Genes that enhance rice resistance to rice blast and their applications

Abstract

This invention belongs to the field of crop genetics and breeding, specifically relating to the rice blast resistance gene OsK8 (Os04g0658700 gene) and its uses. This invention discloses the rice blast resistance gene OsK8, the nucleotide sequence of which is shown in SEQ ID NO: 1. This invention also discloses the application of the aforementioned rice blast resistance gene OsK8: knocking out the OsK8 gene in rice effectively enhances the resistance of rice plants to rice blast fungus; while overexpression of the Os04g0658700 gene in rice reduces the resistance of rice to rice blast fungus.

Description

Technical Field

[0001] This invention belongs to the field of crop genetics and breeding, specifically relating to a gene OsK8 that resists rice blast and its uses. Background Technology

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops, with more than half of the global population relying on it as a staple food. However, rice is often attacked by various pathogens during the production process, which seriously affects its high and stable yield as well as the quality of the rice, endangering global and national food security.

[0003] Rice blast is a devastating fungal disease caused by Magnaphalthe oryzae, often referred to as "rice cancer," and is a major threat to global food security. 【1】 .

[0004] Currently, the main methods for controlling rice blast include breeding disease-resistant varieties, chemical control, and strengthening field management. While the widespread use of pesticides has mitigated the occurrence and severity of rice diseases to some extent, it has also seriously impacted human health and ecological safety. Chemical agents pollute the environment upon which humans depend; large quantities of pesticides flow into water bodies, settle and accumulate in the soil, and are transferred to humans through bioaccumulation in the food chain, causing a series of health hazards. Practice has proven that breeding and promoting disease-resistant varieties is one of the safest and most effective methods for controlling rice blast.

[0005] The key to breeding disease-resistant rice varieties lies in utilizing their inherent disease-resistance genes. Traditional breeding often employs NB-LRR type disease-resistance genes, which provide strong resistance, but generally only target specific pathogen races, resulting in a narrow resistance spectrum and easy loss of resistance due to pathogen mutations. Protein kinases play an indispensable role in various plant physiological processes, including responding to various biotic and abiotic stresses. Protein kinases influence the biochemical activity and function of proteins by regulating the phosphorylation of their substrate proteins. As important genes regulating plant resistance, protein kinases have enormous potential applications in disease-resistant breeding.

[0006] The sequence of the Os04g0658700 gene is available in Gramene (https: / / www.gramene.org / ), but its function in disease resistance is unknown. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a rice blast resistance gene OsK8 and its uses. The present invention improves rice resistance to rice blast through single gene editing.

[0008] To address the aforementioned problems, this invention provides a rice blast resistance gene, OsK8 (Os04g0658700 gene), the nucleotide sequence of which is shown in SEQ ID NO: 1. Its encoded amino acid sequence is shown in SEQ ID NO: 2.

[0009] This invention provides a method for applying the aforementioned rice blast resistance gene OsK8: Knocking out the OsK8 (Os04g0658700) gene in rice using CRISPR-Cas9 gene editing technology can effectively enhance the resistance of rice plants to rice blast fungus. Specifically, this involves causing a frameshift mutation in the OsK8 gene of the rice cultivar Nipponbare to obtain gene-knockout rice plants, thereby increasing the rice's resistance to rice blast.

[0010] An improvement to the use of the rice blast resistance gene OsK8 of the present invention: sgRNA sequence of CRISPR / Cas9 editing target was designed in the rice blast resistance gene OsK8 (Os04g0658700 gene): 5'-TCCACCAGAAGAATCAGCTC-3', primers were artificially synthesized based on the sgRNA sequence and constructed into the CRISPR / Cas9 vector.

[0011] As a further improvement to the use of the rice blast resistance gene OsK8 of the present invention: the synthesized primers are:

[0012] Upstream 5'-TCCACCAGAAGAATCAGCTCgttttagagctagaaat-3'

[0013] Downstream 5'-GAGCTGATTCTTCTGGTGGACggcagccaagccagca-3'.

