Application of rice OsSOG1 protein and coding gene thereof in regulation and control of plant disease resistance

Abstract

The invention discloses an application of a rice OsSOG1 protein and a coding gene thereof in regulation and control of plant disease resistance. The application is specifically as follows: the rice OsSOG1 protein coding gene LOC4340715 is knocked out at a fixed point to obviously reduce the rice blast resistance, and the LOC4340715 gene is overexpressed to obviously enhance the rice blast resistance. The invention provides a new gene and an application method for creating plant breeding based on the disease-resistant function of the OsSOG1 protein, and the gene 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 method in creating disease-resistant materials in rice varieties. Background Art

[0002] Rice blast, caused by the rice blast fungus (Magnaporthe oryzae) infecting rice, is a fungal disease that has attracted much attention in rice production. It poses a devastating threat to rice yields and can even cause total crop failure in severe cases. Planting disease-resistant varieties is the most economical, effective and environmentally friendly prevention and control solution for rice blast. However, with global warming, rice blast is becoming more and more common in rice-growing areas, and correspondingly, the resistance cycle of most disease-resistant varieties promoted in agricultural production is shortened. Therefore, enhancing the persistent resistance of rice blast fungus has become a priority option for improving rice disease resistance.

[0003] Production practice has proved that conventional genetic breeding techniques based on major 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 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 the study of plant functional genomics due to its high efficiency, rapidity, and site-directed mutagenesis. The present invention screened a new rice blast resistance gene through gene editing, which can inhibit the growth and reproduction of rice blast fungi 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 shown in SEQ ID NO: 1;

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

[0009] (a3) a protein derived from rice that has 98% or more identity with (a1) and has the function of regulating plant resistance to rice blast;

[0010] (a4) a protein having the function of regulating plant resistance to rice blast disease 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 WSHQ c-myc 10 EQKLISEEDL HA 9 YPYDVPDYA

[0013] The present invention also provides the application of the above protein, which is 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 the plant's resistance to rice blast fungus.

[0019] In a second aspect, the present invention provides the 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 rice LOC4340715 gene, its encoded protein or rice LOC4340715 gene inhibitor or OsSOG1 protein encoding nucleic acid molecule 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 lesion area of ​​rice blast.

[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 having the same functional protein obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown in SEQ ID NO.1;

[0036] (3) An amino acid sequence having at least 80% homology with 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 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 replace, delete and / or add one or more amino acids without affecting its activity based on the amino acid sequence disclosed in the present invention and conventional technical means in the art such as conservative substitution of amino acids, to obtain a mutant 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 of the present invention can be any nucleotide sequence that can encode the rice OsSOG1 protein. Considering the degeneracy of codons and the preference of codons of different species, those skilled in the art can use codons suitable for expression of a specific 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 of A, T, G, and 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 LOC4340715 gene encoded protein can be used in the form of rice LOC4340715 gene, its encoded protein, rice LOC4340715 gene inhibitor or nucleic acid molecule containing the LOC4340715 gene encoded protein itself, or in the form of expression cassette, vector containing rice LOC4340715 gene or its inhibitor, or host cell containing the expression cassette or the 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 is obtained by a large number of screenings in the present invention. The present invention proves through experiments that CRISRP / Cas9-mediated gene editing using the above-mentioned gRNA can efficiently obtain rice plants edited with the LOC4340715 gene, and insertion or deletion at positions 2598 to 2617 of the sequence shown in SEQID NO.3 will lead to the destruction of the biological function of the rice LOC4340715 gene, so that the rice exhibits the trait of 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 comprises 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 resistance to rice blast or cultivating transgenic plants, comprising: regulating the biological function of rice OsSOG1 protein in the plant; the rice OsSOG1 protein has any one of the following amino acid sequences:

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

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

[0059] (3) An amino acid sequence having at least 80% homology with 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, the pMDC43-LOC4340715 recombinant vector contains the CDS sequence of the LOC4340715 gene shown in SEQ ID NO.2 in the sequence list 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 CRISRP / Cas9 system can be used to cut the XXXNGG nucleotide sequence in the rice LOC4340715 gene 3bp upstream of NGG to produce a blunt end, thereby causing premature termination of translation of the gene or change in protein conformation, thereby destroying the biological function of the protein encoded by the gene; wherein 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) transferring 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 introduced into the 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 means 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 rice 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) transferring 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 for 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 of the transformed plant genome and sequence it; if an insertion or deletion mutation occurs in the nucleic acid sequence from position 2598 to position 2617 of the sequence shown in SEQ ID NO.3, it indicates that the biological function of the plant LOC4340715 gene 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 found that the rice LOC4340715 gene and the OsSOG1 protein it encodes are involved in regulating the immune response of rice to rice blast fungus, and that the resistance of rice to rice blast fungus can be significantly improved by enhancing the biological function of the OsSOG1 protein. Experimental verification showed that after the LOC4340715 gene-overexpressing rice plants were infected with rice blast fungus, the lesion area was reduced by 26.4% to 28.3%, proving that enhancing the biological function of the OsSOG1 protein increases the resistance of rice materials to rice blast fungus.

