Application of protein TaWCR1 and related biological materials thereof in regulation and control of plant low-temperature stress response

Abstract

The invention discloses application of protein TaWCR1 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 amino acid sequence of the protein TaWCR1-A provided by the invention is shown as a sequence 2 in a sequence table, and the amino acid sequence of TaWCR1-D is shown as a sequence 4 in the sequence table. Experiments prove that when the TaWCR1 is knocked out of a wheat variety Fielder, the survival rate of a TaWCR1 knockout plant under a low-temperature condition can be increased. Therefore, the protein TaWCR1 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] The present invention belongs to the field of mutation or genetic engineering, and specifically relates to the application of protein TaWCR1 and related biological materials in regulating low temperature stress response. Background Art

[0002] Wheat is one of the world's most important food crops, and ensuring its yield and quality is crucial to maintaining global food security and social stability. However, frequent extreme cold snaps in recent years have caused widespread freezing damage in my country's major wheat-producing areas, seriously threatening safe wheat production. Therefore, it is crucial to explore wheat's genetic resources for cold tolerance and to breed and cultivate new cold-tolerant wheat varieties. Previous studies have primarily identified a few genetic loci for cold tolerance in wheat, but few specific genes associated with cold tolerance have been identified, severely limiting wheat breeding for cold resistance. Summary of the Invention

[0003] The technical problem to be solved by the present invention is how to regulate the low temperature response of wheat.

[0004] To solve the above technical problems, the present invention first provides an application of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein, wherein the application may be any one of A1) to A5): A1) Application in regulating plant cold tolerance, A2) Application in the preparation of products for regulating plant cold tolerance, A3) Application in cultivating plants with altered cold tolerance, A4) use in the preparation of products for cultivating plants with altered cold tolerance, A5) Application in plant breeding; The TaWCR1 protein may be any one of the following B1)-B5): B1) a protein having an amino acid sequence of SEQ ID NO: 2, B2) a protein having an amino acid sequence of SEQ ID NO: 4, B3) A protein having the same function as the amino acid sequence shown in B1) or B2) by substitution and / or deletion and / or addition of one or more amino acid residues, B4) A protein having an amino acid sequence identity of 80% or more to any of B1) to B3) and having the same function, B5) A fusion protein obtained by ligating a tag to the end of any of the proteins defined in B1) to B4).

[0005] In the above application, the TaWCR1 protein may be derived from wheat.

[0006] The plant breeding index includes plant cold resistance traits, and the plant breeding purpose includes cultivating plants with altered cold resistance traits.

[0007] In the present invention, the substance that regulates the expression of the gene encoding the TaWCR1 protein may be a substance that inhibits, reduces or downregulates the expression of the gene, and the regulation of the cold tolerance trait of the plant is to improve the cold tolerance of the plant.

[0008] In the above application, the regulation may be at least one of the following six types of regulation: 1) regulation at the transcription 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 above proteins can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

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

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

[0012] In the present invention, the above 80% identity may 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 299 amino acid residues, specifically as follows: MSSRSSGGGGASQMMAFSEHSLPKPIAGHPQPQPSPPSSPSERPAARGRRRAQEPGRFLGVRRRPWGRYAAEIRDPTKERHWLGTFDTAQEAALAYDRAALSMKGAQARTNFVYAHAAYNNYPPFLAPFHAQHQPAAYAASSAMPYGG QQQHAGAGPPHIGSSSYHHGHGYHQQGPGECSMPVPSAADHGASGPMDVRGSSGHDFLFPSADDNSGYLSSVVPESCLRPRGGDLQDARRYSVSDADAYGLGLREDVDDLATMVAGFWGGADAPYGGGHDMVASSQGSDNGYSPFSFLSH.

[0014] The SEQ ID NO: 4 consists of 313 amino acid residues, specifically as follows: MSIRSSSGGSGGGHASQMMAFSEHSLPKPIAGHPQPQPSPPSSPSERPAPRGRRRAQEPGRFLGVRRRPWGRYAAAEIRDPTTKERHWLGTFDTAQEAALAYDRAALSMKGAQARTNFVYAHAAYNNYPPFLAPFHAQQQPAAYASSTMPYAGQQHAA PHIGSSYHHGHGHGGLGYHQQGPGAGAGECSMPVPNAADHGASSPMDVRGSSGHDFLFPSADDNSGYLSSVVPESCLRPRGDLQDARRYSVSDADAYGLGLREDVDDLASMVAGFWGGADAAYGGFAPANGGGHDMVASSQGSDNGYSPFSFLSH.

