Application of OsRbohI gene in regulating disease resistance in rice

Abstract

The present invention discloses the application of the OsRbohI gene in regulating rice disease resistance, belonging to the field of biotechnology. The present invention verified multiple OsRbohI mutants with different genetic backgrounds and found that they almost did not produce reactive oxygen species bursts induced by plant immune elicitors, and their resistance to the most destructive rice blast fungus was significantly weakened; Arabidopsis transgenic strains were obtained using the floral dipping transformation method. The OsRbohI protein driven by the Arabidopsis AtRbohD promoter can cross-species complement the reactive oxygen species burst phenotype of the dicotyledonous model plant atrbohd mutant, indicating that OsRbohI can play a cross-species role. The present invention proves the important role of OsRbohI in rice disease resistance, provides new gene resources for rice disease resistance breeding, and lays a foundation for the selection of disease-resistant varieties for other species.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to application of the OsRbohI gene in regulating rice disease resistance. Background Art

[0002] Rice (Oryza sativa) is one of the most important food crops, providing food for over half of the world's population. Its safe production is crucial for ensuring human survival and social stability. Plant diseases pose a serious threat to rice production safety, and the extensive use of pesticides pollutes the environment and poses a food safety risk. Therefore, harnessing the plant's natural immune system and identifying and utilizing rice's own disease-resistance genes to breed resistant varieties is currently the most cost-effective, environmentally friendly, and effective means of preventing and controlling diseases.

[0003] The plant innate immune system primarily consists of two levels: PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity). Extracellular reactive oxygen species (ROS) bursts are one of the most prominent immune responses in plants, mediated by the plasma membrane-localized NADPH oxidase, Rboh (respiratory burst oxidase homologs). ROS can directly kill pathogens but also serve as important signaling molecules to transmit and amplify other immune events in plants, playing a crucial role in plant resistance to pathogen invasion. In the dicotyledonous model plant Arabidopsis thaliana, AtRbohD is the primary protein responsible for ROS bursts during immunity. Atrbohd mutants exhibit virtually undetectable PAMP-induced ROS production and significantly diminished resistance to a variety of pathogens. In response to pathogen infection, PTI promotes full activation of the AtRbohD protein, while ETI enhances AtRbohD protein expression. These two proteins work synergistically to enhance plant disease resistance.

[0004] The rice genome encodes nine Rboh members (OsRbohA to I). Previous studies have reported that several OsRboh proteins may be involved in regulating rice reactive oxygen species burst and disease resistance. OsRbohB has been the most studied, but genetic evidence is less robust. Furthermore, the role of OsRbohI, the protein with the highest homology to Arabidopsis AtRbohD, in rice immunity and disease resistance remains unexplained. Given the importance of reactive oxygen species burst in plant immunity, identifying and characterizing the key proteins involved in the reactive oxygen species burst in rice innate immunity is crucial for rice resistance breeding. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of the OsRbohI gene in regulating rice disease resistance to solve the problems existing in the above-mentioned prior art. The OsRbohI gene positively regulates rice resistance to rice blast fungus.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an application of the OsRbohI gene in regulating rice disease resistance. The OsRbohI gene positively regulates rice disease resistance. The nucleotide sequence of the OsRbohI gene is shown in SEQ ID NO: 1.

[0008] The present invention also provides use of a protein encoded by the OsRbohI gene in regulating rice disease resistance. The OsRbohI gene positively regulates rice disease resistance. The nucleotide sequence of the OsRbohI gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein is shown in SEQ ID NO: 2.

[0009] The present invention also provides use of a recombinant vector comprising the OsRbohI gene in regulating rice disease resistance. The OsRbohI gene positively regulates rice disease resistance. The nucleotide sequence of the OsRbohI gene is shown in SEQ ID NO: 1.

[0010] The present invention also provides the use of a recombinant engineering bacterium containing the recombinant vector in regulating rice disease resistance.

[0011] Preferably, the OsRbohI gene positively regulates rice disease resistance, including positively regulating rice resistance to rice blast.

[0012] The present invention also provides use of a reagent for detecting the expression level of the OsRbohI gene in rice disease resistance breeding. The nucleotide sequence of the OsRbohI gene is shown in SEQ ID NO: 1.

