Application of OsGG1 gene in constructing disease-resistant rice

Abstract

This invention discloses the application of the OsGG1 gene in constructing disease-resistant rice. This invention identifies a novel OsGG1 gene that enhances broad-spectrum disease resistance in rice, and overexpression of OsGG1 can improve rice's disease resistance. The method of this invention can be extended to other methods involving promoter mutations of the OsGG1 gene to increase OsGG1 gene expression and thus enhance broad-spectrum disease resistance in rice. Furthermore, based on the experimental results of this study, haplotypes with high OsGG1 gene expression can be screened in rice genetic resource banks or in nature to identify new disease-resistant rice varieties. This invention is of great significance in the face of severe crop diseases in my country and worldwide, and provides a powerful reference for breeding highly disease-resistant rice varieties.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and specifically to a method for obtaining rice with resistance to rice blast and bacterial blight through molecular breeding. Background Technology

[0002] There are over 300 infectious diseases that harm rice, some distributed worldwide and others confined to specific regions. In my country, 29 rice diseases occur annually. Among them, rice blast, which has a wide spread and poses a significant threat; sheath blight, which has a high incidence rate and a major impact on production; and bacterial leaf blight, which causes regional epidemics and disasters, are collectively known as the three major rice diseases in my country. In addition, there are rice false smut, rice smut, leaf tip blight, and viral diseases that have been intermittently prevalent for the past 40 years, which have gradually spread in recent years. The occurrence and prevalence of these diseases are major limiting factors affecting high and stable rice yields.

[0003] Rice blast is a disease of rice caused by the pathogen Magnaphalthe oryzae. It can occur throughout the entire rice growth cycle, damaging seedlings, leaves, panicles, and nodes, and is respectively called seedling blast, leaf blast, panicle blast, and node blast. Rice blast is distributed throughout rice-growing regions worldwide and is a major disease in rice production, with the most severe outbreaks in Asian and African rice-growing areas. In China, it is generally more severe in mountainous areas than in plains, and more severe in japonica and glutinous rice than indica rice. Except in the South China rice-growing region where early rice is more severely affected than late rice, late rice is more severely affected than early rice in other rice-growing areas. In epidemic years, yields are generally reduced by 10-20%, and in severe cases by 40-50%, with some fields even experiencing total crop failure. Control methods for rice blast mainly include selecting resistant varieties, cultivating high-quality seedlings, implementing effective fertilizer and water management techniques, strengthening field management, implementing control measures, and using chemical pesticides. After the outbreak of rice blast, it is essential to thoroughly understand the symptoms, patterns, and causes of rice blast, and, in conjunction with planting conditions, adopt scientific and feasible rice blast control techniques, establish sound prevention concepts, reduce the likelihood of rice blast occurrence, and avoid losses to rice yield and quality.

[0004] Rice bacterial blight is one of the most important bacterial diseases of rice worldwide, caused by a variant of Xanthomonas oryza pv. oryzae. While not as variable as rice blast, bacterial blight pathogens still exhibit differentiation in pathogenicity. IRRI (1993) identified nine physiological races in the Philippines, and seven races were identified in my country in the 1980s. In my country, the distribution of dominant bacterial blight races varies depending on the ecological region, and their pathogenicity decreases from south to north. Similar to rice blast, resistance to bacterial blight in rice varieties is highly specialized and largely controlled by major genes, making resistance relatively easy to utilize. However, unlike rice blast, resistance to bacterial blight in rice varieties shows significant differences in growth stage. Resistance to bacterial blight can be categorized into seedling stage resistance, mature plant stage resistance, and resistance throughout the entire growth cycle. Because the bacterial blight pathogen itself exhibits relatively little variation, the resistance of resistant varieties is also relatively stable. However, cases have been found abroad, such as in the Philippines, where resistance has been lost due to the emergence of new pathogenic races. Domestically, there are also instances where the resistance of resistant varieties weakens after long-term planting. How to effectively utilize bacterial blight resistance genes has become a crucial issue in the breeding and application of disease-resistant rice varieties.

[0005] Molecular breeding applies molecular biology techniques to breeding at the molecular level. Genetic modification breeding (transgenic breeding) is a common molecular breeding technique that applies genetic engineering to breeding work. Using biological techniques, traits can be altered without changing the crop's genes, or superior varieties can be selected simply through molecular markers. Molecular breeding technology can achieve direct gene selection and effective aggregation, significantly improving breeding efficiency, shortening breeding time, and achieving "precision breeding." On January 24, 2022, the Ministry of Agriculture and Rural Affairs formulated and published the "Guidelines for Safety Evaluation of Gene-Edited Plants for Agricultural Use (Trial)," mainly targeting gene-edited plants without the introduction of exogenous genes, requiring safety evaluation based on potential risks. Further standardizing the safety evaluation management of gene-edited plants in agriculture is a milestone for the research and development and industrial promotion of bio-breeding technology in my country.

[0006] In recent years, how to combine disease resistance with high-yield and high-quality traits to cultivate new high-yielding, high-quality, and multi-resistant varieties has become a pressing issue for rice breeders, pathologists, and molecular biologists. The rapid development of modern cytogenetics and cytogenetic manipulation techniques, as well as molecular genetics and molecular biotechnology, has facilitated the continuous expansion and in-depth development of rice disease resistance breeding research. This has driven the rapid expansion of rice disease resistance breeding technology from conventional breeding to cell engineering breeding and genetic engineering breeding. The organic combination and scientific application of these three levels of breeding technology will undoubtedly propel rice disease resistance breeding into a new era worldwide and in my country. Summary of the Invention

[0007] This technology newly discovered a rice disease resistance gene, OsGG1, and increased its expression in plants through transgenic technology. This increased the plant's resistance to the fungal pathogen *Oryza sativa* and the bacterial pathogen *Bacillus oryzae*. When rice was infected with *Oryza sativa*, the OsGG1-overexpressing lines accumulated higher levels of phytoserotonin (SA) compared to the wild-type *Nipponbare*. SA is a plant hormone associated with disease resistance. Furthermore, SA pathway-dependent disease resistance genes were also expressed at higher levels in the OsGG1-overexpressing lines. This indicates that OsGG1 enhances broad-spectrum disease resistance in rice by regulating the SA signaling pathway.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides an application of the OsGG1 gene in constructing disease-resistant rice, wherein the amino acid sequence encoded by the OsGG1 gene is shown in SEQ ID NO:3.

