Application of oscyp75b4 gene related biological products in improving rice disease resistance

Abstract

This invention relates to the field of plant genetic engineering technology, specifically to OsCYP75B4 The application of gene-related biological products in improving disease resistance in rice, the OsCYP75B4 The genomic nucleotide sequence of the gene is shown in SEQ ID NO.4; OsCYP75B4 The open reading frame sequence of the gene is shown in SEQ ID NO.5. This invention involves knocking out the gene in the rice variety ZH11. OsCYP75B4, Obtaining knockout oscyp75b4 It increases resistance to rice blast and rice bacterial blight, and the plant immune elicitors chitin and flg22 induce... oscyp75b4 The reactive oxygen species (ROS) burst levels and defense-related gene expression levels in [the specific organism] were significantly higher than those in ZH11. Therefore, in this invention... OsCYP75B4 Genes and their proteins are of great significance for breeding new rice varieties with broad-spectrum disease resistance and stable yield, and are suitable for widespread application.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to OsCYP75B4 Application of gene-related bioproducts in improving disease resistance in rice. Background Technology

[0002] Rice is frequently threatened by a variety of 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] In existing technologies, several R genes for resistance to rice blast and bacterial blight have been cloned, and attempts have been made to apply them to variety improvement. However, existing disease resistance genes generally suffer from the following problems in practical applications: First, most R genes have narrow resistance spectra, exhibiting resistance only to specific physiological races. Meanwhile, the composition of pathogen populations in the field is complex and constantly changing, making it easy for new virulent races to overcome the resistance of a single R gene after only a few years of promotion, thus hindering its long-term sustainability. Second, there is a significant contradiction between disease resistance and agronomic traits such as yield. For example, broad-spectrum disease resistance genes... PigmR Or positive immune regulatory genes NPR1 While overexpression of certain substances can enhance resistance, it is often accompanied by suppressed plant growth and development and reduced yield. Although lesion-like mutants exhibit broad-spectrum resistance, they are difficult to apply directly to production due to defects such as reduced plant height and reduced fruit set caused by constitutive autoimmunity.

[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 OsCYP75B4 Application of gene-related bioproducts in improving disease resistance in rice.

[0006] This invention is achieved through the following technical solution: OsCYP75B4 The application of gene-related biological products in improving disease resistance in rice, the OsCYP75B4 The genomic nucleotide sequence of the gene is shown in SEQ ID NO.4; OsCYP75B4 The open reading frame sequence of the gene is shown in SEQ ID NO. 5.

[0007] Preferably, the OsCYP75B4 The amino acid sequence of the protein expressed by the gene is shown in SEQ ID NO.6.

[0008] Preferably, the biological product is OsCYP75B4Gene 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. OsCYP75B4 The expression level of genes is adjusted to improve the disease resistance of rice.

[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. rice Caused by ).

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first discovery of rice OsCYP75B4 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 with wild-type ZH11, rice... OsCYP75B4 Knockout oscyp75b4 Increased 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) and chitin, oscyp75b4 The knockout mutant exhibited significantly higher levels of reactive oxygen species (ROS) bursts and defense-related gene expression. However, under field growing conditions, compared to ZH11, oscyp75b4 There were no significant differences in plant height and tiller number between the knockout mutants. This indicates that the rice disease resistance-related genes... OsCYP75B4 Its proteins have potential targets for development into new rice varieties with broad-spectrum disease resistance and stable yield. 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 to wild-type ZH11, oscyp75b4 Figure A shows the resistance results of knockout mutants to rice bacterial blight and rice blast; Figure B shows typical disease symptoms after inoculation with rice bacterial blight pathogen; Figure C shows ZH11 and... oscyp75b4 C is a statistical graph of leaf lesion length after inoculation with rice bacterial blight pathogens following the removal of the knockout mutant; D is a graph of typical disease symptoms after inoculation with rice blast pathogens; and D is a graph of ZH11 and... oscyp75b4 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 Under the induction of plant immune elicitors, compared with ZH11, oscyp75b4 Figure showing the reactive oxygen species (ROS) burst levels in the knockout mutant; Figure 2 In the diagram, A represents the effect of flg22 induction compared to ZH11. oscyp75b4 Figure showing the reactive oxygen species (ROS) burst levels in the knockout mutant; B represents the level under chitin-induced reaction compared to ZH11. oscyp75b4 A graph showing the reactive oxygen species (ROS) burst levels in the knockout mutant.

