Rice blast resistance regulating gene uvase2 of rice and application thereof

Abstract

The application provides a rice smut fungus gene UvASE2 for regulating rice resistance and an application thereof, and relates to the technical field of plant genetic engineering.The nucleotide sequence of the rice smut fungus gene UvASE2 is shown as SEQ ID NO:1, and the amino acid sequence of the protein coded by the rice smut fungus gene UvASE2 is shown as SEQ ID NO:2.The application finds that the transgenic rice can significantly improve the resistance to rice smut and bacterial leaf blight and significantly induce the burst of active oxygen by constructing a transgenic rice plant heterologously expressing UvASE2.The heterologous expression of the rice smut fungus UvASE2 gene has the function of positively regulating the disease resistance of rice, and the UvASE2 gene can be used to improve the disease resistance of rice, and has important significance for the creation of rice disease-resistant germplasm.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically a gene for regulating rice false smut pathogen resistance. UvASE2 And its applications. Background Technology

[0002] Currently, the major diseases affecting rice include rice blast, rice bacterial blight, sheath blight, and rice false smut. Early research on rice false smut was limited, but it has now risen from a minor rice disease to one of the major ones. The false smut fungus that infects rice panicles in the later stages contains oryzin and smut toxins, which are harmful to humans, livestock, and poultry.

[0003] In recent decades, with climate change, the widespread adoption of high-quality, high-yield hybrid rice, and the excessive application of nitrogen fertilizer, fungal and bacterial diseases have occurred extensively in rice-producing areas, with a trend of increasing year by year. Therefore, the discovery and effective utilization of key genes to improve rice varieties and enhance rice's resistance to various diseases is urgently needed. Plant disease resistance is a complex process regulated by multiple genes. Current research focuses primarily on major resistance genes and resistance-related genes involved in plant disease resistance responses. Several resistance-related genes have been cloned; these genes encode different types of proteins that participate in the rice's resistance to different pathogens by regulating different response pathways within the rice plant. In recent years, functional studies on pathogen-secreted effector proteins have revealed that pathogens can secrete a class of proteins that can induce plant immunity, thereby enhancing plant disease resistance. However, the application of these effector protein genes capable of inducing plant immunity in breeding is currently limited to some extent. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a gene for regulating rice false smut disease resistance. UvASE2 Heterologous expression of rice false smut pathogen UvASE2 Genes have the function of positively regulating the disease resistance of rice.

[0005] Another object of the present invention is to provide a rice blast fungus gene. UvASE2 Application in improving resistant rice varieties.

[0006] Another objective of this invention is to provide a method for improving the resistance of rice to rice false smut and bacterial blight.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a gene for regulating rice false smut pathogen resistance. UvASE2 The rice false smut gene UvASE2 The nucleotide sequence of the rice false smut gene is shown in SEQ ID NO:1. UvASE2 The amino acid sequence encoding the protein is shown in SEQ ID NO:2.

[0008] The present invention also provides the aforementioned rice false smut gene. UvASE2 Application in improving resistant rice varieties.

[0009] Preferably, the resistance includes resistance to rice blast and bacterial blight.

[0010] Preferably, the rice false smut gene is overexpressed in rice recipient material. UvASE2 They obtained transgenic rice plants resistant to rice false smut and bacterial blight.

[0011] Preferably, the rice false smut gene UvASE2 It acts as an elicitor to induce immunity in rice.

[0012] Preferably, the rice false smut gene UvASE2 It can induce a burst of reactive oxygen species in transgenic rice plants.

[0013] This invention also provides a method for improving the resistance of rice to rice false smut and bacterial blight by overexpressing the aforementioned rice false smut gene in rice recipient material. UvASE2 This improves the resistance of rice recipient materials to rice false smut and bacterial blight.

[0014] Preferably, the rice false smut gene is used. UvASE2 The pCAMBIAI1305-UvASE2 plant overexpression vector was constructed by ligating it into the plant expression vector pCAMBIAI1305. The constructed pCAMBIAI1305-UvASE2 overexpression vector was introduced into rice recipient material using Agrobacterium-mediated transformation to cultivate transgenic rice plants resistant to rice false smut and bacterial blight.

[0015] Preferably, the Agrobacterium is Agrobacterium EHA105.

[0016] Preferably, the rice acceptor material is Nipponbare.

[0017] Compared with the prior art, the present invention has the following advantages: This invention discloses a gene from rice false smut fungus that regulates rice resistance. UvASE2(Atypical secreted effector protein 2) This gene can act as a promoter to induce plant immunity. In this invention, by artificially injecting a suspension of rice false smut fungus, observation 28 days after inoculation revealed that the number of rice false smut balls on the transgenic rice panicles was significantly lower than that of the wild-type Nipponbare, indicating that transgenic rice heterologously expressing UvASE2 improved resistance to rice false smut. In this invention, by inoculating bacterial blight fungus suspension using the leaf-cutting method, photographs and measurements of lesion length 14 days after inoculation showed that the lesion length on the leaves of transgenic rice was significantly lower than that of the wild-type Nipponbare. The results indicate that heterologous expression... UvASE2 The genetically modified rice improved its resistance to bacterial blight.

