Application of Rice OsSOG1 Protein and Its Encoding Gene in Regulating Plant Disease Resistance

Abstract

The present invention discloses the use of a rice OsSOG1 protein and its encoding gene for regulating plant disease resistance. Specifically, site-directed knockout of the rice OsSOG1 protein-encoding gene LOC4340715 significantly reduces rice blast resistance, while overexpression of LOC4340715 significantly enhances blast resistance. This invention provides a new gene and application method for developing plant breeding based on the disease resistance function of OsSOG1 protein, and has great application value in agricultural production.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering and relates to the application of rice OsSOG1 protein and its encoding gene, and specifically to the application of rice OsSOG1 protein and its encoding gene LOC4340715 in regulating plant disease resistance and the application of gene editing methods in creating disease-resistant materials in rice varieties. Background Art

[0002] Rice blast, caused by the rice blast fungus (Magnaporthe oryzae), is a highly regarded fungal disease in rice production, posing a devastating threat to rice yields and, in severe cases, causing total crop failure. Planting resistant rice varieties is the most economical, effective, and environmentally friendly approach to controlling rice blast. However, with global warming, rice blast is becoming increasingly common in rice-growing areas, and the resistance cycle of most resistant varieties promoted in agricultural production is correspondingly shortened. Therefore, enhancing durable resistance to rice blast has become a priority for improving rice resistance.

[0003] Production practice has shown that conventional genetic breeding techniques based on major-effect resistance genes are difficult to effectively construct durable resistance to rice blast in rice varieties. Basic biological research has shown that durable resistance to rice blast is closely related to quantitative resistance composed of multiple genes. Therefore, rapidly and extensively discovering and cloning minor-effect resistance genes to rice blast is a key goal in solving the problem of durable disease resistance in rice.

[0004] Gene editing technology is widely used in plant functional genomics research due to its high efficiency, rapidity, and ability to generate site-directed mutagenesis. This study screened a new rice blast-resistance gene through gene editing, which can inhibit the growth and reproduction of the rice blast fungus in infected rice leaves. Summary of the Invention

[0005] The technical problem to be solved by the present invention is the regulation and application of plant disease resistance genes. The purpose is to provide the application of rice OsSOG1 protein and its encoding gene LOC4340715 in regulating plant disease resistance.

[0006] The present invention provides a protein derived from rice (Oryzae sativa L.), named OsSOG1 protein, which is as follows (a1) or (a2) or (a3) ​​or (a4):

[0007] (a1) the protein represented by SEQ ID NO: 1;

[0008] (a2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of (a1);

[0009] (a3) a protein derived from rice that has 98% or greater identity with (a1) and functions to regulate plant blast resistance;

[0010] (a4) A protein having the function of regulating plant blast resistance by replacing and / or deleting and / or adding one or more amino acid residues in (a1). The specific tags are shown in Table 1.

[0011] Table 1 Tag sequences

[0012] Label residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL HA 9 YPYDVPDYA

[0013] The present invention also provides applications of the above protein, which are any of the following:

[0014] D1) Application in regulating (increasing or decreasing) plant disease resistance;

[0015] D2) Use in the preparation of products for regulating (increasing or decreasing) plant disease resistance;

[0016] D3) Application in breeding disease-resistant plants;

[0017] D4) Use in the preparation of products for breeding disease-resistant plants.

[0018] Specifically, in a first aspect, the present invention provides an application of the above protein, wherein the disease resistance is preferably a plant's resistance to rice blast fungus.

[0019] In a second aspect, the present invention provides a use of the protein-related biomaterial, wherein the biomaterial is any one of the following B1) to B4):

[0020] B1) a nucleic acid molecule encoding the protein according to claim 1;

[0021] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0022] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0023] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing

[0024] B3) a recombinant microorganism containing the recombinant vector.

[0025] Preferably, the nucleic acid molecule is the following b1) or b2):

[0026] b1) the coding sequence is the DNA molecule shown in SEQ ID No. 2;

[0027] b2) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 2.

[0028] More specifically, the present invention provides the use of the rice LOC4340715 gene, its encoded protein or an inhibitor of the rice LOC4340715 gene or a nucleic acid molecule encoding the OsSOG1 protein in genetic breeding for improving plant disease resistance.

[0029] Preferably, the disease resistance of the present invention is the plant's resistance to rice blast fungus.

[0030] Preferably, the genetic breeding is to construct transgenic plants resistant to rice blast.

[0031] Preferably, in the above application, the disease resistance of the plant is improved by enhancing the biological function of the rice OsSOG1 protein.

