Application of TaUBC13A protein and gene thereof in wheat stem rot resistance

Abstract

The invention belongs to the field of biological breeding, and particularly relates to application of TaUBC13A and a gene thereof in wheat stem rot resistance. The invention provides TaUBC13A protein with an amino acid sequence as shown in SEQ ID NO: 1, a nucleic acid molecule for coding the TaUBC13A protein, and application of a biological material containing the nucleic acid molecule in regulation and control of wheat stem rot resistance. The biological material is an expression cassette, a transposon, a plasmid vector, a virus vector or a host cell. TaUBC13A silent wheat is prepared and can be used for researching functional analysis of the protein, and TaUBC13A overexpressed wheat is prepared and can be used for developing stem rot resistant germplasm.

Description

Technical Field

[0001] The invention belongs to the field of biological breeding, and particularly relates to the application of TaUBC13A protein and a gene thereof in wheat stem base rot resistance. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the most widely cultivated and productive grasses in the Gramineae family worldwide. Fungal diseases are among the primary biotic stresses faced by wheat, including rust, head blight, leaf blight, powdery mildew, and Fusarium crown rot (FCR). Fusarium crown rot is a common soil-borne disease in actual production, and its impact on wheat production is increasing year by year. It not only causes severe yield losses but also produces toxins, posing a threat to human and livestock health.

[0003] At present, the mechanism by which Fusarium graminearum causes wheat stem rot is still unclear. The ubiquitin-proteasome pathway (UPP), as a core protein degradation pathway, is widely present in eukaryotes and plays a key role in maintaining cellular homeostasis, resisting external environmental stress, regulating growth and differentiation, mediating hormone responses, and influencing the aging process. In recent years, research has focused on the role of ubiquitin-conjugating enzymes (E2s) in enhancing plant resistance to biotic and abiotic stresses. Many studies have shown that proteins with UBC domains (primarily E2 ubiquitin-conjugating enzymes) play a key regulatory role in plant growth and development, stress responses, and various physiological processes. Studies have shown that ubiquitin-conjugating enzymes (E2s) can positively or negatively regulate plant disease resistance. For example, ectopic expression of rice OgUBC1 can make Arabidopsis thaliana resistant to UV-B radiation and Botrytis infection (Jeon EH, Pak JH, Kim MJ, et al. Ectopice expression of ubiquitin-conjugating enzyme gene from wild rice, OgUBC1, confers resistance against UV-B radiation and Botrytis infection in Arabidopsisthaliana[J]. Biochemical and Biophysical Research Communications, 2012, 427(2).); using virus-induced gene silencing to silence TaU4 in wheat leaves can delay the development of wheat leaf blight symptoms and reduce the growth and reproduction of the pathogen, proving that TaU4 can regulate the process of wheat resistance to leaf blight (Millyard L, Lee J, Zhang C, et al. The ubiquitin conjugating enzyme, TaU4 regulates wheat defense against the phytopathogen Zymoseptoria tritici[J]. Scientific Reports, 2016, 6: 35683.). There are currently no reports on ubiquitin-conjugating enzyme E2 mediating FCR resistance in wheat. Summary of the Invention

[0004] The present invention provides an E2 ubiquitin-conjugating enzyme TaUBC13A and application of a gene thereof in wheat stem base rot resistance.

[0005] To achieve the above objectives, the present invention provides the following technical solutions:

[0006] In the first aspect, the present invention provides a TaUBC13A protein having an amino acid sequence as shown in SEQ ID NO: 1, a nucleic acid molecule encoding the TaUBC13A protein, and the use of a biological material containing the nucleic acid molecule in regulating wheat stem rot resistance, wherein the biological material is an expression cassette, a transposon, a plasmid vector, a viral vector or a host cell.

[0007] Furthermore, the sequence of the nucleic acid molecule is shown in SEQ ID NO: 2.

[0008] In a second aspect, the present invention provides a TaUBC13A protein having an amino acid sequence as shown in SEQ ID NO: 1, a nucleic acid molecule encoding the TaUBC13A protein, and a biological material containing the nucleic acid molecule for cultivating wheat with improved or reduced resistance to stem rot.

