A wheat disease-susceptibility related gene, protein and application

Abstract

The present invention belongs to the technical field of genetic engineering, and discloses a wheat disease-susceptibility related gene, protein and application. The wheat disease-susceptibility related gene is TaBRG10 a gene derived from the wheat variety Suwon 11, and its nucleotide sequence is as shown in SEQ ID NO:1. TaBRG10 The amino acid sequence of the protein encoded by the gene is as shown in SEQ ID NO:2. TaBRG10 The gene is induced to express by stripe rust, and under the infection of stripe rust TaBRG10 the area of the gene-silenced plants infected by stripe rust is significantly lower than that of the control plants, indicating that silencing TaBRG10 the gene improves the resistance of plants to wheat stripe rust. The present invention provides a novel germplasm material for the cultivation of wheat varieties resistant to stripe rust.

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 striiformis f. sp . tritici 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 The application of the gene in wheat resistance to stripe rust, and the TaBRG10 The nucleotide sequence of the gene is shown in SEQ ID NO: 1. The TaBRG10 Gene is induced to express by stripe rust fungus.

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

[0009] Furthermore, silencing the TaBRG10 Gene enhances the resistance of plants to wheat stripe rust; silencing the TaBRG10 Silencing sequence of the gene enhances the resistance of plants to wheat stripe rust. The silencing sequence is silencing sequence TaBRG10 -S1 or silencing sequence TaBRG10 -S2; the nucleotide sequence of the silencing sequence TaBRG10 -S1 is the 94th to 235th nucleotides of the TaBRG10 Gene from the 5' end; the nucleotide sequence of the silencing sequence TaBRG10 -S2 is the 533rd to 694th nucleotides of the TaBRG10 Gene from the 5' end.

[0010] Specifically, the present invention detected TaBRG10 The relative expression level of the TaBRG10 Gene in gene - silenced plants and found that after inoculating with the physiological race of stripe rust fungus, in the plants transformed with BSMV: TaBRG10 -S1 and the plants transformed with BSMV: TaBRG10 -S2, the TaBRG10 Expression level of the gene was significantly lower than that in the plants transformed with BSMV:γ, indicating TaBRG10 The expression level of the gene was successfully inhibited in the silenced plants, and the silencing sequences TaBRG10 -S1 and TaBRG10 -S2 are both effective, and the silencing sequence TaBRG10 -S1 or TaBRG10 -S2 can significantly down - regulate the TaBRG10 Expression of the gene.

[0011] The present invention further analyzed TaBRG10The stripe rust resistance of the gene-silenced plants was found. Under the infection of Puccinia striiformis f. sp. tritici, TaBRG10 the infected area of the gene-silenced plants by Puccinia striiformis f. sp. tritici was significantly lower than that of the control plants, indicating that silencing TaBRG10 the gene improved the resistance of wheat to Puccinia striiformis f. sp. tritici.

[0012] In this invention, TaBRG10 histological observation of the gene-silenced plants found that under the infection condition of Puccinia striiformis f. sp. tritici CYR31, visible uredinia were observed on the leaves of all treatments. TaBRG10 The severity of stripe rust in the gene-silenced plants was weaker than that of the control plants, indicating that the wheat leaves with down-regulated TaBRG10 gene expression had improved resistance to the physiological race CYR31 of Puccinia striiformis f. sp. tritici, and the number of uredinia produced on the wheat leaves was significantly reduced.

[0013] Moreover, this invention claims a method for cultivating a wheat variety resistant to stripe rust, which silences TaBRG10 the gene in the plant, and the nucleotide sequence of the TaBRG10 gene is shown as SEQ ID NO:1.

[0014] Furthermore, the cultivation method includes constructing a silencing vector, transferring the silencing vector into wheat to obtain the wheat variety resistant to stripe rust. The silencing vector includes the silencing sequence TaBRG10 -S1 or the silencing sequence TaBRG10 -S2; the nucleotide sequence of the silencing sequence TaBRG10 -S1 is the 94th to 235th nucleotides from the 5'-end of the TaBRG10 gene; the nucleotide sequence of the silencing sequence TaBRG10 -S2 is the 533rd to 694th nucleotides from the 5'-end of the TaBRG10 gene.