[0020] Figure 3 Under the induction of plant immune elicitors, compared with ZH11, oscyp75b4 Figure showing the expression levels of defense-related genes in knockout mutants; Figure 3 In the diagram, A represents the effect of flg22 induction compared to ZH11. oscyp75b4 Knockout mutants of defense-related genes OsPR5 Expression level results; B represents the expression level under flg22 induction compared to ZH11. oscyp75b4 Knockout mutants of defense-related genes OsPR10 Expression level results; C represents the expression level under flg22 induction compared to ZH11. oscyp75b4 Knockout mutants of defense-related genes OsPBZ1 Expression level results; D represents the chitin-induced expression level compared to ZH11. oscyp75b4 Knockout mutants of defense-related genes OsPR5 Expression level results; E represents the expression level under chitin-induced conditions compared to ZH11. oscyp75b4Knockout mutants of defense-related genes OsPR10 Expression level results; F represents the chitin-induced expression level compared to ZH11. oscyp75b4 Knockout mutants of defense-related genes OsPBZ1 The results of the expression level are shown in the figure; ** indicates p <0.01, *** indicates p <0.001.

[0021] Figure 4 Under field growing conditions, oscyp75b4 Figure showing the plant height and effective tiller number of the knockout mutant compared to ZH11; Figure 4 In the text, A represents the mature ZH11 and... oscyp75b4 Photographs of knockout mutant plants; B represents ZH11 and... oscyp75b4 Plant height statistics of knockout mutants; C represents ZH11 and oscyp75b4 Statistical graph of effective tiller number in knockout mutants; ns indicates no significant difference. Detailed Implementation

[0022] 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.

[0023] 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.

[0024] 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.

[0025] oscyp75b4 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'-GCACATGGCGTACAACGGCCGGG-3'. The pCBSG032 vector (preserved by Weimi Biotechnology Co., Ltd.) was linearized by BsaI digestion. The target fragment containing the knockout target sequence was then ligated into the linearized vector via homologous recombination. After transformation into E. coli, the vector was screened with kanamycin and sequenced to confirm the correct recombinant knockout vector.

[0026] 2. Callus induction and subculture Select mature rice seeds (preferably newly harvested seeds from the current year), remove the husks, 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] Select a sufficient number of callus tissues (good callus condition, bright yellow color, round and firm texture, with a particle diameter of about 3 mm) and place them in a 100 mL sterile Erlenmeyer flask. Add an appropriate amount of Agrobacterium suspension (ensure sufficient bacterial suspension to contact the material). Incubate at room temperature for 20 minutes, shaking occasionally. Discard the bacterial suspension, place the callus tissues on sterile filter paper to absorb excess bacterial suspension, and then transfer them to a solid co-culture medium lined with a layer of sterile filter paper. Incubate at 26°C in the dark for 3 days.

[0031] 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.

[0032] 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. Generally, green spots will appear on the calluses in about 10 days, and seedlings will differentiate after another 10 days.

[0033] 7. Seedling rooting Once the differentiated seedlings reach 3cm in height and have a visible root system, they can be transferred 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. oscyp75b4 Gene knockout mutant.

