Application of protein TaWCR2 and related biological materials thereof in regulation and control of plant low-temperature stress response

Abstract

The invention discloses application of protein TaWCR2 and related biological materials thereof in regulation and control of low-temperature stress response of plants. In order to solve the technical problem of how to regulate and control the cold resistance of plants, the invention provides application of protein or a substance for regulating and controlling the expression of a protein coding gene or a substance for regulating and controlling the activity or content of the protein, the protein is TaWCR2, and the amino acid sequence of the protein is shown as a sequence 2 in a sequence table. Experiments prove that when TaWCR2 is knocked out from a wheat variety Fielder, the survival rate of a TaWCR2 knockout plant under a low-temperature condition can be increased. Therefore, the protein TaWCR2 has an important application value in regulating the low-temperature stress response of the wheat, and has a wide prospect in cultivating wheat varieties.

Description

Technical Field

[0001] This invention belongs to the field of mutation or genetic engineering, specifically relating to the application of the protein TaWCR2 and related biomaterials in regulating the response to low temperature stress. Background Technology

[0002] Wheat is one of the world's most important food crops, and ensuring its yield and quality is crucial for maintaining global food security and social stability. However, in recent years, frequent extreme cold waves have caused widespread low-temperature freezing damage in my country's main wheat-producing areas, seriously threatening wheat production. Therefore, exploring wheat's low-temperature tolerance genetic resources and breeding and cultivating new low-temperature resistant wheat varieties is of great significance. In previous studies, researchers mainly identified some wheat low-temperature tolerance genetic loci, but very few specific wheat low-temperature tolerance-related genes have been discovered, severely limiting wheat cold-resistant breeding. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to use gene CRISPR / Cas9 technology to knock out the TaWCR2 gene and thereby regulate the low temperature response of wheat.

[0004] To address the aforementioned technical problems, the present invention first provides the application of a protein or a substance regulating the expression of the protein-encoding gene or a substance regulating the activity or content of the protein, wherein the application may be any one of A1)-A5): A1) Application in regulating plant cold resistance A2) Application in the preparation of products that regulate plant cold resistance A3) Application in cultivating plants with altered cold hardiness. A4) Application in the preparation of products containing plants with altered cold resistance A5) Applications in plant breeding; The protein is TaWCR2 protein, and is any one of the following (B1)-B4): B1) The amino acid sequence of this protein is SEQ ID NO: 2. B2) A protein with the same function as the amino acid sequence shown in B1) by substitution and / or deletion and / or addition of one or more amino acid residues. Proteins that share more than 80% identity with any of the amino acid sequences defined in B3 and B1-B2) and have the same function. B4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in B1)-B3).

[0005] In the above applications, the TaWCR2 protein can be derived from wheat.

[0006] The indicators for plant breeding include cold hardiness traits, and the purpose of plant breeding includes cultivating plants with altered cold hardiness traits.

[0007] In this invention, the substance that regulates the expression of the TaWCR2 protein-encoding gene can be a substance that inhibits, reduces, or downregulates the expression of the gene, and the regulation of plant cold resistance traits means improving plant cold resistance.

[0008] In the above applications, the regulation can be at least one of the following six types of regulation: 1) regulation at the transcriptional level of the coding gene, 2) regulation after transcription of the coding gene, 3) regulation of RNA transport of the coding gene, 4) regulation of translation of the coding gene, 5) regulation of mRNA degradation of the coding gene, and 6) post-translational regulation of the gene.

