Application of TaSLAH2-B gene or TaSLAH2-B protein in improving wheat stripe rust resistance and recombinant vector

Abstract

The invention relates to the technical field of biology, and particularly discloses application of a TaSLAH2-B gene or a TaSLAH2-B protein in improving wheat stripe rust resistance and a recombinant vector, the nucleotide sequence of the TaSLAH2-B gene is shown as SEQ ID NO.1, the CDS sequence of the TaSLAH2-B gene is shown as SEQ ID NO.2, and the amino acid sequence of the protein coded by the TaSLAH2-B gene is shown as SEQ ID NO.3. The invention further discloses a preparation method of the TaSLAH2-B gene. The TaSLAH2-B gene provided by the invention achieves the purpose of improving stripe rust resistance through overexpression in wheat plants.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a TaSLAH2-B gene or a TaSLAH2-B protein in improving wheat stripe rust resistance and a recombinant vector. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the world's major food crops, and its yield and quality are seriously threatened by wheat rust. Wheat rust diseases mainly include stripe rust (YR), leaf rust (LR) and stem rust (SR). Stripe rust is an airborne low-temperature fungal disease caused by the wheat-specific form of the stripe rust fungus (Puccinia striiformis West.f.sp.tritici Eriks.&Henn.). It has the characteristics of rapid transmission and frequent mutations, and is one of the epidemic diseases that seriously harm wheat production. The prevalence of stripe rust is affected by both climatic conditions and pathogen mutations, and the disease is more serious in warm, cool and humid climatic conditions. The disease mainly harms wheat leaves. In severe cases, it affects the leaf sheaths, stems and ears, resulting in premature aging of the leaves, poor filling, and thin grains. Wheat resistance is divided into "quality resistance" and "quantity resistance". "Qualitative resistance" is controlled by one or a few major genes, conferring strong resistance to a specific pathogen or its physiological race. This can trigger hypersensitive necrosis to rapidly control the disease, but it is typically not long-lasting. Due to the frequent variation in stripe rust races, resistance in resistant varieties controlled by major genes can easily be lost, leading to significant yield declines. Therefore, improving wheat resistance to stripe rust is of great significance. Summary of the Invention

[0003] To develop a new approach to improve wheat resistance to stripe rust, the present invention provides the use of the TaSLAH2-B gene or TaSLAH2-B protein in improving wheat stripe rust resistance and a recombinant vector. The present invention improves wheat resistance to stripe rust by increasing the expression of the TaSLAH2-B gene.

[0004] The present invention provides a use of a TaSLAH2-B gene or a TaSLAH2-B protein in improving resistance to wheat stripe rust. The amino acid sequence of the TaSLAH2-B protein is shown in SEQ ID NO. 3. The TaSLAH2-B gene encodes the TaSLAH2-B protein, and the TaSLAH2-B gene is selected from any one of the following DNA molecules:

[0005] (1) The CDS sequence is shown in SEQ ID NO. 2;

[0006] (2) The nucleotide sequence is shown in SEQ ID NO. 1;

[0007] (3) a DNA molecule having at least 90% homology to the CDS sequence shown in SEQ ID NO. 2;

[0008] (4) A DNA molecule having at least 70% homology to the nucleotide sequence shown in SEQ ID NO.1.

[0009] The present invention improves the resistance of wheat to stripe rust by increasing the expression level of the TaSLAH2-B gene in wheat plants.

[0010] Furthermore, genetic engineering methods are used to increase the expression level of the TaSLAH2-B gene in wheat, thereby improving the resistance of wheat to stripe rust.

[0011] Furthermore, the genetic engineering method includes: replacing the DNA fragment between the restriction endonucleases BamH I and Sac I of the vector pLGY with the CDS sequence of the TaSLAH2-B gene shown in SEQ ID NO.2 to obtain the recombinant plasmid pUbi-SLAH-B; then transforming the recombinant plasmid pUbi-SLAH-B into Agrobacterium to obtain recombinant Agrobacterium; and transforming the recombinant Agrobacterium into wheat to obtain wheat with the TaSLAH2-B gene that is resistant to stripe rust.

[0012] Furthermore, the amplification primers for the CDS sequence of the TaSLAH2-B gene are shown as SEQ ID NO.10 to SEQ ID NO.11.

[0013] Furthermore, the Agrobacterium is Agrobacterium tumefaciens GV3101+pSoup.

