Application of TaSnRK2.8 gene in regulating disease resistance of wheat

Abstract

This invention belongs to the field of genetic engineering breeding technology and provides a TaSnRK2.8 The application of genes in regulating wheat disease resistance. TaSnRK2.8 The nucleotide sequence of the gene is shown in SEQ ID NO:1. This gene encodes wheat cytoplasmic serine / threonine protein kinase TaSnRK2.8, ​​and the amino acid sequence of TaSnRK2.8 is shown in SEQ ID NO:2. Overexpression TaSnRK2.8 Genes can significantly enhance wheat's resistance to stripe rust and weaken the pathogenicity of stripe rust fungus; knockout TaSnRK2.8 The gene weakens wheat's resistance to disease, confirming that it is positively regulating wheat resistance to stripe rust. This invention provides important genetic resources and technical support for the genetic improvement of wheat resistance to rust, and can be used to breed wheat varieties resistant to stripe rust.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering breeding technology, and relates to a... TaSnRK2.8 Application of genes in regulating wheat disease resistance. Background Technology

[0002] During wheat cultivation, wheat faces the threat of infection from various pathogens, among which wheat stripe rust (… Puccinia striiformis. f. sp. tritici , Pst As one of the most destructive pathogenic fungi, it has caused serious losses to wheat production. This pathogen is prone to mutation, producing new physiological races, which lead to symptoms such as chlorosis and decreased photosynthetic efficiency in wheat plants after infection. In severe cases, it can cause significant yield reductions, posing a continuous threat to the global wheat industry.

[0003] In the practice of controlling wheat stripe rust, while reasonable field management and pesticide application can mitigate the impact of the disease to some extent, excessive pesticide use can lead to environmental pollution, increased pathogen resistance, and high control costs. In contrast, breeding and promoting disease-resistant wheat varieties is the most economical, environmentally friendly, and long-term control method, and has become the recognized preferred solution in the agricultural field. However, existing disease-resistant varieties generally have a key drawback: their resistance is easily challenged by new variants of the pathogen, and they often lose effective resistance to stripe rust after a period of promotion and planting, making it difficult to achieve long-term stable disease control, which brings continuous risks to wheat production.

[0004] From the current research on plant immune mechanisms, it is known that when plants are infected by pathogens, they release pathogen-associated molecular patterns (P / MAMPs) and damage-associated molecular patterns (DAMPs). These signaling molecules can be sensed by pattern recognition receptors (PRRs) on the plasma membrane, thereby activating a series of intracellular signal transduction pathways, such as calcium ion influx, reactive oxygen species (ROS) production, and MAPK cascade activation, ultimately initiating an immune response to resist pathogen invasion. The sucrose nonfermenting-1-related protein kinase (SnRK) family, as important members of serine / threonine protein kinases, can participate in various signal regulation through phosphorylation modification of target proteins, playing a crucial role in plant immune responses. However, current research on the specific functions, regulatory mechanisms, and application value of SnRK family members in wheat's resistance to stripe rust is still insufficient, and key gene resources that can be effectively used for genetic improvement of wheat resistance to stripe rust have not yet been identified.

[0005] Therefore, the current field of wheat stripe rust control urgently needs to identify key genes with clear disease resistance functions and systematically analyze their resistance mechanisms to provide theoretical support and gene resources for breeding new wheat varieties with broad-spectrum and durable resistance. Conducting research on wheat stripe rust resistance-related genes, disease resistance mechanisms, and the breeding of resistant varieties can not only overcome the limitations of existing resistant varieties and effectively address the challenges posed by pathogen mutations, but also reduce pesticide use and protect the ecological environment. This has crucial practical significance and urgency for ensuring wheat production safety and promoting green and sustainable agricultural development. Summary of the Invention

[0006] The technical problem this invention aims to solve is that existing disease-resistant varieties are easily affected by new variant races of pathogens and rapidly lose their resistance, making long-term stable control difficult. Meanwhile, in current research on plant immune mechanisms, the specific functions, regulatory mechanisms, and application value of wheat sucrose non-fermentation protein kinase (SnRK) family members in stripe rust resistance remain unclear. There is a lack of key gene resources that can be directly used for genetic improvement of wheat stripe rust resistance, leading to technical bottlenecks in the breeding of disease-resistant varieties due to insufficient gene supply and unclear mechanisms.