[0087] (2) The present invention uses CRISPR / Cas9 technology to perform genome-targeted modification of the LOC4340715 gene, achieving efficient site-specific editing of LOC4340715. The present invention found that the nucleotide sequence from position 2598 to position 2617 in the rice LOC4340715 gene was used as the target sequence, which could efficiently achieve site-specific editing of LOC4340715, and that insertion or deletion from position 2598 to position 2617 of the sequence shown in SEQ ID NO.3 would lead to the destruction of the biological function of the rice LOsSOG1 protein, causing the rice to show a decreased level of resistance to rice blast. Experimental verification showed that after the LOC4340715 gene site-specifically edited rice plants were infected with rice blast fungi, 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 from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0089] Figure 1 The mutation type of the nucleotide sequence and the encoded amino acid sequence of the LOC4340715 gene in the ossog1-edited plant under 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 overexpressed 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 is the statistical comparison of the 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 only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, 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 pathogen 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 rice OsSOG1 protein encoding gene LOC4340715 based on CRISPR / Cas9 system Editing Method

[0095] 1. Sequence analysis and target sequence screening of 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 shows that the gene includes 6 exons, which are located at positions 177-184, 1909-2050, 2433-2619, 3073-3554, 3724-4104 and 4185-4241 of the SEQ ID NO.3 sequence, respectively.

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

[0098] After a large number of screenings, 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 the 2598th to 2617th positions 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 a primer annealing method, and were inserted into the downstream of the rice U3 promoter of the CRISPR / Cas9 vector (the CRISPR / Cas9 vector is also called 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, which is publicly available from China Agricultural University) to obtain a 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 Center, 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 of the protein encoded by the LOC4340715 gene

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

[0112] Cloning primers containing the CDS of the LOC4340715 gene were designed, and 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 of the protein encoded by the LOC4340715 gene

[0117] The cloning and overexpression primers of the CDS of the LOC4340715 gene were designed, and 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 rice variety Nipponbare and reverse transcribed to obtain cDNA. The cDNA was used as a template and PCR amplification was performed using forward primer LOC4340715-cloning-F and reverse primer LOC4340715-cloning-R to obtain the primary amplification product. The primary amplification product was used as a template and PCR amplification was performed using forward primer LOC4340715-OE-F and reverse primer LOC4340715-OE-R to obtain the 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 overexpression of LOC4340715 gene

[0124] The recovered product OE-LOC4340715 was subjected to homologous recombination reaction with the target 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 enzymes to obtain a recombinant vector containing the correct CDS nucleotide sequence shown in SEQ ID NO.2 and named pMDC43-LOC4340715. Homologous recombination reaction system: 3.75 μL (50-100 ng) of vector pMDC43 treated with Pac I and Asc I enzymes, 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, transform the reaction system into E. coli DH5α, screen the positive clones, and obtain the correct expression vector containing the OE-LOC4340715 nucleotide sequence, named pMDC43-LOC4340715. pMDC43-LOC4340715 contains the CDS sequence of the LOC4340715 gene shown in SEQ ID NO.2 in the sequence table and the 35S promoter, and can express the 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.

[0126] 3. Obtaining recombinant Agrobacterium tumefaciens

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

[0128] Example 3: Site-directed editing method or transgenic overexpression technology based on CRISPR / Cas9 technology in rice varieties Applications

[0129] Recombinant Agrobacterium EHA105-Cas9-LOC4340715 or EHA105-pMDC43-LOC4340715 was used to infect callus tissue induced by mature embryos of rice variety Nipponbare, and the obtained rice transformed 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), respectively, and cultured at 28°C and 200 rpm with shaking until the OD600nm was 0.6-0.8; centrifuged at 5000 rpm and 4°C for 5 min, and the bacterial precipitate 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 the mature seeds of the rice variety Nipponbare, soak them in 75% ethanol for 1 min, and then oscillate and sterilize them in 3.3% NaClO solution for 20 min, repeating twice. Rinse with sterile water several times until there is no odor, inoculate the sterilized Nipponbare seeds on NBD2 medium to induce callus, culture them in the dark at 26°C for 8-10 days, remove the roots and residual endosperm, subculture for 10 days, and obtain embryonic callus.

[0132] 3. The embryo callus obtained in step 2 was immersed in the recombinant Agrobacterium resuspension obtained in step 1. After 20-30 minutes, the embryo callus group was removed and inoculated on a co-cultivation medium containing two layers of filter paper (acetosyringone concentration was 100 μM, pH 5.2), and co-cultivated at 26° C. in the dark for 2 days.