[0018] Chitin, a component of the fungal cell wall, can trigger a strong innate immune response in leaves, such as a burst of reactive oxygen species (ROS). This invention aims to further explore this topic. UvASE2 The effect on the host rice defense response was investigated by taking leaf samples with a punch, soaking them in sterile water for 16 hours, and then measuring the ROS level within 25 minutes. The results showed that after chitin treatment, transgenic rice lines 1 and 2 could significantly induce ROS bursts.

[0019] The above experimental results indicate that heterologous expression of the UvASE2 gene of rice false smut fungus has a positive regulatory function on rice disease resistance and can be used for rice disease resistance improvement, which is of great significance for the creation of new disease-resistant rice varieties. Attached Figure Description

[0020] Figure 1 The rice false smut gene of the present invention UvASE2 A schematic diagram illustrating the secretion verification process; Figure 2 The rice false smut gene of the present invention UvASE2 Schematic diagram of induced tobacco cell necrosis, where A represents UvASE2 Results of the tobacco cell necrosis induction assay; B represents the ion leakage detection assay results. represent P <0.001 indicates a significant difference.

[0021] Figure 3 This is a schematic diagram illustrating the detection and verification of protein expression in transgenic rice with heterologous expression of the rice false smut gene UvASE2.

[0022] Figure 4 This diagram illustrates the results of resistance testing of the transgenic rice to rice false smut of this invention. Figure A shows the disease incidence in the transgenic rice and Nipponbare rice 28 days after injection of rice false smut fungus, while Figure B shows the statistical results of the number of rice false smut pellets 28 days after injection of rice false smut fungus. This indicates a significant difference in the average number of rice blasts per inoculated panicle between transgenic rice and Nipponbare (NBP) wild-type rice. P< 0.001.

[0023] Figure 5 This diagram illustrates the results of resistance testing of the transgenic rice to bacterial blight. Figure A shows the bacterial blight incidence in the transgenic rice and Nipponbare rice 14 days after inoculation with the bacterial blight pathogen using the leaf-cutting method. Figure B shows the statistical analysis of bacterial blight lesion length 14 days after inoculation with the bacterial blight pathogen using the leaf-cutting method. This indicates a significant difference in the length of lesions on leaves between transgenic rice and Nipponbare wild-type (NBP) inoculated rice. P< 0.001.

[0024] Figure 6 This is a schematic diagram showing the detection results of reactive oxygen species bursts in the transgenic rice of this invention. Detailed Implementation

[0025] This invention provides a gene for regulating rice false smut pathogen resistance. UvASE2 The NCBI accession number for this gene is XM_043144736.1. This is the rice false smut gene. UvASE2 UvASE2 The amino acid sequence encoding the protein is MKLSSTMLVVAARMAMAGRVANLFSPRIIPRQDETELHDRSVAIQGMSGYRISPNALSEVIIIWVNLGNGSPVTTVNQAMTVTKTVTAGPGGAVTPLPGYGASTTAAAPAKCATHTVKVGGPGVLTFQPSELNNIPVGDIVVFEFFAQNHTVTQSSFNIPCKALAGGMDSGFLANPNNTVSPPPQVAMQVMTAKPLWFYCRQKGHCGKGMVFSINPTADKTHAMFRELAIAQNGTGSATPVSGGKSSVAPPVQAPQQTPDGGQRCSGTSVAGPQSSVAPPGPAPQQTPYGEQGGSGLTYGKGTVGADGSCTCVVACSAGSFPAVQPQGVGACGGMAGSLPVMDSMS (SEQ ID NO:2), in this sequence This represents the stop codon.

[0026] This invention uses the pSUC2 yeast secretion system to verify the gene of rice false smut. UvASE2 The experiment demonstrated that UvASE2 can guide the secretion of SUC2 protein, successfully directing the protein into the secretory pathway, enabling the YTK12 transformant carrying UvASE2 to grow on CMD-W and YPRAA media. In a further colorimetric reaction, glucose, the enzyme activity product of SUC2 carrying UvASE2, reduced 2,3,5-triphenyltetrazolium chloride (TTC) to insoluble red 1,3,5-triphenylcarboxylic acid (TPF), indicating that the *Saccharomyces cerevisiae* gene... UvASE2 It has secretory function.