[0014] The present invention also provides a method for improving wheat stripe rust resistance, comprising the following steps:

[0015] The CDS sequence of the TaSLAH2-B gene shown in SEQ ID NO. 2 was amplified, and the amplified product was ligated between the BamHI and SacI restriction sites of the vector pLGY to obtain the recombinant plasmid pUbi-SLAH-B;

[0016] The recombinant plasmid pUbi-SLAH-B was transformed into Agrobacterium and then transformed into wheat to obtain wheat with the TaSLAH2-B gene that was resistant to stripe rust.

[0017] The present invention also provides a method for breeding stripe rust-resistant wheat, comprising the following steps:

[0018] The CDS sequence of the TaSLAH2-B gene shown in SEQ ID NO. 2 was amplified, and the amplified product was ligated between the BamHI and SacI restriction sites of the vector pLGY to obtain the recombinant plasmid pUbi-SLAH-B;

[0019] The recombinant plasmid pUbi-SLAH-B was transformed into Agrobacterium to obtain recombinant Agrobacterium, which was inoculated into a culture medium containing spectinomycin to obtain a recombinant Agrobacterium culture solution. Wheat embryos were infected with the recombinant Agrobacterium culture solution and cultured in the dark for 3 to 4 days. The embryos were then transferred to a selection culture medium and cultured for 8 to 9 days. The embryos were then screened with different concentrations of glufosinate and finally transferred to a differentiation culture medium. After differentiation, the embryos were transferred to a rooting culture medium for culture, ultimately obtaining T0 generation wheat transgenic with the TaSLAH2-B gene.

[0020] The present invention also provides a recombinant vector, which contains the CDS sequence of the TaSLAH2-B gene shown in SEQ ID NO.2.

[0021] The present invention also provides an application of the recombinant vector in improving wheat stripe rust resistance.

[0022] Furthermore, the recombinant vector is obtained by inserting the CDS sequence of the TaSLAH2-B gene shown in SEQ ID NO. 2 between the BamH I and Sac I restriction sites of the vector pLGY.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention provides a broad-spectrum multi-resistance gene TaSLAH2-B for wheat in the adult stage. This gene fully exerts its disease resistance function after inoculation and induction in the adult stage, making wheat show significant stripe rust resistance.

[0025] 2. The present invention overexpresses the TaSLAH2-B gene in the wheat variety Kenong 199, which can improve wheat's resistance to stripe rust, indicating that the protein TaSLAH2-B can improve plant disease resistance. This invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 The subcellular localization of the TaSLAH2-B gene in the present invention; Figure 1 Panel A in the figure is TaSLAH2-GFP; Figure 1Figure B is TaSLAH2-GFP; TaSLAH2-B is localized at the subcellular membrane in wheat protoplasts; GFP is a fluorescent tag; FM4-64: a cell membrane-selective red fluorescent dye; Merged: a merger of bright field and fluorescence; DIA: bright field.

[0028] Figure 2 is the relative expression level of the TaSLAH2-B gene transgenic strain in the present invention.

[0029] Figure 3 This is the phenotype of the TaSLAH2-B gene transgenic strain in the adult stage of the present invention;

[0030] In the figure, the wild type is the susceptible variety Kenong 199, and OE#1 to OE#4 are overexpression lines;

[0031] Figure 4 The phenotype of the near-isogenic line containing the TaSLAH2-B gene in the present invention during the adult stage and the microhistological observation of stripe rust;

[0032] In the figure, A shows the phenotypes of the adult plant of the near-isogenic line (NIL19) containing the TaSLAH2-B gene and the near-isogenic line (NIL18) not containing the TaSLAH2-B gene. Different samples from the same line are parallel test samples.

[0033] B is the microhistological observation of stripe rust at different stages after inoculation in the adult stage of the near-isogenic line containing the TaSLAH2-B gene (NIL19) and the near-isogenic line not containing the TaSLAH2-B gene (NIL18).

[0034] Figure 5 It is the phenotype of the adult plant of the Jing 411 mutant containing the TaSLAH2-B gene in the present invention; among them, CK is the control phenotype of Jing 411, and M1-M6 are the phenotypes of the TaSLAH2-B gene mutant in Jing 411. DETAILED DESCRIPTION

[0035] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0036] Example 1: Cloning of the TaSLAH2-B gene.

[0037] 1. Experimental Methods

[0038] 1. DNA extraction from Jimai 44 seedlings

[0039] Seeds of Jimai 44 were vernalized at 4°C for 14 days. The germinated seeds were then transferred to nutrient soil and incubated at 25°C for two weeks to obtain Jimai 44 seedlings. DNA was extracted from the Jimai 44 seedlings. The Jimai 44 seeds were provided by the National Key Laboratory of Crop Stress Tolerance and Efficient Production.

