TaROD1 gene for regulating disease resistance and application thereof in wheat breeding

Abstract

The application discloses a TaROD1 gene (ROD1, RESISTANCE OF RICE TO DISEASES 1) for regulating disease resistance and application thereof in wheat breeding, and belongs to the technical field of disease-resistant variety breeding. The application obtains a TaROD1-Crispr plant by constructing a gene knockout vector and knocking out the gene TaROD1. Pathogenicity determination shows that the TaROD1-Crispr plant has a significantly higher resistance to wheat scab and stripe rust than a wild type. The application aims to provide application of the wheat C2 domain protein coding gene TaROD1 in wheat scab and stripe rust genetic engineering, so as to provide a gene resource for plant scab and stripe rust genetic engineering.

Description

Technical Field

[0001] This invention belongs to the field of disease-resistant variety breeding technology, specifically involving a TaROD1 gene that regulates disease resistance and its application in wheat breeding. Background Technology

[0002] Wheat is an important food crop in my country, rich in nutrients. Fusarium head blight caused by *Fusarium graminearum* and wheat stripe rust caused by *Strombus styracifolium* are significant diseases affecting wheat production. The former severely damages the wheat ears, while the latter infects the leaves and affects photosynthesis. Both often lead to a decline in wheat yield and quality, causing significant economic losses to wheat production. Currently, production mainly relies on agricultural control and the use of chemical agents to mitigate the occurrence and spread of these diseases. However, these control methods are not very efficient and can cause pesticide residues, posing a significant threat to food safety and public health. Therefore, breeding disease-resistant varieties is the fundamental technical means of disease control, and the discovery and development of disease-resistant gene resources is one of the important means to solve the problem of wheat resistance to Fusarium head blight and stripe rust. However, wheat is an allohexaploid (AABBDD) with a large genome (16,000 Mb) and a high content of repetitive sequences (>80%), making direct gene cloning from wheat difficult and time-consuming.

[0003] TaROD1 is a protein encoding a 120-amino acid C2 domain located on the plasma membrane and involved in the binding of calcium-dependent proteins. It was initially discovered as a susceptibility gene in rice. TaROD1 encodes a C2 domain of calcium-dependent proteins. 2+ Sensors, these C2-domain proteins, appeared in a relatively late stage of plant evolution. Proteins with a C2 domain constitute the largest family of calcium-binding proteins. Intracellularly, Ca... 2+ It is an important secondary messenger that influences various cellular responses and stimuli, such as pathogen attack, and causes cytoplasmic calcium deficiency. 2+ The increase in concentration, these concentrations through Ca 2+ The binding protein attaches to downstream signal transduction pathways. Current research indicates that proteins with a C2 domain are primarily involved in signal transduction and cell membrane transport.

[0004] ROD1-like proteins exhibit high sequence similarity in monocotyledonous crops, but have differentiated in cruciferous plants such as Arabidopsis thaliana. ROD1 and its orthologs constitute a specific type of susceptibility gene, playing a conserved role in cereal crops. The ROD1 protein encoded by ROD1, containing a C2 domain, is Ca... 2+The sensor can act as a global regulator of immunity, promoting the degradation of H2O2 by activating catalase CatB, and its stability is fine-tuned by a pair of E3 ubiquitin ligases RIP1 and APIP6. Summary of the Invention

[0005] The purpose of this invention is to provide a TaROD1 gene (ROD1, RESISTANCE OF RICETO DISEASES1) that regulates disease resistance and its application in wheat breeding. This application utilizes the principles and methods of comparative genomics, and refers to the research results on genes related to immune resistance balance in model plants, to clone the ROD1 homologous gene in wheat. The disease resistance of the TaROD1 gene in wheat is verified by reverse genetics. It can be applied to the genetic engineering of wheat resistance to Fusarium head blight and stripe rust, in order to provide genetic resources for the genetic engineering of plant resistance to Fusarium head blight and stripe rust.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the use of the TaROD1 gene in either (1) or (2) below:

[0008] (1) Regulating plant disease resistance;

[0009] (2) Plant breeding;

[0010] The TaROD1 gene includes: TaROD1-7D located on wheat chromosome 7D, with its nucleotide sequence as shown in SEQ ID NO: 1; TaROD1-7A located on wheat chromosome 7A, with its nucleotide sequence as shown in SEQ ID NO: 2; and TaROD1-4A located on wheat chromosome 4A, with its nucleotide sequence as shown in SEQ ID NO: 3.

[0011] The disease resistance includes resistance to Fusarium head blight and resistance to stripe rust.

[0012] In a second aspect, the present invention provides the application of the TaROD1 gene in regulating wheat resistance to abiotic stresses, wherein the TaROD1 gene comprises: TaROD1-7D located on wheat chromosome 7D, the nucleotide sequence of which is shown in SEQ ID NO: 1; TaROD1-7A located on wheat chromosome 7A, the nucleotide sequence of which is shown in SEQ ID NO: 2; and TaROD1-4A located on wheat chromosome 4A, the nucleotide sequence of which is shown in SEQ ID NO: 3.

[0013] The abiotic stresses mentioned are those caused by salt, high temperature, and drought.

[0014] In a third aspect, the present invention provides the use of the protein encoded by the gene TaROD1 in any of the following (1)-(4):

[0015] (1) Prevention and control of Fusarium head blight caused by Fusarium graminearum;

[0016] (2) Develop drugs to target and control Fusarium head blight;

[0017] (3) Control wheat stripe rust caused by *Stripetra rust*;

[0018] (4) Develop drugs to target and prevent stripe rust.

[0019] The amino acid sequence of the protein encoded by the TaROD1 gene is shown in any one of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0020] In a fourth aspect, the present invention provides a method for improving the disease resistance of wheat, comprising: knocking out the TaROD1 gene in wheat or reducing the activity of the protein encoded by the TaROD1 gene in wheat;

[0021] The disease resistance refers to resistance to Fusarium head blight and stripe rust.

