Disease-resistant gene nbstomrh and application thereof in improving plant disease resistance and disease prevention and control

Abstract

This invention belongs to the field of genetic engineering technology and discloses a disease-resistant gene. NbSTOMRh The gene and its application in improving plant disease resistance and disease control are described. The protein sequence encoded by this gene is shown in SEQ ID NO.2, and the nucleotide sequence encoding the protein is shown in SEQ ID NO.1. This invention involves knocking out [a specific gene] in tobacco. NbSTOMRh Following gene therapy, STOM-induced cell death and reactive oxygen species production were significantly reduced. NbSTOMRh The tobacco knockout variant of the gene showed significantly reduced resistance to *Sclerotinia sclerotiorum*, while expression of the gene significantly enhanced resistance of *Nicotiana benthamiana* to *Sclerotinia sclerotiorum*. Therefore, the present invention provides… NbSTOMRh It can be applied to disease-resistant breeding in tobacco and disease control against Sclerotinia sclerotiorum. Simultaneously, this gene can be transferred across species to rice, enhancing rice's resistance to rice blast and false smut, and can be applied to disease-resistant breeding and disease control in rice.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to disease-resistant genes. NbSTOMRh And its application in improving plant disease resistance and disease control. Background Technology

[0002] Rice is a major food crop in my country and for more than 50% of the world's population. In recent years, with changes in farming methods and climate change, rice blast disease has become increasingly serious. This disease not only reduces rice yields but also produces toxins that harm human health. Furthermore, rice blast, caused by *Bacillus oryzae*, is often referred to as the "cancer" of rice and is considered one of the most important diseases affecting rice. Although genes resistant to rice blast have been reported, genes resistant to rice false smut, and genes resistant to both diseases simultaneously, are rarely reported.

[0003] Rice false smut specifically damages the panicle of rice, is difficult to inoculate, has a long disease cycle, and is greatly affected by the environment. These characteristics pose significant challenges to the identification of rice false smut resistance genes. Currently, very few stable rice materials with high resistance to rice false smut have been identified. Identifying resistance genes from non-host plants and applying them to the control of rice false smut in rice can overcome the dependence on rice germplasm resources resistant to rice false smut.

[0004] Since multiple diseases often occur simultaneously in rice fields, especially rice blast which occurs year-round, the discovery of disease-resistant genes that resist multiple diseases, especially important ones, has a wider range of application value.

[0005] The plant immune system can be divided into two main categories of response mechanisms: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is an immune response triggered by the recognition of pathogen-associated molecular patterns (PAMPs) when a plant encounters a pathogen. A key class of receptors on the surface of plant cells, called pattern recognition receptors (PRRs), are responsible for recognizing these PAMPs. In plants, PRRs initiate defense responses by activating intracellular signaling pathways, such as inducing cell death, reactive oxygen species (ROS) production, and the expression of defense-related genes, thereby effectively preventing further pathogen infection. PRRs can recognize PAMPs conserved in different fungi, and are therefore considered to have broad-spectrum disease resistance potential. The effective functioning of PRRs often requires co-receptors to activate and transmit immune signals; therefore, co-receptors are an important component of PTI immunity.

[0006] Ben's tobacco ( Nicotiana benthamianaAs a widely used model plant, tobacco has been extensively applied in plant immunology research due to its short life cycle and ease of transgenic development. Meanwhile, tobacco is also an important economic crop in my country, susceptible to pathogens such as Sclerotinia sclerotiorum. Selecting tobacco to discover disease-resistant genes not only holds promise for solving the disease resistance problem of tobacco itself, but also for applying non-host disease-resistant genes through cross-species transfer to rice disease-resistant breeding and disease control. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention utilizes STOM, a conserved model molecule in pathogens, to identify the pattern recognition receptor in *Nycium benthamianum* that can recognize it. NbSTOMR At the same time, an important co-receptor involved in this recognition process was identified. NbSTOMRh Knocking out this co-receptor decreased its ability to recognize STOM-induced immunity, while overexpression of this gene enhanced its ability to recognize STOM-induced immunity. Meanwhile, NbSTOMRh It also participates in tobacco resistance to Sclerotinia sclerotiorum; knocking out this gene weakens tobacco resistance to Sclerotinia sclerotiorum, while overexpressing it enhances resistance. Furthermore, [the following text is incomplete and requires further context: "will..."] NbSTOMRh After the gene was transferred to rice, it significantly improved the rice's resistance to rice blast and rice false smut.

