Gene for enhancing rice blast resistance of rice and application thereof

Abstract

The invention belongs to the field of crop genetic breeding, and particularly relates to a rice blast resistant gene OsK8 (Os04g0658700 gene) and application thereof. The invention discloses a rice blast resistance gene OsK8. The nucleotide sequence of the gene is shown as SEQ ID NO: 1. The invention also discloses application of the rice blast-resistant gene OsK8. The OsK8 gene is knocked out from rice, so that the resistance of rice plants to pyricularia oryzae can be effectively enhanced; and the gene Os04g0658700 is overexpressed in the rice, so that the resistance of the rice to the pyricularia grisea is reduced.

Description

Technical Field

[0001] The invention belongs to the field of crop genetic breeding, and in particular relates to a rice blast resistance gene OsK8 and a use thereof. Background Art

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

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

[0004] At present, the main methods for preventing and controlling rice blast include breeding disease-resistant varieties, chemical control and strengthening field management. The extensive use of pesticides has slowed down the occurrence and harm of rice diseases to a certain extent, but it also seriously affects human health and ecological environment safety. Chemical agents will pollute the environment on which humans depend for survival. A large amount of pesticides will flow into water bodies, settle and accumulate in the soil, and transfer to the human body through the enrichment of the food chain, which will cause a series of hazards. Practice has proved that breeding and promoting disease-resistant varieties is one of the safest and most effective methods for preventing and controlling rice blast.

[0005] The key to breeding disease-resistant rice varieties is to utilize its own disease-resistant genes. In traditional breeding, a type of NB-LRR disease-resistant gene is widely used. It provides strong resistance, but generally only targets specific physiological subspecies of pathogens, has a narrow disease resistance range, and is easily lost due to pathogen mutations. Protein kinases play an indispensable role in many physiological processes of plants, including responding to various biological and abiotic stresses. Protein kinases affect the biochemical activity and function of proteins by regulating the phosphorylation of their substrate proteins. As an important gene for regulating plant resistance, protein kinases have great potential for application in disease-resistant breeding.

[0006] The sequence of the Os04g0658700 gene is available on 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 gene OsK8 for resistance to rice blast and its use. The present invention improves the resistance of rice to rice blast through single gene editing.

[0008] In order to solve the above problems, the present invention provides a rice blast resistance gene OsK8 (Os04g0658700 gene), the nucleotide sequence of which is shown in SEQ ID NO: 1. The amino acid sequence encoded by the gene is shown in SEQ ID NO: 2.

[0009] The present invention provides an application method of the rice blast resistance gene OsK8: knocking out the OsK8 (Os04g0658700) gene in rice by CRISPR-Cas9 gene editing technology can effectively enhance the resistance of rice plants to rice blast fungi. Specifically, the OsK8 gene in the rice cultivar Nipponbare undergoes a frameshift mutation to obtain a gene-knockout rice plant, thereby increasing the resistance of rice to rice blast.

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

[0011] As a further improvement of 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 of 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 of the use of the rice blast resistance gene OsK8 of the present invention: two mutant plants obtained by knocking out the rice blast resistance gene OsK8 are Osk8-1 plant and Osk8-2 plant.

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

[0018] The present invention found a gene OsK8 encoding an intracellular receptor-like kinase in rice that negatively regulates rice resistance to rice blast. The OsK8 gene was knocked out in rice using CRISPR-Cas9 gene editing technology, which significantly improved rice resistance to rice blast fungus. In addition, the gene mutation had no significant effect on the growth and yield of rice. On the contrary, overexpression of OsK8 led to increased susceptibility of rice to rice blast. Therefore, the gene has important application value in rice disease resistance breeding.

[0019] In summary, the present invention discloses for the first time that knocking out the OsK8 disease-susceptibility gene has the effect of improving the resistance of rice to rice blast. The purpose of the rice gene OsK8 is that after knocking out the gene, the resistance to rice blast is significantly enhanced, and the growth and development of rice is not significantly affected. The present invention has direct application value in the rapid genetic improvement of rice varieties with good quality but poor resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

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

[0022] Figure 2 Comparison of the growth status of wild type and mutants in the greenhouse for 6 weeks:

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

[0024] The growth conditions were 24°C, 16 h of light, 8 h of darkness, and 6 weeks of growth.

[0025] Figure 3 Comparison of the growth and heading status of the wild type and mutant under field conditions:

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

[0027] The growing conditions were field growth for 2 months until the heading stage.

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

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

[0030] Figure 6 The relative growth of the pathogen Guy11 on rice leaves of wild-type rice varieties NIP, osk8-1 and osk8-2 mutant plants was determined by quantitative PCR to detect the relative growth of the rice blast fungus 28S rDNA relative to the rice OsActin reference gene and calculate the multiple to reflect the relative growth of the rice blast fungus.

