Application of F-box protein gene OsFBX235 in improving resistance to bacterial blight

Abstract

The application belongs to the technical field of genetic engineering, and discloses an F-box protein gene OsFBX235 application in improving white leaf blight resistance. The application specifically discloses a knockout OsFBX235 application of the gene in improving rice white leaf blight resistance or cultivating rice varieties resistant to white leaf blight. The application separates and clones an F-box protein coding gene OsFBX235 from rice, and through functional analysis, it is first confirmed that the gene is involved in the defense reaction of rice to white leaf blight bacteria and is an important negative regulatory factor for regulating rice white leaf blight resistance. Through target gene screening and CRISPR / Cas9 technology, the gene is knocked out OsFBX235 , and the importance of the gene to the regulation of rice white leaf blight resistance is proved OsFBX235 . Meanwhile, the knockout of the gene can be used for creating rice germplasm resources with high white leaf blight resistance, and can be applied to the breeding of new rice varieties resistant to white leaf blight.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the F-box protein gene. OsFBX235 Application in improving resistance to bacterial blight. Background Technology

[0002] Rice bacterial blight is caused by the Gram-negative bacterium Xanthomonas oryzae (a pathogenic rice species). Xanthomonas oryzae pv. Oryzae, Xoo Bacterial bacterial blight (BFB) is a bacterial vascular disease that seriously threatens rice yield and food security. The immune response of rice to BFB is controlled by a complex signaling regulatory network. Related research focuses on improving rice resistance to BFB by identifying genetic resources such as resistance (R) genes, susceptibility (S) genes, and quantitative trait loci (QTLs). However, the resistance of existing resistant varieties is often limited by the specificity of a single resistance gene, making it difficult to cope with the loss of resistance caused by rapid mutation of the pathogen. Therefore, further research into genes that effectively resist BFB in rice is crucial for elucidating the network of rice resistance mechanisms and breeding superior resistant varieties.

[0003] The ubiquitin-26S proteasome pathway (UPS) is a crucial mechanism for regulating protein homeostasis and signal transduction within cells. The Skp1-Cullin-F-box (SCF) complex is a major class of E3 ubiquitin ligases. As the substrate recognition subunit of the SCF complex, the F-box protein binds to Skp1-like proteins via a conserved F-box domain at its N-terminus, and to the target substrate via a specific protein-protein interaction domain at its C-terminus, thereby mediating substrate ubiquitination and degradation. Previous studies have found that F-box proteins play a key role in plant disease defense; for example, in rice, F-box proteins... OsFBX156 It can mediate the degradation of OsHSP71.1 protein to increase the expression of disease-related genes, thereby enhancing rice resistance to rice blast. However, the function of F-box protein in the regulation of rice bacterial blight resistance still needs further investigation.

[0004] Identifying functional genes related to plant disease resistance is crucial for understanding the immune regulatory mechanisms of rice against bacterial blight and the interaction between rice and the pathogen. Analyzing the functions of these resistance-related genes and applying them to the breeding or molecular improvement of resistant varieties can help effectively control and reduce the damage caused by bacterial blight to rice production, thereby enhancing rice's disease resistance and production stability. Therefore, the discovery and utilization of resistance-related genes have significant application value in rice gene function research and disease-resistant breeding. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art described above. It provides a rice F-box protein-related gene.OsFBX235 The gene was isolated, cloned, functionally validated, and applied. This gene belongs to the F-box protein gene family, and its expression level decreases after induction by the bacterial blight pathogen. Deletion of this gene increases resistance to bacterial blight in rice.

[0006] The first aspect of the present invention is to provide a knockout OsFBX235 Application of genes in improving rice resistance to bacterial blight or in breeding bacterial blight-resistant rice varieties.

[0007] The second aspect of the present invention is to provide OsFBX235 The application of inhibitors.

[0008] A third aspect of the present invention is to provide a gRNA.

[0009] The fourth aspect of this invention aims to provide biological materials related to gRNA in the third aspect of this invention.