[0010] In one embodiment of the present invention, the rice is Nipponbare rice (Oryza sativa L. japonica, cv Nipponbare).

[0011] In one embodiment of the present invention, the nucleotide sequence of the OsGG1 gene is as shown in SEQ ID NO: 1 (including introns) or SEQ ID NO: 2 (excluding introns).

[0012] Furthermore, the application is as follows: an overexpression recombinant plasmid is constructed using the OsGG1 gene, and the overexpression recombinant plasmid is transformed into rice using Agrobacterium-mediated transformation to obtain disease-resistant rice.

[0013] In one embodiment of the present invention, the vector for the recombinant plasmid is pTF101. Further, the promoter used for the OsGG1 gene on the overexpression recombinant plasmid is the CaMV35S promoter or the promoter shown in SEQ ID NO:4 (the promoter of the genome itself).

[0014] Specifically, this invention provides methods for constructing overexpression recombinant plasmids with two different promoters.

[0015] In one embodiment of the present invention, the overexpression recombinant plasmid is constructed using the following method:

[0016] (1) Using the genomic DNA of Nipponbare rice (Oryza sativa L. japonica, cv Nipponbare) as a template, PCR amplification was performed using primer pairs pTF101-proOsGG1-OsGG1-GFP-F and pTF101-proOsGG1-OsGG1-GFP-R to obtain the OsGG1 gene fragment carrying the promoter;

[0017] pTF101-proOsGG1-OsGG1-GFP-F:

[0018] 5'-actgaatcaaaggccatgGGAATTATTCTGAATGATAT-3'

[0019] pTF101-proOsGG1-OsGG1-GFP-R:

[0020] 5'-cgactctagaggatcccccTTTATCAGTTGCCAGAACAC-3'

[0021] (2) The vector pTF101 was double-digested with restriction endonucleases SacI and SmaI to obtain a linearized vector (without CaMV35S promoter).

[0022] (3) The OsGG1 gene fragment carrying the promoter described in step (1) is seamlessly ligated with the linearized vector described in step (2) to obtain the overexpression recombinant plasmid.

[0023] In one embodiment of the present invention, the overexpression recombinant plasmid is constructed using the following method:

[0024] 1) Using total cDNA from Nipponbare rice (Oryza sativa L. japonica, cv Nipponbare) as a template, PCR amplification was performed using primer pairs pTF101-pro35S-OsGG1-GFP-F and pTF101-pro35S-OsGG1-GFP-R to obtain the OsGG1 gene fragment.

[0025] pTF101-pro35S-OsGG1-GFP-F:

[0026] 5'-tagatcagcccacgagctATGGCTCTCCTCGACCCAG-3'

[0027] pTF101-pro35S-OsGG1-GFP-R:

[0028] 5'-cgactctagaggatcccccTTTATCAGTTGCCAGAACAC-3'

[0029] 2) The vector pTF101 was double-digested with restriction endonucleases NcoI and SmaI to obtain a linearized vector (with the CaMV35S promoter).

[0030] 3) Seamlessly ligate the OsGG1 gene fragment described in step 1) with the linearized vector described in step 2) to obtain the overexpression recombinant plasmid.

[0031] In the embodiments of the present invention, seamless cloning is achieved using the homologous recombination kit ClonExpress MultiS OneStep Cloning Kit (Vazyme). TM (catalog number: C113-01) Completed.

[0032] This invention identifies a novel gene, OsGG1, that enhances disease resistance in rice. Overexpression of OsGG1 improves the broad-spectrum disease resistance of rice. The genomic sequence of the key gene OsGG1 is shown in SEQ ID NO: 1, the coding sequence is shown in SEQ ID NO: 2, and the promoter sequence of OsGG1 is shown in SEQ ID NO: 3.

[0033] SEQ ID NO:1

[0034]

[0035] SEQ ID NO:2

[0036]

[0037] SEQ ID NO:3

[0038] MASSTQGQVITCKAAVAWEANRPMTIEDVQVAPPQAGEVRVKILFTALCHTDHYTWSGKDPEGLFPCILGHEAAGIVESVGEGVTEVQPGDHVIPCYQAECRECKFCKSGKTNLCGKVRAATGVGVMMNDRKSRFSINGKPIYHFMGTSTFSQYTVVHDVVSVAKINPQAPLDKVCLLGCGVSTGLGAVWNTAKVEAGSIVAIFGLGTVGLAVAEGAKSAGASRIIGIDIDSKKFDVAKNFGVTEFVNPKDHDKPIQQVIVDLTDGGVDYSFECIGNVSVMRSALECCHKGWGTSVIVGVAASGQEISTRPFQLVTGRVWKGTAFGGFKSRSQVPWLVEKYLNKEIKVDEYVTHSMNLTDINKAFDLLHEGGCLRCVLATDK.

[0039] SEQ ID NO:4

[0040]

[0041] Furthermore, the application is as follows: an overexpression recombinant plasmid is constructed using the OsGG1 gene, and the overexpression recombinant plasmid is transformed into rice using Agrobacterium-mediated transformation to obtain disease-resistant rice.

[0042] Furthermore, the Agrobacterium-mediated transformation method is as follows:

[0043] S1: The overexpression recombinant plasmid was transformed into Agrobacterium tumefaciens strain EHA105 by electroporation to obtain transgenic Agrobacterium;

[0044] S2: Using seeds of Nipponbare rice (Oryza sativa L. japonica, cv Nipponbare) as explants to induce callus tissue, the transgenic Agrobacterium described in step S1 was used as the infecting bacterium to transfer the target gene expression cassette into the rice genome using the Agrobacterium infection transformation method. The resulting infected rice callus tissue was cultured in a selection medium containing 50 mg / mL hygromycin for 40 days, and the selection medium was changed to fresh every 20 days to obtain resistant rice callus tissue.

