Target gene for regulating and controlling wheat stalk rust resistance and application of target gene in regulating and controlling wheat stalk rust resistance

Abstract

The invention provides a target gene for regulating and controlling wheat Puccinia graminis resistance and application of the target gene in regulating and controlling the wheat Puccinia graminis resistance, and belongs to the technical field of molecular biology. The invention provides a gene for regulating and controlling the resistance of wheat to Puccinia stelleriana. The gene encodes a protein with an amino acid sequence as shown in SEQ ID No.1. Through a gene silencing method, the encoding gene of the protein is inhibited in wheat, and transgenic wheat with improved wheat stem rust resistance is obtained. Through overexpression of the gene, transgenic wheat with reduced wheat stem rust resistance is obtained. The gene is more resistant to wheat stem rust after being silenced; after the gene is over-expressed, the plant is more susceptible to wheat stem rust. Therefore, the TaSWEET5c gene participates in negative regulation of the resistance of the wheat to the Puccinia graminis.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and particularly relates to a target gene for regulating wheat stem rust resistance and an application of the target gene in regulating wheat stem rust resistance. Background Art

[0002] Wheat stem rust is caused by the wheat-specific form of the fungus Puccinia graminis f.sp. tritici (Pgt), a fully rust-forming fungus with a life cycle consisting of five developmental stages: pycniospore, aeciospore, urediospore, teliospore, and basidiospore. Wheat stem rust, caused by Puccinia graminis f.sp. tritici, is a widespread fungal disease worldwide that can be spread long distances by air currents. It has a long history, widespread distribution, high prevalence, and severe damage. Of the three wheat rust diseases, wheat stem rust causes the most severe yield losses, given the same natural prevalence.

[0003] Wheat stem rust primarily damages the stems and leaf sheaths, but in severe cases can also affect leaves and ears. In average years, it can cause yield reductions of 20% to 30%, and in endemic years, it can result in yield losses of over 70%, with the most severe cases nearly eliminating all crops. In recent years, with the continued promotion of improved wheat varieties in my country, improvements in growing environments, and a reduction in the severity of stem rust damage, wheat yields have increased significantly. However, the frequent virulence variations of the wheat stem rust fungus, the emergence of new and highly pathogenic races, coupled with unstable climate and environmental conditions and inappropriate planting and management practices, pose significant risks to wheat production safety. Extensive experience has shown that the most cost-effective approach to controlling wheat rust is to leverage cultivar resistance. Future research on wheat stem rust should explore the molecular mechanisms of wheat-stem rust interactions and develop effective control strategies.

[0004] The SWEET gene family is a newly discovered class of sugar transporters that bidirectionally transport sugars across plant membranes along a concentration gradient. Current research on their functions has primarily focused on Arabidopsis thaliana and rice. Although research on the SWEET gene family is relatively recent, existing results suggest that they participate in key physiological processes in plant growth and development by regulating the transport, distribution, and storage of sugar compounds within the plant body. However, the relationship between SWEET genes and wheat stem rust resistance is less well-documented. Summary of the Invention

[0005] The present invention provides a target gene for regulating wheat stem rust resistance and its application in regulating wheat stem rust resistance. The TaSWEET5c gene is involved in negatively regulating wheat stem rust resistance, providing a possible strategy for the genetic improvement of disease-resistant high-quality wheat germplasm resources.

[0006] The present invention provides a gene for regulating wheat resistance to stem rust. The gene encodes a protein whose amino acid sequence is shown in SEQ ID No. 1.

[0007] In a preferred embodiment of the present invention, the nucleotide sequence of the gene includes the sequence shown in SEQ ID No. 2.

[0008] The present invention also provides a set of primer pairs for amplifying the above gene, comprising an upstream primer having a nucleotide sequence as shown in SEQ ID No. 3 and a downstream primer having a nucleotide sequence as shown in SEQ ID No. 4.

[0009] The present invention also provides a biological material comprising the above gene, wherein the type of the biological material includes any one of the following: an expression cassette, a vector and a host bacteria.

[0010] The present invention also provides the use of the above gene or the above biological material in regulating wheat resistance to stem rust.

[0011] The present invention also provides the use of the above gene or the above biological material in breeding wheat with high resistance to stem rust.

[0012] The present invention also provides a method for improving wheat resistance to stem rust, comprising inhibiting the expression of the above gene in the wheat genome, or knocking out the above gene.

[0013] The present invention also provides a material for inhibiting the expression of the above gene, with V-TaSWEET5c as the silencing target fragment, and the amplification primer pair of the silencing target fragment includes an upstream primer with a nucleotide sequence as shown in SEQ ID No. 5 and a downstream primer as shown in SEQ ID No. 6.

[0014] The present invention also provides a method for cultivating wheat resistant to stem rust, comprising inhibiting the expression of the above gene in wheat.

[0015] The present invention also provides a method for cultivating a wheat plant model with reduced stem rust resistance, comprising overexpressing the above gene in the wheat genome.

[0016] Beneficial Effects: The present invention provides a gene for regulating wheat stem rust resistance, wherein the gene encodes a protein having an amino acid sequence as shown in SEQ ID No. 1. In an embodiment of the present invention, gene silencing is used to inhibit the expression of the gene encoding the protein in wheat, thereby obtaining transgenic wheat with enhanced resistance to wheat stem rust. Simultaneously, by overexpressing the gene, transgenic wheat with reduced resistance to wheat stem rust is obtained.

