Wheat susceptibility related gene, protein and application

Abstract

The invention belongs to the technical field of gene engineering, and discloses a wheat susceptibility related gene, a protein and application. The wheat susceptibility related gene is a TaBRG10 gene and is derived from a wheat variety water source 11, the nucleotide sequence of the gene is as shown in SEQ ID NO: 1, and the amino acid sequence of protein encoded by the TaBRG10 gene is as shown in SEQ ID NO: 2. The TaBRG10 gene is induced to express by stripe rust, and under the infection of stripe rust, the infection surface of a TaBRG10 gene silent plant infected by stripe rust is positively and obviously lower than that of a control plant, which indicates that the silent TaBRG10 gene improves the resistance of the plant to wheat stripe rust. The invention provides a novel germplasm material for the cultivation of the stripe rust resistant wheat variety.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to a wheat disease susceptibility-related gene, protein and application. Background Art

[0002] Wheat Stripe Rust is caused by Puccinia striatum, a wheat-specific pathogen. Puccinia striiform f. sp . wheat The fungal disease caused by Pst has the characteristics of wide spread and high epidemic frequency. It may occur from wheat seedling to maturity, mainly harming wheat leaves, followed by leaf sheaths and stems. The pathogen of wheat stripe rust mainly infects wheat with asexual summer spores to complete the annual disease cycle, and can also complete its sexual generation on barberry. It can be spread over long distances with air currents, affecting wheat yield and quality.

[0003] At present, the comprehensive control measures for this disease mainly include the cultivation of disease-resistant varieties and the rational use of fungicides. However, in production, the pathogen will continuously mutate to form new pathogenic types or physiological subspecies, causing effective disease-resistant varieties to lose resistance. Susceptibility genes refer to host genes that promote pathogen infection and support affinity interactions. The strategy of improving plant disease resistance by mutating susceptible genes improves the efficiency of disease-resistant breeding and has broad application prospects. Therefore, the exploration of a susceptible gene resource related to wheat stripe rust is of great significance for green disease prevention and control and the cultivation of disease-resistant wheat varieties. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a wheat disease susceptibility-related gene, protein and application. The wheat disease susceptibility-related gene is TaBRG10 Gene, derived from wheat ( Triticum aestivum ) Variety water source 11, whose nucleotide sequence is shown in SEQ ID NO: 1, TaBRG10 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO:2. TaBRG10 Gene expression induced by stripe rust, silenced TaBRG10 The gene improves the resistance of plants to wheat stripe rust. The invention provides a new type of germplasm material for the cultivation of wheat stripe rust resistant varieties.

[0005] To achieve the technical purpose of the present invention, on the one hand, the present invention provides a wheat disease susceptibility-related gene, the wheat disease susceptibility-related gene is TaBRG10 Gene, TaBRG10 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0006] On the other hand, the present invention provides a TaBRG10 protein, comprising the above TaBRG10Gene encoding; the amino acid sequence of the TaBRG10 protein is shown in SEQ ID NO:2.

[0007] On the other hand, the present invention claims TaBRG10 Application of genes in wheat stripe rust resistance, the TaBRG10 The nucleotide sequence of the gene is shown in SEQ ID NO: 1. TaBRG10 The gene expression is induced by stripe rust.

[0008] Specifically, the present invention analyzes TaBRG10 The expression profile of genes in the interaction between wheat and stripe rust found that after wheat water source 11 was inoculated with stripe rust non-compatible race CYR23 and stripe rust compatible race CYR31, TaBRG10 The gene showed an up-regulated expression trend in both the non-compatible and compatible combinations during the early stage of stripe rust infection (3-12h), indicating TaBRG10 The gene expression was induced by stripe rust infection.