[0015] In the above application, the substance may be a biological material, and the biological material may specifically be any one of the following C1) to C8): C1) a nucleic acid molecule that inhibits, reduces or downregulates the expression of the gene encoding the protein, C2) expressing a gene encoding the nucleic acid molecule described in C1), C3) an expression cassette containing 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) a recombinant microorganism containing the gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism 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) a transgenic plant tissue containing the gene described in C2), or a transgenic plant tissue containing the expression cassette described in C3), or a 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).

[0016] In the above application, the nucleic acid molecule in C1) may be a gRNA targeting the protein encoding gene in claim 1.

[0017] The target sequence of the sgRNA may be nucleotides 381 to 402 and / or nucleotides 512 to 533 of sequence 1 in the sequence listing.

[0018] In the above applications, the nucleic acid molecule described in C1) is introduced into the recipient plant by, specifically, transforming plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, or Agrobacterium-mediated transfection, and then cultivating the transformed plant tissues into plants. The term "transformed cells, tissues, or plants" is understood to include not only the final product of the transformation process, but also material obtained through asexual reproduction and transgenic progeny thereof.

[0019] In the above C4), the recombinant vector may be a plant gene editing vector, and the plant gene editing vector may be a plasmid pLGY-E003 vector.

[0020] The recombinant vector is pLGY-E003- TaWCR1- sgRNA. The recombinant vector pLGY-E003- TaWCR1- sgRNA can produce Cas9 and sgRNA targeting T1 and sgRNA targeting T2.

[0021] The T1 is 5'-CCTCGCGCCGTTCCACGCGCAG-3' (corresponding to nucleotides 381 to 402 of SEQ ID NO: 1 and nucleotides 390 to 411 of SEQ ID NO: 3); T2 is 5′-GCTACCACCAGCAGGGCCCGGG-3′ (corresponding to nucleotides 512 to 533 of SEQ ID NO: 1 and nucleotides 521 to 542 of SEQ ID NO: 3).

[0022] In the above application, the microorganism in C5) can be yeast, bacteria, algae or fungi, and can specifically be Agrobacterium tumefaciens EHA105.

[0023] In the above applications, the transgenic plants can be plants obtained through biological methods such as recombinant DNA technology of genetic engineering.

[0024] 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 materials.

[0025] In the above application, the plant tissue in C7) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

[0026] In the above application, the transgenic plant organ in C8) can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

[0027] The present invention also provides a method for regulating plant cold resistance, which may include regulating the cold resistance of the target plant through step M, and the step M may include 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] The present invention also provides a method for cultivating plants with altered cold resistance, which may include obtaining plants with altered cold resistance through step M, wherein step M includes 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.

[0029] In the present invention, the expression of the gene encoding the aforementioned protein and / or the activity and / or content of the aforementioned protein can be inhibited, reduced or down-regulated to obtain a plant with improved cold tolerance.

[0030] The cold resistance may specifically refer to the survival rate of plants at low temperatures.

[0031] The step S can be achieved by gene knockout or gene silencing.

[0032] Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing its DNA sequence.

[0033] The gene silencing refers to the phenomenon of not expressing or underexpressing a gene without damaging the original DNA. Gene silencing is based on the premise that the DNA sequence is not changed, so that the gene is not expressed or underexpressed. Gene silencing can occur at two levels. One is gene silencing at the transcriptional level due to DNA methylation, heterochromatinization and position effects, and the other is post-transcriptional gene silencing, that is, gene inactivation by specifically inhibiting the target RNA at the level after gene transcription, including antisense RNA, co-suppression, gene repression (quelling), RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.

[0034] In the above method, the step M comprises introducing into the target plant a gene knockout vector targeting nucleotides 381 to 402 of sequence 1 in the sequence listing and / or nucleotides 512 to 533 of sequence 1 in the sequence listing.

