Application of biological products related to osntl5 gene in improving disease resistance of rice

Abstract

This invention relates to the field of plant genetic engineering technology, specifically to OsNTL5 The application of gene-related biological products in improving disease resistance in rice, the OsNTL5 The genomic nucleotide sequence of the gene is shown in SEQ ID NO.4; the open reading frame sequence of the OsNTL5 gene is shown in SEQ ID NO.5. This invention involves knocking out the OsNTL5 gene in the rice variety ZH11. OsNTL5 Gene, obtain knockout mutant osntl5 It increases resistance to rice blast and rice bacterial blight, and is induced by the plant immune elicitor flg22. osntl5 The reactive oxygen species (ROS) burst level is significantly higher than that of ZH11. Therefore, the reactive oxygen species (ROS) in this invention... OsNTL5 Genes and their proteins are of great significance for breeding new varieties of broad-spectrum disease-resistant rice and are suitable for widespread application.

Description

Technical Field

[0001] This invention relates to plant genetic engineering, specifically to... OsNTL5 Application of gene-related bioproducts in improving disease resistance in rice. Background Technology

[0002] Rice is frequently threatened by various pathogens, especially by the fungus *Oryza sativa* (or *Oryza sativa*). Magnaporthe oryzae Rice blast caused by Xanthomonas oryzae and pathogenic species of Xanthomonas oryzae ( Xanthomonas oryzae pv. oryzae, Xoo This poses a threat to rice bacterial blight caused by [a disease / pathogen]. Therefore, discovering effective defense genes is crucial for the survival of rice.

[0003] To achieve effective and long-lasting resistance to diseases, existing technologies involve introducing resistance into superior rice varieties. R Genes, such as Xa21 Pi series genes, these genes mediate race-specific immunity, ultimately resulting in broad-spectrum resistance. BSR (This refers to a specific strain.) However, this strategy suffers from a long development cycle and poor resistance persistence.

[0004] Therefore, there is an urgent need to discover new genes in rice to provide a new strategy for improving rice's disease resistance. Summary of the Invention

[0005] To solve the above problems, the present invention provides OsNTL5 Application of gene-related bioproducts in improving disease resistance in rice.

[0006] This invention is achieved through the following technical solution: OsNTL5 The application of gene-related biological products in improving disease resistance in rice, the OsNTL5 The genomic nucleotide sequence of the gene is shown in SEQ ID NO.4; the open reading frame sequence of the OsNTL5 gene is shown in SEQ ID NO.5.

[0007] Preferably, the OsNTL5 The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.6.

[0008] Preferably, the biological product is OsNTL5 Gene knockout vectors or recombinant engineered bacteria.

[0009] Preferably, the recombinant knockout vector comprises a backbone plasmid and a nucleotide sequence as shown in SEQ ID NO.1.

[0010] Preferably, the backbone plasmid is pCBSG032.

[0011] Preferably, the recombinant engineered bacteria are obtained by introducing Agrobacterium into the recombinant knockout vector.

[0012] Preferably, the Agrobacterium is EHA105.

[0013] Preferably, the recombinant knockout vector or recombinant engineered bacteria are used to reduce the concentration of certain substances in rice. OsNTL5 Increasing gene expression levels to enhance rice disease resistance.

[0014] Preferably, improving rice disease resistance refers to improving rice's resistance to rice blast and / or bacterial blight.

[0015] Preferably, the rice blast disease is caused by the fungus *Magnaporthe oryzae* (…). Magnaporthe oryzae The bacterial blight is caused by the pathogenic strain of Xanthomonas oryzae (…). Xanthomonas oryzae pv. oryzae Caused by ).