[0008] In a first aspect, the present invention provides a tobacco anti-sclerotinia protein NbSTOMRh, wherein the protein NbSTOMRh is a protein as described in 1) or 2) or 3) or 4). 1) A protein consisting of the amino acid sequence of SEQ ID NO:2 in the sequence listing; 2) Proteins derived from SEQ ID NO:2 whose amino acid sequence of SEQ ID NO:2 in the sequence listing has been substituted and / or deleted and / or added with one or more amino acid residues, but still retain the function of recognizing pathogen-related molecular patterns or enhancing plant disease resistance; 3) Proteins that have more than 95% homology with the amino acid sequence of 1) or 2) and encode proteins that can recognize pathogen-related molecular patterns or enhance tobacco disease resistance; 4) Proteins derived from the protein shown in SEQ ID NO:2 that retain the ability to recognize pathogen-related molecular patterns or enhance plant disease resistance even after deletion or recombination of the amino acid sequence of 1) or 2).

[0009] Secondly, the present invention also provides a tobacco anti-sclerotinia gene. NbSTOMRh The gene NbSTOMRh It is a nucleic acid molecule that is either 1), 2), or 3) as follows: 1) Its nucleotide sequence is shown in SEQ ID NO.1; 2) The nucleotide sequence encoding the protein as described in the first aspect; 3) Hybridizes under stringent conditions to the nucleotide sequence specified in 1) or 2) and encodes the disease resistance-related protein. NbSTOMRh Nucleic acid molecules.

[0010] Thirdly, the present invention also provides a product containing the ingredients described in the second aspect. NbSTOMRh Biological materials containing nucleic acid molecules, wherein the biological material is any one of the following: 1) Contains the content described in the second aspect NbSTOMRh Nucleic acid expression cassettes; 2) Contains the content described in the second aspect NbSTOMRh Recombinant expression vectors for nucleic acid molecules; 3) An expression carrier containing the expression box described in 1); 4) Recombinant microorganisms containing the recombinant expression vector described in 2); 5) Recombinant microorganisms containing the recombinant expression vector described in 3).

[0011] Fourthly, the present invention also provides a composition for improving tobacco resistance to *Sclerotinia sclerotiorum* or improving rice resistance to rice blast and rice false smut, said composition containing the protein NbSTOMRh described in the first aspect and the gene described in the second aspect. NbSTOMRh Or the biological materials described in the third aspect.

[0012] Fifthly, the present invention provides the protein NbSTOMRh described in the first aspect and the gene described in the second aspect. NbSTOMRh The application of the biomaterials described in the third aspect or the composition described in the fourth aspect, wherein the application is any of the following: 1) Application in the breeding of tobacco resistant to sclerotinia or in the control of sclerotinia disease in tobacco; 2) Application in rice breeding for resistance to rice blast and false smut or in the control of rice blast and false smut.

[0013] Sixthly, the present invention also provides a method for improving tobacco resistance to diseases caused by Sclerotinia sclerotiorum, the method comprising: incorporating the protein NbSTOMRh described in the first aspect and the gene described in the second aspect... NbSTOMRh The steps described in the third aspect, involving the transfer of biological materials into tobacco through gene editing, transgenic technology, or hybridization.

[0014] In some embodiments, the method specifically involves taking the gene described in the second aspect. NbSTOMRh Alternatively, a portion of the sequence may be ligated into a plant expression vector and introduced into tobacco; or the gene containing the gene described in the second aspect may be... NbSTOMRh Tobacco plants transmit resistance genes through sexual hybridization. NbSTOMRh It is then transferred to other tobacco products.

[0015] In a seventh aspect, the present invention also provides a method for improving the resistance of rice to rice blast and rice false smut, the method comprising: incorporating the protein NbSTOMRh described in the first aspect and the gene described in the second aspect... NbSTOMRh The steps described in the third aspect, involving the transfer of biological materials into rice through gene editing or transgenic technology.

[0016] In some embodiments, the method specifically involves taking the gene described in the second aspect. NbSTOMRh Or the biological materials described in the third aspect may be transferred into rice through gene editing or transgenic technology.

[0017] Compared with the prior art, the advantages and beneficial effects of this application are as follows: (1) This invention identified a novel resistance-related gene against Sclerotinia sclerotiorum in tobacco. NbSTOMRh It serves as a pattern recognition receptor for identifying pathogen-associated molecular patterns (STOMs). NbSTOMR The co-receptor induces a plant immune response. Simultaneously, the resistance-related gene described in this invention... NbSTOMRh It can enhance the resistance of tobacco to Sclerotinia sclerotiorum.