[0017] Specifically, in an embodiment of the present invention, a TaSWEET5c silencing vector BSMVγ:TaSWEET5c was constructed by virus-induced gene silencing technology, and a TaSWEET5c overexpression vector pLGY-02:TaSWEET5c was constructed by Agrobacterium-mediated transient expression technology. Gene silencing and transient overexpression of wheat TaSWEET5c were performed respectively. The experimental results showed that in gene silencing, compared with BSMVγ empty vector control plants, wheat TaSWEET5c silenced plants (BSMVγ:TaSWEET5c) were more resistant to wheat stem rust; in transient overexpression, compared with pLGY-02 empty vector control plants, wheat TaSWEET5c overexpressing plants (pLGY-02:TaSWEET5c) were more susceptible to wheat stem rust. Therefore, the TaSWEET5c gene is involved in negatively regulating wheat resistance to stem rust. The gene described in the present invention provides a possibility for the genetic improvement of disease-resistant high-quality germplasm resources of wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The electrophoresis results of total RNA and cDNA extraction from wheat leaves are shown in Figure 1. A: The electrophoresis results of wheat total RNA quality detection; B: The electrophoresis results of internal reference (TaGAPDH) amplification in wheat cDNA;

[0019] Figure 2 The following are the results of electrophoresis of vector enzyme digestion: A: single enzyme digestion of BSMVγ vector electrophoresis result; B: double enzyme digestion of pLGY-02 vector electrophoresis result;

[0020] Figure 3Figure 2 shows the results of enhancing wheat resistance to stem rust after silencing TaSWEET5c, where A: electrophoresis result of amplification of TaSWEET5c silencing fragment; B: electrophoresis result of ligation of TaSWEET5c silencing fragment with BSMVγ vector; C: symptoms of Nicotiana benthamiana leaves after injection of different BSMVs (BSMVγ:NbPDS represents the positive control in tobacco, BSMVγ:TaPDS represents the positive control in wheat, BSMVγ represents the empty vector control, and BSMVγ:TaSWEET5c represents TaSWEET5c). ET5c silenced plants); D: Symptoms of wheat leaves after inoculation with different BSMVs (BSMVγ:TaPDS represents the positive control in wheat, BSMVγ represents the empty vector control, and BSMVγ:TaSWEET5c represents the TaSWEET5c silenced plants); E: Phenotypes of BSMVγ empty vector control plants and BSMVγ:TaSWEET5c silenced plants inoculated with fungi; F: qPCR detection of the TaSWEET5c silencing effect in wheat TaSWEET5c silenced plants (**P<0.01);

[0021] Figure 4 Figure 2 shows the expression of genes related to the course of disease in wheat TaSWEET5c-silenced plants inoculated with wheat stem rust. AE: changes in the expression of TaPR1, TaPR2, TaPR5, TaSOD, and TaCAT after TaSWEET5c silencing (*P<0.05, **P<0.01, ***P<0.001); BSMVγ represents empty vector control plants, and BSMVγ:TaSWEET5c represents wheat TaSWEET5c-silenced plants.

[0022] Figure 5 Figure 2 shows the hyphal growth of wheat TaSWEET5c-silenced plants after inoculation, where A: hyphal growth at different time points after inoculation (GT: germ tube, SV: substomatal sac, IH: primary infective hyphae, HMC: haustorium mother cell, H: haustorium); B: colony growth area (96 h) (***P<0.001); BSMVγ represents empty vector control plants, and BSMVγ:TaSWEET5c represents wheat TaSWEET5c-silenced plants.

[0023] Figure 6Figure 3: Transient overexpression of TaSWEET5c weakened wheat resistance to stem rust. A: Electrophoresis results of amplified TaSWEET5c overexpression fragments; B: Electrophoresis results of ligated TaSWEET5c overexpression fragments with pLGY-02 vector; C: Semi-quantitative RT-PCR detection of TaSWEET5c overexpression effect; D: qPCR detection of TaSWEET5c overexpression effect (***P < 0.001); E: Phenotypes of pLGY-02 empty vector control plants and pLGY-02:TaSWEET5c overexpression plants inoculated with fungi.

[0024] Figure 7 Figure 2 shows the expression of genes related to the course of disease in wheat TaSWEET5c-overexpressing plants inoculated with wheat stem rust. AE: changes in the expression of TaPR1, TaPR2, TaPR5, TaSOD, and TaCAT after TaSWEET5c overexpression (*P<0.05, **P<0.01, ***P<0.001); pLGY-02 represents empty vector control plants, and pLGY-02:TaSWEET5c represents wheat TaSWEET5c-overexpressing plants.

[0025] Figure 8 Figure 3 Mycelial growth of wheat TaSWEET5c-overexpressing plants after inoculation, where A: mycelial growth at different time points after inoculation (GT: germ tube, SV: substomatal sac, IH: primary infective hyphae, HMC: haustorium mother cell, H: haustorium); B: colony growth area (96 h) (**P<0.01); pLGY-02 represents empty vector control plants, and pLGY-02:TaSWEET5c represents wheat TaSWEET5c-overexpressing plants. DETAILED DESCRIPTION

[0026] The present invention provides a gene for regulating wheat resistance to stem rust. The gene encodes a protein whose amino acid sequence is shown in SEQ ID No. 1.

[0027] In the embodiment of the present invention, the cDNA obtained by reverse transcription of wheat RNA is used as a template, and the TaSWEET5c gene is obtained after amplification, and the expression of the gene TaSWEET5c is upregulated under the induction of wheat stem rust.

[0028] The nucleotide sequence of the open reading frame (ORF) of the TaSWEET5c gene of the present invention is shown in SEQ ID No. 2 (702 bp), and the amino acid sequence of the protein encoded thereby is shown in SEQ ID No. 1 (233 aa).

[0029] The ORF sequence of the TaSWEET5c gene of the present invention is shown in SEQ ID No. 2:

[0030] ATGGTTTCGGCCGACGCAGCTCGCAACGTCGTCGGCATCATGGGCAATTGCATCTCCTTTTGCCTCTTCCTCTCCCCTGCGCAGACATTTGACCGGATTTGCAAGAACAAGGACGTGGAGCAGTTCACGCCGGACCCCTACCTGGCGACGCTCATGAACTGCCTGCTCTGGTTCTTCTACGGCCTCCCAATCGTCCACCCGAACAGCCTCCTCGTCATCACCATCAACAGCATCGGCATCATCATCGAGACAATCTACCTCTCCATCTTCTTCCTCTACTCGCCCCCAAAGAAGCGGCTAAAGATACTTCTCGTTGTTGGCTTTGAGGTGGCGTTCGTGGCGTCCGTGGTGGTCGGAGTGCTCCTCAGCGCCCACACCTACGAGGACCGCTCCAGGATCGTCGGCATCATCTGCATCGTCTTCGGCACGATTATGTATGCCGCCCCGCTCACAGTCATGGGGAAAGTTATCAAGACAAAGAGCGTGGAGTATATGCCATTCACCGTGTCTCTGGTGAACACTATCAACGGCTGCTGCTGGCTAGCCTATGGGCTGATAGGGAACGACCCCTATGTGACGATCCCTAATGCCATCGGTACAGTTTTGTGCATCTTCCAGCTGATCCTTTACGTGTGCTACTACAAGTCGACCCCCGTCAAGGAGCAGAATGTCGAGTTGCCCGCCGCCGCCACAGAGAACTAA;

[0031] The amino acid sequence of the protein encoded by the TaSWEET5c gene is shown in SEQ ID No.1:

[0032] MVSADAARNVVGIMGNCISFCLFLSPAQTFDRICKNKDVEQFTPDPYLATLMNCLLWFFYGLPIVHPNSLLVITINSIGIIIETIYLSIFFLYSPPKKRLKILLVVGFEVAFVASVV VGVLLSAHTYEDRSRIVGIICIVFGTIMYAAPLTVMGKVIKTKSVEYMPFTVSLVNTINGCCWLAYGLIGNDPYVTIPNAIGTVLCIFQLILYVCYYKSTPVKEQNVELPAAATEN.

