Application of single-site mutation of disease resistance-related protein OsRLCK176 in improving rice disease resistance

Abstract

The present invention discloses an application of a rice disease resistance-related protein OsRLCK176 single-site mutation in improving rice resistance to pathogenic bacteria, wherein the mutation site of OsRLCK176 is a mutation from S to D at the 83rd position of the amino end of the amino acid sequence of OsRLCK176, which can enhance rice resistance to bacterial leaf streak and bacterial leaf blight. The single-site mutation is used to regulate the disease resistance of rice, and is of great significance for breeding new disease-resistant rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an application of a disease resistance-related protein OsRLCK176 single-site mutation in improving the resistance of rice to pathogenic bacteria. Background Art

[0002] Rice (Oryza sativa) is one of the most important staple crops, providing food for about half of the world's population, and plant diseases have always been a major threat to rice production. Bacterial leaf blight and bacterial stripe disease are common major bacterial diseases of rice in many rice-growing areas in my country. Among them, bacterial stripe disease has not yet found a major disease-resistant gene in rice, making it difficult to prevent and control. Using disease-resistant genes to improve and breed new crop varieties is the most environmentally friendly and cost-effective measure to control the occurrence of diseases, and it is also the development trend of green agriculture in the future. Among them, breeding technologies represented by gene editing have shortened the breeding cycle of varieties and created crop germplasm with broad-spectrum resistance and high-yield traits, showing great application prospects (Li, S., Lin, D., Zhang, Y., Deng, M., Chen, Y., Lv, B., Li, B., Lei, Y., Wang, Y., Zhao, L., Liang, Y., Liu, J., Chen, K., Liu, Z., Xiao, J., Qiu, J., and Gao, C. (2022). Genome-edited powderymildew resistance in wheat without growth penalties. NATURE 602, 455-460). By studying the important functional sites of key rice disease resistance genes, candidate targets and theoretical basis can be provided for the creation of disease-resistant germplasm.

[0003] Plants perceive and respond to various signal molecules through receptor proteins on their cell surfaces. Receptor kinase (RK) is the most important component of plant cell receptors. Receptor kinases are composed of an extracellular domain responsible for sensing signals, a transmembrane domain, and an intracellular kinase domain. Plant receptor kinases perceive and respond to various disease invasions and abiotic stress environments in the natural environment. There are also a large number of cytoplasmic receptor kinase (RLCK) family proteins in plant cells. These proteins are very similar to the intracellular domain of receptor kinases, but lack extracellular domains. More and more studies have found that cytoplasmic receptor kinases combine with receptor kinases to achieve regulation of downstream signal pathways through phosphorylation relays, and are the core hubs for receptor kinases to regulate cell activities (Liang, X., and Zhou, JM (2018). Receptor-like cytoplasmic kinases: central players in plant receptor kinase-mediated signaling. ANNU REV PLANT BIOL 69, 267-299). For example, the Arabidopsis RLCK protein AtBIK1 is an immune regulatory factor associated with multiple important receptor kinases (such as FLS2, EFR and LYK5). After the plant pattern recognition receptor complex senses the presence of pathogens, RLCK participates in plant disease resistance signal transduction as a downstream element, thus playing an important role (Sun, L., and Zhang, J. (2020). Regulatory role of receptor-like cytoplasmic kinases in early immune signaling events in plants. FEMSMICROBIOL REV 44, 845-856).Rice OsRLCK176, OsRLCK57, OsRLCK107 and OsRLCK118 are all homologous proteins of Arabidopsis AtBIK1. Current studies have shown that OsRLCK176 may play a major function in regulating rice disease resistance: in the rice immune response triggered by fungal chitin and peptidoglycan, OsRLCK176 plays an immune signaling role as an essential element downstream of the co-receptor kinase OsCERK1 (Ao, Y., Li, Z., Feng, D., Xiong, F., Liu, J., Li, JF, Wang, M., Wang, J., Liu, B., and Wang, HB (2014). OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity. PLANT J 80, 1072-1084). In addition, the rice-specific receptor kinase SDS2 interacts with OsRLCK176 and OsRLCK118 to regulate downstream immune signals. The osrlck176 mutant has significantly reduced resistance to rice blast (Fan, J., Bai, P., Ning, Y., Wang, J., Shi, X., Xiong, Y., Zhang, K., He, F., Zhang, C., Wang, R., Meng, X., Zhou, J., Wang, M., Shirsekar, G., Park, CH, Bellizzi, M., Liu, W., Jeon, J., Xia, Y., Shan, L., and Wang, G. (2018). The monocot-specific receptor-like kinase SDS2controls cell death and immunity in rice. CELL HOST MICROBE 23, 498–510).

[0004] OsRLCK176 has been found to play a key role in rice disease resistance as a downstream element of multiple important receptor kinases. However, it is still unclear how OsRLCK176 is regulated in rice immunity. Therefore, there has been no in-depth research based on the regulatory mechanism to discover the amino acid sites that regulate OsRLCK176. In other words, the existing technology has not found the association between mutants of key functional sites of OsRLCK176 and rice disease resistance. Summary of the invention

[0005] The purpose of the present invention is to explore and transform the functional sites of OsRLCK176 through in-depth research on the regulatory mechanism of OsRLCK176, and to create rice materials with enhanced disease resistance.

[0006] The present invention is conceived as follows: Although OsRLCK176 has been found to be a positive regulatory factor for rice disease resistance, disease-resistant rice strains that overexpress OsRLCK176 are currently difficult to apply to production and promotion because they contain transgenic components, resulting in limitations in application. To this end, the present invention has found through research that OsRLCK176 regulates protein stability by being phosphorylated by the kinase OsCPK17; it has been identified that the 32nd, 83rd and 209th serines of OsRLCK176 are the main sites for OsCPK17 phosphorylation of OsRLCK176; after the 83rd serine of OsRLCK176 is mutated to aspartic acid to simulate the phosphorylated state, OsRLCK176 S83D Protein stability is enhanced, and pRLCK176::OsRLCK176 S83D The mutant transgenic rice has enhanced resistance to bacterial leaf streak and bacterial leaf blight. This indicates that the mutation of the 83rd amino acid at the amino terminus of the OsRLCK176 protein in rice from S to D can enhance the resistance of rice to bacterial leaf streak and bacterial leaf blight. This provides candidate sites and theoretical basis for the subsequent creation of disease-resistant rice germplasm without transgenic components through fine gene editing technology.