[0035] In the above method, the genome of the target plant contains a DNA molecule whose nucleotide sequence is Sequence 1 and / or Sequence 3 in the sequence listing, and the gene knockout comprises performing any of the following operations on the genome of the target plant: D1) deleting 140 bp of deoxyribonucleotides from positions 399 to 538 of sequence 1 in the sequence listing in the genome of the target plant, and deleting 139 bp of deoxyribonucleotides from positions 399 to 537 of sequence 3 in the sequence listing in the genome of the target plant; D2) deleting 140 bp of deoxyribonucleotides from positions 399 to 538 of sequence 1 in the sequence listing in the genome of the target plant, and deleting 140 bp of deoxyribonucleotides from positions 399 to 538 of sequence 3 in the sequence listing in the genome of the target plant.

[0036] In the present invention, the plant may be any one of the following E1) to E4): E1) dicotyledonous plant, E2) monocotyledonous plant, E3) grass plant, E4) wheat.

[0037] The wheat may specifically be Fielder.

[0038] The present invention also provides a substance, which may be the above-mentioned protein or biological material.

[0039] Experiments have shown that knocking out genes in Fielder TaWCR1, which can improve the survival rate of wheat under low temperature stress. Therefore, the protein TaWCR1 has important application value in regulating the response of wheat to low temperature stress and has broad prospects in breeding wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] 图1 for TaWCR1 Genotyping analysis of knockout mutants. “-” indicates missing nucleotides, and numbers indicate the number of missing nucleotides.

[0041] 图2 for TaWCR1 Temperature phenotype analysis of gene knockout mutants. a shows the phenotype of gene knockout mutants. After wheat seedlings at the three-leaf stage were treated at -6℃ for 10 hours, the mutant line tawcr1-ad#1 and tawcr1-ad#2 Compared with the control, Fielder is more resistant to low temperature. The scale bar is 5 cm; b is TaWCR1 Survival statistics of gene knockout mutants, tawcr1-ad# 1 and tawcr1-ad#2 Compared with the control, the survival rate of Fielder was increased under low temperature treatment. P <0.05(*). DETAILED DESCRIPTION

[0042] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0043] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0044] The following examples were processed using GraphPad Prism 8 statistical software. The experimental results are expressed as mean ± standard deviation and tested using One-way ANOVA. P <0.05 (*) indicates significant difference. P <0.01 (**) indicates a very significant difference.

[0045] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0047] Example 1, knockout TaWCR1 Genetically enhancing wheat cold tolerance 1. Wheat TaWCR1 gene knockout wheat TaWCR1-A The genomic sequence of the gene is Sequence 1 (SEQ ID NO. 1, 900bp) in the sequence listing. Since this gene has no introns, the corresponding coding sequence (CDS) is the same as Sequence 1, as follows: 5′-ATGAGCTCTCGCAGCAGCAGCGGCGGCGGCGGTGCCTCCCAGATGA TGGCCTTCTCGGAGCATTCGCTGCCGAAGCCGATCGCCGGTCACCCGCAGCCGCAGCCGTCCCCGCCGTCGTCGCCGAGCGAGCGGCCGGCGGCGCGCGGCAGGCGGCGCGCGCAGGAGCCCGGGCGCTTCCTGGGCGTGCGCCGGCGGCCGTGGGGCCGGTACGCGGCCGAGATACGCGACCCGACCACCAAGGAGCGGCACTGGCTCGGCACCTTCGACACGGCGCAGGAGGCCGCCCTGGCCTACGACCGCGCCGCGCTCTCCATGAAGGGCGCGCAGGCGCGCACCAACTTCGTCTACGCGCACGCCGCCTACAACAACTACCCGCCCTTCCTCGCGCCGTTCCACGCGCAGCACCAGCCCGCCGCCTACGCCGCGTCCTCGGCCATGCCGTACGGCGGCCAGCAGCAGCACGCGGGCGCGGGGCCGCCGCACATTGGCAGCTCGTACCACCACGGCCACGGCTACCACCAGCAGGGCCCGGGCGAGTGTTCCATGCCGGTGCCCAGTGCCGCGGATCACGGCGCCAGCGGCCCGATGGACGTGCGCGGCAGCAGCGGCCACGACTTCCTCTTCCCCAGCGCCGACGACAACTCCGGGTACCTGAGCAGCGTGGTGCCGGAGAGCTGCCTCCGGCCCCGCGGCGGCGACCTGCAGGACGCGCGGCGCTACTCCGTGTCCGACGCCGACGCCTACGGGCTGGGCCTCCGGGAGGACGTGGACGACCTGGCGACGATGGTGGCCGGCTTCTGGGGCGGCGCCGACGCGCCGTACGGCGGCGGCCACGACATGGTCGCCTCGTCGCAGGGCTCGGACAACGGCTACTCCCCCTTCAGCTTCCTCTCCCACTGA-3′。