[0032] The improvement in disease resistance can be manifested as a reduction in the area of ​​rice blast lesions.

[0033] In the present invention, the protein encoded by the rice LOC4340715 gene has any of the following amino acid sequences:

[0034] (1) the amino acid sequence shown in SEQ ID NO. 1;

[0035] (2) an amino acid sequence of a protein having the same function as the amino acid sequence shown in SEQ ID NO. 1 obtained by replacing, inserting or deleting one or more amino acids;

[0036] (3) An amino acid sequence having at least 80% homology to the amino acid sequence shown in SEQ ID NO. 1; preferably, the homology is at least 90%; more preferably, 95%.

[0037] In the present invention, the rice LOC4340715 gene has any one of the following nucleotide sequences:

[0038] (1) the nucleotide sequence shown in SEQ ID NO. 2;

[0039] (2) A nucleotide sequence encoding a protein with the same function obtained by replacing, inserting or deleting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO. 2.

[0040] The amino acid sequence shown in SEQ ID NO. 1 is the protein sequence of rice OsSOG1. Those skilled in the art can, based on the amino acid sequence disclosed in the present invention and conventional techniques in the art such as conservative substitution of amino acids, replace, delete and / or add one or more amino acids without affecting its activity to obtain mutants having the same activity as the rice OsSOG1 protein disclosed in the present invention.

[0041] The nucleotide sequence shown in SEQ ID NO. 2 is the protein coding sequence of the rice LOC4340715 gene. The rice LOC4340715 gene described herein can be any nucleotide sequence capable of encoding the rice OsSOG1 protein. Taking into account codon degeneracy and codon preferences across species, those skilled in the art can utilize codons appropriate for expression in a particular species as needed.

[0042] In the present invention, the inhibitory factor of the rice LOC4340715 gene is a nucleic acid capable of destroying the biological function of the rice OsSOG1 protein.

[0043] Preferably, the nucleic acid is gRNA or interfering RNA.

[0044] Preferably, the target sequence of the gRNA is a nucleotide sequence of the form XXXNGG in the rice LOC4340715 gene, wherein N is any one base of A, T, G, or C, and XXX is a nucleic acid sequence of 19-20 bp.

[0045] More preferably, the target sequence of the gRNA is from position 2598 to position 2617 of the rice LOC4340715 gene.

[0046] In the present invention, the nucleic acid molecule containing the protein encoded by the LOC4340715 gene is a nucleotide sequence capable of encoding the OsSOG1 protein.

[0047] Preferably, the nucleic acid sequence is CDS.

[0048] Preferably, the CDS is the nucleotide sequence of the exon of the rice LOC4340715 gene.

[0049] More preferably, the CDS is at positions 177-184, 1909-2050, 2433-2619, 3073-3554, 3724-4104 and 4185-4241 of the rice LOC4340715 gene.

[0050] The above-mentioned rice LOC4340715 gene, its encoded protein, rice LOC4340715 gene inhibitor or nucleic acid molecule containing the protein encoded by the LOC4340715 gene can be used in the form of the rice LOC4340715 gene, its encoded protein, rice LOC4340715 gene inhibitor or nucleic acid molecule containing the protein encoded by the LOC4340715 gene itself, or in the form of an expression cassette, vector containing the rice LOC4340715 gene or its inhibitor, or a host cell containing the expression cassette or vector.

[0051] In a third aspect, the present invention provides a gRNA for editing the rice LOC4340715 gene, wherein the target sequence of the gRNA is from position 2598 to position 2617 of the rice LOC4340715 gene.

[0052] Preferably, the gRNA comprises the sequence shown in SEQ ID NO.4.

[0053] The above-mentioned gRNA can work in conjunction with the CRISRP / Cas9 gene editing tool to edit the rice LOC4340715 gene and destroy the biological function of the rice OsSOG1 protein.

[0054] The above-mentioned gRNA was obtained through large-scale screening in the present invention. Experiments in the present invention have demonstrated that CRISRP / Cas9-mediated gene editing using the above-mentioned gRNA can efficiently obtain rice plants edited with the LOC4340715 gene, and that insertions or deletions at positions 2598 to 2617 of the sequence shown in SEQ ID NO. 3 will disrupt the biological function of the rice LOC4340715 gene, resulting in a reduced level of resistance to rice blast.

[0055] In a fourth aspect, the present invention also provides a biological material comprising the gRNA for editing the rice LOC4340715 gene, wherein the biological material includes an expression cassette, a vector, a host cell, an engineered bacterium or a transgenic plant cell line.