[0013] Preferably, the expression of the OsRbohI gene is detected in the rice, indicating that the rice is resistant to rice blast fungus.

[0014] The present invention also provides the use of the OsRbohI gene in regulating disease resistance in Arabidopsis thaliana. By driving the expression of the protein encoded by the OsRbohI gene in Arabidopsis thaliana across species, the reactive oxygen species burst phenotype of the Arabidopsis thaliana is restored, thereby enhancing the immune response of the Arabidopsis thaliana and thereby improving disease resistance.

[0015] The nucleotide sequence of the OsRbohI gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein is shown in SEQ ID NO: 2.

[0016] The present invention also provides a method for regulating disease resistance of rice, comprising the step of positively regulating the expression of the OsRbohI gene in rice, wherein the nucleotide sequence of the OsRbohI gene is shown in SEQ ID NO: 1.

[0017] Preferably, the resistance of rice to rice blast is improved by positively regulating the expression of the OsRbohI gene in rice.

[0018] The present invention discloses the following technical effects:

[0019] The present invention experimentally verified that multiple OsRbohI mutants with different genetic backgrounds almost do not respond to pathogen-associated molecular pattern molecules (PAMPs, hereinafter referred to as PAMPs)-induced reactive oxygen species burst, demonstrating that OsRbohI is the main effector protein in the important process of reactive oxygen species burst in the rice PTI immune system; OsRbohI mutants have significantly reduced resistance to rice blast, further demonstrating its importance to the rice innate immune system; in addition, transgenic Arabidopsis lines were obtained by the floral dip method, and the OsRbohI protein driven by the Arabidopsis AtRbohD promoter can cross-species complement the reactive oxygen species burst phenotype of the dicotyledonous model plant atrbohd mutant, indicating that OsRbohI can act across species to enhance the immune response of Arabidopsis, further demonstrating the important role of OsRbohI in plant disease resistance. The present invention proves that the OsRbohI gene can positively regulate rice resistance to rice blast fungus and can also enhance the immune response of Arabidopsis thaliana by complementing the gene. The present invention provides a new gene resource for rice disease-resistant breeding and can also be applied to the selection of disease-resistant varieties of other plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1Figure 3. T-DNA insertion sites or editing patterns of four different types of OsRbohI mutants (osrbohi-1 to osrbohi-4). A: Genetic diagram of OsRbohI. Gray rectangles represent exons, black lines represent introns, transparent rectangles represent noncoding regions, black triangles represent CRISPR knockout targets, and transparent triangles represent T-DNA insertion sites. B: Editing patterns of two CRISPR knockout mutants. Blue letters represent target sequences, red represents PAM sequences, and black dashes represent deletion sequences.

[0022] Figure 2 Detection of gene expression levels of two T-DNA-inserted OsRbohI mutants; A: RT-PCR detection results, B: qRT-PCR detection results;

[0023] Figure 3 Figure 2. PAMP-induced ROS bursts in four OsRbohI mutants and the corresponding wild type. A: The curve represents the mean ± SE of ROS generated in real time. B: The total ROS generated in 45 minutes in the curve of Figure A.

[0024] Figure 4 Three OsRbohI mutants and the corresponding wild type were inoculated with rice blast fungus; A: Photos of lesions, percentage of lesions, and biomass of rice blast fungi were taken after inoculation with DJ and osrbohi-1 in race RB22 of rice blast fungus; B: Photos of lesions, percentage of lesions, and biomass of rice blast fungi were taken after inoculation with ZH11, osrbohi-3, and osrbohi-4 in race RB22 of rice blast fungus;

[0025] Figure 5 Figure 3. Reactive oxygen burst in Arabidopsis atrbohd mutant complemented by OsRbohI; A: Schematic diagram of the vector for exogenous expression of OsRbohI in transformed Arabidopsis; B: Detection of OsRbohI gene expression in transformed positive plants; C: Detection of reactive oxygen burst in transformed positive plants, wild type Col-0, and mutant atrbohd. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] The OsRbohI gene disclosed in this invention is named LOC_Os11g33120 and is based on the majority of literature reporting its function. Its CDS sequence is SEQ ID NO. 1 in the sequence listing, and the protein encoded by this gene is named OsRbohI, with the amino acid sequence of this protein being SEQ ID NO. 2 in the sequence listing. The cDNA of the OsRbohI gene is 2811 base pairs long and encodes a protein containing 936 amino acids.