[0040] The method for extracting the genome (DNA) of Jimai 44 seedlings is as follows:

[0041] (1) Sampling and grinding: Put two steel balls in a 2 mL centrifuge tube, take a leaf of Jimai 44 seedling and put it in the tube, and grind it with a sample crusher.

[0042] (2) Add 1 mL of 65°C DNA extraction solution (CTAB) and let it stand for 2 hours.

[0043] (3) Add 700 μL of Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1) and shake vigorously. Let it stand for 5 minutes and then centrifuge at 12,000 rpm for 10 minutes.

[0044] (4) Transfer the supernatant to a 2.0 mL centrifuge tube, add 0.8 times the volume of isopropanol, and place at -20°C for 20 min.

[0045] Supernatant ratio: 800:640:50 (sodium acetate).

[0046] (5) Centrifuge at 12000 rpm for 10 min, discard the supernatant, add 1 mL of 70% ethanol by volume to wash twice, and centrifuge at 12000 rpm for 2 min.

[0047] (6) Centrifuge at 12000 rpm for 2 min, air dry, and add 100 μL ddH2O to obtain Jimai 44 DNA.

[0048] 2. Cloning of the TaSLAH2-B gene

[0049] Using the extracted DNA from Jimai 44 as a template, three pairs of primers were used for segmented PCR amplification, yielding three PCR amplification products. Each PCR amplification product was sequenced. The sequences of the three amplification products were spliced ​​together to obtain the TaSLAH2-B gene. The nucleotide sequence of the TaSLAH2-B gene is shown in SEQ ID NO. 1, the CDS sequence of the TaSLAH2-B gene is shown in SEQ ID NO. 2, and the amino acid sequence of the protein encoded by the TaSLAH2-B gene is shown in SEQ ID NO. 3.

[0050] The three pairs of primers include Pro1-F / Pro1-R, Pro2-F / Pro2-R and gDNA1-F / gDNA1-R, wherein the nucleotide sequence of Pro1-F is shown in SEQ ID NO.4, and the nucleotide sequence of Pro1-R is shown in SEQ ID NO.5; the nucleotide sequence of Pro2-F is shown in SEQ ID NO.6, and the nucleotide sequence of Pro2-R is shown in SEQ ID NO.7; the nucleotide sequence of gDNA1-F is shown in SEQ ID NO.8, and the nucleotide sequence of gDNA1-R is shown in SEQ ID NO.9.

[0051] SEQ ID NO.1:

[0052]

[0053] SEQ ID NO.2:

[0054]

[0055] SEQ ID NO.3:

[0056] MASRDGDSICSIQMATEVLDESPGALQDQDEATCSVLFSMPASPSGLHLAQGMAGGAKVEVHDPARPRLMKHARFHSQPSMLIGGGGEAPAMPRSESTREWDRRFDHFRTFSGRLERQLSILRGAVPHEPPTDDMECNAAAKISVEHTDEDNDIPSADSYFAALEGPELETLRPAEVAVLPNGEPWPFLLRFPISAFGMCLGVSSQAMLWKTLSSEHSTGFLGVHPAVNRVLWWASVALTVIVSITYLLKVVFYFEAVRREFHHPVRVNFFFAPWIACLFLVKGLPRPEREINHIVWYLLMTPILCLDLKIYGQWMSSGERQLSKVANPSNHLAVVGNFVGALLGAKMGLRELPIFFFAVGLAHYLVLFVTLYQRLPTNVQLAKELHPIFFFFVTVPNVASMAWATISGEFGHGPKLLYFVSLFLYASLVVRINLFRGVRFSLTWWAYTFPLTSAALATVLYASEVDNMLTRALAVGLAGIATVTVIGVMVNTVYHAFVSKDLFPNDVCIAITRQRPKFNKILAHLRLSSTDEATI。

[0057] SEQ ID NO.4:5’-GGGTGTTGACGTATAAATGT-3’。

[0058] SEQ ID NO.5:5’-GCTATCTCGATTGCCTCGAGTCGTT-3’。

[0059] SEQ ID NO.6:5’-CCGGAGAAAGCTAGCGCACG-3’。

[0060] SEQ ID NO.7:5’-ACATTTATACGTCAACACCC-3’。

[0061] SEQ ID NO.8:5’-TGAAAGACTACATCTTTTAA-3’。

[0062] SEQ ID NO.9:5’-CTATCTCGATTGCCTCGAGT-3’。

[0063] The reaction procedure of PCR amplification is shown in Table 1, and the reaction system of PCR amplification is shown in Table 2.