[0018] (2) The resistance-related gene described in this invention NbSTOMRh It is a pattern recognition receptor NbSTOMR The co-receptor of STOM, a pathogen-associated molecular pattern, is present in various pathogens, thus possessing broad-spectrum anti-disease potential and promising application prospects.

[0019] (3) Disease resistance-related genes identified in this invention NbSTOMRh It not only enhances resistance to Sclerotinia sclerotiorum in this species, but can also enhance rice's resistance to rice blast and false smut through cross-species transfer. Attached Figure Description

[0020] Figure 1 Disease resistance-related genes NbSTOMRh With pattern recognition receptors NbSTOMR Interacting.

[0021] Figure 2 In order to [the purpose of] Ben's tobacco NbSTOMRh Genes are knocked out.

[0022] Figure 3 To knock out disease resistance-related genes NbSTOMRh Attenuate pathogen-associated molecular pattern STOM-induced cell death and reactive oxygen species on Nicotiana benthamiana.

[0023] Figure 4 To replenish disease-resistant genes NbSTOMRhEnhanced pathogen-associated molecular model STOM-induced cell death and reactive oxygen species on Nicotiana benthamiana.

[0024] Figure 5 To knock out disease resistance-related genes NbSTOMRh It weakens the resistance of Nicotiana benthamiana to Sclerotinia sclerotiorum.

[0025] Figure 6 To overexpress or reintroduce disease resistance-related genes NbSTOMRh Enhance the resistance of Nicotiana benthamiana to Sclerotinia sclerotiorum.

[0026] Figure 7 To obtain transfer NbSTOMRh Genetically modified rice.

[0027] Figure 8 To overexpress in rice NbSTOMRh Gene-enhanced resistance to rice blast.

[0028] [[ID=…]] To overexpress in rice ​ Gene-enhanced resistance to rice false smut. Detailed Implementation

[0029] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention is further described below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0030] Example 1: Co-receptor ​ With pattern recognition receptors ​ Interaction co-receptors ​ Its nucleotide sequence is shown in SEQ ID NO:1 in the sequence listing, and its amino acid sequence is shown in SEQ ID NO:2.

[0031] (1) Construction of prokaryotic expression vector Designing and encoding co-receptors ​Extracellular domain-specific primers were used: forward primer: 5'-ggatccccaggaattcccgggATGCAGACTAGTAGCTCCAATAAGTGT-3' (SEQ ID NO:3), reverse primer: 5'-tcagtcagtcacgatgcggccgcCTAATCTCTTTTAAACTTAGTTCGATGTCTA-3' (SEQ ID NO:4). The extracellular domain product was amplified (94℃ 5min; 94℃ 30s, 58℃ 30s, 72℃ 30s, 34 cycles; 72℃ 5min). After digesting the pGEX4T vector with Sma I and Not I, the amplified product was cloned into the pGEX4T vector using recombinant cloning.

[0032] Similarly, design and encode Benn's smoke pattern recognition receptors. ​ Extracellular domain-specific primers were used: forward primer: 5'-ctgtattttcagggcgaattcATGCAAACCAGAAATGATGTGAAA-3' (SEQ ID NO:5), reverse primer: 5'-caggtcgactctagaggatccTCAAGTATCTTGATTTTTCTTTTTTGG-3' (SEQ ID NO:6), and amplification products were generated. These products were cloned into the undigested pHMTc vector containing EcoRI and BamHI using recombinant cloning.

[0033] The recombinant vector was transformed into Escherichia coli DH5α strain, plated on plates containing ampicillin, and positive clones were screened by colony PCR. Plasmids were extracted from the screened positive clones by shaking to obtain the recombinant vector for prokaryotic expression.

[0034] Results: The prokaryotic expression vector was obtained through colony PCR and sequencing verification.

[0035] (2) Inducing protein expression The recombinant plasmid was introduced into *E. coli* Rosetta expression host and plated on selective medium containing ampicillin and chloramphenicol. Positive clones were screened by colony PCR. Positive strains were picked and inoculated into 4 mL of LB medium containing both antibiotics, incubated overnight at 37°C with shaking, and then transferred to 200 mL of LB liquid medium with the same antibiotic conditions. The culture was continued until the OD600 reached 0.6–0.8. Subsequently, 200 μL of 0.1 M IPTG was added, and the culture was induced overnight at 28°C with shaking.

