Application of biological preparation of overexpressed osarf7 gene in improving disease resistance of rice

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically relating to an overexpression method. OsARF7 The application of gene-based bioproducts in improving disease resistance in rice, the aforementioned OsARF7 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. This invention constructs an overexpression gene using transgenic methods. OsARF7 Rice lines. Experiments revealed that overexpression... OsARF7 It can significantly enhance the immunity of rice, such as increasing the expression level of disease resistance-related genes, significantly inducing reactive oxygen species bursts, and significantly improving the resistance of rice to rice false smut and bacterial blight. This invention demonstrates... OsARF7 It has a positive regulatory effect on rice immunity and can be used to improve the disease resistance of rice, which is of great significance for creating disease-resistant rice germplasm resources.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to an overexpression method. OsARF7 Application of gene-based bioproducts in improving disease resistance in rice. Background Technology

[0002] Rice production has long faced threats from various diseases, including rice blast, bacterial blight, sheath blight, and rice false smut. In recent years, influenced by factors such as climate change, large-scale planting of high-yield varieties, and increased application of nutrients, the range of these fungal and bacterial diseases has continued to expand, and the severity of their damage has intensified year by year.

[0003] Against this backdrop, in-depth exploration and full utilization of rice's endogenous disease resistance genes have become an important approach to enhancing rice's overall disease resistance. Plant disease resistance responses involve complex networks of multi-gene synergistic regulation, and current research focus is increasingly on identifying and analyzing key disease resistance genes and related regulatory genes in rice. With the development of molecular biology techniques, several disease resistance-related genes have been successfully cloned. These genes encode proteins with different functions and, through participation in different signal transduction pathways and physiological processes, jointly regulate rice's immune response to various pathogens. Therefore, systematically exploring rice's endogenous disease resistance gene resources, elucidating their functional mechanisms, and applying them to the breeding of disease-resistant varieties are of great significance for achieving green and sustainable control of rice diseases.

[0004] Genetic improvement using endogenous disease-resistant genes in rice is a fundamental strategy for breeding green, disease-resistant varieties. Currently, existing technologies have successfully cloned several disease-resistant genes, mainly falling into two categories: one is major disease-resistant genes encoding NBS-LRR receptor proteins, and the other is key regulatory genes involved in disease-resistant signaling pathways, such as transcription factors and hormone-related elements. However, existing disease-resistant genes still have the following drawbacks: resistance is usually race-specific and easily becomes ineffective due to pathogen mutations; the disease resistance effect of existing genes is not good, leaving room for improvement in their application efficiency in practical breeding. Against this backdrop, systematically discovering key genes with disease-resistant potential in rice and elucidating their molecular mechanisms is of great significance for overcoming current bottlenecks in disease-resistant breeding. Summary of the Invention

[0005] This invention provides an overexpression OsARF7 The application of gene bioproducts in improving rice disease resistance provides a novel gene resource with significant regulatory potential for improving broad-spectrum disease resistance in rice.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides an overexpression OsARF7The application of gene-based bioproducts in improving disease resistance in rice, the aforementioned OsARF7 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0008] Preferably, the biological product is any one of the following:

[0009] A: Includes OsARF7 Gene overexpression vectors;

[0010] B: Includes OsARF7 Agrobacterium gene.

[0011] Preferably, the overexpression vector and / or Agrobacterium are used to enhance... OsARF7 Gene expression levels were adjusted to construct overexpression plants, thereby breeding disease-resistant rice.

[0012] Preferably, the method for constructing overexpression plants is as follows:

[0013] RNA was extracted from rice and reverse transcribed into cDNA;

[0014] Using cDNA as a template, the primers shown in SEQ ID NO.3 and SEQ ID NO.4 were used to clone the sample. OsARF7 Gene;

[0015] Will OsARF7 The gene is ligated into the enzyme-digested starting vector to obtain the overexpression vector;

[0016] Overexpression plants were obtained by infecting rice with an overexpression vector via Agrobacterium-mediated transformation.

[0017] Preferably, the launching carrier is pCAMBIAI1305.

[0018] Preferably, the restriction endonucleases used to digest the starting vector are KpnⅠ and HindⅢ.