[0031] Figure 7 PCR identification of rice Os04g0658700 gene overexpression plants.

[0032] Figure 8 The disease conditions of leaves of wild-type rice variety NIP and two lines of OsK8-overexpressing plants (Ubi::OsK8-1 and -2) after infection with rice blast fungus Guy11; scale bar 1 cm.

[0033] Fig. 9 The relative growth of the pathogen on rice leaves after the wild-type rice variety NIP and two strains overexpressing OsK8 were infected with the pathogen Guy11 was determined. The relative growth of the rice blast fungus MoPot2 relative to the rice OsActin reference gene was detected by quantitative PCR and the multiple was calculated to reflect the relative growth of the rice blast fungus.

[0034] The wild-type and mutant plants were observed five days after inoculation with the rice blast pathogen Guy11; Guy11 spore concentration: 1.5×10 5 The inoculation experiment was repeated 3 times and the results were reproducible. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

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

[0037] According to the OsK8 gene sequence (SEQ ID NO: 1), the targeting sgRNA sequence for CRISPR-Cas9 knockout was designed:

[0038] 5'-TCCACCAGAAGAATCAGCTC-3', the corresponding primers were synthesized according to this sequence

[0039] 5'-TCCACCAGAAGAATCAGCTCGTTTTAGAGCTAGAAAT-3' and

[0040] 5'-GAGCTGATTCTTCTGGTGGACGGCAGCCAAGCCAGCA-3'.

[0041] The knockout vector of OsK8 gene was constructed by Golden Gate cloning method through Bsa1 restriction site. 【2】 to perform the operation.

[0042] The constructed vector was transferred into Agrobacterium strain EHA105, which was used to infect tissue culture callus of O. sativa sp. japonica cv Nipponbare (NIP), and transgenic callus was screened out by the hygromycin resistance gene carried by the vector. Subsequent tissue culture differentiation and regeneration steps were continued to obtain transgenic rice plant osk8 (Os04g0658700) knockout mutant plants.

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

[0044] DNA extraction: About 0.02 g of leaves from wild-type rice variety Nipponbare and mutant plants were taken, ground with liquid nitrogen, and 600 μL of SDS extraction solution (0.1 mol / L Trizma base, 0.05 mol / L EDTA-Na 2 , 0.5mol / L NaCl, 15g / LSDS), incubate at 65℃ for 1h, shake 2-3 times, add 200μL 5M potassium acetate, shake well and place on ice at -20℃ for 20min, add 500μL chloroform, mix well, let stand for 5min, centrifuge at 10000r / min, 4℃ for 7min, take 500μL supernatant and transfer it to a new 1.5mL centrifuge tube, add 500μL pre-cooled isopropanol, place at -20℃ for 30min, take out, centrifuge at 12000r / min, 4℃ for 3min, discard the supernatant, add 500μL 75% ethanol, centrifuge at 12000r / min for 1min, discard the supernatant, add 500μL anhydrous ethanol, centrifuge at 12000r / min for 1min, discard the supernatant, invert at room temperature to air dry, add 100μL ddH 2 O, mix well, and 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 NIP and mutant plants of Rhizoma Ospermi used as templates and amplified using 2×taq PCR reagent (Congwei Century, product number CW2849M).

[0046] The PCR amplification system was as follows: 1 μL template DNA, 5 μL 2× Taq Plus MasterMix (Dye), 0.4 μL each of F primer and R primer, ddH 2 Add 0.1% HO to make up to 10 μL.

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

[0048] After sequencing analysis of the PCR products, two mutant plants of the Os04g0658700 gene were successfully identified: osk8-1 plant (a base T was inserted into the Os04g0658700 gene) and osk8-2 plant (a base A was inserted into the Os04g0658700 gene). Both mutations resulted in a frameshift mutation in the Os04g0658700 gene. Figure 1 shown.

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

[0050] Example 3: Comparison of the growth of the wild type and the osk8 mutant at 6 weeks showed 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 growth conditions of 24°C light: dark = 16h: 8h. The growth conditions of the wild-type and osk8 mutant plants were similar ( Figure 2 ), indicating that knocking out the Os04g0658700 gene had no significant effect on plant height.

[0052] Example 4: Comparison of the growth of the wild type and the osk8 mutant at the heading stage showed no significant difference 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 conditions of the wild-type and osk8 mutant plants were similar ( Figure 3 ), indicating that knocking out the Os04g0658700 gene had no significant effect on rice plant height and heading.

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

[0055] The seed sizes of wild-type rice variety Nipponbare and osk8-1 and osk8-2 mutants are similar ( Figure 4 ), indicating that knocking out the Os04g0658700 gene in rice had no significant effect on seed size.