[0007] Based on this, the present invention provides an application of a single point mutation of a disease resistance-related protein OsRLCK176 in improving rice disease resistance; the single point mutation is a mutation of the 83rd amino acid at the amino terminal of the disease resistance-related protein OsRLCK176 from S to D. The sequence of the OsRLCK176 protein is shown in Sequence 4 in the sequence table.

[0008] The purpose of improving rice disease resistance can be achieved by point mutation of the rice OsRLCK176 protein gene, wherein the disease resistance is resistance to bacterial leaf streak and / or bacterial blight. The coding gene sequence of the rice OsRLCK176 protein gene is the nucleotide sequence shown in sequence 3 in the sequence table.

[0009] The present invention also provides a method for improving the disease resistance of rice, which comprises mutating the 83rd amino acid at the amino terminal of the OsRLCK176 protein in rice from S to D, wherein the sequence of the OsRLCK176 protein is shown in sequence 4 in the sequence table.

[0010] The method can achieve point mutation of amino acids through existing methods such as gene editing, gene knockout followed by transgenic, homologous recombination, etc.

[0011] In one embodiment of the present invention, an OsRLCK176 gene knockout mutant is used as a material, and the coding gene of the OsRLCK176 protein in which the 83rd amino acid at the amino terminal is mutated from serine (S) to aspartic acid (D) is introduced into the OsRLCK176 gene knockout mutant, and transgenic rice expressing the OsRLCK176 protein in which the 83rd amino acid at the amino terminal is mutated from S to D is screened. Compared with rice expressing wild OsRLCK176 protein, the resistance of the rice to bacterial leaf streak and / or white leaf blight is significantly improved.

[0012] Preferably, the gene encoding the OsRLCK176 protein in which the 83rd amino acid at the amino terminal is mutated from S to D is expressed under the guidance of the native promoter of the OsRLCK176 protein in wild-type rice, and the sequence of the promoter is shown in Sequence 5 in the sequence listing.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] pRLCK176::OsRLCK176, which is expressed in the osrlck176 knockout mutant background and driven by the OsRLCK176 promoter, was used to S83D Rice can enhance rice resistance to bacterial leaf blight and bacterial leaf streak. The mutation of the threonine site 83 of the OsRLCK176 protein to aspartic acid can promote the stability of the OsRLCK176 protein. Among the known plant disease resistance-related RLCKs, this phosphorylation site only exists in some rice RLCKs and does not exist in the homologous proteins of Arabidopsis, tobacco and pepper. This gene mutant provides new gene resources and candidate sites for rice disease resistance breeding. Related biological materials can be used to regulate rice disease resistance, which is of great significance for breeding new disease-resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 OsCPK17 phosphorylates OsRLCK176 in vitro;

[0016] Figure 2 LC-MS / MS analysis after in vitro phosphorylation reaction showed that OsRLCK176 was phosphorylated by OsCPK17 at Ser32, Ser35, Ser83, and Ser209;

[0017] Figure 3 It was shown that Ser32, Ser83, and Ser209 were the main sites of OsRLCK176 phosphorylation by OsCPK17 in vitro;

[0018] Figure 4 This showed that OsRLCK176 transiently expressed in protoplasts of rice oscpk17 mutant S83DThe protein stability is enhanced compared to OsRLCK176;

[0019] Figure 5 Sequence alignment analysis showed that the S83 site on OsRLCK176 is unique to rice RLCK VII subfamily proteins;

[0020] Figure 6 pRLCK176::OsRLCK176 was found for rice pathogenic bacteria inoculation S83D Genetically modified materials have enhanced disease resistance;

[0021] A, pRLCK176::OsRLCK176 and pRLCK176::OsRLCK176 driven by the OsRLCK176 promoter in the osrlck176 knockout mutant background inoculated with bacterial leaf streak race RS105. S83D Genetically complemented rice lines; B, pRLCK176::OsRLCK176 and pRLCK176::OsRLCK176 driven by the OsRLCK176 promoter in the osrlck176 knockout mutant background inoculated with Xanthomonas oryzae race PXO99A. S83D Genetically complemented rice lines;

[0022] Figure 7 pRLCK176::OsRLCK176 and pRLCK176::OsRLCK176 S83D Protein expression detection of transgenic materials. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0024] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0025] In the present invention, rice Nipponbare (Oryza sativa L.ssp.japonica cv.Nipponbare) is described in the literature: Stephen A. Goff et al. A Draft Sequence of the Rice Genome (Oryza sativa L.ssp.japonica). Science. 2002, (296): 92, which is publicly available from China Agricultural University.

[0026] In the present invention, the osrlck176 T-DNA insertion mutant was purchased from the rice mutant library of Pohang University of Science and Technology, South Korea ( www.postech.ac.kr / life / pfg / risd ), accession number 2C-10348, referred to as osrlck176 hereafter, and the mutant background is Dongjin rice.

[0027] In the present invention, the oscpk17T-DNA insertion mutant was purchased from the rice mutant library of Pohang University of Science and Technology in South Korea ( www.postech.ac.kr / life / pfg / risd ), accession number 4A-50582, referred to as oscpk17 hereafter, and the mutant background is Dongjin rice.