[0064] Table 1 PCR amplification reaction program

[0065]

[0066] Table 2 PCR amplification reaction system

[0067]

[0068]

[0069] Example 2: Subcellular localization of TaSLAH2-B-GFP fusion protein.

[0070] The source / method of obtaining Chinese Spring protoplasts is:

[0071] 1. Solution preparation

[0072] 1. Preparation of enzyme solution

[0073] The configuration information of the enzyme solution is shown in Table 3.

[0074] Table 3 Enzyme solution configuration table

[0075] Components Final concentration Molar mass Per 50ml final volume 0.4M Mannitol 0.4M 182.17 3.6434g 20 mM KCl 20mM 74.55 0.07455g 20mM MES 20mM 213.2 0.2132g

[0076] Prepare the solution according to the dosage of each component per 50 mL in Table 3. After the prepared solution is dissolved and the volume is fixed, adjust the pH to 5.7 with 1M KOH and sterilize at 121°C for 20 minutes.

[0077] 2. Preparation of wheat protoplast suspension W5 solution

[0078] The configuration information of wheat protoplast suspension W5 solution is shown in Table 4.

[0079] Table 4 Configuration of W5 solution

[0080] Components Final concentration Molar mass Per 50ml final volume 154mM NaCl 154mM 58.44 1.8g <![CDATA[125mM CaC l2 ·2H2O]]> 125mM 147.02 3.68g 5 mM KCl 5mM 74.55 0.075g 2mM MES 2mM 213.2 0.085g

[0081] Prepare the solution according to the dosage of each component per 200 mL in Table 4. After the prepared solution is dissolved and the volume is fixed, adjust the pH to 5.7 with 1 M KOH and sterilize at 121°C for 20 min.

[0082] 3. Preparation of mannitol MgCl2 solution

[0083] The configuration information of mannitol MgCl2 solution is shown in Table 5:

[0084] Table 5 Configuration table of mannitol MgCl2 solution

[0085]

[0086]

[0087] Prepare the solution according to the dosage of each component per 50 mL in Table 5. After the prepared solution is dissolved and the volume is fixed, adjust the pH to 5.7 with 1M KOH and sterilize at 121°C for 20 minutes.

[0088] 4. Preparation of polyethylene glycol 4000 solution

[0089] The configuration information of polyethylene glycol 4000 solution is shown in Table 6.

[0090] Table 6 Configuration of polyethylene glycol 4000 solution

[0091] Components Final concentration Molar mass Per 50ml final volume 0.2M Mannitol 0.2M 182.14 0.3643g <![CDATA[100mM CaC l2 ]]> 100mM <![CDATA[1mL 1MCaCl2 <!-- 8 -->]]> 40% PEG 40％ 4g <![CDATA[ddH2O]]> Dilute to 10 mL

[0092] Prepare the solution according to the dosage of each component per 50 mL in Table 6. After the prepared solution is dissolved and the volume is fixed, filter it with a 0.22uM filter membrane for sterilization.

[0093] 2. Preparation of Protoplasts

[0094] 1. Preparation of enzymatic hydrolysate

[0095] The configuration information of the enzymatic solution is shown in Table 7.

[0096] Table 7 Configuration of enzymatic solution

[0097] Components 50mL final volume Enzyme hydrolysate 50mL 1.0% (wt%) cellulase R10 0.5g 0.2% (wt%) macerozyme R10 0.1g <![CDATA[10mMCaCl2]]> 500uL 0.1% (w / v) BSA 0.05g β-me 17.5uL

[0098] To prepare the enzymatic solution, after adding the two enzymes, incubate the solution in a 55°C water bath for 10 minutes (shake well during this time), then cool to room temperature (an ice bath is sufficient). Once cooled to room temperature, add CaCl2, BSA, and β-Me. After addition, filter the solution through a 0.45µM filter membrane and transfer it to a plate.

[0099] 2. Take 10 first leaves of wheat, place the single leaves on A4 paper, and cut them gently horizontally with a blade. Place them in a petri dish within 40 minutes so that the leaves cover the entire surface of the liquid in the petri dish.

[0100] 3. Vacuum for 15 minutes, wrap with tin foil and observe after 65 minutes of enzymatic hydrolysis, and light green color begins to collect.

[0101] 4. Filter the protoplasts through a 200-mesh filter and wash the filter four times with W5 (pre-cooled W5 with an equal volume of enzymatic solution). Collect the protoplasts into two 50 mL round-bottom centrifuge tubes.