[0009] In a third aspect, the present invention provides a method for cultivating wheat with improved resistance to stem base rot, comprising: overexpressing a nucleic acid molecule encoding TaUBC13A protein in a recipient wheat to obtain transgenic wheat; the transgenic wheat has higher resistance to stem base rot than the recipient wheat.

[0010] Furthermore, the overexpression method is: transferring a vector containing a nucleic acid molecule encoding TaUBC13A protein into a recipient wheat, and screening to obtain positive transgenic wheat.

[0011] In a fourth aspect, the present invention provides a method for cultivating wheat with reduced resistance to stem base rot, comprising: reducing the expression level and / or activity of TaUBC13A protein in recipient wheat to obtain TaUBC13A-silenced wheat; the TaUBC13A-silenced wheat has lower resistance to stem base rot than the recipient wheat.

[0012] Furthermore, the method for reducing the expression level and / or activity of TaUBC13A protein in the recipient wheat is: transiently or stably silencing the nucleic acid molecule encoding TaUBC13A protein in the recipient wheat.

[0013] Furthermore, the silencing method is: introducing a BSMV-VIGS vector system containing a nucleic acid molecule silencing fragment encoding TaUBC13A protein into the recipient wheat.

[0014] The present invention cloned the sequence of the TaUBC13A gene from wheat and studied its expression after infection with the pseudofusarium graminearum fungus, finding that its expression was significantly induced by the fungus. A BSMV-VIGS vector containing the gene was constructed and, after in vitro transcription and infection of wheat, the BSMV:γ-TaUBC13A strain exhibited reduced resistance to stalk rot after infection with the fungus. The results indicate that TaUBC13A positively regulates wheat resistance to stalk rot. Furthermore, a TaUBC13A gene overexpression vector was constructed and, after infection with wheat, overexpression of the TaUBC13A gene was found to enhance wheat resistance to stalk rot.

[0015] The beneficial effects of the present invention are:

[0016] The present invention discovered that the E2 ubiquitin-conjugating enzyme gene TaUBC13A positively regulates wheat resistance to stem rot. TaUBC13A-silenced wheat can be used to study the protein's function, while TaUBC13A-overexpressing wheat can be used to develop stem rot-resistant germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This figure shows the resistance of TaUBC13A-silenced wheat to stem rot. A: Stem base phenotype of BSMV-silenced wheat inoculated with spores of Fusarium graminearum; B: Disease index of BSMV-silenced wheat inoculated with spores of Fusarium graminearum. WT represents the wild-type control, BSMV:γ represents BSMV:γ-silenced wheat, and BSMV:TaUBC13A represents BSMV:γ-TaUBC13A-silenced wheat.

[0018] Figure 2 Resistance to stem rot in wheat overexpressing TaUBC13A. A: Stem base phenotype of wheat overexpressing TaUBC13A after inoculation with Fusarium graminearum spores. B: Disease index of wheat overexpressing TaUBC13A after inoculation with Fusarium graminearum spores. WT represents the wild-type control, and pCAMBIA1301:TaUBC13A represents wheat overexpressing TaUBC13A. DETAILED DESCRIPTION

[0019] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods. The test reagents used in the following embodiments, unless otherwise specified, are purchased from conventional biochemical reagent stores.

[0020] Example 1

[0021] 1. Experimental Materials

[0022] The experimental materials selected in the embodiment include wheat dwarf resistance 58 (AK58) and Bainong 207, which were planted in the Gene Editing Center of Henan Institute of Science and Technology.

[0023] 2. Experimental reagents and consumables

[0024] Enzymes and kits: Max DNA Polymerase high-fidelity enzyme, restriction endonuclease, 2×Es Taq MasterMix, 2×RealStar Fast SYBR qPCR Mix fluorescence quantitative kit, StarScriptIII All-in-one RT Mix with gDNA Remover reverse transcription kit, gel recovery kit, plasmid small amount extraction kit, EZ-TA / Blunt Zero pTOPOIICloning Kit cloning vector, DNA Marker, Reagent total RNA extraction reagent, RiboMAX TM LargeScale RNAProduction System-T7 In Vitro Transcription Kit.