[0033] The present invention also provides a set of primer pairs for amplifying the above gene, comprising an upstream primer having a nucleotide sequence as shown in SEQ ID No. 3 and a downstream primer having a nucleotide sequence as shown in SEQ ID No. 4.

[0034] In one embodiment of the present invention, cDNA obtained by reverse transcription of wheat RNA was used as a template and primer pairs OE-TaSWEET5c-F and OE-TaSWEET5c-R were used for amplification. The PCR reaction protocol for the amplification included: 94°C pre-denaturation for 2 minutes; 94°C denaturation for 15 seconds, Tm°C annealing for 30 seconds, and 68°C extension for 1 minute / kb, for 35 cycles; and storage at 4°C. The Tm value in the PCR reaction protocol was set to 3-5°C lower than the primer Tm value.

[0035] OE-TaSWEET5c-F (SEQ ID No. 3): GGTACCATGGTTTCGGCCGACGCAG;

[0036] OE-TaSWEET5c-R (SEQ ID No. 4): GAGCTCTTAGTTCTCTGTGGCGGCG.

[0037] The present invention also provides a biological material comprising the above gene, wherein the type of the biological material includes any one of the following: an expression cassette, a vector and a host bacteria.

[0038] The expression cassette of the present invention comprises a promoter, the TaSWEET5c gene and a transcription termination sequence, wherein the promoter is capable of initiating the expression of the TaSWEET5c gene.

[0039] The vector of the present invention may be a cloning vector or an expression vector, wherein the expression vector may be a suitable plant expression vector known in the art.

[0040] The host bacteria of the present invention are host bacteria for cloning or for expression.

[0041] The present invention also provides the use of the above gene or the above biological material in regulating wheat resistance to stem rust.

[0042] The TaSWEET5c gene described in the present invention can regulate wheat resistance to stem rust and exhibits a negative regulatory effect, that is, after inhibiting the expression of the TaSWEET5c gene, the wheat resistance to stem rust is improved, and after overexpressing the TaSWEET5c gene, the wheat resistance to stem rust is reduced. Therefore, based on the TaSWEET5c gene described in the present invention, the wheat resistance to stem rust can be regulated.

[0043] The present invention also provides the use of the above gene or the above biological material in breeding wheat with high resistance to stem rust.

[0044] The method of the present invention, especially the method for inhibiting the expression of the TaSWEET5c gene or the method for knocking out the TaSWEET5c gene, can be used to improve the resistance of wheat to stem rust.

[0045] The present invention also provides a method for improving wheat resistance to stem rust, comprising inhibiting the expression of the above gene in the wheat genome, or knocking out the above gene.

[0046] The present invention does not particularly limit the method of inhibition, which can be RNAi, shRNA or VIGS. In one embodiment, the TaSWEET5c gene is inhibited in wheat by virus-induced gene silencing technology (BSMV-VIGS) based on barley stripe mosaic virus (BSMV).

[0047] The present invention constructs a TaSWEET5c silencing vector BSMVγ:TaSWEET5c through virus-induced gene silencing technology. The BSMV-VIGS vector system includes: a pCa-γbLIC plasmid (referred to as BSMVγ vector), a pCaBS-α plasmid and a pCaBS-β plasmid. The pCa-γbLIC plasmid contains a DNA sequence corresponding to the γb RNA in the three chains of the barley streak mosaic virus (BSMV). Inserting the transcription sequence of the gene segment to be silenced into the pCa-γbLIC plasmid and further combining it with the pCaBS-α plasmid and the pCaBS-β plasmid will lead to the synthesis of viral dsRNA, thereby activating the antiviral RNA silencing pathway and silencing the target gene.

[0048] The present invention also provides a material for inhibiting the expression of the above gene, with V-TaSWEET5c as the silencing target fragment, and the amplification primer pair of the silencing target fragment includes an upstream primer with a nucleotide sequence as shown in SEQ ID No. 5 and a downstream primer as shown in SEQ ID No. 6.

[0049] The invention uses wheat cDNA synthesized by reverse transcription of wheat RNA as a template and uses primers V-TaSWEET5c-F and V-TaSWEET5c-R to amplify a TaSWEET5c silencing target fragment V-TaSWEET5c; the length of the silencing target fragment V-TaSWEET5c is 285 bp.

[0050] The primers designed for amplification and verification in the examples of the present invention were designed using Snap Gene software and commissioned to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The information is shown in Table 1.

[0051] Table 1 Primer sequences used in the examples

[0052]

[0053]

[0054] The present invention also provides a method for cultivating wheat resistant to stem rust, comprising inhibiting the expression of the above gene in wheat.

[0055] In the embodiment of the present invention, the expression of the TaSWEET5c gene was suppressed in wheat by the BSMV-VIGS method. Specifically, the amplified 285bp silencing target fragment V-TaSWEET5c was ligated with the linearized vector obtained by digesting the BSMVγ vector with Q.CutApaⅠ. After transformation into Escherichia coli DH5α, the colony was PCR-positive and the correct plasmid was sequenced, which was named recombinant plasmid BSMVγ:TaSWEET5c. The recombinant plasmid BSMVγ:TaSWEET5c, pCaBS-α, and pCaBS-β were used to transform Agrobacterium GV3101 (pJIC SA-Rep) competent cells, respectively, and screened using Ka-Rif-LB solid medium. The positive recombinant Agrobacterium introduced with the plasmid was named GV3101 / BSMVγ:TaSWEET5c, GV3101 / pCaBS-α, and GV3101 / pCaBS-β, respectively. The plants were then infected with GV3101 / BSMVγ:TaSWEET5c, GV3101 / pCaBS-α and GV3101 / pCaBS-β.