[0010] The fifth aspect of this invention aims to provide a CRISPR / Cas system.

[0011] The sixth aspect of this invention aims to provide the use of the gRNA of the third aspect of this invention, the biological material of the fourth aspect of this invention, the CRISPR / Cas system of the fifth aspect of this invention, or reagents containing the gRNA of the third aspect of this invention, the biological material of the fourth aspect of this invention, or the CRISPR / Cas system of the fifth aspect of this invention.

[0012] The seventh aspect of this invention aims to provide a method.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] The first aspect of the invention provides knockout OsFBX235 Application of genes in a1) or a2):

[0015] a1) Improve resistance to bacterial blight in rice;

[0016] a2) Develop rice varieties resistant to bacterial leaf blight;

[0017] The OsFBX235 The nucleotide sequence is shown in SEQ ID NO:5.

[0018] Should OsFBX235 The gene is located on chromosome 7, with the locus number LOC_Os07g26000 (MSU accession number). It belongs to the F-box protein gene family. Its expression level decreases after induction by bacterial blight pathogens, and its deletion increases rice resistance to bacterial blight. This gene is of great significance for improving rice resistance and breeding superior rice varieties.

[0019] In some embodiments of the present invention, the rice includes the japonica rice variety Nipponbare.

[0020] A second aspect of the present invention provides OsFBX235 The use of the inhibitor in at least one of b1)-b4):

[0021] b1) Improve resistance to bacterial blight in rice;

[0022] b2) Prepare products that enhance resistance to bacterial blight in rice;

[0023] b3) Develop rice varieties resistant to bacterial leaf blight;

[0024] b4) Prepare products for breeding rice varieties resistant to bacterial blight;

[0025] The OsFBX235 The nucleotide sequence is shown in SEQ ID NO:5;

[0026] The OsFBX235 Inhibitors include at least one of c1)-c13):

[0027] c1) Target OsFBX235 The CRISPR / Cas system;

[0028] c2) encodes the nucleic acid molecule of c1);

[0029] c3) An expression cassette containing the nucleic acid molecule described in c2);

[0030] c4) A recombinant vector containing the nucleic acid molecule described in c2);

[0031] c5) A recombinant vector containing the expression cassette described in c3);

[0032] c6) Recombinant cells containing the nucleic acid molecules described in c2);

[0033] c7) Recombinant cells containing the expression cassette described in c3);

[0034] c8) Recombinant cells containing the recombinant vector described in c4);

[0035] c9) Recombinant cells containing the recombinant vector described in c5);

[0036] c10) Recombinant microorganisms containing the nucleic acid molecules described in c2);

[0037] c11) Recombinant microorganisms containing the expression cassette described in c3);

[0038] c12) Recombinant microorganisms containing the vector described in c4);

[0039] c13) Recombinant microorganisms containing the vector described in c5);

[0040] The CRISPR / Cas system includes a target OsFBX235 gRNA;

[0041] The nucleotide sequence of the gRNA is shown in SEQ ID NO:7 and SEQ ID NO:8.

[0042] In some embodiments of the present invention, the rice includes the japonica rice variety Nipponbare.

[0043] In some embodiments of the present invention, the gRNA-related biological material includes at least one of d1)-d12):

[0044] d1) Nucleic acid molecules encoding gRNA;

[0045] d2) An expression cassette containing the nucleic acid molecule described in d1);

[0046] d3) A recombinant vector containing the nucleic acid molecule described in d1);

[0047] d4) A recombinant vector containing the expression cassette described in d2);

[0048] d5) Recombinant cells containing the nucleic acid molecules described in d1);

[0049] d6) Recombinant cells containing the expression cassette described in d2);

[0050] d7) Recombinant cells containing the recombinant vector described in d3);

[0051] d8) Recombinant cells containing the recombinant vector described in d4);

[0052] d9) Recombinant microorganisms containing the nucleic acid molecules described in d1);

[0053] d10) Recombinant microorganisms containing the expression cassette described in d2);

[0054] d11) Recombinant microorganisms containing the recombinant vector described in d3);

[0055] d12) Recombinant microorganisms containing the recombinant vector described in d4).