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

[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°C (16h day / 8h night). After growing for about 3 weeks, the Guy11 bacterial solution was evenly sprayed on the leaves of the above plants.

[0058] The specific implementation steps are as follows: on a clean bench, place a filter paper with rice blast spores on a CM culture medium and seal it with a sealing film; place the culture medium in a 28°C constant temperature incubator and culture it for about 7 days; on a clean bench, cut the hyphae into small pieces with a sterile scalpel, transfer them to a rice bran culture medium, seal it with a sealing film, and place the culture medium in a 28°C constant temperature incubator; after the hyphae grow all over the rice bran culture medium, scrape the hyphae with a sterile glass slide, and then move it to a 28°C light incubator to stimulate spore production for three days; wash the rice blast spores with 0.02% Tween 20, and then filter the spores with filter paper to make the resuspended spore concentration be 1.5×10 5 / mL; Use a spray bottle to evenly spray the diluted spore solution on the surface of rice leaves that have grown for about 3 weeks, and then place the rice in a humid environment in the dark for 48 hours; After 48 hours, gently place the rice in the inoculation room (cultured at 28℃, 16h day / 8h night), and regularly spray water on the seedlings to keep them moist. After 5 days of culture, observe and record the disease phenotype.

[0059] The results showed that the susceptibility of the Os04g0658700 gene mutants osk8-1 and osk8-2 plants was significantly lower than that of the wild-type rice variety Nipponbare ( Figure 5 ), indicating that knocking out the Os04g0658700 gene in rice can effectively improve the plant's resistance to the 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: 0.02 g of rice leaves inoculated with rice blast fungus were placed in a 2 mL centrifuge tube, and ground by liquid nitrogen freezing; 800 μL of CTAB extraction buffer was added, and the mixture was incubated at 65°C for 1 hour, and the mixture was mixed several times every 15 minutes; 800 μL of phenol: chloroform: isopropanol (25:24:1) was added, and the mixture was mixed by inversion, and the mixture was centrifuged at 12000 rpm for 10 minutes, and the upper aqueous phase was transferred to a new 2 mL centrifuge tube; an equal volume of chloroform: isopropanol (24:1) was added, and the mixture was mixed by inversion, and the mixture was centrifuged at 12000 rpm for 10 minutes, and the supernatant was transferred to a new 1.5 mL centrifuge tube; 0.6 times the volume of isopropanol was added, and the mixture was precipitated at -20°C for more than 30 minutes; the mixture was centrifuged at 12000 rpm for 10 minutes, the supernatant was discarded, and 75% ethanol was added for washing twice; the mixture was dried in a vacuum dryer for about 40 minutes, and water was added to dissolve the DNA.

[0062] Quantitative PCR: The rice blast fungus 28S rDNA specific primers 28S rDNA-F: 5'-TGTATGCCAGTGGTCGTACCA-3' and 28S rDNA-R: 5'-CCAGCAAGGTCGAGACGAA-3' were used. The rice OsActin gene was used as an internal reference, and the primers were OsActin-F: 5'-TACGAGAGGAACCGCTCATTCAGATAATTA-3' and OsActin-R: 5'-TCAGCAGATCGTAACGATAAAGCTACTC'. The PerfectStartGreen qPCR SuperMix kit from TransGen Biotech was used, and the reaction system was: 10μL 2×PerfectStart Green qPCRSuperMix, 1μL genomic DNA template, 0.4μL upstream and downstream primers (10μM each), 0.4μL Passive ReferenceDye (50×), and water was added to make up to 20μL. The PCR program was: 94°C pre-denaturation for 30 seconds; 94°C denaturation for 5 seconds, 60°C annealing for 15 seconds, 72°C extension for 10 seconds, 40 cycles. Each sample was repeated 3 times with 2 -ΔΔCT The method processes the data. The results are as follows Figure 6 As shown, the amount of rice blast fungi in the leaves of osk8 mutant rice was significantly less than that of the wild type, indicating that knocking out the gene Os04g0658700 can effectively improve the resistance of rice to rice blast.

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

[0064] According to the OsK8 gene sequence (SEQ ID NO: 1), primers were designed.

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

[0066] The Golden Gate cloning method was used to construct an OsK8 gene overexpression vector driven by the Ubiquitin promoter (Ubi). 【2】 to perform the operation.