[0036] (3) Purification of recombinant proteins The induced bacterial cells were collected in 50 mL centrifuge tubes and centrifuged at 8000 rpm for 10 min at 4 °C. The cells were resuspended in 10–20 mL of lysis buffer (NaH₂PO₄ 50 mM, NaCl 300 mM, Imidazole 10 mM, pH 8.0) and 20 μL of lysozyme was added. The mixture was incubated on ice for 30 min. The cells were then sonicated (1 s / 3 s intervals, 30 min), followed by centrifugation at 10000 rpm for 70 min at 4 °C. The supernatant was collected. A 400 μL nickel column was washed twice with 10 mL of lysis buffer, and the supernatant was added. The column was incubated on a shaker at 4 °C for 2 h. After incubation, the supernatant was removed by centrifugation at 3210 rpm for 5 min. The sample was then washed three times sequentially with 20 mM elution buffer (NaH₂PO₄ 50 mM, NaCl 300 mM, Imidazole 20 mM, pH 8.0), followed by three washes with 50 mM elution buffer (Imidazole 50 mM), and finally eluted 10 times consecutively with 250 μL elution buffer (Imidazole 250 mM). 10 μL of the eluent was added to 6× SDS loading buffer, and the mixture was boiled in a water bath for 10 min before SDS-PAGE analysis. The purification was assessed using Coomassie Brilliant Blue R250 staining. The purified product was then transferred to imidazole-free Tris buffer via ultrafiltration (Millipore Amicon Ultra-15c) for subsequent experiments.

[0037] Result: Purified GST-labeled GST was successfully obtained. ​ Extracellular domain and MBP-labeled NbSTOMR extracellular domain.

[0038] (4) Pull-Down Detection Interaction Equal amount of GST marked ​ Extracellular domain and MBP markers ​The extracellular domains were incubated in a buffer containing Glutathione Sepharose 4B (GS4B, Cytiva) resin (50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 10 mM MgCl2, and 1% NP-40). After incubation at 4°C for 1 hour, the cells were washed 5 times with elution buffer (50 mM Tris-HCl (pH 8.0), 400 mM NaCl, 50 mM reduced glutathione, 1 mM EDTA, and 1 mM DTT), with GST used as a negative control. The loaded and eluted proteins were treated with 6× SDS loading buffer and boiled for 10 min before SDS-PAGE and Western blot analysis. GST and MBP antibodies were used for detection. ​ and ​ Expression of the extracellular domain.

[0039] result: ​ extracellular domain and ​ Extracellular domains interact, while GST itself cannot interact with extracellular domains. ​ Extracellular domain interactions (see) ​ ).

[0040] Example 2: Knockout in Benedictine Smoke ​ Genes do not affect its growth phenotype (1) Constructing a knockout vector Design Targets ​Gene targets: t1: 5'-ATTTACGAGCATTGGCACGT TGG-3' (SEQ ID NO:7); t2: 5'-AAGTTGACGAGTGGTGCAGG AGG-3' (SEQ ID NO:8); t3: 5'-AAAGGAGGACTGATCACTAA AGG-3' (SEQ ID NO:9); then the three fragments were fused together via fragment synthesis to serve as a template for subsequent amplification. Amplification primers were designed: forward primer: 5'-cagtGGTCTCatgcaatttacgagcattggcacgtgttttcagag-3' (SEQ ID NO:10), reverse primer: 5'-cagtGGTCTCaaaacttagtgatcagtcctccttttgcaccag-3' (SEQ ID NO:11). The fused fragment was amplified and then constructed into a BsaI / Eco31I-digested K5-KRSN vector. The recombinant vector was transformed into E. coli, and positive clones were screened by colony PCR. Finally, the knockout vector was obtained by extracting the plasmid through shaking.

[0041] (2) Plasmid was transformed into Agrobacterium GV3101 Add 1 µL of plasmid to 50 µL of GV3101 Agrobacterium competent cells and transform them using liquid nitrogen. Add the successfully transformed GV3101 cells to 1 mL of LB liquid medium and incubate at 30°C and 180 rpm for 30 min on a shaker. Then, inoculate 50 µL of the activated Agrobacterium culture onto LB solid medium and incubate in the dark at 30°C for 48 h.

