Wheat stem rust resistance protein TaERF60 and its encoding gene and application

Abstract

The present invention relates to the field of biotechnology, and specifically to a wheat stem rust resistance protein TaERF60 and its encoding gene and application. The present invention provides a protein having an amino acid sequence as shown in SEQ ID NO: 2, a nucleic acid molecule encoding the protein, and an application of the protein or the nucleic acid molecule in regulating wheat resistance to wheat stem rust. The present invention provides a new target gene for molecular improvement breeding of wheat stem rust resistance, and has good application prospects in breeding of disease-resistant wheat varieties.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a wheat stem rust resistance protein TaERF60 and a coding gene and application thereof. Background Art

[0002] Wheat stem rust is a disease caused by wheat stem rust fungus (Puccinia graminis f.sp.tritici Erikss.&E.Henn.) on wheat (Triticum aestivum L.) plants, mainly harming the stems and leaf sheaths of wheat, but also the leaves and spikes. The summer spores of wheat stem rust fungus are large, oblong to narrow, reddish brown, and scattered irregularly. When mature, the epidermis cracks in large pieces and turns outward, emitting rust-brown summer spore powder; later, black winter spores are produced, and the epidermis breaks and emits black rust-like winter spores.

[0003] Wheat stem rust is a major catastrophic disease in wheat production, and occurs in countries and regions where wheat is grown. The prevention and control methods of this disease include agricultural prevention and control and chemical prevention and control. Agricultural prevention and control includes planting disease-resistant varieties and strengthening cultivation management, such as through the selection, introduction and promotion of rust-resistant varieties and the rational layout of different rust-resistant varieties, so as to effectively control the prevalence of stem rust. Chemical prevention and control includes seed dressing and spraying of pesticides, such as the use of agents such as triadimefon, propiconazole and tebuconazole for seed dressing or spraying. Although chemical prevention and control can achieve significant disease prevention and yield protection effects, it is easy to pollute the environment. Planting disease-resistant varieties is the most economical, safe and effective measure to prevent and control stem rust. Therefore, it is necessary to explore wheat disease-resistant genes to promote the cultivation of wheat stem rust-resistant varieties. Summary of the invention

[0004] The purpose of the present invention is to provide a protein related to wheat stem rust resistance and a coding gene and application thereof.

[0005] The present invention provides a protein having an amino acid sequence as shown in SEQ ID NO: 2. The protein is named TaERF60.

[0006] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0007] The present invention also provides a nucleic acid molecule encoding the protein.

[0008] The nucleic acid molecule may be DNA, such as genomic DNA or cDNA; the nucleic acid molecule may also be RNA, such as mRNA.

[0009] In some embodiments of the present invention, the nucleic acid molecule is a gene encoding the protein, and its nucleotide sequence is shown in SEQ ID NO:1.

[0010] The expression cassette, vector or host bacteria containing the nucleic acid molecule also fall within the protection scope of the present invention.

[0011] The expression cassette consists of a promoter capable of initiating the expression of the gene, the gene, and a transcription termination sequence.

[0012] The vector is a cloning vector or an expression vector; the host bacteria is a cloning host bacteria or an expression host bacteria.

[0013] The expression vector may be any suitable plant expression vector.

[0014] The use of the protein or the nucleic acid molecule in regulating wheat resistance to wheat stem rust also falls within the protection scope of the present invention.

[0015] The use of the protein or the nucleic acid molecule in breeding wheat varieties with improved resistance to wheat stem rust also falls within the protection scope of the present invention.

[0016] The present invention also provides a method for cultivating transgenic wheat with improved wheat stem rust resistance, comprising: overexpressing the protein encoding gene in wheat to obtain transgenic wheat with improved wheat stem rust resistance.

[0017] In some embodiments of the present invention, the coding gene of the protein is introduced into a plant overexpression vector, and the obtained recombinant expression vector is then introduced into wheat plants to obtain transgenic wheat with improved resistance to wheat stem rust. The plant overexpression vector may be a pLGY-02 vector.

[0018] The present invention also provides a method for cultivating transgenic wheat with reduced wheat stem rust resistance, comprising: inhibiting the expression of the protein encoding gene in wheat to obtain transgenic wheat with reduced wheat stem rust resistance.

[0019] In some embodiments of the present invention, the protein encoding gene is inhibited in wheat by using the virus-induced gene silencing technique (BSMV-VIGS) based on barley stripe mosaic virus (BSMV). Barley stripe mosaic virus is a positive-sense single-stranded RNA virus, whose genome contains three strands of RNAα, RNAβ and RNAγ.

[0020] In the above application or method, the wheat stem rust is caused by wheat stem rust fungus (Puccinia graminisf.sp.tritici Erikss.&E.Henn.).

[0021] The present invention constructed TaERF60 silencing vectors BSMVγ:TaERF60-1 and BSMVγ:TaERF60-2 and used virus-induced gene silencing technology to silence the wheat TaERF60 gene. Meanwhile, an overexpression vector pLGY-02:TaERF60 of TaERF60 was constructed and transient overexpression of wheat TaERF60 was carried out using Agrobacterium-mediated transient expression technology. The experimental results showed that compared with the BSMVγ empty vector control plants, the wheat BSMVγ:TaERF60-1 and BSMVγ:TaERF60-2 silenced plants were more susceptible to wheat stem rust; compared with the pLGY-02 empty vector control plants, the wheat pLGY-02:TaERF60 overexpressing plants had stronger resistance to wheat stem rust. Therefore, the TaERF60 gene is involved in positively regulating wheat resistance to stem rust.

[0022] The present invention provides a new target gene for molecular improvement breeding of wheat resistant to stem rust and has good application prospects in the cultivation of wheat disease-resistant varieties. Brief Description of the Drawings

[0023] Figure 1 It shows that the resistance of wheat to Puccinia graminis f. sp. tritici is weakened after TaERF60 silencing; among them, A-B: agarose gel electrophoresis map of the TaERF60 silencing target fragment amplified by PCR (A: TaERF60-1; B: TaERF60-2); C-D: electrophoresis map of the recombinant plasmid obtained by ligating the TaERF60 silencing target fragment with the BSMVγ vector (C: BSMVγ:TaERF60-1; D: BSMVγ:TaERF60-2); E: symptoms of tobacco after injecting different BSMV vectors into Nicotiana benthamiana leaves; F: phenotypes of wheat TaERF60 silenced plants after inoculating with Puccinia graminis f. sp. tritici; G: qPCR detection of the TaERF60 silencing effect in wheat TaERF60 silenced plants, where the vertical coordinate is the relative expression level of TaERF60, and the data includes the mean ± standard error of three repeated results (**P<0.01, ***0.0001<P≤0.001); BSMVγ:NbPDS represents the positive control, BSMVγ represents the empty vector control, and BSMVγ:TaERF60-1 and BSMVγ:TaERF60-2 represent TaERF60 silenced plants.