[0009] Furthermore, silence TaBRG10 Gene increases plant resistance to wheat stripe rust; Silencing the TaBRG10 The silencing sequence of the gene increases the resistance of the plant to wheat stripe rust. The silencing sequence is a silencing sequence TaBRG10 -S1 or Silent Sequence TaBRG10 -S2; the silent sequence TaBRG10 -S1 nucleotide sequence is as described TaBRG10 The 94th to 235th nucleotides from the 5' end of the gene; the silencing sequence TaBRG10 -S2 nucleotide sequence is as described TaBRG10 The gene is from nucleotides 533 to 694 from the 5' end.

[0010] Specifically, the present invention detects TaBRG10 Gene silencing in plants TaBRG10 The relative expression of genes was found to be: TaBRG10 -S1 plants and BSMV transfection: TaBRG10 -S2 plants TaBRG10 The expression level of the gene was significantly lower than that of the BSMV:γ transgenic plants, indicating that TaBRG10 The expression of the gene was successfully suppressed in the silenced plants, and the silenced sequence TaBRG10 -S1 and TaBRG10 -S2 is valid, silent sequence TaBRG10 -S1 or TaBRG10 -S2 can be significantly downregulated TaBRG10 Gene expression.

[0011] The present invention further analyzes TaBRG10Gene silenced plants are resistant to stripe rust. TaBRG10 The infection area of ​​stripe rust in gene-silenced plants was significantly lower than that in control plants, indicating that TaBRG10 Gene improves wheat's resistance to stripe rust.

[0012] The present invention is TaBRG10 Histological observation of gene silenced plants revealed that under the infection condition of stripe rust CYR31, visible summer spores were observed on all treated leaves. TaBRG10 The severity of stripe rust in gene-silenced plants was weaker than that in control plants, indicating that downregulation TaBRG10 Wheat leaves expressing the gene had improved disease resistance to the physiological race CYR31 of stripe rust, and the number of summer spores produced on the wheat leaves was significantly reduced.

[0013] In addition, the present invention claims a method for cultivating a wheat stripe rust resistant variety, which is silent in the plant. TaBRG10 Gene, the TaBRG10 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0014] Furthermore, the breeding method includes constructing a silencing vector, transferring the silencing vector into wheat, and obtaining the wheat stripe rust resistant variety. The silencing vector includes a silencing sequence TaBRG10 -S1 or Silent Sequence TaBRG10 -S2; the silent sequence TaBRG10 -S1 nucleotide sequence is as described TaBRG10 The 94th to 235th nucleotides from the 5' end of the gene; the silencing sequence TaBRG10 -S2 nucleotide sequence is as described TaBRG10 The gene is from nucleotides 533 to 694 from the 5' end.

[0015] Those skilled in the art can easily adopt known methods, such as directed evolution and point mutation methods, to modify the present invention. TaBRG10 Gene mutation. Those artificially modified, with the separation of the present invention TaBRG10 Nucleotides with 75% or higher identity to the nucleotide sequence of the gene are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode TaBRG10 protein and have the same function.

[0016] The term "identity" as used herein refers to sequence similarity to a naturally occurring nucleic acid sequence. "Identity" includes sequences similar to those of the present invention. TaBRG10The nucleotide sequence of a gene has a nucleotide sequence with 75% or more, or 85% or more, or 90% or more, or 95% or more identity. Identity can be evaluated with the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0017] The above-mentioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0018] TaBRG10 Gene expression cassettes are capable of expressing TaBRG10 The DNA of the gene may include not only the promoter TaBRG10 The promoter of gene transcription may also include the terminator TaBRG10 The terminator of gene transcription. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters; tissue, organ and development-specific promoters and inducible promoters. Transcription terminators that can be used in the present invention include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcSE9 terminator, and nopaline and octopine synthase terminators.

[0019] Existing expression vectors can be used to construct TaBRG10Recombinant vector of gene expression cassette. The plant expression vector includes binary Agrobacterium vector and vectors that can be used for plant genetic transformation, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA1305, pCAMBIA1300, pBI121 or pCUB, etc. The plant expression vector may also contain the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos), the 3' non-translated region of the plant gene (such as the soybean storage protein gene) has similar functions. When using the gene construction plant expression vector of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The source of the translation control signal and the start codon is extensive and can be natural or synthetic. The translation initiation region can come from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptⅡ gene that confers resistance to kanamycin and related antibiotics, bar gene that confers resistance to the herbicide phosphinothricin, hph gene that confers resistance to the antibiotic hygromycin, dhfr gene that confers resistance to methotrexate, EPSPS gene that confers resistance to glyphosate), or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0020] The above-mentioned vector or expression vector can be a plasmid, cosmid, phage or virus vector.