[0042] (3) Agrobacterium-mediated stable transformation of Nicotiana benthamiana Sterile tobacco leaves were cut into small pieces with a scalpel and inoculated onto pre-culture medium. An Agrobacterium resuspension with an OD600 of 0.2 was prepared. Tobacco leaves pre-cultured for 2-3 days were inoculated into this suspension for 10-15 minutes. The inoculated tobacco leaves were then inoculated onto filter paper, dried, and then transferred to a co-culture medium for dark incubation for 48-72 hours. After 2 days of incubation, the leaves were transferred to induction medium to induce callus growth, which took approximately 10 days. Callus tissue meeting the criteria was selected and inoculated onto the corresponding resistance selection medium for 15-30 days. Vigorous positive callus tissue from the second screening was inoculated onto differentiation medium, 4-5 calluses per dish, and cultured at 23°C under 16h / 8h light / dark conditions for 15-30 days. If seedlings formed during differentiation, they were inoculated onto a seedling growth medium and grown for 7-10 days. Tobacco genomic DNA was extracted using the CTAB method and detected by PCR.

[0043] Result: A frameshift mutation caused by the insertion of one base or the deletion of four bases was successfully obtained. ​ (See ​ (See Figure A in the middle), and the growth phenotypes of the two mutants did not change significantly (see Figure A in the middle). ​ (Figure B in the middle)

[0044] Example 3: Knocking out the disease resistance-related gene NbSTOMR weakens the pathogen-associated molecular pattern STOM-induced cell death and reactive oxygen species in Nicotiana benthamiana. (1) Agrobacterium-mediated transient transformation of Nicotiana benthamiana Plant expression vectors containing the pathogen-associated molecular pattern STOM and the control GFP were transformed into Agrobacterium competent cells GV3101 via liquid nitrogen method. Positive clones were screened by colony PCR, and then the positive clones were added to liquid medium containing 4 ml LB and cultured at 30°C and 180 rpm for 36 days. After washing the bacteria and adjusting the OD value to 0.5, the clones were injected from the underside of leaves of Nicotiana benthamiana using a 1 ml needleless syringe.

[0045] (2) Observation of cell death and reactive oxygen species phenotype Cell death phenotypes can be observed and statistically analyzed visually 3 days after Agrobacterium injection. Reactive oxygen species (ROS) phenotypes require DAB staining observation: Injected leaves are placed in DAB staining solution (1 mg / ml, pH=3.8) and treated at room temperature in the dark for 8 hours. The staining solution is then removed, and anhydrous ethanol is added for destaining. Once all the green color has been removed from the leaves, they are removed and photographed.

[0046] Results: Pathogen-associated molecular pattern of action (STOM) was observed in... ​ In the knockout strain, induced cell death and reactive oxygen species were significantly reduced compared to non-knockout Nicotiana benthamiana (WT) (see [link to relevant documentation]). ​ This indicates that NbSTOMRh is involved in recognizing pathogen-associated molecular patterns and eliciting plant immune responses.

[0047] Example 4: Replenishment ​ Enhanced pathogen-associated molecular modeling of STOM-induced cell death and reactive oxygen species in *Nyctalus benthamiana* (1) Construction of plant expression vectors Design coding ​Gene-specific primers were used: forward primer: 5'-gaacgatagggtacccccgggATGAATTGTTTCAAAATATCAGTGTTAATT-3' (SEQ ID NO: 12), reverse primer: 5'-gtaaggcctactagtggatccAAGATGGACCGACTCAACTCTTG-3' (SEQ ID NO: 13). The gene was amplified (94℃ 5min; 94℃ 30s, 58℃ 30s, 72℃ 30s, 34 cycles; 72℃ 5min). The pBINHA vector was digested with Sma I and BamHI, and the amplified product was cloned into the pBINHA vector using recombinant cloning. The recombinant vector was transformed into *Escherichia coli* DH5α strain, plated on agar plates containing kanamycin, and positive clones were screened by colony PCR. Plasmids were extracted from the screened positive clones by shaking to obtain the plant expression recombinant vector.

[0048] (2) Agrobacterium-mediated transient transformation of Nicotiana benthamiana Will contain ​ Plant expression vectors containing loss-of-function mutants were transformed into Agrobacterium competent cells GV3101 via liquid nitrogen method. Positive clones were screened by colony PCR, and then added to liquid medium containing 4 ml LB. After incubation at 30°C and 180 rpm for 36 hours, the cells were washed and the OD value was adjusted to 0.5. The culture was then injected into the abaxial surface of Nicotiana benthamiana leaves using a 1 ml needleless syringe. Twelve hours later, plant expression vectors containing STOM were injected.