[0056] In some embodiments of the present invention, the recombinant vector is a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a phage, an F phage, or an artificial chromosome.

[0057] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, the viral vector may be an optional virus, and the cell does not include reproductive material.

[0058] In some embodiments of the present invention, the CRISPR / Cas system further includes Cas proteins and / or biomaterials associated with Cas proteins;

[0059] Biomaterials associated with Cas proteins include at least one of g1)-g12):

[0060] g1) A nucleic acid molecule encoding the Cas protein;

[0061] g2) An expression cassette containing the nucleic acid molecule described in g1);

[0062] g3) A recombinant vector containing the nucleic acid molecule described in g1);

[0063] g4) A recombinant vector containing the expression cassette described in g2);

[0064] g5) Recombinant cells containing the nucleic acid molecules described in g1);

[0065] g6) Recombinant cells containing the expression cassette described in g2);

[0066] g7) Recombinant cells containing the recombinant vector described in g3);

[0067] g8) Recombinant cells containing the recombinant vector described in g4);

[0068] g9) Recombinant microorganisms containing the nucleic acid molecules described in g1);

[0069] g10) recombinant microorganisms containing the expression cassette described in g2);

[0070] g11) Recombinant microorganisms containing the recombinant vector described in g3);

[0071] g12) Recombinant microorganisms containing the recombinant vector described in g4).

[0072] In some embodiments of the present invention, the Cas protein is selected from Cas9, Cas12a, Cas12e, Cas12b, Cas12i, Cas12h, Cas12c, Cas12d, Cas12f, Cas12g, Cas12k, Cas12j, Cas13a, Cas13b, Cas13c, Cas13d, or Cas14, including any recombinant variant thereof; particularly selected from Cas9, including any recombinant variant thereof.

[0073] In some embodiments of the present invention, the product includes, but is not limited to, reagents and kits.

[0074] In some embodiments of the present invention, the product further includes a pharmaceutically acceptable carrier, including but not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, binders, sprays, transdermal absorbents, humectants, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, or adsorbents.

[0075] A third aspect of the present invention provides a gRNA, the nucleotide sequence of which is shown in SEQ ID NO:7 and SEQ ID NO:8.

[0076] In some embodiments of the present invention, the gRNA targeting OsFBX235 It can also be co-knocked out with the Cas protein. OsFBX235 .

[0077] In some embodiments of the present invention, the OsFBX235 The nucleotide sequence is shown in SEQ ID NO:5.

[0078] A fourth aspect of the invention provides biological materials related to the gRNA of the third aspect of the invention, said biological materials comprising at least one of d1)-d12):

[0079] d1) A nucleic acid molecule encoding the gRNA of the third aspect of this invention;

[0080] d2) An expression cassette containing the nucleic acid molecule described in d1);

[0081] d3) A recombinant vector containing the nucleic acid molecule described in d1);

[0082] d4) A recombinant vector containing the expression cassette described in d2);

[0083] d5) Recombinant cells containing the nucleic acid molecules described in d1);

[0084] d6) Recombinant cells containing the expression cassette described in d2);

[0085] d7) Recombinant cells containing the recombinant vector described in d3);

[0086] d8) Recombinant cells containing the recombinant vector described in d4);

[0087] d9) Recombinant microorganisms containing the nucleic acid molecules described in d1);

[0088] d10) Recombinant microorganisms containing the expression cassette described in d2);

[0089] d11) Recombinant microorganisms containing the recombinant vector described in d3);

[0090] d12) Recombinant microorganisms containing the recombinant vector described in d4).

[0091] In some embodiments of the present invention, the cells do not include reproductive material.

[0092] A fifth aspect of the present invention provides a CRISPR / Cas system comprising gRNA from the third aspect of the present invention and / or biological material from the fourth aspect of the present invention.