[0102] 5. Centrifuge at 500 rpm for 10 min at 4°C (set the acceleration / deceleration of the centrifugal force to 1). Remove the supernatant as much as possible (pool the two tubes into one tube) and resuspend the protoplasts in 10 mL of W5 solution for microscopic examination.

[0103] 6. Repeat step 5.

[0104] 7. Place in an ice bath for 40 minutes, centrifuge, and aspirate as much of the supernatant as possible.

[0105] 8. Add appropriate amount of MMG to the protoplasts to complete the preparation.

[0106] 3. Protoplast Transformation

[0107] 1. Add 10uL of recombinant plasmid PGL486-TaSLAH2-B-GFP into a 2mL centrifuge tube.

[0108] 2. Add 100uL of protoplasts.

[0109] 3. Add 110uL of fresh PEG to the mixture and mix thoroughly by adding dropwise with a pipette. The number of additions is 25.

[0110] 4. Incubate in the dark at room temperature for 25 minutes, then add 440 μL of W5 solution and mix thoroughly with a pipette (approximately 7 to 10 times) to stop the conversion.

[0111] 5. Cycle at 500 rpm for 5 min at 4°C and discard the supernatant.

[0112] 6. Add 500uL of W5 solution and mix thoroughly by adding dropwise with a syringe. The number of additions should be 3.

[0113] 7. Cycle at 500 rpm for 5 min at 4°C and discard the supernatant.

[0114] 8. Finally, suspend the wheat protoplasts in 300uL W5 solution and incubate at 26℃ for 16h.

[0115] The small DNA fragment between the restriction endonucleases XhoI and BamHI in the vector PGL486GFP was replaced with the CDS sequence of the TaSLAH2-B gene, as shown in SEQ ID NO. 2, to generate the recombinant plasmid PGL486-TaSLAH2-B-GFP. The recombinant plasmid PGL486-TaSLAH2-B-GFP expresses the TaSLAH2-B-GFP fusion protein. PGL486GFP is a commonly used GFP fusion protein expression vector in plants. It contains multiple cloning sites for constructing the vector using restriction endonucleases. The vector is kanamycin-resistant and was provided by the National Key Laboratory of Crop Stress Tolerance and Efficient Production.

[0116] The recombinant plasmid PGL486-TaSLAH2-B-GFP or vector PGL486GFP (as a control) was transformed into Chinese Spring protoplasts and then observed under a laser confocal microscope.

[0117] Test results are shown in Figure 1 , wherein PGL486GFP is the vector and PGL486-TaSLAH2-B-GFP is the recombinant plasmid.

[0118] Figure 1 The results showed that the TaSLAH2-B-GFP fusion protein was expressed in the plasma membrane of wheat protoplasts, that is, the TaSLAH2-B protein was localized in the plasma membrane.

[0119] Example 3: Obtaining TaSLAH2-B Transgenic Wheat

[0120] 1. Obtaining the recombinant vector pLGY-Ubi-SLAH-B

[0121] On Snapgene, the DNA fragment between the restriction endonucleases BamHI and SacI in the pLGY vector was replaced with the CDS sequence of the TaSLAH2-B gene shown in SEQ ID NO. 2. Specifically, primers F shown in SEQ ID NO. 10 and R shown in SEQ ID NO. 11 were designed as PCR primers and PCR amplification was performed. The PCR amplification reaction system and procedure are shown in Table 8 and Table 9, respectively. Simultaneously, the pLGY vector was enzymatically digested, and the PCR fragment product and the digested vector were homologously recombined. The recombinant product was transformed into E. coli and sequenced to obtain the recombinant plasmid pLGY-Ubi-SLAH-B.

[0122] The vector pLGY was provided by the National Key Laboratory of Crop Stress Resistance and Efficient Production.

[0123] SEQ ID NO. 10: 5'-gccctaggcctactagggatccGCTATCTCGATTGCCTCGAG-3'. SEQ ID NO. 11: 5'-tacgaacgaaagctctgagctcTGAAAGACTACATCTTTTAA-3'.

[0124] Table 8 PCR amplification reaction system

[0125] Components volume Template (SLAH2-B cDNA sequence template) 2uL Primer F 2uL Primer R 2uL <![CDATA[KOD One TM PCR Master Mix]]> 25uL sterile water 19uL Total volume 50uL

[0126] Table 9 PCR amplification reaction program

[0127]

[0128]

[0129] 2. Obtaining recombinant Agrobacterium tumefaciens

[0130] The recombinant plasmid pUbi-SLAH-B was transformed into Agrobacterium tumefaciens GV3101+pSoup strain to obtain recombinant Agrobacterium X containing the recombinant vector pUbi-SLAH-B, referred to as recombinant Agrobacterium X.

