Wheat sheath blight resistance gene TaGST_B3 and its application

Abstract

The present invention belongs to the technical field of plant genetic engineering, and specifically relates to a wheat sheath blight resistance gene. TaGST_B3 And its application. TaGST_B3 Its nucleotide sequence is shown in SEQ ID NO: 1; its full-length cDNA nucleotide sequence is shown in SEQ ID NO: 2, and its CDS sequence is shown in SEQ ID NO: 3. TaGST_B3 The amino acid sequence of the encoded protein is shown in SEQ ID NO: 4. The present invention is the first to use transgenic technology to introduce a wheat sheath blight resistance gene TaGST_B3 Transformed into common wheat for overexpression, TaGST_B3 The role of genes in wheat sheath blight resistance breeding. The present invention helps to reveal the molecular genetic basis of wheat sheath blight, and at the same time plays an important role in detecting and improving wheat varieties using genetic engineering technology, providing a new way to breed new disease-resistant and safe wheat varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular relates to a wheat sheath blight resistance-related gene TaGST_B3, an expression product thereof, an expression vector thereof and applications. Background Art

[0002] Wheat sheath blight caused by Rhizoctonia cerealis (Rc) is a very destructive fungal disease at the base of wheat stems, which has a great impact on wheat yield and quality. The average yield reduction in the disease-affected area is 10% to 40%. In severe cases, it can cause withered and white ears, and even cause grain failure (Mcbeath JH, Mcbeath J. Plant Diseases, Pests and Food Security: Environmental Change and Food Security in China. 2010.; Hamada MS, Yin YN, Chen HG, Ma ZH. The escalating threat of Rhizoctonia cerealis, the causal agent of sharp eyespot in wheat. Pest Manag Sci. 2011, 67(11): 1411-1419.). According to statistics, the average annual occurrence area of ​​wheat sheath blight in my country from 2015 to 2022 is about 8.2 million hm2. 2 , posing a major threat to wheat production (https: / / www.natesc.org.cn / ). Since Rc has a wide host range and the pathogen can survive for a long time in the soil or infected crop residues, the wheat-corn rotation and straw return to the field farming methods have led to the increasing severity of wheat sheath blight in my country, and it has become one of the most important diseases in many major wheat producing areas in my country.

[0003] Wheat eyelash disease is a quantitative trait (Quantitative trait locus, QTL) controlled by multiple genes (Chen J, Li GH, Du ZY et al. Mapping of QTL conferring resistance to sharp eyespot (Rhizoctonia cerealis) in bread wheat at the adult plant growth stage. Theor Appl Genet, 2013, 126: 2865-2878). There is no germplasm in common wheat materials that is immune or highly resistant to eyelash disease (Ren Y, Yu PB, Wang Y, Hou WX, Yang X, Fan JL, Wu XH, Lv XL, Zhang N, Zhao L, Dong ZD, Chen F. Development of a rapid approach for detecting sharp eyespot resistance in seedling-stage wheat and its Application in Chinese wheat cultivars. Plant Dis. 2020, 104(6): 1662-1667.). Currently, research on wheat sheath blight is mostly focused on the identification of materials' sheath blight resistance and the preliminary positioning of disease-resistant genes. Compared with other plant diseases, such as wheat stripe rust, powdery mildew, and rice sheath blight, there is much less research on the genetics of wheat sheath blight resistance.Since 2002, about 27 QTLs for sheath blight resistance have been located using different populations and different types of molecular markers, distributed on 14 chromosomes of wheat (Cai Shibin, Ren Lijuan, Yan Wei, et al. Preliminary study on innovation of wheat sheath blight resistance germplasm and QTL positioning. Chinese Agricultural Science, 2006, 39(5): 928-934; Chen J, Li GH, Du ZY et al. Mapping of QTL conferring resistance to sharp eyespot (Rhizoctonia cerealis) in bread wheat at the adult plant growth stage. Theor Appl Genet, 2013, 126: 2865-2878; Wu XJ, Cheng K, Zhao RH. Quantitative trait lociresponsible for sharp eyespot resistance in common wheat CI12633. Scientific Reports, 2017, 7(1): 11799). These QTLs are mainly concentrated in the A and B genomes of wheat, and there are relatively few QTLs in the D group. According to the physical location of these markers in the Chinese Spring genome (http: / / 202.194.139.32 / ), it can be seen that there are three QTLs controlling sheath blight resistance on chromosome 3B of common wheat. In addition, there are more than two QTLs controlling sheath blight resistance on chromosomes 1A, 2B, 5A, 6B and 7D of wheat. However, no QTL for sheath blight resistance has been positionally cloned so far, and the molecular mechanism of sheath blight resistance genes is still not very clear. Therefore, the exploration and cloning of important genes regulating sheath blight resistance will enrich human understanding of the molecular mechanism of crop sheath blight resistance and lay the foundation for the utilization of wheat sheath blight resistance genes and disease-resistant breeding. Summary of the invention