[0056] In a fifth aspect, the present invention provides a method for regulating plant blast resistance or cultivating transgenic plants, comprising: regulating the biological function of a rice OsSOG1 protein in a plant; the rice OsSOG1 protein having any of the following amino acid sequences:

[0057] (1) the amino acid sequence shown in SEQ ID NO. 1;

[0058] (2) an amino acid sequence of a protein having the same function as the amino acid sequence shown in SEQ ID NO. 1 obtained by replacing, inserting or deleting one or more amino acids;

[0059] (3) An amino acid sequence having at least 80% homology to the amino acid sequence shown in SEQ ID NO. 1; preferably, the homology is at least 90%; more preferably, 95%.

[0060] Preferably, the rice blast resistance of the plant is increased or decreased by enhancing or inhibiting the biological function of the rice OsSOG1 protein.

[0061] The above-mentioned enhancement of the biological function of rice OsSOG1 protein can be achieved by conventional technical means in the art.

[0062] Preferably, the PMDC43 plant binary expression system can be used to enhance the biological function of the rice OsSOG1 protein; the PMDC43 plant binary expression system can be a pMDC43-LOC4340715 recombinant vector, which contains the CDS sequence of the LOC4340715 gene shown in SEQ ID NO.2 in the sequence listing and a 35S promoter, and can express a fusion protein of GFP and OsSOG1 shown in SEQ ID NO.1, and the expression of the fusion protein is driven by the 35S promoter.

[0063] The above-mentioned inhibition of the biological function of rice OsSOG1 protein can be achieved by conventional technical means in the art.

[0064] Preferably, the biological function of the rice OsSOG1 protein is destroyed by using the CRISRP / Cas9 system; in the CRISRP / Cas9 system, the target sequence of the gRNA is from position 2598 to position 2617 of the rice LOC4340715 gene.

[0065] More preferably, the gRNA comprises the sequence shown in SEQ ID NO.4.

[0066] The CRISPR / Cas9 system can be used to cut the XXXNGG nucleotide sequence in the rice LOC4340715 gene 3bp upstream of NGG to produce blunt ends, thereby causing premature translation termination of the gene or protein conformation changes, thereby destroying the biological function of the gene-encoded protein; where N is any base among A, T, G, and C, and XXX is a 19-20bp nucleic acid sequence.

[0067] As a preferred embodiment of the present invention, the method for enhancing plant resistance to rice blast or cultivating transgenic plants comprises the following steps:

[0068] (1) transferring the gene shown in SEQ ID NO. 2 into a plant binary expression vector plasmid to obtain a recombinant expression vector expressing the gene shown in SEQ ID NO. 2;

[0069] (2) Transforming the recombinant expression vector constructed in step (1) into rice;

[0070] (3) Transgenic rice with enhanced resistance to rice blast was obtained through screening and identification.

[0071] Preferably, in the above step (1), the plant binary expression vector system can be a PMDC43 plant binary expression vector plasmid;

[0072] Preferably, in the above step (2), the recombinant expression vector constructed in step (1) is transformed into a rice variety by means of Agrobacterium tumefaciens-mediated method.

[0073] Preferably, in the above step (3), the screening method is specifically: using primers for the backbone vector sequence of the PMDC43 plant binary expression vector plasmid to perform PCR amplification. If a target band of 1504 bp is amplified, it indicates that the constructed recombinant expression vector has been successfully transformed into the plant, that is, a rice plant overexpressing the gene shown in SEQ ID NO.2 is obtained.

[0074] Preferably, in the above step (3), the identification method is specifically: using specific primers for the LOC4340715 gene to detect the transcriptional expression level of the LOC4340715 gene; if the transcription abundance of the nucleotide sequence shown between SEQ ID NO. 17 and SEQ ID NO. 18 increases, resulting in overexpression of the LOC4340715 gene, it indicates that the biological function of the LOC4340715 gene in the plant is enhanced.

[0075] By enhancing the biological function of the LOC4340715 gene in rice, the resistance of rice to rice blast was improved.

[0076] As another preferred embodiment of the present invention, the method for inhibiting plant blast resistance or cultivating transgenic plants comprises the following steps:

[0077] (1) Constructing a CRISRP / Cas9 gene editing plasmid containing the gRNA shown in SEQ ID NO.4;

[0078] (2) Transforming the CRISRP / Cas9 gene editing plasmid constructed in step (1) into rice;

[0079] (3) Rice blast-resistant materials were obtained through screening and identification.