[0007] To address the aforementioned technical problems, this invention utilizes reverse genetics to screen and identify a wheat cytoplasmic serine / threonine protein kinase gene, and extensive experiments have confirmed its application value in wheat breeding. Specifically, this invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a wheat TaSnRK2.8 The application of genes in regulating wheat disease resistance, the aforementioned TaSnRK2.8 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0009] Furthermore, in the above applications, the... TaSnRK2.8 The gene plays a positive regulatory role in the interaction between wheat and stripe rust fungus, and overexpression TaSnRK2.8 Genetic enhancement of wheat's resistance to stripe rust.

[0010] Furthermore, in the above applications, overexpression TaSnRK2.8 Genes are generated through Agrobacterium-mediated genetic transformation.

[0011] Furthermore, in the above applications, the... TaSnRK2.8 The gene encodes the TaSnRK2.8 protein, the amino acid sequence of which is shown in SEQ ID NO:2.

[0012] Furthermore, in the above applications, the TaSnRK2.8 protein is a wheat cytoplasmic serine / threonine protein kinase.

[0013] Secondly, the present invention provides a method for breeding wheat varieties resistant to stripe rust, the method comprising: overexpressing in wheat materials... TaSnRK2.8 Genes, the ones mentioned TaSnRK2.8 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0014] Furthermore, in the above-mentioned breeding method, the recipient of the genetic transformation is a wheat embryo.

[0015] To ensure a complete and unambiguous understanding of the technical solution of this invention, it should be noted that the encoding gene of wheat cytoplasmic serine / threonine protein kinase TaSnRK2.8 described in this invention is indicated by italicized text. TaSnRK2.8 The term "TaSnRK2.8" indicates that the wheat cytoplasmic serine / threonine protein kinase TaSnRK2.8 is represented by the non-italicized font "TaSnRK2.8". Of course, those skilled in the art can clearly and completely understand the meaning of the relevant expressions based on the description in this invention.

[0016] Compared with the prior art, the present invention has the following significant advantages:

[0017] It has been clarifiedTaSnRK2.8 The study of the stripe rust resistance function and regulatory mechanism of the gene fills a gap in the application of wheat SnRK family genes in stripe rust resistance. This invention confirms, through expression profiling analysis, that TaSnRK2.8 is induced by stripe rust infection. Combined with overexpression and knockout verification, it clarifies that TaSnRK2.8 plays a positive regulatory role in wheat stripe rust resistance, providing new theoretical basis for the functional study of cytoplasmic serine / threonine protein kinases in plant immune signal transduction.

[0018] It provides a brand-new wheat stripe rust resistance gene resource, solving the problems of scarcity of existing disease resistance gene resources and easy loss of resistance. TaSnRK2.8 As a key gene for specific resistance to stripe rust, the overexpression of this gene showed significant resistance to the main prevalent races of stripe rust in the plants, with a significant reduction in spore biomass compared to the wild type. The knockout of the gene resulted in a significant weakening of resistance. The verification results are fully reliable and provide a highly efficient functional gene for the genetic improvement of wheat disease resistance.

[0019] The cultivation method is highly efficient and rapid, overcoming the limitations of traditional breeding techniques. This invention employs Agrobacterium-mediated genetic transformation technology, which can achieve [the desired results] in a relatively short time. TaSnRK2.8 Targeted overexpression of genes can rapidly create wheat germplasm materials resistant to rust, significantly shortening the breeding cycle compared to traditional methods, and the improvement targets are clear, enabling precise regulation of disease resistance traits.

[0020] The control effect is green and environmentally friendly, meeting the needs of sustainable agricultural development. The disease-resistant wheat varieties bred using the technology of this invention can significantly enhance resistance to stripe rust, reduce environmental pollution and pathogen resistance problems caused by pesticide application, and lower control costs. This provides a new solution for long-term green control of wheat stripe rust, which is of great significance for ensuring wheat production safety and the agricultural ecological environment. Attached Figure Description

[0021] Figure 1 for TaSnRK2.8 This diagram illustrates the gene expression profile analysis, showing the relative expression levels of the gene at different time points after wheat infection by different races of stripe rust. The horizontal axis represents the time after stripe rust inoculation, and the vertical axis represents the relative gene expression level; CYR23 represents the non-virulent race of stripe rust, and CYR31 represents the virulent race. This indicates a significant difference in gene expression levels at this time point compared to the 0-hour infection time (P < 0.05). This indicates a highly significant difference with P < 0.01.