[0032] SEQ ID NO.1, full-length coding nucleotide sequence of gene OsRbohI:

[0033]

[0034] SEQ ID NO.2: OsRbohI full-length protein amino acid sequence

[0035] MHHTRAGAADGAGGGGEDIVEAGAEAPPPQRERLVPHSGPLSKRSGMRKSARFAESVSAPLSAPSGVAAARRAAAAANDDDEEDYVEITLDVRDDSVAVHSVKPAGGGGEDSDVTLLARTLEKRSSSFGHSVIRNASSRIKQVSQELRRLASVNRRGGGGGGPRFDRSKSAAAHALKGLKFISRADGGAGWPAVEKRFDDLAKDGLLPRSKFGQCIGMKELEFAGELFDALARRRNISGDSISKAELLEFWDQISDTSFDSRLQTFFDMVDKNADGRITEEEVKEIITLSASANKLSKVQEQSEEYARLIMEELDPSNLGYIELYNLEMLLLQAPSQSVRIGTTNSRNLSQMLSQNLRPTAEPNPLRRWWRRASYFLEDNWRRVWVLLLWLAICAGLFTYKFIQYRHRAVFHVMGYCVCVAKGGAETLKFNMALILLPVCRNTVTWIRNRAAVARVVPFDDNLNFHKVIAVGITVGAGLHVISHLTCDFPRLLHATDAEYEPMKRFFGDTRPPNYWWFVKGTEGWTGLVMLVLMAVAFTLATPWFRRGRLRLPRPLNRLTGFNAFWYSHHCFVIVYALLIVHGYYLFLTKDWYKKTTWMYLAVPMFLYACERLTRALRSSVRPVKILKVAVYPGNVLSLHFSKPQGFKYKSGQYIFVNCAAVSPFQWHPFSITSAPQDDYVSVHIRTLGDWTRELKNVFSRVCRPPTEGKSGLLRAEYDRDGAMTNPSFPKVLIDGPYGAPAQDYKQYDIVLLVGLGIGATPMISIIKDIINNMRQLDGDLEDGDGNDNSVSSSSAAFKTRRAYFYWVTREQGSFEWFRGVMDEVAETDKKGVIELHNYCTSVYEEGDARSALIAMLQSLNHAKHGVDVVSGTRVKTHFARPNWRNVYKRIALNHRDQRVGVFYCGAPVLTKELRELAQDFSRKTSTKFDFHKENF

[0036] The following verification focuses on the correlation between the OsRbohI gene and rice disease resistance, mainly through two T-DNA insertion mutants and two knockout mutants for specific verification and explanation.

[0037] Example 1 Plant material cultivation

[0038] Rice seeds were surface-sterilized with 70% alcohol (v / v) for 3 minutes, then sterilized with 50% sodium hypochlorite solution by shaking at 100 rpm for 40 minutes. The seeds were then rinsed 10 times with sterile water and evenly plated on 1 / 2 MS solid culture medium. After 7-10 days, the seeds were transferred to long pots and grown in an artificial greenhouse with a 12-hour light / 12-hour dark cycle, 80% relative humidity, and a room temperature of 28°C.

[0039] Arabidopsis seeds were disinfected with 75% alcohol (v / v) for 10 minutes and then washed five times with sterile water. They were then imbibed in a dark place at 4°C for three days and germinated on 1 / 2MS solid culture medium. After seven days, they were transferred to small pots and grown in an artificial greenhouse at 22°C with a 16-hour light / 8-hour dark cycle and 65% relative humidity.