[0025] Other drugs: agarose, peptone, yeast extract, chloroform, isoamyl alcohol, ethanol, isopropanol, NH4NO3, KH2PO4, sodium carboxymethyl cellulose CMC, MgSO4·7H2O, sodium chloride, ampicillin, kanamycin, and Escherichia coli competent cells.

[0026] Culture medium: LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl); LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl), 15 g / L agar powder, dilute to 1 L; LB selective medium: after the medium is autoclaved and cooled to 55°C, add the corresponding concentration of antibiotics and shake well to obtain liquid or solid selective medium; sodium carboxymethyl cellulose (CMC) conidiation medium: 0.5 g NH4NO3, 0.5 g KH2PO4, 7.5 g CMC, 1 g yeast extract, 0.25 g MgSO4·7H2O, dilute to 500 mL with distilled water, autoclave at 121°C for 20 min, and store in a 4°C refrigerator until used.

[0027] Main instruments: PCR amplifier (BIO-RAD), high-speed centrifuge (eppendorf), electrophoresis equipment (BIO-RAD), gel imaging system (BIO-RAD), fluorescence quantitative PCR instrument (ABIQuantStudio6), high-pressure sterilizer (Shanghai Sanshen), electric constant temperature incubator (Shanghai Xianxiang), constant temperature culture oscillator (Shanghai Zhicheng), nucleic acid detector (NanoDrop 2000), artificial climate chamber.

[0028] 3. Experimental Methods and Results

[0029] 1. Obtaining wheat cDNA

[0030] RNA was extracted from wheat AK58 and reverse transcribed to obtain cDNA.

[0031] 2. Cloning of the wheat TaUBC13A gene

[0032] The gene sequence of TaUBC13A was obtained from the Wheatdb website, and primers were designed to clone the CDS sequence of TaUBC13A from the cDNA of wheat AK58 obtained in the above steps.

[0033] Table 1. Primer sequence information for amplifying the CDS of the TaUBC13A gene

[0034] Primer name Sequence (5'-3') TaUBC13A-F ATGGCCAACAGCAACCTTCC TaUBC13A-R TCATGCACCACTGGCAT

[0035] The specific steps include:

[0036] (1) Prepare the following system on ice and amplify the target gene TaUBC13A using AK58 cDNA as template. Max DNA Polymerase high-fidelity enzyme instructions, PCR reaction system is as follows:

[0037] Table 2. PCR amplification reaction system

[0038] Reagent name Reagent dosage 2×PrimeSTAR Max Premix 25 μL Primer 1 (10 μM) 1 μL Primer 2 (10 μM) 1 μL template 2μL Sterile distilled water up to 50μL

[0039] The PCR amplification program was as follows: 98°C for 10 s; 55°C for 15 s; 72°C for 2 min, for 35 cycles. The reaction was then stored at 4°C.

[0040] (2) Use a gel recovery kit to excise the target fragment.

[0041] (3) The product recovered from the gel was ligated to construct a vector and transformed into Escherichia coli according to the EZ-TA / Blunt Zero pTOPOIICloning Kit.

[0042] (4) Culture overnight at 37°C, pick a single clone from the LB selection medium containing kanamycin resistance, and culture with shaking at 37°C.

[0043] (5) Positive clone samples were picked and sequenced after PCR verification of the bacterial solution. A certain amount of glycerol was added to the bacterial solution with correct sequencing to make the final glycerol concentration around 20% and stored at -70°C.

[0044] Based on the amplification results, the open reading frame of TaUBC13A is 462 bp, encoding 153 amino acids, the relative molecular weight of the protein is 17.17 kDa, and the isoelectric point is 7.14.

[0045] The amino acid sequence encoded by TaUBC13A is shown in SEQ ID NO: 1:

[0046] MANSNLPRRIIKETQRLLSEPAPGISASPSEENMRYFNVMILGPAQSPYEGGVFKLELFLPEE

[0047] YPMAAPKVRFLTKIYHPNIDKLGRICLDILKDKWSPALQIRTVLLSIQALLSAPNPDDPLSENIAKHWKSNEAEAVETAKEWTRLYASGA.