[0080] Preferably, in the above step (1), the CRISPR / Cas9 gene editing plasmid is a class II CRISPR / Cas vector system;

[0081] Preferably, in the above step (2), the CRISRP / Cas9 gene editing plasmid constructed in step (1) is introduced into the rice variety by Agrobacterium tumefaciens-mediated method.

[0082] Preferably, in the above step (3), the screening method is specifically: using primers targeting the backbone vector sequence of the CRISRP / Cas9 gene editing plasmid to perform PCR amplification. If a target band of 397 bp is amplified, it indicates that the CRISRP / Cas9 gene editing plasmid has been successfully transformed into the plant.

[0083] Preferably, in the above step (3), the identification method is specifically: using specific primers of the LOC4340715 gene to amplify the LOC4340715 gene fragment in the genome of the transformed plant and sequence it; if the nucleic acid sequence from position 2598 to position 2617 of the sequence shown in SEQ ID NO.3 undergoes an insertion or deletion mutation, it indicates that the biological function of the LOC4340715 gene in the plant is damaged.

[0084] In the present invention, the plant may be a monocotyledonous plant or a dicotyledonous plant, preferably a recipient plant for the rice blast fungus, including but not limited to rice.

[0085] The beneficial effects of the present invention are:

[0086] (1) The present invention discovered that the rice gene LOC4340715 and the protein it encodes, OsSOG1, participate in regulating the rice immune response to the rice blast fungus. By enhancing the biological function of the OsSOG1 protein, the rice's resistance to the blast fungus can be significantly improved. Experimental verification showed that the lesion area of ​​rice plants overexpressing the LOC4340715 gene decreased by 26.4% to 28.3% after infection with the blast fungus, demonstrating that enhancing the biological function of the OsSOG1 protein increases the rice material's resistance to blast.

[0087] (2) The present invention utilizes CRISPR / Cas9 technology to perform genome-targeted modification of the LOC4340715 gene, achieving efficient site-specific editing of LOC4340715. The present invention discovered that using the nucleotide sequence from positions 2598 to 2617 in the rice LOC4340715 gene as the target sequence can efficiently achieve site-specific editing of LOC4340715, and that insertions or deletions from positions 2598 to 2617 in the sequence shown in SEQ ID NO. 3 will disrupt the biological function of the rice LOsSOG1 protein, causing the rice to exhibit a reduced level of resistance to rice blast. Experimental verification showed that after the LOC4340715 gene-site-edited rice plants were infected with the rice blast fungus, the lesion area increased by 22.0% to 34.8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0089] Figure 1 The mutation type of the nucleotide sequence and encoded amino acid sequence of the LOC4340715 gene in the ossog1-edited plant in the rice Nipponbare background in Example 3 of the present invention; wherein, Figure 1 A in the middle is the mutation type of the nucleotide sequence of the LOC4340715 gene in the ossog1-edited plant; Figure 1 Figure B shows the mutation type of the amino acid sequence encoded by the LOC4340715 gene in the ossog1-edited plant.

[0090] Figure 2 The identification and lesion phenotype of ossog1-edited plants and OsSOG1 OE-overexpressing plants in the rice Nipponbare background in Example 3 of the present invention. Figure 2 A in the middle shows the expression level of LOC4340715 gene in ossog1-edited plants and OsSOG1 OE-overexpressing plants; Figure 2 Middle B shows the lesion phenotype of ossog1-edited plants and OsSOG1 OE-overexpressing plants after inoculation with rice blast fungus RB22; Figure 2 Middle C shows the statistical comparison of lesion area between ossog1-edited plants and OsSOG1 OE-overexpressing plants after inoculation with rice blast fungus RB22; Figure 2 D shows the statistical comparison of the growth of rice blast fungus RB22 in infected leaves of ossog1-edited plants and OsSOG1OE-overexpressing plants. DETAILED DESCRIPTION

[0091] The present invention will be further described below in conjunction with specific examples. These examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. In the following examples, all experimental methods, unless otherwise specified, are conventional methods.

[0092] Nipponbare (Oryza sativa ssp. Japonica): recorded in “Yongqing Jiao, Yonghong Wang, Dawei Xue, Jing Wang, Meixian Yan, Guifu Liu, Guojun Dong, Dali Zeng, Zefu Lu, Xudong Zhu, Qian Qian and Jiayang Li. Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice. Nature Genetics, 2010, 42, 541-544”.

