Wheat thermo-sensitive stripe rust resistant gene TaEDR2-7B, recombinant vector, application and method

Abstract

The invention belongs to the technical field of biotechnology, and particularly relates to a wheat thermo-sensitive stripe rust resistant gene TaEDR2-7B, a recombinant vector, application and a method. The nucleotide sequence of the gene TaEDR2-7B is as shown in SEQ ID NO. 1 (sequence identifier number 1). The invention provides the gene TaEDR-7B related to the temperature-sensitive stripe rust resistance of the wheat as well as the encoding protein and the application of the gene TaEDR-7B for the first time. The invention discloses that the gene TaEDR2-7B plays an important role in the temperature-sensitive disease resistance of the wheat.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology technology and specifically relates to a wheat temperature-sensitive stripe rust resistance gene. TaEDR2-7B , recombinant vectors and applications and methods. Background Art

[0002] Wheat is one of the main food crops. Wheat stripe rust is an airborne parasitic fungal disease caused by Puccinia striiformis. It has the characteristics of fast transmission speed, long transmission distance and wide damage range. It has now developed into one of the diseases with greater harm and the widest damage area. The cultivation and use of disease-resistant varieties is the most economical, safe and fundamental way to prevent and control wheat stripe rust, and it is also an important strategy for implementing green disease prevention and control. Therefore, discovering and cloning new genes for resistance to stripe rust, determining their resistance effects, and clarifying their disease resistance mechanisms are of great significance to achieving disease-resistant breeding.

[0003] In the long-term evolution of wheat and stripe rust, wheat has evolved high-temperature resistance to stripe rust, which enables it to resist the infection of stripe rust at relatively high temperatures. High-temperature disease resistance refers to the property that the host is susceptible to disease at lower temperatures (16±1℃) but resistant to disease at relatively high temperatures (25±1℃). This type of resistance is called "temperature-sensitive resistance". After wheat was treated with high temperature (25±1℃), the activity of phenylalanine ammonia lyase (PAL), a key enzyme in lignin synthesis, was significantly enhanced, and the content of phenolic acid in the leaves increased, indicating that when high-temperature rust resistance is expressed, the activity of PAL enzyme is significantly enhanced, lignin accumulates in large quantities, and the cells around the infection point are lignified, forming a mechanical barrier to prevent the spread of pathogens; the expression of high-temperature stripe rust resistance does not affect the stripe rust fungus to produce attachment cells and invade tissue cells on the surface of the host plant leaves, but it can induce rapid necrosis of cells and adjacent cells at the infection point. There are obvious differences in histopathological characteristics before and after the expression of high-temperature stripe rust resistance. Before the expression of high-temperature disease resistance, the colony area in the host plant was large and expanded rapidly, with a large number of haustorium mother cells. In the later stage, the amount of necrotic cells produced was small, but it could not inhibit the expansion of hyphae. After the expression of high-temperature stripe rust resistance, typical cell necrosis rapidly occurred around the infection point, and with the passage of time, the number of cell necrosis and lignification continued to increase. In the late stage of infection, the expansion of the colony was significantly inhibited, and the necrotic cells of the host were connected.

[0004] At present, genes related to high temperature resistance have been clearly reported. Yr36 , Yr39 , Yr52 , Yr59 , Yr62 and Yr79 These genes play a unique role in maintaining the persistence of stripe rust resistance, and different genes have different resistance to stripe rust. Therefore, in order to breed new wheat varieties resistant to stripe rust, it is urgent to discover and clone new genes with temperature-sensitive stripe rust resistance in wheat. Summary of the invention

[0005] In order to discover and clone new genes with thermosensitive stripe rust resistance in wheat, the present invention explores the mechanism of thermosensitive stripe rust resistance in wheat and provides a novel thermosensitive stripe rust resistance gene for wheat. TaEDR2-7B , recombinant vector and application and method. To achieve the above object, the present invention adopts the following technical scheme.