[0027] The present invention also provides the aforementioned rice false smut gene. UvASE2 Application in improving resistant rice varieties. In this invention, the resistance preferably includes resistance to rice false smut and bacterial blight. This invention, through constructing transgenic rice heterologously overexpressing the rice false smut gene UvASE2, found that transgenic rice lines can significantly improve resistance to rice false smut and bacterial blight.

[0028] In this invention, the rice false smut gene UvASE2 It acts as a promoter to induce immunity in rice. To determine the rice false smut pathogen gene... UvASE2The function of UvASE2 in plant immunity was verified using a transient expression system in tobacco. After injecting Agrobacterium fusion with the UvASE2 protein into tobacco leaves for 5 days, it was found that UvASE2 induced cell death in tobacco leaves, with the degree of death approaching that induced by the positive control BAX (a cell death-promoting gene in the mouse Bcl-2 gene family), while the negative control GFP (pGR107-GFP) could not induce cell death in tobacco. This invention also quantitatively analyzed cell death on *N. benthamiana* leaves using an ion leakage experiment. The results showed that BAX and UvASE2-induced cell death in *N. benthamiana* resulted in severe ion leakage, while ion leakage was significantly reduced after GFP expression. These results indicate that UvASE2 can induce cell necrosis in *N. benthamiana*.

[0029] In this invention, the rice false smut gene UvASE2 This study investigated the effect of UvASE2 on the defense response of transgenic rice plants. Leaf samples were collected using a perforator and soaked in sterile water for 16 h. Then, samples were induced with 10 µg / mL chitin, and ROS levels were measured within 25 min. The results showed that transgenic rice lines significantly induced ROS bursts. Chitin, a component of the fungal cell wall, can induce strong innate immune responses, such as ROS bursts, in rice callus, suspension cell lines, and leaves.

[0030] This invention also provides a method for improving the resistance of rice to rice false smut and bacterial blight by overexpressing the aforementioned rice false smut gene in rice recipient material. UvASE2 This method can improve the resistance of rice recipient materials to rice false smut and bacterial blight. In this invention, the method preferably includes the following steps: transferring the rice false smut pathogen gene... UvASE2 A plant overexpression vector, pCAMBIAI1305-UvASE2, was constructed by ligating it into the plant expression vector pCAMBIAI1305. Using Agrobacterium-mediated transformation, the constructed pCAMBIAI1305-UvASE2 overexpression vector was introduced into rice recipient material, resulting in transgenic rice plants resistant to rice false smut and bacterial blight. This invention is preferably based on the rice false smut pathogen gene. UvASE2 Sequence, design primers for PCR, perform PCR amplification, and then ligate to... KpnI and HindⅢA plant overexpression vector, pCAMBIAI1305-UvASE2, was constructed in the digested pCAMBIAI1305 vector. This overexpression vector was then introduced into Nipponbare rice recipient material via Agrobacterium transformation to obtain transgenic rice heterologously overexpressing the rice false smut gene UvASE2. In this invention, Agrobacterium is preferably EHA105. Through resistance verification of the constructed transgenic rice plants heterologously expressing UvASE2, this invention found that the transgenic rice plants significantly improved resistance to rice false smut and bacterial blight.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0033] Example 1 This embodiment uses the pSUC2 yeast secretion system to verify the secretory function of UvASE2. YTK12 is an auxotrophic yeast lacking the sucrose invertase gene (SUC2) and the tryptophan synthase gene. The pSUC2 vector can synthesize tryptophan and carries a sucrose invertase gene fragment lacking a signal peptide. When the secretory gene is linked to the N-terminus of the pSUC2 vector and transformed into YTK12 yeast, YTK12 yeast can synthesize tryptophan and convert raffinose into glucose, thus enabling normal growth on CMD-W and YPRAA media. The results can be further verified using a colorimetric reaction, as glucose, the enzyme product of SUC2, can reduce 2,3,5-triphenyltetrazolium chloride (TTC) to insoluble red 1,3,5-triphenylcarboxylic acid (TPF). In this embodiment, pSUC2-Avr1b, pSUC2 empty vector, and pSUC2-Mg87 were used as controls. pSUC2-Avr1b can grow on both CMD-W and YPRAA media and can reduce TTC; therefore, pSUC2-Avr1b was used as a positive control, while the pSUC2 empty vector and pSUC2-Mg87 were used as negative controls. The specific verification method is as follows: (1) Construct a vector containing the sucrase transformation gene SUC2. ①Based on genes UvASE2Based on the sequence SEQ ID NO:1, primers for PCR were designed. The forward primer was TTTAATTAAGAATTCATGAAGCTCTCGTCGACAATGC (SEQ ID NO:3), and the reverse primer was AGGGAGAACGAGCTCTGACATGGAGTCCATGACTGG (SEQ ID NO:4). Using the cDNA of rice false smut fungus P1FZ as a template, the reaction mixture was prepared in a 200 μL centrifuge tube. The reaction mixture is shown in Table 1. Table 1 Reaction System