[0021] The microorganisms may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0022] The above transgenic plant cell lines do not include propagation materials.

[0023] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (1) The present invention cloned a gene related to wheat stripe rust from the wheat variety Shuiyuan 11. TaBRG10 A gene, the nucleotide sequence of which is shown in SEQ ID NO: 1, TaBRG10The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO: 2. TaBRG10 The gene was induced to express by stripe rust. After wheat water source 11 was inoculated with stripe rust non-compatible race CYR23 and stripe rust compatible race CYR31, TaBRG10 The gene showed an up-regulated expression trend in both the non-compatible and compatible combinations during the early stage of stripe rust infection (3-12h), indicating TaBRG10 The gene expression was induced by stripe rust infection.

[0024] (2) The present invention provides two silent TaBRG10 The silent sequence of the gene is the silent sequence TaBRG10 -S1 or Silent Sequence TaBRG10 -S2. Silent sequence TaBRG10 -S1 nucleotide sequence is as described TaBRG10 The 94th to 235th nucleotides from the 5' end of the gene; silent sequence TaBRG10 -S2 nucleotide sequence is as described TaBRG10 The gene is from nucleotide 533 to 694 from the 5' end. After inoculation with stripe rust physiological race, transfer to BSMV: TaBRG10 -S1 plants and BSMV transfection: TaBRG10 -S2 plants TaBRG10 The expression level of the gene was significantly lower than that of the BSMV:γ transgenic plants, indicating that TaBRG10 The expression of the gene was successfully suppressed in the silenced plants, and the silenced sequence TaBRG10 -S1 and TaBRG10 -S2 is valid, silent sequence TaBRG10 -S1 or TaBRG10 -S2 can be significantly downregulated TaBRG10 Gene expression.

[0025] (3) Silence TaBRG10 The present invention uses transient silencing technology to obtain TaBRG10 Gene silenced plants, infected with stripe rust, TaBRG10 The infection area of ​​stripe rust in gene-silenced plants was significantly lower than that in control plants, indicating that TaBRG10 The gene improves the resistance of wheat to stripe rust. TaBRG10 The gene provides a basis for the control of wheat stripe rust and provides a new type of germplasm material for the breeding of wheat stripe rust-resistant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 for TaBRG10Analysis of gene expression profiles in the interaction between wheat and stripe rust. Incompatible interactions were inoculated with wheat stripe rust physiological race CYR23; compatible interactions were inoculated with wheat stripe rust physiological race CYR31; "*" indicates that compared with 0h, p Significance at the <0.05 level; "**" indicates that compared with 0h p <0.01 level of significance.

[0027] Figure 2 For the silent sequence TaBRG10 Gene sequence alignment.

[0028] Figure 3 for TaBRG10 Gene silenced plants inoculated with stripe rust races CYR23 and CYR31 TaBRG10 Relative gene expression results. Figure 3 A in the figure is the phenotype diagram of successful virus inoculation; Figure 3 B in the figure is the time after inoculation with stripe rust physiological races CYR23 and CYR31. TaBRG10 Relationship diagram of relative gene expression; MOCK is blank control; BSMV: TaPDS For silence TaPDS gene plants; BSMV:γ is the negative control plant inoculated with BSMV:γ; BSMV: TaBRG10 -S1 and BSMV: TaBRG10 -S2 respectively use silent sequence TaBRG10 -S1 and TaBRG10 -S2 downregulation TaBRG10 Plants expressing the gene; “*” indicates p <0.05 level of significance.