[0040] Example 2 Identification and Expression Level Detection of T-DNA Insertion Mutants of OsRbohI

[0041] To identify the function of the OsRbohI gene in rice disease resistance, two T-DNA insertion mutants of OsRbohI with different backgrounds, osrbohi-1 (type is a T-DNA insertion mutant, the target is the OsRbohI gene, the mutant number is 3A-05869, the background is Dongjin, abbreviated as DJ) and osrbohi-2 (type is a T-DNA insertion mutant, the target is the OsRbohI gene, the mutant number is 1A-24742, the background is Hwayoung, abbreviated as HY), were purchased from Pohang University of Science and Technology, South Korea. T-DNA insertion identification was performed on the mutant rice materials, and finally homozygous mutants were obtained (see Figure 1 Total RNA from the mutants and the corresponding wild-type samples was extracted using a plant RNA rapid extraction kit (MiniBEST Universal RNA Extraction Kit from TaKaRa). cDNA was synthesized by reverse transcription using a reverse transcription kit (First-strand cDNA synthesis kit from TOYOBO), and amplified using the following primers:

[0042] OsRbohI upstream primer (OsRbohI-RT-F): 5′-CGGATGGCAGGATCACAGAA-3′, SEQ ID NO. 3;

[0043] OsRbohI downstream primer (OsRbohI-RT-R): 5′-CGATGCGCTTGTAGACGTTG-3′, SEQ ID NO. 4;

[0044] Internal reference gene OsActin upstream primer (OsActin-RT-F): 5'-ATCCTTCGTCTCGACCTTGC-3', SEQ ID NO. 5;

[0045] Internal reference downstream primer (OsActin-RT-R): 5'-TGGCAGTCTCCATTTCCTGG-3', SEQ ID NO.6.

[0046] Reaction system (20 μL): 10 μL of 2×Hieff PCR Master Mix from Shanghai Yisheng Company; 0.5 μL each of 10 μM upstream primer and downstream primer; 1.0 μL of cDNA template; add water to 20 μL.

[0047] PCR amplification was performed on a Bio-Rad PCR instrument, and the expression of the OsRbohI gene in the mutant was detected by semi-quantitative RT-PCR, as shown in Figure 2 As shown in center A, OsRbohI is not expressed in either osrbohi-1 or osrbohi-2 homozygous mutants.

[0048] Two pairs of qRT-PCR primers were further used for more accurate expression detection:

[0049] Upstream primer (q-OsOsRbohI-RT-F1): 5'-GACGGCAACGATAACTCGGT-3', SEQ ID NO. 7; Downstream primer (q-OsRbohI-RT-R1): 5'-TTGAGCGACTGGAGCATAGC-3', SEQ ID NO. 8;

[0050] Upstream primer (q-OsOsRbohI-RT-F3): 5'-AACTGGCGCAACGTCTACAA-3', SEQ ID NO. 9; downstream primer (q-OsRbohI-RT-R3): 5'-AACTGGCGCAACGTCTACAA-3', SEQ ID NO. 10.

[0051] The reaction system was prepared using a kit from Nanjing Novozymes: 10 μL of 2× ChamQ Universal SYBR qPCR Master Mix; 0.4 μL each of 10 μM upstream and downstream primers; 2.0 μL of cDNA template; and water was added to 20 μL. PCR amplification was performed using a two-step method on a real-time fluorescence quantitative PCR instrument. The results are shown in the figure. Figure 2 As shown in B, the expression of OsRbohI was almost completely undetectable in the two mutants.

[0052] Example 3 Identification of CRISPR knockout mutants of OsRbohI

[0053] In order to obtain more mutant strains, CRISPR knockout mutant seeds were obtained from the research group of Zhao Fangjie of Nanjing Agricultural University and Hangzhou Baige Biotechnology Co., Ltd. The genomic DNA of the mutants was extracted by CTAB method and used as a template. PCR amplification (Shanghai Yisheng Company, 2×HieffPCRMaster Mix) was performed for three different targets. The obtained products were sent to Shanghai Qingke Company for DNA sequencing, and the following were obtained: Figure 1 The two homozygous lines in B are osrbohi-3 and osrbohi-4. The sequencing primers used for the three knockout targets are:

[0054] Target 1: upstream primer OsRbohI-T1-F: 5'-ATCAAGCAGGTGTCGCAGGA-3' (SEQ ID NO. 11) and downstream primer OsRbohI-T1-R: 5'-CGAAGCTGGTGTCGGAGATTT-3' (SEQ ID NO. 12);

[0055] Target 2 and 3: upstream primer OsRbohI-T2 / 3-F: 5'-CAGGAGCAGTCCGAGGAGTA-3' (SEQ ID NO. 13) and downstream primer OsRbohI-T2 / 3-R: 5'-GTAGCCCATGACGTGGAACA-3' (SEQ ID NO. 14), PCR amplification reagents and equipment used are the same as in Example 1. The results are shown in FIG. Figure 1 As shown in B, both osrbohi-3 and osrbohi-4 are homozygous CRISPR knockout mutants.

