3 gene. Compared with the wild - type rice variety Nipponbare, up - regulating the expression of the rice blast resistance gene OsNPR3 can effectively improve the resistance of plants to the rice blast pathogen.

[0057] The sixth object of the present invention is to provide the application of the knockout vector of the OsNPR3 gene in improving the susceptibility of rice to rice blast.

[0058] Furthermore, the knockout vector is an sgRNA sequence designed for the CRISPR / Cas9 editing target of the rice blast resistance gene OsNPR3: 5’-TCTCGAGGTAACGATTGTCC-3’, SEQ ID NO.12, and is constructed by artificially synthesizing primers according to this sgRNA sequence and constructing them into the CRISPR / Cas9 vector.

[0059] The present invention takes OsNPR3 as the research object, obtains the OsNPR3 - MYC transgenic material and the OsNPR3 mutant material Osnpr3 - 1 by up - regulating the expression of OsNPR3, and inoculates these materials with rice blast. It is found that the lesion length of the OsNPR3 - MYC transgenic plants is significantly shorter than that of NIP, while the lesion length of the Osnpr3 - 1 mutant is significantly longer than that of NIP. Thus, it can be seen that the OsNPR3 transgenic rice can significantly enhance the resistance of rice to rice blast; the OsNPR3 over - expression transgenic plants have good application value in the field of transgenic research. The present invention has practical guiding significance for cultivating transgenic plants resistant to rice blast and also has important application prospects in the field of plant disease control.

[0060] The beneficial effects of the present invention are as follows:

[0061] The present invention conducts a functional identification of the OsNPR3 gene for blast resistance, and for the first time discovers and proves the positive regulatory role of the OsNPR3 gene in rice blast resistance; and through experiments, it is demonstrated that the resistance of rice to blast is weakened after the knockout of the OsNPR3 gene, while the overexpression of OsNPR3 can enhance the resistance of rice to blast, thereby indicating that the OsNPR3 gene positively regulates the resistance of rice to blast. The above results all show that the OsNPR3 gene is an excellent candidate gene for creating transgenic rice with enhanced blast resistance and molecular crop design, and has important theoretical value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Editing positions and target information of the knockout of OsNPR3 in Example 1 of the present invention ( Figure 1 A) and the map information of the construction of the OsNPR3 overexpression vector ( Figure 1 B).

[0063] Figure 2 Genotype identification of OsNPR3 and the OsNPR3 knockout mutant in the OsNPR3-MYC plant in Example 2 of the present invention, where:

[0064] Figure 2 A is the PCR identification of the overexpressed plants of the rice OsNPR3-MYC gene;

[0065] Figure 2 B is the Western blot identification of the OsNPR3-MYC transgenic rice;

[0066] Figure 2 C is the dCAPs digestion identification of the osnpr3-1 mutant.

[0067] Figure 3 Blast resistance identification of the OsNPR3-MYC plant and the OsNPR3 knockout mutant plant in Example 3 of the present invention ( Figure 3 A) and statistics ( Figure 3 B). DETAILED DESCRIPTION OF THE INVENTION

[0068] The following examples are used to further explain the present invention, but the examples do not limit the present invention in any form.

[0069] In order to make the purpose, technical solution and advantages of the present invention clearer, the following specific examples are used to further elaborate on the present invention in detail. The specific examples described here are only used to explain the present invention and are not used to limit the present invention. The raw materials involved in the following examples are commercially available unless otherwise specified, and the detection methods involved are conventional methods unless otherwise specified.