[0006] The invention provides a wheat temperature-sensitive stripe rust resistance gene TaEDR2-7B , the gene TaEDR2-7B Derived from wheat, it is a DNA molecule as shown below (b1), (b2), (b3), (b4) or (b5): (b1) A DNA molecule whose coding region is shown in SEQ ID NO.1.

[0007] (b2) A DNA molecule whose nucleotide sequence is shown in SEQ ID NO.1.

[0008] (b3) hybridizes with the DNA molecule defined in (b1) or (b2) under stringent conditions and is derived from a DNA molecule of wheat.

[0009] (b4) A DNA molecule derived from wheat and having at least 70% to 99% homology with the DNA molecule defined in (b1).

[0010] (b5) A DNA molecule derived from wheat and having at least 70% to 99% homology with the DNA molecule defined in (b2).

[0011] The DNA molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0012] The nucleotide sequence shown in SEQ ID NO.1 consists of 15723 nucleotides.

[0013] The purpose of the present invention is to discover and clone a new gene with the characteristics of temperature-sensitive resistance to stripe rust in wheat, and to provide a new gene with the characteristics of temperature-sensitive resistance to stripe rust in wheat. TaEDR2-7B The gene provided by the present invention TaEDR2-7B It is a targeted gene for the temperature-sensitive stripe rust resistance of wheat. This gene functions through the protein it encodes under specific external temperature stimulation, making the wheat containing this gene show significant stripe rust resistance. It can meet the needs of accurately screening stripe rust-resistant wheat varieties under different temperature environments (i.e. in the range of 10℃~25℃).

[0014] Among them, the gene provided by the present invention TaEDR2-7BAt 18°C ​​to 25°C, it can better play the role of resisting stripe rust. One of the purposes of cloning this gene in the present invention is to select the target germplasm or wheat family without temperature stimulation. In actual production or research, it is only necessary to know which wheat varieties or wheat families contain this gene based on the gene sequence. Those containing this gene will definitely play the role of resisting stripe rust at 18°C ​​to 25°C. Therefore, the different temperature ranges referred to here can be any temperature that meets the normal growth of wheat.

[0015] The present invention also provides the wheat temperature-sensitive stripe rust resistance gene TaEDR2-7B The expressed protein has an amino acid sequence as shown in SEQ ID NO. 2. The protein is protein TaEDR2-7B, also known as TaEDR2-7B protein.

[0016] The TaEDR2-7B protein is the following protein (a1), (a2), (a3), (a4) or (a5): (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO.2.

[0017] (a2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.2.

[0018] (a3) A protein derived from wheat and associated with stripe rust resistance and / or yield, obtained by substituting, deleting and / or adding at least one amino acid residue of the protein described in (a1).

[0019] (a4) A protein derived from wheat and associated with stripe rust resistance and / or yield, obtained by substituting, deleting and / or adding at least one amino acid residue of the protein described in (a2).

[0020] (a5) Proteins encoded by other genes that have an amino acid sequence composition that is more than 50% similar to that of SEQ ID NO.2 and have the activity of the protein of SEQ ID NO.2.

[0021] The amino acid sequence shown in SEQ ID NO.2 consists of 725 amino acid residues.

[0022] In the protein of (a3) ​​above, the substitution, deletion and / or addition of at least one amino acid residue is a substitution, deletion and / or addition of no more than 10 amino acid residues.

[0023] The protein in (a3) ​​above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0024] A person skilled in the art can easily adopt known methods, such as directed evolution and point mutation, to modify the gene encoding the present invention. TaEDR2-7B The nucleotide sequence of the artificially modified gene is mutated. TaEDR2-7B Nucleotides with 75% or higher identity to the nucleotide sequence of the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein TaEDR2-7B.

[0025] The term "identity" as used herein refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein TaEDR2-7B of the present invention encoding the amino acid sequence shown in SEQ ID NO.2. Identity can be evaluated by the naked eye or by 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.

[0026] The present invention also provides a method for preparing a wheat thermosensitive stripe rust-resistant gene. TaEDR2-7B recombinant vector or recombinant bacteria.

[0027] Preferably, the recombinant vector comprises the gene TaEDR2-7B , also comprising a method for connecting the gene TaEDR2-7B of the plasmid.