[0034] ② The mixed reaction system was used for gene amplification in a PCR instrument, and the PCR reaction conditions were set as shown in Table 2: Table 2 PCR reaction system

[0035] (2) Expression vector digestion Perform the required pSUC2 vector EcoRI and XhoI Double enzyme digestion was performed, and the reaction system was mixed in a 200 μL centrifuge tube (see Table 3). The mixture was then incubated at 37°C for 3-4 hours. Table 3 Enzyme digestion reaction system

[0036] (3) Obtaining recycled rubber products After the products from steps (1) and (2) are obtained, 10× Loading Buffer is added to the tube, and electrophoresis is performed on a 1% agarose gel at 150V to verify the fragment size. The target fragment band in the gel is then recovered and purified using a gel extraction kit, and the recovered product is stored at -20℃. The experimental method is based on the agarose gel DNA recovery kit from Kangwei Century Biological Reagent Co., Ltd., and the steps are as follows: ① Weigh the cut target gel block in a 2.0 mL centrifuge tube, add 1 volume of PG Buffer, and solv in a 65℃ dry bath. During this process, continuously invert and mix until completely dissolved, then remove and cool to room temperature.

[0037] ② Adsorption column equilibration: Add 200µL PS Buffer to the adsorption column and centrifuge at 12000rpm for 1min.

[0038] ③ Remove the waste liquid, add the gel solution to the adsorption column, let it stand at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute.

[0039] ④ Remove the waste liquid, add 500µL PW Buffer to the adsorption column for rinsing, centrifuge at 12000rpm for 1min, and rinse again.

[0040] ⑤ Remove waste liquid by centrifuging an empty column at 12,000 rpm for 3 minutes to remove residual ethanol.

[0041] ⑥ Place the adsorption column in a new 1.5 mL centrifuge tube and open the cap. Let it stand at room temperature for 5 minutes to allow the ethanol to evaporate.

[0042] ⑦ Add 30~50µL of EB Buffer (preheated to 65℃) to the adsorption column, let stand for 1 min, centrifuge at 12000rpm for 1 min to obtain DNA solution, and store at -20℃ for later use.

[0043] (4) Homologous recombination to obtain expression vectors The experimental method referenced the cloning kit from Nanjing Novizan Biotechnology Co., Ltd., and the steps are as follows: ①Use DNA ligase to ligate the digested vector and the target fragment, and mix the reaction system in a 200 μL centrifuge tube (see Table 4); incubate at 37℃ for 30 min; Table 4 Connection Reaction System

[0044] ② Thaw on ice to clone competent cells (DH5α competent cells, Vazyme#C502).

[0045] ③ Add 10 μL of the recombinant product to 100 μL of competent cells, gently tap the tube wall to mix (do not shake to mix), and let stand on ice for 30 min.

[0046] ④ After heat shock in a 42℃ water bath for 45 seconds, immediately place it on ice to cool for 2~3 minutes.

[0047] ⑤ Add 900 μL LB (without antibiotics) and shake at 37°C for 1 hour (200-250 rpm).

[0048] ⑥ Preheat the LB solid culture medium plates containing the corresponding antibodies in an incubator at 37°C.

[0049] ⑦ Centrifuge at 5000 rpm (2400 × g) for 5 min, and remove 900 µL of supernatant in a clean bench. Resuspend the bacteria in the remaining culture medium and spread it evenly on a plate containing the correct antibiotic using a sterile spreader.

[0050] ⑧ Incubate upside down in a 37℃ constant temperature incubator for 12~16 hours.

[0051] ⑨ Pick a single-clone transformant colony from the plate, add 5 mL of LB liquid medium containing antibiotics to the test tube, and place it slanted in a constant temperature shaker at 37℃ and 180 rpm for 12 hours to propagate. ⑩ Take a portion of the bacterial culture and purify the plasmid using a plasmid mini-prep kit (Tiangen Biotech (Beijing) Co., Ltd., DP103). Use PCR and first-generation sequencing technologies to verify whether the target gene has been successfully and correctly ligated into the plasmid vector. If the construction is successful, mix the remaining bacterial culture with 40% glycerol at a 1:1 ratio and store it in a -80℃ freezer for long-term storage.