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first discovery of rice OsNTL5 Negative regulation of rice immunity provides a foundation for studying the molecular mechanisms by which this gene family regulates rice immunity. The results of this invention indicate that, compared to wild-type ZH11, rice... OsNTL5 Knockout osntl5 Increases resistance to rice blast and bacterial blight, important diseases of rice. Furthermore, compared to ZH11, under the induction of the plant immune elicitor flg22 (the most conserved 22 amino acid epitopes at the N-terminus of bacterial flagellin), osntl5 The knockout mutant exhibited a significantly stronger reactive oxygen species (ROS) burst, indicating that the rice disease resistance-related genes... OsNTL5 Its proteins have potential targets for development into new rice varieties with broad-spectrum disease resistance. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Compared with wild-type ZH11, this invention... osntl5 Figure showing the resistance of knockout mutants to rice bacterial blight and rice blast; Figure 1 In the diagram, A shows typical symptoms of rice bacterial blight after inoculation; B shows ZH11 and... osntl5C is a statistical diagram of leaf lesion length after inoculation with rice bacterial blight pathogens following the removal of the knockout mutant; D is a diagram of typical disease symptoms after inoculation with rice blast pathogens; and D is a diagram of ZH11 and... osntl5 A statistical chart of the relative biomass of rice blast fungus after inoculation of the knockout mutant with rice blast fungus; ** indicates p <0.01, *** indicates p <0.001.

[0019] Figure 2 This invention, under the induction of the plant immune elicitor flg22, compared with ZH11, osntl5 The knockout mutant exhibits a higher level of reactive oxygen species (ROS) bursts. Detailed Implementation

[0020] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0022] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the beneficial effects of this invention are illustrated below through specific embodiments. In the description of this invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0023] osntl5 The knockout mutant was purchased from Weimi Biotechnology Co., Ltd., and constructed using the CRISPR-Cas9 method. The specific construction method is as follows: 1. Knockout vector construction The target knockout site (SEQ ID NO.1) was selected as 5'-AACCATTCTGAAGACGTGGATGG-3'. The pCBSG032 vector (preserved by Weimi Biotechnology Co., Ltd.) was linearized by BsaI restriction enzyme digestion. The target fragment containing the knockout target sequence was ligated into the linearized vector by homologous recombination. After transformation into E. coli, the vector was screened by kanamycin and sequenced to obtain the correct recombinant knockout vector.

[0024] 2. Callus induction and subculture Select mature rice seeds, remove the husks, and pour them into 50mL centrifuge tubes. Add 75% ethanol (v / v) for 1 min to sterilize, discard the ethanol, rinse once with sterile water, discard the ethanol, then add 30% sodium hypochlorite (v / v) for 20 min to sterilize, discard the sodium hypochlorite, and rinse 6 times with sterile water. Use a pipette to remove excess water (or use sterile filter paper to blot dry), and transfer the seeds to induction medium, 25 seeds per dish.

[0025] After callus grows, it can be directly transformed using the protoembryo. The small particles growing next to the protoembryo can be picked and placed on a new induction medium for subculture. When they grow to a suitable size, they can also be transformed.

[0026] 3. Agrobacterium culture Agrobacterium EHA105 containing the target gene vector was streaked on a plate containing the corresponding antibiotic and incubated in the dark at 28°C for 2 days until a single colony appeared.

[0027] 4. Agrobacterium infection Prepare the infection solution. Use a pipette to draw up the infection solution and wash off the Agrobacterium on the plate to obtain the Agrobacterium suspension used for co-culture transformation of rice.

[0028] Select callus tissue (good callus condition, bright yellow color, smooth and firm texture, with a particle diameter of 3 mm or less) and place it in a 100 mL sterile Erlenmeyer flask. Add Agrobacterium suspension and incubate at room temperature for 20 minutes. Discard the bacterial suspension, place the callus tissue on sterile filter paper to absorb excess bacterial suspension, and then transfer it to a solid co-culture medium lined with a layer of sterile filter paper. Incubate at 26°C in the dark for 3 days.

[0029] 5. Screening and Cultivation After 3 days of co-culture, the callus tissue needs to be cleaned. Using a 1mL blue pipette tip, the callus on the co-culture medium is transferred to a sterile Erlenmeyer flask. Sterile water is added and the flask is rinsed twice. A third rinse is made with sterile water containing 500µl / L carbenicillin. Excess water is removed with a pipette, and the callus is transferred to sterile filter paper. The callus is then dried using the airflow in a laminar flow hood for approximately 30 minutes. Once dry, the callus is transferred to selection medium for selection culture at 30°C in the dark. The selection process lasts for 4 weeks.