[0028] The recombinant vector can be a recombinant plasmid obtained by inserting the CDS sequence of the DNA molecule shown in SEQ ID NO.1 into the multiple cloning site of the expression vector.

[0029] The recombinant vector may be specifically a recombinant vector pUbi-EDR2-B. The recombinant vector pUbi-EDR2-B may be a restriction endonuclease that converts the vector pCub BamH The small DNA fragment between I is replaced with the CDS sequence in SEQ ID NO.1 to obtain a recombinant plasmid.

[0030] The recombinant bacteria are also called recombinant microorganisms, and the recombinant microorganisms can be recombinant bacteria obtained by introducing the recombinant vector into the starting microorganism.

[0031] Wherein, the starting microorganism may be Agrobacterium or Escherichia coli. The Agrobacterium may be Agrobacterium tumefaciens. The Agrobacterium tumefaciens may be Agrobacterium tumefaciens GV3101.

[0032] The recombinant microorganism may be specifically GV3101 / pUbi-EDR2-B. The GV3101 / pUbi-EDR2-B may be a recombinant Agrobacterium obtained by introducing the recombinant vector pUbi-EDR2-B into Agrobacterium tumefaciens GV3101.

[0033] The present invention also provides the gene TaEDR2-7B , the application of the protein or the recombinant vector, expression cassette or recombinant bacteria in increasing plant disease resistance and / or increasing plant yield. Specifically including at least one of the following applications:

[0034] Improve wheat stripe rust resistance.

[0035] Breeding transgenic wheat with improved resistance to stripe rust.

[0036] Preferably, genetic engineering is used to increase the TaEDR2-7B The expression level of α was increased, thereby obtaining transgenic wheat with high resistance to stripe rust.

[0037] The present invention also provides a method for cultivating transgenic wheat, comprising the following steps: Improving the gene in wheat TaEDR2-7B The expression level and / or activity of the protein is increased, or the expression level and / or activity of the protein in the starting plant is increased to obtain transgenic wheat with improved stripe rust resistance.

[0038] Preferably, by introducing the gene into the wheat TaEDR2-7B To achieve the improvement of the gene in wheat TaEDR2-7B expression level and / or activity.

[0039] In the above method, the "increasing the expression level and / or activity of the protein in the starting plant" and "increasing the gene in the starting plant" TaEDR2-7B The expression level and / or activity of the gene in the recipient plant can be increased by methods well known in the art such as transgenic, multiple copies, changing promoters, and regulatory factors. TaEDR2-7B The expressed protein or gene TaEDR2-7B expression level and / or activity.

[0040] Preferably, by introducing into the wheat TaEDR2-7B The recombinant vector is used to increase the gene expression in wheat TaEDR2-7B The expression level of the gene TaEDR2-7B The recombinant vector comprises the gene TaEDR2-7B , further comprising a method for connecting the gene TaEDR2-7B of the plasmid.

[0041] Furthermore, the gene TaEDR2-7BThe recombinant vector may specifically be the recombinant vector pUbi-EDR2.

[0042] The present invention also provides a wheat breeding method, which increases the gene TaEDR2-7B Or the content of the protein, thereby improving disease resistance.

[0043] Compared with the prior art, the present invention has the following beneficial effects: 1. The purpose of the present invention is to discover and clone a new gene with characteristics of temperature-sensitive resistance to stripe rust in wheat, and to provide a new gene with characteristics of temperature-sensitive resistance to stripe rust in wheat. TaEDR2-7B The gene fully exerts its function after being induced by temperature (15°C-28°C), making wheat show obvious resistance to stripe rust. TaEDR2-7B It is a targeted gene for the temperature-sensitive stripe rust resistance of wheat. This gene functions through the protein it encodes under specific external temperature stimulation, causing the wheat containing this gene to show significant stripe rust resistance. The target germplasm or wheat family can be selected without temperature stimulation, and can meet the needs of accurately screening stripe rust-resistant wheat varieties under the temperature environment of normal wheat growth.