[0015] Those of ordinary skill in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the TaBRG10 gene of this invention. Those nucleotides that have been artificially modified and have a nucleotide sequence identity of 75% or higher with the nucleotide sequence of the TaBRG10 gene isolated from this invention, as long as they encode the TaBRG10 protein and have the same function, are all derived from the nucleotide sequence of this invention and are equivalent to the sequence of this invention.

[0016] The term "identity" used here refers to the sequence similarity with the natural nucleic acid sequence. "Identity" includes the TaBRG10A nucleotide sequence of a gene having 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity. Identity can be evaluated by 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 identity of 75% or more can be 80%, 85%, 90% or 95% or more identity.

[0018] TaBRG10 A gene expression cassette refers to DNA that can express a gene in a host cell. TaBRG10 The DNA of the gene may not only include a promoter for initiating gene transcription, but also include TaBRG10 a promoter for initiating gene transcription, and may also include a terminator for TaBRG10 terminating 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 terminator.

[0019] An existing expression vector can be used to construct a vector containing the above-mentioned TaBRG10Recombinant vector of gene expression cassette. The plant expression vector includes binary Agrobacterium vectors and vectors that can be used for plant genetic transformation, etc., such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA1305, pCAMBIA1300, pBI121 or pCUB, etc. The plant expression vector may also contain the 3'-untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor. For example, the non-translated regions transcribed at the 3' end of the Agrobacterium tumefaciens Ti plasmid gene (such as the nopaline synthase gene Nos) and plant genes (such as soybean storage protein genes) have similar functions. When constructing a plant expression vector using the gene of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the start codon in the adjacent region, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene. For the convenience of identifying and screening transgenic plant cells or plants, the used plant expression vector can be processed, such as adding a gene encoding an enzyme or a luminescent compound that can produce a color change and can be expressed in plants (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (such as the nptⅡ gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, the hph gene that confers resistance to the antibiotic hygromycin, and the dhfr gene that confers resistance to methotrexate, the EPSPS gene that confers resistance to glyphosate) or a marker gene for anti-chemical reagents (such as an anti-herbicide gene), and a mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened by adversity.

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

[0021] The above-mentioned microorganism can be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0022] The above-mentioned 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:

[0024] (1) In the present invention, a gene related to wheat stripe rust was cloned from the wheat variety Shuaiyuan 11, TaBRG10 and its 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 The gene is induced by Puccinia striiformis f. sp. tritici. After inoculating the wheat cultivar Suyuan 11 with the non-compatible race CYR23 and the compatible race CYR31 of Puccinia striiformis f. sp. tritici, TaBRG10 the gene showed an up-regulated expression trend during the early stage (3 - 12 h) of Puccinia striiformis f. sp. tritici infection in both non-compatible and compatible combinations, indicating that TaBRG10 the gene is induced by Puccinia striiformis f. sp. tritici infection.

[0025] (2)The present invention provides two silencing TaBRG10 sequences for the gene, which are silencing sequence TaBRG10 -S1 or silencing sequence TaBRG10 -S2. The nucleotide sequence of silencing sequence TaBRG10 -S1 is the 94th to 235th nucleotides of the TaBRG10 gene starting from the 5'-end; the nucleotide sequence of silencing sequence TaBRG10 -S2 is the 533rd to 694th nucleotides of the TaBRG10 gene starting from the 5'-end. After inoculating with the physiological race of Puccinia striiformis f. sp. tritici, in the plants transformed with BSMV: TaBRG10 -S1 and the plants transformed with BSMV: TaBRG10 -S2, the TaBRG10 gene expression level was significantly lower than that in the plants transformed with BSMV:γ, indicating that TaBRG10 the gene expression was successfully inhibited in the silenced plants, and both silencing sequences TaBRG10 -S1 and TaBRG10 -S2 are effective. Either silencing sequence TaBRG10 -S1 or TaBRG10 -S2 can significantly down-regulate the TaBRG10 gene expression.