[0049] (3) Observation of cell death and reactive oxygen species phenotype Cell death phenotypes can be observed and statistically analyzed visually 3 days after Agrobacterium injection. Reactive oxygen species (ROS) phenotypes require DAB staining observation: Injected leaves are placed in DAB staining solution (1 mg / ml, pH=3.8) and treated at room temperature in the dark for 8 hours. The staining solution is then removed, and anhydrous ethanol is added for destaining. Once all the green color has been removed from the leaves, they are removed and photographed.

[0050] Result: In ​ The knockout body expresses ​ Or loss-of-function mutants, found to express ​ It can restore pathogen-associated molecular pattern-induced cell death and reactive oxygen species (see ​ ), further indicating ​ It participates in identifying pathogen-associated molecular patterns and stimulating plant immune responses.

[0051] Example 5: Knockout of disease resistance-related genes ​ Weakening the resistance of Nicotiana benthamiana to Sclerotinia sclerotiorum. Inoculate with Sclerotium sclerotiorum separately ​ Knockout cells and WT cells were cultured in a moist environment, and the area of ​​the lesions was counted.

[0052] Results: Sclerotinia sclerotiorum in ​ The lesion area of ​​the knockout body was significantly larger than that of the WT, indicating that ​ Positive regulation of resistance to Sclerotinia sclerotiorum.

[0053] Example 6: Complementation / Overexpression of Disease Resistance-Related Genes ​ Enhance the resistance of Nicotiana benthamiana to Sclerotinia sclerotiorum. exist ​ Transient expression in knockout bodies and WT Benedictine smoke ​ as well as ​ The function loss mutant was then inoculated with Sclerotinia sclerotiorum and cultured in a moist environment, and the lesion area was counted.

[0054] Result: In ​ Knockout and overexpression in WT Benedictine fumes ​ The lesion area was significantly smaller than WT, indicating that ​ Positive regulation of resistance to Sclerotinia sclerotiorum (see...) ​ ).

[0055] Example 7 ​ Transferred to rice (1) Construction of plant expression vectors Specific primers encoding the NbSTOMRh gene were designed: forward primer: 5'-tacttctgttgcaacggtaccATGAATTGTTTCAAAATATCAGTGTTAATT-3' (SEQ ID NO: 14), reverse primer: 5'-cacgatacaggatccactagtAAGATGGACCGACTCAACTCTTG-3' (SEQ ID NO: 15). The gene was amplified (94℃ 5min; 94℃ 30s, 58℃ 30s, 72℃ 30s, 34 cycles; 72℃ 5min). The pBWA vector was digested with Kpn I and Spe I, and the amplified product was cloned into the pBWA vector using recombinant cloning. The recombinant vector was transformed into *Escherichia coli* DH5α strain, plated on agar plates containing kanamycin, and positive clones were screened by colony PCR. Plasmids were extracted from the screened positive clones by shaking to obtain the plant expression recombinant vector.

[0056] (2) Rice conversion Will contain ​1 µL of the plant expression vector plasmid was added to 50 µL of EHA105 Agrobacterium competent cells. After thorough mixing, the mixture was transferred to an electroporation cuvette. After electroporation, 1 mL of LB liquid medium was added, and the mixture was thoroughly mixed. The mixture was then transferred to a 1.5 mL centrifuge tube and incubated at 30 °C and 180 rpm for 30 min. 50 µL of the activated Agrobacterium culture was then inoculated onto LB solid medium and incubated in the dark at 30 °C for 48 h. Positive clones were verified by colony PCR and used for further processing.

[0057] Select rice grains without mold spots and with normal sprouts. Disinfect with 75% alcohol for 1 min, rinse with sterile water for 1 min each time; disinfect with 15% sodium hypochlorite for 20 min, rinse with sterile water 3 times for 1 min each time; inoculate the disinfected rice grains into induction medium and culture at 26℃ under light for 20 days.

[0058] Pick Agrobacterium and place it in the infection solution to prepare an Agrobacterium resuspension with OD600 = 0.2. Pick the callus in an Erlenmeyer flask, add the Agrobacterium resuspension, infect for 10-15 min, discard the bacterial solution, and inoculate the callus into a co-culture medium. Co-culture at 20℃ for 48-72 h.