[0044] 2. The experimental results of the present invention prove that: in the wheat variety Zhengmai 7698, the gene is overexpressed TaEDR2-7B , can improve the resistance of wheat to stripe rust, and the protein TaEDR2-7B can improve the disease resistance of plants. The invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 For the present invention TaEDR2-7B Subcellular localization of genes; Figure 1 Figure A in the figure is TaEDR2-GFPmCherry; Figure 1 Figure B shows TaEDR2-GFP; TaEDR2-7B Subcellular localization in wheat protoplasts; GFP is a fluorescent tag; mCherry: empty vector; Merged: merge of bright field and fluorescence; DIA: bright field.

[0046] Figure 2 For the present invention TaEDR2-7B Relative expression levels of genes in transgenic lines.

[0047] Figure 3 For the present invention TaEDR2-7B High temperature phenotype of genetically modified strains; among them. Figure 3 Figure A shows the disease response phenotypes of the susceptible wild-type wheat variety Zhengmai 7698 and its transgenic materials after inoculation with stripe rust under low temperature (10℃~16℃), all of which are susceptible; different samples from the same strain are all parallel test samples;Figure 3 Figure B shows the disease response phenotype of Zhengmai 7698 and its transgenic materials after inoculation with stripe rust under high temperature (18℃~25℃) conditions, in which the wild type is susceptible and the transgenic strain is resistant; different samples under the same strain are all parallel test samples. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0049] Example 1: Gene TaEDR2-7B Clone 1. Take the seeds of Dwarf Kang 58 and germinate them at 25℃ for 3 days. Then transfer the germinated seeds to nutrient soil and culture them at 25℃ for two weeks to obtain Dwarf Kang 58 seedlings.

[0050] Extract DNA from Aikang 58 seedlings.

[0051] Among them, the seeds of Dwarf 58 come from the National Key Laboratory of Crop Stress Resistance and Efficient Production.

[0052] The method for extracting genome (DNA) is as follows: (1) Sampling and grinding: Put two steel balls in a 2 mL centrifuge tube, take a leaf of the dwarf 58 seedling and put it in the centrifuge tube, and grind it with a sample crusher.

[0053] (2) Add 1 mL of 65°C DNA extraction solution (CTAB) and incubate for 2 h.

[0054] (3) Add 700 μL of Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1) and shake vigorously. Let stand for 5 minutes and then centrifuge at 12,000 rpm for 10 minutes.

[0055] (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.

[0056] Supernatant ratio: 800:640:50 (sodium acetate).

[0057] (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.

[0058] (6) Centrifuge at 12000 rpm for 2 min, dry, and add 100 μL ddH2O to obtain dwarf anti-58 DNA.

[0059] 2. Using the DNA of Dwarf Anti-58 as a template, 6 pairs of PCR primers were used to perform PCR amplification in segments, and a PCR amplification product of about 3000 bp was obtained in each segment.

[0060] The PCR amplification products were sequenced.

[0061] Among them, 6 pairs of PCR primers are as follows: Pro: The nucleotide sequence of F of Pro is shown in SEQ ID NO.3: 5′-CTCTGTTGCGCCTGTTGTTC-3′.

[0062] The nucleotide sequence of R of Pro is shown in SEQ ID NO.4: 5′-GCTTTCTCGGCGCTTCAAC-3′.

[0063] gDNA1: The nucleotide sequence of F of gDNA1 is shown in SEQ ID NO.5: 5′-CCGAAGGACCCGCCACTTT-3′.

[0064] The nucleotide sequence of R of gDNA1 is shown in SEQ ID NO.6: 5'-GTTGACAAGGTAGAACACGCAAT-3'.

[0065] gDNA2: The nucleotide sequence of F of gDNA2 is shown in SEQ ID NO.7: 5'-GTTGACAACCACTCCCCTGT-3'.

[0066] The nucleotide sequence of R of gDNA2 is shown in SEQ ID NO.8: 5'-ACAGTTACTCTGTATCTTCAAGGCA-3'.

[0067] gDNA3: The nucleotide sequence of F of gDNA3 is shown in SEQ ID NO.9: 5'-TACGATATTGGCGGCGTAATG-3'.