[0014] As a further improvement to the use of the rice blast resistance gene OsK8 of the present invention:

[0015] Compared with the wild-type rice variety Nipponbare, knocking out the rice blast resistance gene OsK8 can effectively improve the plant's resistance to the rice blast pathogen.

[0016] As a further improvement to the use of the rice blast resistance gene OsK8 of the present invention: the two mutant plants obtained by knocking out the rice blast resistance gene OsK8 are Osk8-1 plants and Osk8-2 plants.

[0017] In this invention, the following are also disclosed: overexpression of the gene Os04g0658700 in rice reduces the resistance of rice to rice blast fungus; the susceptibility of Os04g0658700 overexpressing plants Ubi::OsK8-1 and Ubi::OsK8-2 is higher than that of the control wild-type rice variety Nipponbare.

[0018] This invention discovers that the gene OsK8, encoding an intracellular receptor-like kinase, negatively regulates rice resistance to rice blast. Knocking out the OsK8 gene in rice using CRISPR-Cas9 gene editing technology significantly improved the rice's resistance to rice blast fungus. Furthermore, this gene mutation had no significant impact on rice growth, development, or yield. Conversely, overexpression of OsK8 led to increased susceptibility to rice blast. Therefore, this gene has important application value in rice disease resistance breeding.

[0019] In summary, this invention discloses for the first time that knocking out the OsK8 susceptibility gene can enhance the rice's resistance to rice blast. The purpose of knocking out the OsK8 gene is to significantly enhance resistance to rice blast without significantly affecting rice growth and development. This invention has direct application value in the rapid genetic improvement of rice varieties with good quality but poor resistance. Attached Figure Description

[0020] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0021] Figure 1 Sequence analysis of mutation sites in rice OsK8 gene mutants. Nipponbare (NIP): wild-type control variety; osk8-1 and osk8-2: two different insertion mutants of the Os04g0658700 gene.

[0022] Figure 2 Comparison of growth status of wild-type and mutant strains in a greenhouse over 6 weeks:

[0023] Among them, NIP is a wild-type plant, and osk8-1 and osk8-2 are Os04g0658700 insertion mutant plants; scale bar 10cm.

[0024] The growing conditions are 24℃, light duration: 16h, darkness duration: 8h, and growth time: 6 weeks.

[0025] Figure 3 A comparison of the growth and heading characteristics of the wild type and the mutant under field conditions:

[0026] Among them, NIP is a wild-type plant, and osk8-1 and osk8-2 are Os04g0658700 insertion mutant plants; scale bar 10cm.

[0027] The growing conditions are two months of field growth until the heading stage.

[0028] Figure 4 Seed morphology of wild-type rice varieties NIP, osk8-1, and osk8-2 mutants; scale bar 1 cm.

[0029] Figure 5 The leaf disease status of wild-type rice varieties NIP, osk8-1, and osk8-2 mutant plants after infection with the rice blast fungus Guy11; scale bar 1cm.

[0030] Figure 6 To determine the relative growth of the pathogen on rice leaves after wild-type rice varieties NIP, osk8-1, and osk8-2 mutant plants were infected with the pathogen Guy11, quantitative PCR was used to detect the relative growth of the blast fungus 28S rDNA relative to the rice OsActin internal reference gene and the fold increase was calculated to reflect the relative growth of the blast fungus.

[0031] Figure 7 To identify rice plants overexpressing the Os04g0658700 gene by PCR.

[0032] Figure 8 The leaf disease incidence of two rice lines (Ubi::OsK8-1 and -2) after infection with Guy11, a fungus that causes rice blast, is shown in the wild-type rice variety NIP and OsK8 overexpression. Scale bar: 1 cm.

[0033] Figure 9 To determine the relative growth of rice blast fungus on rice leaves after two strains of wild-type rice variety NIP and overexpressing OsK8 were infected with Guy11, quantitative PCR was used to detect the relative growth of rice blast fungus MoPot2 relative to the rice OsActin internal reference gene and calculate the fold to reflect the relative growth of rice blast fungus.