[0022] The TaROD1 gene in wheat was knocked out using gene editing technology;

[0023] The knockout method uses CRISPR dual-target knockout of the wheat TaROD1 gene.

[0024] The dual targets are target 1 and target 2, and their nucleotide sequences are shown in SEQ ID NO.7-SEQ ID NO.8. TaROD1-F / F0 is synthesized based on target 1, and TaROD1-R / R0 is synthesized based on target 2. The nucleotide sequences of TaROD1-F and TaROD1-R are shown in SEQ ID NO.9-SEQ ID NO.10, and the nucleotide sequences of TaROD1-F0 and TaROD1-R0 are shown in SEQ ID NO.11-SEQ ID NO.12.

[0025] TaROD1-F / F0 and TaROD1-R / R0 are expression cassette primers carrying the TaROD1 gene CRISPR dual target sites.

[0026] In a fifth aspect, the present invention provides the use of the substance described in (1) or (2) in the preparation of a medicament for preventing and controlling wheat disease resistance:

[0027] (1) Substances that block or inhibit the expression of the TaROD1 gene;

[0028] (2) Substances that inhibit the activity of the protein encoded by the TaROD1 gene;

[0029] The disease resistance refers to resistance to Fusarium head blight and stripe rust.

[0030] Preferably, the substance that blocks or inhibits the expression of the TaROD1 gene is the antisense RNA or siRNA of the TaROD1 gene.

[0031] The beneficial effects of this invention are:

[0032] This invention constructs a gene knockout vector to knock out the TaROD1 gene, obtaining TaROD1-Crispr plants. Pathogenicity tests show that TaROD1-Crispr plants have significantly higher resistance to wheat scab and stripe rust than the wild type. The purpose of this discovery is to provide an application of the wheat C2 domain protein-coding gene TaROD1 in wheat scab and stripe rust resistance genetic engineering, in order to provide gene resources for plant scab and stripe rust resistance genetic engineering. Attached Figure Description

[0033] Figure 1 A is a diagram of the TaROD1 gene structure; Figure 1 B is a distribution map of the TaROD1 gene on chromosomes; Figure 1 C is a diagram of the spatiotemporal expression pattern of amplified TaROD1 with three copies; Figure 1 D represents the gene expression pattern of TaROD1 under stress conditions, where Fg is an abbreviation for Fusarium graminearum, and Fg-2d indicates 2 days after inoculation with Fusarium graminearum. Figure 1 E represents the expression pattern of the TaROD1 gene under stress conditions caused by Fusarium graminearum and Fusarium head blight, while Fhb1 represents Fusarium head blight resistant mutant wheat.

[0034] Figure 2 To identify the editing type of TaROD1-Crispr plants; where A is the gene editing type identification of Crispr-ROD1 T0 generation and B is the gene editing type identification of Crispr-ROD1 T1 generation.

[0035] Figure 3 Phenotypic identification of CRISpr-ROD1 Fusarium head blight inoculation: A is the phenotypic identification of CRISpr-ROD1 T2 generation Fusarium head blight inoculation 7 days after inoculation, and B is the phenotypic identification of CRISpr-ROD1 T2 generation Fusarium head blight inoculation 14 days after inoculation.

[0036] Figure 4Phenotypic identification of wild-type and Crispr-ROD1T2 generation stripe rust 21 days after inoculation; where A is leaf phenotypic identification; B is leaf disease and inoculation area phenotypic identification; C is DAB staining after WT and Crispr-ROD1 stripe rust inoculation treatment; D is a schematic diagram of biomass ratio. Detailed Implementation

[0037] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0038] To determine the copy number of the wheat C2 domain protein-coding gene TaROD1 in wheat and its chromosome distribution, the amplified TaROD1 sequence was analyzed using BLASTN on ensembl plants (http: / / plants.ensembl.org / index.html). Conserved domains of the sequence were analyzed using Pfam software (http: / / pfam.xfam.org / ).

[0039] The wheat C2 domain protein-coding gene TaROD1 was mainly obtained by BLASTing the amino acid sequence corresponding to OsROD1 on NCBI. The orthologous gene TaROD1 in wheat has three copies in hexaploid wheat, located on chromosomes Chr7D, Chr7A, and Chr4A, respectively. Therefore, these three copies are named TaROD1-7D, TaROD1-7A, and TaROD1-4A. Figure 1 The CDS sequences encoded by these sequences were cloned, with lengths of 645bp, 657bp, and 645bp, and their nucleic acid sequences are shown in SEQ ID NO: 1-SEQ ID NO: 3, respectively.