[0009] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0010] In this invention, the protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0011] In this invention, the identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0012] In this invention, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0013] The SEQ ID NO: 2 consists of 354 amino acid residues, as detailed below: MFADAGFSFSAYSSSPPGPAYSYSYSYSSSPTVLSHPHPDHSSFSSPPSSAAPAQPPPPPLPLPLLHQQHQHQHQHQAAAAVATASNDDDDDAMYHHLGDMGQPSLISEYDLGAEGDLFKAPEPIIEEPLLALDPVAAAISMMSGGDNAMDDSIKVSDMGLSEVLYECEKELMEK SAIEETISELLDVKIPMLQVEDVPGELRASSSSTVAAGTGECSLQKSVSSGCLSSGDWMNGSAVRPNFLDFQGLDFEAAFGLRRAYSEGDIQKLGANTPRPGIAANVQASGERLVTISDLKSEERKQKLNRYRKKKIQRNFGRKIKYACRKALADSQPRVRGRFAKMDDGDMLKPRK.

[0014] In the above applications, the substance may be a biological material, specifically any one of the following C1) to C8): C1) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein. C2) expresses the gene encoding the nucleic acid molecule described in C1). C3) contains the expression cassette of the gene described in C2). C4) A recombinant vector containing the gene described in C2), or a recombinant vector containing the expression cassette described in C3). C5) Recombinant microorganisms containing the gene described in C2), or recombinant microorganisms containing the expression cassette described in C3), or recombinant microorganisms containing the recombinant vector described in C4). C6) A transgenic plant cell line containing the gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4). C7) Transgenic plant tissue containing the gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4). C8) A transgenic plant organ containing the gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

[0015] In the above applications, the nucleic acid molecule described in C1) may be a gRNA that targets the protein-coding gene described in claim 1.

[0016] The target sequence of the sgRNA may be nucleotides 2188 to 2207 and / or nucleotides 2305 to 2324 of sequence 1 in the sequence listing.

[0017] In the above applications, the nucleic acid molecule described in C1) is introduced into the recipient plant, specifically by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and then culturing the transformed plant tissues into plants. The transformed cells, tissues, or plants are understood to include not only the final products of the transformation process but also the materials obtained through asexual reproduction and transgenic progeny.

[0018] In C4) above, the recombinant vector can be a plant gene editing vector. The plant gene editing vector can be the plasmid pLGY-E003 vector.

[0019] The recombinant vector is pLGY-E003- TaWCR2- sgRNA. The recombinant vector pLGY-E003- TaWCR2 - sgRNA can produce Cas9 and sgRNA targeting T1 and T2.

[0020] The T1 is 5'-GTCCTCGACCTGCAGCATG-3' (corresponding to nucleotides 2188 to 2207 of SEQ ID NO:1); T2 is 5'-GGGCGACTGGATGAACGGGT-3' (corresponding to nucleotides 2305 to 2324 of SEQ ID NO:1).

[0021] In the above applications, the microorganism described in C5) can be yeast, bacteria, algae, or fungi. Specifically, it can be Agrobacterium tumefaciens EHA105.

[0022] In the above applications, the transgenic plant can be a plant obtained through biological methods such as recombinant DNA technology in genetic engineering.

[0023] In the above applications, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs described in C6) may or may not include propagation material.

[0024] In the above applications, the plant tissue described in C7) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers.

[0025] In the above applications, the transgenic plant organs described in C8) can be the roots, stems, leaves, flowers, fruits, and seeds of the transgenic plant.

[0026] The present invention also provides a method for regulating the cold resistance of plants, the method comprising regulating the cold resistance of a target plant through step M, wherein step M may comprise knocking out or inhibiting or reducing or downregulating the expression of the encoding gene of the protein described above in the target plant, and / or the activity and / or content of the protein described above; the target plant contains the encoding gene of the protein described above.

[0027] The present invention also provides a method for cultivating plants with altered cold tolerance, the method comprising obtaining plants with altered cold tolerance through step M, wherein step M comprises knocking out or inhibiting or reducing or downregulating the expression of the gene encoding the protein described above in the target plant, and / or the activity and / or content of the protein described above; the target plant contains the gene encoding the protein described above.

[0028] In this invention, by inhibiting or reducing or downregulating the expression of the gene encoding the protein described above and / or the activity and / or content of the protein described above, plants with improved cold resistance can be obtained.