[0024] Figure 2Show the expression of related genes in wheat TaERF60-silenced plants after inoculation with wheat stem rust fungus; among them, A-E respectively show the changes in the expression levels of TaPR1, TaPR2, TaPR5, TaSOD, and TaCAT in wheat plants after TaERF60 silencing. The abscissa is the time (hours) after wheat plants are inoculated with wheat stem rust fungus, and the ordinate is the relative expression level of the corresponding gene in wheat plants. The data include the mean ± standard error of three repeated results (*P<0.05, **P<0.01, ***0.0001<P≤0.001); BSMVγ represents the empty vector control plants, and BSMVγ:TaERF60-1 and BSMVγ:TaERF60-2 represent wheat TaERF60-silenced plants.

[0025] Figure 3 Show the hyphal growth of wheat TaERF60-silenced plants after inoculation with wheat stem rust fungus; among them, A is the hyphal growth of wheat stem rust fungus in wheat TaERF60-silenced plants observed under a laser confocal microscope (GT: germ tube; SV: substomatal vesicle; IH: primary infection hypha). 12hpi, 48hpi, and 96hpi in the figure indicate 12h, 48h, and 96h after TaERF60-silenced plants are inoculated with wheat stem rust fungus; B is the colony area of stem rust fungus in wheat TaERF60-silenced plants 96h after inoculation with wheat stem rust fungus. The data include the mean ± standard error of three repeated results (**P<0.01); BSMVγ represents the empty vector control plants, and BSMVγ:TaERF60-1 and BSMVγ:TaERF60-2 represent wheat TaERF60-silenced plants.

[0026] Figure 4 Show the infection phenotypes of wheat plants with transient overexpression of TaERF60 after inoculation with wheat stem rust fungus; among them, A: electrophoresis pattern of the full-length coding sequence (CDS) of TaERF60 gene amplified by PCR; B: relative expression level of TaERF60 detected by qRT-PCR. The data include the mean ± standard error of three repeated results (***0.0001<P≤0.001); C: phenotypes of different wheat plants after inoculation with wheat stem rust fungus; pLGY-02 represents the empty vector control plants, pLGY-02:TaERF60 represents wheat TaERF60-overexpressing plants, and CK represents healthy wheat plants.

[0027] Figure 5Show the expression of related genes in wheat TaERF60 overexpression plants after inoculation with wheat stem rust fungus; among them, A-E: the changes in the expression levels of TaPR1, TaPR2, TaPR5, TaCAT, and TaSOD in wheat plants after overexpression of TaERF60. The abscissa represents the time (hours) after wheat plants were inoculated with wheat stem rust fungus, and the ordinate represents the relative expression levels of the corresponding genes in wheat plants. The data include the mean ± standard error of three repeated results (*P<0.05, **P<0.01, ***0.0001<P≤0.001); pLGY-02 represents the empty vector control plants, and pLGY-02:TaERF60 represents wheat TaERF60 overexpression plants.

[0028] Figure 6 Show the hyphal growth of wheat TaERF60 overexpression plants after inoculation with wheat stem rust fungus; among them, A: the hyphal growth of wheat stem rust fungus in wheat TaERF60 overexpression plants observed under a laser confocal microscope (GT: germ tube; SV: substomatal vesicle). In the figure, 12hpi, 48hpi, and 96hpi represent 12 hours, 48 hours, and 96 hours after wheat TaERF60-silenced plants were inoculated with wheat stem rust fungus; B: the colony area of stem rust fungus in wheat TaERF60 overexpression plants 96 hours after inoculation with wheat stem rust fungus. The data include the mean ± standard error of three repeated results (***0.0001<P≤0.001); pLGY-02 represents the empty vector control plants, and pLGY-02:TaERF60 represents wheat TaERF60 overexpression plants.

[0029] Figure 7 Show the results of subcellular localization analysis of TaERF60-GFP; among them, RFP represents the field of view under 555nm excitation light, GFP represents the field of view under 488nm excitation light, DIC represents the bright field, and Merge represents the composite of the GFP field of view, RFP field of view, and bright field; H2B-mRFP is a nuclear localization marker that emits red fluorescence under 555nm excitation light. Detailed implementation manners

[0030] The present invention will be elaborated in detail below in combination with embodiments. It should be understood that the following embodiments are only for the explanation and illustration of the present invention and do not limit the scope of the present invention in any way.

[0031] Biomaterials

[0032] "Little Club" (LC) is a known wheat variety susceptible to stem rust, and "Mianzi 52" is a known wheat variety resistant to stem rust, both of which are recorded in "Cao Yuanyin, Chen Wanquan. Evolution of host identification and naming methods for physiological races of wheat stem rust [J]. Journal of Wheat Crops, 2010, 30(1): 167-172." The public can obtain the above wheat varieties from the applicant for the sole purpose of repeating the present invention.

[0033] 34MKGQM is a prevalent wheat stem rust fungus species in China, which is recorded in the article “Li Tianya, Chen Si, Cao Yuanyin, et al. Analysis of wheat varieties (lines) resistance to stem rust in Yunnan Province [J]. Journal of Wheat Crops, 2014, 34(2): 267-271.” The public can obtain the above wheat stem rust fungus species from the applicant for the sole purpose of repeating the present invention.

[0034] The barley stripe mosaic virus (BSMV) VIGS vector system (pCa-γbLIC plasmid, pCaBS-α plasmid and pCaBS-β plasmid) is a vector system constructed by the laboratory of Professor Li Dawei of China Agricultural University, and is described in the article "Yuan C, Li C, Yan L, Jackson AO, Liu Z, Han C, Yu J, Li D (2011) A High Throughput Barley Stripe Mosaic Virus Vector for Virus Induced Gene Silencing in Monocots and Dicots. PLoS One, Volume 6, Issue 10, e26468". The pCa-γbLIC plasmid contains the DNA sequence corresponding to the γb RNA in the three chains of the barley stripe mosaic virus (BSMV), and at the same time, a LIC connection site is introduced downstream of the γb gene in the plasmid for cloning exogenous gene fragments. In the following examples, the pCa-γbLIC plasmid is referred to as the BSMVγ vector. The above-mentioned vector system is available to the public from the applicant for the sole purpose of replicating the present invention.