[0056] The present invention also provides a method for cultivating a wheat plant model with reduced stem rust resistance, comprising overexpressing the above gene in the wheat genome.

[0057] In the present invention, when performing the overexpression, a conventional overexpression vector is used, the ORF fragment is connected to a basic vector, and then transformed into Agrobacterium GV3101 competent cells, and then the TaSWEET5c gene is overexpressed based on an Agrobacterium-mediated method. In one embodiment of the present invention, the basic vector is a pLGY-02 vector.

[0058] The stem rust disease described in the present invention is caused by wheat stem rust fungus (Puccinia graminis f.sp.tritici Erikss.&E.Henn.).

[0059] To further illustrate the present invention, a target gene for regulating wheat stem rust resistance and its application in regulating wheat stem rust resistance provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0060] The wheat varieties used in the following examples include Little Club (LC) and Mianzi 52, both known wheat varieties. The wheat stem rust fungus 34MKGQM, a prevalent race of wheat stem rust in China, forms an affinity combination with Little Club LC and a non-affinity combination with Mianzi 52. Both wheat and stem rust fungi were provided by our laboratory and are available to the public from the applicant for the sole purpose of reproducing the present invention.

[0061] The barley streak mosaic virus (BSMV) VIGS vector system (pCa-γbLIC plasmid is referred to as BSMVγ vector, pCaBS-α plasmid and pCaBS-β plasmid), BSMVγ-NbPDS (tobacco positive control for gene silencing) and BSMVγ-TaPDS (wheat positive control for gene silencing) used in the following examples were provided by this laboratory and are available to the public from the applicant for the sole purpose of repeating the present invention.

[0062] The barley streak mosaic virus (BSMV) VIGS vector system includes: (pCa-γbLIC plasmid, pCaBS-α plasmid and pCaBS-β plasmid), among which the pCa-γbLIC plasmid contains the DNA sequence corresponding to the γb RNA in the three chains of barley streak mosaic virus (BSMV). Inserting the transcription sequence representing the gene segment to be silenced into the pCa-γbLIC plasmid and further combining it with the pCaBS-α plasmid and pCaBS-β plasmid will lead to the synthesis of viral dsRNA, thereby activating the antiviral RNA silencing pathway and causing the target gene to be silenced.

[0063] 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 it from the applicant and it is only used to repeat the present invention.

[0064] The applicant declares that the above biological materials may be distributed to the public within twenty years from the date of application for the sole purpose of necessary verification experiments.

[0065] In the following examples, the reagents, consumables and solution formulations used are as follows:

[0066] 50×TAE Buffer, 2×Taq Master Mix (Dye Plus), high-fidelity enzyme KOD-Plus, DNA purification and recovery kit, T4 DNA Polymerase, T4 DNA Ligase, antibiotics such as Kanamycin and Rifampin, NaCl, Yeast Extract, Typtone, agar powder, rapid plasmid extraction kit, dimethyl sulfoxide, chloral hydrate, potassium hydroxide powder, WGA-Alexa488, fast-cutting enzyme, etc.

[0067] (1) 1×TAE Buffer: Add ddH2O to 20 mL of 50×TAE Buffer and adjust the volume to 1000 mL.

[0068] (2) LB medium: Weigh 1 g of NaCl, 0.5 g of Yeast Extract, and 1 g of Typtone, add ddH2O to make up to 100 mL, and sterilize at 120°C for 20 min (2 g of agar powder should be added to the solid medium).

[0069] (3) Antibiotic (kanamycin and other antibiotics) storage solution (50 mg / L): Weigh 2.5 g of antibiotics and place them in a 50 mL centrifuge tube. First, add 40 mL of ddH2O. Mix thoroughly to dissolve, then add ddH2O to make the volume up to 50 mL. Filter and sterilize with a 0.22 μm filter membrane. Aliquot and store at -20°C.

[0070] (4) Rifampicin stock solution (20 mg / L): Weigh 0.2 g of rifampicin into a 50 mL centrifuge tube, add 10 mL of DMSO (dimethyl sulfoxide), filter sterilize, and aliquot. Store at -20°C.

[0071] (5) Antibiotic-LB medium: Add 100 μL of the corresponding antibiotic to 100 mL of liquid LB medium (2 g of agar powder should be added to solid medium).

[0072] (6) Potassium hydroxide solution (0.5 mol / L): Weigh 1.4 g of potassium hydroxide powder, add 50 mL of ddH2O, mix well and set aside.

[0073] (7) 50 mM Tris-HCl: Measure 2.5 mL of 1 M Tris-HCl, add 47.5 mL of ddH2O, and adjust the pH to 7.0-7.4 with HCl.

[0074] (8) WGA-Alexa 488 fluorescent dye: Weigh 1 mg of WGA-Alexa 488 powder and add it to 50 mL of 50 mM Tris-HCl buffer, mix well and set aside.

[0075] In the following examples, the main instruments used in the experiments are:

[0076] GI54DS automatic pressure steam sterilizer, manufactured by Zealway. Centrifuge 5418R refrigerated centrifuge, manufactured by Eppendorf. S1000 TM PCR instrument, manufactured by BIO-RAD. QuantStudio real-time fluorescence quantitative PCR instrument, manufactured by Applied Biosystems. Electronic analytical balance, manufactured by Sartorius. TGL ice maker, manufactured by Shanghai Medical Analytical Instrument Factory. Vortex shaker, manufactured by Thermo Fisher Scientific. DYY electrophoresis apparatus, manufactured by Beijing Liuyi Company. Alliance 4.7 Chroma UV gel imager, manufactured by Uvitec. Artificial climate chamber, manufactured by Shanghai Yiheng Scientific Instrument Co., Ltd. Digital constant temperature water bath, manufactured by Guohua Electric Co., Ltd. H2O3-100°C metal bath, manufactured by Kayoudi Biotechnology Co., Ltd. Spectrophotometer, manufactured by Shanghai Youke Instrument Co., Ltd. Electric constant temperature incubator, manufactured by Shanghai Jinghong Laboratory Equipment Co., Ltd. Constant temperature oscillator, manufactured by Shanghai Yiheng Scientific Instrument Co., Ltd. FV3000 laser scanning confocal microscope (OLYMPUS), manufactured by Olympus.