[0052] (5) Construct the pSUC2 vector and transform it into yeast YTK12 pSUC2-Avr1b, pSUC2-Mg87, pSUC2-EV (empty vector), and pSUC2-UvASE2 were transferred to YTK12. 100 µL of YTK12 competent yeast cells were taken from a -80°C freezer and thawed on ice. 2000 μL of the constructed pSUC2 expression vector (c is the expression vector concentration, in ng / µL) and 500 µL of EZ3 were slowly added. The mixture was gently stirred with a pipette tip and then incubated in a 30°C water bath for 45 min. During this time, the cells were inverted and mixed several times every 15 min. Afterward, the cells were centrifuged at 8000 rpm for 1 min, plated on CMD-W medium, and incubated upside down in a 28°C incubator for 3 days to obtain single-clone transformants.

[0053] (6) YPRAA culture medium plate verification method The single-clone transformants in (3) were streaked on CMD-W and YPRAA medium, inverted and cultured in a 28℃ constant temperature incubator for 3 days, and the growth of the colonies was observed.

[0054] (7) TTC Reduction Verification Method ① The single-clone transformant in step (3) was transferred into 5 mL of CMD-W liquid medium and cultured in a constant temperature shaker at 28℃ and 200 rpm for 12 h. YTK12 yeast that was not transferred into any vector was cultured in 5 mL of YPD liquid medium. ② Collect bacterial culture in a 2mL centrifuge tube, centrifuge at 11000rpm for 1min, discard the supernatant, add sterile ddH2O, gently aspirate to resuspend the bacterial cells for washing, centrifuge at 11000rpm for 1min, discard the supernatant, and repeat the washing process once. ③ Add 750µL ddH2O, 250µL HAc-NaAc buffer (10mM, pH=4.7) and 500µL 10% sucrose solution to the tube, mix well, and incubate in a 37℃ water bath for 10 min. ④ Centrifuge at 11000 rpm for 1 min, take 100 µL of supernatant into a clean test tube, add 900 µL of 0.1% TTC solution (adjust to an alkaline environment using NaOH solution), let stand at room temperature for 5 min, and observe the color change of the solution.

[0055] according to Figure 1 The results showed that UvASE2 can guide the secretion of SUC2 protein, successfully directing the protein into the secretory pathway, enabling the YTK12 transformant carrying UvASE2 to grow on CMD-W and YPRAA media. In a further colorimetric reaction, glucose, the enzyme activity product of SUC2 carrying UvASE2, reduced 2,3,5-triphenyltetrazolium chloride (TTC) to insoluble red 1,3,5-triphenylcarboxylic acid (TPF), demonstrating that UvASE2 possesses secretory function.

[0056] Example 2 This embodiment uses a tobacco transient expression system to verify the effect of the atypical effector protein UvASE2 on plant immunity, and finds that UvASE2 can induce tobacco cell necrosis. The specific operation is as follows: (1) Construction of pGR107 (Potato virus X, PVX) expression vector Based on genes UvASE2 Based on the sequence SEQ ID NO:1, primers for PCR were designed: the forward primer was CTAGAACTAGTGGATCCCCCGGGATGAAGCTCTCGTCGACAATG (SEQ ID NO:5), and the reverse primer was TTAACCGTTCATCGGCGGTCGACTCATGACATGGAGTCCATGAC (SEQ ID NO:6). PCR amplification was performed, followed by ligation into... SmaⅠ and Sal Ⅰ The pGR107-UvASE2 expression vector was constructed from the enzyme-digested pGR107 vector, and the specific method is described in Example 1.

[0057] (2) pGR107-UvASE2 expression was transformed into Agrobacterium GV3101 and a single clone of the transformed strain was obtained. ① Take 100µL of Agrobacterium GV3101 competent cells from a -80℃ freezer and thaw them on ice. Slowly add 10µL of the correctly constructed pGR107-UvASE2 expression vector, gently mix with a pipette tip, and incubate on ice for 30 min. Then, rapidly freeze them in liquid nitrogen for 1 min, and then heat shock them in a 37℃ water bath for 90 s without shaking the bacterial culture. Then, quickly and gently transfer them to an ice bath for 3 min. Finally, add 500µL of LB liquid medium and incubate horizontally in a 28℃, 150rpm constant temperature shaker for 3 h. ② Centrifuge at 8000 rpm for 1 min, remove 500 µL of supernatant in a clean bench, gently mix the remaining 100 µL of supernatant with the precipitated bacterial cells, and then evenly spread the mixture on LA medium plates containing Kanamycin (working concentration 50 µg / mL) and Rifampicin (working concentration 25 µg / mL) using a sterilized and cooled spreader. Invert the plates and incubate them in a 28°C incubator for 3 days. ③ Pick a single-clone transformant colony from the plate, add 5 mL of LB liquid medium containing both Kanamycin and Rifampicin to the test tube, and incubate at 37°C and 180 rpm in a constant temperature shaker until the concentration reaches OD500. 600 =1, thus obtaining Agrobacterium tumefaciens bacterial solution.