[0022] Figure 2 for TaSnRK2.8Phenotypic identification results (left) and relative biomass statistics (right) of wheat plants overexpressing the gene and control plants after inoculation with stripe rust fungus. In the figure, TaSnRK2.8-OE represents... TaSnRK2.8 Wheat plants overexpressing the gene were represented by four different lines: L2, L3, L6, and L8. Fielder was the wild-type wheat control variety. The stripe rust fungus used for inoculation was physiological race CYR33, and the culture time after inoculation was 14 days. The vertical axis of the right figure represents the relative biomass of the stripe rust fungus. This indicates a significant difference in stripe rust biomass between knockout plants and control plants. P <0.01. This indicates a highly significant difference in stripe rust biomass between knockout plants and control plants. P <0.001.

[0023] Figure 3 for TaSnRK2.8 A graph validating the gene editing status of gene knockout plants. In the graph, TaSnRK2.8-sgRNA represents the target gene. TaSnRK2.8 Guide RNA sequences of the gene; Fielder-5A, Fielder-5B, and Fielder-5D represent wild-type wheat varieties from the Fielder cultivar. TaSnRK2.8 The copy sequences of the gene on chromosomes 5A, 5B, and 5D; tasnrk2.8-ko-L1-5A, tasnrk2.8-ko-L2-5A, and tasnrk2.8-ko-L3-5A correspond to the edited sequences on chromosomes 5A, 5B, and 5D of the three knockout strains, respectively; "-1 bp" and "-8 bp" indicate a base deletion at the corresponding position, with deletion lengths of 1 base and 8 bases, respectively; "+1 bp" and "+4 bp" indicate a base insertion at the corresponding position, with insertion lengths of 1 base and 4 bases, respectively.

[0024] Figure 4 for TaSnRK2.8 Phenotypic identification results (left) and relative biomass statistics (right) of gene knockout wheat plants and control plants after inoculation with stripe rust fungus. (Figure) TaSnRK2.8 -KO representative TaSnRK2.8 Gene knockout wheat plants, L1, L2, and L3 are three different lines of knockout plants; Fielder is a wild-type wheat control variety; the stripe rust fungus inoculated is physiological race CYR23, and the culture time after inoculation is 14 days; the vertical axis of the right figure represents the relative biomass of stripe rust fungus; This indicates a highly significant difference in stripe rust biomass between knockout plants and control plants. P <0.001. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental and detection methods in the following embodiments are conventional methods; the reagents and materials mentioned are commercially available unless otherwise specified; and the index data are measured using conventional methods unless otherwise specified.

[0027] The main experimental materials, reagents, and equipment used in this example are as follows:

[0028] I. Experimental Materials

[0029] 1. Plant materials

[0030] Wheat variety Fielder, provided by Northwest A&F University; wheat T1 generation TaSnRK2.8 overexpressing plant lines (L2, L3, L6, L8), constructed in this invention; wheat TaSnRK2.8 knockout plant lines (… TaSnRK2.8 -ko#L1, L2, L3), obtained through gene editing in this invention.

[0031] 2. Pathogenic materials

[0032] The non-toxic race CYR23 and the prevalent toxic race CYR33 of wheat stripe rust were preserved and propagated in our laboratory.

[0033] 3. Carrier material

[0034] The plant transgenic expression vector pANIC6 and its intermediate vector Donor were preserved and propagated in our laboratory; the gene editing vector pCL4B-Cas9 and its intermediate vector sgA... + It is preserved and propagated by this laboratory.

[0035] 4. Microbial materials

[0036] Agrobacterium strain EHA105 was purchased from Shanghai Weidi Biotechnology Co., Ltd. (Catalog No.: AC1013); Escherichia coli DH5α competent cells (Catalog No.: DL1001) were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0037] II. Experimental Reagents

[0038] 1. Enzymes: T4 DNA ligase (Catalog No.: C301-01), PCR amplification enzyme 2×Taq Plus Master Mix (DyePlus) (Catalog No.: P212), real-time quantitative PCR enzyme ChamQ SYBR Color qPCR Master Mix (Catalog No.: Q411), purchased from Nanjing Novizan Biotechnology Co., Ltd.; restriction endonucleases BtgZ Ⅰ (Item No.: R0703S) was purchased from New England Biolabs; Bsa Ⅰ (Item No.: ER0291) was purchased from Thermo Fisher.