[0004] The present invention provides a wheat sheath blight resistance-related gene TaGST_B3, its expression product, and its expression vector, and applies them to the preparation of transgenic plant cells and plant breeding to solve the technical problem of the lack of wheat sheath blight resistance-related genes and provide more options for wheat breeding.

[0005] The present invention adopts the following technical solution:

[0006] The wheat sheath blight resistance-related gene TaGST_B3 of the present invention has a nucleotide sequence as shown in SEQ ID NO: 1, specifically:

[0007]

[0008] The full-length cDNA nucleotide sequence of gene TaGST_B3 is shown in SEQ ID NO: 2, specifically:

[0009]

[0010] The CDS sequence of gene TaGST_B3 is shown in SEQ ID NO:3, specifically:

[0011] 5'--3'.

[0012] The protein TaGST_B3 encoded by the wheat sheath blight resistance-related gene of the present invention has an amino acid sequence as shown in SEQ ID NO: 4, specifically:

[0013] MSPVKVFGHPMLTNVARVLLFLEEVGAEYELVPMDFVAGEHKRPQHVQLNPFAKMPGFQDGDLVLFESRAIAKYILRKYGGTAGLDLLGENSGIEELAMVDVWTEVEAQQYYPAI SPVVFECIIIPFIIPGGGAAPNQTVVDESLERLRGVLGIYEARLEKSRYLAGDSISFADLNHIPFTFYFMTTPYAKVFDDYPKVKAWWEMLMARPAVQRVCNHMPTEFKLGAQY.

[0014] A specific primer pair for identifying the wheat sheath blight gene TaGST_B3, the specific primer pair comprising:

[0015] The nucleotide sequence of the upstream primer is shown in SEQ ID NO:5 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:6.

[0016] The method for detecting wheat sheath blight alleles using the above-mentioned specific primers comprises the following steps:

[0017] Step 1: Extract the genomic DNA of the wheat to be tested;

[0018] Step 2: Using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using the upstream primer shown in SEQ ID NO:5 and the downstream primer shown in SEQ ID NO:6 to obtain an amplified product, and then the following judgment is performed:

[0019] The amplified product was judged to have 4 bases of Idel at the 363rd position before ATG in the promoter region to determine the gene type; among them, the deletion type (----) and its corresponding allele type were named TaGST_B3a; the insertion type (TACT) and its corresponding allele type were named TaGST_B3b.

[0020] Further phenotypic identification results showed that the corresponding disease index of the deletion type (----) plants was lower and the plants had stronger disease resistance, while the corresponding disease index of the insertion type (TACT) plants was higher and the plants had weaker disease resistance; therefore, the TaGST_B3 gene plays a very important role in the resistance to wheat stripe blight, and it also contributes to deeper research on the genetics and molecular biology of wheat disease-resistant breeding.

[0021] The present invention also provides a recombinant expression vector constructed by the above gene, and a recombinant bacterium constructed by the above recombinant expression vector.

[0022] The recombinant expression vector can be specifically a recombinant plasmid obtained by inserting the nucleic acid sequence shown in SEQ ID NO: 3 between the restriction sites BamH I and SacI of the plant expression vector pLGY-OE3-GAI.

[0023] Plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. 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 the addition of polyadenylic acid to the 3′ end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the carmine synthase Nos gene) and the 3′ non-translated region of the plant gene (such as the soybean storage protein gene) have similar functions. When using the target gene to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, corn ubiquitin promoter (Ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of 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 come from the transcription initiation region or the 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 markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0024] The present invention also provides the use of the TaGST_B3 gene in preparing transgenic plants. That is, the gene is introduced into the target plant to obtain a transgenic plant with adjustable sheath blight resistance. The expression vector carrying the gene can be used to transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, gene gun, etc., and the transformed plant tissue is cultivated into a plant.