[0035] The pLGY-02 used in the following examples is a wheat overexpression vector, which is recorded in the article "Liang Fang, Liu Yifei, Cui Zhongchi, et al. Rapid identification of the function of wheat pathogenesis-related protein gene TaPR1 using Agrobacterium-mediated method [J]. Journal of Hebei Agricultural University, 2019, 42(2): 12-17." The pLGY-02 vector used in the following examples was donated by Professor Wang Haiyan of the College of Plant Protection, Hebei Agricultural University. The public can obtain the pLGY-02 vector from the applicant for the sole purpose of repeating the present invention.

[0036] The pEH19 vector used in the following examples is the pEH19 vector described in “Shuo Yang, Yuwen Fu, Yang Zhang et al. Rhizoctonia solani transcriptional activator interacts with rice WRKY53 and grassy tiller 1 to activate SWEET transporters for nutrition. 2022, 5: 1-12. DOI: 10.1016 / j.jare.2022.10.001”, which is publicly available from the applicant and is only used to repeat the present invention.

[0037] The recombinant Agrobacterium GV3101 / P19 and GV3101 / H2B-mRFP used in the following examples are recorded in the article “Yuan P, Yang S, Feng L, Chu J, Dong H, Sun J, Chen H, Li Z, Yamamoto N, Zheng A, Li S, Yoon HC, Chen J, Ma D, Xuan YH. Red-light receptor Phytochrome B inhibits BZR1-NAC028-CAD8B signaling to negatively regulate rice resistance to sheath blight. 2023, 46(4): 1249-1263. DOI: 10.1111 / pce.14502.”, which is available to the public from the applicant for the sole purpose of repeating the present invention.

[0038] The applicant declares that the above biological materials may be distributed to the public within twenty years from the filing date for the sole purpose of verifying the invention.

[0039] Medium formulation

[0040] LB medium: weigh 2 g sodium chloride, 1 g yeast extract powder, and 2 g tryptone, make up to 200 mL, and sterilize at 120°C for 20 min (4 g agar powder should be added to the solid medium).

[0041] PCR primers

[0042] Table 1 PCR primers used in the following examples

[0043]

[0044]

[0045] Main instruments

[0046] GI54DS automatic pressure steam sterilizer, manufactured by Zealway. Centrifuge 5418R refrigerated centrifuge, manufactured by Eppendorf. S1000 TM PCR instrument, produced by BIO-RAD. QuantStudio real-time fluorescence quantitative PCR instrument, produced by Applied Biosystems. Electronic analytical balance, produced by Sartorius. TGL ice machine, produced by Shanghai Medical Analytical Instrument Factory. Vortex shaker, produced by Thermo Fisher Scientific. DYY electrophoresis instrument, produced by Beijing Liuyi Company. Alliance 4.7Chroma UV gel imager, produced by Uvitec. H2O3-100C metal bath, produced by Kayoudi Biotechnology. Spectrophotometer, produced by Shanghai Youke Instrument Co., Ltd. 30℃ shaker, produced by Dalong Xingchuang Experimental Instrument (Beijing) Co., Ltd. FV3000 laser scanning confocal microscope (OLYMPUS), produced by Olympus.

[0047] If not otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure. If not otherwise specified, the experimental methods and conditions used in the following examples are all conventional experimental methods and conditions in the art, and reference can be made to relevant experimental manuals, known documents or manufacturer specifications. Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0048] Example 1. Cloning and functional verification of wheat TaERF60 gene

[0049] 1. Wheat RNA Extraction and cDNA Synthesis

[0050] The total RNA of wheat (Little Club) was extracted by Trizol method. The specific steps are as follows: put wheat leaves into a mortar, grind them into powder in liquid nitrogen, put them into a pre-cooled 1.5mL centrifuge tube and place them on ice, quickly add 1mL RNAiso Plus (Takara), mix them upside down until the sample is completely dissolved in RNAiso Plus; add 200μL chloroform, shake for 15s, centrifuge at 12000rpm for 5min, take the supernatant (about 530μL) and transfer it to a new 1.5mL centrifuge tube; add an equal volume of isopropanol, mix them upside down by hand, let them stand at 4℃ for 20min, centrifuge at 12000rpm for 20min, discard the supernatant and keep the white precipitate; add 350μL 75% ethanol, blow, centrifuge at 12000rpm for 5min, and aspirate the ethanol; open the lid and place it in a clean bench and blow for 5min; add 20μL sterile water and mix to obtain RNA solution, which is stored in a -20℃ refrigerator.

[0051] The obtained total RNA of wheat was reverse transcribed using the RNA reverse transcription kit (Cat. No. R423-01) of Novozymes according to the kit instructions to synthesize the first strand of cDNA as a template for gene cloning. The obtained wheat cDNA was stored in a -20°C refrigerator.

[0052] 2. Cloning of target gene

[0053] According to previous research data, we found a gene TaERF60 in wheat that is upregulated under the induction of wheat stem rust. The nucleotide sequence of the open reading frame (ORF) of the wheat TaERF60 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO:2.

[0054] Open reading frame sequence of wheat TaERF60 gene (846 bp):