[0028] Example 1. Cloning of OsCPK17 and OsRLCK176 cDNA

[0029] Leaves of rice 'Nipponbare' seedlings grown under normal photoperiod for 30 days were taken and ground with liquid nitrogen. Total RNA of the samples was extracted using a plant RNA rapid extraction kit (Beijing Aidelai Biotechnology Co., Ltd.), and cDNA was synthesized by reverse transcription using a reverse transcription kit (One-StepgDNA Removal and cDNA Synthesis SuperMix, Beijing Quanshijin Company). Specific primers were designed: upstream primer OsCPK17-F: 5'-AAGGATCCATGGGGAACACCTGCGTCGG-3'; downstream primer OsCPK17-R: 5'-AACTCGAGACCAAGCTTCAATGCTTCCC-3'. Upstream primer OsRLCK176-F: 5'-AAGGATCCATGGGGAATTGCTGGGGCGCCA-3'; downstream primer OsRLCK176-R: 5'-AACTCGAGTTAAACCAGCCTCGCATTTGCGGG-3'. Reaction system (25 μL): 10×PCR buffer 2.5 μL; 2.5 mM dNTPs 1.0 μL; 10 μM upstream and downstream primers 0.5 μL each; Novozymes Phanta Max Super-Fidelity DNA polymerase 0.2 μL; cDNA template 1.0 μL; ddH2O supplemented to 25 μL. Using rice 'Nipponbare' cDNA as a template, PCR amplification was performed on an Applied Biosystems veritithermal cycler. Amplification conditions were: 95°C pre-denaturation for 5 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute for a total of 35 cycles, and finally 72°C extension for 10 minutes. The recovered PCR product was connected to the His-MBP prokaryotic expression vector (Escherichia coli expression vector, containing His-MBP tag and kanamycin resistance, obtained from Professor Chen Zhongzhou of China Agricultural University.Liu Y, Ma L, Li M, Tian Z, Yang M, Wu X, Wang X, Shang G, Xie M, Chen Y, Liu X, Jiang L, Wu W, Xu C, Xia L, Li G, Dai S, Chen Z (2023) Structures of human TR4LBD-JAZF1 and TR4DBD-DNA complexes reveal the molecular basis of transcriptional regulation. Nucleic Acids Research 51:1443-1457, publicly available from China Agricultural University), the constructed vectors were named MBP-OsCPK17 and MBP-OsRLCK176 and sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0030] The sequencing results show that the PCR products have the nucleotide sequences of SEQ ID NO.1 and SEQ ID NO.2 in the sequence list, respectively, and the genes of the PCR products are named OsCPK17 and OsRLCK176. The proteins encoded by the genes are named OsCPK17 and OsRLCK176, and the amino acid sequences of the proteins are SEQ ID NO.3 and SEQ ID NO.4 in the sequence list. The cDNA of the OsCPK17 gene is 1707bp in total length (SEQ ID NO.1), encoding a protein containing 568 amino acids (SEQ ID NO.3). The cDNA of the OsRLCK176 gene is 1188bp in total length (SEQ ID NO.2), encoding a protein containing 395 amino acids (SEQ ID NO.4). The promoter nucleotide sequence of the OsRLCK176 gene is SEQ ID NO.5, totaling 1050bp.

[0031] MBP-OsRLCK176 KM The point mutation protein expression vector was constructed based on MBP-OsRLCK176, in which the lysine residues at positions 108 and 109 were replaced by alanine. Plasmid site-directed mutagenesis PCR (Site-Directed Mutagenesis) technology was used for amplification. KM -F:5'-GATGGTCATTGCTGTAGCGGCGTTGAATCAGGAAGGCCAC-3'; and OsRLCK176 KM -R:5'-GTGGCCTTCCTGATTCAACGCCGCTACAGCAATGACCATC-3'.

[0032] Point mutation PCR amplification was performed using MBP-OsRLCK176 as a template.

[0033] The reaction system was: 2×Phanta Max Buffer 12.5μL, 10mM dNTP Mix 1μL, 10μM Forward Primer 0.65μL, 10μM Reverse Primer 0.65μL, DMSO 1.5μL, Phanta Max Super-Fidelity DNA Polymerase (1U / μL) 0.5μL, template DNA 50ng, and ddH2O to make up to 25μl.

[0034] PCR amplification program: 95°C for 2 min, then 95°C for 30 s, 55°C for 1 min, 68°C at 1 kb / min for 18 cycles, and stored at 16°C.

[0035] After PCR amplification, 0.5 μL DpnI was added to digest the template DNA at 37°C overnight, and heat-shock transformation was performed into BL21 (DE3). The plasmid was extracted and the expression vector in which the lysine residues at positions 108 and 109 of the correct OsRLCK176 protein were replaced by alanine was identified and named MBP-OsRLCK176 KM .

[0036] Example 2: In vitro kinase phosphorylation assay of OsCPK17 and OsRLCK176 and LC-MS / MS analysis

[0037] The in vitro phosphorylation reaction system was prepared as follows: Kinase protein 1 μg, Substrate 3 μg, Tris-HCl (pH = 7.5) 25 mM, MgCl2 10 mM, CaCl2 10 mM, DTT 1 mM, Cold ATP 50 μM, ddH2O to 20 μL, [γ- 32 P]ATP 1μCi.

[0038] MBP-OsRLCK176 KM and MBP-OsCPK17 were reacted in the above system at 30℃ for 1h. 5×SDS Loading Buffer was added to terminate the reaction, and the samples were boiled at 95℃ for 5min to denature the proteins, and then electrophoresed on 10% SDS-PAGE gel. After the electrophoresis, the phosphor screen was placed overnight and the phosphor screen was scanned with Typhoon for imaging. KM Can be phosphorylated by MBP-OsCPK17 in vitro ( Figure 1 ). Figure 1 In the upper part, 32P-labeled MBP-OsCPK17 has an autophosphorylation band at 130 kDa, and MBP-OsCPK17 does not phosphorylate the MBP protein used as a control. KM No phosphorylated bands, but MBP-OsRLCK176 KM When incubated with MBP-OsCPK17, MBP-OsRLCK176 was observed at 100 kDa. KM Phosphorylated bands. Figure 1 In the lower part, the amount of added protein is shown by Coomassie blue staining.