[0029] The cold resistance can specifically refer to the survival rate of plants under low temperatures.

[0030] Step S can be achieved through gene knockout or gene silencing.

[0031] Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout inactivates a specific target gene by altering its DNA sequence.

[0032] Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing presupposes no change in the DNA sequence, resulting in the absence or reduction of gene expression. Gene silencing can occur at two levels: transcriptional silencing due to DNA methylation, heterochromatinization, and position effects; and post-transcriptional gene silencing, which inactivates the gene at the post-transcriptional level through specific inhibition of target RNA. This includes antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0033] In the above method, step M includes introducing a gene knockout vector into the target plant targeting nucleotides 2188 to 2207 of sequence 1 in the sequence listing and / or nucleotides 2305 to 2324 of sequence 1 in the sequence listing.

[0034] In the above method, the genome of the target plant contains DNA molecules whose nucleotide sequence is a sequence in the sequence listing, and the gene knockout includes inserting 1 bp deoxyribonucleotide A between positions 2190 and 2191 of sequence 1 in the sequence listing of the target plant genome.

[0035] In this invention, the plant can be any one of the following D1)-D4): D1) dicotyledonous plant, D2) monocotyledonous plant, D3) grass, D4) wheat.

[0036] The wheat in question can specifically be Fielder.

[0037] The present invention also provides a substance, which may be the aforementioned protein or biological material.

[0038] Experiments have shown that gene knockout in Fielder TaWCR2 This study found that TaWCR2 protein can improve the survival rate of wheat under low-temperature stress. Therefore, TaWCR2 has important application value in regulating the low-temperature stress response of wheat and has broad prospects in wheat variety breeding. Attached Figure Description

[0039] Figure 1 for TaWCR2 Genotypic analysis of gene knockout mutants.

[0040] Figure 2 for TaWCR2 Temperature phenotypic analysis of gene knockout mutants. 'a' represents the phenotype of the gene knockout mutant. After wheat seedlings at the three-leaf stage were treated at -6℃ for 10 hours, the mutant lines... TaWCR2 Compared to the control Fielder, it is more resistant to low temperatures; the scale bar is 5 cm; b is... TaWCR2 Survival statistics of gene knockout mutants TaWCR2 Compared to the control group Fielder, the survival rate increased under low-temperature treatment. P <0.05 (*). Detailed Implementation

[0041] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0043] The following examples used GraphPad Prism 8 statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and one-way ANOVA was used. P <0.05 (*) indicates a significant difference.

[0044] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0045] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0046] Example 1, Knockout TaWCR2 Gene-enhanced wheat cold resistance 1. Wheat TaWCR2 Gene knockout wheat TaWCR2 The genome sequence of the gene is sequence 1 (SEQ ID NO: 1, 3407 bp) in the sequence listing. Positions 1-300, 1910-2416, 3025-3186, and 3312-3407 bp are exons, as follows:

[0047] wheat TaWCR2 The amino acid sequence of the protein TaWCR2 encoded by the gene is sequence 2 in the sequence listing (SEQ ID NO: 2, 354 aa), as follows: MFADAGFSFSAYSSSPPGPAYSYSYSYSSSPTVLSHPHPDHSSFSSPPSSAAPAQPPPPPLPLPLLHQQHQHQHQHQAAAAVATASNDDDDDAMYHHLGDMGQPSLISEYDLGAEGDLFKAPEPIIEEPLLALDPVAAAISMMSGGDNAMDDSIKVSDMGLSEVLYECEKELMEK SAIEETISELLDVKIPMLQVEDVPGELRASSSSTVAAGTGECSLQKSVSSGCLSSGDWMNGSAVRPNFLDFQGLDFEAAFGLRRAYSEGDIQKLGANTPRPGIAANVQASGERLVTISDLKSEERKQKLNRYRKKKIQRNFGRKIKYACRKALADSQPRVRGRFAKMDDGDMLKPRK.