[0029] Figure 4 for TaBRG10 Histological observation of gene silenced plants after inoculation with stripe rust. Figure 4 A in the figure is the mycelial growth at 48h and 120h after inoculation of stripe rust; Figure 4 B in the figure is the length of the haustorium of the stripe rust at the infection point 48 hours after inoculation of the stripe rust; Figure 4 C in the figure is the number of haustoria mother cells of stripe rust at the infection point 48 hours after inoculation of stripe rust; Figure 4 D in the figure is the infection area of ​​stripe rust at the infection point 48h and 120h after inoculation. p <0.01 level of significance.

[0030] Figure 5 for TaBRG10 Phenotypic analysis of gene silenced plants after inoculation with stripe rust races CYR23 and CYR31. Figure 5 A in TaBRG10Phenotype of gene silenced plants after inoculation with CYR23; Figure 5 B in TaBRG10 Phenotype of gene silenced plants after inoculation with CYR31; Figure 5 C in TaBRG10 Biomass analysis of gene silenced plants inoculated with CYR23 and CYR31. BSMV:γ is the negative control plant inoculated with BSMV:γ; BSMV: TaBRG10 -S1 and BSMV: TaBRG10 -S2 respectively use silent sequence TaBRG10 -S1 and TaBRG10 -S2 downregulation TaBRG10 “*” indicates plants expressing p <0.05 level of significance. DETAILED DESCRIPTION

[0031] The technical scheme of the present invention is now described in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0032] Example 1 This embodiment provides TaBRG10 Acquisition of gene and TaBRG10 protein.

[0033] 1. TaBRG10 Gene acquisition Wheat seedlings from Shuiyuan 11 that had grown normally for 7 days were taken, quickly frozen with liquid nitrogen, and stored at -80°C for later use. The total RNA of wheat leaves was extracted using the Trizol method (TianGen), and the first-strand cDNA was synthesized using reverse transcriptase XL (AMV). The cDNA was synthesized using the SMART method, and amplification primers (TaBRG10-F: 5'-ATGGCCTTCTTCTCCCACCA-3'; TaBRG10-R: 5'-CATATATATCTCCATGCCAA-3') were designed for PCR amplification. The PCR products were detected by 1.0% agarose gel electrophoresis. A PCR product of 1017 bp was obtained, which was TaBRG10 gene, after sequencing, the PCR product has a nucleotide sequence as shown in SEQ ID NO: 1, TaBRG10 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO:2.

[0034] Example 2 This embodiment provides TaBRG10 Expression profiling of genes in the interaction between wheat and stripe rust.

[0035] Wheat was inoculated with stripe rust at the one-leaf and one-heart stage. The inoculation method was based on the reference "Discovery of a New Normal-Temperature Pathogenic Bacterial Strain of Lovelin 10" (Kang Zhensheng, Li Zhenqi). Wheat water source 11 leaves were inoculated with non-compatible race CYR23 and compatible race CYR31, respectively, and sterile water was used as the control group. Samples were taken at 0h, 3h, 6h, 9h, 12h, 18h, 24h, 48h, 72h, 96h, and 120h after inoculation, and the sampling time point of the control group was consistent with that of the treatment group. When sampling, fresh leaves were cut, wrapped in tin-platinum paper, and quickly frozen in liquid nitrogen, and then stored at -80℃ for later use. The Trizol method (TianGen) was used to extract total RNA from wheat leaves, and reverse transcriptase XL (AMV) was used for first-strand cDNA synthesis. The SMART method was used to synthesize cDNA. According to the wheat TaBRG10 elongation factor gene TaEF -1α sequence-specific quantitative PCR primers were designed.

[0036] Before using quantitative PCR primers, the specificity and amplification efficiency of their amplification products must be tested (≥90%). TaEF -1α was used as an internal reference gene in qRT-PCR analysis. AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and Bio-Rad CFX Manager quantitative PCR instrument (Bio-rad, Hercules, California) were used to perform real-time quantitative PCR amplification using cDNA from each treatment sampling point as template. Each reaction was repeated at least 3 times, and the Ct value of each repeat and its mean and standard deviation were generated by the quantitative PCR instrument by manually adjusting the baseline. Each reaction was repeated 3 times, and the Ct value was averaged. The experimental data were analyzed using the Delta Delta Ct method to determine TaBRG10 The relative expression of genes is shown in Figure 1 shown.