[0068] The nucleotide sequence of R of gDNA3 is shown in SEQ ID NO.10: 5'-TACATGGAAGATGCTTGTGTGTTTC-3'.

[0069] gDNA4: The nucleotide sequence of F of gDNA4 is shown in SEQ ID NO.11: 5′-ACGGCATCTATGGCCCTTTT-3′.

[0070] The nucleotide sequence of R of gDNA4 is shown in SEQ ID NO.12: 5′-TCGAGATGGCATGCTTTTGC-3′.

[0071] gDNA5: The nucleotide sequence of F of gDNA5 is shown in SEQ ID NO.13: 5'-GCAAAAACATACTCCCTCCGT-3'.

[0072] The nucleotide sequence of R of gDNA5 is shown in SEQ ID NO.14: 5'-GGCGTTGTAGTTGAAGAACCT-3'.

[0073] The reaction procedure of PCR amplification is shown in Table 1: Table 1 PCR amplification reaction procedure The reaction system for PCR amplification is shown in Table 2: Table 2 PCR amplification reaction system 3. The sequences of each sequencing result were spliced ​​together, and the nucleotide sequence of the final PCR amplification product was shown in SEQ ID NO.1:

[0074] 4. Name the gene shown in SEQ ID NO.1 TaEDR2-7B Gene. TaEDR2-7B Wheat temperature-sensitive stripe rust resistance gene TaEDR2-7B ,Gene TaEDR2-7B .

[0075] Gene TaEDR2-7B The amino acid sequence of the encoded protein is shown in SEQ ID NO.2: .

[0076] Example 2: Subcellular localization of TaEDR2-7B-GFP fusion protein 1. Insert the vector PCAM1305GFP restriction endonuclease Bamh The small DNA fragment between I was replaced with the CDS sequence in SEQ ID NO.1 to obtain the recombinant plasmid PCAM1305GFP-TaEDR2-7B.

[0077] The recombinant plasmid PCAM1305GFP-TaEDR2-7B expresses TaEDR2-7B-GFP fusion protein.

[0078] Among them, the vector PCAM1305GFP comes from the National Key Laboratory of Crop Stress Resistance and Efficient Production.

[0079] 2. The recombinant plasmid PCAM1305GFP-TaEDR2-7B or vector PCAM1305GFP (as a control) was transformed into Chinese Spring protoplasts and then observed under a laser confocal microscope.

[0080] The source / method of obtaining Chinese Spring protoplasts is: 1. Solution preparation The configuration information of the enzyme solution is shown in Table 3: Table 3 Enzyme solution configuration table Prepare according to the amount 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 incubate at 121°C for 20 minutes.

[0081] The configuration information of wheat protoplast suspension W5 solution is shown in Table 4: Table 4 Configuration of W5 solution Prepare the solution according to the amount 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 1M KOH and incubate at 121℃ for 20 minutes.

[0082] The configuration information of mannitol MgCl2 solution is shown in Table 5: Table 5 Configuration table of mannitol MgCl2 solution Prepare according to the amount 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 incubate at 121℃ for 20 minutes.

[0083] The configuration information of polyethylene glycol 4000 solution is shown in Table 6: Table 6 Configuration of polyethylene glycol 4000 solution Prepare according to the amount of each component per 50 mL in Table 6, and filter and sterilize with a 0.22uM filter membrane after the prepared solution is dissolved and fixed to volume.

[0084] 2. Preparation of Protoplasts 1. First, prepare the enzymatic hydrolysate. The configuration information of the enzymatic hydrolysate is shown in Table 7.

[0085] Table 7 Enzyme solution configuration table 2. After adding the two enzymes, place in a 55°C water bath for 10 minutes (shake the liquid during this period), then cool to room temperature (ice bath is fine), and then add CaCl2, BSA and β-Me after cooling to room temperature. After adding everything, filter with a 0.45uM filter membrane and filter into a plate.