[0093] In some embodiments of the present invention, the CRISPR / Cas system further includes Cas protein and / or Cas protein-related biomaterials;

[0094] Biomaterials associated with Cas proteins include at least one of g1)-g12):

[0095] g1) A nucleic acid molecule encoding the Cas protein;

[0096] g2) An expression cassette containing the nucleic acid molecule described in g1);

[0097] g3) A recombinant vector containing the nucleic acid molecule described in g1);

[0098] g4) A recombinant vector containing the expression cassette described in g2);

[0099] g5) Recombinant cells containing the nucleic acid molecules described in g1);

[0100] g6) Recombinant cells containing the expression cassette described in g2);

[0101] g7) Recombinant cells containing the recombinant vector described in g3);

[0102] g8) Recombinant cells containing the recombinant vector described in g4);

[0103] g9) Recombinant microorganisms containing the nucleic acid molecules described in g1);

[0104] g10) recombinant microorganisms containing the expression cassette described in g2);

[0105] g11) Recombinant microorganisms containing the recombinant vector described in g3);

[0106] g12) Recombinant microorganisms containing the recombinant vector described in g4).

[0107] In some embodiments of the present invention, the Cas protein is selected from Cas9, Cas12a, Cas12e, Cas12b, Cas12i, Cas12h, Cas12c, Cas12d, Cas12f, Cas12g, Cas12k, Cas12j, Cas13a, Cas13b, Cas13c, Cas13d, or Cas14, including any recombinant variant thereof; particularly selected from Cas9, including any recombinant variant thereof.

[0108] A sixth aspect of the invention provides the application of any one of e1)-e4) in any one of b1)-b4):

[0109] e1) gRNA of the third aspect of the present invention;

[0110] e2) The biomaterial of the fourth aspect of this invention;

[0111] e3) The CRISPR / Cas system of the fifth aspect of the present invention;

[0112] e4) Reagents containing gRNA of the third aspect of the present invention, biological material of the fourth aspect of the present invention, or CRISPR / Cas system of the fifth aspect of the present invention;

[0113] b1) Improve resistance to bacterial blight in rice;

[0114] b2) Prepare products that enhance resistance to bacterial blight in rice;

[0115] b3) Develop rice varieties resistant to bacterial leaf blight;

[0116] b4) Prepare products for breeding rice varieties resistant to bacterial blight.

[0117] In some embodiments of the present invention, the product includes, but is not limited to, reagents and kits.

[0118] In some embodiments of the present invention, the product further includes a pharmaceutically acceptable carrier, including but not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, binders, sprays, transdermal absorbents, humectants, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, or adsorbents.

[0119] In some embodiments of the present invention, the rice includes the japonica rice variety Nipponbare.

[0120] A seventh aspect of the invention provides a method comprising knocking out [a substance] in rice. OsFBX235 Steps:

[0121] The method is f1) or f2):

[0122] f1) A method to improve resistance to bacterial blight in rice;

[0123] f2) A method for breeding rice varieties resistant to bacterial leaf blight;

[0124] The OsFBX235 The nucleotide sequence is shown in SEQ ID NO:5.

[0125] In some embodiments of the present invention, the knockout of rice OsFBX235 The step involves introducing a reagent containing gRNA from the third aspect of this invention, biological material from the fourth aspect of this invention, or a CRISPR / Cas system from the fifth aspect of this invention into rice.

[0126] In some embodiments of the present invention, the method includes using gene editing technology to knock out [a specific gene in rice]. OsFBX235 The steps.

[0127] In some embodiments of the present invention, the gene editing technology includes ZFNs, TALENs, or CRISPR / Cas technology.

[0128] In some embodiments of the present invention, CRISPR / Cas9 technology is used to knock out... OsFBX235 Gene.