[0058] (3) Transient expression of tobacco by single-clonal transformed strains This invention uses Agrobacterium GV3101 transformed with pGR107-BAX as a positive control and Agrobacterium GV3101 transformed with pGR107-GFP as a negative control. Subsequently, the desired Agrobacterium was activated by streak plating on LA medium plates supplemented with two antibiotics, Kanamycin (working concentration 50 μg / mL) and Rifampicin (working concentration 25 μg / mL), and incubated upside down at 28℃ for 3 days. The activated Agrobacterium was then cultured in liquid LB medium with shaking for 12-16 hours. The cultured Agrobacterium underwent further processing as follows: ① Prepare the buffer suspension; see Table 5 for the specific formula: Table 5 Buffer Suspension Formulation

[0059] ② Collect bacterial culture using a 2mL centrifuge tube, centrifuge at 9000rpm for 1min, discard the supernatant, add sterile ddH2O, gently aspirate to resuspend the bacterial cells for washing, centrifuge at 9000rpm for 1min, discard the supernatant, and repeat the washing process once. ③ Resuspend the bacterial cells in 1 mL of buffer suspension, and then measure the OD using a spectrophotometer. 600 Adjust the bacterial concentration to 1.0 using buffer suspension and let it stand at 28℃ for 4-6 hours; ④ Use a 1mL disposable syringe (without needle) to draw up Agrobacterium tumefaciens solution and inject it into the lower epidermis of Tobacco Benzovia leaves under pressure. Observe the tissue necrosis at the injection site one week later.

[0060] (4) Conductivity measurement ① Three days after the target protein was expressed on tobacco, holes were punched at the inoculation site using a 9mm diameter punch to obtain a sufficient number of leaf discs.

[0061] ② Add 5 mL of ddH2O to a 10 mL centrifuge tube, place 5 leaf discs in one tube, and shake at 80 rpm for 3 hours.

[0062] ② Remove the impeller, use a conductivity meter to test the liquid and record the data; put the impeller back into the liquid and treat it in a boiling water bath for 25 minutes.

[0063] ③ Test the liquid again with a conductivity meter and record the readings.

[0064] ④ The ratio of the two values ​​for the bladed disk conductivity.

[0065] To determine the function of UvASE2 in plant immunity, this invention utilized a transient expression system in tobacco for verification. After injecting Agrobacterium fusion with UvASE2 protein into tobacco leaves for 5 days, it was found that UvASE2 could induce cell death in tobacco leaves, with the degree of death being similar to that induced by the positive control group BXA (a gene promoting apoptosis in the mouse Bcl-2 gene family), while the negative control GFP (pGR107-GFP) could not induce cell necrosis in tobacco leaves. Figure 2 (A) This invention also uses ion leakage experiments to quantitatively analyze cell death on tobacco leaves. According to Figure 2 Results B showed that BAX and UvASE2-induced cell death in *Nicotiana benthamiana* resulted in severe ion leakage, while GFP expression significantly reduced ion leakage in leaf cells. These results indicate that UvASE2 can induce cell necrosis in *Nicotiana benthamiana*.

[0066] Example 3 In this embodiment, transgenic rice plants heterologously overexpressing the rice false smut gene UvASE2 were constructed. The specific steps are as follows: (1) Construction of plant overexpression vector pCAMBIAI1305-UvASE2 Based on genes UvASE2Based on the sequence SEQ ID NO:1, primers for PCR were designed: the forward primer was TGTACAGAGCTCGGTACCATGAAGCTCTCGTCGACAATGC (SEQ ID NO:7), and the reverse primer was GTCTTTGTAGTCAAGCTTTGACATGGAGTCCATGACTGG (SEQ ID NO:8). PCR amplification was performed, followed by ligation into... KpnI and HindⅢ The pCAMBIAI1305-UvASE2 overexpression vector was constructed from the enzyme-digested pCAMBIAI1305 vector, and the specific method is described in Example 1.

[0067] (2) Obtaining transgenic rice plants expressed heterologously At Wuhan Boyuan Biotechnology Co., Ltd. (Project No. TRGEN_31639C23F668, Order No. T78569), transgenic rice plants that heterologously overexpressed the UvASE2 gene of rice false smut fungus were obtained by transforming Boyuan Nipponbare rice with Agrobacterium tumefaciens. The Agrobacterium tumefaciens was EHA105.