[0077] Example 1: Functional verification of TaSWEET5c in the interaction between wheat and stem rust

[0078] 1. Wheat RNA extraction and cDNA synthesis

[0079] Wheat RNA was extracted using the Trizol method and the RNA quality was tested by 1.0% agarose gel electrophoresis (200 V, 15 min). Figure 1 As shown in center A, the 25S, 18S, and 5S bands are clearly visible, indicating good integrity of the extracted total RNA. Total RNA was measured using a nucleic acid protein detector, revealing A260 / 280 and A260 / 230 ratios between 1.90 and 2.30, indicating good total RNA purity. Both purity and integrity meet the requirements of subsequent experiments.

[0080] The obtained wheat total RNA was reverse transcribed using the Novozymes RNA Reverse Transcription Kit (Cat. No. R423-01) according to the kit instructions to synthesize first-strand cDNA. To ensure the quality of the cDNA, RT-PCR was used to further detect the expression of TaGAPDH in the cDNA. Using the reverse-transcribed wheat cDNA as a template, the TaGAPDH gene was amplified using primers TaGAPDH-F and TaGAPDH-R (Tm 58°C). The GenBank accession number for TaGAPDH is LOC123058820.

[0081] 10 μL PCR reaction system: 2×TaqMasterMix (Dye Plus) 5 μL, Forward Primer 1 μL, Reverse Primer 1 μL and cDNA 3 μL.

[0082] The PCR reaction program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, and annealing at 50-72°C. a 15 s, 72°C extension b 15 s / kb, 35 cycles; final extension at 72°C for 5 min, storage at 4°C. a. The annealing temperature needs to be adjusted based on the primer Tm value, generally set 3-5°C below the primer Tm value. The annealing temperature for TaGAPDH is 58°C. b. The extension time needs to be adjusted based on the fragment size. The extension time for TaGAPDH is 5 s.

[0083] After the reaction, the TaGAPDH band was detected by gel electrophoresis. Figure 1 As shown in Figure B, TaGAPDH was amplified with uniform band brightness, indicating successful reverse transcription and consistent cDNA concentration. The resulting wheat cDNA can be used as a template for subsequent gene cloning and stored at -20°C until needed.

[0084] 2. Cloning of target genes

[0085] The present invention discovered a gene TaSWEET5c whose expression is upregulated under the induction of wheat stem rust. The nucleotide sequence of the ORF of the TaSWEET5c gene is shown in SEQ ID No. 2, and the amino acid sequence of the protein encoded by the TaSWEET5c gene is shown in SEQ ID No. 1.

[0086] The wheat cDNA synthesized by reverse transcription was used as a template and primers V-TaSWEET5c-F and V-TaSWEET5c-R (Tm of 58°C) were used to amplify the TaSWEET5c silencing target fragment V-TaSWEET5c (285 bp);

[0087] The full-length TaSWEET5c gene CDS (702 bp) was amplified using primers OE-TaSWEET5c-F and OE-TaSWEET5c-R (Tm of 58°C).

[0088] 50 μL PCR reaction system: 10× KOD buffer 5 μL, dNTPs 5 μL, MgSO4 2 μL, Forward Primer 1.5 μL, Reverse Primer 1.5 μL, cDNA 2 μL, KOD Plus 1 μL, and the balance ddH2O.

[0089] The PCR reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, Tm°C a Anneal for 30 seconds, extend at 68°C for 1 minute / kb b , 35 cycles; store at 4°C. a. The annealing temperature needs to be adjusted according to the primer Tm value, generally set to 3-5°C lower than the primer Tm value; b. The extension time should be adjusted accordingly based on the fragment size.

[0090] After the reaction is completed, the PCR products are detected by gel electrophoresis. Figure 3 China A and Figure 6 As shown in Figure A, the product size is correct. The target bands were recovered using a DNA purification and recovery kit (Takara, Cat. No. 9761) according to the kit instructions to obtain the V-TaSWEET5c silencing target gene fragment and the OE-TaSWEET5c overexpression gene fragment, which were then stored at -20°C for future use.

[0091] 3. Enzyme digestion of vector

[0092] (1) Single enzyme digestion of BSMVγ vector

[0093] The enzyme digestion system was configured according to the instructions, and the BSMVγ vector was digested with Q.CutApaⅠ (Takara). The enzyme digestion reaction system was placed in a metal bath at 37°C (the optimal temperature for Q.CutApaⅠ reaction) for 1 hour. After the reaction, a DNA purification and recovery kit was used according to the kit instructions for recovery. The recovered product was subjected to gel electrophoresis to obtain the BSMVγ linear vector ( Figure 2 Store in a -20℃ refrigerator until use.

[0094] 30 μL enzyme digestion system: 10×Q.CutBuffer 3 μL, Q.CutApaⅠ1 μL, BSMVγ5 μL and the balance ddH2O.

[0095] (2) Double enzyme digestion of pLGY-02 vector

[0096] The pLGY-02 vector was double-digested with Q.CutKpnⅠ and Q.CutSac I (Takara). The digestion procedure and recovery of the pLGY-02 vector were the same as those of the BSMVγ vector. The recovered product was subjected to gel electrophoresis to obtain the pLGY-02 linear vector ( Figure 2 B), and stored in a -20℃ refrigerator for later use.

[0097] The 30 μL enzyme digestion system is as follows: 10×Q.CutBuffer 3 μL, Q.CutKpnⅠ1 μL, Q.Cut Sac I1 μL, pLGY-025 μL and the balance ddH2O.

[0098] 4. Ligation of target gene and vector

[0099] (1) BSMVγ-Gene ligation

[0100] Vazyme's T4 DNA Polymerase was used to connect the enzyme-digested and purified BSMVγ linear vector with the V-TaSWEET5c target gene fragment.

[0101] 20 μL ligation system: T4 DNApol Buffer 2 μL, BSA 1 μL, dTTP / dATP 1 μL, T4 DNApol 0.5 μL, single-enzyme-digested BSMVγ / silencing fragment V-TaSWEET5c 200 ng / 100 ng, and the balance ddH2O.