[0045] S3: The resistant rice callus obtained in step S2 is transferred to a differentiation medium and cultured for 40 days, with the medium being replaced with fresh differentiation medium every 20 days; the resulting differentiated rice seedlings are transferred to a rooting medium and cultured until roots emerge, and then identified by PCR to obtain the disease-resistant rice.

[0046] Transgenic identification of the rice transgenic plants described in this invention:

[0047] (1) Transgenic rice plants were identified by immunoblotting. This confirmed whether an extra copy of the target gene was integrated into the rice genome and expressed.

[0048] (2) Transgenic lines with high expression of OsGG1 were identified by quantitative PCR.

[0049] Disease resistance identification of the transgenic rice plants described in this invention:

[0050] (1) Transgenic rice T0 generation self-pollination yields T1 generation, T1 generation self-pollination yields T2 generation;

[0051] (2) Inoculate with rice blast fungus GUY11 and 2539 to observe the disease resistance between transgenic rice lines and transgenic recipient control (Nipponbare).

[0052] (3) Inoculate with bacterial blight pathogen and observe the disease resistance between transgenic rice lines and transgenic recipient control (Nipponbare).

[0053] This invention verifies that OsGG1 overexpression lines exhibit strong resistance to rice blast and bacterial blight. Following reinfection with the pathogen, the transgenic lines accumulate more salicylic acid, which plays an important positive role in biotic stress defense. The expression of salicylic acid-dependent disease resistance genes in the transgenic lines is also upregulated after rice infection.

[0054] Compared with existing technologies, the beneficial effects of this invention are as follows: a novel gene, OsGG1, has been identified to enhance broad-spectrum disease resistance in rice. Overexpression of OsGG1 can improve rice's disease resistance. The method of this invention can be extended to other methods involving promoter mutations of the OsGG1 gene to increase its expression and thus enhance broad-spectrum disease resistance in rice. Furthermore, based on the experimental results of this study, haplotypes with low OsGG1 gene expression can be screened in rice genetic resource banks or in nature to identify new disease-resistant rice varieties. This invention is of great significance in the face of severe crop diseases in my country and worldwide, and provides a powerful reference for breeding highly disease-resistant rice varieties. Attached Figure Description

[0055] Figure 1 Identification of OsGG1 overexpression lines.

[0056] A. Gene vector used for transgenic creation of OsGG1 overexpressing plants. B. OsGG1 expression level in the obtained OsGG1 transgenic lines detected by qRT-PCR. C. OsGG1-GFP fusion protein in the obtained OsGG1 transgenic lines detected by Western blotting. Nipponbare is an OsGG1 transgenic rice variety. OsACTIN (LOC_Os03g50885) was detected as an internal reference gene and a control gene in qRT-PCR and Western blotting, respectively. The left side of the figure shows transgenic material with the OsGG1 promoter driving the OsGG1 gene sequence fused with the GFP sequence, and the right side shows transgenic material with the CaMV35S promoter driving the OsGG1 coding sequence fused with the GFP sequence. Lines OE-1 and OE-4 were selected for further experimental analysis.

[0057] Figure 2 OsGG1 overexpression enhances resistance to rice blast (Magnaporthe oryzae).

[0058] Overexpression of OsGG1 enhanced rice blast resistance. Rice lines were infected with the virulent isolate GUY11 and the non-virulent isolate 2539, respectively. Fifteen two-week-old rice plants were treated with 1 ml of a solution containing 2 × 10⁻⁶ OsGG1. 5Inoculate rice blast fungus with a spray solution containing one conidia per milliliter of bacterial suspension. One week later, photograph the leaf phenotype. Scale bar: 5 cm. B. Quantify the severity of the disease on rice lines in A. Number and diameter of fungal patches on each leaf. Data were analyzed using three biological replicates. * indicates a significant difference compared to the wild type (Nipponbare) (P, 0.05, t-test).

[0059] Figure 3 Overexpression of OsGG1 enhanced resistance to bacterial blight in rice.

[0060] Overexpression of Nipponbare and OsGG1 strains revealed bacterial blight phenotypes on plant leaves. Phenotypic development was observed 14 days after inoculation with bacterial blight. Scale bar: 2 cm.

[0061] Figure 4 OsGG1 regulates the level of nitric oxide (NO) in plants.

[0062] A. NO levels were reduced in the OsGG1 overexpression material. NO in plant roots was stained using the fluorescent nitric oxide dye DAF-FM DA. cPTIO was used as a nitric oxide scavenger. Scale bar: 10 μm. B. Quantification of root fluorescence intensity in material A. Data were quantified using ImageJ software. Three biological replicates were used. * indicates a significant difference compared to the wild type (Nipponbare) (P, 0.05, t-test).

[0063] Figure 5 OsGG1 positively regulates salicylic acid (SA) pathway-dependent disease resistance genes in rice.

[0064] Expression of the AB SA pathway resistance genes was induced in OsGG1 overexpression materials. The transcriptional abundance of SA pathway-dependent resistance genes PR1a A and PR1b B after GUY11 inoculation was detected by qRT-qPCR. c represents the concentration of SA in rice lines after GUY11 inoculation. Data were analyzed using three biological replicates. * indicates a significant difference compared to the wild type (Nipponbare) (P, 0.05, t-test). Detailed Implementation

[0065] Example 1: Constructing pTF101-pro35S-OsGG1-GFP and pTF101-proOsGG1-OsGG1-GFP vectors.

[0066] 1. Primer design

[0067] According to the Rice Genome Annotation Project ( http: / / rice.uga.edu / The OsGG1 (LOC_Os02g57040) gene sequence and coding sequence (CDS) were obtained from the database, and 2000bp upstream of the OsGG1 start codon was used as the OsGG1 gene promoter. PCR amplification primers were designed, which contained 15-20bp homologous sequences of the linearized vector backbone for homologous recombination assembly of the vector. The OsGG1 gene fragment (including introns) was amplified using rice (Oryza sativa L. japonica, cv Nipponbare) genomic DNA as a template, using pTF101-proOsGG1-OsGG1-GFP-F and pTF101-proOsGG1-OsGG1-GFP-R primers. The OsGG1 coding sequence was amplified using total cDNA from rice (Oryza sativa L. japonica, cv Nipponbare) as a template, using pTF101-pro35S-OsGG1-GFP-F and pTF101-pro35S-OsGG1-GFP-R primers. The primer sequences are as follows:

[0068] pTF101-proOsGG1-OsGG1-GFP-F:

[0069] 5'-actgaatcaaaggccatgGGAATTATTCTGAATGATAT-3'.