[0070] Example 1: Construction of Overexpression Vector of Rice OsNPR3

[0071] Cloning of Rice OsNPR3 Gene

[0072] Primers OsNPR3-F and OsNPR3-R were designed according to the full-length sequence SEQ ID NO.1 of OsNPR3, and the primer sequences used are as follows:

[0073] OsNPR3-F: 5’-GGGGTACCGATGTCCATCACTACTTGTACA-3’, SEQ ID NO.4;

[0074] OsNPR3-R: 5’-GACTAGTCCGTGATAGCTTCCCTTTCTTG-3’, SEQ ID NO.5;

[0075] PCR Amplification System: The total volume is 50 μL, including 5 μL of 10xPCR buffer, 2 μL of each of the F / R primers, 1 μL of dNTP, 2 μL of cDNA template, 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and 37 μL of ddH2O.

[0076] PCR Amplification Program: Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 2 min, 33 cycles, and final extension at 72°C for 5 min. The PCR product was subjected to gel extraction and ligated to the pCAMBIA1305-3xMYC vector (a plant expression vector based on the pCAMBIA1305.1 vector, with the 3xMYC tag sequence added at the multiple cloning site (MCS) after modification, Figure 1 between the SpeI and KpnI restriction sites of B). Single colonies were picked for colony PCR and then sequenced to obtain the correct pCAMBIA1305-OsNPR3-3xMYC recombinant plasmid. The correct recombinant plasmid was transferred into Agrobacterium tumefaciens EHA105 by the liquid nitrogen transformation method and sent to Changzhou Baige Transgenic Company for genetic transformation.

[0077] Example 2: Construction of Osnpr3 Mutant Plants Using CRISPR-Cas9 Technology

[0078] According to the OsNPR3 gene sequence (SEQ ID NO.1), the most suitable sgRNA sequence of the OsNPR3 gene was designed and screened using the online tool http: / / crispr.hzau.edu.cn / CRISPR2 / ( Figure 1(A), Design primers by adding specific adapters according to the pCBSG03 vector instruction manual. The sgRNA sequence for CRISPR-Cas9 knockout: 5’-TCTCGAGGTAACGATTGTCC-3’, SEQ ID NO.12; then synthesize the corresponding primers according to this sequence:

[0079] OsNPR3-CRISPR-F: 5’-TCAGTCTCGAGGTAACGATTGTCC-3’, SEQ ID NO.6;

[0080] OsNPR3-CRISPR-R: 5’-AAACGGACAATCGTTACCTCGAGA-3’, SEQ ID NO.7.

[0081] After primer synthesis, prepare the following reaction system: 2 μL of F primer, 2 μL of R primer, 96 μL of ddH2O, anneal at 37 °C for 30 minutes, and then ligate the annealed product into the pYLCRISPR / Cas9-MH vector linearized by BsaI digestion.

[0082] Ligation system:

[0083]

[0084] Make up to 10 μL with ddH2O and carry out a 2-hour ligation reaction at 25 °C. Subsequently, transform the ligation product into DH5α competent cells, coat it on an LB plate, and incubate it upside down at 37 °C for 12 - 16 hours. Pick single colonies for PCR verification, screen out positive clones, shake the bacteria to extract plasmids and send them for sequencing to obtain the correct recombinant plasmid. Send the verified recombinant plasmid to the company for genetic transformation to obtain Osnpr3 transgenic knockout materials. Through PCR identification and sequencing analysis, screen out the correct mutant Osnpr3-1 (insert a T in the second exon) for subsequent experimental research.

[0085] Example 3: Identification of transgenic rice

[0086] (1) Identify overexpression materials:

[0087] DNA extraction (CTAB method):

[0088] 1. Use an automatic sample grinder to grind at a frequency of 60 Hz for 90 seconds. Place an appropriate amount of rice leaves, 3-mm diameter steel beads, and 300 μL of CTAB solution in a 2.0 mL centrifuge tube and break the cells thoroughly.

[0089] 2. Incubate in an oven at 65 °C for 10 minutes to completely lyse the samples in the centrifuge tube.

[0090] 3. After cooling to room temperature, add 200 μL of chloroform to the centrifuge tube and mix the solution thoroughly by inverting the tube up and down.

[0091] 4. Centrifuge at 10,000 RPM for 10 minutes to separate the phases of the sample.