[0129] In some embodiments of the present invention, CRISPR / Cas9 technology is used to knock out... OsFBX235 Genes include the following steps:

[0130] (1) Target selection: Targets were selected based on the target design website (http: / / skl.scau.edu.cn / ). OsFBX235 The nucleotide sequence of the gene target is shown in SEQ ID NO:7-8;

[0131] (2) Target primer design: The nucleotide sequences of the target primers are shown in SEQ ID NO:9-12;

[0132] (3) Target sequence amplification. Using pCBC-MT1T2 as a template, amplification was performed using primers shown in SEQ ID NO:9-12 to obtain the target sequence containing pCBC-MT1T2. OsFBX235 MT1T2-PCR of the target sequence of the gene gRNA;

[0133] (4) Target sequence ligation with vector. BsaI The pHUE411 vector was digested with enzymes, and the gel-recovered product of MT1T2-PCR was constructed into the pHUE411 vector using homologous recombination to obtain the pHUE411+MT1T2-PCR vector.

[0134] (5) Rice callus transformation and induction of regenerated plants. The pHUE411+MT1T2-PCR vector constructed in step 4 was transformed into Agrobacterium. Rice callus tissue was placed in Agrobacterium solution for infection. The callus tissue was then placed on differentiation medium to induce differentiation of regenerated plants.

[0135] (6) Identification of positive plants. DNA was extracted from fresh leaves of regenerated plants. Primers (sequences shown in SEQ ID NO:13-14) were designed upstream and downstream of the genomic target site for PCR amplification. The amplified products were sequenced and detected. Wild-type plants were used as controls. Plants whose sequencing results at the target site differed from those of wild-type plants were considered positive plants with gene editing.

[0136] In some embodiments of the present invention, the rice includes the japonica rice variety Nipponbare.

[0137] The beneficial effects of this invention are:

[0138] This invention isolates and clones an F-box protein-coding gene from rice. OsFBX235 Functional analysis confirmed for the first time that this gene participates in the defense response of rice against bacterial blight pathogens and is an important negative regulator of rice resistance to bacterial blight. Target gene screening combined with CRISPR / Cas9 knockout technology was used to further investigate its role. [[ID= Genes prove ​ The gene plays a crucial role in regulating resistance to bacterial blight in rice. Furthermore, knocking out this gene can be used to create rice germplasm resources with high resistance to bacterial blight, and can be applied to the breeding of new rice varieties resistant to bacterial blight.

[0139] This invention successfully obtained CRISPR / Cas9 technology in rice. ​ A gene knockout mutant rice plant exhibited high resistance to bacterial blight. This suggests that this gene could serve as a target gene for molecular breeding, and gene knockout could enhance rice resistance to bacterial blight, providing new genetic material and insights for rice molecular breeding.

[0140] This invention helps to better understand ​ The regulatory mechanism ​ The cloning and functional verification of the rice provides new molecular evidence for further understanding the rice-pathogen interaction and its disease resistance signal transduction pathway, and has high breeding application value. Attached Figure Description

[0141] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0142] ​ For rice ​ The gene expression results in rice after being induced by bacterial blight.

[0143] ​ For rice ​ A schematic diagram of the full-length genome structure; the black parts represent exons.

[0144] ​ For rice ​ Verification of gene mutation sites.

[0145] ​ For rice ​ The gene mutation sequence was compared with the original sequence.

[0146] ​ For rice ​ Phenotypic identification of mutant plants against bacterial blight. Among them, WT is a Nipponbare rice variety; ​ , ​ This is a rice mutant plant. (Image shown) represent P <0.01. Detailed Implementation

[0147] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0148] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0149] The primers and sequencing used in the examples were performed by Beijing Qingke Biotechnology Co., Ltd.; various restriction endonucleases and DNA ligases used in the experiments were purchased from New England Biolabs; the genome extraction kit was purchased from Zhengzhou Suling Biotechnology Co., Ltd.; the reverse transcription kit and high-fidelity enzyme were purchased from Nanjing Novizan Biotechnology Co., Ltd.; the plasmid extraction kit and gel extraction kit were purchased from Tiangen Biotech (Beijing) Co., Ltd.; and the methods were all performed in accordance with the manufacturer's instructions.