[0034] 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. rice )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 information for *Blastomyces oryzae* RB22 is disclosed in the references: [1] Zhu Yongsheng, Cai Qiuhua, Xue Weimin, et al. Preliminary analysis of blast resistance genotypes in Yunyin rice 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 rice in Rice. Molecular Plant. 2015;8(11):1679-1682. DOI: 10.1016 / j.molp.2015.08.004. Example 1: Identification of rice OsCYP75B4 Gene knockout mutant oscyp75b4 Knockout type 1. In OsCYP75B4 Design and identification of 5'-3' primers for upstream and downstream detection of gene knockout target oscyp75b4 Knockout type of knockout mutant. Upstream detection primer F oscyp75b4 (SEQ ID NO.2): 5'-CCCTGAGATAATAGACGC-3'; Downstream detection primer R oscyp75b4 (SEQ ID NO.3): 5'-GACATTCACCTCCTTTCC-3', the amplified target fragment is 633bp in size.

[0035] Includes OsCYP75B4 The full-length nucleotide sequence of the rice genome (from the first bolded start codon ATG to the second bolded stop codon TAA) is shown in SEQ ID NO.4.

[0036]

[0037] 2. Prepare a 2% (w / v) CTAB Buffer for extracting rice genome. Table 1. Preparation method of 2% CTAB Buffer for extracted genome volume fraction. 3. Extract ZH11 and oscyp75b4 Experimental steps for knocking out the genome of a mutant 1) Planting ZH11 and oscyp75b4 In the South Pond of the Greenhouse of China Agricultural University, when the plants grew to the stage of three leaves and one heart, the leaves were cut off with scissors and placed in 2mL centrifuge tubes. 3mm steel balls were added, and the tubes were quickly frozen with liquid nitrogen. The samples were then ground at a frequency of 45Hz using an electric grinder.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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 min, aspirate and discard the supernatant, and air dry the precipitated genome in a clean bench.

[0043] 7) Dissolve the dried genome in ddH2O, then store at -20℃ for later use.

[0044] 4. Use the primers designed in step 1 to perform PCR amplification of ZH11 and oscyp75b4 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.

[0045] Table 2 PCR amplification target fragment formulation 2) Amplify the target fragment in the PCR instrument according to the procedure in Table 3 below.

[0046] 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).

[0047] 5. The target fragment was recovered using the Gel Extraction Kit from Jiangsu Kangwei Century Biotechnology Co., Ltd.

[0048] 1) Precisely cut out the gel containing the target DNA band and place it in a 2 mL centrifuge tube.

[0049] 2) Add 500 µL PG and dissolve the gel block in a 50°C water bath.

[0050] 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.

[0051] 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.

[0052] 5) Add 450 μL of PW rinsing solution, centrifuge at 13000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0053] 6) Repeat step 5) once.

[0054] 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.

[0055] 6. The recovered target fragment was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and ZH11 and oscyp75b4 By comparing the sequencing results of the knockout mutant, a determination can be made. oscyp75b4 Knockout type, the result is in the original OsCYP75B4 The gene has a single guanine base missing from its genome and is homozygous, which causes premature termination of translation of the OsCYP75B4 protein.

[0056] OsCYP75B4 The open reading frame of the gene is shown in SEQ ID NO.5.

[0057]

[0058] The amino acid sequence of the OsCYP75B4 protein is shown in SEQ ID NO. 6: .

[0059] Example 2, ZH11 wild type and oscyp75b4 Knockout mutant leaf-cutting method inoculation of Xanthomonas oryzae pathogenic species ( Xoo This study compares the resistance of these two materials to bacterial blight in rice. 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) Wild-type rice ZH11 was grown in Greenhouse No. 1 at China Agricultural University. oscyp75b4 Knockout mutant for 40 days.

[0060] 2) Xoo Strain PXO99 A Remove from the -80°C freezer and streak onto NA solid agar plates for activation.

[0061] 3) Pick the activated monoclonal plaques into NA liquid medium and incubate at 28°C and 200 rpm for 16 h.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 7) Dip clean scissors in the bacterial solution and apply it to rice ZH11 and oscyp75b4 Cut the second leaf from the tip of the knockout mutant 3 cm away.

[0066] 8) Under high temperature and humidity conditions, 14 days after the onset of the disease, the length of the lesions was counted and photographed for recording. Results are as follows: Figure 1 As shown in A and B, oscyp75b4 The knockout mutant exhibits enhanced resistance to bacterial blight.