[0070] pTF101-proOsGG1-OsGG1-GFP-R:

[0071] 5'-cgactctagaggatcccccTTTATCAGTTGCCAGAACAC-3'.

[0072] pTF101-pro35S-OsGG1-GFP-F:

[0073] 5'-tagatcagcccacgagctATGGCTCTCCTCGACCCAG-3'.

[0074] pTF101-pro35S-OsGG1-GFP-R:

[0075] 5'-cgactctagaggatcccccTTTATCAGTTGCCAGAACAC-3'.

[0076] 2. Enzyme digestion vector pTF101

[0077] According to the product instructions, use restrictive internal NcoI (Thermo Scientific) TM,catalog number:FD0573) / SmaI(Thermo Scientific TM (catalog number: FD0663) The enzyme digestion vector pTF101 was used to construct the pTF101-proCaMV35S-OsGG1-GFP vector.

[0078] Using the restriction endonuclease SacI (Thermo Scientific) TM The pTF101 vector (catalog number: FD0573) was digested with SmaI to remove the pro35S sequence, which was used to construct the pTF101-proOsGG1-OsGG1-GFP vector. The digestion reaction was incubated at 37°C for 30 minutes.

[0079] 3. Constructing a carrier

[0080] According to the product instructions, use the homologous recombination kit ClonExpress MultiS One Step Cloning Kit (Vazyme) TM (catalog number: C113-01), the gene fragment obtained in the first step of PCR and the vector obtained in the second step were ligated at 45℃ for 15 minutes. The ligation product was then transformed into E. coli competent DH5α cells (100 μL, Weidi Biotechnology). TM The culture was placed on ice for 30 minutes, then incubated in a 42°C water bath for 45 seconds. 500 μL of LB liquid medium was added, and the culture was incubated at 37°C for 200 rpm for 1 hour. 100 μL of the bacterial culture was then evenly spread onto LB solid medium containing 50 mg / L kanamycin and incubated in the dark at 37°C for 12 hours. After single colonies grew, 2-3 single colonies were picked, activated, and sequenced using the vector sequencing primer M13R. The sequence was verified to be correct, yielding the pTF101-pro35S-OsGG1-GFP and pTF101-proOsGG1-OsGG1-GFP vectors. Figure 1 A).

[0081] Example 2: Rice Conversion

[0082] 1. Agrobacterium-mediated transformation

[0083] The constructed pTF101-pro35S-OsGG1-GFP and pTF101-proOsGG1-OsGG1-GFP vector plasmids were transformed into Agrobacterium strain EHA105, respectively. 100 μL of Agrobacterium strain EHA105 competent cells (Vitamin B1) were then introduced into the cells. TMAdd 500 ng of plasmid to the medium, place on ice for 5 minutes, then perform liquid nitrogen shock for 10 seconds. After condensation, place in a 37°C water bath for 5 minutes, then place on ice for 2 minutes. Add 0.5 mL of antibiotic-free YEP liquid medium, and incubate at 28°C with shaking at 250 rpm for 3 hours. Centrifuge at 3500 rpm for 3 minutes, and spread the bacterial pellet evenly on YEP solid medium containing 30 mg / L kanamycin and 20 mg / L rifampin. Incubate at 28°C for 2 days to obtain Agrobacterium carrying the pTF101-pro35S-OsGG1-GFP vector and Agrobacterium carrying the pTF101-proOsGG1-OsGG1-GFP vector.

[0084] 2. Inducing callus formation

[0085] Disinfection: Select mature and plump Nipponbare rice seeds (Oryza sativa L. japonica, cv Nipponbare), remove the husk; disinfect with 75% alcohol for 1 minute; rinse 3 times with sterile distilled water; soak in 0.15% mercuric chloride for 15-18 minutes, shaking every 5 minutes; rinse 3 times with sterile distilled water. Inoculate the disinfected seeds into the induction medium and culture at 32℃ under light for 5-10 days until callus tissue is induced.

[0086] 3. Infection and co-culture

[0087] Before infection, Agrobacterium was activated by streak plating. The activated Agrobacterium was then cultured in suspension medium with shaking until the bacterial culture reached OD. 600 =0.8~1.0 (28℃, 200rpm for 3~3.5 hours). Then adjust the bacterial concentration to OD600 = 0.1~0.2 with suspension medium. Immerse the callus tissue in Agrobacterium suspension for 5 minutes. Discard the bacterial suspension and blot dry the surface of the callus tissue with sterile filter paper. Transfer the callus tissue to co-culture medium and incubate in the dark at 25℃ for 3~4 days.

[0088] 4. Clean bacteria

[0089] After co-culturing, soak the callus tissue in sterile distilled water containing 500 mg / L ticarcillin in a sterile bottle for 30 minutes. Rinse the callus repeatedly with sterile distilled water 3-5 times. Use sterile filter paper to absorb as much moisture as possible from the callus surface and place it in a laminar flow hood to air dry.

[0090] 5. Screening and Cultivation

[0091] After cleaning, the callus was transferred to selection medium and cultured at 32°C under light for 2 weeks. During this period, the selection medium could be changed according to the infection status.

[0092] 6. Differentiation culture

[0093] Fourteen days after screening, the surviving callus was transferred to differentiation medium and cultured at 28°C (photoperiod of 16 hours light / 8 hours dark) until rice seedlings differentiated.

[0094] 7. Rooting Culture

[0095] When the rice seedlings grow to 3-4 cm in height on the differentiation medium, they are transferred to the rooting medium for further culture until roots are induced and complete rice plants are formed (T0 generation).

[0096] Solution formulation:

[0097] 1. N6max mother liquor: 28.3g KNO3, 4.0g KH2PO4, 4.63g (NH4)2SO4, 1.85g MgSO4·7H2O,

[0098] 1.66g CaCl2·2H2O; dissolve each piece individually, then add distilled water to bring the volume to 1L.