[0055] A TGGCGACGACGGTGGA CTGGCGCAGCTATAGGCCCGATCTTCCGGCGGCGATGTACCA

[0056] CATGGTGGACGGCAGAGACCAGGTAATGCACGCGTTCGCTCCGCCGACGGCGCAGGGC

[0057] GCGGCGCCGACCATCCTCTCTCCTTCCCCTGCCCCGGCGCGGAGCAGAGCGCCGGCCT

[0058] GCTTCGTGGCGCCAGCTACCTCACTCCCGCGCAAATCCTCCAGCTCCAGTCGCAGCTCC

[0059] ACC ACGTGCGCCGGGC GCCGGGCGCGGCCATGGCAGTGGCGGGGCAGCCCATGAAGCG

[0060] GCACGGCGTTGCGGCGCTCCCGGCGCAGCCGGCGGCCAAGCTGTACCGCGGCGTGCGG

[0061] CAGCGGCATTGGGGGAAGTGGGTCGCCGAGATCCGCCTGCCCCGCAACCGCACCCGCC

[0062] TCTGGCTCGGCACCTTCGACACCGCCGACGAGGCCGCGCTGGCCTACGACGCCGCCGC

[0063] CTTCCGGCTCCGCGGCGAGTCCGCCAGGCTCAACTTCCCCGAGCTCAGGCGCGGCGGC

[0064] GAGCACCACGGCCCGCCGCTCGACGCCGCGATCGACGCCAAGCTCCGCTCCATCTGCC

[0065] ACGGGGAG GACCTGCCGCAGAGCCAG AGCAATGCGACGCCGGCGCCGACGCCGACCC

[0066] TGACGCCGAGCTCTTTCCCGGACGTCAAGAGCGAGCCAGGCTGCTCCGTCTCCGAGAG

[0067] CTCGTCGTCGGCCGACGGCGAGGTGTCCTCGTGCTCCGACGTCGTCCCGGAGATGCAGC

[0068] TTCTTGATTTCTCGGA GGCTCCATGGGACGAGTC CCTGCTGCGCAAGTACCCGTCGCTCGAGATCGACTGGGACGCGATCCTTTCTTGA(SEQ ID NO:1)

[0069] Amino acid sequence of the protein encoded by wheat TaERF60 gene (281aa):

[0070] MATTVDWRSYRPDLPAAMYHMVDGRDQVMHAFAPPTAQGAAPTISFSFPCPGAEQSAGLL

[0071] RGASYLTPAQILQLQSQLHHVRRAPGAAMAVAGQPMKRHGVAALPAQPAAKLYRGVRQRH

[0072] WGKWVAEIRLPRNRTRLWLGTFDTADEAALAYDAAAFRLRGESARLNFPELRRGGEHHGP

[0073] PLDAAIDAKLRSICHGEDLPQSQSNATPAPTPTLTPSSFPDVKSEPGCSVSESSSSADGEVSSCSDVVPEMQLLDFSEAPWDESLLRKYPSLEIDWDAILS(SEQ ID NO:2)

[0074] Using the wheat cDNA synthesized by reverse transcription as the template, primers V-TaERF60-1-F and V-TaERF60-1-R (Tm is 58°C) were used to amplify the TaERF60 silencing target fragment V-TaERF60-1 (200 bp), primers V-TaERF60-2-F and V-TaERF60-2-R (Tm is 58°C) were used to amplify the TaERF60 silencing target fragment V-TaERF60-2 (250 bp), and primers OE-TaERF60-F and OE-TaERF60-R (Tm is 58°C) were used to amplify the TaERF60 gene coding sequence (CDS) full-length OE-TaERF60 (846 bp). The nucleotide sequences of the primers are shown in Table 1. Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the primers. The PCR reaction system is shown in Table 2, where the high-fidelity enzyme KOD Plus was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.

[0075] Table 2 PCR reaction system

[0076]

[0077]

[0078] The PCR reaction program was: 94°C for 2 min; 94°C for 15 s, Tm for 30 s, 68°C for 1 kb / 60 ​​s, 35 cycles; stored at 4°C (the reaction program was set according to the primer annealing temperature Tm and the fragment size). The PCR product was detected by 1.0% agarose gel electrophoresis (200 V, 15 min). The results were as follows: Figure 1A. Figure 1 B. Figure 4 As shown in A, the PCR product size is correct. The target band was recovered using a DNA purification recovery kit (Takara, Cat. No. 9761) according to the kit instructions to obtain TaERF60 silencing target fragments V-TaERF60-1 and V-TaERF60-2, as well as the full-length TaERF60 gene CDS OE-TaERF60 (overexpressed gene fragment), which were stored in a -20°C refrigerator.

[0079] 3. Construction of TaERF60 Silencing Vector

[0080] The BSMVγ vector was digested with ApaⅠ endonuclease (Takara). The digestion system and conditions were as follows:

[0081] Table 3 ApaⅠ enzyme digestion system

[0082] Reagents Dosage <![CDATA[ddH2O]]> 21μL 10×Buffer 3μL ApaⅠ 1μL BSMVγ 5μL

[0083] The prepared enzyme digestion reaction system was placed in a 37°C metal bath for 1 hour. After the reaction was completed, the digestion product was detected by 1.0% agarose gel electrophoresis. The BSMVγ linear vector was obtained by using a DNA purification recovery kit (Takara, Cat. No. 9761) according to the kit instructions and stored in a -20°C refrigerator.

[0084] The purified BSMVγ linear vector was connected with the TaERF60 silencing target fragments V-TaERF60-1 and V-TaERF60-2 using Vazyme's T4 DNA Polymerase as follows.

[0085] Table 4 Treatment of BSMVγ linear vector

[0086]

[0087]

[0088] Add the above system into a PCR tube, incubate at 25°C for 90 min; or at 72°C for 20 min. Cool the resulting reaction solution on ice for 5-10 min.

[0089] Table 5. Processing of TaERF60 silencing target fragments

[0090]

[0091] Add the above system into a PCR tube, incubate at 25°C for 90 min; or at 72°C for 20 min. Cool the resulting reaction solution on ice for 5-10 min.

[0092] Pipette 10 μL of the treated BSMVγ linear vector and 20 μL of the treated TaERF60 silencing target fragment, mix thoroughly, and gradually heat to 66°C for 2 minutes, then place at room temperature for 10 minutes to obtain the ligation product. The ligation product was transformed into Escherichia coli DH5α competent cells by heat shock method and screened with kanamycin. Pick 6 to 8 monoclonal clones, use 2×Taq Master Mix (Dye Plus) of Nanjing Novozyme Biotechnology Co., Ltd., and perform bacterial liquid PCR verification according to the following reaction system and reaction procedure. The PCR primers are BSMV-11 and BSMV-32, and their nucleotide sequences are shown in Table 1.

[0093] Table 6 Bacterial liquid PCR reaction system

[0094] Reagents Dosage 2×Taq Master Mix (Dye Plus) 10μL 10 μM forward primer 1μL 10 μM reverse primer 1μL Bacterial liquid 1μL <![CDATA[ddH2O]]> Make up to 20 μL

[0095] The PCR reaction program was: 98°C for 30s; 98°C for 30s, 58°C for 30s, 72°C for 10s, 35 cycles; 72°C for 5min. After the reaction, the amplified product was sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing verification.