[0039] Point mutation PCR amplification with MBP-OsRLCK176 KM As a template, MBP-OsRLCK176 KMS32A 、MBP-OsRLCK176 KMS35A 、MBP-OsRLCK176 KMS83A 、MBP-OsRLCK176 KMS209A PCR amplification of point mutations. Expression of OsRLCK176 KM The expression vector MBP-OsRLCK176 in which the 32nd position of the amino terminal is mutated from S to A KMS32A Point mutation primer: OsRLCK176 KMS32A -F:5'-ATCCAAGTATGCTAGCAACGCCAGTGTGTCTGCAGCCTCG-3'; and OsRLCK176 KMS32A -R:5'-CGAGGCTGCAGACACACTGGCGTTGCTAGCATACTTGGAT-3'. Expression of OsRLCK176 KM The expression vector MBP-OsRLCK176 in which the 35th position of the amino terminal is mutated from S to A KMS35A Point mutation primer: OsRLCK176 KMS35A -F:5'-TGCTAGCAACTCCAGTGTGGCTGCAGCCTCGGTGCCGCC-3'; and OsRLCK176 KMS35A -R:5'-GGCGGCACCGAGGCTGCAGCCACACTGGAGTTGCTAGCA-3'. Expression of OsRLCK176 KM The expression vector MBP-OsRLCK176 in which the 83rd position of the amino terminal is mutated from S to A KMS83A Point mutation primer: OsRLCK176 KMS83A-F:5'-GGTGAGGGAGGCTTTGGAGCAGTCTTCAAAGGGTGGATC-3'; and OsRLCK176 KMS83A -R:5'-GATCCACCCTTTGAAGACTGCTCCAAAGCCTCCCTCACC-3'. Express OsRLCK176 KM The expression vector MBP-OsRLCK176 in which the 209th position of the amino terminal is mutated from S to A KMS209A Point mutation primer: OsRLCK176 KMS209A -F:5'-CTACCGTGATTTCAAGACCGCTAATGTCCTTTTGGATGCG-3'; and OsRLCK176 KMS209A -R:5'-CGCATCCAAAAGGACATTAGCGGTCTTGAAATCACGGTAG-3'.

[0040] The reaction system was: 2×Phanta Max Buffer 12.5μL, 10mM dNTP Mix 1μL, 10μM Forward Primer 0.65μL, 10μM Reverse Primer 0.65μL, DMSO 1.5μL, PhantaMax Super-Fidelity DNA Polymerase (1U / μL) 0.5μL, template DNA 50ng, and ddH2O to make up to 25μL.

[0041] PCR amplification program: 95°C for 2 min, then 95°C for 30 s, 55°C for 1 min, 68°C at 1 kb / min for 18 cycles, and stored at 16°C.

[0042] After PCR amplification, 0.5 μL DpnI was added to digest the template DNA at 37°C overnight, and then heat-shocked and transformed into BL21 (DE3). KMS32A 、MBP-OsRLCK176 KMS35A 、MBP-OsRLCK176 KMS83A 、MBP-OsRLCK176 KMS209A .

[0043] MBP-OsRLCK176 KM 、MBP-OsRLCK176 KMS32A 、MBP-OsRLCK176 KMS35A 、MBP-OsRLCK176 KMS83A 、MBP-OsRLCK176 KMS209ARespectively react with MBP-OsCPK17 in the phosphorylation reaction system at 30℃ for 1h. Add 5×SDS Loading Buffer to terminate the reaction, boil the sample at 95℃ for 5min to denature the protein, and then electrophoresed on 10% SDS-PAGE gel. After the electrophoresis, place the sample on the phosphor screen overnight and scan the phosphor screen with Typhoon for imaging. The results of in vitro phosphorylation analysis showed that Ser32, Ser83 and Ser209 were the main sites of OsRLCK176 phosphorylated by OsCPK17 in vitro ( Figure 3 ), Figure 3 In the upper part, 32 P-labeled MBP-OsCPK17 has an autophosphorylation band at 130 kDa. MBP-OsRLCK176 KM When incubated with MBP-OsCPK17, we can see MBP-OsRLCK176 at 100 kDa. KM The phosphorylated band. And MBP-OsRLCK176 KMS32A 、MBP-OsRLCK176 KMS83A 、MBP-OsRLCK176 KMS209A The phosphorylation intensity of MBP-OsRLCK176 was significantly weaker than that of MBP-OsRLCK176 KM , while MBP-OsRLCK176 KMS35A With MBP-OsRLCK176 KM Consistent. Single MBP-OsRLCK176 KM There is no phosphorylated band. Figure 3 In the lower part, the amount of added protein is shown by Coomassie blue staining.

[0044] In an in vitro kinase assay, MBP-OsRLCK176 KM After incubation with MBP-OsCPK17 and 50 μM cold ATP at 30°C for 1 h, OsRLCK176 was phosphorylated. After protein enrichment with ultrafiltration tubes, trypsin was used for digestion at 37°C overnight, and then LC-MS / MS was performed to analyze OsRLCK176. KM The phosphorylated sites are shown. Figure 2 pS32), Ser35( Figure 2 pS35), Ser83( Figure 2 pS83) and Ser209 ( Figure 2 pS209 in OsRLCK176 is the main site of OsCPK17 phosphorylation in vitro ( Figure 2 ).

[0045] Example 3: Stability detection of OsRLCK176-HA protein in rice protoplasts

[0046] 3.1 Protoplast preparation and transformation

[0047] 1) Take 40 green rice seedlings of Dongjin (DJ) and oscpk17 mutant cultured for about 10-14 days, remove the roots and leaves, cut the rice seedlings (stems and leaf sheaths) into 0.5 mm wide strips with a sharp blade, and add 0.6 M mannitol to maintain the cell osmotic pressure during cutting;

[0048] 2) Place the tissue in a 25 mL sterile triangular flask and immediately add 20 mL of enzymatic solution; place in a shaker at 28°C and 80 rpm for 4-6 hours away from light;

[0049] 3) The enzymatic hydrolyzate was filtered through 40 μM nylon mesh and centrifuged at 1,000 rpm for 6 min (slow rise and slow fall, the same below). 30 mL of W5 solution (9 g of sodium chloride, 18.4 g of calcium chloride, 0.37 g of potassium chloride, 200 mM pH = 5.7 MES 2 mL, ddH2O was added to the residue and then filtered through 40 μM nylon mesh again and centrifuged at 1,000 rpm for 6 min to collect rice protoplasts.