[0099] 2. N6min stock solution: 0.44g MnSO4·4H2O, 0.15g ZnSO4·7H2O, 0.16g H3BO3, 0.08g KI; dissolve each ingredient individually, then add distilled water to bring the volume to 1L.

[0100] 3. Fe2+-EDTA stock solution: Dissolve 3.73g Na2EDTA·2H2O in hot water at 70℃, then add 2.78g FeSO4·7H2O to dissolve; bring the volume to 1L, chelate at 70℃ for 2 hours; store at 4℃ protected from light.

[0101] 4. Vitamin stock solution: 0.1g nicotinic acid, 0.1g VB6, 0.1g VB1, 0.2g glycine, 10g inositol;

[0102] Bring the volume to 1L and store at 4℃.

[0103] Culture medium formulation:

[0104] 1. Induction medium (pH=5.8): 100mL N6max stock solution (10x), 10mL N6min stock solution (100x),

[0105] Fe 2+ 10 mL of EDTA stock solution (100x), 10 mL of Vitamin stock solution (100x), 2.5 mL of 1 mg / mL 2,4-D stock solution, 0.06% proline, 0.08% CH, 3% sucrose, and 0.3% Phytagel; with water as the solvent, bring the volume to 1 L and autoclave.

[0106] 2. Suspension culture medium (pH=5.2): 100 mL N6max stock solution (10x), 10 mL N6min stock solution (100x), Fe 2+ - 10 mL of EDTA stock solution (100x), 10 mL of Vitamin stock solution (100x), 2.5 mL of 1 mg / mL 2,4-D stock solution, 0.06% proline, 0.08% CH, and 2% sucrose; the solvent is water, and the volume is adjusted to 1 L. The solution is then autoclaved. When using, add 5 mL of 50% glucose and 250 μL of 0.2 M AS stock solution.

[0107] 3. Co-culture medium (pH=5.6): 50 mL N6max stock solution (10x), 5 mL N6min stock solution (100x), Fe 2+ - 5 mL of EDTA stock solution (100x), 5 mL of Vitamin stock solution (100x), 0.625 mL of 1 mg / mL 2,4-D stock solution, 0.015% proline, 0.02% CH, 0.75% sucrose, and 0.2% agar powder; the solvent is water, and the volume is adjusted to 1 L. The mixture is then autoclaved. Before use, 5 mL of 50% glucose and 250 μL of 0.2 M AS stock solution are added.

[0108] 4. Screening medium (pH=6.0): 100 mL N6max stock solution (10x), 10 mL N6min stock solution (100x), Fe 2+ 10 mL of EDTA stock solution (100x), 10 mL of Vitamin stock solution (100x), 0.625 mL of 1 mg / mL 2,4-D stock solution, 0.015% proline, 0.02% CH, 0.75% sucrose, and 0.2% agar powder; the solvent is water, and the volume is adjusted to 1 L. The mixture is then autoclaved. 50 mg / mL ticarcillin is added before use.

[0109] 5. Differentiation medium (pH=5.8): 100 mL N6max stock solution (10x), 10 mL N6min stock solution (100x), Fe 2+ - 10 mL of EDTA stock solution (100x), 10 mL of Vitamin stock solution (100x), 2.0 mL of 1 mg / mL KT stock solution, 0.2 mL of 1 mg / mL NAA stock solution, 0.06% proline, 0.08% CH, 3% sorbitol, 3% sucrose, 0.3% Phytagel; solvent is water, bring to a final volume of 1 L, and autoclave.

[0110] 6. Rooting medium (pH=5.8): 50 mL N6max stock solution (10x), 5 mL N6min stock solution (100x), Fe 2+- 5 mL of EDTA stock solution (100x), 5 mL of Vitamin stock solution (100x), 2% sucrose, 0.3% Phytagel; solvent is water, bring to a final volume of 1 L, and autoclave.

[0111] 7. LB liquid medium (pH=7.0): 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride; solvent is water, bring to a final volume of 1 L, and autoclave.

[0112] 8. LB solid medium: Add 12 g / L agar powder to LB liquid medium, use water as solvent, and autoclave.

[0113] 9. YEP liquid culture medium (pH=7.0): 5 g / L yeast extract, 10 g / L tryptone, 5 g / L sodium chloride; solvent is water, bring to a final volume of 1 L, and autoclave.

[0114] 10. YEP solid medium: Add 12 g / L agar powder to YEP liquid medium, use water as solvent, and autoclave.

[0115] Example 3: Quantitative analysis and protein identification of transgenic lines

[0116] 1. Quantitative PCR identification of transgenic lines.

[0117] Take 20-50 mg of leaves from OsGG1 transgenic lines (OE-1 to 6; where OE-1 to 3 are pTF101-proOsGG1-OsGG1-GFP transgenic lines; OE-4 to 6 are pTF101-pro35S-OsGG1-GFP transgenic lines) and apply RNA-easy Isolation Reagent (Vazyme). TM The kit (catalog number: R701-01) extracts total RNA from samples.

[0118] Take 1 μL of the extracted total RNA as a template and use the RT reagent Kit with gDNA Eraser (PrimeScript) TMThe kit (catalog number: RR047A) was used for reverse transcription to synthesize cDNA. Step 1: gDNA removal reaction: Take a 1.5 mL RNase-free centrifuge tube and add 2 μL of 5×gDNA Eraser Buffer, 1 μL of gDNA Eraser, 2 μg of RNA, and RNase-free ddH2O to a total volume of 10 μL on ice. Vortex to mix, centrifuge for 20 seconds, and incubate at 42°C for 2 minutes. Step 2: Reverse transcription reaction: On ice, add 1 μL of PrimeScript RTEnzyme Mix I, 1 μL of RT Primer Mix, 24 μL of 5×PrimeScript Buffer, and 4 μL of RNase-free dH2O to the reaction mixture from Step 1. Incubate the mixture at 37°C for 15 minutes, then incubate at 85°C for 5 seconds to obtain the sample cDNA.