[0034] The above wild-type and mutant plants were observed five days after inoculation with the rice blast fungus Guy11; Guy11 spore concentration: 1.5 × 10⁻⁶. 5 CFU / mL; the inoculation experiment was repeated 3 times, and the results were reproducible. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0036] Example 1: Construction of osk8 (Os04g0658700) mutant plants using CRISPR-Cas9 technology

[0037] Based on the OsK8 gene sequence (SEQ ID NO: 1), a CRISPR-Cas9 knockout targeting sgRNA sequence was designed:

[0038] 5'-TCCACCAGAAGAATCAGCTC-3', synthesize the corresponding primers based on this sequence.

[0039] 5'-TCCACCAGAAGAATCAGCTCGTTTTAGAGCTAGAAAT-3' and

[0040] 5'-GAGCTGATTCTTCTGGTGGACGGCAGCCAAGCCAGCA-3'.

[0041] Using the "Kinmen cloning" method, a knockout vector for the OsK8 gene was constructed via the Bsa1 restriction site, which can be followed according to the literature. 【2】 Perform the operation.

[0042] The constructed vector was transferred into Agrobacterium strain EHA105, which was then used to infect tissue culture callus of Nipponbare rice (O. sativassp. japonica cv Nipponbare, NIP). Transgenic callus was selected by screening for the hygromycin resistance gene carried by the vector. Subsequent tissue culture differentiation and regeneration steps were then performed to obtain transgenic rice plants osk8 (Os04g0658700) knockout mutant plants.

[0043] Example 2: Identification of Os04g0658700 knockout mutant plants

[0044] DNA Extraction: Approximately 0.02 g of leaves from wild-type rice variety Nipponbare and its mutant were ground with liquid nitrogen and then added to 600 μL of SDS extraction buffer (0.1 mol / L Trizma base, 0.05 mol / L EDTA-Na2, 0.5 mol / L NaCl, 15 g / L SDS). The mixture was incubated at 65°C for 1 hour, shaking 2-3 times during this period. 200 μL of 5M potassium acetate was added, and the mixture was shaken well. The mixture was then incubated at -20°C for 20 minutes. 500 μL of chloroform was added, and the mixture was mixed thoroughly. After standing for 5 minutes, the mixture was centrifuged at 10000 rpm for 7 minutes at 4°C. 500 μL of the supernatant was transferred to a new 1.5 mL centrifuge tube, and 500 μL of pre-chilled isopropanol was added. The tube was placed at -20°C for 30 minutes, then centrifuged at 12000 rpm for 3 minutes at 4°C. The supernatant was discarded, and 500 μL of... Centrifuge at 12000 rpm for 1 min with 75% ethanol, discard the supernatant, add 500 μL of anhydrous ethanol, centrifuge at 12000 rpm for 1 min, discard the supernatant, invert and air dry at room temperature, add 100 μL of ddH2O and mix well, store at -20℃ for later use.

[0045] PCR amplification: Synthesized primers were used for PCR amplification of the OsK8 (Os04g065870) gene: OsK8-F: 5'-GACAGCAACAATGGCGAACC-3', OsK8-R: 5'-CAGGTCCAAAGCCTCCTCTC-3'. DNA from Nipponbare rice NIP and mutant plants was used as templates for amplification using 2×taq PCR reagent (Kangwei Century, product number CW2849M).

[0046] The PCR amplification system consisted of 1 μL template DNA, 5 μL 2×taq Plus MasterMix (Dye), 0.4 μL each of F primer and R primer, and ddH2O to a final volume of 10 μL.

[0047] The PCR amplification program was as follows: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 sec, 58℃ annealing for 30 sec, 72℃ extension for 30 sec, 35 cycles; 72℃ final extension for 2 min.

[0048] Sequencing analysis of the PCR products successfully identified two mutant plants of the Os04g0658700 gene: osk8-1 (with a T base inserted into the Os04g0658700 gene) and osk8-2 (with an A base inserted into the Os04g0658700 gene). Both mutations resulted in a frameshift mutation in the Os04g0658700 gene. Figure 1 As shown.