[0050] 4) Discard the supernatant, wash the precipitate three times with W5, centrifuge at 1,500 rpm for 6 min at room temperature, and discard the supernatant;

[0051] 5) Add 3 mL of MMG solution to suspend the collected protoplasts (mannitol 7.29 g, magnesium chloride 0.305 g, potassium chloride 0.37 g, 200 mM pH = 5.7 MES 2 mL, water to 100 mL), and incubate on ice for 30 min;

[0052] 6) 50 μg OsRLCK176-HA (Wang J, Wang S, Hu K, Yang J, Xin X, Zhou W, Fan J, Cui F, Mou B, Zhang S, Wang G, Sun W (2018) The kinase OsCPK4 regulates a buffering mechanism that fine-tunes innate immunity. Plant Physiol 176: 1835-1849, available to the public from China Agricultural University) or OsRLCK176 were added to a 10 mL centrifuge tube. S83D -HA (point mutation construction, constructed on the basis of OsRLCK176-HA. Plasmid site-directed mutagenesis PCR (Site-Directed Mutagenesis) technology was used for amplification. OsRLCK176 S83D-F:5'-TGGTGAGGGAGGCTTTTGGAGACGTCTTCAAAGGGTGGATC-3'; and OsRLCK176 S83D -R:5'-GATCCACCCTTTGAAGACGTCTCCAAAGCCTCCCTCACCA-3'. Amplification system: 2×Phanta Max Buffer 12.5μL, 10mM dNTP Mix 1μL, 10μM Forward Primer

[0053] 0.65μL, 10μM Reverse Primer 0.65μL, DMSO 1.5μL, Phanta Max Super-Fidelity DNA Polymerase (1U / μL) 0.5μl, template DNA 50ng, ddH2O to 25μL. PCR amplification program: 95℃ for 2min, then 95℃ for 30s, 55℃ for 1min, 68℃

[0054] 1kb / min, 18 cycles, 16℃ storage. After PCR amplification, 0.5μl DpnI was added to digest the template DNA at 37℃ overnight, heat-shocked and transformed into DH5α, plasmid was extracted, and OsRLCK176 was obtained. S83D -HA vector. ) plasmid, 1 mL protoplasts, 1.1 mL PEG-CaCl2, react at 28°C in the dark for 10 min;

[0055] 7) Add 5 mL of W5 solution to terminate the reaction, centrifuge at 1,000 rpm for 6 min, and discard the supernatant;

[0056] 8) Add 1 mL of W5 solution to the precipitate, shake gently, and incubate at 28°C overnight;

[0057] 9) Centrifuge at 1,000 rpm for 6 min to collect protoplasts for subsequent related experiments.

[0058] 3.2 Protein degradation experiment

[0059] The protoplasts were treated with 50uM CHX (Cycloheximide, actinomycin: protein synthesis inhibitor), incubated in a 28°C incubator for 2h, centrifuged to remove the supernatant, added with 100μL 2×SDS Loading Buffer, boiled at 95°C for 10min, and then subjected to SDS-PAGE protein electrophoresis.

[0060] 3.3 Western-blot hybridization

[0061] 1) Transfer protein sample to PVDF membrane

[0062] Use scissors to cut out a PVDF membrane with a pore size of 0.45μm and two filter papers of (5.5cm×9cm). Treat the PVDF membrane with methanol for 15s, ddH2O for 2-3min, and finally soak it in the transfer solution for use. After the electrophoresis is completed, disassemble the glass plate, carefully remove the concentrated gel, remove the separation gel and place it in the transfer solution. Place the filter paper, separation gel, and PVDF membrane in the non-woven fabric in order, clamp them and put them in the transfer tank, with the separation gel side facing the negative pole of the power supply and the PVDF membrane side facing the positive pole of the power supply. Pour the 4℃ pre-cooled transfer solution flush with the transfer tank, place the transfer tank in ice water, voltage 80V, current 200mA, transfer for 2-2.5h.

[0063] 2) Western-blot hybridization

[0064] Take a new 10cm square culture dish, pour 30mL of blocking solution (5% skim milk powder dissolved in 1×TBS / T solution containing 1‰ Tween 20), place the PVDF membrane after transfer in it, and incubate it on a shaker at 70rpm at room temperature for 2h. Then add 30mL of anti-HA antibody (Roche China), anti-GFP antibody (Cangwei Century) or anti-β-Actin antibody (Cangwei Century) antibody dilution diluted at 1:2000 and incubate overnight at 4℃, then wash the membrane 3 times with 1×TBS / T containing 1‰ Tween 20, 10min each time. Then use 5% skim milk powder solution prepared in 1×TBS / T containing 0.5‰ Tween 20, dilute the secondary antibody Goat-anti-Mouse Antibody at a ratio of 1:5000, incubate for 1h, and then wash the membrane 3 times with 1×TBS / T solution containing 0.5‰ Tween 20, 10min each time.

[0065] The incubated and washed PVDF membrane was added with HRP substrate luminol reagent and HRP peroxide matrix solution (Immobilon Western chemiluminescent HRP substrate) in a ratio of 1:1, and the reaction was carried out in the dark for 5 minutes before development.

[0066] Total proteins extracted from CHX-treated transfected protoplasts were analyzed by immunoblotting, and the results showed that OsRLCK176-HA protein was more susceptible to degradation in oscpk17 protoplasts than in wild-type protoplasts.

[0067] Figure 4In the results, OsRLCK176-HA was expressed in Dongjin (DJ) protoplasts, and there was no significant difference in the stability of OsRLCK176 protein after CHX treatment. OsRLCK176-HA was expressed in oscpk17 protoplasts, and the stability of OsRLCK176 protein was significantly reduced after CHX treatment, indicating that OsCPK17 positively regulates the stability of OsRLCK176 protein. S83D -HA protein, the stability of OsRLCK176 protein was restored, indicating that the S83D phosphorylation mutation enhanced the stability of OsRLCK176-HA protein. GFP as an internal control indicated that the protoplast transformation efficiency was consistent.