[0150] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0151] Example 1: After inoculation with bacterial blight pathogen ​ Gene expression trend analysis

[0152] Quantitative RT-qPCR was used to detect the bacterial blight pathogen in Nipponbare rice after inoculation. ​ Gene expression patterns were analyzed. Specifically, using the wild-type Nipponbare rice variety, bacterial blight infection experiments were conducted on 4-week-old rice seedlings at 25°C using the hole-injection method. The bacterial blight infection concentration was OD0.05. 600 =1.0. Rice leaves were collected at each treatment stage (0, 6, 12, 24, 48, 72, 96 hpi, i.e., 0, 6, 12, 24, 48, 72, 96 h after inoculation with Bacillus subtilis). After being frozen in liquid nitrogen, the leaves were stored at -80℃, and three samples were taken at each time point. Total RNA was extracted from each sample using the OMEGA HP Plant RNA Kit (R6837-02). The RNA was reverse transcribed into cDNA using the HiScript® II Reverse Transcriptase System (Vazyme Biotech Co., Ltd). cDNA was then used as a template for detection and analysis by quantitative real-time PCR. Quantitative Real-Time PCR Detection ​ The primer sequences are as follows: 5'-TCCCAGAGGACATAGTGTCAAGG-3' (SEQ ID NO:1) and 5'-CGATGTCGAGCTCATTACAACAGC-3' (SEQ ID NO:2). Rice was used. ​ The primer sequences used as internal reference genes are as follows: 5'-AGGAAGGCTGGAAGAGGACC-3' (SEQ ID NO:3) and 5'-CGGGAAATTGTGAGGGACAT-3' (SEQ ID NO:4).

[0153] The results showed that in rice infected with bacterial blight, ​ The relative decrease in expression levels suggests that rice may respond to pathogen stress by downregulating [a specific gene]. ​ The expression of [a specific substance] regulates resistance to bacterial blight ([a specific substance]). ​ ).

[0154] Example 2 Rice ​ Gene cloning and mutant construction

[0155] The rice shown in SEQ ID NO:5 was obtained using the Rice MSU7.0 database (http: / / rice.uga.edu / index.shtml). ​ The CDS nucleotide sequence and amino acid sequence shown in SEQ ID NO:6 of the gene ( ​ This rice ​ The gene is located on chromosome 7, and its locus number is LOC_Os07g26000 (MSU accession number).

[0156]

[0157] MDATATSTVAGGGFTGTTISARRAHMDGICLVNKKRRLTLRPCVEVDHSSKRVRSRCAKFESLPEDIVSRIISQLTLKEAVVMSSTSTKLRRAWIYHPNLYLDTSIVFGSSDRQKRVPSTETFID TVNFILRTHSGLGVNKLAVMFELRKEHAHDIDGWVSFAVTSKARVVTLNFSPYHGSHDRSYNFPCHLFNGKSGSHLQVLQLDTVTLGPSPPGFCGFANLTMLTLENVLVLGDLQFLLKCPALEWL TIRMCSQLHNLYAPEPLPRLTFLCVQDCAIDKIDVHAPNLTTFKYRGRFKVIIALRECLKLKTASIVSPIEDNLYYIFTELPNGLPHVERLHVNVFVKTQIPGFTQAPYKFINLRHLTMRITYEI AKRFGRNAVLQLAYFSEAAPFLVDLHLDMLCLDFYESRPARDVIMNRPHYSLKRACITGFNGNGGQVALVKFILKNAVKLEEMVIDPKGRITNQMMGEHKGRRMIKEKLVPKYKNGLLVIL (SEQ ID NO:6).