[0026] (3)Silencing the TaBRG10 gene enhances the resistance of plants to wheat stripe rust. The present invention obtained TaBRG10 gene-silenced plants by using transient silencing technology. Under the infection of Puccinia striiformis f. sp. tritici, the infection area of the TaBRG10 gene-silenced plants by Puccinia striiformis f. sp. tritici was significantly lower than that of the control plants, indicating that silencing the TaBRG10 gene enhanced the resistance of wheat to Puccinia striiformis f. sp. tritici. The TaBRG10 gene provided by the present invention provides a basis for the control of wheat stripe rust and a new germplasm material for the breeding of wheat varieties resistant to stripe rust. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 For TaBRG10Expression profile analysis diagram of genes in the interaction between wheat and Puccinia striiformis f. sp. tritici. Incompatible interaction: inoculation with the physiological race CYR23 of Puccinia striiformis f. sp. tritici; Compatible interaction: inoculation with the physiological race CYR31 of Puccinia striiformis f. sp. tritici; "*" indicates significance at the p 0.05 level compared with 0 h; "**" indicates significance at the p 0.01 level compared with 0 h.

[0028] Figure 2 For the alignment diagram of the silencing sequence and the TaBRG10 gene sequence.

[0029] Figure 3 For TaBRG10 relative expression level results diagram of the TaBRG10 gene in plants with Figure 3 gene silencing after inoculation with the physiological races CYR23 and CYR31 of Puccinia striiformis f. sp. tritici. Figure 3 In A of TaBRG10 is the phenotype diagram of successful virus inoculation; TaPDS In B of TaPDS is the relationship diagram between time and the relative expression level of the TaBRG10 -S1 and BSMV: TaBRG10 -S2 are plants with down-regulated TaBRG10 -S1 and TaBRG10 -S2 of the TaBRG10 gene expression using the silencing sequences p respectively; "*" indicates significance at the

[0030] Figure 4 For TaBRG10 histological observation diagram of plants with Figure 4 gene silencing after inoculation with Puccinia striiformis f. sp. tritici. Figure 4 In A of Figure 4 is the hyphal growth diagram at 48 h and 120 h after inoculation with Puccinia striiformis f. sp. tritici; Figure 4 In B of p is the haustorium length of Puccinia striiformis f. sp. tritici at the infection site at 48 h after inoculation;

[0031] Figure 5 For TaBRG10 phenotype analysis diagram of plants with Figure 5 gene silencing after inoculation with the physiological races CYR23 and CYR31 of Puccinia striiformis f. sp. tritici. TaBRG10 Figure 5 In A of TaBRG10Phenotype diagram of gene-silenced plants inoculated with CYR23; Figure 5 B in TaBRG10 Phenotype diagram of gene-silenced plants inoculated with CYR31; Figure 5 C in TaBRG10 Biomass analysis diagram 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 are respectively plants with down-regulated TaBRG10 -S1 and TaBRG10 -S2 using the silencing sequence TaBRG10 gene expression. "*" indicates significance at the p <0.05 level. Specific implementation mode

[0032] The technical solutions of the present invention will now be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments. The experimental methods and detection methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials are all commercially available unless otherwise specified.

[0033] Example 1

[0034] This example provides TaBRG10 obtaining of the

[0035] 1. TaBRG10 Obtaining of the gene

[0036] Take the seedlings of wheat Shuiai 11 that have grown normally for 7 days, quickly freeze them with liquid nitrogen, and store them at -80°C for later use. Extract the total RNA of wheat leaves using the Trizol method (TianGen), and use reverse transcriptase XL (AMV) for the synthesis of the first-strand cDNA. Synthesize cDNA using the SMART method, design amplification primers (TaBRG10-F: 5'-ATGGCCTTCTTCTCCCACCA-3'; TaBRG10-R: 5'-CATATATATCTCCATGCCAA-3'), perform PCR amplification, and detect the PCR product by 1.0% agarose gel electrophoresis. A 1017bp PCR product is obtained, which is the TaBRG10 gene. After sequencing, this PCR product has the nucleotide sequence shown in SEQ ID NO:1, TaBRG10 and the amino acid sequence of the protein encoded by the

[0037] gene is shown in SEQ ID NO:2.