[0068] Example 4: Sequence Alignment Analysis of the S83 Site on OsRLCK176

[0069] Phylogenetic tree of homologous proteins of OsRLCK176 in the RLCKVII subfamily constructed in rice, Arabidopsis, pepper and tobacco using MEGA 7.0 ( Figure 5 Left). Multiple sequence alignment of OsRLCK176 homologous proteins from different plant species ( Figure 5 Right). The software performed 500 bootstrap replicates and calculated evolutionary distances using the Poisson correction method.

[0070] Figure 5 It was shown that the highest homology with OsRLCK176 is OsRLCK57, OsRLCK107, and OsRLCK118, and the serine (S) at position 83 is conserved among OsRLCK176, OsRLCK57, OsRLCK107, and OsRLCK118. When S at position 83 is mutated to D, the site becomes a simulated phosphorylation state.

[0071] Example 5: pRLCK176::OsRLCK176 and pRLCK176::OsRLCK176 driven by the promoter of OsRLCK176 in the osrlck176 knockout mutant background S83D Creation and expression detection of rice transgenic materials

[0072] 5.1 OsRLCK176 expression vector and OsRLCK176 S83D Construction of expression vector

[0073] The pRGEcHA vector was constructed, and the primers pRGEcHA-OsRLCK176-F: 5'-CCGGGTGAGCTCGGTACCACAAAAAATATAAAATTCTG-3'; and pRGEcHA-OsRLCK176-R: 5'-GTACATAGCGGCCGCACTAGTAACCAGCCTCGCATTTGCGGG-3'; were used to amplify the OsRLCK176 self-promoter (sequence 5 in the sequence list) and its own sequence (sequence 2 in the sequence list) using the rice genome as a template, and the fragments were connected to the pRGEcHA vector using homologous recombinase (Beijing Novozymes Co., Ltd.) to obtain the OsRLCK176 expression vector.

[0074] OsRLCK176 S83D The point mutation protein expression vector was constructed based on pRGEcHA-OsRLCK176. The plasmid site-directed mutagenesis PCR (Site-Directed Mutagenesis) technology was used for amplification. S83D -F:5'-TGGTGAGGGAGGCTTTTGGAGACGTCTTCAAAGGGTGGATC-3'; and OsRLCK176 S83D -R:5'-GATCCACCCTTTGAAGACGTTCCCAAAGCCTCCCTCACCA-3'.

[0075] Point mutation PCR amplification was performed using pRGEcHA-OsRLCK176 as a template.

[0076] Amplification system: 2×Phanta Max Buffer 12.5μL, 10mM dNTP Mix 1μL, 10μM Forward Primer 0.65μL, 10μM Reverse Primer 0.65μL, DMSO1.5μL, Phanta Max Super-Fidelity DNA Polymerase (1U / μL) 0.5μL, template DNA 50ng, ddH2O to 25μL.

[0077] PCR amplification program: 95°C for 2 min, then 95°C for 30 s, 55°C for 1 min, 68°C at 1 kb / min, for 18 cycles, and stored at 16°C.

[0078] After PCR amplification, 0.5 μL DpnI was added to digest the template DNA at 37°C overnight, and heat-shock transformation was performed to DH5α. The plasmid was extracted and the OsRLCK176 was verified. S83DIn the expression vector, the OsRLCK176 promoter (sequence 5 in the sequence list) drives the single-site mutation OsRLCK176 S83D The gene (nucleotides at positions 247-249 at the 5' end of sequence 2 in the sequence list are TCA, which mutate to GAC), i.e., sequence 6 in the sequence list, encodes the amino acid sequence shown in sequence 7 in the sequence list (i.e., the nucleotide at position 83 of amino acid sequence 4 in the sequence list mutates from serine to aspartic acid).

[0079] 5.2 Transgenic rice pRLCK176::OsRLCK176 and pRLCK176::OsRLCK176 S83D Acquisition

[0080] 5.2.1 Induction of rice callus

[0081] After the mature seeds of the rice osrlck176 mutant were shelled, they were placed in a sterile Erlenmeyer flask and treated with 75% ethanol for 3 minutes. The waste liquid was poured out, and 50% sodium hypochlorite solution was poured in. The seeds were shaken at 150 rpm for 30-40 minutes at room temperature. The waste liquid was then poured out, and sterile water was added to wash them repeatedly for 12 times. The seeds were then spread on filter paper to dry, and then transferred to the induction medium and cultured at 28°C for 4 weeks. Note that all operations must be sterile.

[0082] 5.2.2 Subculture

[0083] Carefully pick the grown rice callus to the subculture medium and culture it at 28°C under light for 1-2 weeks. At this time, the rice callus is golden yellow and granular.

[0084] 5.2.3 Transgenic rice transformation

[0085] 1) Compare the successfully constructed OsRLCK176 with OsRLCK176 S83D The expression vector plasmid was transferred into the Agrobacterium EHA105 strain. The specific transformation method was as follows: 1-2 μL of plasmid was added to 30-50 μL of EHA105 competent cells, placed on ice for 30 min, treated with liquid nitrogen for 1 min, incubated in a 37°C water bath for 90 s, and placed on ice for 3 min. Then, 500 μL of LB medium was added, and the culture was shaken at 28°C and 180 rpm for 3-4 h, then spread on a Rif+ vector-resistant LA plate and cultured at 28°C for 2-3 d.

[0086] 2) Pick a single colony of Agrobacterium grown in LB medium containing the corresponding antibiotics, incubate overnight at 28°C, centrifuge at 4000 rpm for 10 min to collect multiple tubes of bacteria, add 1 mL of AAM (+150 μM As) medium to resuspend the bacteria, then take an appropriate amount of the suspended bacterial solution and add it to 30 mL of AAM (+150 μM As), and adjust the bacterial solution concentration to OD 600=0.1~0.2.

[0087] 3) Pick several dishes of subcultured callus tissue into a sterile conical flask, add the prepared Agrobacterium solution, make sure the callus is completely immersed in the Agrobacterium solution, and shake at 28℃ and 120 rpm for 15 minutes.