[0158] Building an editor using CRISPR / Cas9 methods ​ The recombinant vector for the gene is as follows: Log in to the website http: / / skl.scau.edu.cn / targetdesign / and filter... ​ The gene target, the nucleotide sequence of gRNA target sequence 1 is TTACCATCCTAATCTTTACT (SEQ ID NO:7), and the nucleotide sequence of gRNA target sequence 2 is TTGGACACTTCAATAGTGTT (SEQ ID NO:8). ​Primers were designed based on the target sequences. The primers for gRNA target sequence 1 were F: 5'-AATAATGGTCTCAGGCGTACCATCCTAATCTTTACT-3' (SEQ ID NO:9) and R: 5'-GTACCATCCTAATCTTTACTGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO:10); the primers for gRNA target sequence 2 were F: 5'-AACACTATTGAAGTGTCCACGCTTCTTGGTGCC-3' (SEQ ID NO:11) and R: 5'-ATTATTGGTCTCTAAACAACACTATTGAAGTGTCCA-3' (SEQ ID NO:12). Using the pCBC-MT1T2 vector as a template, PCR amplification was performed using the two primer pairs shown in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12. The amplification system consisted of: 1 μL pCBC-MT1T2, 1 μL each of the primers (10 μM), 15 μL 2 × Phanta Flash Master Mix (P510), and 10 μL ddH2O. The amplification program was as follows: 98℃ for 30 s, 98℃ for 10 s, 58℃ for 5 s, 72℃ for 5 s, 32 cycles, followed by 72℃ for 1 min. The PCR product was purified and recovered to obtain the product containing... ​ The target sequence of the gene gRNA was obtained by MT1T2-PCR. Then, the gel-recovered product of MT1T2-PCR was constructed into the pHUE411 vector using an enzyme digestion-ligation system to obtain the pHUE411+MT1T2-PCR vector. The enzyme digestion-ligation system is shown in Table 1.

[0159] Table 1 Enzyme digestion-ligation system and reaction conditions

[0160]

[0161] The obtained pHUE411+MT1T2-PCR vector was transformed into Agrobacterium ( ​ Agrobacterium carrying the transformation plasmid pHUE411+MT1T2-PCR vector was used to transform the target sequence into the wild-type japonica rice variety Nipponbare by infecting rice callus tissue. ​ ssp ​(cv. Nipponbare) (Specific methods are referred to "Hiei Y, Ohta S, Komari T, Kumashiro T. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant journal: for cell and molecular biology 6: 271-82"); using PCR amplification products for sequencing and comparison with wild type, two [types] were screened under Nipponbare background. ​ Mutants, respectively ​ , ​ ( ​ and ​ ).filter ​ The upstream and downstream primers for the homozygous CRISPR / Cas9 mutant are 5'-ACAGCAGCAAGCGGGTAAG-3' (SEQ ID NO:13) and 5'-TCAAGGGCAGGGCATTTC-3' (SEQ ID NO:14), respectively.

[0162] Example 3 ​ Phenotypic identification of resistance to bacterial blight

[0163] The background of Japan Sunny Day constructed using Example 2 ​ , ​ Mutants and wild-type Nipponbare (WT) rice seeds were planted, and 8-week-old rice leaves were inoculated with bacterial blight pathogens using the leaf-cutting method. Two weeks later, bacterial blight resistance phenotypes were identified.

[0164] The results are as follows ​ As shown, compared to the wild type, ​ and ​ The lesion length was significantly shortened in both mutants, which indicates that... ​ Negative regulation of bacterial blight resistance in rice.

[0165] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Knockout OsFBX235 Application of genes in a1) or a2): a1) Improve resistance to bacterial blight in rice; a2) Develop rice varieties resistant to bacterial leaf blight; The OsFBX235 The nucleotide sequence is shown in SEQ ID NO:

5.