[0092] 5. Pipette 200 μL of the supernatant into a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix well, and place at -20 °C for 20 minutes to precipitate the DNA.

[0093] 6. Centrifuge at 12,000 RPM for 10 minutes, discard the supernatant, and collect the DNA precipitate.

[0094] 7. Wash the precipitate with 500 μL of 70% ethanol, centrifuge at 12,000 RPM for 5 minutes, discard the supernatant, repeat the washing once, and invert to air dry to remove residual ethanol.

[0095] 8. Add 300 μL of ddH2O to the centrifuge tube to dissolve the DNA precipitate and store at -20 °C for later use.

[0096] PCR Amplification:

[0097] Synthesize primers for PCR identification of OsNPR3-MYC. The primers are as follows:

[0098] OsNPR3-F: 5’-ATGCGCATAGCACAAGCT-3’, SEQ ID NO.8;

[0099] OsNPR3-R: 5’-GTATAATTGCGGGACTCTAATC-3’, SEQ ID NO.9.

[0100] After PCR identification, the results are as Figure 2 shown in A. There is no band in the wild-type NIP, while there is an obvious PCR band in the transgenic material, indicating that the transgenic material is correct.

[0101] At the same time, we also identified the transgenic material by Western blot experiment and detected it with Anti-MYC antibody. The results are as Figure 2 shown in B. The signal of the MYC-tagged protein can be detected in the transgenic material, but not in the NIP material. This further indicates that the transgenic material is correct and the target protein OsNPR3-MYC is expressed in rice.

[0102] (2) Identification of mutant materials:

[0103] Using the DNA of NIP and mutant plants as templates, amplification was performed with 2×Rapid Taq Plus Master Mix (Vazyme, P223-01). The primers were as follows:

[0104] Osnpr3-1-F: 5’-AGCCGCTTCTCGAGGTAACGATTGT-3’, SEQ ID NO.10;

[0105] Osnpr3-1-R: 5’-TGTCGCAGCGCCGATTATCTGAT-3’, SEQ ID NO.11.

[0106] The PCR amplification system was: 1 μL of template DNA, 5 μL of 2×Rapid Taq Plus Master Mix, 0.4 μL each of F primer and R primer, and supplemented with ddH2O to 10 μL. The PCR amplification program was: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 30 sec, for 35 cycles; final extension at 72°C for 2 min. The obtained PCR products were identified by digestion with PshAI (NEB, R0593). The homozygous Osnpr3-1 mutant could not be cut by the endonuclease, and the band was larger, while the control material NIP could be cut by PshAI, and the band was smaller ( Figure 2 C). Finally, the homozygous Osnpr3-1 mutant was obtained for subsequent inoculation experiments. Example 4: Analysis of rice blast inoculation and resistance of OsNPR3 overexpression and mutant materials

[0107] Materials and methods:

[0108] The steps for cultivating rice seedlings were as follows: Soak rice seeds in water for 2 days and then germinate them at 37°C under moisturizing conditions for 2 days. After the rice showed white tips, transfer them to the soil. After transplanting, transfer the seedlings to a long-day incubator and continue to cultivate until they had five leaves and one heart (pay attention to maintaining water and fertilizer supply during this period). The leaves of the grown seedlings could be used for inoculation.

[0109] The cultivation method of Magnaporthe oryzae is as follows: The tested Magnaporthe oryzae strain FJ81278 was provided by Professor Wang Zonghua of Fujian Agriculture and Forestry University (Chen et al., 2013) and was stored in this laboratory on dry filter paper in a 4°C refrigerator. When in use, the strain was transferred to SDC medium and activated in an incubator at 28°C for 7 days. The activated strain was transferred to a new SDC medium and continued to be cultured at 28°C for 5 - 7 days. Subsequently, the mycelia and spores growing on the surface of the medium were scraped off and then placed in an incubator at 28°C for about 2 days for inoculation experiments. Preparation method of SDC medium: Take 100 g of rice straw, boil it for 20 minutes, filter it through two layers of gauze, and discard the residue. Add 40 g of corn flour and 15 g of agar powder to the filtrate, and make up the volume to 1 L with ddH2O. Sterilize it at 115°C for 15 minutes, pour the plates, and store them at 4°C for later use.