[0086] 3. Take 10 first leaves of wheat, place the single leaf on A4 paper, cut it lightly with a blade, and put it into a petri dish within 40 minutes so that the leaves cover the liquid surface of the petri dish.

[0087] 4. Vacuum for 15 minutes. Wrap with tin foil and observe after 65 minutes of enzymatic hydrolysis, and light green begins to collect.

[0088] 5. Filter the protoplasts through a 200-mesh filter and wash the filter four times with W5 (pre-cooled W5 with an equal volume as the enzymatic solution). Collect the protoplasts into two 50-mL round-bottom centrifuge tubes.

[0089] 6. Centrifuge at 500 rpm for 10 min at 4°C (acceleration / deceleration of centrifugal force ACC is set to 1), remove the supernatant as much as possible (pool the two tubes into one tube), resuspend the protoplasts in 10 mL of W5 solution, and examine under a microscope.

[0090] 7. Repeat step 6 8. Place in ice bath for 40 minutes, centrifuge, and aspirate out as much supernatant as possible.

[0091] 9. Add appropriate amount of MMG.

[0092] 3. Protoplast Transformation 1. Prepare 20uL of transformation mixture in a 2mL centrifuge tube and transfer 10uL per transfer.

[0093] 2. 100uL protoplasts.

[0094] 3. Add 110uL of fresh PEG with a gun and mix thoroughly. The number of drops is 25.

[0095] 4. Invert in the dark at room temperature for 25 minutes, then add 440uL W5 solution and mix with a gun (about 7 to 10 times) to stop the conversion.

[0096] 5. 500 rpm for 5 min at 4°C, discard the supernatant.

[0097] 6. Add 500uL W5 solution and mix thoroughly by adding dropwise with a spray gun. The number of drops is 3.

[0098] 7. 500 rpm for 5 min at 4°C, discard the supernatant.

[0099] 8. Finally, suspend the wheat protoplasts in 300uL W5 solution and incubate at 26°C for 16h.

[0100] Test results are shown in Figure 1 , wherein PCAM1305GFP is a vector and PCAM1305GFP-TaEDR2-7B is a recombinant plasmid.

[0101] Figure 1 The results showed that the TaEDR2-7B-GFP fusion protein was expressed in the endoplasmic reticulum of wheat protoplasts, that is, the TaEDR2-7B protein was localized in the endoplasmic reticulum.

[0102] Example 3: Transfer TaEDR2-7B Obtaining genetically modified wheat 1. Obtaining the recombinant vector pCub-EDR2 The restriction endonucleases of the vector pCub were inserted into Snapgene BamH The small DNA fragment between I was replaced with the CDS sequence in SEQ ID NO.1, PCR primers were designed, PCR amplification was performed, the vector was digested at the same time, the PCR fragment product and the digested vector were homologously recombined, the recombinant product was transformed into Escherichia coli, and it was sequenced to finally obtain the recombinant plasmid pUbi-EDR2-B.

[0103] Among them, the vector pCub comes from the National Key Laboratory of Crop Resistance and Efficient Production.

[0104] PCR primers are: The nucleotide sequence of F is shown in SEQ ID NO.15: 5'-gtgttacttctgcagggatccATGTCGTCATCCTCGTCGAC-3'.

[0105] The nucleotide sequence of R is shown in SEQ ID NO.16: 5'-agctcggtacccggggatccTTAATCCTCAAGCACAGGAAC-3'.

[0106] The reaction system for PCR amplification is shown in Table 8: Table 8 Reaction system for PCR amplification The reaction program of PCR amplification is shown in Table 9: Table 9 PCR amplification reaction program 2. Obtaining recombinant Agrobacterium tumefaciens The recombinant plasmid pUbi-EDR2-B was transformed into Agrobacterium tumefaciens GV3101+pSoup strain to obtain recombinant Agrobacterium X containing the recombinant vector pUbi-EDR2-B, referred to as recombinant Agrobacterium X.

[0107] Among them, the Agrobacterium tumefaciens GV3101+pSoup strain came from Shanghai Weidi Biotechnology Co., Ltd.