[0030] 6. Differentiation and regeneration One month after selection, bright yellow positive calluses will appear. At this time, the positive calluses can be picked and transferred to differentiation medium for differentiation and regeneration. Place 16 positive calluses on each differentiation dish and culture them under light in a 30℃ greenhouse. Green spots will appear on the calluses after 10 days, and seedlings will differentiate after about 10 days.

[0031] 7. Seedling rooting Once the differentiated seedlings reach 3cm in height and have a distinct root system, transfer them to a rooting medium to allow them to grow. The rooting medium should be poured into relatively tall bottles or tubes to provide sufficient space for the rooted seedlings to grow taller. Rooting culture conditions are 30℃ under sterile light. osntl5 Gene knockout mutant.

[0032] ZH11 wild type: This wild type rice material is the japonica rice variety Zhonghua 11, bred by the Institute of Crop Science, Chinese Academy of Agricultural Sciences (National Rice Data Center, http: / / www.ricedata.cn / variety / varis / 601422.htm). Xanthomonas oryzae pathogenic species ( Xanthomonas oryzae pv. oryzae )for Xoo Strain PXO99 A Published in the reference: Salzberg SL, Sommer DD, Schatz MC, et al. Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv. oryzae PXO99 A .BMC Genomics. 2008;9:204. DOI: 10.1186 / 1471-2164-9-204. Rice blast fungus ( Magnaporthe oryzae The reference for RB22 of rice blast fungus is: [1] Zhu Yongsheng, Cai Qiuhua, Xue Weimin, et al. Preliminary analysis of the genotype of rice blast resistance in Yunyin and construction of near-isogenous lines [J]. Fujian Journal of Agricultural Sciences, 2012, 27(1): 1-6. [2] Ning Y, Shi X, Wang R, Fan J, Park CH, Zhang C, Zhang T, OuyangX, Li S, Wang GL. OsELF3-2, an Ortholog of Arabidopsis ELF3, Interacts with the E3 Ligase APIP6 and Negatively Regulations Immunity against Magnaporthe oryzae in Rice. Molecular Plant. 2015;8(11):1679-1682. DOI: 10.1016 / j.molp.2015.08.004. Example 1: Identification of rice OsNTL5 Gene knockout mutant osntl5 Knockout type OsNTL5 Gene knockout mutant 1. In OsNTL5 Design and identification of 5'-3' primers for upstream and downstream detection of gene knockout target osntl5 Knockout type of knockout mutant. Upstream detection primer F osntl5 (SEQ ID NO.2): 5'-AGAGGAGCGTATTGTTCG-3'; Downstream detection primer R osntl5 (SEQ ID NO.3): 5'-GGATTCATTGGCTAAAGG-3', the amplified target fragment is 472bp in size.

[0033] Include OsNTL5 The full-length nucleotide sequence of the rice genome (from the first bolded start codon ATG to the second bolded stop codon TAG) is SEQ ID NO.4.

[0034]

[0035] 2. Prepare a 2% (w / v) CTAB Buffer for extracting rice genome.

[0036] Table 1. Preparation method of 2% CTAB Buffer for extracted genome samples (volume fraction). 3. Extraction of ZH11 wild type and osntl5 Experimental steps for knocking out the genome of a mutant 1) Cultivating ZH11 (wild type) and osntl5 (Knockout mutant) When the sample grew to the three-leaf-one-heart stage in the South Pond of the Greenhouse of China Agricultural University, the leaves were cut off with scissors and placed in a 2mL centrifuge tube. A 3mm steel ball was added, and the sample was flash-frozen in liquid nitrogen. The sample was then ground at a frequency of 45Hz using an electric grinder.

[0037] 2) Add 1 mL of 2% CTAB Buffer, mix well, and incubate at 65°C for 30 min, inverting and mixing once every 10 min. Then centrifuge at 12000 rpm for 10 min.