[0025] The target plant can be either a monocot or a dicot. The monocot can be specifically wheat, such as wheat Fielder.

[0026] Likewise, the above gene and / or the above protein can also be used in plant breeding.

[0027] The beneficial effects of the present invention are:

[0028] 1. The present invention discloses for the first time and confirms a new wheat sheath blight resistance-related gene, named TaGST_B3. The gene is located on wheat chromosome 3B, and the protein it expresses can regulate wheat sheath blight resistance.

[0029] 2. The present invention clarifies the DNA sequence, CDS sequence and protein encoding sequence of the wheat sheath blight resistance-related gene TaGST_B3, laying a foundation for the application practice of wheat sheath blight resistance breeding.

[0030] 3. The specific primers SEQ ID NO:5, the upstream primer, and SEQ ID NO:6, the downstream primer, can be directly used for the detection of wheat sheath blight resistance genotypes, which is beneficial to the cultivation of disease-resistant wheat varieties and the creation of excellent germplasm resources, achieving early screening and saving time and resources.

[0031] 4. The present invention is the first to use transgenic technology to transfer the wheat sheath blight resistance gene TaGST_B3 into common wheat for overexpression, verifying the role of the TaGST_B3 gene in wheat sheath blight resistance breeding.

[0032] 5. The present invention helps to reveal the molecular genetic basis of wheat sheath blight, and at the same time plays an important role in detecting and improving wheat varieties using genetic engineering technology, providing a new approach for breeding new disease-resistant and safe wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the frequency distribution diagram of the disease index (DI) of wheat sheath blight in the PM population. In the figure, a: DI of sheath blight in the field; b: DI of sheath blight in the greenhouse; DI: disease index; I: replication 1; II: replication 2.

[0034] Figure 2 QTL mapping of wheat sheath blight resistance.

[0035] Figure 3 The expression levels of TaGST_B3, TaFAOα and TaPEF1 in the resistance and sensitive pools. In the figure, RB1 is the resistance pool 1, RB2 is the resistance pool 2; SB1 is the sensitive pool 1, SB2 is the sensitive pool 2.

[0036] Figure 4 The relative expression levels of TaGST_B3 in the resistant parent Pingyuan 50 and the susceptible parent Mingxian 169 after 5 days of inoculation. BJJ indicates no inoculation with Rhizoctonia solani, and JJ indicates inoculation with Rhizoctonia solani.

[0037] Figure 5 The sequence polymorphism of TaGST_B3 between the resistant parent Pingyuan 50 and the susceptible parent Mingxian 169.

[0038] Figure 6 This is a bar graph of the relative expression level of the TaGST_B3 gene in the VIGS silencing experiment.

[0039] Figure 7 These are photos comparing the resistance of different treatment materials to wheat sheath blight in the VIGS silencing experiment.

[0040] Figure 8 The figure is a phenotypic comparison of sheath blight resistance between the wild type KWT and the TaGST_B3 mutant (K1027) at the seedling stage and the adult stage. In the figure, TaGST_B3 represents the TaGST_B3 gene mutant (K1027).

[0041] Fig. 9 This is the plasmid map of the pLGY-OE3-GAI vector.

[0042] Fig.10 The figure shows the phenotypic comparison between the overexpression transgenic lines (T1-1, T1-2 and T1-10) and the empty vector control (Fielder).

[0043] Fig.11 is the expression level of TaGST_B3 gene in overexpression transgenic lines (T1-1, T1-2, T1-10) and wild-type Fielder. DETAILED DESCRIPTION

[0044] The present invention is described in more detail below through specific implementation modes to facilitate understanding of the technical solution of the present invention, but is not intended to limit the protection scope of the present invention.

[0045] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; unless otherwise specified, the reagents involved are all commercially available conventional reagents; unless otherwise specified, the test methods involved are all conventional methods. The quantitative tests in the following examples were repeated three times, and the results were averaged. Primer synthesis and sequencing were completed by Sangon Biotech (Shanghai) Co., Ltd.