[0019] Preferably, the recipient material used in Agrobacterium-mediated infection of rice is callus tissue.

[0020] Preferably, improving rice disease resistance refers to improving rice's resistance to rice bacterial blight and / or rice false smut.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention provides an overexpression OsARF7 The application of gene-based bioproducts in improving disease resistance in rice, the aforementioned OsARF7 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. This invention utilizes the construction of an overexpression... OsARF7 Rice lines that expressed the gene were observed to have overexpressed *Strombus haemolyticus* spores after artificial inoculation of immature panicles. OsARF7 The number of rice blasts on the panicle of the genetically modified rice is significantly lower than that of the wild-type Nipponbare, indicating that... OsARF7 Overexpression of the gene enhances the resistance of rice to rice false smut. This invention involves inoculating rice with bacterial blight fungal suspension using the leaf-cutting method. Two weeks later, photographs were taken and the length of lesions was measured. The results showed that overexpression of the gene... OsARF7 On rice leaves, the lesion length of the gene was significantly shorter than that of the wild-type Zhonghua 11, indicating that overexpression of the gene was present. OsARF7 Genes can enhance rice's resistance to bacterial blight.

[0023] Furthermore, this invention also knocked out the CRISPR-Cas9 system in rice. OsARF7 Genes, used to assist in proof OsARF7 The function is missing. Experiments revealed that: OsARF7 The expression levels of disease resistance-related genes, the level of induced reactive oxygen species bursts, and the resistance to rice false smut and bacterial blight pathogens in genetically modified rice were all significantly reduced compared to the wild type.

[0024] In summary, this invention not only provides a basis for disease-resistant rice breeding, but also... OsARF7 This novel gene resource possesses broad-spectrum and highly efficient disease resistance characteristics. More importantly, it has sufficient experimental evidence and clear mechanistic support, which can effectively overcome the key bottlenecks in existing technologies where disease-resistant genes are prone to failure and disease resistance is poor. This is of great significance for creating disease-resistant rice germplasm resources. Attached Figure Description

[0025] Picture 1 For overexpression OsARF7 Detection of expression levels of disease-related genes in rice lines. A represents the expression levels of chitin-treated groups. OsPR1b The results of relative gene expression level detection; B represents the chitin treatment group. OsPR10a The results of relative gene expression level detection; C represents the expression levels of each treatment group after flg22 treatment. OsPR1b The results of relative gene expression level detection; D represents the expression levels of each treatment group after flg22 treatment. OsPR10a The results of relative gene expression level detection. WT represents the wild-type rice variety Zhonghua 11, n=3. The error bar is represented by the standard deviation SD. One-way ANOVA-Duncan analysis was used for post-hoc comparison analysis of differences. a, b, c: P <0.05.

[0026] Picture 2 For overexpression OsARF7Detection of reactive oxygen species (ROS) bursts in rice lines. A shows the ROS burst detection results for each group after chitin treatment; B shows the ROS burst detection results for each group after flg22 treatment. WT represents the wild-type rice variety Zhonghua 11, n=6, and the error bars represent the standard error SE.

[0027] Picture 3 For overexpression OsARF7 Results of resistance testing of rice lines to bacterial blight. A shows the incidence of bacterial blight in each experimental group 14 days after inoculation with bacterial blight pathogen using the leaf-cutting method; B shows the statistical analysis of lesion length of bacterial blight in each experimental group 14 days after inoculation with bacterial blight pathogen using the leaf-cutting method. EV represents the blank control group transformed with pCAMBIAI1305 under the same genetic background, n=10. Error bars are represented as standard deviation SD, using Student's algorithm. t The difference analysis was performed to test the results. P <0.01.

[0028] Picture 4 For overexpression OsARF7 Results of resistance testing of rice lines to rice false smut. A shows the incidence of rice false smut in each experimental group after injection inoculation with rice false smut fungus; B shows the statistical results of the number of rice false smut pellets in each experimental group after injection inoculation with rice false smut fungus. WT represents the wild-type rice variety Zhonghua 11, n=20. The error bars are represented by the standard deviation SD, using Student's Law. t The difference analysis was performed to test the results. P <0.01.