[0119] Take 0.5 μL of the above cDNA as a template and use TB Green qPCR Master Mix (Takara) TM (catalog number: 639676) qRT-PCR was performed on a LightCycler 480 (Roche Diagnostics) amplification condition: preheating (94℃ for 5 min), amplification (94℃ for 10 s, 58℃ for 10 s, 72℃ for 10 s, 40 cycles). OsACTIN was used as an internal control, and the formula for calculating the relative expression level was 2. -△△Ct All experiments were performed in three biological replicates and two technical replicates. OsGG1 overexpression lines all showed good overexpression efficacy. Figure 1 B). The lines with the highest OsGG1 expression levels in the two overexpression materials (OE-1 and OE-4) were selected for further experiments.

[0120] OsGSNOR1-qRT-F: 5'-CAGTTGGACTTGCAGTTGCTG-3'

[0121] OsGSNOR1-qRT-R:5'-TGTAGTCCACAACCACCGTCT-3'

[0122] OsACTIN-qRT-F:5'-CATTGCTGACAGGATGAGCAAG-3'

[0123] OsACTIN-qRT-R:5'-TGGCAATCCACATCTGCTGGAA-3'

[0124] 2. Immunoblotting detection of transgenic strains

[0125] Total protein was extracted from transgenic lines OE-1–6. 20–50 mg of plant leaf tissue was ground into powder in liquid nitrogen and dissolved in a total protein extract containing 2% (w / v) SDS, 60 mM Tris-HCl, pH 8.5, 2.5% (v / v) glycerol, 0.13 mM EDTA, 1 mM benzyl sulfonyl fluoride (PMSF), and a mixture of protease inhibitors (Sigma). The supernatant was collected once or twice to remove tissue residue.

[0126] Protein samples (30-50 mg) were loaded onto SDS-PAGE gels for protein electrophoresis. Subsequently, the samples were transferred using an eBlot analyzer. TM L1 (catalog number: L00686C) transfers protein samples from SDS-PAGE gels to polyvinylidene fluoride (PVDF, Millipore). TM (catalog number: ISEQ00010) membrane. β-actin antibody (1:5000, ABclonal) was used. TM (catalog number: AC006) and GFP antibody (1:2000, Themo Fisher) TM PVDF membranes were incubated with FDbio-Femto ECL (catalog number: 33-2600) and then incubated with FDbio-Femto ECL (Femto Biotechnology). TM Colorimetric analysis was performed using a kit (catalog number: FD8030). The ChemDoc XRS+ system (Bio-Rad) was used. TM (catalog number: 1708265) Immunoblotting results showed that the exogenous fusion protein could be detected in all OsGG1 overexpression lines. Figure 1 C).

[0127] Example 4: Pathogenicity analysis of rice blast fungus

[0128] 1. Methods for preserving strains of rice blast fungus

[0129] This experiment used pathogenic isolate GUY11 and non-pathogenic isolate 2539 of rice blast fungus. A modified dry filter paper method was adopted: sterilized filter paper was laid on CM medium plates. When rice blast fungus colonies covered the entire filter paper, the filter paper containing rice blast fungus was peeled off and placed in a desiccator for about two weeks. Then, the completely dried filter paper was placed in cryovials, sealed with sealing film, numbered, and stored in cryovial boxes at -20℃ for long-term storage.

[0130] The rice blast fungus strain was inoculated onto CM solid medium and cultured at 25°C for 16 hours in light and 8 hours in darkness. When the strain nearly covered the entire medium, it was sealed and stored in a 4°C refrigerator. When needed for future use, a portion of the strain was inoculated onto fresh CM solid medium using an inoculation loop and cultured.

[0131] 2. Culture medium formulation

[0132] CM medium: 10 g / L D-Glucose, 2 g / L Peptone, 1 g / L Casamino acids, 1 g / L Yeastextract, 6 g / L NaNO3, 1.52 g / L KH2PO4, 0.52 g / L KCl, 0.52 g / L MgSO4·7H2O, 0.1% (v / v) trace elements α ,0.1%(v / v)Vitamin solution β Adjust the pH to 6.5 with NaOH. If preparing solid culture medium, add 15g of agar powder per liter of medium and autoclave at 121℃ for 15 minutes.

[0133] α 1000×Trace elements (100mL): 5g Na4EDTA, 2.2g ZnSO4·7H2O, 1.1g H3BO3, 0.5g MnCl2·4H2O, 0.5g FeSO4·7H2O, 0.17g CoCl2·6H2O, 0.16g CuSO4·5H2O, 0.15gNaMoO4·5H2O.

[0134] β 1000×Vitamin solution (100ml): 0.01g of each of the following substances: Vitamin H (Biotin), Vitamin B6 (Pyridoxin), Vitamin B1 (Thiamine), Vitamin B2 (Riboflavin), p-aminobenzoic acid (PABA), and nicotinic acid.

[0135] 3. Pathogenicity analysis of rice blast fungus

[0136] Rice blast fungus GUY11 and 2539 were cultured on CM plates. After approximately 12 days of spreading, the spores were washed with about 4 mL of water and scraped off using a triangular stick sterilized with an alcohol lamp. The spores were then transferred to 10 mL centrifuge tubes and filtered through three layers of sterile lens paper to obtain a clear liquid. The solution was centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected. The spores were counted using a hemocytometer to bring the spore suspension to a concentration of 2 × 10⁻⁶.5 Species / ml. Prepare a 0.4% gelatin solution and mix it with the spore suspension at a 1:1 ratio until the spore suspension concentration is 1×10⁻⁶. 5 per ml.

[0137] Two milliliters of spore solution were evenly sprayed onto 14-day-old rice seedlings (wild-type Nipponbare control and OsGG1 overexpression lines OE-1 and OE-4; OE-1 was a pTF101-proOsGG1-OsGG1-GFP transgenic line, and OE-4 was a pTF101-pro35S-OsGG1-GFP transgenic line) using a small spray bottle. An equal volume of spore solution was used per seedling, with three replicates per treatment. The control group was sprayed with an equal volume of 0.2% gelatin solution, with three replicates per treatment. After spraying, the seedlings were covered with a cola bottle to prevent cross-contamination.