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

[0134] 5. After repeated screening for 2 times, transfer the resistant callus to differentiation medium (24 hours of light / day) to induce differentiation; when new rootless seedlings are generated, transfer the regenerated seedlings to 1 / 2MS medium to induce rooting; when the seedlings grow strong, move them into the artificial climate chamber for nutrient solution cultivation. Ossog1 regenerated plants (plants obtained by transfection with recombinant Agrobacterium EHA105-Cas9-LOC4340715) and OsSOG1 OE regenerated plants (plants obtained by infecting rice Nipponbare with recombinant Agrobacterium EHA105-pMDC43-LOC4340715) were obtained respectively.

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

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

[0137] Regeneration of 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. The genome of the obtained ossog1 regenerated plant was used as a template, and the specific primers LOC4340715-Dection-F (sequence as shown in SEQ ID NO.15) and LOC4340715-Dection-R (sequence as shown in SEQ ID NO.16) of the rice OsSOG1 protein encoding gene LOC4340715 were used for PCR amplification, and the obtained 201bp amplification product was sequenced for verification. The sequencing verification results showed that among the 27 positive transformed plants, there were 7 transformed plants with gene editing in the LOC4340715 gene (referred to as ossog1 edited plants). The number of positive transformed plants detected, the number of edited transformed plants, and the percentage of the number of edited transformed plants to the number of regenerated plants detected were statistically analyzed, that is, the editing efficiency (%), and the results are shown in Table 2.

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

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

[0141] 8. Collect seeds of ossog1-edited plants and screen homozygous edited plants by self-segregation. After screening, two homozygous edited types (ossog1-1d and ossog1-27d) were obtained. Their nucleotide and amino acid sequences are shown in 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 of LOC4340715 gene was detected using a primer pair consisting of forward primer LOC4340715-qRTF (sequence as shown in SEQ ID NO.17): 5'-ACCAAAGAAGTGACCGCGAA-3' and reverse primer LOC4340715-qRTR (sequence as shown in SEQ ID NO.18): 5'-AAGCAGTCTTCCAACCACCG-3'. The internal reference used was Actin, forward primer Actin-F (sequence as shown in SEQ ID NO.19): 5'-CCTGACGGAGCGTGGTTAC-3' and reverse primer Actin-R (sequence as shown in SEQ ID NO.20): 5'-CCAGGGCGATGTAGGAAAGC-3'. The results are shown in Figure 9. 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, respectively, and reverse transcribed. The expression level of the LOC4340715 gene was detected using a primer pair consisting of a forward primer LOC4340715-qRTF (sequence as shown in SEQ ID NO.17): 5'-ACCAAAGAAGTGACCGCGAA-3' and a reverse primer LOC4340715-qRTR (sequence as shown in SEQ ID NO.18): 5'-AAGCAGTCTTCCAACCACCG-3'. The internal reference used was Actin, the forward primer Actin-F (sequence as shown in SEQ ID NO.19): 5'-CCTGACGGAGCGTGGTTAC-3' and the reverse primer Actin-R (sequence as shown in SEQ ID NO.20): 5'-CCAGGGCGATGTAGGAAAGC-3'. Figure 2As shown, 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 and analyzed. The lesion data of at least 14 independent plants were counted for each line. The results were as follows: Figure 2 As shown: compared with the wild type (Nipponbare), both ossog1-1d and ossog1-27d showed a decreased resistance to rice blast, and the lesion area caused by the rice blast fungus RB22 increased by more than 22.0%; OsSOG1 OE-13 and OsSOG1 OE-17 plants showed an increased resistance to rice blast, and the lesion length caused by the rice blast fungus RB22 was reduced by more than 26.4%; the above 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 modifications or variations 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 regulating plant disease resistance; D2) Use in the preparation of products for regulating plant disease resistance; D3) Application in breeding disease-resistant plants; D4) Use in the preparation of products for breeding disease-resistant plants; The protein is the following A1) or A2): A1) a protein whose amino acid sequence is SEQ ID No. 1; A2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1); A3) an amino acid sequence of a protein having the same function obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence 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. The use of a biomaterial related to the protein of claim 1, characterized in that: The application is any of the following: D1) Application in regulating plant disease resistance; D2) Use in the preparation of products for regulating plant disease resistance; D3) Application in breeding disease-resistant plants; D4) 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 the recombinant vector described in B3).

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.2; b3) 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.

5. The method according to claim 4, characterized in that The enhancing of the content and / or activity of the protein of claim 1 in the target plant is achieved by increasing the expression level of the gene encoding the protein in the target plant.

6. The method according to claim 5, characterized in that The coding gene of the protein 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.2; b3) The nucleotide sequence is the DNA molecule shown in SEQ ID No.

3.

7. Use of the method according to any one of claims 4 to 6 in producing rice resistant to rice blast.

Images