[0029] Picture 5 for osarf7 Detection of expression levels of disease-related genes in gene knockout mutant lines. A represents the expression levels of chitin-treated groups. OsPR1b The results of relative gene expression level detection; B represents the chitin treatment group. OsPR10a The results of relative gene expression level detection. WT represents the wild-type rice variety Zhonghua 11, n=3. The error bar is represented by the standard deviation SD. One-way ANOVA-Duncan analysis was used for post-hoc comparison analysis of differences. a, b, c: P <0.05.

[0030] Picture 6 for osarf7 Detection of reactive oxygen species (ROS) bursts in gene knockout mutant lines. A shows the ROS burst detection results for each treatment group after chitin treatment; B shows the ROS burst detection results for each treatment group after flg22 treatment. WT represents the wild-type rice variety Zhonghua 11, n=6, and the error bars represent the standard error SE.

[0031] Picture 7 for osarf7Results of resistance testing for bacterial blight in gene knockout mutant lines. A shows the incidence of bacterial blight in each experimental group 14 days after inoculation with bacterial blight pathogen using the leaf-cutting method; B shows the lesion length statistics of bacterial blight in each experimental group 14 days after inoculation with bacterial blight pathogen using the leaf-cutting method. WT represents the wild-type rice variety Zhonghua 11, n=20, and the error bars are expressed as standard deviation (SD). Student's algorithm was used. t The difference analysis was performed to test the results. P <0.01.

[0032] Picture 8 for osarf7 Results of resistance testing of gene knockout mutant lines to rice false smut. A shows the incidence of rice false smut in each experimental group after injection inoculation with *Strombus oryzae*; B shows the statistical results of the number of rice false smut pellets in each experimental group after injection inoculation with *Strombus oryzae*. WT represents the wild-type rice variety Zhonghua 11, n=40. Error bars are represented by standard deviation SD. Student's score was used. t Perform a difference analysis, *: P <0.05. Detailed Implementation

[0033] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0034] The inventive concept of this invention is as follows:

[0035] This invention discovers an endogenous gene that regulates disease resistance in rice. OsARF7 The MSU number of the gene is LOC_Os02g35140, and the nucleic acid sequence that the gene can express is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0036] SEQ ID NO.1:

[0037]

[0038] SEQ ID NO.2:

[0039] .

[0040] Pathogen-associated molecular patterns, such as the bacterial flagellar protein flg22 or the fungal cell wall component chitin, can trigger innate immune responses in rice, including the high expression of disease-related genes and reactive oxygen species bursts. This invention is for discussion purposes. OsARF7 The effect of genes on the immune response of rice was investigated using the wild rice variety Zhonghua 11. Genes were first cultured and overexpressed in a sterile environment. OsARF7 Rice seedlings were bred to two weeks old, then transferred to a liquid medium containing PAMPs and soaked for 6 hours. Equal weights of rice leaves were then harvested to extract total RNA. Two types of disease-related genes in the RNA samples were identified using qRT-PCR. PR1b and PR10a Content testing revealed that, compared to the wild-type Zhonghua 11, overexpression... OsARF7 In rice with genetics PR1b and PR10a The expression level was significantly increased. This invention also used a 5cm diameter punch to prepare rice leaf samples for testing. After PAMP treatment, the ROS level of the samples was detected within 25 minutes. It was found that compared to wild-type Zhonghua 11, overexpression was significantly increased. OsARF7 The ROS level in the rice samples was significantly increased.

[0041] All the above results demonstrate that OsARF7 Genes can positively regulate the immunity of rice and can be used to improve the disease resistance of rice, which is of great significance for the creation of new disease-resistant rice varieties.

[0042] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0043] The rice bacterial blight PXO99A strain used in this invention is disclosed in the reference: Zeng Chen. Construction of MCP-free mutant of rice bacterial blight PXO99A strain and study on chemokine receptor gene [D]. Guangxi University, 2019.