[0039] 2. Molecular cloning reagents, including the Gateway reaction reagent for homologous recombination (catalog number: 11791020), were purchased from ThermoFisher; DNA extraction reagent (catalog number: DC112) and plasmid extraction reagent FastPure EndoFree Plasmid Mini Kit (catalog number: DC203) were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0040] 3. Cell culture reagents: LB liquid medium for Agrobacterium and Escherichia coli culture: Weigh 10 g / L peptone (Oxoid, UK, catalog number: LP0042B), 5 g / L yeast extract (Oxoid, UK, catalog number: LP0021B), and 10 g / L sodium chloride (Thermo Fisher, catalog number: BP358-212), dissolve in deionized water, adjust pH to 7.0, and autoclave at 121℃ for 20 minutes. Kanamycin stock solution: Weigh kanamycin sulfate powder (Sigma-Aldrich, USA, catalog number: 60615), dissolve in sterile ultrapure water to prepare a 50 mg / mL stock solution, sterilize by filtration through a 0.22 μm filter membrane, store at -20℃, and add to the medium to a final concentration of 50 μg / mL before use.

[0041] 4. Genetic transformation reagents and wheat callus induction and differentiation culture media were provided by the National Key Laboratory of Crop Stress Resistance and High-Efficiency Production. All chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0042] III. Experimental Equipment

[0043] Nucleic acid amplification and detection equipment: PCR instrument, real-time fluorescence quantitative PCR instrument; gel imaging system, electrophoresis apparatus; microbial culture equipment: constant temperature shaker, constant temperature incubator, sterile operating table; molecular manipulation equipment: centrifuge, pipette, vortex mixer; plant culture box (dark and humidified device), all provided by the National Key Laboratory of Crop Stress Resistance and High-Efficiency Production.

[0044] Example 1

[0045] This embodiment describes the wheat cytoplasmic serine / threonine protein kinase gene (i.e. TaSnRK2.8 Acquisition of genes.

[0046] I. Experimental Methods

[0047] 1. Primer design: Using the EnsemblPlants database, we found... TaSnRK2.8 The nucleotide sequence of the gene (accession number: TraesCS5B02G406400), based on TaSnRK2.8 Based on the nucleotide sequence of the gene (as shown in SEQ ID NO:1), design full-length amplification primers for this gene, where the forward primer is... TaSnRK2.8 -F, the reverse primer is TaSnRK2.8 -R, the primer nucleotide sequence is:

[0048] TaSnRK2.8 -F: 5'-ATGGCAGGGGCGGCG-3' (SEQ ID NO: 3);

[0049] TaSnRK2.8 -R: 5'-TCACATCGCATACACGATCTCTCC-3' (SEQ ID NO: 4).

[0050] 2. Template preparation: Using Fielder wheat plants as material, total RNA was extracted using an RNA extraction kit, and then cDNA was synthesized using a reverse transcription kit. The obtained cDNA was used as a template for PCR amplification.

[0051] 3. PCR amplification: Prepare the reaction solution with the designed primers, cDNA template and PCR amplification reagents (reaction system: 1 μL template DNA, 0.5 μL each of forward and reverse primers, 10 μL 2×PCR buffer, and sterile deionized water to 20 μL). The PCR reaction conditions are: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; and finally, 72℃ for a full extension for 10 min.

[0052] 4. Sequencing verification: The PCR amplification products were detected by agarose gel electrophoresis, and the target fragment was recovered using a gel recovery kit. The recovered fragment was sent to a sequencing company (Beijing Qingke Biotechnology Co., Ltd.) for sequencing.

[0053] II. Test Results

[0054] Sequencing results showed that the amplified gene fragment sequence was consistent with... TaSnRK2.8The gene sequences are completely identical, and their nucleotide sequences are shown in SEQ ID NO:1, indicating that... TaSnRK2.8 The full-length gene sequence was successfully amplified. TaSnRK2.8 The gene encodes wheat cytoplasmic serine / threonine protein kinase TaSnRK2.8, ​​and its amino acid sequence is shown in SEQ ID NO:2.

[0055] Example 2

[0056] This embodiment describes TaSnRK2.8 Gene expression profiles under stripe rust infection.

[0057] I. Experimental Methods

[0058] 1. Preparation of experimental materials: Using the wheat variety Fielder as the experimental material, wheat leaves were inoculated with the non-toxic race CYR23 and the toxic race CYR31 of stripe rust at the two-leaf-one-heart stage. Wheat leaf samples were collected at 0, 6, 12, 24, 48, and 72 h after inoculation. Total RNA was extracted from the samples at each time point using an RNA extraction kit, and then the total RNA was reverse transcribed into cDNA using a reverse transcription kit to obtain cDNA templates of wheat-stripe rust interaction at different time points. The cDNA templates were stored at -20℃ for later use.