[0067] Example 3, ZH11 wild type and oscyp75b4 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.

[0068] 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.

[0069] 2) When the rice seedlings grown in the greenhouse reach the three-leaf stage, start inoculating them with rice blast fungus. Place the rice seedlings in a box filled with water, spray the spores evenly onto the rice seedlings, seal the box with plastic wrap, and cover it with black plastic film for 24 hours.

[0070] 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.

[0071] 4) Place ZH11 and oscyp75b4 Genome samples were extracted from diseased leaves in which the knockout mutant was found, following step 3 of Example 1. The extracted ZH11 and... oscyp75b4 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 oscyp75b4 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.

[0072] 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'.

[0073] MoPot2 -qPCR-F (SEQ ID NO.9): 5'-ACGACCCGTCTTTACTTATTTGG-3'; MoPot2 - qPCR-R (SEQ ID NO. 10): 5'-AAGTAGCGTTGGTTTTGTTGGAT-3'.

[0074] qPCR experiments used Polymerex 2 × M5 HiPer Realtime PCR Super mix with Low Rox (SYBRgreen, with anti-Taq) (catalog number MF797-01); Novizan 0.1 mL Semi-Skirted 96-Well PCR Plates (catalog number PCR09611C-01); and Thermo Fisher ABI QuantStudio 6 Flex instruments from the College of Plant Protection, China Agricultural University.

[0075] 5) The qPCR experimental system using Polymerase reagent is shown in Table 6 below.

[0076] Table 6 qPCR system 6) The procedure for the qPCR experiment is shown in Table 7 below.

[0077] Table 7 qPCR program The results are as follows Figure 1 As shown in C and D, oscyp75b4 Knockout mutants exhibit enhanced resistance to rice blast.

[0078] Example 4: Induction of ZH11 and ZH11 using plant immune elicitors flg22 and chitin. oscyp75b4 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.

[0079] 2. Prepare a 1 mg / mL chitin stock solution: Dissolve 50 mg chitin (product number Sigma, C9752) in 50 mL of sterile ddH2O, break it up with an ultrasonic cell disruptor, shake it evenly on a shaker, filter it through a 0.22 µm filter for sterilization, and dispense it.

[0080] 3. 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.

[0081] 4. 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.

[0082] 5. 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.

[0083] 2) Wrap the white opaque 96-well plate with aluminum foil and allow it to darken for 8 hours.

[0084] 3) Prepare flg22 reaction solution according to Table 8 below.

[0085] Table 8. Preparation method of flg22 activating solution 4) Prepare the chitin reaction solution according to Table 9 below.

[0086] Table 9. Preparation method of chitin activation solution 5) After thoroughly mixing the flg22 and chitin reaction solutions, remove the equilibrated 96-well plate and add the samples to the wells using a multi-channel pipette. Read the fluorescence values ​​using the microplate reader at the public experimental platform of the College of Plant Protection, China Agricultural University. Simultaneously, a negative control group (mock) was set up, with an equal volume of sterile ddH2O added to the mock group to replace either flg22 or chitin elicitors. Results are as follows: Figure 2 As shown, flg22 and chitin induce oscyp75b4 The knockout mutant had a significantly higher level of reactive oxygen species than the wild-type ZH11.

[0087] Example 5: Detection of flg22 and chitin-induced ZH11 and oscyp75b4 Knockout mutant defense-related genes OsPR5 , OsPR10 and OsPBZ1 Differences in expression levels 1. Prepare 1 / 2 MS medium for culturing rice tissue culture seedlings according to Table 10. Table 10 Preparation method of 1 / 2 MS medium 2. Preparation of sterile rice ZH11 and oscyp75b4 Knockout mutant tissue culture seedlings 1) Select ZH11 grains that are plump and healthy. oscyp75b4 Remove the mutant seeds, remove the shells using a shelling machine, and prepare sterile tissue culture flasks, ddH2O, filter paper, and 200 mL Erlenmeyer flasks. 2) Prepare 70% ethanol (volume fraction) and 70% sodium hypochlorite (mass volume fraction) in a clean bench. First, wash the seeds with 70% ethanol for 2 min, and then wash the seeds with 70% sodium hypochlorite in a shaker at 28℃ and 140 rpm for 30 min.