[0038] 3) Transfer 500 µL of the supernatant to a new 1.5 mL centrifuge tube, add 500 µL of a mixture of chloroform and isoamyl alcohol (24:1), mix well, and centrifuge at 12000 rpm for 10 min. At this point, the sample will separate into layers.

[0039] 4) Pipette 450 µL of the supernatant into a new 1.5 mL centrifuge tube, add 2 / 3 volume of isopropanol, mix well, and then cover the centrifuge tube plate with a resealable bag and let it stand overnight at -20°C.

[0040] 5) Centrifuge the overnight samples at 12,000 rpm for 10 min, discard the supernatant, add 1 mL of anhydrous ethanol to each sample tube, mix by inversion, and centrifuge at 12,000 rpm for 10 min.

[0041] 6) Carefully aspirate and discard the supernatant, leaving the white precipitate. Add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 12000 rpm for 10 m, aspirate and discard the supernatant, and air dry the precipitated genome in a clean bench.

[0042] 7) Dissolve the dried genome in ddH2O, then store at -20℃ for later use.

[0043] 4. Use the primers designed in step 1 to perform PCR amplification of ZH11 and osntl5 Knockout target fragment in mutant genome 1) PCR was performed using the Phanta Max Super-Fidelity DNA Polymerase kit from Nanjing Novizan Biotechnology Co., Ltd. First, the samples were mixed according to Table 2 below.

[0044] Table 2 PCR amplification target fragment formulation 2) Amplify the target fragment in the PCR instrument according to the procedure in Table 3 below.

[0045] Table 3. PCR Procedure for Amplifying the Target Fragment 3) Use a 1% (w / v) agarose gel to electrophoretically separate the target fragment from the PCR stock solution in step 2).

[0046] 5. The target fragment was recovered using the Gel Extraction Kit from Jiangsu Kangwei Century Biotechnology Co., Ltd.

[0047] 1) Precisely cut out the gel containing the target DNA band and place it in a 2 mL centrifuge tube.

[0048] 2) Add 500 µL PG and dissolve the gel block in a 50°C water bath.

[0049] 3) Add 200 μL PS to the adsorption column, centrifuge at 13000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0050] 4) Take the solution from step 2) and add it to the adsorption column. Centrifuge at 13000 rpm for 1 min and discard the waste liquid in the collection tube.

[0051] 5) Add 450 μL of PW rinsing solution, centrifuge at 13000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0052] 6) Repeat step 5) once.

[0053] 7) Discard the waste liquid in the collection tube, centrifuge at 13000 rpm for 2 min, place the adsorption column in a 1.5 mL centrifuge tube, add 50 μL Buffer EB, let stand for 3 min, and centrifuge at 13000 rpm for 1 min.

[0054] 6. The recovered target fragment was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and ZH11 and osntl5 By comparing the sequencing results of the knockout mutant, a determination can be made. osntl5 Knockout type, the result is in the original OsNTL5 The gene is homozygous for a single cytosine base deletion in its genome, which causes premature termination of translation of the OsNTL5 protein.

[0055] OsNTL5 The open reading frame of the gene is shown in SEQ ID NO.5:

[0056] The amino acid sequence of the OsNTL5 protein is shown in SEQ ID NO.6: .

[0057] Example 2, ZH11 wild type and osntl5 Knockout mutant leaf-cutting method inoculation of Xanthomonas oryzae pathogenic species Xoo Strain PXO99 A The resistance of these two materials to bacterial blight in rice was compared.

[0058] 1. Prepare NA solid and liquid culture media according to Table 4. Table 4. Preparation methods of NA solid and liquid culture media 2. Xoo Inoculation experiment 1) Rice ZH11 and rice were grown in Greenhouse No. 1, South Campus, China Agricultural University. osntl5 Knockout mutant for 40 days.

[0059] 2) Xoo Strain PXO99 A Remove from the -80°C freezer and streak onto NA solid agar plates for activation.

[0060] 3) Pick the activated monoclonal plaques into NA liquid medium and incubate at 28°C and 200 rpm for 16 h.

[0061] 4) Collect the bacterial cells by centrifugation at 9000 rpm for 1 min, discard the waste liquid, add sterile ddH2O to resuspend and wash the bacterial cells, centrifuge at 9000 rpm for 1 min, and discard the waste liquid.