[0059] 2. Primer design and synthesis: Select wheat elongation factor gene TaEF Design specific primers for the internal reference gene, where the forward primer is: TaEF -F, the reverse primer is TaEF -R, the primer nucleotide sequence is:

[0060] ​ -F: 5'-tggtgtcatcaagcctggtatggt-3' (SEQ ID NO: 5);

[0061] ​ -R: 5'-actcatggtgcatctcaacggact-3' (SEQ ID NO: 6).

[0062] according to ​ Gene design qRT-PCR quantitative primers:

[0063] ​ -qRT-F: 5'-CCAGATGCAGGCACCTAAT-3' (SEQ ID NO: 7);

[0064] ​ -qRT-R: 5'-ATCGTCCATCAGATCAGCGG-3' (SEQ ID NO: 8).

[0065] All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0066] 3. Real-time quantitative PCR detection: Samples were taken at various time points after inoculation, and total RNA was extracted using the Trizol method. RevertAid was used. TM RNA reverse transcription was performed using the Master Mix reverse transcription kit (Thermo Scientific, America): 0.5–5 μg RNA, 10.0 μL 2×RT Buffer Mix, 1.0 μL 20×RT Enzyme Mix, and sterile water to a final volume of 20.0 μL. The reaction was carried out at 42°C for 30 min, and the cDNA was stored at -80°C. Based on the cDNA templates from each time point, the reaction system was prepared according to the instructions of the real-time quantitative PCR kit. The total reaction volume was 20 μL, containing 2 μL cDNA template, 0.8 μL each of forward and reverse primers, 10 μL of real-time PCR mixture, and 7.2 μL sterile water. The reaction conditions were: 95°C pre-denaturation for 1 min; 95°C denaturation for 10 s, 60°C annealing for 30 s, 72°C extension for 1 min, for a total of 35 cycles; and a final extension at 72°C for 10 min. Three biological replicates were performed for each sample, with cDNA from wheat leaves uninoculated with stripe rust (0 h) as a control.

[0067] 4. Data processing: using 2 -ΔΔCt The method is used to analyze real-time quantitative PCR data and calculate... ​ The relative expression levels of genes at different time points were analyzed for significance using Graphpad 8.0.2 statistical software. P < 0.05 was considered significant, and P < 0.01 was considered highly significant.

[0068] II. Test Results

[0069] The test results are as follows ​ As shown, by ​ It can be seen that when stripe rust was not inoculated (0 h). ​ The gene is basally expressed in wheat leaves; after inoculation with the non-toxic race CYR23 and the toxic race CYR31 of stripe rust, ​ Gene expression levels showed a clear trend of induction and upregulation, exhibiting regular changes at different time points. Specifically, after CYR23 inoculation, gene expression levels peaked at 6 h, at which point the relative expression level was significantly higher than at 0 h; after CYR31 inoculation, gene expression levels peaked at 12 h, also showing a highly significant difference from 0 h.

[0070] The above results indicate that ​The gene expression was induced by stripe rust infection and responded to different types of stripe rust races, suggesting that the gene may be involved in the immune response of wheat to stripe rust, providing an important basis for further verification of the gene's anti-rust function.

[0071] Example 3

[0072] ​ Cultivation and disease resistance identification of gene-overexpressing plants.

[0073] I. Experimental Methods

[0074] 1. Construction of overexpression vector: as shown in SEQ ID NO:1 ​ Using the full-length gene sequence as a template, primers were designed to construct an overexpression vector, with the forward primer being... ​ -6E-F, reverse primer is ​ -6E-R, the primer nucleotide sequence is:

[0075] ​ -6E-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTACCCATACGACGTCCCAGACTACGCTATGGCGCTGACCCGGCTG-3' (SEQ ID NO: 9);

[0076] ​ -6E-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTTAAGCGTAGTCTGGGACGTCGTATGGGTATCAAGGGAACAACAATGC-3' (SEQ ID NO: 10).

[0077] The full-length gene fragment was obtained by PCR amplification (reaction system: 1 μL template DNA, 0.5 μL each of forward and reverse primers, 10 μL 2×PCR buffer, and sterile deionized water to a final volume of 20 μL; reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; final extension at 72℃ for 10 min) TM LR Clonase TM The enzyme mixture (catalog number: 11791020) was used to insert the amplified fragment into the plant transgenic expression vector pANIC6E to construct the TaSnRK2.8-pANIC6E overexpression vector.