[0048] Its corresponding coding sequence (CDS) is sequence 3 (SEQ ID NO: 3, 1065bp) in the sequence listing, as follows:

[0049] 1.2 Primer Design and Amplification For target genes TaWCR2 Gene editing targets were designed based on conserved sequences, and the targets are as follows: T1: 5'- GTCCTCGACCTGCAGCATG-3' (corresponding to nucleotides 2188 to 2207 of SEQ ID NO:1); T2: 5'-GGGCGACTGGATGAACGGGT-3' (corresponding to nucleotides 2305 to 2324 of SEQ ID NO:1); Forward primer F (5'-ccgaggtctcgggcgCGTCCTCGACCTGCAGCATGgtttcagagctatgctggaaac-3') and reverse primer R (5'-acctcggtctccaaacACCCGTTCATCCAGTCGCCCcaagtctgatgcagcaagc-3') were designed and synthesized. Using the intermediate vector pMETaU6.1 as a template, PCR amplification was performed. The PCR amplification system is shown in Table 1, and the PCR amplification program is shown in Table 2. The PCR product containing the 808 bp target band was recovered. The obtained PCR product contained two target sequences and the TaU6.1 promoter sequence. This PCR product contained an sgRNA expression cassette targeting T1 and an sgRNA expression cassette targeting T2, and was named sgRNA1-TaU6.1-snRNA2-sgRNA2. The nucleotide sequence of the PCR product is sequence 4 in the sequence listing (SEQ ID NO: 4, 808 bp), where bp 1-15 is the BsaI restriction site and its protective bases, bp 16-35 is the T1 sequence, bp 36-62 is the regulatory sequence, bp 63-127 is the gRNA scaffold, bp 128-412 is the regulatory sequence, bp 413-774 is the wheat U6 promoter, bp 775-794 is the T2 sequence, and bp 795-812 is the BsaI restriction site and its protective bases. The specific sequence is as follows: 5’-CCGAGGTCTCGGGCGGTCCTCGACCTGCAGCATGGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTTTCGTTTTGCATTGAGTTTTCTCCGTCGCATGTTTGCAGTTTTATTTTCCGTTTTGCATTGAAATTTCTCCGTCTCATGTTTGCAGCGTGTTCAAAAAGTACGCAGCTGTATTTCACTTATTTACGGCGCCACATTTTCATGCCGTTTGTGCCAACTATCCCGAGCTAGTGAATACAGCTTGGCTTCACACAACACTGGTGACCCGCTGACCTGCTCGTACCTCGTACCGTCGTACGGCACAGCATTTGGAATTAAAGGGTGTGATCGATACTGCTTGCTGCTACCAAGCCCGTTATTCTGACAGTTCTGGTGCTCAACACATTTATATTTATCAAGGAGCACATTGTTACTCACTGCTAGGAGGGAATCGAACTAGGAATATTGATCAGAGGAACTACGAGAGAGCTGAAGATAACTGCCCTCTAGCTCTCACTGATCTGGGTCGCATAGTGAGATGCAGCCCACGTGAGTTCAGCAACGGTCTAGCGCTGGGCTTTTAGGCCCGCATGATCGGGCTTTTGTCGGGTGGTCGACGTGTTCACGATTGGGGAGAGCAACGCAGCAGTTCCTCTTAGTTTAGTCCCACCTCGCCTGTCCAGCAGAGTTCTGACCGGTTTATAAACTCGCTTGCTGCATCAGACTGGGCGACTGGATGAACGGGTGTTTGGAGACCGAGGT-3’。

[0050] Table 1. PCR Amplification System

[0051] Table 2. PCR Amplification Program

[0052] 3. Ligation of PCR Product and Gene Editing Vector The gene editing backbone vector pLGY-E003 (WPA1 encodes a vWA domain protein that regulates wheat plant architecture, ISSN 2214-5141, https: / / doi.org / 10.1016 / j.cj.2024.05.008.) was digested with Bsa1, and the vector was recovered from the gel. The PCR product was ligated with the aforementioned digested vector to obtain the recombinant plasmid pLGY-E003- TaWCR2- sgRNA. The ligation reaction conditions were as follows: 37℃ for 5 min, 16℃ for 10 min, for a total of 60 cycles; then 16℃ for 1 h. The ligation reaction system is shown in Table 3. Recombinant vector pLGY-E003- TaWCR2- sgRNA can produce Cas9 and sgRNA targeting T1 and T2.