[0038] This example provides TaBRG10Expression Profile Analysis of Genes in the Interaction between Wheat and Puccinia striiformis f. sp. tritici

[0039] At the one-leaf and one-heart stage of wheat, inoculate with Puccinia striiformis f. sp. tritici. The inoculation method refers to the literature "Discovery of a New Virulent Strain of Puccinia striiformis f. sp. tritici on Lovrin 10 at Normal Temperature" (Kang Zhensheng, Li Zhenqi). Leaves of wheat variety Shuiai 11 were inoculated with the non-affinity race CYR23 and the affinity race CYR31 respectively, and the group inoculated with sterile water was used as the control group. Samples were taken at 0 h, 3 h, 6 h, 9 h, 12 h, 18 h, 24 h, 48 h, 72 h, 96 h, and 120 h after inoculation respectively, and the sampling time points of the control group were consistent with those of the treatment group. When sampling, cut fresh leaves, wrap them with tin foil and quickly freeze them in liquid nitrogen, and then store them at -80 °C for later use. Total RNA of wheat leaves was extracted by the Trizol method (TianGen), and the first-strand cDNA was synthesized using reverse transcriptase XL (AMV). cDNA was synthesized by the SMART method. According to the wheat TaBRG10 gene and the elongation factor gene TaEF -1α sequences, specific quantitative PCR primers were designed.

[0040] Before using the quantitative PCR primers, the specificity and amplification efficiency (≥90%) of their amplification products need to be detected. TaEF -1α was used as an internal reference gene in qRT-PCR analysis. Using AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and Bio-Rad CFX Manager quantitative PCR instrument (Bio-rad, Hercules, California), real-time quantitative PCR amplification was carried out using the cDNA of each treatment sampling point as a template respectively. Each reaction was repeated at least 3 times, and the Ct values, their averages and standard deviations of each repetition were generated by the quantitative PCR instrument by manually adjusting the baseline. Each reaction was repeated 3 times, and the average Ct value was taken. The experimental data was analyzed by the Delta Delta Ct method to determine TaBRG10 the relative expression level of the gene, and the results are as Figure 1 shown.

[0041] The qRT-PCR primer sequences are as follows:

[0042] Q TaBRG10 -F: 5’-CAGTGAGGTAGACGATACAGCATCC–3’;

[0043] Q TaBRG10 -R: 5’-ACGCCTCGCTTGATTTACAGACC-3’.

[0044] Q TaEF -F: 5’-TGGTGTCATCAAGCCTGGTATGGT-3’;

[0045] Q TaEF -R:5'-ACTCATGGTGCATCTCAACGGACT-3'.

[0046] 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.

[0047] Example 3

[0048] This example provides a method for using VIGS technology to TaBRG10 Role of genes in wheat stripe rust resistance.

[0049] 1. TaBRG10 Construction of BSMV-VIGS vector system

[0050] (1) Acquisition of silent sequences

[0051] 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.

[0052] 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 recognition site of the restriction endonuclease) was used for PCR amplification 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 was named the silencing sequence TaBRG10 -S2.

[0053] (2) Construction of the silencing vector

[0054] γ- TaBRG10 -S1 silencing vector construction: The silencing sequence TaBRG10 -S1 and the BSMV-VIGS viral vector γ were digested with PacI and NotI respectively. The digested silencing sequence TaBRG10 -S1 was ligated to the backbone of the digested BSMV-VIGS viral vector γ to obtain the recombinant vector γ- TaBRG10 -S1 (the fragment between the PacI and NotI cleavage sites in the BSMV-VIGS viral vector γ was replaced by the silencing sequence TaBRG10 -S1). In the recombinant vector, TaBRG10 -S1 and TaBRG10 have opposite gene expression directions. Using the primer pair γ-F (5'-AAAGTGAGGTTAACGCAATACG-3') and γ-R (5'-TCAGGCATCGTTTTCAAGTT-3') for PCR amplification and sequencing identification of the recombinant vector γ- TaBRG10 -S1, the positive clone vector is the cDNA sequence of the TaBRG10 gene from nucleotides 94 to 235 from the 5' end inserted in the opposite direction of gene expression between the PacI and NotI cleavage sites of the γ strand of the BSMV-VIGS viral vector, and the other sequences of the BSMV-VIGS viral vector γ remain unchanged.