[0093] Rice blast fungus strain RB22: described in “Chan Ho Park, Gautam Shirsekar, Maria Bellizzi, Songbiao Chen, Pattavipha Songkumarn, Xin Xie, Xuetao Shi, Yuese Ning, BoZhou, Pavinee Suttiviriya, Mo Wang, Kenji Umemura, Guo-Liang Wang. The E3 LigaseAPIP10 Connects the Effector AvrPiz-t to the NLR Receptor Piz-t in Rice. PLoS Pathogens, 2016, 12(3): e1005529”.

[0094] Example 1: Site-directed editing of the rice OsSOG1 protein-encoding gene LOC4340715 based on the CRISPR / Cas9 system Editing Method

[0095] 1. Sequence analysis and target sequence screening of the rice OsSOG1 protein-encoding gene LOC4340715

[0096] The genomic sequence of the rice OsSOG1 protein gene LOC4340715 is shown in SEQ ID NO.3, the coding sequence (CDS) is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein OsSOG1 is shown in SEQ ID NO.1. Sequence analysis showed that the gene includes six exons, located at positions 177-184, 1909-2050, 2433-2619, 3073-3554, 3724-4104, and 4185-4241 of SEQ ID NO.3, respectively.

[0097] According to sequence analysis, the sequence on the third exon of the rice OsSOG1 protein-coding gene LOC4340715 (positions 2598-2617 of SEQ ID NO. 3) was used as the LOC4340715-T target sequence for the site-directed editing method of the rice OsSOG1 protein-coding gene LOC4340715 based on CRISPR / Cas9 technology.

[0098] After extensive screening, the present invention determined that the CRISPR / Cas9 technology was used to target the positive strand of the third exon of the rice LOC4340715 gene, and positions 2598 to 2617 of SEQ ID NO.3 were used as the LOC4340715-T target sequence. The LOC4340715-T target sequence is shown in SEQ ID NO.4.

[0099] 2. CRISPR / Cas9 vector primer design and construction of recombinant expression vector

[0100] (1) Design and synthesis of CRISPR / Cas9 target sequence primers

[0101] Primers targeting the LOC4340715-T target sequence of the LOC4340715 gene were designed based on CRISPR / Cas9 technology. The sequences of the LOC4340715-T target sequence primers LOC4340715-TF and LOC4340715-TR are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

[0102] SEQ ID NO.5: LOC4340715-TF: 5'-GGCAACCCATCCACAGAAACTTCC-3';

[0103] SEQ ID NO. 6: LOC4340715-TR: 5'-AAACGGAAGTTTCTGTGGATGGGT-3'.

[0104] Primers LOC4340715-TF and LOC4340715-TR were synthesized respectively.

[0105] (2) Construction of CRISPR / Cas9 technology recombinant expression vector

[0106] The LOC4340715-T target sequence primers LOC4340715-TF and LOC4340715-TR were synthesized into a double-stranded target sequence by primer annealing method, and then inserted into the downstream of the rice U3 promoter of the CRISPR / Cas9 vector (the CRISPR / Cas9 vector is also referred to as pHUN4C12 in the following literature: Xu, Rongfang., Li, Hao., Qin, Ruiying., Wang, Lu., Li, Li., Wei, Pengcheng., and Yang, Jianbo. (2014). Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice. Rice 7, 5-9, publicly available from China Agricultural University) to obtain the Cas9-LOC4340715 recombinant expression vector; sequencing confirmed that the rice U3 promoter downstream of the Cas9-LOC4340715 recombinant expression vector was inserted as shown in SEQ ID The sequence shown in NO.4.

[0107] 3. Obtaining recombinant Agrobacterium tumefaciens

[0108] The recombinant expression vector Cas9-LOC4340715 constructed in 2 above was heat-shock transformed into Agrobacterium EHA105 (BioVector NTCC Type Culture Collection, commercially available) to obtain recombinant Agrobacterium containing the recombinant expression vector Cas9-LOC4340715, which was named EHA105-Cas9-LOC4340715.

[0109] Example 2: Overexpression of rice OsSOG1 protein encoding gene LOC4340715

[0110] 1. Cloning of the nucleotide sequence encoding the protein of the LOC4340715 gene

[0111] (1) Design and synthesis of amplification primers containing the nucleotide sequence of the protein encoded by the LOC4340715 gene

[0112] Cloning primers containing the CDS of the LOC4340715 gene were designed. The sequences of LOC4340715-cloning-F and LOC4340715-cloning-R are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively.

[0113] SEQ ID NO.7: LOC4340715-cloning-F: 5'-CCCTCGTCGGATCCCTAGTA-3';

[0114] SEQ ID NO. 8: LOC4340715-cloning-R: 5'-CTCAGCCCTGCTTTACCACA-3'.