[0044] The rice smut fungi used in the present invention are disclosed in the literature: Li GB, He JX, Wu JL, Wang H, Zhang XJ, Xu ZJ, Wang WM, Fan J. Overproduction ofOsRACK1A, an effector-targeted scaffold protein promoting OsRBOHB-mediatedROS production, confers rice floral resistance to false smut disease without yield penalty. Mol Plant. 2022, 15(11):1790-1806. doi: 10.1016 / j.molp.2022.10.009.

[0045] The abbreviations related to this invention are as follows:

[0046] PAMPs, pathogen-associated molecular pattern markers; chitin; PR Disease-related genes; ROS (reactive oxygen species).

[0047] Example 1

[0048] overexpression OsARF7 The application of gene-based bioproducts in improving disease resistance in rice is as follows:

[0049] 1. Using qRT-PCR technology to target overexpression OsARF7 The expression levels of disease-related genes in rice lines were detected to make a preliminary judgment. OsARF7 The influence of genes on rice immunity. The specific procedures are as follows:

[0050] (1) Constructing a bacterial strong promoter 35S OsARF7 Overexpression vector.

[0051] based on OsARF7The gene sequence SEQ ID NO.1 was used to design primers for PCR operations. The sequences of the forward and reverse primers are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The plasmid used for the expression vector is pCAMBIAI1305.

[0052] Forward primer, SEQ ID NO.3: tgtacagagctcggtaccATGGCTGGCTCCGTCGTTGC.

[0053] Reverse primer, SEQ ID NO.4: gtctttgtagtcaagcttGCAATCCTTATCGGTGACGG.

[0054] RNA was extracted from wild-type rice Nipponbare using a high-fidelity PCR kit manufactured by Nanjing Novizan Biotechnology Co., Ltd., and reverse transcribed into cDNA. Using the cDNA as a template, the reaction mixture was prepared in 200 μL centrifuge tubes. The reaction mixture is shown in Table 1, and the PCR program is shown in Table 2. After the reaction, 10× Loading Buffer was added to the tubes, and fragments were separated by agarose gel electrophoresis at 150 V to verify fragment size.

[0055] Table 1 PCR reaction system

[0056]

[0057] Table 2 PCR reaction procedure

[0058]

[0059] In Table 2, " / " indicates that this item is not present.

[0060] This invention uses a DNA agarose gel extraction kit manufactured by Kangwei Century Biological Reagent Co., Ltd. to recover PCR products. A gel block containing the target band is cut, weighed, and placed in a 2mL centrifuge tube. An equal weight of PG Buffer is added, and the tube is placed in a 65℃ water bath to dissolve the gel. The mixture is continuously inverted and mixed until the gel is completely dissolved, then cooled to room temperature. During this process, 200µL of PS Buffer is added to the adsorption column, and the tube is centrifuged at 12000rpm for 1 min, discarding the waste liquid. Gel solution is added to the adsorption column, incubated at room temperature for 2 min, and centrifuged at 12000rpm for 1 min, discarding the waste liquid. 500µL of PW Buffer is added to the adsorption column for rinsing, and the tube is centrifuged at 12000rpm for 1 min, discarding the waste liquid. After rinsing twice, the tube is centrifuged at 12000rpm for 2 min, and the adsorption column is transferred to a new 1.5mL centrifuge tube. The tube is then opened and allowed to stand for 5 min to remove residual ethanol. Add 50µL of EBBuffer preheated to 65℃ to the adsorption column, let stand for 1 min, and then centrifuge at 12000rpm for 1 min to obtain the final PCR product.

[0061] This invention uses the restriction endonucleases KpnⅠ and HindⅢ produced by NEB to perform double digestion of the desired pCAMBIAI1305 plasmid. The reaction mixture was prepared in 200 μL centrifuge tubes, as shown in Table 3. The mixture was incubated at 37°C for 2 hours. After the reaction, the digested products were verified by electrophoresis and recovered. The purification method for the digested products was the same as that for PCR product recovery.

[0062] Table 3 Enzyme digestion reaction system

[0063]

[0064] This invention uses the seamless cloning kit from Nanjing Novizan Biotechnology Co., Ltd. to ligate PCR products and enzyme digestion products to obtain an overexpression vector. Specifically, the reaction mixture was placed in a 200 μL centrifuge tube as shown in Table 4. The mixture was then incubated at 37°C for 30 min.