[0062] 5) Preparation of 100mM magnesium chloride solution: Dissolve 2.033 g of magnesium chloride hexahydrate powder (product number Sigma, M2670) in 100 mL ddH2O, autoclave at 121℃ for 20 min. Store at room temperature. Dilute with ddH2O before use.

[0063] 6) Resuspend the bacterial cells in 10 mM magnesium chloride solution, dilute 10 times, and measure the OD of the bacterial suspension. 600 According to the detected OD 600 As a result, the bacterial culture was diluted to the final OD using 10 mM magnesium chloride solution. 600 It is 0.8.

[0064] 7) Dip clean scissors in the bacterial solution and apply it to rice ZH11 and osntl5 Cut the second leaf from the tip of the knockout mutant 3 cm away.

[0065] 8) Under high temperature and humidity conditions, 14 days after the onset of the disease, the length of the lesions should be recorded and photographed. For example... Figure 1 As shown in A and B, osntl5 The knockout mutant exhibits significantly enhanced resistance to bacterial blight.

[0066] Example 3, ZH11 and osntl5 The knockout mutants were inoculated with rice blast fungus using a spray method to compare the rice blast resistance of the two materials. 1. Prepare the tomato oat culture medium (TOA) according to Table 5 below.

[0067] Table 5. Preparation method of TOA culture medium 2. Rice blast fungus inoculation experiment 1) Incubate *Blastomyces oryzae* RB22 on TOA medium for 14 days, add sterile ddH2O and spread it on the surface of the medium. Collect spores by filtration using a magic cloth filter, count the number of spores using a hemocytometer, and dilute the spore concentration to 1.0 × 10⁻⁶. 5 CFU / mL, add 0.01% Tween 20.

[0068] 2) Greenhouse cultivation of rice seedlings (ZH11 and osntl5 The rice seedlings (knockout mutants) grew to the three-leaf stage and were then inoculated with rice blast fungus. The seedlings were placed in a water-filled box, and the spores were sprayed evenly onto the seedlings. The box was then sealed with plastic wrap and covered with black plastic film for 24 hours. Each seedling was sprayed with the same amount of spores.

[0069] 3) 24 hours after vaccination, remove the black plastic film and allow the disease to develop naturally for 7 days. Observe the disease situation and take pictures for record.

[0070] 4) Place ZH11 and osntl5 Genome samples were extracted from diseased leaves in which the knockout mutant was found, following step 3 of Example 1. The extracted ZH11 and... osntl5 The mutant genome was diluted to 100 ng / µL and used as a template for real-time quantitative PCR (qPCR) experiments, using rice as a sample. OsUbi As an internal reference gene, ZH11 and osntl5 Rice blast fungus gene in mutant samples MoPot2 The expression level of [a specific substance] is used as one of the indicators to measure the severity of rice blast disease.

[0071] Gene OsUbi and MoPot2 The qPCR primers are as follows: OsUbi -qPCR-F (SEQ ID NO.7): 5'-TTCTGGTCCTTCCACTTTCAG-3'; OsUbi - qPCR-R (SEQ ID NO. 8): 5'-ACGATTGATTTAACCAGTCCATGA-3'.

[0072] MoPot2 -qPCR-F (SEQ ID NO.9): 5'-ACGACCCGTCTTTACTTATTTGG-3'; MoPot2 - qPCR-R (SEQ ID NO. 10): 5'-AAGTAGCGTTGGTTTTGTTGGAT-3'.

[0073] qPCR experiments used the following reagents: 2×M5 HiPer Realtime PCR Super mix with Low Rox (SYBRgreen, with anti-Taq) (catalog number MF797-01); 0.1 mL of Novizan Semi-Skirted 96-Well PCR Plates (catalog number PCR09611C-01); and an ABI QuantStudio 6 Flex instrument from Thermo Fisher Scientific, College of Plant Protection, China Agricultural University.