[0088] 3) Wash the seeds 5 times with sterile water, then spread them on sterile filter paper to dry.

[0089] 4) Heat the solid 1 / 2 MS medium and pour it into a sterilized tissue culture bottle. Spread the dried seeds evenly in the tissue culture bottle and label them.

[0090] 5) Grow to the required size in the tissue culture room.

[0091] 3. flg22 and chitin induce ZH11 and oscyp75b4 Knockout mutant detection OsPR5 , OsPR10 and OsPBZ1 Expression level 1) In a windless clean bench, pour 40 mL of liquid 1 / 2 MS into a 10 cm square dish. Select rice seedlings of uniform length and gently pick up the seed part of the seedling from the bottle and transfer it into the dish with sterile tweezers cooled to room temperature. Place three seedlings in each dish, ensuring that the roots, stems and leaves are in full contact with the culture medium. After sealing, let it stand for 2 days. Process the seedlings after the second flag leaf has fully unfolded and the third flag leaf has just emerged. 2) Add 1 mM flg22 at a ratio of 1:1000 or 1 mg / mL chitin at a ratio of 1:100 to a new liquid 1 / 2 MS, then add surfactant Silwet-77 at a ratio of 1:20000, mix thoroughly, discard the equilibrated old medium, add 40 mL of reaction solution, and use a pipette tip to adjust the seedlings to be in full contact with the medium. React for 6 h.

[0092] 3) After the reaction time is over, quickly take the leaves, flash freeze them in liquid nitrogen, and prepare for RNA extraction.

[0093] 4. Extract ZH11 and oscyp75b4 Knockout RNA The procedure was performed using the Polymer Plant Total RNA Extraction Kit (catalog number MF456).

[0094] 1) Load the sample into a 2 mL RNase-free centrifuge tube containing 3 mm steel balls, freeze it with liquid nitrogen, pre-cool the grinder rotor with liquid nitrogen, and grind the sample at 40 Hz for 60 s.

[0095] 2) Add 1 mL of lysis buffer RL to each sample tube, tighten the cap, and shake vigorously for 20 s to ensure complete lysis. Let stand at room temperature for 5 min.

[0096] 3) Add 250 µL of RNase-free chloroform to the fume hood, shake vigorously for 15 seconds to mix the sample, and then let it stand at room temperature for 3 minutes.

[0097] 4) Centrifuge at 12000 rpm for 10 min at 4℃. The sample will separate into three layers.

[0098] 5) Transfer the top aqueous phase to a freshly prepared RNase-free 1.5 mL centrifuge tube, add 1 / 2 volume of RNase-free anhydrous ethanol, and mix thoroughly.

[0099] 6) Add the solution obtained in step 5 to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0100] 7) Add 500 µL of solution RE, centrifuge at 12000 rpm for 1 min, and discard the waste liquid in the collection tube.

[0101] 8) Add 500 µL of solution RW, centrifuge at 12000 rpm for 45 s, and discard the waste liquid in the collection tube.

[0102] 9) Repeat step 8.

[0103] 10) Discard the waste liquid in the collection tube, centrifuge at 13000 rpm for 3 min, transfer the column head to an RNase-free 1.5 mL centrifuge tube, open the cap and let it stand to dry the ethanol for 3 min.

[0104] 11) Add 70 µL of RNase-free ddH2O, let stand for 2 min, centrifuge at 12000 rpm for 1 min, collect RNA, detect RNA concentration, and store at -80℃.

[0105] 5. Place ZH11 and oscyp75b4 Knockout mutant RNA is reverse transcribed into cDNA.