[0040] SEQ ID NO: 1: TaROD1-7D CDS

[0041] ATGCCGTCGTCGAGGCTCGTGCCCAAGGACGGCGAGTGGGACGGCGGCGTGGCGCT

[0042] GGAGGTCACCGTGCTGTCGGCCGAGTCGCTGCGCCTGCCGCCCACCTACTCGCCTCT

[0043] CCCGCGCCGGCTGCGGCCCTACGTCGCCGTCTCGTCCTCTCCTCCTCCTCCTCCGCC

[0044] GGCTGCAGCACGGGCGTGGCGCCCGCGTCGTCCGGCGCGGGGGAGCACTCTTGGGC

[0045] CGACGTCGGCGAGGACGCGCGCCTCGTGGTGCCCGTGGGCGCGGGGTTCCTGGAGG

[0046] GCCGCGACGACGTGCGCGTGGCGGTGCTCTCGGAGTCGGGCTGCGCGCGGCTCGTG

[0047] GGCGACACGCCGCTGGGCTGGTGCCGCGTGCCCGCCGCCGACGTGCTGGATGGCCT

[0048] CCGCCCGCCCCGCGCGCTCCGCAGGCTCAGCTACTCGCTCCGCTGCCCGCGCCGCG

[0049] GCGGGCCGGGACACGGCGTCGTCCACCTCGCCGTGCGCGTCCTCGGCGACGTCCAC

[0050] GTCCACGTCGCCCGCCCGGACCCCGCCCCGCCCGCGCAGCCCGGCTGGTGCCGCGT

[0051] CGCCATGGGCATCCCGGTCTCCGGCGCCGCGGCCGTCGTCGGCACACCGTCTCCCTGGGCATGGAGCCAGACGTCGCGGTGA。

[0052] SEQ ID NO:2:TaROD1-7A CDS

[0053] ATGCCGTCGTCGAGGCTCGTGCCCAAGGACGGCGAGTGGGGAGGCGGCGTGGCGCT

[0054] GGAGGTCACCGTGCTGTCGGCCGAGTCGCTGCGCCTGCCGCCCACCTACTCGCCGCT

[0055] CCCGCGCCGGCTGCGGCCCTACGTCGCCGTCTCCTCCTCCTCCTCGTCCTCGTCCGC

[0056] CGGCTGCAGCACGGGCGTGGCCCCCGCGTCGTCCGGCGCGGGCGAGCACTCGTGGG

[0057] AGGACGTCGGCGAGGACGCGCGCCTGGTGGTGCCCGTGGGCGCGGGGTTCCTGGAG

[0058] GGCCGCGACGACGTGCGCGTGGCGGTGCTCTCGGAGTCCGGCTGCGCGCGGCTCGT

[0059] CGGCGACACGCCGCTGGGCTGGTGCCGCGTGCCCGCCGCCGACGTGCTGGACGGCC

[0060] TCCACCCGCCGCGCGCGCTCCGCAGGCTCAGCTACTCGCTCCGCTGCCCGCGCCGCG

[0061] GCGGGCCCGGACACGGCGTCGTCCACCTCGCCGTGCGCGTCCTCGGCGACGTCCAC

[0062] GTCCACGTCGCCCGCCCGGACCCGGCCCCGCCAGCGCAGCCCGGCTGGTGCCGCGT

[0063] CGCCATGGGCATCCCGGTCTCCGGCCCCTCCGCCGCCGCCGCGGCCGTCGTCGGCACGCCGTCCCCCTGGGCGTGGAGCCAGGCGTCGCGCTGA。

[0064] SEQ ID NO:3:TaROD1-4A CDS

[0065] ATGCCGTCGTCGAGGCTCGTGCCCCCCAAGGACGGCGAGTGGGGCGGCGGCGGCGT

[0066] CGCGCTGGAGGTCACCGTGCTCTCGGCCGAGTCGCTGCGCCTGCCGCCCACCTACTC

[0067] GCCGCTCCCGCGCCGCCTGCGGCCCTACGTCGCCGTCTCCTCCTCCTCCTCCGCCGG

[0068] CTGCAGCACGGGCGTGGCGCCCGCGACGTCCGGCGCGGGCGAGCACTCGTGGGAG

[0069] GACGCCGGCGAGGACGCGCGCCTCGTGGTGCCCGTGGGGGCCGGGTTCCTGGAGGG

[0070] CCGCGACGACGTGCGCGTGGCGGTGCTCTCGGAGTCCGGCTGCGCGCCTCGTCG

[0071] GCGACACGCCGCTGGGGTGGTGCCGCGTGCCCGCCGCCGACGTGCTCGACGGCCTC

[0072] CGCCCGCCGCGCGCGCTGCGCAGGCTCAGCTACTCGCTCCGCTGCCCGCGCCGCGG

[0073] CGGGCCGGGACACGGCCGTCGTCCACCTCGCCGTGCGCGTCCTCGGCGACGTCCGCG

[0074] TCGCCCCCCGCCCGGACCCCGCCCCGCCCGCGCAGCCCGGCTGGTGCCGCGTCGCC

[0075] ATGGGCATACCGGTCTCCGGCCCCTCCGCCGCCGCCGTCGTCGGCACGCCGTCGCCCTGGGCGTGGAGCCAGACATCGCGGTGA.

[0076] Analysis of the gene structure of these three copies showed that TaROD1-7D, TaROD1-7A, and TaROD1-4A each contain only one exon.

[0077] The three copies, TaROD1-7D, TaROD1-7A, and TaROD1-4A, encode protein sequences of 214, 218, and 214 amino acids, respectively, as shown in SEQ ID NO: 4-SEQ ID NO: 6.

[0078] SEQ ID NO: 4: TaROD1-7D

[0079] MPSSRLVPKDGEWDGGVALEVTVLSAESLRLPPTYSPLPRRLRPYVAVSSSSSSAGCS

[0080] TGVAPASSGAGEHSWADVGEDARLVVPVGAGFLEGRDDVRVAVLSESGCARLVGDTP

[0081] LGWCRVPAADVLDGLRPPRALRRLSYSLRCPRRGGPGHGVVHLAVRVLGDVHVHVAR

[0082] PDPAPPAQPGWCRVAMGIPVSGAAAVVGTPSPWAWSQTSR

[0083] SEQ ID NO: 5: TaROD1-7A

[0084] MPSSRLVPKDGEWGGGVALEVTVLSAESLRLPPTYSPLPRRLRPYVAVSSSSSSSSAGCS

[0085] TGVAPASSGAGEHSWEDVGEDARLVVPVGAGFLEGRDDVRVAVLSESGCARLVGDTP

[0086] LGWCRVPAADVLDGLHPPRALRRLSYSLRCPRRGGPGHGVVHLAVRVLGDVHVHVAR

[0087] PDPAPPAQPGWCRVAMGIPVSGPSAAAAAVVGTPSPWAWSQASRSEQ ID NO: 6: TaROD1-4A

[0088] MPSSRLVPPKDGEWGGGGVALEVTVLSAESLRLPPTYSPLPRRLRPYVAVSSSSSAGCS

[0089] TGVAPATSGAGEHSWEDAGEDARLVVPVGAGFLEGRDDVRVAVLSESGCARLVGDTP

[0090] LGWCRVPAADVLDGLRPPRALRRLSYSLRCPRRGGPGHGVVHLAVRVLGDVRVAPRP

[0091] DPAPPAQPGWCRVAMGIPVSGPSAAAVVGTPSPWAWSQTSR

[0092] The predicted results of the secondary and tertiary structures of the TaROD1 protein show that its structure contains a large number of random coils.