[0046] The wheat materials involved are:

[0047] PM population: A DH population consisting of 117 families formed by the F1 generation of Pingyuan 50 and Mingxian 169 through colchicine-induced doubling. This population was used for QTL mapping and verification of the TaGST_B3 allele effect.

[0048] Pingyuan 50: Its genotype and phenotype are resistant to wheat sheath blight, and it is used in VIGS experiments to verify the effect of TaGST_B3 gene;

[0049] Kronos mutants: The mutant system of wild-type Kronos-WT and Kronos1027 (missense mutation of TaGST_B3 gene) was used to compare the effect of TaGST_B3 gene; (Kronos mutant materials came from the mutant library of tetraploid wheat Kronos created by the research group of Academician Jorge Dubcovsky at the University of California, Davis (Krasileva et al., PNAS, 2017, 114:913-921)).

[0050] Fielder: The genotype and phenotype are susceptible to wheat stripe blight, and it is a receptor material that overexpresses the transgenic TaGST_B3 gene.

[0051] Example 1: Location and acquisition of wheat sheath blight-related gene TaGST_B3

[0052] The greenhouse diseased wheat grains mixed with soil method and field toothpick inoculation method (Ren Yan et al., Development of arapid approach for detecting sharp eyespot resistance in seedling-stage wheat and its application in Chinese wheat cultivars. Plant Disease, 2020, 104(6): 1662-1667) were used to identify the resistance of PM population to sheath blight at the seedling and milky stages. The phenotypic identification results showed that the sheath blight DI of the DH population showed a continuous distribution at both the milky and seedling stages and had super affinity analysis ( Figure 1 ), indicating that the sheath blight resistance gene contained in Pingyuan 50 is a typical quantitative trait inheritance.

[0053] Based on the phenotypic data of the PM population in the field and greenhouse, extremely resistant and susceptible families were selected to construct a resistant and susceptible pool. The wheat 660K SNP array (Sun Congwei et al., The Wheat 660K SNP array demonstrates great potential for marker-assisted selection in polyploid wheat, Plant Biotechnology Journal, 2020(18):1354-1360) was used to perform BSA analysis of the parents and the mixed pool. The differential SNPs with consistent polymorphism between the parents and the resistant and susceptible mixed pools were obtained, which were mainly located on chromosomes 3B and 6B. Then, 35 KASP markers were uniformly developed in the above-mentioned positioning regions for population genotype analysis. Combined with the detection information of SSR markers, a QTL locus related to sheath blight resistance was finally detected on chromosome 3B of wheat, named QSe.hnau-3BS ( Figure 2 ), located between KASP markers A011839 and A011841, corresponding to a physical segment of approximately 7.4 Mb. Phenotypic contribution analysis showed that this QTL could significantly reduce the DI value of the PM population regardless of milky stage or seedling stage (Table 1). This QTL has not been reported before and is a new QTL locus for sheath blight resistance.

[0054] Table 1 Effects of the presence and absence of QSe.hnau-3BS on resistance to sheath blight

[0055]

[0056] Further combined with the transcriptome results of the individuals in the resistant pool of the PM population after inoculation with wheat sheath blight, it was found that there were three differentially expressed genes (TaGST_B3, TaFAOα and TaPEF1) in the positioning segment of the above QTL, but only one gene annotated as glutathione transferase (GST) TaGST_B3 was significantly upregulated in the resistant pool ( Figure 3 ), while the other two genes were significantly down-regulated in the resistant pool. The results of fluorescence quantitative analysis also showed that the expression level of TaGST_B3 in the resistant parent Pingyuan 50 was significantly higher than that in the susceptible parent Mingxian 169 ( Figure 4 ). Therefore, this gene was predicted to be a gene related to wheat sheath blight resistance.

[0057] Two pairs of PCR primers were designed with reference to Primer5 to amplify the promoter and coding region sequences of the TaGST_B3 gene.

[0058] The primer sequences are as follows:

[0059] Primer pair 1:

[0060] SEQ ID NO:5(TaGST-F1):5'-GCGAGGTGACTGGATGACG-3';

[0061] SEQ ID NO: 6 (TaGST-R1): 5'-ACGGCTCACCGAACAGGAC-3'.

[0062] Primer pair 2:

[0063] SEQ ID NO:7(TaGST-F2):5'-CTTACTTCCTTCGCACCACA-3';

[0064] SEQ ID NO: 8 (TaGST-R2): 5'-ACCTATCGTGCTTCTGATGTT-3'.