[0115] Primers LOC4340715-cloning-F and LOC4340715-cloning-R were synthesized respectively.

[0116] (2) Design and synthesis of cloning primers for the nucleotide sequence encoding the protein of the LOC4340715 gene

[0117] The cloning and overexpression primers for the CDS of the LOC4340715 gene were designed. The sequences of LOC4340715-OE-F and LOC4340715-OE-R are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively.

[0118] SEQ ID NO.9: LOC4340715-OE-F: 5'-tgaactatacaaaggcgcgccaATGACCGGGACATCCTGGAT-3';

[0119] SEQ ID NO. 10: LOC4340715-OE-R: 5'-ctctagaactagttaattaaTCAATCGATCACCTTGCCAC-3'.

[0120] Primers LOC4340715-OE-F and LOC4340715-OE-R were synthesized respectively.

[0121] (3) Cloning of the nucleotide sequence encoding the protein of the LOC4340715 gene

[0122] Total RNA was extracted from seedlings of the rice variety Nipponbare and reverse transcribed to obtain cDNA. PCR amplification was performed using the cDNA as a template with forward primer LOC4340715-cloning-F and reverse primer LOC4340715-cloning-R to obtain a primary amplification product. PCR amplification was also performed using the primary amplification product as a template with forward primer LOC4340715-OE-F and reverse primer LOC4340715-OE-R to obtain a secondary amplification product. The secondary amplification product was identified by band size and recovered by gel purification, and the recovered product was named OE-LOC4340715.

[0123] 2. Construction of recombinant expression vector for LOC4340715 gene overexpression

[0124] The recovered product, OE-LOC4340715, was homologously recombined with the destination vector pMDC43 (Mark Curtis & Ueli Grossniklaus. A Gateway cloning vector set for high-throughput functional analysis of genes in plants. Plant Physiology, 2003, 133, 462-469, publicly available from China Agricultural University) treated with Pac I and Asc I. The resulting recombinant vector, named pMDC43-LOC4340715, contained the correct CDS nucleotide sequence of SEQ ID NO. 2. The homologous recombination reaction system consisted of 3.75 μL (50-100 ng) of the Pac I and Asc I-treated vector pMDC43, 1.25 μL (10-30 ng) of OE-LOC4340715, and 5 μL of 2× Seamless Cloning Mix.

[0125] Homologous recombination reaction conditions: Incubate at 37°C for 30 minutes. The reaction system was transformed into Escherichia coli DH5α, and positive clones were screened. An expression vector containing the correct OE-LOC4340715 nucleotide sequence was obtained and named pMDC43-LOC4340715. pMDC43-LOC4340715 contains the CDS sequence of the LOC4340715 gene as shown in SEQ ID NO. 2 in the sequence listing and the 35S promoter. It can express the GFP and OsSOG1 fusion protein as shown in SEQ ID NO. 1, and expression of this fusion protein is driven by the 35S promoter.

[0126] 3. Obtaining recombinant Agrobacterium tumefaciens

[0127] The recombinant expression vector pMDC43-LOC4340715 constructed in the above 2 was heat-shock transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium containing the recombinant expression vector pMDC43-LOC4340715, which was named EHA105-pMDC43-LOC4340715.

[0128] Example 3: CRISPR / Cas9-based site-directed editing or transgenic overexpression in rice varieties Application

[0129] Recombinant Agrobacterium EHA105-Cas9-LOC4340715 or EHA105-pMDC43-LOC4340715 was used to infect callus tissue induced from mature embryos of the rice variety Nipponbare. The resulting transformed rice plants were named ossog1 and OsSOG1OE, respectively. The specific experimental methods are as follows:

[0130] 1. The recombinant Agrobacterium EHA105-Cas9-LOC4340715 and EHA105-pMDC43-LOC4340715 obtained in Examples 1 and 2 were inoculated into YEB liquid medium (containing 50 μg / ml kanamycin and 20 μg / ml rifampicin), and cultured with shaking at 28°C and 200 rpm until the OD600nm was 0.6-0.8; centrifuged at 5000 rpm and 4°C for 5 min, and the bacterial pellet was resuspended in AAM liquid medium (acetosyringone concentration was 200 μM, pH 5.2) to a concentration of OD 600 is 0.6-0.8, and two recombinant Agrobacterium resuspensions are obtained.