[0078] 2. Genetic transformation to obtain overexpressing plants: The successfully constructed TaSnRK2.8-pANIC6E overexpression vector was transformed into Agrobacterium tumefaciens strain EHA105. Agrobacterium-mediated transformation was performed using the immature embryo of the wheat variety Fielder as the transformation recipient. ​ -OE transgenic overexpression plants.

[0079] 3. Transgenic Positive Detection: Genomic DNA was extracted from the above-mentioned transgenic overexpressing plants and PCR detection was performed using universal detection primers for the pANIC6E vector. The forward primer was 6E-F, and the reverse primer was 6E-R. The primer nucleotide sequences are as follows:

[0080] 6E-F: 5'-TTAGCCCTGCCTCATACG-3' (SEQ ID NO: 11);

[0081] 6E-R: 5'-CTATCATAGATGTCGCTATAAACC-3' (SEQ ID NO: 12).

[0082] PCR reaction system: 1 μL template DNA, 0.5 μL each of forward and reverse primers, 10 μL 2×PCR buffer, and sterile deionized water to a final volume of 20 μL; reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; final extension at 72℃ for 10 min, and PCR-positive transgenic overexpression plants were screened.

[0083] 4. Disease Resistance Identification: The T1 generation of positive transgenic plants was selected, and four lines (L2, L3, L6, and L8) were obtained through screening, with wild-type wheat Fielder as the control. All lines and control plants were inoculated with the major prevalent stripe rust race CYR33. After inoculation, they were cultured in a dark, humid environment for 24 h, and then transferred to conventional light cultivation (temperature 20–25℃, light duration 12–16 h / day). Fourteen days after inoculation, the disease phenotype of each plant was observed and recorded.

[0084] Simultaneously, DNA was extracted from the leaf sites inoculated with stripe rust fungus on each plant, diluted to 400 ng / μL as a template, and the relative biomass of stripe rust fungus was detected using real-time quantitative PCR (reaction conditions as in Example 2). The internal reference gene for stripe rust fungus was [missing information]. ​ The wheat internal reference gene is ​ (The primer sequence is shown in Example 2), the ​ The gene primer sequences are as follows:

[0085] ​-F: 5'-TTCGCCGTCCGTGATATGAGACAA-3' (SEQ ID NO: 13);

[0086] ​ -R: 5'-ATGCGTATCATGGTGGTGGAGTGA-3' (SEQ ID NO: 14).

[0087] II. Test Results

[0088] PCR testing confirmed that the sample was successfully obtained. ​ The presence of positive gene overexpression plants indicates that the TaSnRK2.8-pANIC6E overexpression vector has been successfully integrated into the wheat genome.

[0089] Phenotypic identification results 14 days after inoculation with stripe rust fungus CYR33 are as follows ​ As shown in the left figure, TaSnRK2.8-OE in the figure represents ​ Wheat plants overexpressing the gene were divided into four different overexpression lines: L2, L3, L6, and L8, with Fielder representing the wild-type control. The figures show that the wild-type control plants exhibited a higher number and larger coverage area of ​​stripe rust spore masses on their leaves, indicating more severe disease. In contrast, the four overexpression lines showed a significantly reduced number of spore masses and a much smaller coverage area, indicating a significantly milder disease phenotype compared to the control.

[0090] Real-time quantitative PCR results showed that the relative biomass of stripe rust fungus in the leaves of overexpressing plants L2, L3, L6, and L8 was significantly higher than that of other strains. ​ The right figure in the figure is significantly lower than that of wild-type Fielder, consistent with phenotypic observations.

[0091] The above results indicate that overexpression ​ The gene can significantly enhance wheat's resistance to the prevalent stripe rust race CYR33, while effectively inhibiting the growth and development of stripe rust and reducing its spore biomass.

[0092] Example 4

[0093] ​ Cultivation and disease resistance identification of gene knockout plants.

[0094] I. Experimental Methods

[0095] 1. Gene editing target design: based on wheat ​ Based on the gene's genomic sequence and the target design requirements of CRISPR / Cas9 technology, two specific targets were designed. ​ The guide RNAs (gRNAs) for the gene are gRNA1 and gRNA2, with the following nucleotide sequences:

[0096] gRNA1: 5'-GTCATGGAATATGCATCTGG-3' (SEQ ID NO: 15);

[0097] gRNA2: 5'-AAATCACATATCTTCAAGCG-3' (SEQ ID NO: 16).