[0055] γ- TaBRG10 -S2 silencing vector construction: The silencing sequence TaBRG10 -S2 and the BSMV-VIGS viral vector γ were digested with PacI and NotI respectively. The digested silencing sequence TaBRG10 -S2 was ligated to the backbone of the digested BSMV-VIGS viral vector γ to obtain the recombinant vector γ- TaBRG10 -S2 (the fragment between the PacI and NotI cleavage sites in the BSMV-VIGS viral vector γ was replaced by the silencing sequence TaBRG10 -S2). In the recombinant vector, TaBRG10 -S2 and TaBRG10The gene expression direction is opposite. Using the primer pair γ-F and γ-R to perform PCR amplification and sequencing identification on the recombinant vector γ- TaBRG10 -S2, the positive clone vector is the one in which the cDNA sequence of the TaBRG10 gene from the 533rd to 694th nucleotides at the 5' end is inserted into the PacI and NotI restriction sites of the γ chain of the BSMV-VIGS virus vector in the opposite direction of gene expression, while keeping the other sequences of the BSMV-VIGS virus vector γ unchanged.

[0056] 2. In vitro transcription of BSMV

[0057] Linearization of the vector: Use MluI to digest the BSMV virus vectors α and γ respectively, use BssHⅡ to digest the recombinant vectors γ- TaBRG10 -S1 and the recombinant vector γ- TaBRG10 -S2, and use SpeI to digest the β chain of the BSMV virus vector to obtain linearized plasmids respectively.

[0058] In vitro transcription of BSMV: Using the above linearized plasmids as templates for in vitro transcription to obtain in vitro transcribed BSMV virus vectors α, β, γ, γ- TaBRG10 -S1 and γ- TaBRG10 -S2. The in vitro transcription is carried out according to the instructions of RiboMAX™ Large Scale RNA Production System-T7 (purchased from Promega, catalog number: P1300). The reaction system and reaction conditions for in vitro transcription are: the total reaction volume is 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, react at 37 °C for 4 h, and store the transcription products at -80 °C for standby.

[0059] 3. Inoculation of BSMV

[0060] Dilute the in vitro transcribed BSMV-VIGS vectors α, β and γ 3-fold with DEPC water and mix them equally, then add 6 volumes of 1× FES Buffer to obtain the BSMV:γ recombinant virus vector solution. Dilute the in vitro transcribed BSMV-VIGS vectors α, β and γ- TaBRG10 -S1, γ-TaBRG10-S2 3-fold with DEPC water and mix them equally, then add 6 volumes of 1× FES Buffer to obtain the BSMV: TaBRG10 -S1 recombinant virus vector solution or BSMV: TaBRG10 -S2 recombinant virus vector solution.

[0061] The wheat seeds of water source 11 were sown in nutrient soil. When they grew to the two-leaf stage, 10 μL of BSMV:γ, BSMV: TaBRG10 -S1 and BSMV: TaBRG10 -S2 recombinant virus vector solutions were respectively smeared and inoculated on the second flat leaf of wheat. After 10 min, DEPC water was sprayed, and the temperature was adjusted to 25 °C for moisturizing for 24 h. Then, they were cultured under normal conditions at 25 °C to obtain BSMV:γ-transformed plants, BSMV: TaBRG10 -S1-transformed plants and BSMV: TaBRG10 -S2-transformed plants. Some plants were smeared with 1×FES Buffer to obtain mock-inoculated plants.

[0062] 4. TaBRG10 Relative expression level of the TaBRG10 gene in gene-silenced plants

[0063] The above-mentioned BSMV:γ-transformed plants, BSMV: TaBRG10 -S1-transformed plants and BSMV: TaBRG10 -S2-transformed plants and mock-inoculated plants (MOCK) were cultured under normal conditions for 10 days, and then inoculated with the stripe rust pathogen physiological races CYR23 and CYR31. The phenotypic diagrams after inoculation are as shown in Figure 3 A in it. Samples were taken at 0 h, 24 h, 48 h, and 120 h after inoculation for RNA extraction and reverse transcription to synthesize cDNA. Using the synthesized cDNA as a template, qRT-PCR detection was carried out according to the method of Example 1 to detect TaBRG10 the relative expression level of the gene ( Figure 3 B in it).