[0037] The qRT-PCR primer sequences are: Q TaBRG10 -F: 5'-CAGTGAGGTAGACGATACAGCATCC–3'; Q TaBRG10 -R:5'-ACGCCTCGCTTGATTTACAGACC-3'.

[0038] Q TaEF -F:5'-TGGTGTCATCAAGCCTGGTATGGT-3'; Q TaEF -R:5'-ACTCATGGTGCATCTCAACGGACT-3'.

[0039] Depend on Figure 1 It can be seen that after wheat water source 11 was inoculated with the non-compatible stripe rust race CYR23 and the compatible stripe rust race CYR31, TaBRG10 The gene showed an up-regulated expression trend in both the non-compatible and compatible combinations during the early stage of stripe rust infection (3-12h), indicating TaBRG10 The gene expression was induced by stripe rust infection.

[0040] Example 3 This example provides a method for using VIGS technology to TaBRG10 Role of genes in wheat stripe rust resistance.

[0041] 1. TaBRG10 Construction of BSMV-VIGS vector system (1) Acquisition of silent sequences Silent sequence TaBRG10 -S1 acquisition: amplified in Example 1 TaBRG10 Gene as template, using primer pair TaBRG10 -S1F (5'-TAGCTAGCTGATTAATTAAAGGAACGCGCTGCCGGTGCC-3', where TTAATTAA is a restriction endonuclease recognition site) and TaBRG10 -S1R (5'-TTGCTAGCTGAGCGGCCGCACCCCATACCCGCCGCAGCC-3', where GCGGCCGC is the restriction endonuclease recognition site) was PCR amplified to obtain a PCR amplification product of 142 bp (corresponding to nucleotides 94 to 235 from the 5' end in SEQ ID NO: 1) ( Figure 2 ), which is named as the silent sequence TaBRG10 -S1.

[0042] Silent sequence TaBRG10 -S2 acquisition: amplified in Example 1 TaBRG10 Gene as template, using primer pair TaBRG10 -S2F (5'-TAGCTAGCTGATTAATTAAAGCAGTTTGAAGCGCTGGCC-3', where TTAATTAA is a restriction endonuclease recognition site) and TaBRG10 -S2R (5'-TTGCTAGCTGAGCGGCCGCTGTACTTAGCTCGTTGCTGC-3', where GCGGCCGC is the restriction endonuclease recognition site) was PCR amplified to obtain a PCR amplification product of 162 bp (corresponding to nucleotides 533 to 694 from the 5' end in SEQ ID NO: 1) ( Figure 2 ), which is named as the silent sequence TaBRG10 -S2.

[0043] (2) Construction of silencing vector γ- TaBRG10 - Construction of S1 silencing vector: Use PacI and NotI to digest the silencing sequence respectively TaBRG10 -S1 and BSMV-VIGS virus vector γ, the silencing sequence after enzyme cutting TaBRG10 -S1 was connected with the BSMV-VIGS virus vector γ vector backbone after enzyme digestion to obtain the recombinant vector γ- TaBRG10 -S1 (silent sequence TaBRG10 -S1 replaces the fragment between PacI and NotI restriction sites in BSMV-VIGS viral vector γ), in the recombinant vector TaBRG10 -S1 with TaBRG10 The recombinant vector γ- TaBRG10 -S1 was amplified by PCR and sequenced for identification. The positive clone vector was TaBRG10 The gene cDNA sequence is inserted between the PacI and NotI restriction sites of the γ chain of the BSMV-VIGS viral vector in the opposite direction of gene expression from the 94th to 235th nucleotides of the 5' end, and the other sequences of the BSMV-VIGS viral vector γ remain unchanged.