[0096] The positive colonies with correct sequencing were extracted with a rapid plasmid extraction kit (Takara, catalog number: 9760) to obtain the TaERF60 silencing vector BSMVγ: TaERF60-1 ( Figure 1 C) and BSMVγ:TaERF60-2( Figure 1 D).

[0097] In the previous stage, our laboratory constructed the recombinant plasmid BSMVγ:NbPDS used as a positive control for BSMV-VIGS. Its construction method is the same as that of BSMVγ:TaERF60-1 and BSMVγ:TaERF60-2. NbPDS is the tobacco phytoene dehydrogenase PDS gene, and its GenBank accession number is ABE99707.1. The primers used to amplify the NbPDS silencing target fragment are V-NbPDS-F and V-NbPDS-R, and the template is the leaf cDNA of Nicotiana benthamiana. The nucleotide sequences of the primers are shown in Table 1.

[0098] The nucleotide sequence of the NbPDS silencing target fragment is as follows:

[0099] ATTTGCACCCGCAGAAGAGTGGATAAATCGTAGTGACTCAGAAATTATTGATGCTAC

[0100] AATGAAGGAACTAGCGAAGCTTTTCCCTGATGAAATTTCGGCAGATCAGAGCAAAGCAA

[0101] AAATATTGAAGTATCATGTTGTCAAAACCCCAAGGTCTGTTTATAAAACTGTGCCAGGTT

[0102] GTGAACCCTGTCGGCCCTTGCAAAGATCCCCTATAGAGGGTTTTTATTTAGCTGGTGACT

[0103] ACACGAAACAGAAG (SEQ ID NO:29).

[0104] 4. Construction of TaERF60 overexpression vector

[0105] The pLGY-02 vector and the overexpressed gene fragment OE-TaERF60 were double-digested with SacⅠ and BamH I endonucleases (Takara) and purified and recovered. The digestion system and conditions are as follows:

[0106] Table 7 SacⅠ and BamH I restriction enzyme digestion reaction system

[0107] Reagents Dosage <![CDATA[ddH2O]]> 21μL 10×Buffer 3μL SacⅠ 0.5μL BamH 0.5μL pLGY-02 vector / OE-TaERF60 fragment 5μL

[0108] The prepared enzyme digestion reaction system was placed in a 37°C metal bath for 1 hour. After the reaction, the digestion product was detected by 1.0% agarose gel electrophoresis. The DNA purification recovery kit (Takara, Cat. No. 9761) was used for recovery according to the kit instructions to obtain the pLGY-02 linear vector and the double-digested OE-TaERF60 fragment, which were stored in a -20°C refrigerator.

[0109] Vazyme's T4 DNA Ligase was used to connect the pLGY-02 linear vector purified by double restriction digestion with the OE-TaERF60 fragment purified by double restriction digestion.

[0110] Table 8 Ligation reaction system of pLGY-02 vector and OE-TaERF60 fragment

[0111]

[0112]

[0113] Reaction conditions: 22°C, 3h; 70°C, 10min.

[0114] The ligation product was transformed into Escherichia coli DH5α competent cells by heat shock method and screened with ampicillin. 6 to 8 single clones were picked and verified by bacterial liquid PCR using 2×Taq Master Mix (DyePlus) of Nanjing Novozyme Biotechnology Co., Ltd. according to the bacterial liquid PCR reaction system and reaction procedure shown in Table 6. The PCR primers were OE-TaERF60-F and OE-TaERF60-R, and their nucleotide sequences were shown in Table 1.

[0115] After the reaction, the amplified product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing verification. The positive colonies with correct sequencing were extracted with a rapid plasmid extraction kit (Takara, catalog number: 9760) to obtain the TaERF60 overexpression vector pLGY-02:TaERF60.

[0116] 5. Transformation of Agrobacterium Competent Cells

[0117] Agrobacterium GV3101 competent cells were transformed with plasmids BSMVγ, BSMVγ:TaERF60-1, BSMVγ:TaERF60-2, BSMVγ:NbPDS, pCaBS-α, pCaBS-β, pLGY-02 and pLGY-02:TaERF60, respectively. The Agrobacterium transformation method is as follows: take out GV3101 competent cells from the -80°C refrigerator, thaw on ice, add 5 μL of plasmid, gently pipette to mix, and let stand on ice for 20 minutes; quick freeze with liquid nitrogen for 1 minute, 37°C for 5 minutes, and let stand on ice for 5 minutes; add 400 μL LB liquid culture medium to the clean bench, shake at 200 rpm at 30°C for 2.5 hours; centrifuge at 5000 rpm for 1 minute, discard most of the supernatant, resuspend the bacteria with the remaining small amount of liquid and spread on LB solid culture medium containing the corresponding antibiotics (the vector resistance of BSMVγ, pCaBS-α and pCaBS-β is kanamycin, and the vector resistance of pLGY-02 is ampicillin), seal and culture inverted at 30°C for 48 hours.

[0118] According to the introduced plasmids, the obtained recombinant Agrobacterium were named GV3101 / BSMVγ, GV3101 / BSMVγ:TaERF60-1, GV3101 / BSMVγ:TaERF60-2, GV3101 / BSMVγ:NbPDS, GV3101 / pCaBS-α, GV3101 / pCaBS-β, GV3101 / pLGY-02 and GV3101 / pLGY-02:TaERF60.

[0119] 6. TaERF60 gene silencing

[0120] The above-mentioned recombinant Agrobacterium GV3101 / BSMVγ, GV3101 / BSMVγ:TaERF60-1, GV3101 / BSMVγ:TaERF60-2, GV3101 / BSMVγ:NbPDS, GV3101 / pCaBS-α and GV3101 / pCaBS-β were inoculated into LB liquid medium containing kanamycin (50 mg / L) and cultured in a shaking incubator at 30°C until OD 600nm The value is 1-1.5. Take out the Agrobacterium solution, centrifuge it at 4000rpm for 10min at room temperature, discard the supernatant, wash the bacteria three times with the prepared Agrobacterium suspension in a dark place, suspend the bacteria, and then adjust the OD of the bacterial solution. 600nm The formula of Agrobacterium suspension is as follows: take 500 μL 10 mM MES, 50 μL 150 mM As, 5 mL 10 mM MgCl2, add sterile water to make up to 50 mL, mix well and set aside.