[0108] The conditions for the above transformation are as follows: 1. Add 1 μL of recombinant plasmid pUbi-EDR2-B to 30 μL of GV3101+pSoup competent medium and let stand on ice for 20 min.

[0109] 2. After standing, place the competent state in liquid nitrogen for 3 minutes.

[0110] 3. Then take the competent cells out of the liquid nitrogen and immediately place them in a 37°C water bath for 5 min.

[0111] 4. Take out the competent cells from the water bath and immediately place them on ice for 5 minutes.

[0112] 5. After standing, add 1 mL of LB liquid medium to the competent medium, and then place it in a 28°C shaker for 1h40min.

[0113] 6. Remove the competent medium from the shaker, take 1 mL of the competent medium that has been added to the LB liquid medium to the YEP solid medium and place it in a 30°C incubator.

[0114] 3. Transfer TaEDR2-7B Obtaining genetically modified wheat The recombinant Agrobacterium X was sent to the National Key Laboratory for genetic modification, and 12 transgenic TaEDR2-7B Genetically modified wheat.

[0115] Agrobacterium-mediated gene transformation was used to introduce GV3101 / pUbi-EDR2-B into the wheat variety Zhengmai 7698 to obtain the T0 generation of wheat to be transformed with the TaEDR2-7B gene. The specific steps are as follows:

[0116] (1) Inoculate GV3101 / pUbi-EDR2-B into YEB liquid medium containing 25 mg / L spectinomycin and culture at 28°C with shaking until the OD600nm value reaches 0.5 to obtain recombinant Agrobacterium culture liquid.

[0117] (2) Place the immature embryos of the wheat variety Zhengmai 7698 in a centrifuge tube (2 mL) filled with preservation solution (dissolve 10 mL 10×LS Major, 1 mL 100×LS Minor, 1 mL 100×FeEDTA, 1 mL 100×Vitamin, 10 mL Glucose and 0.5 g MES in an appropriate amount of water, and then dilute to 1 L with water), heat treat at 46°C for 3 min, and centrifuge at 4°C and 2000 rpm for 10 min to obtain the treated immature embryos.

[0118] (3) After completing step (2), add the recombinant Agrobacterium solution to the treated immature embryos, culture them in the dark at 22°C for 3 days, then transfer them to a selection medium and culture them in the dark at 28°C for 8 days; then screen them with different concentrations of glufosinate ammonium, and finally transfer them to a differentiation medium. After differentiation, transfer them to a rooting medium, and after they reach a certain size, transfer them to nutrient soil to obtain T0 generation wheat to be transfected with the TaEDR2-7B gene.

[0119] Example 4: Real-time fluorescence quantitative detection of transfection TaEDR2-7B Genetic wheat TaEDR2-7B Gene expression 1. Transfer 12 TaEDR2-7B The leaves of Zhengmai 7698 seedlings grown for 2 weeks were stored in liquid nitrogen to obtain 12 samples to be tested, namely OE1-OE12.

[0120] 2. Take the wheat variety Zhengmai 7698, and culture it at 16°C with alternating light and dark for 10 days to obtain the wheat seedlings to be tested; store the wheat seedlings to be tested in liquid nitrogen to obtain the corresponding test sample WT.

[0121] 3. Extract the total RNA of the above OE1-OE12 and WT samples, then reverse transcribe the first-strand cDNA, and perform real-time quantitative PCR detection. TaEDR2-7B The relative expression of genes ( TaEF1α gene as the internal reference gene).

[0122] The method for extracting total RNA is the same as above.

[0123] Detection TaEDR2-7B The primers for the genes are: The nucleotide sequence of F is shown in SEQ ID NO.17: 5′-CGTAAGCCCCAGCACAAGAT-3′.

[0124] The nucleotide sequence of R is shown in SEQ ID NO.18: 5′-TACCATCGGGCGACACTACA-3′.

[0125] Detection TaEF1α The primers for the genes are: The nucleotide sequence of F is shown in SEQ ID NO.19: 5'-TGGTGTCATCAAGCCTGGTATGGT-3'.