[0059] Inoculate the callus into the selection medium and incubate in the dark at 26°C for 20-30 days; inoculate the positive callus into the secondary selection medium, and be sure to select single-clonal callus during the callus selection process, and incubate in the dark at 26°C for 7-10 days. Positive callus was inoculated onto differentiation medium and cultured at 25-27℃ under light for 15-20 days. After the shoots differentiated into 2-5cm long, they were inoculated onto rooting medium and cultured at 30℃ under light for 7-10 days. Rice genomic DNA was extracted using the CTAB method and detected by PCR. Result: Transfer successfully completed. ​ Rice (see) ​ ).

[0060] Example 8 Overexpression in rice ​ Enhance rice's resistance to rice blast After 7 days of growth of *Magnapordica oryzae* on sporulation medium, the cells were irradiated with ultraviolet light for 3 days, washed with sterile water, and the concentration was adjusted to 5 × 10⁻⁶. 4 Spores / mL, inoculated into rice seedlings about 14 days old, and the disease severity was assessed after 5-7 days of moist culture following inoculation.

[0061] Result: Overexpression ​ The disease severity of rice seedlings was significantly lower than that of the ZH11 variety that had not been genetically modified (see...). ​ This indicates that rice has been transferred into ​ Afterwards, it significantly enhanced the rice's resistance to rice blast.

[0062] Example 9 Overexpression in rice ​Enhancing the resistance of rice to rice false smut After growing Aspergillus oryzae on PSA medium for 7 days, it was cultured in liquid for 5-7 days, then crushed using a crusher to form a mixture of spores and mycelia, and the concentration was adjusted to 1×10⁻⁶. 6 Spores / mL were inoculated onto the rice panicle using a syringe with a needle, and the severity of disease was assessed approximately 30 days later.

[0063] Result: Overexpression ​ The incidence of rice panicle blast disease was significantly lower in the ZH11 variety that had not been genetically modified (see...). ​ This indicates that rice has been transferred into ​ Afterwards, it significantly enhanced the resistance of rice to rice false smut.

[0064] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A tobacco anti-sclerotinia protein NbSTOMRh, characterized in that, The amino acid sequence of the protein NbSTOMRh is shown in SEQ ID NO:2 in the sequence listing.

2. A tobacco anti-Sclerotinia sclerotiorum gene NbSTOMRh Its characteristics are, The gene NbSTOMRh It is a nucleic acid molecule that is either 1) or 2) below: 1) Its nucleotide sequence is shown in SEQ ID NO:1; 2) The nucleotide sequence encoding the protein as described in claim 1.

3. A biomaterial containing the nucleic acid molecule of claim 2, characterized in that, The biomaterial is any of the following: S1) An expression cassette containing the nucleic acid molecule described in claim 2; S2) A recombinant expression vector containing the nucleic acid molecule described in claim 2; S3) Recombinant microorganisms containing the recombinant expression vector described in S2).

4. A composition for improving tobacco resistance to Sclerotinia sclerotiorum or improving rice resistance to rice blast and rice false smut, characterized in that, The composition contains the protein NbSTOMRh as described in claim 1 and the gene as described in claim 2. NbSTOMRh Or the biomaterial described in claim 3.

5. The gene according to claim 2 NbSTOMRh The application of the biomaterial of claim 3 or the composition of claim 4, characterized in that, The application is any of the following: 1) Application in the breeding of tobacco resistant to sclerotinia or in the control of sclerotinia disease in tobacco; 2) Application in rice breeding for resistance to rice blast and false smut or in the control of rice blast and false smut.

6. A method for improving tobacco resistance to diseases caused by Sclerotinia sclerotiorum, characterized in that, The method includes applying the gene according to claim 2. NbSTOMRh The steps involved in introducing genetically modified organisms (GMOs) or hybridization into tobacco.

7. The method according to claim 6, characterized in that, The method specifically involves using the gene described in claim 2. NbSTOMRh Linked to a plant expression vector and introduced into tobacco; or containing the gene as described in claim 2. NbSTOMRh Tobacco plants transmit resistance genes through sexual hybridization. NbSTOMRh It is then transferred to other tobacco products.

8. A method for improving the resistance of rice to rice blast and rice false smut, characterized in that, The method includes applying the gene according to claim 2. NbSTOMRh The steps involved in transferring genetically modified organisms (GMOs) into rice.

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