[0102] The digested BSMVγ and the silencing fragment V-TaSWEET5c were added to PCR tubes according to the above system. The ligation process was performed according to the following protocol: 25°C for 90 minutes, followed by 72°C for 20 minutes. After completion of the reaction, the resulting reaction solution was cooled on ice for 5-10 minutes. Then, 10 μL of the treated BSMVγ linearized vector and 20 μL of the treated TaSWEET5c silencing target fragment were aspirated and mixed thoroughly. The mixture was then gradually heated to 66°C for 2 minutes and then allowed to stand at room temperature for 10 minutes to obtain the ligation product. The ligation product was transformed into competent E. coli DH5α cells using the heat shock method and cultured using Ka-LB solid medium for selection.

[0103] (2) Ligation of pLGY-02 vector and TaSWEET5c

[0104] The pLGY-02 linearized vector, purified by double digestion with Vazyme's T4 DNA Ligase, was mixed with the OE-TaSWEET5c target gene fragment to prepare a ligation reaction system. The ligation reaction was performed according to the following protocol: 22°C for 3 hours, then 70°C for 10 minutes. The ligation product was heat-shocked and transformed into competent Escherichia coli DH5α cells, which were then screened and cultured using Ka-LB solid medium.

[0105] 10 μL ligation system: 1.5 μL of double-digested pLGY-02, 6.5 μL of target gene fragment OE-TaSWEET5c, 1 μL of T4 DNA Ligase Buffer, and 1 μL of T4 DNA Ligase.

[0106] (3) Escherichia coli DH5α colony PCR

[0107] Six to eight colonies were selected for PCR verification. The PCR reaction procedure was as follows: initial denaturation at 95°C for 2 min; 35 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 15 s / kb; a final extension at 72°C for 5 min, followed by storage at 4°C. The primers for colony PCR for BSMVγ:TaSWEET5c were BSMV-11 and BSMV-32; and the primers for colony PCR for pLGY-02:TaSWEET5c were OE-TaSWEET5c-F and OE-TaSWEET5c-R.

[0108] After the reaction was completed, the target bands were detected by gel electrophoresis, and the electrophoresis patterns of the BSMVγ vector, pLGY-02 vector and TaSWEET5c were obtained ( Figure 3 Middle B and Figure 6 (B) Positive colonies were picked and cultured in Ka-LB liquid medium at 37°C on a constant-temperature shaker for 12–16 hours. Plasmids were extracted using a rapid plasmid extraction kit and sent to Suzhou Genewise Biotechnology Co., Ltd. for sequencing verification. The recombinant plasmids BSMVγ:TaSWEET5c and pLGY-02:TaSWEET5, which were sequenced correctly, were stored at -20°C until further use.

[0109] 5. Transformation of Agrobacterium Competent Cells

[0110] (1) Transformation of Agrobacterium GV3101 competent cells

[0111] Plasmids BSMVγ, BSMVγ:TaSWEET5c, BSMVγ:NbPDS, BSMVγ:TaPDS, pCaBS-α, pCaBS-β, pLGY-02, and pLGY-02:TaSWEET5c were transformed into competent Agrobacterium tumefaciens GV3101 (pJIC SA-Rep) cells using the freeze-thaw method and screened using Ka-Rif-LB solid medium. NbPDS is the tobacco phytoene dehydrogenase PDS gene (GenBank accession number ABE99707), and TaPDS is the wheat phytoene dehydrogenase PDS gene (GenBank accession number LOC100270658). Both were constructed using the same method as described above for BSMVγ:TaSWEET5c.

[0112] (2) Agrobacterium spp. GV3101 colony PCR

[0113] The colony PCR method is the same as that for E. coli DH5α. The colony PCR primers for BSMVγ, BSMVγ:TaSWEET5c, BSMVγ:NbPDS, and BSMVγ:TaPDS are BSMV-11 and BSMV-32; the colony PCR primers for pCaBS-α and pCaBS-β are BSMV-8 and BSMV-9; and the colony PCR primers for pLGY-02 and pLGY-02:TaSWEET5c are OE-TaSWEET5c-F and OE-TaSWEET5c-R.

[0114] According to the introduced plasmid, the positive recombinant Agrobacterium obtained by screening was named GV3101 / BSMVγ, GV3101 / BSMVγ:TaSWEET5c, GV3101 / BSMVγ:NbPDS, GV3101 / BSMVγ:TaPDS, GV3101 / pCaBS-α, GV3101 / pCaBS-β, GV3101 / pLGY-02 and GV3101 / pLGY-02:TaSWEET5c.

[0115] 6. TaSWEET5c gene silencing

[0116] (1) Agrobacterium injection into tobacco

[0117] 1) Recombinant Agrobacterium GV3101 / BSMVγ, GV3101 / BSMVγ:TaSWEET5c, GV3101 / BSMVγ:NbPDS, GV3101 / BSMVγ:TaPDS, GV3101 / pCaBS-α and GV3101 / pCaBS-β were inoculated into Ka-Rif-LB liquid medium respectively and cultured in a constant temperature shaker at 30°C until OD 600 The value is 1 to 1.5.

[0118] 2) Place the Agrobacterium suspension into a sterile Eppendorf tube and centrifuge at 4000 rpm for 10 min at room temperature. Discard the supernatant and collect the precipitate. Wash the bacteria three times with the prepared Agrobacterium suspension in the dark and suspend the bacteria. Then adjust the OD value of the bacterial suspension. 600 It is 0.6~0.7.

[0119] 3) Prepare 10 μL of Agrobacterium suspension: 100 μL of 10 mM MES, 10 μL of 150 mM AAs, 1 mL of 10 mM MgCl2, and the balance of ddH2O.

[0120] 4) Adjust the OD 600 Agrobacterium GV3101 / BSMVγ, GV3101 / BSMVγ:TaSWEET5c, GV3101 / BSMVγ:NbPDS, GV3101 / BSMVγ:TaPDS were mixed with GV3101 / pCaBS-α and GV3101 / pCaBS-β bacterial solutions at a volume ratio of 1:1:1 to obtain 4 mixed bacterial solutions, including:

[0121] ① Empty vector control tobacco (BSMVγ): inject a mixed bacterial solution of GV3101 / BSMVγ, GV3101 / pCaBS-α, and GV3101 / pCaBS-β at a volume ratio of 1:1:1;

[0122] ② Positive control tobacco (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;

[0123] ③ Positive control wheat (BSMVγ:TaPDS): injection of a mixed bacterial solution of GV3101 / BSMVγ:TaPDS, GV3101 / pCaBS-α, and GV3101 / pCaBS-β in a volume ratio of 1:1:1;

[0124] ④ Tobacco silenced plants (BSMVγ:TaSWEET5c): Inject a mixed bacterial solution of GV3101 / BSMVγ:TaSWEET5c, GV3101 / pCaBS-α and GV3101 / pCaBS-β in a volume ratio of 1:1:1.