[0049] The nucleotide sequence of the Os04g0658700 gene in osk8-1 plants is as described in SEQ ID NO:3; the nucleotide sequence of the Os04g0658700 gene in osk8-2 plants is as described in SEQ ID NO:4.

[0050] Example 3: Comparison of growth of wild type and osk8 mutant at 6 weeks, with no significant difference in the aboveground parts.

[0051] The wild-type rice variety Nipponbare and the Os04g0658700 gene mutant plants osk8-1 and osk8-2 were planted in a culture room under the following growing conditions: 24℃, light:dark = 16h:8h. The growth of the wild-type and osk8 mutant plants was similar. Figure 2 The results indicate that knocking out the Os04g0658700 gene does not significantly affect the plant height of rice.

[0052] Example 4: Comparison of growth at the heading stage between wild type and osk8 mutant; no significant difference was found in the aboveground parts.

[0053] The wild-type rice variety Nipponbare and the Os04g0658700 gene mutant plants osk8-1 and osk8-2 were planted in the field for 2 months until the heading stage. The growth of the wild-type and osk8 mutant plants was similar. Figure 3 The results indicate that knocking out the Os04g0658700 gene does not significantly affect the plant height and heading of rice.

[0054] Example 5: There was no significant difference in seed size between wild-type Nipponbare and the osk8 mutant.

[0055] The seeds of the wild-type rice variety Nipponbare and the mutants of osk8-1 and osk8-2 were similar in size. Figure 4 The results indicate that knocking out the Os04g0658700 gene does not significantly affect seed size in rice.

[0056] Example 6: Identification of disease resistance in plants with the Os04g0658700 gene mutant

[0057] The wild-type rice variety Nipponbare and the Os04g0658700 gene mutant plants osk8-1 and osk8-2 were planted in a greenhouse and cultured at 28℃ (16h day / 8h night). After about 3 weeks of growth, Guy11 bacterial solution was evenly sprayed on the leaves of the above plants.

[0058] The specific implementation steps are as follows: In a clean bench, place filter paper containing *Magnapordia oryzae* spores on CM medium and seal it with sealing film; place the medium in a 28℃ constant temperature incubator and incubate for about 7 days; in a clean bench, use a sterile scalpel to cut the mycelium into small pieces, transfer them to rice bran medium, seal with sealing film, and place the medium in a 28℃ constant temperature incubator; after the mycelium has completely covered the rice bran medium, scrape off the mycelium with a sterile glass slide, and then transfer it to a 28℃ light incubator to stimulate spore production for three days; wash off the *Magnapordia oryzae* spores with 0.02% Tween 20, then filter the spores with filter paper to make the resuspended spore concentration 1.5 × 10⁻⁶. 5 The diluted spore solution was evenly sprayed onto the surface of rice leaves that had been growing for about 3 weeks using a spray bottle. The rice was then placed in a humid environment and kept in the dark for 48 hours. After 48 hours, the rice was gently placed in the inoculation room (28℃, 16 hours day / 8 hours night), and the seedlings were regularly sprayed with water to keep them moist. The disease phenotype was observed and recorded after 5 days of cultivation.

[0059] The results showed that the susceptibility of the Os04g0658700 gene mutants osk8-1 and osk8-2 was significantly lower than that of the control wild-type rice variety Nipponbare. Figure 5 The results indicate that knocking out the Os04g0658700 gene in rice can effectively improve the plant's resistance to rice blast pathogen.