[0126] The nucleotide sequence of R is shown in SEQ ID NO.20: 5'-ACTCATGGTGCATCTCAACGGACT-3'.

[0127] Test results are shown in Figure 2 WT is the wheat variety Zhengmai 7698, OE1 and OE12 are both transgenic TaEDR2-7B Genetically modified wheat.

[0128] Figure 2 The results showed that the wheat varieties Zhengmai 7698 and empty vector wheat TaEDR2-7B There was no significant difference in the relative expression of the gene; compared with the wheat variety Zhengmai 7698, the TaEDR2-7B gene-transfected wheat TaEDR2-7B The relative expression levels of genes were significantly increased. TaEDR2-7B Genetic wheat lines TaEDR2-7B The gene with higher relative expression was named OE8 and was used for subsequent experiments.

[0129] Example 5: Wheat stripe rust resistance under high temperature treatment 1. Take Zhengmai 7698 and transgenic OE8 seeds, plant the seeds in nutrient solution, place them in a light incubator at 15°C for two weeks, and obtain Zhengmai 7698 seedlings.

[0130] 2. When the Zhengmai 7698 seedlings grew to two leaves and one heart, they were inoculated with a mixture of stripe rust species (CYR32, CYR33 and CYR34). After culturing in a normal temperature incubator for 8 days, they were transferred to a 25°C incubator for high temperature treatment. After 1 day of high temperature treatment, they were transferred to a 15°C incubator.

[0131] Wheat stripe rust resistance phenotype Figure 3 .

[0132] The results showed that the transgenic OE8 treated with high temperature (25°C) showed stronger resistance to stripe rust than OE8 not treated with high temperature.

[0133] From the above results, we can see that TaEDR2-7BWheat stripe rust resistance is related to temperature.

[0134] In summary, TaEDR2-7B There is obvious resistance to wheat stripe rust, and this resistance is related to temperature.

[0135] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0136] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A wheat temperature-sensitive stripe rust resistance gene TaEDR2-7B , characterized in that, The gene TaEDR2-7B The nucleotide sequence is shown in SEQ ID NO.

1.

2. The wheat temperature-sensitive stripe rust resistance gene according to claim 1 TaEDR2-7B The expressed protein is characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A method comprising the wheat temperature-sensitive stripe rust resistance gene according to claim 1 TaEDR2-7B recombinant vector, expression cassette or recombinant bacteria.

4. The recombinant vector, expression cassette or recombinant bacterium according to claim 3, characterized in that: The recombinant vector includes the gene TaEDR2-7B , further comprising a method for connecting the gene TaEDR2-7B of the plasmid.

5. The wheat temperature-sensitive stripe rust resistance gene according to claim 1 TaEDR2-7B , Use of the protein according to claim 2 or the recombinant vector, expression cassette or recombinant bacteria according to claim 3, characterized in that: Include at least one of the following applications: Improve wheat stripe rust resistance; Breeding transgenic wheat with improved resistance to stripe rust.

6. The use according to claim 5, characterized in that: Using genetic engineering to improve the TaEDR2- 7B The expression level of the gene was increased, thereby obtaining transgenic wheat with high resistance to stripe rust.

7. A method for cultivating transgenic wheat, characterized in that: The steps include: Improving the gene according to claim 1 in wheat TaEDR2-7B The expression level and / or activity of the protein was increased to obtain transgenic wheat with improved stripe rust resistance.

8. The method according to claim 7, characterized in that By introducing the gene into the wheat TaEDR2- 7B , to achieve the improvement of the gene in the wheat TaEDR2-7B expression level and / or activity.

9. The method according to claim 7, characterized in that: By introducing into the wheat TaEDR2-7B The recombinant vector is used to increase the gene expression in wheat TaEDR2-7B expression level and / or activity; Containing the gene TaEDR2-7B The recombinant vector comprises the gene TaEDR2-7B , further comprising a method for connecting the gene TaEDR2-7B of the plasmid.

10. A wheat breeding method, characterized in that: Increase the gene of claim 1 in wheat TaEDR2-7B Or the content of the protein as described in claim 2, thereby improving disease resistance.

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