[0125] 5) Activate the mixed bacterial solution at room temperature in the dark for 2 to 3 hours, and then inject it into Nicotiana benthamiana leaves.

[0126] 6) After the tobacco leaves have dried, place the injected tobacco back into the light incubator for cultivation. After the tobacco leaves show the virus phenotype, inoculate the wheat.

[0127] About 14 days after the tobacco stalks were injected, the tobacco showed virus phenotype ( Figure 3 Middle C), compared with healthy Nicotiana benthamiana, the positive control tobacco (BSMVγ:NbPDS) showed obvious albinism, and the positive control wheat (BSMVγ:TaPDS), empty vector control tobacco (BSMVγ) and silenced tobacco plants (BSMVγ:TaSWEET5c) all showed obvious mottled, curled and chlorotic phenomena, indicating that BSMV has successfully invaded tobacco.

[0128] (2) Virus friction inoculation of wheat

[0129] Add 5 mL of PBS buffer to a sterile mortar, add approximately 1 g of tobacco leaves exhibiting a viral phenotype, and a small amount of quartz sand. Grind thoroughly into a homogenate, transfer into a centrifuge tube, and briefly store on ice. Inoculate the homogenate onto the surface of a single-leaf, single-heart wheat leaf LC. Once the leaf is dry, return it to the illuminated incubator for incubation. Set up wheat plants with an empty vector control (inoculated with BSMVγ), a positive control (inoculated with BSMVγ:TaPDS), and silenced wheat plants (inoculated with BSMVγ:TaSWEET5c).

[0130] (3) TaSWEET5c gene silencing effect

[0131] About 10 days after virus inoculation, wheat showed virus phenotype ( Figure 3 Middle D), compared with healthy wheat, the positive control wheat (BSMVγ:TaPDS) showed obvious albinism, and the empty vector control wheat (BSMVγ) and wheat silenced plants (BSMVγ:TaSWEET5c) showed obvious mottled, curled and chlorotic phenomena, indicating that BSMV has successfully invaded wheat.

[0132] Total RNA was extracted from leaves of wheat plants expressing the virus (BSMVγ) and wheat plants expressing the virus (BSMVγ:TaSWEET5c) with the empty vector as a control and reverse-transcribed into cDNA. After normalization with TaGAPDH, the silencing effect of TaSWEET5c was assessed using real-time quantitative PCR using primers q-TaSWEET5c-F / R (Tm 58°C). The nucleotide sequences of the primers are shown in Table 1.

[0133] 10 μL fluorescence quantitative PCR reaction system: ChamQ Universal SYBR qPCRMasterMix 5 μL, forward primer (F) 1 μL, reverse primer (R) 1 μL and cDNA 3 μL.

[0134] The fluorescence quantitative PCR reaction program was as follows: 95°C, 30 s; 95°C, 10 s; Tm value, 10 s; 60°C, 40 s; 95°C, 1 min; 55°C, 30 s; 95°C, 30 s; steps 2 to 4 required 35 cycles.

[0135] After the reaction is completed, the Ct value is obtained and substituted into 2 -ΔΔCt The calculation formula is used to calculate the relative value. The result is as follows Figure 3 As shown in Figure F, compared with the wheat inoculated with BSMVγ, the relative expression level of TaSWEET5c in the wheat inoculated with BSMVγ:TaSWEET5c was significantly reduced, indicating that the TaSWEET5c gene was successfully silenced.

[0136] 7. TaSWEET5c gene overexpression

[0137] The recombinant Agrobacterium GV3101 / pLGY-02 and GV3101 / pLGY-02:TaSWEET5c were inoculated into Ka-Rif-LB liquid medium in a clean bench and cultured in a constant temperature shaker at 30°C until OD 600 The value is 1 to 1.5. According to the method of injecting tobacco with Agrobacterium, the bacterial solution OD 600 Adjust to 0.6~0.7, adjust OD 600 Agrobacterium was incubated at room temperature in the dark for 2–3 hours to activate. The Agrobacterium was then injected into single-leaf wheat leaves of the strain Maizi 52. Wheat plants injected with GV3101 / pLGY-02 served as controls, while those injected with GV3101 / pLGY-02:TaSWEET5c served as overexpression plants. After the leaves dried, they were returned to a lighted incubator. Samples were collected at 0, 12, 24, 48, 72, and 96 hours after injection. Total RNA was extracted from the wheat leaves and reverse-transcribed into cDNA.

[0138] The relative expression of TaSWEET5c in wheat inoculated with GV3101 / pLGY-02 and wheat inoculated with GV3101 / pLGY-02:TaSWEET5c at different time points was detected by semi-quantitative RT-PCR. It was found that the brightness of the band inoculated with GV3101 / pLGY-02:TaSWEET5c was stronger than that of the band inoculated with GV3101 / pLGY-02 at 24h, 48h, 72h and 96h. Figure 6 C). The overexpression effect of TaSWEET5c was further detected by real-time fluorescence quantitative PCR. The fluorescence quantitative PCR detection method was the same as above. After the reaction, the Ct value was obtained and substituted into 2 -ΔΔCt The calculation formula is used to calculate the relative value. The result is as follows Figure 6As shown in D, compared with the wheat inoculated with GV3101 / pLGY-02, the relative expression level of TaSWEET5c in the wheat inoculated with GV3101 / pLGY-02:TaSWEET5c was significantly increased, indicating that the TaSWEET5c gene was successfully overexpressed.

[0139] 8. Testing the resistance of TaSWEET5c silenced and overexpressing plants to stem rust

[0140] Empty vector control wheat, wheat silenced plants and wheat injected with GV3101 / pLGY-02 and GV3101 / pLGY-02:TaSWEET5c that showed viral phenotype were inoculated with wheat stem rust fungus 34MKGQM.