[0106] The assay was performed using the Century Kangwei HiFiScript All-in-one RT Master Mix for qPCR kit (catalog number CW3371).

[0107] 1) Prepare the reaction solution on ice according to Table 11. (Add samples in the order of water, mix, and RNA).

[0108] Table 11 Preparation method of reverse transcription reaction solution Note: "-" indicates that this item is not available.

[0109] 2) After gently stirring the reaction solution until it is homogeneous, proceed with the reaction according to the procedure in Table 12 below.

[0110] Table 12 Reverse Transcription Procedure 3) After the reaction is complete, store at 4°C or -20°C (long-term). Avoid repeated freeze-thaw cycles for cDNA.

[0111] 6. Perform real-time quantitative PCR (RT-qPCR) experiments.

[0112] 1) Design a protocol suitable for RT-qPCR using the NCBI website. OsActin (Internal reference gene) OsPR5 OsPR10 and OsPBZ1 Upstream and downstream primers. (Their respective primers are listed below.)

[0113] OsActin1 -RT-qPCR-F (SEQ ID NO. 11): 5'-TCCATCTTGGCATCTCTCAG-3'; OsActin1 -RT-qPCR-R (SEQ ID NO. 12): 5'-GTACCCGCATCAGGCATCTG-3'.

[0114] OsPR5 -RT-qPCR-F (SEQ ID NO. 13): 5'-CAACAGCAACTACCAAGTCGTCTT-3'; OsPR5 -RT-qPCR-R (SEQ ID NO. 14): 5'-CAAGGTGTCGTTTTATTCATCAAC-3'.

[0115] OsPR10 -RT-qPCR-F (SEQ ID NO. 15): 5'-CGCCGCAAGTCATGTCCTAA-3'; OsPR10 -RT-qPCR-R (SEQ ID NO. 16): 5'-CCACGATGTCCTTCTCCTTCTC-3'.

[0116] OsPBZ1 -RT-qPCR-F (SEQ ID NO.17): 5'-GACATCGTGGATGGCTACTATGG-3'; OsPBZ1 -RT-qPCR-R (SEQ ID NO. 18): 5'-TCACTCACTCTAGGTGGGATATAC-3'.

[0117] 2) The following reagents were used: Polymerase 2×M5 HiPer Realtime PCR Super mix with Low Rox (SYBRgreen, with anti-Taq) (antibody qPCR mix, low Rox premix) (catalog number MF797-01); Novizan 0.1 mL Semi-Skirted 96-Well PCR Plates (catalog number PCR09611C-01); and Thermo Fisher ABI QuantStudio 6 Flex instrument from the College of Plant Protection, China Agricultural University.

[0118] 3) Prepare samples according to step 5) of step 2 in implementation case 3. 4) After spotting, perform RT-qPCR following step 6) of step 2 in Implementation Case 3. The results are as follows: Figure 3 As shown, flg22 and chitin induce oscyp75b4 The expression levels of defense-related genes in the knockout mutant were significantly higher than those in the wild-type ZH11. The mock mutant served as the negative control group, with an equal volume of sterile ddH2O added to replace the flg22 or chitin elicitor.

[0119] Example 6: Statistical analysis of ZH11 and under field growth conditions oscyp75b4 Agronomic traits of knockout mutants 1) ZH11 and oscyp75b4 Knockout mutants.

[0120] 2) During the rice ripening period, for ZH11 and oscyp75b4 Plant height and effective tiller number were counted and photographed for the knockout mutants. Results are as follows: Figure 4 As shown, oscyp75b4 The knockout mutant showed no significant difference in plant height and tiller number compared to ZH11 under field growing conditions.

[0121] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and 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.

[0122] 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.

[0123] 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. OsCYP75B4 The application of gene-related biological products in improving disease resistance in rice is characterized by, The OsCYP75B4 The genomic nucleotide sequence of the gene is shown in SEQ ID NO.4; OsCYP75B4 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 OsCYP75B4 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. OsCYP75B4 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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