[0098] 2. Gene editing vector construction: gRNA was tandemly constructed into the gene editing vector pCL4B-Cas9 using an in vitro bridging method. The specific steps are as follows:

[0099] 1) Intermediate vector construction (gRNA2 insertion): Using T4 DNA ligase, gRNA2 is inserted into the intermediate vector. ​ Ⅰ Intermediate vector sgA digested by a single enzyme + Ligation was performed overnight at 16°C; the ligation product was transformed into *E. coli* DH5α competent cells, and PCR detection was performed using primers ENTRY4-F and ENTRY4-R to screen for positive single colonies. Plasmids were extracted from positive single colonies by shaking and then sent for sequencing verification. The sequences of the ENTRY4-F and ENTRY4-R primers are as follows:

[0100] ENTRY4-F: 5'-GCGTTTCTACAAACTCTTCCTG-3' (SEQ ID NO: 17);

[0101] ENTRY4-R: 5'-TGGGTCTAGATATCTCGAGTG-3' (SEQ ID NO: 18).

[0102] 2) Intermediate vector construction (gRNA1 insertion): Using T4 DNA ligase, gRNA1 is inserted into the intermediate vector. ​ I. The linearized intermediate vector was ligated overnight at 16°C; the constructed recombinant plasmid was introduced into E. coli DH5α competent cells, positive transformants were screened and verified by monoclonal PCR, and plasmid DNA was extracted after liquid culture amplification.

[0103] 3) Final editing vector construction: The intermediate vector plasmid successfully constructed above was recombinated with the gene editing vector pCL4B-Cas9 using the LR reaction. The recombination product was transformed into E. coli competent cells. PCR detection was performed using primers NOS-F and NOS-R to screen for positive single colonies. Plasmids were extracted by shaking and sent for sequencing verification to obtain wheat germ cells. ​ Gene editing vector. The NOS-F and NOS-R primer sequences are as follows:

[0104] NOS-F: 5'-AAGCACATACGTCAGAAACATTAT-3' (SEQ ID NO: 19);

[0105] NOS-R: 5'-TGGGTGAGATTCCTTGAAGTTGAGTA-3' (SEQ ID NO: 20).

[0106] 3. Agrobacterium-mediated genetic transformation: This involves sequencing to verify correct results. ​ Gene editing vectors were transferred into Agrobacterium strain EHA105 to obtain CRISPR / Cas9-gRNA Agrobacterium transformants. Using the immature embryos of the wheat variety Fielder as transformation recipients, Agrobacterium-mediated genetic transformation of wheat callus was performed (transformation-related media: callus induction MS + 2,4-D medium, AAM infection medium, MS co-culture basal medium, MS resistant callus selection medium, MS callus differentiation medium, 1 / 2 MS rooting medium). Transgenic positive plants were obtained by resistance screening (resistance screening reagent: 200 mg / L glufosinate) and PCR detection.

[0107] 4. Validation of knockout plants: Molecular detection and sequencing analysis are performed on the progeny of transgenic positive plants, and genomic DNA is extracted from the plants for analysis. ​ Specific primers were designed for the gene editing target region for PCR amplification. The amplification products were sent to a sequencing company (Beijing Qingke Biotechnology Co., Ltd.) for sequencing. The gene editing was analyzed, and three genes were selected. ​ Homozygous genes successfully edited on copies of chromosomes 5A, 5B, and 5D. ​ The gene knockout lines are designated as TaSnRK2.8-ko#L1, TaSnRK2.8-ko#L2, and TaSnRK2.8-ko#L3. The specific primer sequences are as follows:

[0108] TaSnRK2.8-g1-hitom-F1: 5'-ggagtgagtacggtgtgcCTAGTTCACCACAAAAGAGGAG-3' (SEQ ID NO: 21);

[0109] TaSnRK2.8-g1-hitom-R1: 5'-gagttggatgctggatggGCAGAGTAACCAGAGAGC-3' (SEQ ID NO: 22);

[0110] TaSnRK2.8-g1-hitom-F1: 5'-ggagtgagtacggtgtgcCTAGTTCACCACAAAAGAGGAG-3' (SEQ ID NO: 21);

[0111] TaSnRK2.8-g1-hitom-R2: 5'-gagttggatgctggatggACTTAGTCCCACCAATGATTC-3' (SEQ ID NO: 23);

[0112] TaSnRK2.8-g2-hitom-F1: 5'-ggagtgagtacggtgtgcTGTCACCGTGACTTGAAG-3' (SEQ ID NO: 24);

[0113] TaSnRK2.8-g2-hitom-R1: 5'-gagttggatgctggatggACTGCATGAACATTGACTTAAAT-3' (SEQ ID NO: 25).