[0064] As can be seen from Figure 3 B in it, after inoculation with the stripe rust pathogen physiological races, the expression levels of the TaBRG10 gene in BSMV: TaBRG10 -S1-transformed plants and BSMV: TaBRG10 -S2-transformed plants were significantly lower than those in BSMV:γ-transformed plants, indicating that TaBRG10 the expression level of the TaBRG10 gene was successfully inhibited in the gene-silenced plants, and the silencing sequences TaBRG10 -S1 and TaBRG10 -S2 were both effective. Either the silencing sequence TaBRG10 -S1 or TaBRG10 -S2 could significantly down-regulate the expression of the

[0065] 5. TaBRG10 Analysis of stripe rust resistance of gene-silenced plants

[0066] The plants with effectively silenced TaBRG10 gene expression (BSMV: TaBRG10-S1 and BSMV: TaBRG10 -S2), and negative control plants (BSMV:γ). After the third leaf unfolded, they were inoculated with stripe rust physiological races CYR23 and CYR31. Samples were taken at 24 h, 48 h, and 120 h after inoculation for WGA staining to observe the development of stripe rust ( Figure 4 ), and 30 infection sites were counted each time, with 3 biological replicates. The disease incidence was observed 14 d after inoculation ( Figure 5 ).

[0067] As can be seen from Figure 4 , under the infection of stripe rust, the infection area of the gene-silenced plants by stripe rust was significantly lower than that of the control plants, indicating that silencing TaBRG10 gene enhanced the resistance of wheat to stripe rust. TaBRG10

[0068] As can be seen from Figure 5 , under the infection condition of stripe rust CYR23, there were no significant differences in the phenotypes on the leaves of all treatments. Under the infection condition of stripe rust CYR31, visible uredinia were observed on the leaves of all treatments. TaBRG10 The severity of stripe rust in the gene-silenced plants was weaker than that of the control plants, indicating that down-regulating TaBRG10 gene expression in wheat leaves enhanced the disease resistance to stripe rust physiological race CYR31, and the number of uredinia produced on wheat leaves decreased significantly.

[0069] As described above, the basic principles, main features, and advantages of the present invention are better described. The above embodiments and the description are only for describing the preferred embodiments of the present invention. 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 those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the present invention.​

Claims

1. TaBRG10 Application of gene in wheat resistance to stripe rust, characterized in that, The said TaBRG10 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; Silencing the TaBRG10 gene enhances wheat resistance to stripe rust; The pathogen of the stripe rust is Puccinia striiformis f. sp. tritici CYR31.

2. The application according to claim 1, characterized in that, The said TaBRG10 gene is induced to express by Puccinia striiformis f. sp. tritici.

3. The application according to claim 1, wherein Silencing the TaBRG10 silencing sequence of the gene improves the resistance of wheat to stripe rust.

4. The application according to claim 3, wherein The silencing sequence is the silencing sequence TaBRG10 -S1 or the silencing sequence TaBRG10 -S2; The silencing sequence TaBRG10 - The nucleotide sequence of S1 is the TaBRG10 94th to 235th nucleotides from the 5' end of the gene; The silencing sequence TaBRG10 - The nucleotide sequence of S2 is the TaBRG10 533rd to 694th nucleotides of the gene starting from the 5' end.

5. A method for cultivating a wheat variety resistant to stripe rust, characterized in that, Silencing in plants TaBRG10 gene, the TaBRG10 nucleotide sequence of the gene is shown in SEQ ID NO: 1; The pathogen of the stripe rust is Puccinia striiformis f. sp. tritici CYR31.

6. The cultivation method according to claim 5, characterized in that, It includes constructing a silencing vector and transferring the silencing vector into wheat to obtain the wheat variety resistant to stripe rust.

7. The cultivation method according to claim 6, characterized in that, The silencing vector includes a silencing sequence TaBRG10 -S1 or a silencing sequence TaBRG10 -S2; The silencing sequence TaBRG10 - The nucleotide sequence of S1 is the TaBRG10 94th to 235th nucleotides from the 5' end of the gene; The silencing sequence TaBRG10 - The nucleotide sequence of S2 is the TaBRG10 533rd to 694th nucleotides from the 5' end of the gene.

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