[0068] (3) Detection of expression in transgenic rice plants ① Cut out the corresponding tissue samples, then freeze them quickly in liquid nitrogen, grind them, and use a kit to extract plant tissue proteins; ② Take 20µL of extracted protein sample, add 5µL of 5×SDS Loading Buffer and mix well. Heat in a dry bath at 100℃ for 10 min. Place the prepared polyacrylamide gel in a BIO-RAD electrophoresis tank and perform sample loading. Electrophoresis at 80V for about 2 h. Cut off the separating gel and soak it in transfer buffer. ③ Cut a PVDF membrane the same size as the separating gel, rinse with anhydrous methanol for 15 seconds, rinse with ddH2O for 2 minutes, and then soak it in the transfer solution for 5 minutes; cut a filter paper of appropriate size and soak it in the transfer solution for 5 minutes; soak the sponge pad that comes with the transfer clamp in the transfer solution for 5 minutes. ④ Open the transfer clamp and cover the membrane in the following order according to the color of the clamps: “negative electrode clamp – sponge pad – filter paper – separating gel – PVDF membrane – filter paper – sponge pad – positive electrode clamp”. During this process, be careful to avoid air bubbles between the layers. Use 80V voltage in the transfer solution to transfer the membrane for about 2 hours.

[0069] ⑤ Remove the transferred PVDF membrane and place it in blocking solution. Block on a shaker at 60 rpm at room temperature for at least 1 hour. Discard the blocking solution, add 20 mL of Anti-FLAG (Beijing TransGen Biotech Co., Ltd., catalog number: HT201-01), and incubate for 1 hour. After incubation, recover the hybridization solution, add 20 mL of 1×TBST, and wash on a shaker at 60 rpm at room temperature for 10 minutes. Repeat the washing step twice. Incubate with the secondary antibody Anti-Mouse (Beijing TransGen Biotech Co., Ltd., catalog number: HS201-01) for 1 hour using the same method, and then wash three times with 1×TBST. ⑥ Place the treated PVDF membrane in a cut transparent self-sealing bag, add the reaction substrate (500µL HRP Substrate Peroxide Solution + 500µL HRP Substrate Luminol Reagent; Immobilon Western, Millipore), react for 5 min, blot dry with absorbent paper, press the X-ray film in a dark room for about 5 min, determine the pressing time according to the signal intensity, then develop in developer (Kangwei Century), remove and fix in fixer (Kangwei Century) to obtain the X-ray film; or treat the PVDF membrane with luminescent solution and then use a chemiluminescence imaging system to directly photograph and observe the luminescence of the protein bands.

[0070] The formulations of some of the reagents required for the experiment are shown in Tables 6 and 7: Table 6 Transfer Buffer Formulation

[0071] Table 7 10×TBS Buffer Recipe

[0072] Based on the formulation in Table 7, dissolve in distilled water to pH 7.5 and bring the volume to 1L. Preparation of 1×TBST solution: 100mL 10×TBS Buffer, 500µL Tween 20, bring the volume to 1L with distilled water.

[0073] Experimental results show that: The rice false smut gene... UvASE2 The SEQ ID NO:1 sequence was fused into the pCAMBIAI1305 plant expression vector. Heterologous overexpression transgenic rice plants were obtained via Agrobacterium-mediated transformation. Since FLAG and UvASE2 are fusion proteins, the expression of UvASE2 protein could be detected using a FLAG antibody. Western blot results showed (see...) Figure 3 ), rice plant 1 ( Figure 3 (UvASE2OE-1) and 2 ( Figure 3UvASE2OE-2 was expressed, thus obtaining two transgenic T0 generation homozygous lines expressing the UvASE2 protein fusion tag FLAG.

[0074] Example 4 After cultivating the T0 generation homozygous rice seedlings 1 and 2 of the transgenic rice that heterologously overexpressed the rice false smut gene UvASE2 obtained in Example 3, T1 generation seeds of rice seedlings 1 and 2 were obtained. The T1 generation was then propagated to obtain T2 generation transgenic rice plants. This example verifies the resistance of the T2 generation transgenic rice plants to rice false smut. The specific steps are as follows: (1) Take the wild-type rice false smut fungus (PJ60-2) from the -80℃ freezer and activate it onto a solid plate. Incubate it in a 28℃ incubator for 10 days. Cut the fungal blocks into 100mL of liquid culture medium and incubate them in a shaker at 180 rpm at 28℃ for 5-7 days. (2) Adjust the concentration of rice false spores to 1×10 6 1 mL of bacterial suspension was injected into the panicles of newly heading T2 generation rice plants 1 and 2 using a syringe. Each sample was replicated three times, with 20 panicles inoculated each time. The number of rice blast balls and the incidence of disease were counted 28 days after inoculation.

[0075] according to Figure 4 The results showed that the rice false smut pathogen invades the rice flower before heading. In the later stages of the disease, a large number of chlamydospores are formed, which encapsulate the infected filaments to form rice false smut balls. Figure 4 (A) To further investigate the immune effects of UvASE2 on rice, rice plants 1 and 2 of the T2 generation at heading stage were artificially injected with a suspension of rice false smut pathogen. Photographs were taken 28 days after inoculation, and the results showed that the number of rice false smut pellets in the transgenic rice (UvASE2OE-1 and UvASE2OE-2) was significantly lower than that in the wild-type Nipponbare. Figure 4 (B in the figure) indicates that transgenic rice heterologously expressing UvASE2 improves resistance to rice false smut.