[0121] Adjust the OD 600nm The bacterial solutions of BSMVγ recombinant Agrobacterium (GV3101 / BSMVγ, GV3101 / BSMVγ: TaERF60-1, GV3101 / BSMVγ: TaERF60-2, GV3101 / BSMVγ: NbPDS), GV3101 / pCaBS-α recombinant Agrobacterium and GV3101 / pCaBS-β recombinant Agrobacterium were mixed in a volume ratio of 1:1:1 to obtain 4 mixed bacterial solutions. The mixed bacterial solutions were placed at room temperature and dark conditions for 2 to 3 hours for activation, and then injected into leaves of Nicotiana benthamiana.

[0122] Empty vector control tobacco (BSMVγ): Inject a mixed bacterial solution of GV3101 / BSMVγ, GV3101 / pCaBS-α and GV3101 / pCaBS-β in a volume ratio of 1:1:1.

[0123] Positive control (BSMVγ:NbPDS): Inject a mixed bacterial solution of GV3101 / BSMVγ:NbPDS, GV3101 / pCaBS-α and GV3101 / pCaBS-β in a volume ratio of 1:1:1.

[0124] Tobacco silenced strain (BSMVγ:TaERF60-1): Inject a mixed bacterial solution of GV3101 / BSMVγ:TaERF60-1, GV3101 / pCaBS-α and GV3101 / pCaBS-β in a volume ratio of 1:1:1.

[0125] Tobacco silenced strain (BSMVγ:TaERF60-2): Inject a mixed bacterial solution of GV3101 / BSMVγ:TaERF60-2, GV3101 / pCaBS-α and GV3101 / pCaBS-β in a volume ratio of 1:1:1.

[0126] After the tobacco leaves have dried, the injected tobacco is placed back into the light incubator for cultivation. When the tobacco leaves show virus phenotypes, wheat is inoculated. Figure 1 As shown in E, compared with healthy Nicotiana benthamiana, the positive control (BSMVγ:NbPDS) showed obvious albinism, and the empty vector control tobacco (BSMVγ) and the tobacco silenced strains (BSMVγ:TaERF60-1 and BSMVγ:TaERF60-2) showed obvious mottled chlorosis, indicating that BSMV has successfully invaded tobacco.

[0127] Add 5mL PBS buffer and about 0.5g tobacco leaves 10 days after virus inoculation into a sterilized mortar, grind into juice, mix thoroughly by pipetting, and place in a 2mL centrifuge tube and store on ice. Sprinkle a little quartz sand on the surface of wheat "Mianzi 52" leaves, then put on latex gloves and dip into the juice of tobacco leaves to inoculate wheat leaves with BSMV virus. Set up empty vector control wheat (wheat inoculated with BSMVγ) and wheat silent strains (wheat inoculated with BSMVγ: TaERF60-1 and wheat inoculated with BSMVγ: TaERF60-2). After inoculation, the wheat was placed in a 24℃ incubator for culture, and the symptoms of BSMV infection were clearly observed after 14 days.

[0128] Total RNA from the leaves of empty vector control wheat and wheat silenced strains 5 days after virus inoculation was extracted and reverse transcribed into cDNA. Using cDNA as a template, real-time fluorescence quantitative PCR was performed using the specific primers q-TaERF60-F / q-TaERF60-R (Tm value of 58°C) of the TaERF60 gene to detect the silencing effect of TaERF60. At the same time, real-time fluorescence quantitative PCR was performed using the primers q-TaGAPDH-F and q-TaGAPDH-R (Tm value of 58°C) of the internal reference gene TaGAPDH (GenBank accession number LOC123160238). Using 2 ^ -ΔΔCt analysis method was used to calculate the relative expression of TaERF60 gene. The nucleotide sequences of the primers are shown in Table 1.

[0129] Fluorescence quantitative PCR reaction system

[0130] Reagents Dosage ChamQ Universal SYBR qPCR Master Mix (Novozyme) 10μL Forward primer (F) 2μL Reverse primer (R) 2μL cDNA 6μL

[0131] Fluorescence quantitative PCR reaction procedure

[0132]

[0133] like Figure 1 As shown in G, compared with the BSMVγ empty vector control wheat, the relative expression levels of TaERF60 in the wheat BSMVγ:TaERF60-1 silenced strain and the wheat BSMVγ:TaERF60-2 silenced strain were significantly reduced, indicating that the TaERF60 gene was successfully silenced.

[0134] 7. TaERF60 gene overexpression

[0135] The recombinant Agrobacterium GV3101 / pLGY-02: TaERF60 and GV3101 / pLGY-02 were inoculated into LB liquid medium containing ampicillin (50 mg / L) in a clean bench and cultured at 30°C in a shaking incubator until OD 600nm The value is 1-1.5. Take out the Agrobacterium solution, centrifuge it at 4000rpm for 10min at room temperature, discard the supernatant, wash the bacteria three times with the prepared Agrobacterium suspension in a dark place, suspend the bacteria, and then adjust the OD of the bacterial solution. 600nm The formula of Agrobacterium suspension is as follows: take 500 μL 10 mM MES, 50 μL 150 mM As, 5 mL 10 mM MgCl2, add sterile water to make up to 50 mL, mix well and set aside.

[0136] Place the bacterial solution at room temperature and in the dark for 2 to 3 hours to activate it, and then inject it into the leaves of wheat "small dense spike" respectively. The wheat injected with GV3101 / pLGY-02:TaERF60 is a TaERF60 overexpression strain. The wheat injected with GV3101 / pLGY-02 is an empty vector control plant. After the wheat leaves become dry, put them back into the light incubator for cultivation. 72 hours after injection, extract the total RNA from the leaves of the wheat TaERF60 overexpression strain and the empty vector control plant and reverse transcribe it into cDNA, and use real-time fluorescence quantitative PCR to detect the effect of TaERF60 overexpression. The fluorescence quantitative PCR detection method is the same as the detection method of the TaERF60 silencing effect mentioned above.

[0137] The results are as follows Figure 4 As shown in B, 72 h after injection, the relative expression level of TaERF60 in the TaERF60 overexpression strain was significantly increased compared with that in the empty vector control plant, indicating that the TaERF60 gene was successfully overexpressed.