[0138] The inoculated seedlings were placed in a 22℃ incubator in the dark and kept moist for 48 hours. Then, the light was turned on, and the seedlings were cultured under 26℃ light conditions. After 5-7 days, the disease development was observed and photographed, and the size and number of plaques were counted using Adobe Photoshop to identify (…). Figure 2 B). The experiment was repeated 2-3 times. OsGG1 overexpression lines showed significant resistance to rice blast compared to the wild type. Figure 2 )

[0139] Example 5: Pathogenicity analysis of bacterial blight pathogen

[0140] 1. Culture of *Bacterium tumefaciens* and *Bacterial leaf streak* of rice

[0141] Take out the bacterial strain stored at -70℃, use a sterile pipette tip to draw the bacterial solution onto the potato slant culture medium on a clean bench, and spread it evenly with a sterile inoculation loop.

[0142] After culturing in a 28°C incubator for 3 days, the bacteria will turn bright yellow. They can then be stored at 4°C for one month. When using, transfer them to a new culture medium according to the method described in step 1.

[0143] After being transferred again, the rooting tubes were placed in a 28°C incubator for 2 days. The bacteria grew to a bright yellow color and could be used to preserve the strain or for inoculation experiments.

[0144] 2. Preservation of strains of *Bacterium tumefaciens* and *Bacterial leaf streak*, the pathogens causing bacterial blight of rice.

[0145] If the bacterial cells cultured in step 3 above need to be preserved for a long time, add 10-30 ml of sterile 30% glycerol to the rooting tube, mix it with the bacterial cells, and then dispense it into sterile 0.5 ml centrifuge tubes and store it at -70℃ for a long time.

[0146] 3. Inoculation and investigation methods for rice bacterial blight pathogen

[0147] After the bacterial cells were cultured at 28°C for 2 days and turned bright yellow, they were diluted with PBS solution, and the concentration was diluted to approximately 9 x 10⁻⁶ using a turbidimetric method. 9 bacteria / ml

[0148] Inoculation is performed using the leaf-cutting method during the rice booting stage (or seedling stage). When inoculating, dip scissors in the bacterial solution and cut off approximately 2 cm of the tip of the flag leaf (for seedling inoculation, select fully extended leaves). Each plant should be inoculated with at least 5 flag leaves.

[0149] Phenotypic observation was conducted 14 to 21 days after vaccination. Figure 3 At least 5 sword leaves were investigated on each individual plant. OsGG1 overexpression lines showed significant resistance to bacterial blight compared to wild-type lines. Figure 3 ).

[0150] 4. PBS solution formulation

[0151] Add 8 g / L NaCl, 0.2 g / L KCl, 3.628 g / L Na₂HPO₄, and 0.24 g / L KH₂PO₄ to sterile deionized water to a final volume of 1 L. Adjust the pH to 7.4 with NaOH or HCl.

[0152] Example 6: Nitric oxide (NO) fluorescent staining.

[0153] 1. DAF-FM DA (Themo Fisher) TM (catalog number: D23842) staining.

[0154] Wild-type Nipponbare control and OsGG1 overexpression lines OE-1 and OE-4 (OE-1 is a pTF101-proOsGG1-OsGG1-GFP transgenic line, and OE-4 is a pTF101-pro35S-OsGG1-GFP transgenic line) were germinated and cultured in rice nutrient solution for 7 days.

[0155] DAF-FM DA staining solution was prepared using fresh rice nutrient solution, with a final concentration of 5 μM.

[0156] The roots were incubated in the prepared staining solution for 30 minutes at 28°C. Then, the roots were washed with fresh nutrient solution to remove excess probe. The rice was then cultured in fresh nutrient solution for 15-30 minutes to allow complete deacetylation of the diacetate within the root cells.

[0157] 2. cPTIO processing.

[0158] Using 0.5mM cPTIO (Sigma) TMThe plants were cultured in nutrient solution (catalog number: C221-10MG) for 30 minutes, and then the roots were stained with DAF-FM DA according to the above experimental conditions.

[0159] 3. Use a two-photon confocal microscope to observe the fluorescence of plant roots.

[0160] The maximum fluorescence excitation and emission values ​​were 495 nm and 515 nm, respectively. Root samples from different strains were photographed under the same excitation intensity and Gain value. Figure 4 A).

[0161] 4. The fluorescence intensity in the fluorescence images was quantified using ImageJ software. The OsGG1 overexpression line had a lower nitric oxide content in vivo compared to the wild type. Figure 4 B).

[0162] Example 7: Detection of disease resistance genes via the salicylic acid pathway

[0163] 1. Detection of disease resistance genes via salicylic acid (SA) pathway: Wild-type Nipponbare control and OsGG1 overexpression lines OE-1 and OE-4 (OE-1 is a pTF101-proOsGG1-OsGG1-GFP transgenic line, and OE-4 is a pTF101-pro35S-OsGG1-GFP transgenic line) were inoculated with rice blast fungus GUY11 according to the above-mentioned method for analyzing the pathogenicity of rice blast.

[0164] 2. Collect 20-50 mg of leaf tissue at 0, 24, 48, and 72 hours after inoculation. Extract total RNA and synthesize cDNA using the method described above.

[0165] 3. qRT-PCR was used to detect the expression levels of salicylic acid pathway-dependent resistance genes PR1a and PR1b in different rice lines. OsACTIN was used as an internal reference gene to calculate relative expression levels. OsGG1 overexpressing lines showed higher transcriptional responses to salicylic acid (SA) pathway resistance genes PR1a and PR1b compared to the wild type after inoculation with rice blast. Figure 5 A and 5B).

[0166] 4. Quantitative primers:

[0167] OsPR1a-qRT-F: 5'-ACGTACGTATGCTGGTGAGA-3'

[0168] OsPR1a-qRT-R: 5'-AGACCATGCATGTAACCACGA-3'

[0169] OsPR1b-qRT-F: 5'-ACTCCCCTCCCAAGCTCAAA-3'

[0170] OsPR1b-qRT-R: 5'-TCTCTGGCTGGCGTAGTTCT-3'

[0171] Example 8: Detection of Salicylic Acid Content

[0172] SA was extracted from 100 mg of plant leaf tissue at 0 and 48 hours after inoculation of wild-type Nipponbare control and OsGG1 overexpression lines OE-1 and OE-4 (OE-1 is a pTF101-proOsGG1-OsGG1-GFP transgenic line, and OE-4 is a pTF101-pro35S-OsGG1-GFP transgenic line) inoculated with GUY11 strain as described above.