[0056] Example 4 Genetic transformation and positive identification of Arabidopsis

[0057] Vector construction: Genomic DNA of Arabidopsis wild type Col-0 was extracted as a template, and the upstream primer AtRBOHD-PF: 5'-CCCGAATTACTGCAGGAATAGTGTATGGTTAGGGTC-3' (SEQ ID NO. 15) and the downstream primer AtRBOHD-PR: 5'-CCCGAATTACTGCAGGAATAGTGTATGGTTAGGGTC-3' (SEQ ID NO. 16) were used to amplify the 2041 bp promoter sequence upstream of its coding region. PCR amplification was performed using Phanta Super-Fidelity DNA Polymerase (NanoVibans, Nanjing). After gel running, the PCR product was recovered using a mini-purification kit (Shanghai Huiling Company). The PCR product and the vector pRHVnHA were digested with restriction enzymes from New Brunswick, purified, and ligated with T4 ligase from New Brunswick to replace the ubiquitin promoter sequence on the original vector to form a new vector (see ). Figure 5 Middle A).

[0058] Appropriate amounts of leaves from rice ZH11 seedlings grown for 30 days under a normal photoperiod were collected and ground with liquid nitrogen. Total RNA was extracted using a plant RNA rapid extraction kit (MiniBEST Universal RNA Extraction Kit from TaKaRa) and reverse transcribed into cDNA using a reverse transcription kit (First-strand cDNA synthesis kit from TOYOBO). The cDNA of rice ZH11 was then used as a template to amplify the CDS sequence of OsRbohI using the upstream primer AtDp-OsRbohI-F: 5'-AGATTACGCTGGATCCATGCACCATACCAGGGCTGC-3' (SEQ ID NO. 17) and the downstream primer AtDp-OsRbohI-R: 5'-CCGCACTAGTAAGCTTCTAGAAATTCTCCTTGTGGAAGTCG-3' (SEQ ID NO. 18). The cDNA was cloned using the same method and homologous recombination enzymes (ClonExpress Ultra One Step cloning kit, Nanjing Novozymes) were used. kit) for seamless cloning. The recombinant plasmid was confirmed by DNA sequencing (Shanghai Qingke Company) and then transformed into Agrobacterium GV3101 competent cells. The Arabidopsis atrbohd mutant was genetically modified by the floral dip method. The mature seeds were screened for positive seedlings on 1 / 2 MS solid medium containing 50 mg / mL hygromycin. Total RNA was extracted and reverse transcribed into cDNA. The upstream primer OsRbohI-RT-F2: 5'-TACTGGTGGTTCGTGAAGGG-3' (SEQ ID NO. 19); the downstream primer OsRbohI-RT-R2: 5'-AGTCGAATTTCGTGCTCGTCT-3' (SEQ ID NO. 20); the upstream primer of the internal reference gene AtActin2: (AtActin2-RT-F): 5'-ATCCTTCGTCTCGACCTTGC-3' (SEQ ID Semi-quantitative RT-PCR detection was performed using the downstream primer for the internal reference gene OsActin (AtActin2-RT-R): 5'-GAGACACACCATCACCAGAAT-3' (SEQ ID NO. 22). DNA extraction, RNA extraction, reverse transcription, PCR amplification, agarose gel electrophoresis, gel excision and recovery, gene fragment digestion, DNA purification, ligation, transformation, and plasmid extraction are all standard molecular experiments. Specific steps are described in the Molecular Cloning Laboratory Manual (Science Press, 2017) and the instructions for each kit.

[0059] The results are as follows Figure 5As shown in B, the expression of the rice OsRbohI gene can be detected in the Arabidopsis atrbohd mutant.