[0065] Table 4 Connection Reaction System

[0066]

[0067] This invention utilizes *E. coli* DH5α competent cells produced by Vazyme for monoclonal transformation of overexpression vectors. 100 μL of *E. coli* DH5α competent cells were thawed on ice, and 10 μL of ligation product was slowly added. The mixture was gently stirred with a pipette tip and incubated on ice for 30 min. Then, the cells were heat-shocked in a 42°C water bath for 45 s without shaking the bacterial culture. The mixture was then quickly and gently transferred to an ice bath and incubated for 3 min. Finally, 500 μL of LB liquid medium was added, and the mixture was incubated at 37°C and 180 rpm in a shaker for 1 h. The cells were centrifuged at 5000 rpm for 3 min. 500 μL of supernatant was removed in a clean bench. The remaining 100 μL of supernatant was gently mixed with the precipitated bacterial cells and evenly spread using a sterilized and cooled spreader onto LA medium plates containing kanamycin antibiotics. The plates were then incubated upside down in a 37°C incubator for 16 h. Pick a single-clone transformant colony from the plate into a test tube, add 5 mL of LB liquid medium with added kanamycin, and place it slanted in a constant temperature shaker at 37℃ and 180 rpm for 12 h to propagate.

[0068] This invention uses a plasmid mini-prep kit produced by Beijing Tiangen Biotech Co., Ltd. to purify plasmids using a small amount of propagated bacterial culture. The extraction method was performed according to the kit instructions. PCR and first-generation sequencing technologies were used to verify whether the target gene was successfully and correctly ligated into the vector. After successful construction, the remaining bacterial culture was mixed with 40% (v / v) glycerol at a 1:1 ratio to prepare a bacterial strain, which was then stored at -80℃ for long-term preservation.

[0069] (2) Acquired overexpression OsARF7 The homozygous rice line.

[0070] Using wild-type rice Zhonghua 11 as a background, asexual T0 generation rice seedlings were cultured by infecting rice callus tissue with Agrobacterium-mediated transformation. T1 generation rice was obtained by harvesting seeds from individual T0 plants. Rice plants successfully transfected with the hygromycin resistance gene were screened on 1 / 2 MS medium plates containing hygromycin, yielding overexpressed rice varieties. OsARF7 We identified homozygous rice lines, analyzed the germination rate of T1 generation seeds, and then used Western blotting to identify protein expression in the homozygous rice seedlings.

[0071] The method for identifying OsARF7 protein using Western blot is as follows:

[0072] Seedling leaves were cut and rapidly frozen in liquid nitrogen, then ground and used to extract plant tissue proteins using IP-Buffer. 20 µL of the extracted protein sample was added to 5 µL of 5×SDS Loading Buffer and mixed thoroughly. The sample was then heated in a dry bath at 100 °C for 10 min. The prepared polyacrylamide gel was placed in a BIO-RAD electrophoresis tank, and the sample was loaded and electrophoresed at 80 V for approximately 2 h. The separating gel was cut and soaked in Transferring Buffer. A PVDF membrane of the same size as the separating gel was cut, rinsed with anhydrous methanol for 15 s, rinsed with ddH2O for 2 min, and then soaked in Transferring Buffer for 5 min. Appropriately sized filter paper pieces were also soaked in Transferring Buffer for 5 min. The sponge pad accompanying the transfer clamp was soaked in Transferring Buffer for 5 min. Open the transfer clamp and, according to the clamp colors, sequentially cover the membrane in the following order: negative electrode clamp – sponge pad – filter paper – separating gel – PVDF membrane – filter paper – sponge pad – positive electrode clamp. During this process, avoid air bubbles between layers. Transfer the membrane using 80V in Transferring Buffer for approximately 2 hours. Remove the transferred PVDF membrane and place it in blocking buffer. Block on a horizontal shaker at 60 rpm for at least 4 hours. Discard the blocking buffer, add 20 mL of hybridization buffer, and add primary antibody according to the antibody instructions. Incubate for 1 hour. After incubation, recover the hybridization buffer, add 20 mL of 1×TBST, and wash on a horizontal shaker at 60 rpm for 10 minutes. Repeat the washing step twice. Incubate with secondary antibody using the same method, and then wash three times with 1×TBST. After treating the PVDF membrane with chemiluminescence solution, directly photograph it using a chemiluminescence imaging system to observe the luminescence of the protein bands.