[0048] The CDS sequence of the TaUBC13A gene is shown in SEQ ID NO: 2:

[0049] ATGGCCAACAGCAACCTTCCGCGACGAATCATCAAGGAGACGCAGAGACTCCTCAGCG

[0050] AGCCAGCACCGGGGATCAGCGCTTCGCCCTCGGAGGAGAACATGCGCTACTTCAACGT

[0051] CATGATCCTTGGTCCGGCGCAGTCGCCCTACGAAGGGGGAGTTTTTAAGCTGGAACTCT

[0052] TTTTACCCGAGGAGTATCCGATGGCTGCCCCAAAGGTTAGGTTTCTGACCAAAATATACC

[0053] ATCCCAACATTGACAAGCTTGGTAGGATATGCCTTGACATTCTCAAGGATAAATGGAGTC

[0054] CGGCTCTTCAGATTCGAACAGTTCTTTTGAGTATCCAGGCACTCCTAAGTGCACCAAATC

[0055] CAGATGACCCTCTCTCTGAAAACATTGCTAAGCACTGGAAGTCCAATGAGGCAGAAGCTGTTGAAACAGCGAAGGAGTGGACTCGTCTGTATGCCAGTGGTGCATGA.

[0056] 2. Construction of BSMV-VIGS Vector and Gene Silencing of TaUBC13A

[0057] (1) Construction of BSMV:TaUBC13A recombinant vector

[0058] Based on the CDS sequence of TaUBC13A, we selected the appropriate target location, designed primers, and introduced a Sma I restriction site and protective bases to amplify the BSMV-VIGS fragment. The primer sequences are as follows:

[0059] Table 3. TaUBC13A gene VIGS primer sequence information

[0060] Primer name Sequence (5'-3') Amplification length vTaUBC13A-F AGCGGCCGCCCCGGGCCCTACGAAGGGGGAGTTTTT 300 vTaUBC13A-R ATTAATTAACCCGGGACGAGTCCACTCCTTCGCTGT 300

[0061] Using high-fidelity enzymes Max DNA Polymerase was used to amplify the VIGS-silencing fragment of the TaUBC13A gene. After purification, the fragment was ligated into the BSMV:γ vector digested with Sma I. The ligation product was transformed into competent E. coli, and the recombinant plasmid was verified by bacterial PCR and sequenced. The sequencing results were correct for the BSMV:γ-TaUBC13A plasmid.

[0062] (2) BSMV vector linearization

[0063] Transfer 400 μL of the bacterial suspension containing the correct sequencing results to a 100 mL conical flask and add 30 mL of LB medium containing ampicillin resistance to expand the culture. Then, extract the BSMV:γ-TaUBC13A plasmid and other viral vector plasmids (BSMV:γ, BSMV:α, BSMV:β, BSMV:γ-PDS).

[0064] The BSMV:α, BSMV:γ, BSMV:γ-PDS, and BSMV:γ-TaUBC13A plasmids were digested with MluI restriction enzyme; the BSMV:β plasmid was digested with SpeI restriction enzyme. After the digestion reaction, 0.5 μL of the digestion product was removed and diluted with an appropriate amount of distilled water. Electrophoresis was then performed to determine whether the plasmid was completely linearized. Finally, the digestion product was incubated at 65°C for 15 minutes to inactivate the enzyme.

[0065] (3) In vitro transcription

[0066] The five linearized plasmids were transcribed in vitro using an in vitro transcription kit. The reaction system was:

[0067] Table 4. In vitro transcription system

[0068] Reagents system 5× T7 Transcription Buffer 4 μL rATP 1 μL rUTP 1 μL rCTP 1 μL rGTP 0.4μL Ribo m7G Cap Analog(40mM) 1.5 μL Enzyme (200U / μL) 2μL

[0069] The above reactants were added to a microcentrifuge tube, which was then incubated at 37°C for 2 hours. After electrophoresis was performed to detect successful in vitro transcription, the tube was stored at -80°C for later use.