[0053] The nucleotide sequence of the sgRNA targeting T1 is as follows: 5'- GUCCUCGACCUGCAGCAUG -3'.

[0054] The nucleotide sequence of the sgRNA targeting T2 is as follows: 5'- GGGCGACUGGAUGAACGGGU -3'.

[0055] Table 3. Connection Reaction System

[0056] 4. Agrobacterium-mediated transformation The recombinant plasmid pLGY-E003- TaWCR2- sgRNA was transformed into Agrobacterium tumefaciens strain EHA105 to obtain the recombinant plasmid pLGY-E003- TaWCR2- Recombinant Agrobacterium tumefaciens EHA105 / pLGY-E003- sgRNA TaWCR2- sgRNA.

[0057] Recombinant Agrobacterium EHA105 / pLGY-E003- TaWCR2- Wheat Fielder (recipient) was infected with sgRNA, and T0 generation transgenic wheat was obtained by following the procedure: Agrobacterium-mediated genetic transformation was used to transform recombinant strain EHA105 / pLGY-E003- TaWCR2- sgRNA was transferred into the scutellum of the Fielder embryo of a wild-type wheat variety and cultured. T0 generation transgenic wheat was planted and T0 generation seeds were harvested. T0 generation seeds were self-pollinated to obtain T1 generation seeds. T1 generation seeds were planted to obtain T1 generation seedlings.

[0058] The formulations of the relevant culture media are as follows: Callus induction medium (1 L): 100 mL MS macronutrient salts (×10), 1 mL L7 micronutrient salts (×1000), 10 mL ferric sodium EDTA (×100), 1 mL MS vitamins (×1000), 100 mg inositol, 0.5 g glutamine, 100 mg casein, 1.95 g MES, 40 g maltose. Mix well and adjust the pH to 5.7. Add 2 g of plant gel and sterilize at 121℃ for 15 min. After the medium cools to 55℃, add the filtered and sterilized reagents according to the stock solution ratio, including 0.25 mg 2,4-D, 1 mg chlorhexidine, and 80 mg timenine.

[0059] Regeneration medium (1 L): 100 mL L7 macronutrient salts (×10), 1 mL L7 micronutrient salts (×1000), 10 mL ferric sodium EDTA (×100), 5 mL vitamin / inositol mixture (×200), 100 mg inositol, 30 g maltose. Mix well and adjust the pH to 5.7. Add 2 g of plant gel and sterilize at high temperature. After the medium cools to 55°C, add the filtered and sterilized reagents according to the stock solution ratio, including 0.05 mg 2,4-D, 80 mg timenine, and 2.5 mg zeatin. The screening medium is the regeneration medium with 1.25 mg glufosinate added.

[0060] MS macronutrient salts (×10): 16.5 g / L ammonium nitrate, 19.0 g / L potassium nitrate, 1.7 g / L potassium dihydrogen phosphate, 3.7 g / L magnesium sulfate heptahydrate, 4.4 g / L calcium chloride dihydrate. Mix well, incubate at 121℃ for 15 min, and store at 4℃.

[0061] L7 Macronutrient Salts (×10): 2.5 g / L ammonium nitrate, 15.0 g / L potassium nitrate, 2.0 g / L potassium dihydrogen phosphate, 3.5 g / L magnesium sulfate heptahydrate, 4.5 g / L calcium chloride dihydrate. Mix well, incubate at 121℃ for 15 min, and store at 4℃.