[0141] The inoculation method is as follows: take out the summer spores of wheat stem rust fungus 34MKGQM from a -80°C refrigerator and activate them in a 40°C water bath for 10 minutes, add 0.1 g of summer spores of wheat stem rust fungus 34MKGQM into a 500 mL spray bottle with Tween: water at a ratio of 20:1000, shake well and inoculate on the leaves of the above-mentioned wheat plants, keep moist at 16-18°C for 16-20 hours, and then move into a light incubator for culture.

[0142] To examine the expression of genes associated with disease progression after TaSWEET5c silencing and overexpression, qRT-PCR was used to measure the expression levels of TaPR1, TaPR2, TaPR5, TaSOD, and TaCAT at 0, 12, 24, 48, 72, and 96 hours after inoculation. The GenBank accession numbers for TaPR1, TaPR2, TaPR5, TaSOD, and TaCAT are LOC543437, LOC123080647, LOC543342, LOC101290631, and LOC123168517, respectively. Fluorescence quantitative PCR was used as described above. The nucleotide sequences of the primers for the relevant genes are shown in Table 1. The results showed that compared with the control, the expression levels of TaPRs genes (TaPR1, TaPR2 and TaPR5) in TaSWEET5c silenced plants were significantly upregulated, and ROS scavenging genes (TaSOD and TaCAT) were significantly downregulated ( Figure 4 In addition, compared with the control, the expression levels of TaPRs genes (TaPR1, TaPR2, and TaPR5) in TaSWEET5c-overexpressing plants were significantly downregulated, while TaSOD and TaCAT were significantly upregulated ( Figure 7 ).

[0143] To examine the mycelial infection of TaSWEET5c-silenced and overexpressed wheat, laser confocal microscopy was used to observe mycelial infection at 12, 24, 48, and 96 hours after inoculation, as well as mycelial area at 96 hours. Samples were prepared as follows: samples were placed in Eppendorf tubes (Eppendorf tubes) with a 1:1 ratio of acetic acid to ethanol and decolorized in a 40°C water bath. The leaves were rinsed three times with ddH2O and further decolorized with chloral hydrate for 20 minutes. The leaves were rinsed three times with ddH2O and softened by soaking in a 0.5 mol / L potassium hydroxide solution for 5 minutes. The leaves were rinsed three times with ddH2O and then stained in WGA-Alexa488 fluorescent stain for 30 minutes in the dark. The prepared samples were then stored in 50% glycerol until further observation. Observation and analysis showed that there was no significant difference between the silenced and overexpressed wheat and the control at the early infection stage (12h), but the difference was more obvious at the late infection stage (24h, 48h and 96h), as shown by the restricted expansion of hyphae in TaSWEET5c silenced plants and the significantly smaller colony area than the control ( Figure 5 ); The hyphae of TaSWEET5c-overexpressing plants expanded faster, and the colony area was significantly larger than that of the control ( Figure 8 ).

[0144] Fourteen days after inoculation with wheat stem rust, the phenotypes of wheat TaSWEET5c silenced and overexpressed plants were observed to further identify the wheat stem rust infection type. The wheat stem rust infection type grading scale, 0-4, is shown in Table 2. The phenotypes indicate that the empty vector control BSMVγ infection type is 4, while the silenced plant BSMVγ:TaSWEET5c infection type is 3. The spore piles of the silenced plants are smaller than those of the control ( Figure 3 The infection type of the empty vector control GV3101 / pLGY-02 was 1, while the infection type of the overexpression plant GV3101 / pLGY-02:TaSWEET5c increased to 2 ( Figure 6 Middle E). This suggests that TaSWEET5c may negatively regulate the expression of certain defense-related genes in the interaction between wheat and stem rust, thereby negatively regulating wheat resistance to stem rust.

[0145] Table 2 Classification standard of wheat stem rust infection type 0-4

[0146] Grading standard 0 No allergic spots 0； No summer spores but yellow-white allergic spots can be seen 1 There are tiny summer spores but they are surrounded by obvious yellow-white allergic dead spots. 2 Small to medium-sized uredia but often inhabit green islands surrounded by spindle-shaped allergic dieback 3 Medium-sized uredia, rarely fused; no allergic dieback around the uredia, but some chlorosis may occur 4 The urediospores are large and often fused. There is no allergic dieback but chlorosis may occur.

[0147] Note: In the infection type levels 1, 2, 3, and 4, if the lesions of the same infection type are larger, they will be marked with "+", and if they are smaller, they will be marked with "-". Among them, 0, 0;, 1-, 1, 1+, 2, and 2+ are classified as low infection type (disease-resistant), and 3-, 3, 3+, and 4 are classified as high infection type (disease-susceptible).

[0148] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A gene for regulating wheat resistance to stem rust, characterized in that: The gene encodes a protein whose amino acid sequence is shown in SEQ ID No.

1.

2. The gene according to claim 1, characterized in that The nucleotide sequence of the gene includes the sequence shown in SEQ ID No.

2.

3. A set of primer pairs for amplifying the gene according to claim 1 or 2, characterized in that: It includes an upstream primer with a nucleotide sequence as shown in SEQ ID No. 3 and a downstream primer with a nucleotide sequence as shown in SEQ ID No.

4.

4. A biological material comprising the gene according to claim 1 or 2, characterized in that: The types of the biological materials include any one of the following: expression cassette, vector and host bacteria.

5. Use of the gene according to claim 1 or 2 or the biological material according to claim 4 in regulating wheat resistance to stem rust.

6. Use of the gene according to claim 1 or 2 or the biological material according to claim 4 in breeding wheat with high resistance to stem rust.

7. A method for improving wheat resistance to stem rust, characterized in that: The method comprises inhibiting the expression of the gene according to claim 1 or 2 in the wheat genome, or knocking out the gene according to claim 1 or 2.

8. A material for inhibiting the expression of the gene according to claim 1 or 2, characterized in that V-TaSWEET5c is used as the silencing target fragment, and the amplification primer pair for the silencing target fragment includes an upstream primer whose nucleotide sequence is shown as SEQ ID No. 5 and a downstream primer shown as SEQ ID No.

6.

9. A method for cultivating wheat resistant to stem rust, characterized in that: The method comprises inhibiting the expression of the gene according to claim 1 or 2 in wheat.

10. A method for cultivating a wheat plant model with reduced resistance to stem rust, characterized in that: The method comprises overexpressing the gene according to claim 1 or 2 in the wheat genome.

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