[0044] γ- TaBRG10 - Construction of S2 silencing vector: Use PacI and NotI to digest the silencing sequence respectively TaBRG10 -S2 and BSMV-VIGS virus vector γ, the silencing sequence after enzyme cutting TaBRG10 -S2 was connected with the BSMV-VIGS virus vector γ vector backbone after enzyme digestion to obtain the recombinant vector γ- TaBRG10 -S2 (silent sequence TaBRG10 -S2 replaces the fragment between PacI and NotI restriction sites in BSMV-VIGS viral vector γ), in the recombinant vector TaBRG10 -S2 with TaBRG10 The gene expression direction is opposite. TaBRG10 -S2 was amplified by PCR and sequenced for identification. The positive clone vector was TaBRG10 The gene cDNA sequence was inserted between the PacI and NotI restriction sites of the γ chain of the BSMV-VIGS viral vector in the opposite direction of gene expression from the 533rd to 694th nucleotides of the 5' end, and the other sequences of the BSMV-VIGS viral vector γ were kept unchanged.

[0045] 2. In vitro transcription of BSMV Linearization of the vector: Use MluI to digest the BSMV viral vector α and γ, and use BssHⅡ to digest the recombinant vector γ- TaBRG10 -S1 and recombinant vector γ- TaBRG10 -S2, the β chain of BSMV viral vector was cut with SpeI to obtain linearized plasmids.

[0046] BSMV in vitro transcription: The above linearized plasmid was used as a template for in vitro transcription to obtain in vitro transcribed BSMV viral vectors α, β, γ, γ- TaBRG10 -S1 and γ- TaBRG10 -S2. In vitro transcription was performed according to the instructions of RiboMAXTM LargeScale RNA Production System-T7 (purchased from Promega, catalog number: P1300). The reaction system and reaction conditions for in vitro transcription were as follows: total reaction volume 20.0 μL, including 6.5 μL of linearized plasmid, 4.0 μL of 5× Transcription Buffer, 1.5 μL of Cap (purchased from Promega, catalog number: P1718), 6.0 μL of rNTP PreMix, 2.0 μL of Enzyme Mix, reaction at 37°C for 4 h, and the transcription product was stored at -80°C for later use.

[0047] 3. BSMV inoculation The in vitro transcribed BSMV-VIGS vectors α, β and γ were diluted 3 times with DEPC water and mixed in equal amounts, and then 6 volumes of 1×FES Buffer were added to obtain the BSMV:γ recombinant viral vector solution. TaBRG10 -S1 and γ-TaBRG10-S2 were diluted 3 times with DEPC water and mixed in equal amounts, and then 6 times the volume of 1×FESBuffer was added to obtain BSMV: TaBRG10 -S1 recombinant viral vector solution or BSMV: TaBRG10 -S2 recombinant viral vector solution.

[0048] Wheat water source 11 was sown in nutrient soil and when it grew to the two-leaf stage, 10 μL of BSMV:γ and BSMV: TaBRG10 -S1 and BSMV: TaBRG10 -S2 recombinant virus vector solution was smeared and inoculated on the flat second leaf of wheat. After 10 minutes, DEPC water was sprayed and the temperature was adjusted to 25℃ to keep moist for 24 hours. Then it was transferred to normal conditions at 25℃ to obtain BSMV:γ transgenic plants and BSMV: TaBRG10 -S1 plants and BSMV transfection: TaBRG10-S2 plants, some plants were coated with 1×FESBuffer to obtain simulated inoculated plants.

[0049] 4. TaBRG10 Gene silencing in plants TaBRG10 Relative expression of genes The above-mentioned BSMV:γ plants and BSMV: TaBRG10 -S1 plants and BSMV transfection: TaBRG10 -S2 plants and mock-inoculated plants (MOCK) were cultured under normal conditions for 10 days and then inoculated with stripe rust physiological races CYR23 and CYR31. The phenotypes after inoculation are shown in Figure 2. Figure 3 As shown in A. Samples were taken at 0h, 24h, 48h, and 120h after inoculation for RNA extraction and reverse transcription to synthesize cDNA. The synthesized cDNA was used as a template and qRT-PCR detection was performed according to the method of Example 1. TaBRG10 The relative expression of genes ( Figure 3 B in the figure).