[0110] The in vitro punching inoculation method is as follows: First, prepare a spore suspension of Magnaporthe oryzae strain FJ81278 with a concentration of 1x10 5 spores / mL, and add 0.02% Tween and mix well. Pipette 10 μL of the suspension and drop it on the pre-punched position of the rice leaf. Place the inoculated leaf in a moist petri dish and put it in an incubator at a temperature of 28°C and a humidity greater than 90%. First, conduct dark cultivation for 24 hours. Subsequently, adjust the cultivation conditions to an alternating light / dark environment (12 h / 12 h) and continue to culture for 7 days, and count the lesion length. Each material was inoculated at least 3 times repeatedly to ensure that the number of lesions counted was not less than 5.

[0111] The results showed that: Compared with the wild type NIP, both transgenic lines of the OsNPR3 - MYC material showed obvious disease resistance, while the Osnpr3 - 1 mutant material showed obvious disease susceptibility ( Figure 3 ). It shows that up - regulating the expression of OsNPR3 can significantly enhance the ability of rice to resist Magnaporthe oryzae, while knocking out or mutating the OsNPR3 gene can improve the susceptibility of rice to Magnaporthe oryzae.

[0112] The above - mentioned is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

[0113] The references involved in the text are as follows:

[0114] [1]C. Chen, et al., Genome comparison of two Magnaporthe oryzae field isolates reveals genome variations and potential virulence effectors. BMC Genomics 14, 887 (2013).

Claims

1. Application of OsNPR3 gene in regulating rice disease resistance, characterized in that: Overexpression of the OsNPR3 gene can improve rice resistance to rice blast, and knocking out or mutating the OsNPR3 gene can increase rice sensitivity to rice blast.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the OsNPR3 gene is shown in SEQ ID NO.1, and the CDS sequence of the OsNPR3 gene is shown in SEQ ID NO.

2.

3. Application of the protein encoded by the OsNPR3 gene in regulating rice resistance to rice blast, characterized in that: Increasing the expression of OsNPR3 protein can improve the resistance of rice to rice blast disease, while decreasing the expression of OsNPR3 protein can increase the sensitivity of rice to rice blast disease. The amino acid sequence of the protein is shown in SEQ ID NO.

3.

4. Application of recombinant vector containing OsNPR3 gene in improving rice resistance to rice blast.

5. The use according to claim 4, characterized in that: The basic vector of the recombinant vector containing the OsNPR3 gene is pCAMBIA1305-3xMYC, and the OsNPR3 gene is inserted between the SpeI and KpnI restriction sites to construct the recombinant vector.

6. Use of a recombinant engineered bacterium comprising the recombinant vector according to claim 4 in improving resistance of rice to rice blast.

7. Use of primers for amplifying the OsNPR3 gene of claim 1 in improving rice resistance to rice blast, characterized in that: The primers are: OsNPR3-F:5'-GGGGTACCGATGTCCATCACTACTTGTACA-3', SEQ ID NO.4; OsNPR3-R: 5'-GACTAGTCCGTGATAGCTTCCCTTTCTTG-3', SEQ ID NO.

5.

8. Application of OsNPR3 gene knockout vector in improving rice susceptibility to rice blast.

9. The use according to claim 8, characterized in that: The knockout vector is constructed by designing a sgRNA sequence of a CRISPR / Cas9 editing target for the rice blast resistance gene OsNPR3: 5'-TCTCGAGGTAACGATTGTCC-3', SEQ ID NO.12, and artificially synthesizing primers based on the sgRNA sequence and constructing them into a CRISPR / Cas9 vector.

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