[0088] 4) Discard the bacterial solution, spread the callus on sterile filter paper and let it air dry for 30 minutes, then transfer it to the co-culture medium and culture it in the dark at 28°C for 3 days.

[0089] 5.2.4 Screening and culture of resistant calli

[0090] 1) Pick rice callus after dark culture for 3 days and put it into a sterile empty bottle, add sterile water and wash it repeatedly for 10-12 times. Add Timentin (working concentration 400 μg / mL) to the water for the last wash. Then spread the callus on sterile filter paper to dry, transfer it to the selection medium, and culture it at 28°C for 3 weeks.

[0091] 2) Pick the resistant callus grown on the selection medium and culture it on the differentiation medium at 28°C for 4 weeks until seedlings grow.

[0092] 3) The grown seedlings are transferred to a rooting tube containing rooting medium and cultured for 1-2 weeks until roots grow.

[0093] 4) For rice seedlings with healthy root systems, open the cover and add sterile water until the rice seedlings can grow upright (repeatedly change the water during this period), and then transfer to a greenhouse or field. S83D The expression line pRLCK176::OsRLCK176 S83D .

[0094] 5) The seeds of the T1 generation were dehulled and disinfected, and cultured on 1 / 2MS medium containing G418 (50 μg / mL) for 1 week. The rooting and germination rates were counted to determine whether it was a single-copy insertion. At the same time, the rooted and germinated rice leaves were taken, and the protein was extracted for Western Blot detection to determine whether it was expressed.

[0095] pRLCK176::OsRLCK176 and pRLCK176::OsRLCK176 S83D The expressed protein was HA-tagged, and immunoblotting showed that pRLCK176::OsRLCK176 and pRLCK176::OsRLCK176 S83DThe band was detected in the strain, but not in the osrlck176 mutant. Actin indicates that the loading amount of plant total protein extract is consistent ( Figure 7 ). Figure 7 ctrl represents the knockout mutant of osrlck176, in which the expression of HA-tagged protein could not be detected. S83D The expression of HA-tagged protein could be detected in all strains.

[0096] 6) Select T1 seeds with single copy insertion and protein expression for seed propagation, remove the shells and disinfect the harvested T2 seeds, culture them on 1 / 2MS medium containing G418 (50 μg / mL) for 1 week, and then count the rooting and germination rates to determine whether they are homozygous lines. After seed propagation and pure heterozygous identification, the complementary rice homozygous line pRLCK176::OsRLCK176 in which the OsRLCK176 promoter drives the expression of the OsRLCK176 gene in the osrlck176 knockout mutant background was obtained, and the complementary rice homozygous line pRLCK176::OsRLCK176 in which the OsRLCK176 promoter drives the expression of the OsRLCK176 gene in the osrlck176 knockout mutant background was obtained. S83D Gene expression complementation in rice homozygous line pRLCK176::OsRLCK176 S83D .

[0097] Complementation rice homozygous line pRLCK176::OsRLCK176 and complementation rice homozygous line pRLCK176::OsRLCK176 S83D The plasmid sequence was connected to the vector by homologous recombination and verified correct by sequencing.

[0098] The culture medium formula used is as follows:

[0099] 1) Induction medium (1L system): 30g sucrose, 0.1g inositol, 2.8g proline, 0.5g glutamine, 0.3g CEH (NA-Amine A), 20×50mL N6 macro, 100×10mL B5 micro, 1000×1mL B5 micro, 100×10mL B5 vitamin, 100×10mL iron salt, 4mL 4-D (0.5g / L), water to 1L. Adjust pH to 5.8 after constant volume, dispense, add 1.2g plant gum to every 400mL culture medium, and sterilize at 121℃ for 20min.

[0100] 2) Subculture medium (1L system): 30g sucrose, 0.1g inositol, 2.8g proline, 0.5g glutamine, 0.3g CEH (NA-Amine A), 20×50mL N6 macro, 100×10mL B5 micro, 1000×1mL B5 micro, 100×10mL B5 vitamin, 100×10mL iron salt, 4mL 2,4-D (0.5g / L), water to 1L. Adjust pH to 5.8 after constant volume, dispense, add 1.4g plant gum per 400mL culture medium, and sterilize at 121℃ for 20min.

[0101] 3) Select culture medium

[0102] Adding timentin antibiotic (400 μg / mL) and hygromycin (50 μg / mL) to the subculture medium makes the selection medium.

[0103] 4) Co-culture medium (1L system): sucrose 30g, inositol 2g, morpholineethanesulfonic acid 3.9g, CEH (NA-Amine A) 0.5g, N6 macro 20×50mL, B5 micro 100×10mL, B5 micro 1000×1mL, B5 vitamin 100×10mL, iron salt 100×10mL, water is added to 1L.

[0104] After constant volume, adjust the pH to 5.5, dispense, add 1.4 g of plant gum per 400 mL of culture medium, and sterilize at 121°C for 20 min. Add 400 μL of 250 mM acetosyringone to 400 mL of culture medium.

[0105] 5) AAM medium (1 L system): 2.95 g potassium chloride, 0.15 g calcium chloride dihydrate, 0.25 g magnesium sulfate heptahydrate, 0.15 g disodium hydrogen phosphate dihydrate, and water to 1 L. Sterilize at 121°C for 20 min.

[0106] 6) Differentiation medium (1L system): MS (containing vitamins) powder 4.43g, naphthaleneacetic acid (1mg / mL), 100μL, kinetin (1mg / mL) 2.5mL, sucrose 30g, sorbitol 20g, inositol 0.1g, CEH (NA-Aminea) 2g, water to 1L. Adjust pH to 5.8 after constant volume, dispense, add 1.6g of plant gum per 400mL of medium, and sterilize at 121℃ for 20min.

[0107] 7) Rooting medium (1L system): 20g sucrose, 0.1g inositol, 4.43g MS (containing vitamins) powder, water to 1L. Adjust pH to 5.8 after constant volume, dispense, add 1.6g plant gum per 400mL medium, and sterilize at 121℃ for 20min. Note that 400μL hygromycin (50μg / mL) and 200μL timentin (400μg / mL) should be added to 400mL medium when pouring the plate.