[0050] Depend on Figure 3 From B, we can see that after inoculation with stripe rust physiological species, BSMV was transformed: TaBRG10 -S1 plants and BSMV transfection: TaBRG10 -S2 plants TaBRG10 The expression level of the gene was significantly lower than that of the BSMV:γ transgenic plants, indicating that TaBRG10 The expression of the gene was successfully suppressed in the silenced plants, and the silenced sequence TaBRG10 -S1 and TaBRG10 -S2 is valid, silent sequence TaBRG10 -S1 or TaBRG10 -S2 can be significantly downregulated TaBRG10 Gene expression.

[0051] 5. TaBRG10 Analysis of Stripe Rust Resistance in Gene Silenced Plants Will effectively silence TaBRG10 Gene expression plants (BSMV: TaBRG10 -S1 and BSMV: TaBRG10 -S2), negative control plants (BSMV:γ), and inoculated with stripe rust physiological races CYR23 and CYR31 after the third leaf was unfolded. Samples were taken at 24h, 48h, and 120h after inoculation and WGA staining was performed to observe the development of stripe rust ( Figure 4 ), 30 infection points were counted each time, and three biological replicates were performed. The disease incidence was observed 14 days after inoculation ( Figure 5 ).

[0052] Depend on Figure 4 It can be seen that under the infection of stripe rust, TaBRG10The infection area of ​​stripe rust in gene-silenced plants was significantly lower than that in control plants, indicating that TaBRG10 Gene improves wheat's resistance to stripe rust.

[0053] Depend on Figure 5 It can be seen that under the infection condition of stripe rust CYR23, there was no significant difference in the phenotype of leaves in all treatments. Under the infection condition of stripe rust CYR31, visible summer spores were observed on the leaves of all treatments. TaBRG10 The severity of stripe rust in gene-silenced plants was weaker than that in control plants, indicating that downregulation TaBRG10 Wheat leaves expressing the gene had improved disease resistance to the physiological race CYR31 of stripe rust, and the number of summer spores produced on the wheat leaves was significantly reduced.

[0054] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of the preferred embodiments of the present invention, and the present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. TaBRG10 The application of the gene in wheat stripe rust resistance is characterized in that: Said TaBRG10 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: Said TaBRG10 The gene expression is induced by stripe rust.

3. The use according to claim 1, characterized in that: Silence TaBRG10 Genes improve wheat resistance to stripe rust.

4. The use according to claim 1, characterized in that: Silence TaBRG10 Gene silencing sequences improve wheat resistance to stripe rust.

5. The use according to claim 4, characterized in that: The silent sequence is a silent sequence TaBRG10 -S1 or Silent Sequence TaBRG10 -S2; The silencing sequence TaBRG10 -S1 nucleotide sequence is as described TaBRG10 The gene is from nucleotides 94 to 235 from the 5' end; The silencing sequence TaBRG10 -S2 nucleotide sequence is as described TaBRG10 The gene is from nucleotides 533 to 694 from the 5' end.

6. A method for breeding wheat varieties resistant to stripe rust, characterized in that: Silence in the plant TaBRG10 Gene, the TaBRG10 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

7. The cultivation method according to claim 6, characterized in that: The method comprises constructing a silencing vector, transferring the silencing vector into wheat, and obtaining the wheat stripe rust resistant variety.

8. The cultivation method according to claim 7, characterized in that: The silencing vector comprises a silencing sequence TaBRG10 -S1 or Silent Sequence TaBRG10 -S2; The silencing sequence TaBRG10 -S1 nucleotide sequence is as described TaBRG10 The gene is from nucleotides 94 to 235 from the 5' end; The silencing sequence TaBRG10 -S2 nucleotide sequence is as described TaBRG10 The gene is from nucleotides 533 to 694 from the 5' end.

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