[0073] Homozygous rice lines OsARF7OE-1 and OsARF7OE-2, expressing the OsARF7 protein, were obtained through hygromycin plate screening and Western blotting. Seeds from individual plants (T1 generation) were harvested to propagate T2 generation rice, which served as validation material. T0 generation rice was constructed by Wuhan Boyuan Biotechnology Co., Ltd. using the pCAMBIAI1305-35S::OsARF7-eGFP vector.

[0074] (3) Disease course-related genes PRs The detection.

[0075] After treating rice seedlings with flg22 or chitin, leaves were cut and cryogenically ground in liquid nitrogen. Total mRNA was extracted from the samples using an RNA extraction kit manufactured by Kangwei Century Biological Reagent Co., Ltd., and then reverse transcribed into cDNA for subsequent quantitative real-time polymerase chain reaction (qRT-PCR) detection. Experimental methods followed the instructions for the FastSYBR Mixture kit manufactured by Kangwei Century Biological Reagent Co., Ltd. The reaction system and conditions for each well in a 96-well plate during the qRT-PCR reaction are shown in Tables 5 and 6. Finally, the raw data were statistically analyzed using computer software, and graphs were plotted for observation. PRs Gene expression status.

[0076] Table 5 qRT-PCR reaction system

[0077]

[0078] Table 6 qRT-PCR reaction procedure

[0079]

[0080] Note: In Table 6, " / " indicates that this item is not present.

[0081] according to Picture 1 The results showed that after treatment with chitin or flg22, overexpression OsARF7 Rice lines OsARF7OE-1 and OsARF7OE-2 in OsPR1b and OsPR10a The expression levels of two disease-related genes in rice were significantly higher than those in wild-type rice Zhonghua 11. This indicates... OsARF7 Increased expression of these genes can promote the expression of disease resistance-related genes in rice, thereby enhancing rice immunity.

[0082] 2. Overexpression OsARF7 Reactive oxygen species (ROS) burst detection was performed on rice lines to determine... OsARF7 The influence of genes on rice immunity.

[0083] The detection principle utilizes horseradish peroxidase (HRP) to continuously degrade peroxide ions generated during reactive oxygen species (ROS) bursts in leaf samples, simultaneously inducing L-012 chemiluminescence, resulting in a light intensity curve that gradually increases and then decreases over time. With constant HRP efficiency, the stronger the ROS burst in the sample, the more peroxide ions accumulate, and the higher the peak value of the curve. T2 generation overexpression was used. OsARF7 The genetically modified rice lines OsARF7OE-1 and OsARF7OE-2 were manipulated. The specific operation process is as follows:

[0084] 1) Before removing the spikelet, take the second leaf below the flag leaf of the mature seedling, make a leaf sample using a 5mm diameter circular punch, and place it in a sterile ddH2O dark place for 16 hours.

[0085] 2) Add the required reagents to a 1.5 mL centrifuge tube. The reaction system is shown in Table 7. The treatment group used flg22 or chitin as PAMPs to induce leaf samples, applied at a ratio of 1:1000 and 1:100, respectively; the mock group used an equal amount of ddH2O instead of flg22 or chitin.

[0086] Table 7 ROS Reaction System

[0087]

[0088] Note: In Table 7, " / " does not have this item.

[0089] 3) After adding the leaf samples, the RLU luminescence of the total system was quickly detected by a chemiluminescence detector once per minute for 25 minutes. Each sample treatment required 6 replicates and 3 independent experiments. Finally, the data were plotted into a curve to compare the differences between transgenic rice and wild-type rice in the treatment groups.