[0131] 2. Remove the husks from mature seeds of the rice variety Nipponbare. Soak in 75% ethanol for 1 minute, then sterilize in a 3.3% NaClO solution with shaking for 20 minutes, repeating twice. Rinse several times with sterile water until odor-free. Inoculate the sterilized seeds on NBD2 medium to induce callus. Incubate in the dark at 26°C for 8-10 days. Remove the roots and residual endosperm, then subculture for 10 days to obtain embryonic callus.

[0132] 3. Immerse the embryonic callus obtained in step 2 in the recombinant Agrobacterium resuspension obtained in step 1. Remove the embryonic callus group after 20-30 minutes and inoculate it on a co-cultivation medium (acetosyringone concentration of 100 μM, pH 5.2) containing two layers of filter paper. Co-cultivate at 26°C in the dark for 2 days.

[0133] 4. Inoculate the callus co-cultivated in step 3 into a screening medium (hygromycin concentration of 50 mg / L, pH 5.8), culture for 14 days in the dark at 28°C, and transfer the resistant callus to a selection medium containing 50 mg / L Hyg for further screening.

[0134] After two rounds of screening, resistant calli were transferred to differentiation medium (24 hours of light per day) for differentiation induction. Once new rootless seedlings were generated, the regenerated seedlings were transferred to 1 / 2 MS medium for rooting induction. Once the seedlings were robust, they were transferred to a nutrient solution culture chamber. Ossog1 regenerated plants (transfected with recombinant Agrobacterium tumefaciens EHA105-Cas9-LOC4340715) and OsSOG1 OE regenerated plants (transfected with recombinant Agrobacterium tumefaciens EHA105-pMDC43-LOC4340715 in the rice strain Nipponbare) were obtained.

[0135] 6. After the regenerated plants survived transplantation, total DNA was extracted from the leaves of the regenerated plants. PCR amplification was performed using primers U3-F (sequence shown in SEQ ID NO. 11) and 4C12-R (sequence shown in SEQ ID NO. 12) based on the Cas9-LOC4340715 recombinant expression vector, and primers pMDC43-F (sequence shown in SEQ ID NO. 13) and NOS-R (sequence shown in SEQ ID NO. 14) based on pMDC43-LOC4340715. The amplified product fragments were 397 bp and 1504 bp, respectively, to screen for positively transformed plants. The number of regenerated plants tested, the number of positively transformed plants, and the percentage of positively transformed plants to the total number of regenerated plants tested (i.e., the positive rate (%)) were calculated. The results are shown in Table 1.

[0136] Table 1. Positive rate test results of LOC4340715 gene-transformed rice varieties

[0137] Regenerated plants Number of regenerated plants Number of positive transformed plants Positive rate (%) ossog1 27 27 100.0 OsSOG1 OE 23 23 100.0

[0138] 7. Using the genome of the obtained ossog1 regenerated plant as a template, PCR amplification was performed using the specific primers LOC4340715-Dection-F (sequence shown in SEQ ID NO. 15) and LOC4340715-Dection-R (sequence shown in SEQ ID NO. 16) for the rice OsSOG1 protein encoding gene LOC4340715. The resulting 201 bp amplification product was sequenced for verification. The sequencing verification results showed that among the 27 positive transformed plants, 7 transformed plants had gene editing in the LOC4340715 gene (referred to as ossog1-edited plants). The number of positive transformed plants tested, the number of edited transformed plants, and the percentage of edited transformed plants to the number of regenerated plants tested, i.e., the editing efficiency (%), were statistically analyzed. The results are shown in Table 2.

[0139] Table 2. Detection results of Cas9-LOC4340715-induced mutations in the rice LOC4340715 gene

[0140] Regenerated plants Number of regenerated plants Number of plants transformed with mutation Mutation efficiency (%) ossog1 27 7 25.9

[0141] 8. Seeds of ossog1-edited plants were collected and homozygous edited plants were screened by self-segregation. Two homozygous edited types (ossog1-1d and ossog1-27d) were obtained after screening. Their nucleotide and amino acid sequences are shown in Figure 2. Figure 1 shown.