[0114] 5. Disease resistance identification: Select stably inherited strains. ​ Gene knockout lines were used, with wild-type wheat Fielder as a control. All lines and control plants were inoculated with the non-toxic race CYR23 of stripe rust. After inoculation, they were cultured in a dark, humid environment for 24 h, followed by conventional culture under light (temperature 20–25℃, light duration 12–16 h / day). Fourteen days after inoculation, the inoculated sites of wheat leaves that had passed the full disease development stage were cut and pasted onto black cardstock, scanned, and photographed. Preliminary identification of disease resistance phenotypes (0–10 grades) was performed. The disease phenotype of each plant was observed and recorded. The relative biomass of stripe rust was detected using real-time quantitative PCR (internal reference gene for stripe rust was [insert gene here]). ​ The wheat internal reference gene is ​ (The primer sequences are the same as in Examples 2 and 3).

[0115] II. Test Results

[0116] Sequencing validation results show that ​ The gene-editing vector was successfully constructed and successfully transformed into Agrobacterium strain EHA105, achieving effective transformation of wheat embryos.

[0117] Sequencing analysis results of progeny of transgenic positive plants are as follows: ​ As shown in the figure, the gene editing status of the three knockout lines TaSnRK2.8-ko#L1, L2, and L3 is illustrated: Fielder-5A, Fielder-5B, and Fielder-5D represent the gene editing of wild-type wheat on chromosomes 5A, 5B, and 5D, respectively. ​ Gene copy sequence; TaSnRK2.8-ko#L1 has 1 base deleted on chromosome 5A, 1 base inserted on chromosome 5B, and 1 base deleted on chromosome 5D; ​-ko#L2 has an 8-base deletion on chromosome 5A, an 1-base insertion on chromosome 5B, and a 1-base deletion on chromosome 5D; TaSnRK2.8-ko#L3 has an 1-base insertion on chromosome 5A, an 1-base deletion on chromosome 5B, and an 4-base insertion on chromosome 5D. All three strains are... ​ Homozygous mutant plants with fully edited genes.

[0118] ​ for ​ Phenotypic identification results (left) and relative biomass statistics (right) of gene knockout wheat plants and control plants after inoculation with stripe rust fungus. Phenotypic observation showed that the wild-type control plant, Fielder, exhibited milder disease severity, while the three knockout lines, TaSnRK2.8-ko#L1, L2, and L3, had more stripe rust spore masses and a wider coverage area on their leaves, indicating a significantly more severe disease phenotype than the control. Real-time quantitative PCR results showed that the relative biomass of stripe rust fungus in the TaSnRK2.8-ko#L1, L2, and L3 lines was significantly higher than that in the wild-type Fielder.

[0119] The results of this embodiment indicate that knockout ​ Following gene modification, wheat's resistance to stripe rust was significantly weakened, while the relative biomass of stripe rust significantly increased, further confirming... ​ Genes play a positive regulatory role in the immune response to wheat stripe rust.

[0120] The above embodiments can well illustrate the technical solution of the present invention, but they are only describing preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, all kinds of changes and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A kind TaSnRK2.8 The application of genes in regulating wheat resistance to stripe rust is characterized by, The TaSnRK2.8 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The TaSnRK2.8 The gene plays a positive regulatory role in the interaction between wheat and stripe rust fungus, and overexpression TaSnRK2.8 Genetic enhancement of wheat's resistance to stripe rust.

2. The application according to claim 1, characterized in that, overexpression TaSnRK2.8 Genes are generated through Agrobacterium-mediated genetic transformation.

3. The application according to claim 2, characterized in that, The TaSnRK2.8 The gene encodes the TaSnRK2.8 protein, the amino acid sequence of which is shown in SEQ ID NO:

2.

4. The application according to claim 3, characterized in that, The TaSnRK2.8 protein is a wheat cytoplasmic serine / threonine protein kinase.

5. A method for breeding a wheat variety resistant to stripe rust, characterized in that, The breeding method includes: overexpression in wheat materials. TaSnRK2.8 Genes, the ones mentioned TaSnRK2.8 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

6. The cultivation method according to claim 5, characterized in that, The recipient of genetic transformation is the wheat embryo.

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