[0138] 8. Test of resistance of TaERF60 silenced strain and TaERF60 overexpression strain to stem rust

[0139] Wheat plants 5 days after inoculation with BSMVγ, BSMVγ: TaERF60-1 and BSMVγ: TaERF60-2, and wheat plants 3 days after injection with GV3101 / pLGY-02 and GV3101 / pLGY-02: TaERF60 were selected and inoculated with wheat stem rust 34MKGQM. The inoculation method was as follows: Tween and water were mixed evenly in a volume ratio of 20:1000 and put into a spray pot, 1g of summer spores of wheat stem rust 34MKGQM were added, and the mixture was shaken and inoculated on the leaves of the above wheat plants, kept moisturized at 16-18°C for 16-20h, and then moved into a greenhouse for cultivation.

[0140] Total RNA from the leaves of the wheat plants was extracted 24h, 48h and 120h after inoculation with wheat stem rust and reverse transcribed into cDNA. Real-time fluorescence quantitative PCR was used to detect the expression levels of related genes such as TaPR1 (GenBank accession number LOC543437), TaPR2 (GenBank accession number LOC123080647), TaPR5 (GenBank accession number LOC543342), TaCAT (GenBank accession number LOC123168517) and TaSOD (GenBank accession number LOC101290631) in the wheat plants 24h, 48h and 120h after inoculation with wheat stem rust. The fluorescence quantitative PCR detection method is the same as the detection method of the TaERF60 silencing effect. The PCR primers for TaPR1 are q-TaPR1-F / q-TaPR1-R (Tm value is 59°C), the PCR primers for TaPR2 are q-TaPR2-F / q-TaPR2-R (Tm value is 64°C), the PCR primers for TaPR5 are q-TaPR5-F / q-TaPR5-R (Tm value is 62°C), the PCR primers for TaCAT are q-TaCAT-F / q-TaCAT-R (Tm value is 58°C), and the PCR primers for TaSOD are q-TaSOD-F / q-TaSOD-R (Tm value is 58°C). The nucleotide sequences of the primers are shown in Table 1.

[0141] Compared with the empty vector control (BSMVγ), the expression of three PR genes (TaPR1, TaPR2 and TaPR5) in wheat TaERF60 silenced strains (BSMVγ:TaERF60-1 and BSMVγ:TaERF60-2) was significantly downregulated ( Figure 2 AC), reactive oxygen species (ROS) scavenging genes (TaSOD and TaCAT) were significantly upregulated ( Figure 2D and E). Compared with the empty vector control (pLGY-02), the expression levels of three PR genes (TaPR1, TaPR2 and TaPR5) in the wheat TaERF60 overexpression strain (pLGY-02:TaERF60) were significantly upregulated ( Figure 5 AC), the expression levels of ROS scavenging genes (TaCAT and TaSOD) were significantly downregulated ( Figure 5 D and E). The above-mentioned PR genes (Pathogenesis-related genes) are a type of genes that are specifically induced by pathogens or exogenous hormones and are closely related to systemic acquired resistance.

[0142] Laser confocal microscopy was used to observe the mycelial infection of wheat TaERF60 silenced strains and TaERF60 overexpression strains at 12h, 48h and 96h after inoculation with wheat stem rust. Compared with the empty vector control (BSMVγ), the length of germ tube (GT), the length of primary infected hyphae (IH) and the number of substomatal vesicles (SV) in wheat TaERF60 silenced strains (BSMVγ:TaERF60-1 and BSMVγ:TaERF60-2) were significantly increased ( Figure 3 A). Compared with the empty vector control (pLGY-02), the length of the germ tube (GT) and the number of substomatal vesicles (SV) in the wheat TaERF60 overexpression strain (pLGY-02:TaERF60) were significantly reduced ( Figure 6 A).

[0143] The colony area of ​​wheat stem rust of TaERF60 silenced strain and TaERF60 overexpression strain was counted 96 hours after inoculation with wheat stem rust. The phenotype of wheat TaERF60 silenced strain and TaERF60 overexpression strain was observed 14 days after inoculation with wheat stem rust to identify the infection type of wheat stem rust. The 0-4 classification standard of wheat stem rust infection type is as follows:

[0144] 0: No allergic spots occur;

[0145] 0;: no summer spores but yellow-white allergic spots can be seen;

[0146] 1: There are tiny summer spores but they are surrounded by obvious yellow-white allergic dead spots;

[0147] 2: Small to medium-sized summer spores but often inhabit green islands surrounded by spindle-shaped allergic dieback;

[0148] 3: Medium-sized summer spores, rarely fused; there should be no allergic dieback around the spores, but there may be chlorosis;

[0149] 4: Summer spores are large, often fused, without allergic dieback, but may also produce chlorosis;

[0150] In the infection type levels 1, 2, 3, and 4, if the lesions of the same infection type are larger, add "+", and if they are smaller, add "-". Among them, 0, 0;, 1-, 1, 1+, 2-, 2 and 2+ belong to low infection types (disease-resistant), and 3-, 3, 3+, 4-, 4 and 4+ belong to high infection types (susceptible).

[0151] The results showed that the infection type of the BSMVγ empty vector control plant was 1, and the infection type of the TaERF60 silenced strains BSMVγ:TaERF60-1 and BSMVγ:TaERF60-2 was 2. The spore piles on the TaERF60 silenced strains were much larger than those on the BSMVγ empty vector control plants ( Figure 1 F), the colony area of ​​stem rust on the TaERF60 silenced strain was significantly larger than that on the BSMVγ empty vector control plant ( Figure 3 B). The infection type of the pLGY-02 empty vector control plant was 4, and the infection type of the TaERF60 overexpression strain pLGY-02:TaERF60 was 2 ( Figure 4 C), the colony area of ​​stem rust on the TaERF60 overexpression strain was significantly smaller than that on the pLGY-02 empty vector control plant ( Figure 6 B). This indicates that the TaERF60 gene has a positive regulatory effect on wheat stem rust resistance.

[0152] Example 2. Subcellular localization of TaERF60

[0153] 1. Cloning of target gene

[0154] Using the recombinant plasmid pLGY-02: TaERF60 constructed in Example 1 as a template, primers TaERF60-GW-F and TaERF60-GW-R were used for PCR to amplify the full length of the TaERF60 gene coding sequence (CDS). The nucleotide sequences of the primers are shown in Table 1. The reaction system is shown in Table 2. The reaction procedure is: 94°C 2min; 94°C 15s, 58°C 30s, 68°C 1kb / 60s, 35 cycles; 4°C storage. After the reaction, 1.0% agarose gel electrophoresis (200V, 15min) was used to detect the PCR product, and a universal DNA purification and recovery kit (Takara, catalog number 9761) was used to recover the TaERF60 gene fragment according to the kit instructions.