[0070] (4) Virus inoculation

[0071] Take 2.5 μL of each in vitro transcribed product, mix them in a ratio of 1:1:1 (α:β:γ, where γ is BSMV:γ, BSMV:γ-PDS or BSMV:γ-TaUBC13A), and dilute them with an equal volume of DEPC-treated water; add 45 μL of FES buffer to every 2.5 μL of the mixture and mix them by pipetting; inoculate AK58 seedlings at the one-leaf and one-heart stage with consistent growth with BSMV virus; spray a little DEPC-treated water after inoculation to maintain humidity; after all wheat is inoculated, quickly cover it with a light-proof plastic cover and cover it for 24 hours to ensure the humidity of the inoculated wheat growth microenvironment, then culture it with a normal 16 / 8h light and dark cycle, and observe the PDS phenotype and virus infection symptoms regularly.

[0072] (5) Identification method of stem base rot

[0073] Ten days after virus inoculation, a distinct albinism phenotype was observed in the BSMV:γ-PDS positive control, while a chlorotic phenotype was observed in the BSMV:γ empty vector and BSMV:γ-TaUBC13A experimental groups, indicating successful virus inoculation and the successful establishment of the silencing system. Real-time fluorescence quantitative PCR was used to measure the expression levels of TaUBC13A in BSMV:γ- and BSMV:γ-TaUBC13A-silenced plants. The results showed that compared with the BSMV:γ-silenced control, the expression of TaUBC13A in BSMV:γ-silenced plants was significantly reduced. This demonstrates that the expression of BSMV:γ-TaUBC13A transcripts was significantly suppressed, confirming that the TaUBC13A gene was effectively silenced. Subsequently, wheat plants transfected with BSMV:γ and BSMV:γ-TaUBC13A were inoculated at the stem base with 200 mL of 1×10 6 After 21 days of inoculation, the wheat stem base was phenotypically observed and disease statistics were performed. The number of plants infected with BSMV:γ-TaUBC13A virus and BSMV:γ virus were counted, and the disease index (DI) was calculated:

[0074]

[0075] The results showed that compared with the stem base of plants transfected with BSMV:γ, the stem base of wheat plants transfected with BSMV:γ-TaUBC13A showed severe browning and some were accompanied by necrosis. There were significant differences between the experimental group and the control group. Figure 1 As shown in A, the disease index of the TaUBC13A gene silenced strain was 45.9%, which was higher than the control group γ (27.3%), proving that silencing of the gene significantly reduced wheat resistance to stem rot. Figure 1 As shown in B.

[0076] 3. Creation of wheat strains overexpressing the TaUBC13A gene

[0077] (1) Construction of TaUBC13A overexpression vector

[0078] According to the restriction sites of the pCAMBIA1301 vector, primers carrying the corresponding restriction sites were added to both ends of the TaUBC13A gene fragment. The primer sequences are shown in the following table:

[0079] Table 5. Primer sequence information for TaUBC13A gene overexpression

[0080] Primer name Sequence (5'-3') Amplification length TaUBC13A OE-F GGTACCCGGGGATCCATGGCCAACAGCAAC 462 TaUBC13A OE-R CAGGTCGACTCTAGATGCACCACTGGCATA 462

[0081] Using AK58 cDNA as a template, amplify the product using the primers listed in Table 5. Detect the amplified product by 1.5% gel electrophoresis. After detection, recover the target band and ligate it with the pCAMBIA1301 vector using homologous recombination. Transform the cell into DH5α competent cells. Plate the transformed cell suspension onto selection medium and incubate at 37°C for 16 hours. Single colonies are selected for verification by PCR and sequencing. After expansion of the positive cell suspension, plasmid extraction is performed. The recombinant plasmid is labeled pCAMBIA1301:TaUBC13A.