[0065] Primer pair 1 amplifies the promoter region sequence of TaGST_B3 gene and a small part of the gene coding region sequence, and primer pair 2 amplifies the entire gene coding region sequence.

[0066] The above two pairs of primers were used to amplify the genomic DNA of wheat materials such as Pingyuan 50 and Mingxian 169. The PCR amplification system is shown in Table 2, and the PCR amplification program is shown in Table 3.

[0067] Table 2 PCR amplification system of primer pair 1 and primer pair 2 for TaGST_B3 gene

[0068] Components volume 2×phanta buffer 12.5μL dNTP 0.5μL Phanta Enzyme 0.5μL Primer F 0.5μL Primer R 0.5μL DNA 2μL <![CDATA[ddH2O]]> To 25μL

[0069] Table 3 PCR amplification procedure of TaGST_B3 gene with primer pair 1 and primer pair 2

[0070]

[0071] The sequences of the promoter and coding regions of TaGST_B3 genes of the resistant parent Pingyuan 50 and the susceptible parent Mingxian 169 were compared. It was found that the polymorphisms in the coding regions of the resistant and susceptible parents did not cause amino acid changes, but there were abundant polymorphisms in the promoter regions of the two parents ( Figure 5 ). The nucleotide sequence of the TaGST_B3 gene of Pingyuan 50 is shown in SEQ ID NO:1, and the full-length cDNA nucleotide sequence is shown in SEQ ID NO:2.

[0072] Example 2: Detection of TaGST_B3 gene-specific marker

[0073] Using DNA of 5 extremely resistant materials and 5 extremely sensitive materials of the PM population as templates, PCR amplification was performed using the upstream primer shown in SEQ ID NO:5 and the downstream primer shown in SEQ ID NO:6 to obtain the amplified product, and then the following judgment was performed:

[0074] The amplified product was judged to have 4 bases of Idel at the 363rd position before ATG in the promoter region to determine the gene type; among them, the deletion type (----) and its corresponding allele type were named TaGST_B3a; the insertion type (TACT) and its corresponding allele type were named TaGST_B3b.

[0075] The results showed that the genotypes of the five extremely resistant DH lines were all TaGST_B3a, and the genotypes of the five extremely resistant DH lines were all TaGST_B3b.

[0076] Example 3: TaGST_B3 gene VIGS silencing test and phenotypic identification

[0077] Primers were designed based on the cDNA sequence of the TaGST_B3a gene, and restriction sites and protective bases were added to the 5' ends of the forward and reverse primers, respectively, to form the upstream primer: TaGST-F3: 5'-TTTTTTTAGCTAGCTGATTAATTAACCCAACACGTCCAGCTAAAC-3' and the downstream primer: TaGST-R3: 5'-CTTCCGTTGCTAGCTGAGCGGCCGCCGGAGGATGTACTTGGCGAT-3'. This primer pair was used to amplify the VIGS silencing fragment of the TaGST_B3 gene and was recovered for later use.

[0078] The VIGS silencing fragment of the TaGST_B3 gene and the γ-PDS-as vector were further double-digested with Pac1 and Not1, the silencing fragment and the corresponding γ-linearized vector fragment were recovered, and the recombinant vector construction (γ-TaGST_B3) was completed using the seamless cloning method.

[0079] (2) Plant VIGS silencing experiment of TaGST_B3 gene

[0080] The viral vector (α, β, γ, γ-PDS) and TaGST_B3 gene silencing recombinant vector (γ-TaGST_B3) plasmids were linearized by restriction enzyme digestion, and the linearized plasmids were further transcribed in vitro using the RiboMAXTM Large Scale RNA Production Systems-T7 kit to obtain different components of viral in vitro transcription products. Take 2.5μL of each in vitro transcription product α, β, γ / γ-PDS / γ-DIR, mix them in a ratio of 1:1:1 and dilute them with an equal volume of DEPC water, take 5μL of the diluted mixture and add it to 90μL FES buffer to mix thoroughly by pipetting. Four different treatments were set up for each experiment, namely: complete blank control group (WT), virus blank control group (α+β+γ), albino positive control group (α+β+γ-PDS) and gene silencing group (α+β+γ-TaGST_B3).