[0155] 2. Ligation of target gene and vector

[0156] First, the TaERF60 gene fragment was connected to the pDONR221 vector of the Gateway system through BP reaction. The reaction system and procedure are as follows:

[0157] Reagents Dosage TaERF60 gene fragment 1μL pDONR221 vector 1μL BP Clonase Enzyme Mix 0.5μL

[0158] 25℃, 3h; 65℃, 15min. The ligation product was transferred into E. coli DH5α competent cells by heat shock method. Single clones were picked and verified by bacterial liquid PCR using the above primers TaERF60-GW-F and TaERF60-GW-R. Single clones identified correctly by PCR were sequenced to obtain positive clones with correct sequences. The plasmids of positive clones were extracted to obtain the recombinant plasmid pDONR221:TaERF60.

[0159] Then, the TaERF60 gene fragment was connected to the expression vector pEH19 by LR reaction. The reaction system and procedure were as follows:

[0160] Reagents Dosage Recombinant plasmid pDONR221:TaERF60 1μL pEH19 vector 1μL LR Clonase Enzyme Mix 0.5μL

[0161] 25℃, 3h; 65℃, 15min. The ligation product was transformed into E. coli DH5α competent cells by heat shock method. Single clones were picked and verified by bacterial liquid PCR using the above primers TaERF60-GW-F and TaERF60-GW-R to obtain positive clones. The plasmid of the positive clone was extracted to obtain the recombinant plasmid pEH19:TaERF60.

[0162] The above-mentioned BP Clonase Enzyme Mix and LR Clonase Enzyme Mix were purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0163] 3. Transformation of Agrobacterium Competent Cells

[0164] The recombinant plasmid pEH19: TaERF60 was used to transform the competent cells of Agrobacterium tumefaciens GV3101 to obtain the recombinant Agrobacterium tumefaciens GV3101 / pEH19: TaERF60. Meanwhile, the pEH19 empty vector was used to transform the competent cells of Agrobacterium tumefaciens GV3101 to obtain the recombinant Agrobacterium tumefaciens GV3101 / pEH19. The Agrobacterium transformation method was the same as in Example 1.

[0165] 4. Transient expression of genes in tobacco

[0166] The recombinant Agrobacterium GV3101 / P19 (Yuan P et al., 2023) was inoculated into LB liquid medium containing kanamycin (50 mg / L), and the recombinant Agrobacterium GV3101 / H2B-mRFP (Yuan P et al., 2023), GV3101 / pEH19 and GV3101 / pEH19: TaERF60 were inoculated into LB liquid medium containing spectinomycin (50 mg / L) respectively, and cultured in a shaking incubator at 30°C for 12-16 hours. The Agrobacterium liquid was taken out, centrifuged at 4000 rpm for 10 minutes at room temperature, the supernatant was discarded, the bacteria were washed three times with the Agrobacterium suspension prepared in the dark, and then the bacteria were suspended, and the OD of the bacterial liquid was adjusted. 600nm The formula of Agrobacterium suspension is as follows: take 500 μL 10 mM MES, 50 μL 150 mM As, 5 mL 10 mM MgCl2, add sterile water to make up to 50 mL, mix well and set aside.

[0167] Place the bacterial solution at room temperature and in the dark for 2 to 3 hours to activate. Mix the bacterial solutions of recombinant Agrobacterium GV3101 / P19, GV3101 / H2B-mRFP and GV3101 / pEH19 in a volume ratio of 1:1:1 to obtain combined bacterial solution C. Mix the bacterial solutions of recombinant Agrobacterium GV3101 / P19, GV3101 / H2B-mRFP and GV3101 / pEH19:TaERF60 in a volume ratio of 1:1:1 to obtain combined bacterial solution T. Use combined bacterial solution C and combined bacterial solution T to inject Nicotiana benthamiana leaves respectively. After culturing for 2 to 3 days after injection, cut part of the tobacco leaf tissue, make slices with the back of the leaf facing up, and observe the fluorescence expression in the leaves using a confocal laser microscope. The pEH19 vector has a green fluorescent protein (GFP) tag, so the recombinant plasmid pEH19:TaERF60 expresses the TaERF60-GFP fusion protein. The excitation wavelength of GFP is 488nm, and the image acquisition wavelength range is 515-540nm. The expression of the TaERF60-GFP fusion protein was observed using 488nm excitation light and photographed. The results showed that in tobacco injected with GV3101 / pEH19, green fluorescence appeared in the nucleus and cell membrane, while in tobacco injected with GV3101 / pEH19:TaERF60, green fluorescence only appeared in the nucleus ( Figure 7 ). This indicates that TaERF60 is localized in the cell nucleus.

Claims

1. Use of a protein having an amino acid sequence as shown in SEQ ID NO: 2 or a nucleic acid molecule encoding the protein in regulating wheat resistance to wheat stem rust.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid molecule encoding the protein is shown in SEQ ID NO:

1.

3. Use of a protein having an amino acid sequence as shown in SEQ ID NO: 2 or a nucleic acid molecule encoding the protein in breeding wheat varieties with improved resistance to wheat stem rust.

4. The use according to claim 3, characterized in that: The nucleotide sequence of the nucleic acid molecule encoding the protein is shown in SEQ ID NO:

1.

5. A method for cultivating transgenic wheat with improved resistance to wheat stem rust, comprising: The gene encoding the protein whose amino acid sequence is shown in SEQ ID NO: 2 is overexpressed in wheat to obtain transgenic wheat with improved resistance to wheat stem rust.

6. The method according to claim 5, characterized in that The nucleotide sequence of the gene encoding the protein is shown in SEQ ID NO:

1.

7. A method for cultivating transgenic wheat with reduced resistance to wheat stem rust, comprising: The expression of a gene encoding a protein having an amino acid sequence as shown in SEQ ID NO: 2 is suppressed in wheat to obtain transgenic wheat having reduced resistance to wheat stem rust.

8. The method according to claim 7, characterized in that The nucleotide sequence of the gene encoding the protein is shown in SEQ ID NO:

1.

9. The use according to any one of claims 1 to 4 or the method according to any one of claims 5 to 8, characterized in that: The wheat stem rust is caused by Puccinia tritici.

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