[0074] 5) The qPCR experimental system using Polymerase reagent is shown in Table 6 below. Table 6 qPCR system 6) The qPCR experimental procedure used is shown in Table 7 below. Table 7 qPCR program The results are as follows Figure 1 As shown in C and D, osntl5 The knockout mutant exhibits significantly enhanced resistance to rice blast.

[0075] Example 4: Induction of ZH11 and ZH11 using the plant immune elicitor flg22 osntl5 Reactive oxygen species burst experiment of knockout mutant 1. Prepare 1mM flg22 stock solution: Synthesize flg22 small peptide (QRLSTGSRINSAKDDAAGLQIA, N-terminal acetylated (Sangon Biotech), dissolve 45.4 mg flg22 small peptide powder in 20 mL sterile ddH2O, filter and sterilize using a 0.22 µm filter, and dispense.

[0076] 2. Prepare a stock solution of 40mM luminol (L-012) as a reactive oxygen species substrate: Dissolve 62.1 mg of L-012 powder (product number: wako, 120-04891) in 5 mL of ddH2O and dispense.

[0077] 3. Preparation of 10 mg / mL horseradish peroxidase (HRP) stock solution as a catalyst for the reaction of reactive oxygen species with luminol: Take 10 mg of HRP powder (product number: Beijing Coollab Technology Co., Ltd., CH6161) and dissolve it in 10 mL of ddH2O for dispensing.

[0078] 4. Reactive oxygen species detection experiment 1) After planting rice for about 30 days, take the second leaf from the bottom, use a 3 mm diameter punch to punch out the leaf disc and place it in a container containing sterile ddH2O. Vacuum for 15 minutes, add 100 µL of sterile ddH2O to the wells of a white opaque 96-well plate, and select the leaf disc into the well.

[0079] 2) Wrap the white opaque 96-well plate with aluminum foil and allow it to darken for 8 hours.

[0080] 3) Prepare flg22 reaction solution according to Table 8. Table 8. Preparation method of flg22 activating solution 0) After mixing the flg22 reaction solution, remove the equilibrated 96-well plate and add the sample to the wells using a multi-pipeline. Read the fluorescence values ​​using a microplate reader from the public experimental platform of the College of Plant Protection, China Agricultural University. Simultaneously, a negative control group (mock) was set up, in which an equal volume of sterile ddH2O was added to replace the flg22 or chitin elicitor. Results are as follows: Figure 2 As shown, flg22-induced osntl5 The knockout mutant had a higher level of reactive oxygen species than the wild-type ZH11.

[0081] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range, as well as any value between the two endpoints, can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0082] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0083] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. OsNTL5 The application of gene-related biological products in improving disease resistance in rice is characterized by, The OsNTL5 The genomic nucleotide sequence of the gene is shown in SEQ ID NO.4; OsNTL5 The open reading frame sequence of the gene is shown in SEQ ID NO.

5.

2. The application according to claim 1, characterized in that, The biological product is OsNTL5 Gene knockout vectors or recombinant engineered bacteria.

3. The application according to claim 2, characterized in that, The recombinant knockout vector comprises a backbone plasmid and a nucleotide sequence as shown in SEQ ID NO.

1.

4. The application according to claim 3, characterized in that, The backbone plasmid is pCBSG032.

5. The application according to claim 2, characterized in that, The recombinant engineered bacteria were obtained by introducing Agrobacterium tumefaciens into the recombinant knockout vector.

6. The application according to claim 5, characterized in that, The Agrobacterium is EHA105.

7. The application according to claim 2, characterized in that, The recombinant knockout vector or recombinant engineered bacteria were used to reduce the concentration of [unclear] in rice. OsNTL5 The expression level of genes is adjusted to improve the disease resistance of rice.

8. The application according to claim 1, characterized in that, Improving rice disease resistance refers to enhancing rice's resistance to rice blast and / or bacterial blight.

9. The application according to claim 8, characterized in that, The rice blast disease is caused by the fungus *Magnaporthe oryzae* (…). Magnaporthe oryzae The bacterial blight is caused by the pathogenic strain of Xanthomonas oryzae (…). Xanthomonas oryzae pv. oryzae Caused by ).

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