[0062] L7 Trace Element Salts (×1,000): 15.0 g / L manganese sulfate, 5.0 g / L boric acid, 7.5 g / L zinc sulfate heptahydrate, 0.75 g / L potassium iodide, 0.25 g / L sodium molybdate dihydrate, 0.025 g / L copper sulfate pentahydrate, 0.025 g / L cobalt chloride hexahydrate. Filter for sterilization and store at 4°C. Vitamin / Inositol mixture (×200): 40.0 g / L Inositol, 2.0 g / L Vitamin B1 Hydrochloride, 0.2 g / L Vitamin B6, 0.2 g / L Niacin, 0.2 g / L Calcium Pantothenate, 0.2 g / L Vitamin C, filtered and sterilized, stored at 4°C.

[0063] 5. Identification of positive plants 20 T1 generation plants were selected TaWCR2 DNA was extracted from the proposed transgenic wheat seedlings. Using genomic DNA from T1 generation seedlings as templates, PCR amplification was performed on the plants using the upstream identification primer: 5'-ggagtgagtacggtgtgcTCCGAGCTGCTGGACGTCAAGA-3' and the downstream identification primer: 5'-gagttggatgctggatggTTCTGAATGTCACCCTCGCTGTAGGCT-3'. The PCR products were then subjected to Hi-Tom sequencing to identify mutations at the target sites in the T1 generation plants. The sequencing results were compared with those of the recipient plants. TaWCR2 Nucleotide sequences are compared to detect the mutation type of the mutant. 。 The specific steps are as follows: 1) Total DNA was extracted from the leaves of 20 T1 generation transgenic wheat seedlings using the CTAB genomic DNA extraction method. The DNA content in the leaves of each T0 generation transgenic wheat was approximately 200 ng / μL.

[0064] 2) Use PCR technology to obtain fragment sequences, including gene editing target sites, from each T1 generation of transgenic wheat.

[0065] The reaction system is 20 μL, consisting of 10 μL of SYBR. ® PremixExTaq TM 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, and 1 μL of T1 generation pseudotransferase. TaWCR2 It consists of genomic DNA from genetically modified wheat and 8.0 μL of nuclease-free water.

[0066] Reaction program: 94℃ pre-denaturation for 3 min; 95℃ denaturation for 3 sec; 55℃ annealing for 30 sec; 35 cycles.

[0067] 3) Hi-tom sequencing detects mutants.

[0068] The PCR products were subjected to Hi-tom sequencing to detect gene editing. TaWCR2 Homozygous mutant plants are named Tawcr2 The seeds were harvested as T2 generation for subsequent experiments. TaWCR2 See gene sequence changesFigure 1 As shown. Compared to wild-type Fielder, Tawcr2 In the genome TaWCR2 The region corresponding to the gene underwent the following change: a 1 bp deoxyribonucleotide A was inserted between positions 2190 and 2191.

[0069] Example 2 T1 generation TaWCR2 Phenotypic identification of gene knockout wheat under low temperature stress Harvesting the mutant in Example 1 Tawcr2 Wheat seeds and wild-type control Fielder seeds were sown separately in soil and placed in a light incubator at 16°C for 16 hours of light / 8 hours of darkness. After approximately 20 days of cultivation, when the seedlings reached the two-leaf-one-heart stage, wheat seedlings with uniform growth were selected for low-temperature treatment. After testing at different temperatures (-4°C, -6°C, -8°C, -10°C) and for different durations (4 hours, 6 hours, 8 hours, 10 hours), it was determined that -6°C for 10 hours was most suitable for phenotypic identification of the experimental materials in this invention. Therefore, after treatment at -6°C for 10 hours, the seedlings were then restored to 16°C for 3 days, with 30 wild-type Fielder and mutant seedlings treated each time. Tawcr2 Seedlings were treated three times. Photos were taken before treatment and after recovery to record survival rates. Photos of wheat before and after recovery are provided. Figure 2 In the middle a, the statistical results are as follows Figure 2 b.