[0090] according to Picture 2 The results showed that after treatment with chitin or flg22, overexpression OsARF7 The reactive oxygen species (ROS) burst levels of the rice lines OsARF7OE-1 and OsARF7OE-2 were significantly higher than those of the wild-type rice Zhonghua 11. This indicates that... OsARF7 Increased expression of this substance can promote the reactive oxygen species burst level in rice when facing pathogen infection, thereby enhancing rice immunity.

[0091] 3. The overexpression was investigated through field artificial inoculation experiments. OsARF7 Immunophenotyping of rice bacterial blight pathogens was performed on rice lines to determine... OsARF7 The influence of genes on rice immunity.

[0092] The T2 generation overexpression obtained above was used OsARF7 The genetically modified rice lines OsARF7OE-1 and OsARF7OE-2 were manipulated. The specific procedures are as follows:

[0093] The PXO99A strain of rice bacterial blight was removed from a -80℃ freezer. It was incubated for 12 hours at 28℃ using NA liquid medium containing cephalexin at 200 rpm. The collected PXO99A cells were then resuspended in a 10 mM MgCl2 solution. OD 600Adjust the concentration to 0.8. Dip sterilized scissors in PXO99A bacterial solution and inoculate the tip of the second leaf after the flag leaf of the rice seedling. 14 days after inoculation, measure the length from the leaf cut to the end of the lesion.

[0094] Picture 3 The results showed that 14 days after artificial inoculation, overexpression... OsARF7 Compared with the blank control group EV, the rice lines OsARF7OE-1 and OsARF7OE-2 showed significantly shorter lesion extension lengths from the leaf cut downwards, a finding supported by statistical results. This indicates that... OsARF7 Increased expression of this substance can inhibit the infection of bacterial blight pathogen PXO99A, thereby enhancing rice's resistance to bacterial bacterial blight.

[0095] 4. The overexpression of [a specific organism] was investigated through field artificial inoculation experiments. OsARF7 Immunophenotypic detection of rice false smut pathogen was performed on rice lines to determine... OsARF7 The influence of genes on rice immunity.

[0096] The T2 generation overexpression obtained above was used OsARF7 The genetically modified rice lines OsARF7OE-1 and OsARF7OE-2 were manipulated. The specific procedures are as follows:

[0097] Wild-type rice blast fungus PJ52 was activated from a -80℃ freezer and placed on PSA agar plates, then incubated at 28℃ for 10 days. Mycelial blocks were then transferred to 100 mL of liquid culture medium and cultured at 28℃ with a shaker at 180 rpm for 7 days to induce sporulation under stress. Spores were filtered using a cell filter under aseptic conditions, and the spore concentration was adjusted to 1×10⁻⁶ using PSB liquid culture medium. 6 1 mL of rice false spore suspension was injected into the panicle of rice plants during the booting stage using a syringe, with 10 replicates per sample. The number of rice false spore balls and the incidence rate were counted 21 days after inoculation. Rice false spores invade rice plants through the floral parts during the booting stage. In the later stages of the disease, numerous chlamydospores form, which encapsulate the infected filaments to form rice false spore balls.

[0098] Picture 4 The results showed that 21 days after artificial inoculation, overexpression... OsARF7 Compared with the wild-type rice Zhonghua 11, the rice lines OsARF7OE-1 and OsARF7OE-2 had significantly fewer rice panicles, a finding supported by statistical results. This indicates that... OsARF7 Increased expression of this substance can inhibit the infection of rice false smut fungus PJ52, thereby enhancing rice's resistance to rice false smut.

[0099] 5. Directed mutation of rice using CRISPR-Cas9 technology OsARF7 Genes, used to assist in proof OsARF7 The function is as follows. Specific operation is as follows:

[0100] (1) Obtain osarf7 Homozygous gene knockout mutant lines.

[0101] Based on the whole genome sequence of wild-type rice Nipponbare and OsARF7 The gene sequence SEQ ID NO.1 was used to design a knockout target site, the sequence of which is shown in SEQ ID NO.5. Forward and reverse sequencing primers were designed based on the target site, and their sequences are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively.

[0102] The construction of the gene knockout line was completed by Weimi Biotechnology Co., Ltd., which obtained T1 generation seeds. The T2 generation rice obtained through propagation underwent DNA extraction and first-generation sequencing of the target site to obtain the insertion unit site T. osarf7 / 1-3 and missing CAACT osarf7 / 1-4 As material for subsequent verification.