[0093] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0094] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions.

[0095] Example 1: TaROD1 Expression Pattern Analysis

[0096] (1) Sample preparation

[0097] Different tissues were collected at various growth and development stages of WT: coleoptile, aboveground parts of 7-day seedlings, roots, stems and leaves during tillering, roots, stems, leaves, flag leaves and ears during flowering, and roots, stems, leaves, flag leaves and ears during grain filling. The tissues were wrapped in tin foil and flash-frozen with liquid nitrogen and stored at -80°C for later use.

[0098] (2) Total RNA extraction

[0099] RNA extraction was performed according to the instructions of the Vazyme Polysaccharide and Polyphenol Plant RNA Extraction Kit. The specific procedures are as follows:

[0100] 1. Grind the sample thoroughly under liquid nitrogen cryogenic conditions;

[0101] 2. Transfer 100g of the ground sample powder into a pre-cooled 2mL centrifuge tube, immediately add 600μL Buffer EL or 600μL Buffer PSL (for spikelet tissue), vortex vigorously for 30sec to ensure the sample and lysis buffer are thoroughly mixed, centrifuge at 12000rpm (13,400×g) for 5min, and immediately proceed with subsequent operations.

[0102] 3. Take about 500 μL of the supernatant into FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III has been placed in the collection tube), centrifuge at 12,000 rpm (13,400×g) for 30 seconds, discard FastPure gDNA-Filter Columns III, and collect the filtrate.

[0103] 4. Add 0.5 times the volume of the filtrate to the collection tube, then shake to mix for 15 seconds.

[0104] 5. Transfer the above mixture to FastPure RNA Columns V (FastPure RNA Columns V has been placed in the collection tube), centrifuge at 12000 rpm (13400×g) for 30 seconds, and discard the filtrate.

[0105] 6. Add 700 μL of Buffer RWA to FastPure RNA Columns V, centrifuge at 12000 rpm (13400×g) for 30 seconds, and discard the filtrate.

[0106] 7. Add 500 μL of Buffer RWB to FastPure RNA Columns V (please check that 48 ml of anhydrous ethanol has been added before use), centrifuge at 12000 rpm (13400 × g) for 30 seconds, and discard the filtrate.

[0107] 8. Repeat step 7.

[0108] 9. Place the FastPure RNA Columns V back into the collection tube and centrifuge at 12,000 rpm (13,400 × g) for 2 min.

[0109] 10. Transfer FastPure RNA Columns V to a new RNase-free Collection Tube 1.5ml centrifuge tube, add 80μL of RNase-free ddH2O to the center of the adsorption column membrane, and let stand at room temperature for 5min; centrifuge at 12,000rpm (13,400×g) for 1min, and add the first elution buffer back to the adsorption column for a second elution.

[0110] 11. Add 400 μL of DEPC-treated water and repeat steps 3-9;

[0111] 12. Perform concentration determination and gel electrophoresis; RNA can be used directly in downstream experiments or stored at -80℃ for later use.

[0112] (3) cDNA first-strand synthesis

[0113] according to The IIQ RT SuperMix for qPCR (+gDNA wiper) instructions describe how to synthesize the first strand of cDNA. Specific steps are shown in Table 1.

[0114] Table 1:

[0115]

[0116] Gently mix with a pipette. Incubate at 42°C for 2 minutes.

[0117] Add 5×HiScript II qRTSuperMix II directly to the reaction tube in step 1 and gently mix with a pipette.

[0118] Table 2:

[0119]

[0120] Perform PCR amplification according to the reaction procedure shown in Table 3. The obtained cDNA first-strand product can be used immediately for qPCR reactions, or stored at -20℃ and used within six months; for long-term storage, it is recommended to aliquot and store at -70℃. Repeated freeze-thaw cycles should be avoided with cDNA.

[0121] Table 3:

[0122]

[0123] (4) Quantitative Real-Time PCR Analysis

[0124] qRT-PCR primers for TaROD1 were designed using the Snapgene tool in the conserved regions of the three copies of TaROD1 (primer sequences are shown as QF-TaROD1 and QR-TaROD1), and synthesized by Shanghai Sangon Biotech. Before using the primers for quantification, their specificity and amplification efficiency needed to be tested. In the qRT-PCR system, wheat elongation factor-1 (TaEF1) was used as an internal reference gene. Following the ChamQ SYBR qPCR Master Mix method, the reaction system shown in Table 4 was prepared on ice. The qPCR reaction was performed under the conditions shown in Table 5. Melting curves and fluorescence value change curves were then generated. Each reaction was repeated three times, and the Ct values ​​were averaged. The results were then used for relative quantification. -ΔΔCt Methods analysis of experimental results. The experimental results were performed in three independent biological replicates.

[0125] QF-TaROD1:GGGTTCCTGGAGGGCCGC;

[0126] QR-TaROD1: AGCCCAGCGGCTGTCGC.

[0127] Table 4:

[0128]

[0129] Table 5:

[0130]

[0131] (5) Expression pattern of TaROD1 gene under stress conditions of Fusarium graminearum and Fusarium head blight.

[0132] Analysis of the TaROD1 gene expression pattern under stress conditions showed that the relative expression level of TaROD1-7D was higher than that of some homologous genes at 2 and 3 days after inoculation with Fusarium graminearum. In wheat resistant to Fusarium head blight Fhb1 mutant, the relative expression level of TaROD1-7D was significantly higher than that of homologous genes, suggesting that TaROD1-7D preferentially responds to the invasion of Fusarium head blight pathogen. Figure 1 D, E).