[0060] Example 7: Quantitative PCR detection of rice blast fungus biomass on leaves of wild-type and mutant plants

[0061] DNA extraction: Take 0.02g of rice leaves infecting rice blast fungus and place them in a 2mL centrifuge tube. Grind the mixture in liquid nitrogen. Add 800μL of CTAB extraction buffer and incubate at 65℃ for 1h, inverting and mixing several times every 15min. Add 800μL of phenol:chloroform:isopropanol (25:24:1), invert and mix, centrifuge at 12000rpm for 10min, and transfer the upper aqueous phase to a new 2mL centrifuge tube. Add an equal volume of chloroform:isopropanol (24:1), invert and mix, centrifuge at 12000rpm for 10min, and transfer the supernatant to a new 1.5mL centrifuge tube. Add 0.6 times the volume of isopropanol and precipitate at -20℃ for at least 30min. Centrifuge at 12000rpm for 10min, discard the supernatant, and wash twice with 75% ethanol. Dry in a vacuum dryer for about 40min, and dissolve the DNA in water.

[0062] Quantitative PCR: 28S rDNA-specific primers for *Blastomyces oryzae* were used: 28S rDNA-F: 5'-TGTATGCCAGTGGTCGTACCA-3' and 28S rDNA-R: 5'-CCAGCAAGGTCGAGACGAA-3'. The rice OsActin gene was used as an internal control, with primers OsActin-F: 5'-TACGAGAGGAACCGCTCATTCAGATAATTA-3' and OsActin-R: 5'-TCAGCAGATCGTAACGATAAAGCTACTC'. The PerfectStartGreen qPCR SuperMix kit from TransGen Biotech was used. The reaction mixture consisted of: 10 μL 2×PerfectStartGreen qPCRSuperMix, 1 μL genomic DNA template, 0.4 μL forward and reverse primers (10 μM each), 0.4 μL Passive Reference Dye (50×), and water to a final volume of 20 μL. The PCR program was as follows: 94℃ pre-denaturation for 30 seconds; 94℃ denaturation for 5 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 10 seconds, for 40 cycles. Each sample was tested in triplicate. -ΔΔCT The method processes the data. The results are as follows: Figure 6 As shown, the amount of rice blast fungus in the leaves of the osk8 mutant rice was significantly less than that in the wild type, indicating that knocking out the Os04g0658700 gene can effectively improve the resistance of rice to rice blast.

[0063] Example 8: Creation and identification of rice plants overexpressing the Os04g0658700 gene

[0064] Primers were designed based on the OsK8 gene sequence (SEQ ID NO: 1).

[0065] X6-F: 5'-CGGTCCCGGGGGATCATGGCAACCCAAATGCG-3' and X6-R: 5'-TGCTCACCATGGATCTATGCCACAGCTCATGTCGTACG-3'.

[0066] An overexpression vector of the OsK8 gene driven by the Ubiquitin promoter (Ubi) was constructed using the "Golden Gate cloning" method, which can be followed according to the literature. 【2】 Perform the operation.

[0067] The constructed vector was transformed into Agrobacterium strain EHA105, which was then used to infect tissue culture callus of Nipponbare rice. Transgenic callus was selected by screening for the hygromycin resistance gene carried by the vector. Subsequent tissue culture differentiation and regeneration steps were performed to obtain transgenic positive plants (Os04g0658700). DNA was extracted from these plants, and overexpression materials were identified using specific primers OsK8-qPCR-F: 5'-GTTGCTGTGAAGCAGCTGAG-3' and GFP-R: 5'-GGACACGCTGAACTTGTGG-3'. The results are as follows: Figure 7 As shown, the lanes representing 2kb markers are 2kb, 1kb, 750bp, 500bp, and 250bp from top to bottom. Specifically, they are divided into two categories: one with a band at 600bp indicates plants overexpressing OsK8, and the other without a band at 600bp indicates wild-type NIPs. Therefore, lanes 1-6, 8, 9, 13, and 14 contain Ubi::OsK8-1 overexpressing plants; lanes 18-23 contain Ubi::OsK8-2 overexpressing plants; and lanes 7, 10-12, 15, 16, 17, and 24 contain non-transgenic wild-type NIPs.