[0067] The constructed vector was transferred into Agrobacterium strain EHA105, which was used to infect tissue culture callus of Nipponbare rice, and transgenic callus was selected by the hygromycin resistance gene carried by the vector. Subsequent tissue culture differentiation and regeneration steps were continued to obtain transgenic positive plants of Os04g0658700, and DNA was extracted. The overexpression material was identified by specific primers OsK8-qPCR-F: 5'-GTTGCTGTGAAGCAGCTGAG-3' and GFP-R: 5'-GGACACGCTGAACTTGTGG-3'. The results are shown in the figure. Figure 7 As shown, the marker represents the lane of 2kb marker, and from top to bottom, it is 2kb, 1kb, 750bp, 500bp, and 250bp. It is specifically divided into the following two categories: one is that there is a band at 600bp, indicating that the plant overexpresses OsK8, and the other is that there is no band at the 600bp position, indicating that it is a wild-type NIP. Therefore, lanes 1-6, 8, 9, 13, and 14 are Ubi::OsK8-1 overexpressing plants, lanes 18-23 are overexpressing plants Ubi::OsK8-2, and lanes 7, 10-12, 15, 16, 17, and 24 are wild-type NIPs without transgenes.

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

[0069] The wild-type rice variety Nipponbare and the Os04g0658700 gene overexpressing plants Ubi::OsK8-1 and Ubi::OsK8-2 were planted in a greenhouse at 28°C (16h day / 8h night). After growing for about 3 weeks, the Guy11 bacterial solution was inoculated in vitro on the leaves of the above plants.

[0070] The specific implementation steps are as follows: The rice blast fungus culture method is the same as that in Example 6. Take the second leaf of rice grown for about 3 weeks, take a 7 cm long leaf from 5 cm below the tip of the leaf, punch the leaf with a hole puncher and insert it on a 0.7% agar plate containing 80 mg / L benzimidazole, and drip 10 μL of 1.5×10 5 / mL of rice blast fungus spore solution, placed in a 26℃ incubator (16h day / 8h night), and observed and recorded the disease resistance phenotype after 5 days of incubation ( 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 biomass on leaves of wild-type and OsK8 overexpressing plants

[0072] DNA was extracted as described in Example 7 above, and the specific primers MoPot2-F: 5'-ACGACCCGTCTTTACTTATTTGG-3 and MoPot2-R: 5'-AAGTAGCGTTGGTTTTGTTGGAT-3 were used to detect the rice blast fungus MoPot2 gene. The rice OsActin gene was used as an internal reference, and the primers used were OsActin-F: 5'-TACGAGAGGAACCGCTCATTCAGATAATTA-3' and OsActin-R: 5'-TCAGCAGATCGTAACGATAAAGCTACTC'.

[0073] The results showed that the Os04g0658700 gene overexpression plants Ubi::OsK8-1 and Ubi::OsK8-2 were more susceptible to disease than the wild-type rice variety Nipponbare ( Fig. 9 ), indicating that overexpression of the gene Os04g0658700 in rice reduced the resistance of rice to rice blast fungus.

[0074] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

[0075] The references mentioned above are as follows:

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

[0077] [2] Zeng DC, Ma XL, Xie XR, 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, et al. A protocol for CRISPR / Cas9-based multi-gene editing vector construction and mutation analysis in plants. Chinese Science: Life Sciences, 2018, 48).

Claims

1. A rice blast resistance gene OsK8, characterized in that: The nucleotide sequence of the rice gene OsK8 is shown in SEQ ID NO:

1.

2. The protein encoded by the rice blast resistance gene OsK8 according to claim 1, characterized in that: The amino acid sequence is shown in SEQ ID NO:

2.

3. The use of the rice blast resistance gene OsK8 as claimed in claim 1, characterized in that: Knocking out the OsK8 gene in rice can enhance rice's resistance to rice blast.

4. The use of the rice blast resistance gene OsK8 according to claim 3, characterized in that: The sgRNA sequence of the CRISPR / Cas9 editing target was designed in the rice blast resistance gene OsK8: 5'-TCCACCAGAAGAATCAGCTC-3'. Primers were artificially synthesized based on the sgRNA sequence and constructed into the CRISPR / Cas9 vector.

5. The use of the rice blast resistance gene OsK8 according to claim 4, characterized in that The synthesized primers are: Upstream 5′-TCCACCAGAAGAATCAGCTCgttttagagctagaaat-3′ Downstream 5′-GAGCTGATTCTTCTGGTGGACggcagccaagccagca-3′.

6. The use of the rice blast resistance gene OsK8 according to claim 5, characterized in that: 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.

7. The use of the rice blast resistance gene OsK8 according to claim 6, characterized in that: The two mutant plants obtained by knocking out the rice blast resistance gene OsK8 are Osk8-1 and Osk8-2.

8. The use of the rice blast resistance gene OsK8 as claimed in claim 1, characterized in that: Overexpression of the gene Os04g0658700 in rice reduced rice resistance to rice blast fungus.

9. The use of the rice blast resistance gene OsK8 according to claim 8, characterized in that: The Os04g0658700 gene overexpression plants Ubi::OsK8-1 and Ubi::OsK8-2 were more susceptible to disease than the control wild-type rice variety Nipponbare.

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