[0142] 9. Total RNA was extracted from leaves of wild-type Nipponbare, ossog1-1d, and ossog1-27d plants and reverse transcribed. The expression level of the LOC4340715 gene was detected using a primer pair consisting of the forward primer LOC4340715-qRTF (sequence shown in SEQ ID NO.17): 5'-ACCAAAGAAGTGACCGCGAA-3' and the reverse primer LOC4340715-qRTR (sequence shown in SEQ ID NO.18): 5'-AAGCAGTCTTCCAACCACCG-3'. The internal control used was Actin, the forward primer Actin-F (sequence shown in SEQ ID NO.19): 5'-CCTGACGGAGCGTGGTTAC-3' and the reverse primer Actin-R (sequence shown in SEQ ID NO.20): 5'-CCAGGGCGATGTAGGAAAGC-3'. The results are shown in Figure 2. Figure 2 As shown, the expression levels of the LOC4340715 gene in ossog1-1d and ossog1-27d plants were significantly downregulated compared with the wild type Nipponbare (NIP), and the expression levels were approximately 0.15 and 0.28 times that of the wild type, respectively.

[0143] 10. Total RNA was extracted from leaves of wild-type Nipponbare and OsSOG1 OE regenerated plants, and reverse transcribed. The expression level of the LOC4340715 gene was detected using a primer pair consisting of the forward primer LOC4340715-qRTF (sequence shown in SEQ ID NO.17): 5'-ACCAAAGAAGTGACCGCGAA-3' and the reverse primer LOC4340715-qRTR (sequence shown in SEQ ID NO.18): 5'-AAGCAGTCTTCCAACCACCG-3'. The internal control used was Actin, the forward primer Actin-F (sequence shown in SEQ ID NO.19): 5'-CCTGACGGAGCGTGGTTAC-3' and the reverse primer Actin-R (sequence shown in SEQ ID NO.20): 5'-CCAGGGCGATGTAGGAAAGC-3'. Figure 2As shown in the figure, the expression levels of LOC4340715 gene in OsSOG1 OE-13 and OsSOG1 OE-17 were significantly upregulated compared with the wild type Nipponbare (NIP), and the expression levels were 6.62 and 12.01 times that of the wild type, respectively.

[0144] 11. The wild-type Nipponbare, ossog1-1d, ossog1-27d, OsSOG1 OE-13, and OsSOG1 OE-17 plants were inoculated with the rice blast fungus RB22, and the resistance of the LOC4340715 gene to the rice blast fungus was evaluated. The lesion data of at least 14 independent plants were collected for each line, and the results were as follows: Figure 2 As shown in the results, compared with the wild type (Nipponbare), both ossog1-1d and ossog1-27d showed a decreased resistance to rice blast, with the lesion area caused by the rice blast fungus RB22 increasing by more than 22.0%. Both OsSOG1 OE-13 and OsSOG1 OE-17 plants showed an increased resistance to rice blast, with the lesion length caused by the rice blast fungus RB22 decreasing by more than 26.4%. These results indicate that the rice OsSOG1 protein-encoding gene LOC4340715 positively regulates rice resistance to the rice blast fungus RB22.

[0145] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. The use of protein, characterized in that The application is any of the following: D1) Application in breeding disease-resistant plants; D2) Use in the preparation of products for breeding disease-resistant plants; The protein is the following A1) or A2): A1) a protein having an amino acid sequence of SEQ ID No. 1; A2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1); The disease resistance is resistance to rice blast, the disease-resistant plant is a plant resistant to rice blast, and the plant is rice.

2. Use of a biomaterial related to the protein of claim 1, characterized in that: The application is any of the following A sort of: D1) Application in breeding disease-resistant plants; D2) Use in the preparation of products for breeding disease-resistant plants; The disease resistance is resistance to rice blast, the disease-resistant plant is a plant resistant to rice blast, and the plant is rice; The biological material is any one of the following B1) to B4): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing B3) a recombinant microorganism containing the recombinant vector.

3. The use according to claim 2, characterized in that The nucleic acid molecule is the following b1) or b2): b1) the coding sequence is the DNA molecule shown in SEQ ID No. 2; b2) The nucleotide sequence is the DNA molecule shown in SEQ ID No.

3.

4. A method for cultivating disease-resistant plants, characterized in that: The method comprises enhancing the content and / or activity of the protein described in claim 1 in a target plant to obtain a disease-resistant plant having higher disease resistance than the target plant; the disease resistance is resistance to rice blast, the disease-resistant plant is a plant resistant to rice blast, and the plant is rice; the enhancing the content and / or activity of the protein described in claim 1 in the target plant is achieved by increasing the expression level of the gene encoding the protein in the target plant.

5. The method according to claim 4, characterized in that The protein encoding gene is as follows b1) or b2): b1) the coding sequence is the DNA molecule shown in SEQ ID No. 2; b2) The nucleotide sequence is the DNA molecule shown in SEQ ID No.

3.

6. Use of the method according to claim 4 or 5 in creating blast-resistant rice.

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