[0131] The Agrobacterium tumefaciens GV3101+pSoup strain was purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0132] The specific steps for transforming the recombinant plasmid pUbi-SLAH-B into the Agrobacterium tumefaciens GV3101+pSoup strain are as follows:

[0133] 1. Add 1 μL of recombinant plasmid pUbi-SLAH-B to 30 μL of GV3101 competent medium from Weidi Company and let it stand on ice for 20 minutes.

[0134] 2. After standing, place the competent state in liquid nitrogen for 3 minutes.

[0135] 3. Then remove the competent cells from liquid nitrogen and immediately place them in a 37°C water bath for 5 minutes.

[0136] 4. Take out the competent cells from the water bath and immediately place them on ice for 5 minutes.

[0137] 5. After standing, add 1 mL of LB liquid medium to the competent medium and then place it in a shaking incubator at 28°C for 1 hour and 40 minutes.

[0138] 6. Remove the competent culture from the shaker, take 1 mL of the competent culture that has been added to LB liquid medium to YEP solid medium and place it in a 30°C incubator.

[0139] 3. Obtaining transgenic wheat with TaSLAH2-B gene

[0140] The recombinant Agrobacterium X was sent to the Shandong Academy of Agricultural Sciences for wheat genetic modification, and 5 transgenic wheat strains with the TaSLAH2-B gene were obtained.

[0141] Agrobacterium-mediated gene transformation was used to introduce the target gene GV3101 / pUbi-SLAH-B into the wheat variety Kenong 199, resulting in the T0 generation of transgenic wheat with the TaSLAH2-B gene. The specific steps are as follows:

[0142] (1) GV3101 / pUbi-SLAH-B was inoculated into YEB liquid medium containing 25 mg / L spectinomycin and cultured at 28°C with shaking until OD 600The nm value was 0.5, and the recombinant Agrobacterium liquid was obtained.

[0143] (2) The immature embryos of the wheat variety Kenong 199 were placed in a 2 mL centrifuge tube filled with preservation solution (10 mL 10×LS Major, 1 mL 100×LS Minor, 1 mL 100× Fe-EDTA, 1 mL 100× Vitamin, 10 mL Glucose and 0.5 g MES were dissolved in an appropriate amount of water and then diluted to 1 L with water), heat treated at 46°C for 3 min, and centrifuged at 4°C and 2000 rpm for 10 min to obtain the treated immature embryos.

[0144] (3) After completing step (2), recombinant Agrobacterium liquid was added to the treated immature embryos, and the embryos were cultured in the dark at 22°C for 3 days, and then transferred to a selection medium and cultured in the dark at 28°C for 8 days; thereafter, the embryos were screened with different concentrations of glufosinate ammonium, and finally transferred to a differentiation medium. After differentiation, the embryos were transferred to a rooting medium, and after reaching a certain size, they were transplanted into nutrient soil to obtain 10 T0 generation transgenic TaSLAH2-B wheat plants, which were respectively designated as OE1, OE1-1, OE1-2, OE1-3, OE2-1, OE2-2, OE3, OE3-1, OE3-2 and OE5.

[0145] Example 4: Real-time fluorescence quantitative detection of the expression level of the TaSLAH2-B gene in transgenic wheat

[0146] Leaves from five two-week-old, transgenic wheat seedlings of Kenong 199, expressing the TaSLAH2-B gene, were stored in liquid nitrogen, yielding a total of five transgenic test samples. Kenong 199 wheat was cultured at 16°C under alternating light and dark conditions for 10 days to obtain the test wheat seedlings. These seedlings were then stored in liquid nitrogen to obtain the corresponding WT test samples. Total RNA was extracted from the transgenic and WT test samples, and first-strand cDNA was reverse-transcribed. Real-time quantitative PCR was used to determine the relative expression of the TaSLAH2-B gene (TaEF1α was used as an internal reference gene).

[0147] The primers for real-time quantitative PCR detection of TaSLAH2-B gene include the upstream primer shown in SEQ ID NO.12 and the downstream primer shown in SEQ ID NO.13.

[0148] SEQ ID NO. 12: 5'-GCCCTGAACTTGAGACCCTT-3'.