[0068] Example 9: Identification of disease resistance in rice plants overexpressing the Os04g0658700 gene

[0069] The wild-type rice variety Nipponbare and its Os04g0658700 gene overexpressing plants Ubi::OsK8-1 and Ubi::OsK8-2 were cultured in a greenhouse at 28℃ (16h day / 8h night). After about 3 weeks of growth, Guy11 bacterial solution was inoculated onto the leaves of these plants in vitro.

[0070] The specific implementation steps are as follows: The rice blast fungus culture method is the same as in Example 6. Take the second leaf from the top of rice plants that have grown for about 3 weeks, and take a 7cm long leaf from 5cm below the leaf tip. Use a punch to make holes in the leaf and insert it into a 0.7% agar plate containing 80mg / L benzimidazole. Add 10μL of a 1.5×10⁻⁶ agar solution to the punched hole. 5 Rice blast fungus spore suspension at 1 / mL was placed in a 26℃ incubator (16h day / 8h night) for 5 days. The disease resistance phenotype was then observed and recorded. Figure 8 ).according to Figure 8 It can be seen that the lesion area of ​​the overexpressing plants Ubi::OsK8-1 and Ubi::OsK8-2 is larger than that of the wild-type plant NIP, indicating that the overexpressing OsK8 is more susceptible to rice blast than the wild-type NIP.

[0071] Example 10: Quantitative PCR detection of rice blast fungus biomass on leaves of wild-type and OsK8-overexpressing plants

[0072] DNA was extracted as described in Example 7 above, and the specific primers for the *Magnapordica oryzae* MoPot2 gene, MoPot2-F: 5'-ACGACCCGTCTTTACTTATTTGG-3 and MoPot2-R: 5'-AAGTAGCGTTGGTTTTGTTGGAT-3, were detected by quantitative PCR. The rice OsActin gene was used as an internal control, with primers OsActin-F: 5'-TACGAGAGGAACCGCTCATTCAGATAATTA-3' and OsActin-R: 5'-TCAGCAGATCGTAACGATAAAGCTACTC'.

[0073] The results showed that the susceptibility of Os04g0658700 gene overexpressing plants Ubi::OsK8-1 and Ubi::OsK8-2 was higher than that of the control wild-type rice variety Nipponbare. Figure 9 The results indicate that overexpression of the gene Os04g0658700 in rice reduces the resistance of rice to rice blast fungus.

[0074] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

[0075] The references mentioned above are as follows:

[0076] [1]Galhano, R. and Talbot, N. J. (2011) The biology of blast: Understanding how Magnaporthe oryzae invades rice plants. Fungal Biology Reviews, 25, 61-67.

[0077] [2]Zeng D C, Ma X L, Xie X R, et al. A protocol for CRISPR / Cas9-based multi-gene editing and sequence decoding of mutant sites in plants (in Chinese). Sci Sin Vitae, 2018, 48, doi:10.1360 / N052018-00069 (Zeng Dongchang, Ma Xingliang, Xie Xianrong, etc. A method for constructing CRISPR / Cas9 multi-gene editing vectors and analyzing mutations in plants. Science China: Life Sciences, 2018, 48).

Claims

1. Knockout of rice blast resistance gene OsK8 The application of the reagent in enhancing rice resistance to rice blast is characterized by: Nucleotide sequence of the rice gene OsK8 is shown as SEQ ID NO:

1.

2. The application according to claim 1, characterized in that: In rice blast resistance gene OsK8 The sgRNA sequence of the CRISPR / Cas9 editing target site in the rice blast resistance gene is 5'-TCCACCAGAAGAATCAGCTC-3', a primer is artificially synthesized according to the sgRNA sequence and constructed into a CRISPR / Cas9 vector.

3. The application according to claim 2, characterized in that... The synthesized primers are: Upstream 5'-TCCACCAGAAGAATCAGCTCgttttagagctagaaat-3' Downstream 5'-GAGCTGATTCTTCTGGTGGACggcagccaagccagca-3'.

4. The application according to claim 3, characterized in that: Compared to the wild-type rice variety Nipponbare, knocking out the rice blast resistance gene... OsK8 It can effectively improve the plant's resistance to rice blast pathogen.

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