[0082] (2) pCAMBIA1301: TaUBC13A for wheat genetic transformation

[0083] pCAMBIA1301:TaUBC13A was transformed into wheat strain Bainong 207 using Agrobacterium tumefaciens. Positive isolates were identified by PCR and GUS staining. Positive isolates were cultured in a climatic chamber maintained at 25°C / 16°C (light / dark), a 16 / 8 h photoperiod (light / dark), and 70% humidity.

[0084] (3) Detection of TaUBC13A overexpression levels and study of its anti-FCR function

[0085] To further confirm the expression of TaUBC13A in wheat overexpressing plants, we collected leaves from seedlings of T1-generation overexpressing plants and wild-type plants (WT). RNA was extracted and reverse-transcribed into cDNA. Real-time quantitative PCR was then used to measure the relative expression levels of TaUBC13A in the two plant types. The results showed that TaUBC13A expression was significantly increased in the overexpressing lines compared to wild-type wheat plants, consistent with the expected results.

[0086] In order to explore whether the TaUBC13A gene can enhance the ability of wheat plants to resist stem rot, the overexpression pure lines selected above were used to identify the resistance to stem rot in the seedling stage. After the wheat grew well, the spores of Fusarium graminearum were inoculated at the base of the stem, and the soil was kept moist. The disease was identified after 21 days. Figure 2 As shown in Figure A, under the same growth conditions, overexpressing wheat plants grew stronger than wild-type wheat. Observation of the stem base revealed that wild-type plants showed obvious browning and slight necrosis, while transgenic plants only showed sporadic spots, demonstrating that overexpressing the TaUBC13A gene can enhance wheat's resistance to stem base rot. Furthermore, the disease index of the TaUBC13A gene-overexpressing strain (40.7%) was significantly lower than that of the wild-type plants (66.7%), demonstrating that overexpression of this gene significantly improved wheat's resistance to stem base rot. Figure 2 As shown in B.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of a TaUBC13A protein having an amino acid sequence as shown in SEQ ID NO: 1, a nucleic acid molecule encoding the TaUBC13A protein, or a biological material containing the nucleic acid molecule in regulating wheat stem rot resistance, characterized in that: The biological material is an expression cassette, a transposon, a plasmid vector, a viral vector or a host cell.

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

2.

3. Use of a TaUBC13A protein having an amino acid sequence as shown in SEQ ID NO: 1, a nucleic acid molecule encoding the TaUBC13A protein, or a biological material containing the nucleic acid molecule in cultivating wheat with improved or reduced resistance to stem rot, characterized in that: The biological material is an expression cassette, a transposon, a plasmid vector, a viral vector or a host cell.

4. A method for cultivating wheat with improved resistance to stem rot, characterized in that: include: A nucleic acid molecule encoding the TaUBC13A protein is overexpressed in a recipient wheat to obtain transgenic wheat; the transgenic wheat has higher resistance to stem base rot than the recipient wheat, and the amino acid sequence of the TaUBC13A protein is shown in SEQ ID NO:

1.

5. The method according to claim 4, characterized in that The overexpression method comprises the following steps: transferring a vector containing a nucleic acid molecule encoding the TaUBC13A protein into a recipient wheat, and screening to obtain positive transgenic wheat.

6. The method according to claim 4, characterized in that The sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

7. A method for cultivating wheat with reduced resistance to stem rot, characterized in that: include: Reducing the expression level and / or activity of TaUBC13A protein in recipient wheat to obtain TaUBC13A silenced wheat; The TaUBC13A silenced wheat has lower resistance to stem base rot than the recipient wheat; the amino acid sequence of the TaUBC13A protein is shown in SEQ ID NO:

1.

8. The method according to claim 7, characterized in that The method for reducing the expression level and / or activity of the TaUBC13A protein in the recipient wheat is: transiently or stably silencing the nucleic acid molecule encoding the TaUBC13A protein in the recipient wheat.

9. The method according to claim 8, characterized in that The silencing method comprises the following steps: introducing a BSMV-VIGS vector system containing a nucleic acid molecule silencing fragment encoding TaUBC13A protein into recipient wheat.

10. The method according to claim 7, characterized in that The sequence of the nucleic acid molecule is shown in SEQ ID NO: 2.

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