2. OsFBX235 The use of the inhibitor in at least one of b1)-b4): b1) Improve resistance to bacterial blight in rice; b2) Prepare products that enhance resistance to bacterial blight in rice; b3) Develop rice varieties resistant to bacterial leaf blight; b4) Prepare products for breeding rice varieties resistant to bacterial blight; The OsFBX235 The nucleotide sequence is shown in SEQ ID NO:5; The OsFBX235 Inhibitors include at least one of c1)-c9): c1) Target OsFBX235 The CRISPR / Cas system; c2) encodes the nucleic acid molecule of c1); c3) An expression cassette containing the nucleic acid molecule described in c2); c4) A recombinant vector containing the nucleic acid molecule described in c2); c5) A recombinant vector containing the expression cassette described in c3); c6) Recombinant cells containing the nucleic acid molecules described in c2); c7) Recombinant cells containing the expression cassette described in c3); c8) Recombinant cells containing the recombinant vector described in c4); c9) Recombinant cells containing the recombinant vector described in c5); The CRISPR / Cas system includes a target OsFBX235 gRNA; The nucleotide sequence of the gRNA is shown in SEQ ID NO:7 and SEQ ID NO:

8.

3. The application according to claim 2, characterized in that, The OsFBX235 Inhibitors include at least one of C10-C13: c10) Recombinant microorganisms containing the nucleic acid molecules described in c2); c11) Recombinant microorganisms containing the expression cassette described in c3); c12) Recombinant microorganisms containing the vector described in c4); c13) Recombinant microorganisms containing the vector described in c5).

4. A gRNA, the nucleotide sequence of which is shown in SEQ ID NO:7 and SEQ ID NO:

8.

5. The biomaterial related to the gRNA of claim 4, characterized in that, The biomaterial includes at least one of d1)-d8): d1) A nucleic acid molecule encoding the gRNA of claim 3; d2) An expression cassette containing the nucleic acid molecule described in d1); d3) A recombinant vector containing the nucleic acid molecule described in d1); d4) A recombinant vector containing the expression cassette described in d2); d5) Recombinant cells containing the nucleic acid molecules described in d1); d6) Recombinant cells containing the expression cassette described in d2); d7) Recombinant cells containing the recombinant vector described in d3); d8) Recombinant cells containing the recombinant vector described in d4).

6. The biomaterial according to claim 5, characterized in that, The biomaterial includes at least one of d9)-d12): d9) Recombinant microorganisms containing the nucleic acid molecules described in d1); d10) Recombinant microorganisms containing the expression cassette described in d2); d11) Recombinant microorganisms containing the recombinant vector described in d3); d12) Recombinant microorganisms containing the recombinant vector described in d4).

7. A CRISPR / Cas system comprising the gRNA of claim 4 and / or the biological material of claim 5 or 6.

8. The CRISPR / Cas system according to claim 7, characterized in that, The CRISPR / Cas system also includes the Cas protein and / or biomaterials associated with the Cas protein.

9. The CRISPR / Cas system according to claim 8, characterized in that, The Cas protein is selected from any one of Cas9, Cas12a, Cas12e, Cas12b, Cas12i, Cas12h, Cas12c, Cas12d, Cas12f, Cas12g, Cas12k, Cas12j, Cas13a, Cas13b, Cas13c, Cas13d and Cas14.

10. Application of any term in e1)-e4) to any term in b1)-b4): e1) The gRNA as described in claim 4; e2) The biomaterial as described in claim 5 or 6; e3) The CRISPR / Cas system according to any one of claims 7-9; e4) A reagent containing the gRNA of claim 4, the biological material of claim 5 or 6, or the CRISPR / Cas system of any one of claims 7-9; b1) Improve resistance to bacterial blight in rice; b2) Prepare products that enhance resistance to bacterial blight in rice; b3) Develop rice varieties resistant to bacterial leaf blight; b4) Prepare products for breeding rice varieties resistant to bacterial blight.

11. A method comprising knocking out rice OsFBX235 Steps: The method is f1) or f2): f1) A method to improve resistance to bacterial blight in rice; f2) A method for breeding rice varieties resistant to bacterial leaf blight; The OsFBX235 The nucleotide sequence is shown in SEQ ID NO:

5.

12. The method according to claim 11, characterized in that, The knockout rice OsFBX235 The step involves introducing a reagent containing the gRNA of claim 4, the biological material of claim 5 or 6, or the CRISPR / Cas system of any one of claims 7-9 into rice.

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