[0173] The rice leaf tissue was thoroughly ground in liquid nitrogen. 1 ml of 90% ethanol was added, and the resulting solution was vortexed for 30 seconds.

[0174] Centrifuge the suspension at 15,000 rpm for 5 minutes. Collect the supernatant. Extract the precipitate with 100% methanol. Then combine the precipitate with the supernatant. Repeat the extraction process once.

[0175] The combined supernatant fractions were dried under high-speed vacuum. The residue was resuspended in 1 ml of 5% trichloroacetic acid.

[0176] Add 1 ml (ethyl acetate:cyclopentane:isopropanol = 50:50:1) and vortex for about 1 minute to perform organic extraction of free SA. Transfer the upper phase (about 1 ml) to a new 2 ml centrifuge tube. Repeat the extraction step and combine the two organic phases (about 2 ml).

[0177] The combined solutions were dried under high-speed vacuum and resuspended in 0.5 mL of 0.01 M H₂SO₄. The suspension was centrifuged at 15,000 rpm for 2 minutes and filtered using a 0.25 μM filter. The samples were diluted 10–100 times with deionized water.

[0178] Salicylic acid (SA) content was detected by HPLC. OsGG1 overexpression lines induced higher SA content after rice blast infection compared to wild-type lines. Figure 5 C).

Claims

1. A kind OsGG1 The application of genes in constructing disease-resistant rice is characterized by: The application is through overexpression OsGG1 Gene-constructed disease-resistant rice, the OsGG1 The amino acid sequence encoded by the gene is shown in SEQ ID NO: 3, and the disease-resistant rice is rice resistant to rice blast or... anti- Rice affected by bacterial leaf blight.

2. The application as described in claim 1, characterized in that: The rice mentioned is Nipponbare rice (Japanese rice). Oryza sativa L. japonica , cv Nipponbare).

3. The application as described in claim 1, characterized in that: The OsGG1 The nucleotide sequence of the gene is shown in SEQ ID NO:1 or SEQ ID NO:

2.

4. The application as described in claim 1, characterized in that... The application is as follows: [The application is described in the original text] OsGG1 Gene overexpression recombinant plasmids were constructed, and the overexpression recombinant plasmids were transformed into rice using Agrobacterium-mediated transformation to obtain disease-resistant rice.

5. The application as described in claim 4, characterized in that... The application is as follows: the vector for the recombinant plasmid is pTF101.

6. The application as described in claim 4, characterized in that... The application is as follows: on the overexpression recombinant plasmid. OsGG1 The promoter used in the gene is CaMV35S A promoter or a promoter as shown in SEQ ID NO:

4.

7. The application as described in claim 4, characterized in that... The application is as follows: the Agrobacterium-mediated transformation method uses Agrobacterium (… Agrobacterium tumefaciens ) strain EHA105.

8. The application as described in claim 4, characterized in that... The application is as follows: the overexpression recombinant plasmid is constructed using the following method: (1) Using the genomic DNA of Nipponbare rice ( Oryza sativa L. japonica , cv Nipponbare) as a template, primer pairs pTF101-proOsGG1-OsGG1-GFP-F and pTF101-proOsGG1-OsGG1-GFP-R were used for PCR amplification to obtain the OsGG1 gene fragment carrying the promoter; pTF101-proOsGG1-OsGG1-GFP-F ： 5'-actgaatcaaaggccatgGGAATTATTCTGAATGATAT-3' pTF101-proOsGG1-OsGG1-GFP-R ： 5'-cgactctagaggatcccccTTTATCAGTTGCCAGAACAC-3' (2) The vector pTF101 was double-digested with restriction endonucleases SacI and SmaI to obtain a linearized vector; (3) The promoter-carrying device described in step (1) OsGG1 The gene fragment is seamlessly ligated with the linearized vector described in step (2) to obtain the overexpression recombinant plasmid.

9. The application as described in claim 4, characterized in that: The overexpression recombinant plasmid was constructed using the following method: 1) Using Nipponbare rice ( Oryza sativa L. japonica Using total cDNA from cv Nipponbare as a template, primer pairs were used... pTF101-pro35S-OsGG1-GFP -F and pTF101-pro35S-OsGG1-GFP -R is used for PCR amplification to obtain OsGG1 Gene fragments; pTF101-pro35S-OsGG1-GFP -F： 5'-tagatcagcccacgagctATGGCTCTCCTCGACCCAG-3' pTF101-pro35S-OsGG1-GFP -R： 5'-cgactctagaggatcccccTTTATCAGTTGCCAGAACAC-3' 2) The vector pTF101 was double-digested with restriction endonucleases NcoI and SmaI to obtain a linearized vector; 3) The steps described in step 1) OsGG1 The gene fragment is seamlessly ligated with the linearized vector described in step 2) to obtain the overexpression recombinant plasmid.

10. The application as described in claim 4, characterized in that... The Agrobacterium-mediated transformation method is as follows: S1: Transfer the over-expression recombinant plasmid into Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) strain EHA105 by electroporation to obtain transgenic Agrobacterium tumefaciens; S2: Japanese dry rice ( Oryza sativa L. japonica Seeds of cv Nipponbare were used as explants to induce callus tissue. The transgenic Agrobacterium described in step S1 was used as the infecting bacterium to transfer the target gene expression frame into the rice genome. The resulting infected rice callus tissue was cultured in a selection medium containing 50 mg / mL hygromycin for 40 days, and the medium was changed to fresh medium every 20 days to obtain resistant rice callus tissue. S3: The resistant rice callus obtained in step S2 is transferred to a differentiation medium and cultured for 40 days, with the medium being replaced with fresh differentiation medium every 20 days; the resulting differentiated rice seedlings are transferred to a rooting medium and cultured until roots emerge, and then identified by PCR to obtain the disease-resistant rice.

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