[0149] SEQ ID NO. 13: 5'-TGGACACAATGACGGTGAGG-3'.

[0150] The primers for detecting the internal reference gene TaEF1α include the upstream primer shown in SEQ ID NO.14 and the downstream primer shown in SEQ ID NO.15.

[0151] SEQ ID NO. 14: 5'-TGGTGTCATCAAGCCTGGTATGGT-3'.

[0152] SEQ ID NO. 15: 5'-ACTCATGGTGCATCTCAACGGACT-3'.

[0153] Test results are shown in Figure 2 Among them, WT is the wheat variety Kenong 199, and OE1~OE5 are all transgenic TaSLAH2-B gene wheat.

[0154] Figure 2 The results showed that there was no significant difference in the relative expression of the TaSLAH2-B gene between the wheat variety Kenong 199 and the empty vector wheat. However, the relative expression of the TaSLAH2-B gene in the transgenic wheat was significantly increased compared with the wheat variety Kenong 199. Among the transgenic wheat lines with the highest relative expression of the TaSLAH2-B gene, the one with the highest relative expression of the TaSLAH2-B gene was named OE2-2 and used in subsequent experiments.

[0155] Example 5: Verification of stripe rust resistance of transgenic wheat with TaSLAH2-B gene

[0156] Take seeds of Kenong 199 and transgenic wheat OE2-2 with TaSLAH2-B gene, plant the seeds in nutrient solution, place them in a light incubator and culture them at a temperature of 15°C until they reach the adult stage.

[0157] When Kenong 199 and the transgenic wheat OE2-2 with the TaSLAH2-B gene reached adulthood, their flag leaves were inoculated with a mixture of stripe rust fungi consisting of CYR32, CYR33, and CYR34. The stripe rust mixture was prepared by sequentially picking equal masses of CYR32, CYR33, and CYR3 with a pipette and adding them to 2 mL of fluoride solution. Mixing was done until the solution turned from colorless to yellow. For inoculation, 100 μL of the prepared stripe rust mixture was evenly applied to the flag leaf using a 100 μL pipette. After inoculation, the plants were kept moist and dark for 24 hours. Subsequently, they were incubated in a 25°C incubator for 14 days before observing the phenotypes of the control and transgenic plants.

[0158] The stripe rust fungi CYR32, stripe rust fungi CYR33 and stripe rust fungi CYR34 were all provided by the National Key Laboratory of Crop Stress Resistance and Efficient Production.

[0159] The results are as follows Figure 3As shown, the transgenic OE2-2 showed significant stripe rust resistance in the adult stage after inoculation.

[0160] Example 5: Phenotype of the near-isogenic line containing the TaSLAH2-B gene at the adult stage and microscopic histological observation of stripe rust under inoculation treatment at the adult stage

[0161] In the early stages of the laboratory, near-isogenic lines containing only and without the TaSLAH2-B gene were created, designated NIL18-R and NIL19-S. These lines were derived from a hybrid combination of the susceptible cultivars Jimai 229 and Jimai 44. After the near-isogenic lines were grown to the adult stage, their flag leaves were inoculated with a mixture of stripe rust fungi (CYR32, CYR33, CYR34, and V5) according to the inoculation procedure described in Example 5. Samples were collected at different time points (3, 5, 8, 12, and 16 days) after inoculation to observe the stripe rust infection process and disease severity in the near-isogenic lines containing the TaSLAH2-B gene. Phenotypic differences between the near-isogenic lines at later stages of disease development were also observed microscopically, further confirming that the TaSLAH2-B gene can provide resistance to wheat in the adult stage.

[0162] The above-mentioned stripe rust species were all provided by the National Key Laboratory of Crop Resistance and Efficient Production.

[0163] The results are as follows Figure 4 As shown, the near-isogenic line NIL19 containing the TaSLAH2-B gene showed slower disease development and stronger stripe rust resistance than the near-isogenic line NIL18 not containing the TaSLAH2-B gene.

[0164] Example 7: Phenotype of a Jing 411 mutant harboring the TaSLAH2-B gene after inoculation at the adult stage. Flag leaves of a TaSLAH2-B-related mutant (TaSLAH2 M1-6) were inoculated with a mixture of stripe rust fungi, using the same procedure as in Example 5. Following inoculation, the stripe rust phenotype of the TaSLAH2-B mutant was observed.

[0165] The results are as follows Figure 5 As shown in Figure 2, the TaSLAH2-B mutant significantly lost wheat resistance during the adult stage compared to the Jing 411 control (J411-CK). The phenotypic data of the Jing 411 mutant also demonstrated that TaSLAH2-B can provide wheat resistance during the adult stage.