[0133] (6) TaROD1 spatiotemporal representation mode

[0134] RNA was extracted from the aboveground parts of WT coleoptiles and 7-day seedlings, roots, stems, and leaves during the tillering stage, roots, stems, leaves, flag leaf, and panicles during the flowering stage, and roots, stems, leaves, flag leaf, and panicles during the grain-filling stage. Real-time PCR was performed to analyze the expression of TaROD1 at different stages and parts of wheat growth and development. TaROD1 expression was highest at the 7-day seedling stage, suggesting that TaROD1 is easily induced by biotic / abiotic stresses during the seedling stage, resulting in relatively high expression levels. TaROD1 expression was relatively higher in leaves than in roots and stems at different growth and development stages, as leaves are common sites of pathogen infection. This is consistent with the fact that ROD1 expression in rice can be induced by pathogen infection. Figure 1 C).

[0135] Example 2: Obtaining TaROD1Crispr plants

[0136] (1) CRISPR dual-target expression cassette primer design

[0137] The sequence and exon position information of TaROD1 (TraesCS7D02G055600) were obtained from the EnsemblPlants wheat database. Target sites were searched based on the exon regions within the DNA sequence, ensuring the target sites were conserved across homologous genes in wheat. The exon sequences of the genes to be edited using CRISPR were sequentially entered into the CRISPR target search website CRISPR Direct (http: / / crispr.dbcls.jp / ) starting from the start codon. The search settings were: PAM sequence requirement, NGG; Specificitycheck, Wheat (Triticum aestivum).

[0138] Genome, IWGSC1.0+popseq (Naito et al. 2015). CRISPR target sites predicted by the CRISPR Direct website underwent three screening tests. First, the conserved nature of the predicted targets across homologous genes was assessed, and parameters such as TM value and GC content were compared to determine their usability. This screening yielded the first batch of relatively reliable candidate targets. Second, the Ensembl Plants and WheatOmics 1.0 websites were used to predict off-target effects on the first batch of target site sequences. DNA sequences approximately 10 bp upstream and downstream of gene fragments with high similarity to the target sites after blast were evaluated, and candidate targets with potential off-target sites were discarded, resulting in the second batch of candidate targets. Finally, the RNAfold online prediction website was used to predict the secondary structure of the sgRNA of the second batch of candidate targets and assess sgRNA stability. Target sequences near the start codon were selected whenever possible. The PAM sequence (NGG) was removed from the obtained target sequence. The corresponding position in the primer before target 1 was replaced to synthesize TaROD1-F / F0. The corresponding position in the primer after target 2 was reverse complementary was replaced to synthesize TaROD1-R / R0. TaROD1-F / F0 and TaROD1-R / R0 are the expression cassette primers carrying the dual targets of the TaROD1 gene.

[0139] Target 1 is present in copies TaROD1-7D and TaROD1-7A, and its nucleotide sequence is shown in SEQ ID NO. 7:

[0140] SEQ ID NO.7: GTCGTCGAGGCTCGTGCCCAAGG

[0141] Target 2 is present in three copies of TaROD1-7D, TaROD1-7A, and TaROD1-4A, and its nucleotide sequence is shown in SEQ ID NO.8:

[0142] SEQ ID NO.8: CCGCAGGCTCAGCTACTCGCTCC

[0143] The nucleotide sequences of TaROD1-F and TaROD1-R are shown in SEQ ID NO.9-SEQ ID NO.10, and the nucleotide sequences of TaROD1-F0 and TaROD1-R0 are shown in SEQ ID NO.11-SEQ ID NO.12.

[0144] SEQ ID NO.9: aataatggtctcAAGCgGTCGTCGAGGCTCGTGCCCAAGG

[0145] SEQ ID NO.10: gGTCGTCGAGGCTCGTGCCCAAGGgttttagagctagaaatagc

[0146] SEQ ID NO.11: CCGCAGGCTCAGCTACTCGCTCCCGCTTTCTTGGTGCC

[0147] SEQ ID NO.12: ATTATTGGTCTCTAAACCCGCAGGCTCAGCTACTCGCTCC

[0148] (2) Synthesis of CRISPR dual-target expression cassette

[0149] Primers TaROD1-F / R and TaROD1-F0 / R0 were diluted to final concentrations of 20 μM and 1 μM, respectively, using ddH2O. The reaction mixtures shown in Table 6 were then prepared on ice for PCR amplification, following the amplification program shown in Table 7. After amplification, the reaction products were detected by electrophoresis on a 1% agarose gel. The target fragment was approximately 960 bp in length, and the fragment was recovered by gel excision.

[0150] Table 6:

[0151]

[0152] Table 7:

[0153]

[0154] (3) Constructing CRISPR final vectors

[0155] The CRISPR dual-target expression cassette and the CRISPR final vector TaU3P411 (preserved in our laboratory) were ligated using a cut-and-ligate method. The ligation system is shown in Table 8 (15 μL), and the PCR reaction program is shown in Table 9. The constructed CRISPR final vector was transformed into E. coli, plated, and cultured overnight. Single clones were then picked for colony PCR screening to identify positive clones.

[0156] Table 8:

[0157]

[0158] Table 9:

[0159]

[0160] (4) Heat shock transformation and verification of Escherichia coli

[0161] 1. Remove competent cells from the -80℃ freezer and thaw them on ice for 2-3 minutes;

[0162] 2. Add 5 μL of CRISPR final vector ligation product, gently mix with a pipette tip, and then incubate on ice for 30 min.

[0163] 3. Heat shock in a 42℃ water bath for 90 seconds, then quickly place on ice to cool for 2 minutes;

[0164] 4. Add 1 mL of LB liquid medium to the centrifuge tube and incubate at 37°C and 220 rpm for 45-60 min to allow for recovery;

[0165] 5. Centrifuge at 12000 rpm for 30 seconds, discard the supernatant, resuspend the bacterial cells in 100 μL of LB liquid medium, spread on LB solid plates containing 50 μg / ml kanamycin, and incubate overnight at 37°C.

[0166] 6. The ligation system was transformed into *E. coli*, plated, and cultured overnight. Single clones were picked for colony PCR screening to identify positive clones. The primers were 411-seq-F / R (SEQ ID NO.13-SEQ ID NO.14). The colony PCR reaction system using a common DNA polymerase (2×Taq Mix, Hangzhou Borui Company) is shown in Table 10 (25 μL).