[0060] Example 5 Determination of active oxygen burst in rice and Arabidopsis materials

[0061] For rice materials, leaf sheaths of the OsRbohI mutant and the corresponding wild type, grown for 10 to 12 days in glass bottles containing 1 / 2 MS solid medium, were uniformly cut into 3 mm long segments. These segments were gently picked up with tweezers and placed in a 96-well microplate containing 100 μL of sterile water, with three leaf sheath segments per well. The plates were covered and incubated in the dark overnight. The next morning, the sterile water was removed with a pipette and replaced with a reaction solution containing 50 mM Tris-Cl, pH 7.5, 10 μg / mL horseradish peroxidase (HRP, Bio-Rad), 10 μM L-012 chemiluminescent probe, and either 50 μM flg22 peptide (Shanghai Sangon Biotechnology) or 20 μg / mL chitin (Sigma). The reaction solution was then placed in a Varioskan Flashmultireader (BioTek) and measured for 45 minutes. The total amount of reactive oxygen species was calculated using the formula:

[0062] ˉˉ

[0063] Total active oxygen content = [sum(y1:y n )×2-y1-y n ]×time interval / measurement time per well / 2, that is, Figure 5 The area of ​​the irregular figure formed by the curve and the horizontal axis shown in A is divided into multiple right-angled trapezoids to calculate the total area. In this experiment, n=45, the time interval is 1 minute, and the measurement time for each well is 400ms.

[0064] The final measurement results are shown in Figure 3 The results showed that reactive oxygen species induced by the two most commonly used PAMPs, flg22 and chitin, were almost undetectable in the T-DNA insertion homozygous mutants of the OsRbohI gene in the Dongjin and Hwayoung backgrounds and the CRISPR knockout homozygous mutant in the ZH11 background (i.e., osrbohi-4).

[0065] For Arabidopsis thaliana materials, take Arabidopsis thaliana leaves that have grown uniformly for 4 weeks in a growth chamber, use a hole punch to take leaf discs with a diameter of 4 mm, and transfer them to a 96-well microplate containing 100 μL of sterile water, with one leaf disc per well. Cover and treat overnight in the dark. The next morning, use a pistol to remove the sterile water and replace it with a reaction solution containing 50 mM Tris-Cl, pH 7.5, 10 μg / mL HRP (Bio-Rad), 10 μM L-012, and the plant immune elicitor 1 μM flg22 (Shanghai Shenggong Biological) or 20 μg / mL chitin (Sigma). Then quickly place it in the microplate reader Varioskan Flash multireader (BioTek) for measurement for 45 minutes. The total amount of reactive oxygen species is calculated according to the formula, and the final measurement results are shown in the table. Figure 5 Center C, Expression of the rice OsRbohI protein in the Arabidopsis atrbohd mutant partially rescued its PAMP-induced ROS burst phenotype.

[0066] Example 6 Inoculation of OsRbohI mutant material with rice blast fungus

[0067] To identify the function of OsRbohI in rice disease resistance, the mutant strains and the corresponding wild type as controls were inoculated with the rice blast fungus Magnaporthe oryzae (M.oryzae) race RB22. Figure 4 As shown in the figure, the lesion area and rice blast fungus biomass of the OsRbohI mutant strain were significantly higher than those of the wild type, indicating that the OsRbohI gene knockout weakened the resistance to bacterial leaf streak disease. OsRbohI positively regulates the resistance of rice to rice blast fungus, which is consistent with its lack of production of PAMP-induced reactive oxygen species.

[0068] 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. Application of the OsRbohI gene in regulating disease resistance in Arabidopsis thaliana, characterized in that: By driving the expression of the protein encoded by the OsRbohI gene in the Arabidopsis thaliana atrbohd mutant across species, the reactive oxygen species burst phenotype induced by pathogen-associated molecular patterns in the Arabidopsis thaliana atrbohd mutant is restored, thereby enhancing the immune response of the Arabidopsis thaliana and thereby improving disease resistance; The pathogen-associated molecular patterns include flg22 and chitin; The nucleotide sequence of the OsRbohI gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein is shown in SEQ ID NO: 2.

Images