[0103] SEQ ID NO. 5: GGCATCTGCTTACAACTGGCTGG.

[0104] SEQ ID NO. 6: GGCAAGAGCTTGTGGCTAGA.

[0105] SEQ ID NO. 7: GCATTGGTGAAGAACTTAGGTACA.

[0106] (2) Verification osarf7 Immunophenotype of gene knockout mutant lines.

[0107] Based on the above method, osarf7 Gene knockout mutant lines were subjected to pathogenesis-related gene detection, reactive oxygen species burst detection, and immunophenotypic detection of rice bacterial blight and rice false smut fungus through field artificial inoculation experiments.

[0108] Picture 5 The results showed that after treatment with chitin, OsARF7 Gene knockout mutant lines osarf7 / 1-3 and osarf7 / 1-4 exist OsPR1b and OsPR10a The expression levels of two disease-related genes in rice were significantly lower than those in wild-type rice Zhonghua 11. This indicates that the genes are missing. OsARF7 The gene will suppress the expression of disease resistance-related genes in rice, thereby affecting rice immunity.

[0109] Picture 6 The results showed that after treatment with chitin or flg22, osarf7Gene knockout mutant lines osarf7 / 1-3 and osarf7 / 1-4 The reactive oxygen species (ROS) bloom rate was significantly lower in the wild-type rice variety Zhonghua 11 than in the wild-type rice. This indicates a deficiency. OsARF7 Genes can reduce the reactive oxygen species (ROS) burst levels in rice when it is infected by pathogens, thereby affecting rice immunity.

[0110] Picture 7 The results showed that 14 days after artificial inoculation, osarf7 Gene knockout mutant lines osarf7 / 1-3 and osarf7 / 1-4 Compared to the wild-type rice Zhonghua 11, its lesions extended significantly longer downwards from the leaf cut, a finding supported by statistical results. This indicates a deficiency. OsARF7 When the gene is modified, the bacterial blight pathogen PXO99A has a stronger infectivity, and rice's resistance to bacterial bacterial blight is weakened.

[0111] Picture 8 The results showed that 21 days after artificial inoculation, osarf7 Gene knockout mutant lines osarf7 / 1-3 and osarf7 / 1-4 Compared to the wild-type rice Zhonghua 11, it had significantly more rice panicles with higher numbers of blasted grains, a finding supported by statistical results. This indicates a lack of... OsARF7 When the gene is modified, the rice false smut fungus PJ52 has a stronger infectivity, and rice's resistance to rice false smut is weakened.

[0112] 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.

[0113] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Overexpression OsARF7 The application of gene-based bioproducts in improving disease resistance in rice is characterized by, The OsARF7 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO.

2. Improving rice disease resistance refers to enhancing rice's resistance to bacterial blight and / or rice false smut.

2. Use according to claim 1, wherein The biological product is any one of the following: A: Includes OsARF7 Gene overexpression vectors; B: Includes OsARF7 Agrobacterium gene.

3. The application as described in claim 2, characterized in that, Using the overexpression vector and / or Agrobacterium to enhance OsARF7 Gene expression levels were adjusted to construct overexpression plants, thereby breeding disease-resistant rice.

4. The application as described in claim 3, characterized in that, The method for constructing overexpression plants is as follows: RNA was extracted from rice and reverse transcribed into cDNA; Using cDNA as a template, the primers shown in SEQ ID NO.3 and SEQ ID NO.4 were used to clone the sample. OsARF7 Gene; Will OsARF7 The gene is ligated into the enzyme-digested starting vector to obtain the overexpression vector; Overexpression plants were obtained by infecting rice with an overexpression vector via Agrobacterium-mediated transformation.

5. The application as described in claim 4, characterized in that, The launch vehicle is pCAMBIAI1305.

6. The application as described in claim 5, characterized in that, The restriction endonucleases used to digest the starting vector are KpnⅠ and HindⅢ.

7. The application as described in claim 4, characterized in that, The recipient material used in Agrobacterium-mediated infection of rice is callus tissue.

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