[0167] SEQ ID NO.13: GGCCGGTGTCATCTATGTTACTA

[0168] SEQ ID NO.14:CCCGACATAGATGCAATAACTTC

[0169] Table 10:

[0170]

[0171] Taking a 25 μL system as an example, prepare the total volume, aliquot it into eight-tube strips, and the final volume is 20 μL. Pick a single colony as template DNA and perform a PCR reaction. The reaction procedure is shown in Table 11:

[0172] Table 11:

[0173]

[0174] Positive clone bacterial cultures were sent to Shanghai Boshan Biotechnology Co., Ltd. for sequencing using the validation primer 411-seq-F / R. Correct positive clones were preserved in bacterial cultures, plasmids were extracted, and stored at -20℃ for later use.

[0175] (5) Transformation and verification of Agrobacterium

[0176] The positive plasmid obtained in (4) was transformed into Agrobacterium using the freeze-thaw method. The wheat was transformed using the Agrobacterium-mediated transformation method, with Fielder as the recipient wheat variety. Wheat embryos were taken two weeks after flowering for transformation. CRISPR plants were obtained by Agrobacterium infection, and the CRISPR vector transformation of the TaROD1 gene was carried out by the wheat genetic transformation technology platform of Shandong Agricultural University.

[0177] The specific steps for the freeze-thaw transformation method using Agrobacterium are as follows:

[0178] 1. Remove Agrobacterium competent cells from the -80℃ freezer and thaw them slowly in an ice bath;

[0179] 2. Add about 1 μg of plasmid to Agrobacterium competent cells in a sterile laminar flow hood, gently stir the bottom of the tube to mix, and let stand in an ice water bath for 5 min.

[0180] 3. Use tweezers to pick up the Agrobacterium competent cell centrifuge tube, let the bottom of the centrifuge tube touch the liquid nitrogen first, and release the tube after the competent cells turn pure white. Let the competent cells freeze in liquid nitrogen for 5 minutes.

[0181] 4. Use tweezers to remove the centrifuge tubes onto the float plate and place them in a 37°C constant temperature water bath for 5 minutes.

[0182] 5. Carefully remove the centrifuge tube and let it stand in an ice water bath for 5 minutes;

[0183] 6. In a sterile laminar flow hood, add 800 μL of antibiotic-free LB liquid culture medium to a centrifuge tube containing competent cells;

[0184] 7. Incubate at 28℃ with shaking for 2-3 hours, centrifuge at 6000 rpm for 1 minute, and retain about 100 μL of supernatant. Use a pipette to mix the bacterial precipitate and supernatant.

[0185] 8. Take an appropriate amount of the bacterial suspension and spread it evenly on LB solid medium containing 50 μg / ml kanamycin. After the water in the bacterial suspension has been fully absorbed by the medium, invert the LB solid medium and incubate at 37℃ for 48-72 hours.

[0186] 9. Pick single colonies and perform colony PCR using 411-seq-F / R. After culturing positive clones in LB liquid medium, extract plasmids and send them for sequencing.

[0187] 10. The correctly sequenced Agrobacterium and glycerol with a final concentration of 50% were stored at a 1:1 ratio in a -80°C freezer.

[0188] This embodiment uses dual-target gene knockout technology to knock out the ROD1 gene in wheat. Compared with single-target gene knockout, dual-target gene knockout has a synergistic effect in improving the gene editing effect.

[0189] Example 3: Identification of TaROD1Crispr wheat plants

[0190] (1) Bar gene testing:

[0191] Genomic DNA was extracted from leaves of each T0 generation CRISPR seedling using the CTAB method. PCR amplification was performed using Bar gene-specific primers F / R (SEQ ID NO.15-SEQ ID NO.16), followed by electrophoresis verification. The correct target band indicated positive expression of the bar gene, signifying successful CRISPR vector transformation.

[0192] SEQ ID NO.15: CGGTCTGCACCATCGTCAACCACT

[0193] SEQ ID NO.16: GAAACCCACGTCATGCCAGTTCCC

[0194] (2) Target sequence editing status:

[0195] Genomic DNA was extracted from leaves of each T0 generation CRISPR seedling using the CTAB method. The target site sequence was amplified using universal primers for different generations, T1seq-F / R (SEQ ID NO.17-SEQ ID NO.18) and T2seq-F / R (SEQ ID NO.19-SEQ ID NO.20). The sequences were then sequenced on Hi-TOM (http: / / www.hi-tom.net / hi-tom / ), a high-throughput tracking platform for CRISPR / Cas system-induced mutations. The editing type was determined by comparing the sequences with those of wild-type wheat.

[0196] SEQ ID NO.17:ggagtgagtacggtgtgcGACCACGCCAGCCCCATT

[0197] SEQ ID NO.18:gagttggatgctggatggCCGACAGCACGGTGACCTCC

[0198] SEQ ID NO.19:ggagtgagtacggtgtgcGGCGACACGCCGCTGGG

[0199] SEQ ID NO.20:gagttggatgctggatggGAGGACGCGCACGGCGAGGT

[0200] (3) TaROD1Crispr wheat plant editing and identification

[0201] T0 generation CRISPR plants of the TaROD1 gene were identified using Bar gene detection and sequencing. The TaROD1 gene was knocked out using CRISPR / Cas9 gene editing technology. The Hi-TOM (http: / / www.hi-tom.net / hi-tom / ) sequencing sequence was compared with wild-type wheat sequences to determine the editing type. Two T0rod1 wheat plants were identified; one plant showed editing only in TaROD1-7D, with a two-base deletion, denoted as rod1-4A / 7A / 7d. (-2) Another strain showed editing on both TaROD1-7D and TaROD1-4A, with a deletion of two bases in each, and was designated as rod1-4a. (-2) / 7A / 7d (-2) Six T1 generation rod1 wheat plants were sequenced and their sequences were compared and analyzed. Multiple editing types were found: rod1-1 had a two-base deletion in TaROD1-7D, denoted as rod1-4A / 7A / 7d. (-2) This results in a frameshift mutation; rod1-2 has a 5-base deletion in TaROD1-7D, denoted as rod1-4A / 7A / 7d. (-5) This causes a frameshift mutation; rod1-3 and rod1-4 have the same editing type, with a one-base insertion on TaROD1-7D, denoted as rod1-4A / 7A / 7d. (+1) This results in a frameshift mutation; rod1-5 has a one-base substitution on TaROD1-7D, denoted as rod1-4A / 7A / 7d, resulting in a point mutation; rod1-6 undergoes editing on both TaROD1-7D and TaROD1-7A, with a one-base deletion on TaROD1-7A and a one-base insertion on TaROD1-7D, denoted as rod1-4a. (-1) / 7A / 7d (+1) ( Figure 2 Seeds of rod1-1, rod1-2, rod1-3 and rod1-4, rod1-6 are harvested from individual T1 generation plants.