[0081] When the virus is infected, spray a small amount of DEPC water on the surface of the plant to be infected (Pingyuan 50), take 8-10μL of FES mixed solution and apply it on clean gloves, rub the second leaf of the seedling from the base to the tip of the leaf 3 times, control the strength during rubbing, and spray a little DEPC water from top to bottom to maintain humidity after rubbing. Clean gloves need to be changed for each treatment. After the virus is inoculated, place it in a 23±2℃ incubator to keep warm, moisturize and avoid light for 24h, and then adjust it to a 16h / 8h light and dark cycle for cultivation, and observe and record phenotypic changes regularly.

[0082] (3) Phenotypic identification of TaGST_B3 gene plants after VIGS silencing

[0083] After VIGS silencing for 2 weeks, qRT-PCR was used to detect the expression of TaGST_B3 gene in silenced plants (BSMV TaGST_B3 ), virus-free plants (BSMV0) and non-silenced plants (WT, plain 50), as shown in Figure 6 As shown, the results showed that the relative expression level of TaGST_B3 gene in silenced plants was significantly downregulated, and the TaGST_B3 gene was effectively silenced.

[0084] Further investigation of the resistance of silenced plants to sheath blight showed that the disease index of TaGST_B3 silenced plants (DI=80) was significantly higher than that of virus-free plants (plain 50, DI=67), and the disease resistance of plants was significantly reduced (see Figure 7 ).

[0085] In summary, the disease resistance of plants was significantly reduced after silencing of the TaGST_B3 gene, indicating that this gene is a positive regulatory gene in the response process of wheat sheath blight.

[0086] Example 4: Detection of EMS mutants

[0087] The tetraploid Kronos mutant was further used to verify the function of the TaGST_B3 gene. The nucleotide at 10 bp after the 5' end ATG of the TaGST_B3 gene sequence on chromosome 3B corresponding to Kronos1027 mutated from G to A, causing a missense mutation in the gene. The mutant Kronos1027 and the wild type Kronos-WT were tested for resistance to wheat sheath blight at the seedling and adult stages. The results showed that compared with Kronos-WT (DI=60) at the seedling stage, the disease index of the TaGST_B3 gene mutant plant Kronos1027 (DI=90) was significantly increased, and the plant's disease resistance was significantly reduced (see Figure 8 ), it is speculated that TaGST_B3 gene plays a positive regulatory role in plants.

[0088] Example 5: Acquisition and identification of TaGST_B3 transgenic wheat

[0089] (1) Construction of TaGST_B3 plant overexpression vector

[0090] Primers were designed based on the full-length CDS sequence of TaGST_B3 in wheat plain 50 (shown in SEQ ID NO: 3), and restriction endonuclease BamH I and Sac I recognition sites and protective bases were introduced at both ends of the primers. The primer sequences are as follows:

[0091] TaGST-F4:5'-GATGACGATGACAAG GGATCC TCTCCGGTGAAGGTGTTCG-3′;

[0092] (The underlined bases are the restriction endonuclease BamH I recognition sites and protection bases);

[0093] TaGST-R4:5'-ACGAACGAAAGCTCT GAGCTC CTAGTACTGCGCACCTAGCTTA-3′;

[0094] (The underlined bases are the recognition sites and protective bases of the restriction endonuclease Sac I).

[0095] Total RNA from Pingyuan 50 seedlings was extracted using TRIZOL reagent, and reverse transcribed using SuperScript III reverse transcriptase (Invitrogen, Cat no. 18080-044) to obtain cDNA. Using this cDNA as a template, PCR was performed using primers TaGSTF-F4 and TaGSTF-R4 to amplify the coding sequence of wheat TaGST_B3 gene.

[0096] Table 4 PCR amplification system of TaGST_B3 gene using primers TaGST-F4 and TaGST-R4

[0097]

[0098]

[0099] The PCR reaction program is:

[0100] 95℃ for 5 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, 35 cycles; finally, extend at 68℃ for 10 min.

[0101] After the reaction was completed, the amplified product was detected by 1% agarose gel electrophoresis, and a 690 bp DNA fragment was recovered and purified for sequencing. The sequencing results showed that the amplified DNA fragment was consistent with the sequence shown in SEQ ID NO:3.

[0102] The expression vector pLGY-OE3-GAI was selected, and its vector map is as follows Fig. 9 shown.