[0108] 8) N6 bulk 20×(2L)

[0109] Solution A: 113.2 g potassium nitrate, 18.52 g ammonium sulfate, 16 g potassium dihydrogen phosphate, and make up to 1 L with water.

[0110] Solution B: 7.4 g of magnesium sulfate heptahydrate, make up to 200 mL with water.

[0111] Solution C: 6.64 g calcium chloride dihydrate, make up to 200 mL with water.

[0112] First, slowly mix solution A and solution B, then add solution C, add water to make up to 2 L, and store at 4°C.

[0113] 9) B5 trace 100× (1L): magnesium sulfate monohydrate 781 mg, zinc sulfate heptahydrate 200 mg, boric acid 300 mg, potassium iodide 75 mg.

[0114] Weigh the first two reagents and the last two reagents separately, dissolve them in 400mL ddH2O, slowly mix the two mixed solutions together, add water to make up to 1L, and store at 4℃.

[0115] 10) B5 trace 1000× (1L): 250 mg sodium molybdate dihydrate, 25 mg copper sulfate pentahydrate, 25 mg cobalt chloride hexahydrate, make up to 1L with water. Store at 4°C.

[0116] 11) Vitamin B5 100× (1L): Vitamin B1 (thiamine hydrochloride) 1g, vitamin B6 (pyridoxine hydrochloride) 0.1g, niacin 0.1g, add water to 1L. Store at 4℃.

[0117] 12) Fe salt 100× (1L): Weigh 2.78g of ferrous sulfate heptahydrate and dissolve it in 400mL ddH2O (liquid A), then weigh 2.78g of ethylenediaminetetraacetic acid and dissolve it in 400mL hot water (liquid B). Mix liquid A and liquid B, add nearly 1L of water, and heat the mixture in a microwave oven to boil. The color of the mixture becomes darker, and then cool to room temperature. Make up to volume and place in a brown reagent bottle and store at 4℃.

[0118] 13) 2,4-D (0.5 g / L): Weigh 0.1 g of 2,4-D reagent, add 15 mL of anhydrous ethanol, then add distilled water to make up to 200 mL, and keep at room temperature for use.

[0119] 14) KT (1 mg / mL): Weigh 0.1 g of KT reagent, add a small amount of dilute hydrochloric acid to dissolve it, and then add distilled water to make up to 100 mL.

[0120] 15) NAA (1 mg / mL): Weigh 0.05 g of naphthylacetic acid, first add a small amount of 1 M potassium hydroxide to aid dissolution, then add distilled water to make up to 50 mL.

[0121] Example 6, osrlck176 to pRLCK176::OsRLCK176 and pRLCK176::OsRLCK176 S83D Pathogen inoculation of complementary rice homozygous lines

[0122] To identify osrlck176, transform pRLCK176::OsRLCK176 and pRLCK176::OsRLCK176 S83D To investigate the function of complementary rice homozygous lines in rice disease resistance, we first compared the pRLCK176::OsRLCK176 complementary rice homozygous lines with the pRLCK176::OsRLCK176 S83D The complementary rice homozygous strains were inoculated with the physiological race RS105 of Xanthomonas oryzae pv.oryzicola (Xoc). The experiment was repeated three times and the quantitative results were averaged. In each repetition, at least 10 rice plants were tested. The results are shown in Figure 6 A, The lesion length of pRLCK176::OsRLCK176 complemented rice was significantly longer than that of pRLCK176::OsRLCK176 S83D Complementation of lesion length in rice indicates that OsRLCK176 S83D Gene-expressing plants showed enhanced resistance to bacterial leaf streak disease.

[0123] To identify osrlck176, transform pRLCK176::OsRLCK176 and pRLCK176::OsRLCK176 S83D To investigate the function of complementary rice homozygous lines in rice disease resistance, we investigated the function of complementary rice homozygous lines pRLCK176::OsRLCK176 and pRLCK176::OsRLCK176 S83DThe complementary rice homozygous strains were inoculated with the physiological race PXO99A of Xanthomonas oryzae (Xoo). The experiment was repeated three times and the quantitative results were averaged. In each repetition, at least 10 rice plants were tested. The results are shown in Figure 6 B, The lesion length of pRLCK176::OsRLCK176 complemented rice was significantly longer than that of pRLCK176::OsRLCK176 S83D Complementation of lesion length in rice indicates that OsRLCK176 S83D Gene-expressing plants have enhanced resistance to bacterial blight ( Figure 6 ).

[0124] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0125] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of single-site mutation of disease resistance-related protein OsRLCK176 in improving rice disease resistance; The amino acid sequence of the disease resistance-related protein OsRLCK176 is shown in SEQ ID NO.4, and the single point mutation is that the 83rd amino acid at the amino terminal of the disease resistance-related protein OsRLCK176 mutates from S to D; the disease resistance is resistance to bacterial leaf streak and / or resistance to bacterial blight.

2. A method for improving the disease resistance of rice is to mutate the 83rd amino acid at the amino terminus of the disease resistance-related protein OsRLCK176 in rice from S to D, wherein: The amino acid sequence of the disease resistance-related protein OsRLCK176 is shown in SEQ ID NO.4; the disease resistance is resistance to bacterial leaf streak disease and / or resistance to bacterial leaf blight disease.

3. An OsRLCK176 single-site mutant protein for improving rice disease resistance, characterized in that: The amino acid sequence of the OsRLCK176 single-site mutant protein is shown in SEQ ID NO.7; the disease resistance is resistance to bacterial leaf streak and / or resistance to bacterial blight.

4. The gene encoding the OsRLCK176 single-site mutant protein for improving rice disease resistance according to claim 3; the nucleotide sequence of the encoding gene is shown as SEQ ID NO.

6.

5. Use of the OsRLCK176 single-site mutant protein according to claim 3 or the encoding gene according to claim 4 in improving rice disease resistance; the disease resistance is resistance to bacterial leaf streak disease and / or resistance to bacterial leaf blight disease.

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