[0202] Example 4: Identification of Fusarium head blight resistance phenotype in wheat plants with TaROD1 gene knockout

[0203] (1) Cultivation of Fusarium graminearum:

[0204] The cultivation of Fusarium graminearum spore suspension requires two culture media: potato dextrose agar (PDA) and CMC liquid medium. First, the Fusarium graminearum strain is transferred to PDA plates for activation and then incubated upside down in a 25°C incubator for 2-3 days until the plates are fully covered with mycelium.

[0205] (2) Collection of Fusarium graminearum conidia

[0206] 1. Take about 100 mL of CMC liquid culture medium in a 250 mL sterile Erlenmeyer flask. Use the large hole of the yellow pipette tip to collect 6-8 mycelial cakes from the edge of the colony on the plate. Transfer them to the CMC liquid culture medium and incubate at 200 rpm and 25 °C with shaking for 3-5 days to prepare conidia.

[0207] 2. Filter the CMC culture medium through 2-4 layers of gauze, collect the filtrate in a 10mL sterile centrifuge tube, centrifuge at 2000rpm for 5-10min, discard the supernatant, and collect the conidial precipitate.

[0208] 3. Resuspend the conidia in ddH2O, detect the bacterial load under a microscope, and set aside for later use.

[0209] 4. Dilute Fusarium graminearum spores to 5 × 10⁻⁶ using an appropriate amount of 0.02% Tween20 under aseptic conditions. 5 -5×10 6 Prepare a suitable concentration of Fusarium graminearum spore suspension by spores / ml and store it in a refrigerator at 4℃ for later use.

[0210] (3) Fusarium graminearum inoculation treatment

[0211] The resistance of wheat materials to wheat scab was determined by single-floret drip inoculation. T2 generation wheat plants with the TaROD1 gene knocked out and in normal growth and development, at the flowering stage of the spikelet, were selected for inoculation treatment. 10 μL of a 5×10⁻⁶ concentration was inoculated onto each of the two basal florets on the left and right sides of the middle spikelet. 5 -5×10 6 Inoculation with *Fusarium graminearum* at a concentration of 1 / ml was performed, followed by sealing in a bag for approximately three days to maintain humidity. The bag was removed after Fusarium head blight symptoms appeared, at which point browning began to appear at the base of the inoculated florets. Phenotypic observations were conducted at 7 and 14 days post-inoculation, and the number of infected florets and the total number of florets were counted. Results showed that 7 days after inoculation, wheat plants with the TaROD1 gene knocked out exhibited whitening of the spikelets, with a significantly smaller spread than the wild type. At 14 days post-inoculation, the difference in spikelet phenotype between the inoculated TaROD1 gene knocked-out wheat plants and the wild type was even more significant. Therefore, silencing the TaROD1 gene may slow the spread of *Fusarium graminearum*, thereby increasing resistance to Fusarium head blight in wheat. Figure 3 ).

[0212] Example 5: Identification of stripe rust resistance phenotype in wheat plants with TaROD1 gene knockout

[0213] (1) Material preparation:

[0214] Selected WT, rod1-4A / 7A / 7d (-2)rod1-4a (-1) / 7A / 7d (+1) (Note: rod1-4A / 7A / 7d) (-2) rod1-4a (-1) / 7A / 7d (+1) The wheat seeds (rod1-1 and rod1-6 single-plant lines of the T1 generation) were first disinfected by soaking in 0.02% Tween 20 for 10 minutes, then rinsed three times with deionized water, soaking for 10 minutes each time. They were then placed in 10cm glass petri dishes and left to stand overnight at 4℃ for low-temperature germination treatment. The germinated seedlings were then transplanted into 5cm flower pots, with about 4 seeds per pot, and cultured under 16 / 22℃ and 16 / 8h light / dark cycle conditions until they reached the two-leaf and one-heart stage.

[0215] (2) Wheat stripe rust inoculation treatment:

[0216] Wheat stripe rust fungus was inoculated using a smear method. CYR34 urediniospores, frozen at -80℃, were removed, wrapped in aluminum foil, and heat-shocked in a 42℃ water bath for 3 minutes. The heat-shocked spores were then mixed with pine pollen at a 1:10 ratio on the aluminum foil. To remove the waxy coating, the top leaf of the wheat leaf was gently rubbed with a finger. A suitable amount of the pollen-spore mixture was then applied gently to the upper surface of the wheat leaf using a cotton swab. After inoculation, the plants were placed in the dark at 10℃ for 24 hours, maintaining 100% relative humidity; then grown under 16 hours of light / 15℃ and 8 hours of darkness / 10℃ conditions. Phenotypic results were recorded after 2–3 weeks. The results showed that 10 days after inoculation, wheat plants with the TaROD1 gene knocked out in the rod1 T2 generation exhibited a significant hypersensitive necrosis response in their leaves. At 21 days, the rod1 T2 generation TaROD1 gene knocked out wheat plants produced fewer uredinia on their leaves than the wild type, and rod1-4a... (-1) / 7A / 7d (+1) The number of uredinia on the plant leaves was significantly less than that on rod1-4A / 7A / 7d. (-2) Plant leaves. This suggests that silencing the TaROD1 gene increases wheat's resistance to stripe rust. Figure 4 ).