[0103] The above 690 bp DNA fragment was cloned into the plant expression vector pLGY-OE3-GAI between the restriction sites BamH I and Sac I to obtain a recombinant expression vector containing the wheat TaGST_B3 gene, named pLGY-OE3-GAI-TaGST_B3.

[0104] (2) Obtaining TaGST_B3 transgenic wheat

[0105] pLGY-OE3-GAI-TaGST_B3 was transformed into immature embryo callus of wheat Fielder by Agrobacterium infection, and positive plants were obtained after screening, pre-differentiation and differentiation. The positive plants were identified by PCR using primers TaGSTF-F5 and TaGSTF-R5 to obtain T0 generation transgenic plants.

[0106] The primer sequences are as follows:

[0107] TaGST-F5: 5'-TTAGCCCTGCCTTCATACGC-3';

[0108] TaGST-R5: 5'-TGCGGAGGATATACTTGGCG-3'.

[0109] Amplification system and PCR procedure:

[0110] Table 5 PCR amplification system of TaGST_B3 gene using primers TaGSTF-F5 and TaGSTF-R5

[0111] Components volume 2×phanta buffer 12.5μL dNTP 0.5μL Phanta Enzyme 0.5μL Primer F 0.5μL Primer R 0.5μL DNA 2μL <![CDATA[ddH2O]]> To 25μL

[0112] Table 6 PCR amplification procedure of TaGST_B3 gene by primers TaGST-F5 and TaGST-R5

[0113]

[0114] (3) PCR identification and phenotypic identification of transgenic wheat

[0115] The TaGST-F5 and TaGST-R5 primer pairs were used to perform PCR screening for positive transgenic plants, and a total of 10 positive transgenic plants were obtained. After additional generations, T1 generation transgenic lines (T1-1, T1-2 and T1-10) were obtained.

[0116] The phenotypes of the overexpressing transgenic lines T1 (T1-1, T1-2 and T1-10) and the wild-type Fielder in the greenhouse were identified by sheath blight inoculation. Fig.10 As shown:

[0117] Compared with wild-type Fielder plants, the sheath blight disease index of overexpressing transgenic lines T1 (T1-1, T1-2 and T1-10) was significantly reduced, and the disease resistance of the plants was significantly improved.

[0118] Using TaGST-F6 and TaGST-R6 primers, fluorescence real-time quantitative PCR was used to detect the expression level of gene TaGST_B3 in the leaves of T1 generation positive transgenic plants.

[0119] The primer sequences are as follows:

[0120] TaGST-F6: 5'-TCGTCCTGTTCGGTGAGC-3';

[0121] TaGST-R6: 5'-TAATTCTTCGATTCCGCTG-3'.

[0122] Amplification system and PCR program: same as Table 5 and Table 6.

[0123] The results of real-time quantitative PCR were as follows Fig.11 As shown, the expression level of the target gene TaGST_B3 in the leaves of the T1 generation positive transgenic plants was significantly higher than that of the wild-type Fielder, and was about 5 to 14 times the expression level in the empty leaves.

[0124] In summary, the protein expressed by the wheat sheath blight resistance-related gene TaGST_B3 of the present invention can regulate wheat sheath blight resistance, can be used to study the molecular mechanism related to controlling wheat sheath blight, and has important application value in wheat disease resistance breeding.

[0125] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, any equivalent changes or modifications made according to the structures, features and principles described in the patent scope of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A method for detecting wheat sheath blight alleles, comprising the following steps: Step 1: Extract the genomic DNA of the wheat to be tested; Step 2: Using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using the upstream primer shown in SEQ ID NO:5 and the downstream primer shown in SEQ ID NO:6 to obtain an amplified product, and then the following judgment is performed: The amplified product was judged to have 4 bases of Idel at the 363rd position before ATG in the promoter region to determine the gene type. The amplified product at the 363rd position before ATG in the promoter region was the 1830th position of the sequence shown in SEQ ID NO: 1; the allele type corresponding to the deletion type was named TaGST_B3a ; The corresponding allele type of the insertion type TACT is named TaGST_B3b ; The disease index of the deletion type plants was lower than that of the insertion type TACT plants, and the disease resistance of the deletion type plants was stronger than that of the insertion type TACT plants.

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