[0076] Example 5 The T2 generation heterologous overexpression of rice false smut gene obtained in Example 4 UvASE2 The transgenic rice plants were tested for resistance to bacterial blight. The specific steps are as follows: (1) Remove the bacterial blight pathogen from the -4℃ freezer ( Xanthomonas oryzae pv. oryzae , Xoo The cells were streaked onto plates, then activated with NA liquid medium containing 25 μg / mL cephalexin. The cells were collected by centrifugation at 9000 rpm, washed twice with 10 mM MgCl2 solution, resuspended, and OD adjusted. 600 It is 0.8.

[0077] (2) Dip scissors in the bacterial solution and inoculate the tip of the second leaf after the flag leaf of the rice seedling. 14 days after inoculation, count the length from the leaf cut to the end of the lesion.

[0078] Inoculate with a suspension of *Rhizoctonia solani* using the leaf-cutting method. Fourteen days after inoculation, photograph and measure the length of the lesions. Figure 5 Results A through B showed that the lesion length in transgenic rice was significantly shorter than that in wild-type Nipponbare rice, indicating heterologous expression. UvASE2 The genetically modified rice has improved resistance to bacterial blight.

[0079] Example 6 Chitin, a component of the fungal cell wall, can trigger a strong innate immune response in leaves, such as a burst of reactive oxygen species (ROS). This example explores the genes of the rice false smut pathogen. UvASE2 The effects on the host rice's defense response were investigated through the following steps: (1) Plant the T2 generation transgenic rice plants obtained in Example 4, take the second leaf after the flag leaf of the seedling before the heading, use a 5mm diameter punch to punch the leaf, select the complete leaf and put it in ddH2O and let it stand in the dark for 16h. (2) Load the samples and reagents listed in Table 8 into 1.5 mL centrifuge tubes: Table 8 Reaction Samples and Reagents

[0080] The final concentration of chitin was 10 μg / mL; the mock treatment involved replacing chitin with an equal volume of ddH2O.

[0081] (3) Use a chemiluminescence detector to detect the luminescence per minute for 25 minutes. Each leaf treatment needs to be repeated 3 times and 3 different sampling treatments need to be performed. Finally, the data are plotted into a curve to compare the differences between transgenic rice and wild type in the treatment group.

[0082] Chitin, a component of the fungal cell wall, can trigger strong innate immune responses, such as reactive oxygen species (ROS) bursts, in rice callus, suspension cell lines, and leaves. According to... Figure 6 The results showed that after induction treatment with chitin, the ROS level in the leaves of UvASE2OE transgenic rice within 25 min was detected, and a significant ROS surge was found in transgenic rice lines 1 (UvASE2OE-1-Chitin) and 2 (UvASE2OE-2-Chitin).

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A rice false smut gene, UvASE2, for enhancing rice resistance, characterized in that, The nucleotide sequence of the rice false smut gene UvASE2 is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the rice false smut gene UvASE2 is shown in SEQ ID NO:

2.

2. The application of the rice false smut gene UvASE2 as described in claim 1 in improving resistant rice varieties, characterized in that, The resistance includes resistance to rice false smut and bacterial blight.

3. The application according to claim 2, characterized in that, By overexpressing the rice false smut gene UvASE2 in rice recipient materials, transgenic rice plants resistant to rice false smut and bacterial blight were obtained.

4. The application according to claim 2, characterized in that, The rice false smut pathogen gene UvASE2 acts as an elicitor to induce immunity in rice.

5. The application according to claim 2, characterized in that, The UvASE2 gene of rice false smut can induce a burst of reactive oxygen species in transgenic rice plants.

6. A method for improving the resistance of rice to rice false smut and bacterial blight, characterized in that, Overexpression of the rice false smut gene UvASE2 as described in claim 1 in rice recipient materials enhances the resistance of rice recipient materials to rice false smut and bacterial blight.

7. The method according to claim 6, characterized in that, The rice false smut gene UvASE2 was ligated into the plant expression vector pCAMBIAI1305 to construct the pCAMBIAI1305-UvASE2 plant overexpression vector; the constructed pCAMBIAI1305-UvASE2 overexpression vector was introduced into rice recipient material using Agrobacterium-mediated transformation to cultivate transgenic rice plants resistant to rice false smut and bacterial blight.

8. The method according to claim 7, characterized in that, The Agrobacterium is Agrobacterium EHA105.

9. The method according to claim 6, characterized in that, The rice acceptor material is Nipponbare.

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