[0217] (3) Ratio of wheat stripe rust to wheat biomass after wheat stripe rust inoculation treatment

[0218] Wheat leaves from WT and rod1 wheat plants that developed disease after inoculation with wheat stripe rust were collected. Total RNA was extracted, synthesized, and cDNA was obtained. This cDNA was then used as a template for quantitative real-time PCR analysis. Quantitative primers (SEQ ID NO.23-SEQ ID NO.24) for wheat and wheat stripe rust elongation factors TaEF (SEQ ID NO.21-SEQ ID NO.22) and PsEF, as reported in the literature, were used for real-time quantitative PCR. The Ct values ​​obtained from the real-time PCR were substituted into the corresponding Ct-copy number standard curve to obtain the corresponding copy numbers of TaEF and PsEF. The ratio of wheat stripe rust biomass to wheat biomass in the sample was calculated as the PsEF copy number / TaEF copy number. The results are shown below. Figure 4 As shown in D.

[0219] SEQ ID NO.21: TGGTGTCATCAAGCCTGGTATGGT

[0220] SEQ ID NO.22: ACTCATGGTGCATCTCAACGGACT

[0221] SEQ ID NO.23: TTCGCCGTCCGTGATATGAGACAA

[0222] SEQ ID NO.24: ATGCGTATCATGGTGGTGGAGTGA

[0223] Example 6: DAB staining treatment after wheat stripe rust inoculation

[0224] To further verify the changes in disease resistance of TaROD1 Crispr plants, the accumulation of reactive oxygen species (ROS) in Crispr plants after stripe rust inoculation was observed. DAB is a commonly used substrate for horseradish peroxidase. Under the catalysis of horseradish peroxidase, DAB produces a brown precipitate. This brown precipitate is insoluble in water and ethanol. DAB staining was performed according to the Solarbio Enhanced DAB Chromogenic Kit (20×) instructions:

[0225] (1) Prepare the DAB working solution by adding 250 μl of solution A, 250 μl of solution B, and 250 μl of solution C to 4.25 ml of distilled water and mixing them thoroughly. Note that the solution should be prepared immediately before use and used within 1 hour.

[0226] (2) Take leaves of WT and rod1 wheat that have been infected with stripe rust 21 days after inoculation. Take a diseased area about 5 cm long and add an appropriate amount of DAB working solution to the leaf tissue to ensure that the sample is fully covered. Incubate at room temperature in the dark for 1-30 minutes. If the color development time is too long, it may cause an increase in background, so the color development process should be closely observed.

[0227] (3) After staining, the color development reaction was terminated by rinsing with running water when the background was light and the appropriate color development intensity was reached. The results showed that CRISpr-ROD1 leaves had significantly more and larger areas of light brown spots than WT leaves (possibly due to the lighter staining time and the presence of stripe rust fungus urediniospores), indicating that CRISpr-ROD1 induces the production of reactive oxygen species (ROS) to resist the invasion of stripe rust fungus, and is stronger than WT. Figure 4 C).

[0228] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Knockout TaROD1 The application of genes in the following (1) or (2): (1) Improve plant disease resistance; (2) Plant breeding; The TaROD1 Genes include those located on wheat chromosome 7D. TaROD1-7D and genes located on wheat chromosome 4A TaROD1-4A ; The knockout TaROD1 Genes include: Knockout TaROD1-7D Or in the knockout TaROD1-7D On this basis, further knockout TaROD1-4A ; The gene TaROD1-7D The nucleotide sequence is shown in SEQ ID NO: 1; The gene TaROD1-4A The nucleotide sequence is shown in SEQ ID NO: 3; The plant is wheat; the disease resistance includes resistance to Fusarium head blight and resistance to stripe rust.

2. Inactivation TaROD1 The gene-encoded protein or reduced TaROD1 The activity of the encoded protein is applied in any of the following (1)-(4): (1) Prevention and control of wheat scab caused by Fusarium graminearum; (2) To develop drugs for the prevention and control of wheat scab as a target; (3) Control of wheat stripe rust caused by *Strombyx mori*; (4) Develop drugs to target and control wheat stripe rust; The TaROD1 Genes include those located on wheat chromosome 7D. TaROD1-7D and genes located on wheat chromosome 4A TaROD1-4A ; Deactivation TaROD1 The proteins encoded by genes include: Knockout TaROD1-7D Or in the knockout TaROD1-7D On this basis, further knockout TaROD1-4A ; The gene TaROD1-7D The nucleotide sequence is shown in SEQ ID NO: 1; The gene TaROD1-4A The nucleotide sequence is shown in SEQ ID NO:

3.

3. A method for improving resistance to wheat scab and stripe rust, characterized in that, include: Knock out wheat TaROD1 Genes or reduction of wheat TaROD1 The activity of gene-encoded proteins; The TaROD1 Genes include those located on wheat chromosome 7D. TaROD1-7D and genes located on wheat chromosome 4A TaROD1-4A ; The knockout of wheat TaROD1 Genes include: knockout of wheat alone TaROD1-7D Or in the process of knocking out wheat TaROD1-7D On this basis, further knockout TaROD1-4A ; The gene TaROD1-7D The nucleotide sequence is shown in SEQ ID NO: 1; The gene TaROD1-4A The nucleotide sequence is shown in SEQ ID NO:

3.

4. The use of the substance described in (1) or (2) below in the preparation of drugs to improve resistance to wheat scab and stripe rust: (1) Block or inhibit TaROD1 Substances involved in gene expression; (2) Inhibition TaROD1 Substances that encode the activity of proteins; The blocking or inhibition TaROD1 The substance expressed by genes is called a gene. TaROD1 Antisense RNA or siRNA; The TaROD1 Genes include those located on wheat chromosome 7D. TaROD1-7D and genes located on wheat chromosome 4A TaROD1-4A ; The gene TaROD1-7D The nucleotide sequence is shown in SEQ ID NO: 1; The gene TaROD1-4A The nucleotide sequence is shown in SEQ ID NO: 3.

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