[0166] From the above results, it can be seen that TaSLAH2-B can significantly enhance the resistance of wheat to stripe rust.

[0167] In summary, TaSLAH2-B has obvious resistance to wheat stripe rust in the adult stage.

[0168] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0169] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. Use of the TaSLAH2-B gene or TaSLAH2-B protein in improving wheat stripe rust resistance, characterized in that: The amino acid sequence of the TaSLAH2-B protein is shown in SEQ ID NO.

3. The TaSLAH2-B gene encodes the TaSLAH2-B protein, and the TaSLAH2-B gene sequence is selected from any one of the following DNA molecules: (1) The CDS sequence is shown in SEQ ID NO. 2; (2) The nucleotide sequence is shown in SEQ ID NO. 1; (3) a DNA molecule having at least 90% homology to the CDS sequence shown in SEQ ID NO. 2; (4) A DNA molecule having at least 70% homology to the nucleotide sequence shown in SEQ ID NO.

1.

2. The use of the TaSLAH2-B gene or TaSLAH2-B protein according to claim 1 in improving wheat stripe rust resistance, characterized in that: Genetic engineering methods are used to increase the expression level of the TaSLAH2-B gene in wheat, thereby improving the resistance of wheat to stripe rust.

3. The use of the TaSLAH2-B gene or TaSLAH2-B protein in improving wheat stripe rust resistance according to claim 2, characterized in that: The genetic engineering method includes: replacing the DNA fragment between the restriction endonucleases BamH I and Sac I of the vector pLGY with the CDS sequence of the TaSLAH2-B gene shown in SEQ ID NO.2 to obtain a recombinant plasmid pUbi-SLAH-B; then transforming the recombinant plasmid pUbi-SLAH-B into Agrobacterium to obtain recombinant Agrobacterium; and transforming the recombinant Agrobacterium into wheat to obtain wheat with a TaSLAH2-B gene that is resistant to stripe rust.

4. The use of the TaSLAH2-B gene or TaSLAH2-B protein in improving wheat stripe rust resistance according to claim 3, characterized in that: The primers for amplifying the CDS sequence of the TaSLAH2-B gene are shown in SEQ ID NO.10 to SEQ ID NO.

11.

5. The use of the TaSLAH2-B gene or TaSLAH2-B protein in improving wheat stripe rust resistance according to claim 3, characterized in that: The Agrobacterium is Agrobacterium tumefaciens GV3101+pSoup.

6. A method for improving wheat stripe rust resistance, characterized in that: The steps include: Amplify the CDS sequence of the TaSLAH2-B gene shown in SEQ ID NO. 2 of claim 1, and ligate the amplified product between the BamH I and Sac I restriction sites of the vector pLGY to obtain the recombinant plasmid pUbi-SLAH-B; The recombinant plasmid pUbi-SLAH-B was transformed into Agrobacterium and then transformed into wheat to obtain wheat with the TaSLAH2-B gene that was resistant to stripe rust.

7. A method for breeding wheat resistant to stripe rust, characterized in that: The steps include: Amplify the CDS sequence of the TaSLAH2-B gene shown in SEQ ID NO. 2 of claim 1, and ligate the amplified product between the BamH I and Sac I restriction sites of the vector pLGY to obtain the recombinant plasmid pUbi-SLAH-B; The recombinant plasmid pUbi-SLAH-B was transformed into Agrobacterium to obtain recombinant Agrobacterium, which was inoculated into a culture medium containing spectinomycin to obtain a recombinant Agrobacterium culture solution. Wheat embryos were infected with the recombinant Agrobacterium culture solution and cultured in the dark for 3 to 4 days. The embryos were then transferred to a selection culture medium and cultured for 8 to 9 days. The embryos were then screened with different concentrations of glufosinate and finally transferred to a differentiation culture medium. After differentiation, the embryos were transferred to a rooting culture medium for culture, ultimately obtaining T0 generation wheat transgenic with the TaSLAH2-B gene.

8. A recombinant vector, characterized in that The recombinant vector contains the CDS sequence of the TaSLAH2-B gene shown in SEQ ID NO. 2 of claim 1.

9. Use of the recombinant vector according to claim 8 in improving resistance to wheat stripe rust, characterized in that: The recombinant vector is obtained by inserting the CDS sequence of the TaSLAH2-B gene shown in SEQ ID NO. 2 between the BamH I and Sac I restriction sites of the vector pLGY.

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