[0070] The phenotypes of the low-temperature stress experiments described above were as follows: compared with the control Fielder, the knockout lines... Ta wcr2 Survival rates increased; Fielder's survival rate was 27.78%, and the mutant... Tawcr2 The survival rate was 66.66%, showing a significant difference.

[0071] The results show that TaWCR2 Gene knockout lines showed increased survival rates at low temperatures, exhibiting a more cold-resistant phenotype.

[0072] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of a protein or a substance regulating the expression of the gene encoding the protein or a substance regulating the activity or content of the protein, characterized in that, The application is any one of A1)-A5): A1) Application in regulating plant cold resistance A2) Application in the preparation of products that regulate plant cold resistance A3) Application in cultivating plants with altered cold hardiness A4) Application in the preparation of products using plants with altered cold resistance A5) Applications in plant breeding; The protein is TaWCR2 protein, and is any one of the following (B1)-B4): B1) The amino acid sequence of this protein is SEQ ID NO:

2. B2) A protein with the same function as the amino acid sequence shown in B1) by substitution and / or deletion and / or addition of one or more amino acid residues. Proteins that share more than 80% identity with any of the amino acid sequences defined in B3 and B1-B2) and have the same function. B4) A fusion protein obtained by attaching a tag to the end of any of the proteins defined in B1)-B3).

2. The application according to claim 1, characterized in that, The TaWCR2 protein is derived from wheat.

3. The application according to claim 1 or 2, characterized in that, The substance is a biological material, and the biological material is any one of the following C1) to C8): C1) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein described in claim 1 or 2. C2) expresses the gene encoding the nucleic acid molecule described in C1). C3) contains the expression cassette of the gene described in C2). C4) A recombinant vector containing the gene described in C2), or a recombinant vector containing the expression cassette described in C3). C5) Recombinant microorganisms containing the gene described in C2), or recombinant microorganisms containing the expression cassette described in C3), or recombinant microorganisms containing the recombinant vector described in C4). C6) A transgenic plant cell line containing the gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4). C7) Transgenic plant tissue containing the gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4). C8) A transgenic plant organ containing the gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

4. The application according to claim 3, characterized in that, C1) The nucleic acid molecule is a gRNA that targets the protein-coding gene described in claim 1.

5. A method for regulating the cold resistance of plants, characterized in that, The method includes regulating the cold resistance of a target plant through step M, wherein step M includes knocking out, inhibiting, reducing or downregulating the expression of the gene encoding the protein of claim 1 or 2 in the target plant, and / or the activity and / or content of the protein of claim 1 or 2; the target plant contains the gene encoding the protein of claim 1 or 2.

6. A method for cultivating plants with altered cold resistance, characterized in that, The method includes obtaining a plant with altered cold resistance through step M, wherein step M includes knocking out, inhibiting, reducing, or downregulating the expression of the gene encoding the protein of claim 1 or 2 in the target plant, and / or the activity and / or content of the protein of claim 1 or 2; the target plant contains the gene encoding the protein of claim 1 or 2.

7. The method as described in claim 5 or 6, characterized in that, Step M includes introducing a gene knockout vector into the target plant targeting nucleotides 2188 to 2207 of sequence 1 in the sequence listing and / or nucleotides 2305 to 2324 of sequence 1 in the sequence listing.

8. The method as described in claim 5 or 6, characterized in that, The genome of the target plant contains a DNA molecule whose nucleotide sequence is sequence 1 in the sequence listing. The gene knockout involves inserting 1 bp of deoxyribonucleotide A between positions 2190 and 2191 of sequence 1 in the sequence listing of the target plant genome.

9. The application according to any one of claims 1-4 or the method according to any one of claims 5-8, characterized in that, The plant is any one of the following D1)-D4): D1) Dicotyledons, D2) Monocotyledons, D3) Gramineae plants, D4) Wheat.

10. A substance, characterized in that, The substance is the protein described in claim 1 or 2 and / or the biological material described in claim 3.

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