[0048] 小麦 TaWCR1-A 基因编码的蛋白质TaWCR1-A的氨基酸序列为序列表中序列2(SEQID NO.2,299 aa),具体如下: MSSRSSGGGGASQMMAFSEHSLPKPIAGHPQPQPSPPSSPSERPAARGRRRAQEPGRFLGVRRRPWGRYAAEIRDPTKERHWLGTFDTAQEAALAYDRAALSMKGAQARTNFVYAHAAYNNYPPFLAPFHAQHQPAAYAASSAMPYGG QQQHAGAGPPHIGSSSYHHGHGYHQQGPGECSMPVPSAADHGASGPMDVRGSSGHDFLFPSADDNSGYLSSVVPESCLRPRGGDLQDARRYSVSDADAYGLGLREDVDDLATMVAGFWGGADAPYGGGHDMVASSQGSDNGYSPFSFLSH.

[0049] wheat TaWCR1-D The genomic sequence of the gene is Sequence 3 (SEQ ID NO. 3, 942 bp) in the sequence listing. Since this gene has no introns, the corresponding coding sequence (CDS) is the same as Sequence 3, as follows: 5′-ATGAGCATCCGCAGCAGCAGCGGCGGCAGCGGCGGCGGCCATGCCTCCCAGATGATGGCGTTCTCGGAGCATTCGCTGCCGAAGCCGATCGCCGGCCACCCGCAGCCGCAGCCGTCCCCGCCGTCGTCTCCGAGCGAGAGGCCGGCGCCGCGTGGCAGGCGGCGCGCGCAGGAGCCCGGGCGCTTCCTGGGCGTGCGCCGGCGGCCGTGGGGCCGGTACGCGGCGGAGATACGCGACCCGACCACCAAGGAGCGGCACTGGCTCGGCACCTTCGACACGGCGCAGGAGGCCGCCCTGGCCTACGACCGCGCCGCGCTCTCCATGAAGGGCGCGCAGGCGCGCACCAACTTCGTCTACGCGCACGCCGCCTACAACAACTACCCGCCCTTCCTCGCGCCGTTCCACGCGCAGCAGCAGCCCGCCGCCTACGCGTCCTCGACCATGCCGTACGCCGGCCAGCAGCACGCGGCGCCGCACATTGGCAGCTCGTACCACCACGGCCACGGCCACGGCGGCCTCGGCTACCACCAGCAGGGCCCGGGCGCCGGCGCGGGCGAGTGCTCCATGCCGGTGCCCAATGCCGCCGATCACGGCGCCAGCAGCCCGATGGACGTGCGCGGCAGCAGCGGCCACGACTTCCTCTTCCCCAGCGCCGACGACAACTCCGGGTACCTGAGCAGCGTGGTGCCGGAGAGCTGCCTCCGGCCCCGCGGCGGCGACCTGCAGGACGCGCGGCGCTACTCCGTGTCCGACGCCGACGCCTACGGGCTGGGCCTCCGGGAGGACGTGGACGACCTGGCGTCCATGGTGGCCGGCTTCTGGGGCGGCGCCGACGCGGCGTACGGCGGGTTCGCCCCCGCGAACGGCGGCGGCCACGACATGGTCGCCTCGTCGCAGGGCTCCGACAACGGCTACTCCCCCTTCAGCTTCCTCTCCCACTGA-3′。

[0050] 小麦 TaWCR1-DThe amino acid sequence of the protein TaWCR1-D encoded by the gene is sequence 4 (SEQ ID NO. 4, 313 aa) in the sequence listing, as follows: MSIRSSSGGSGGGHASQMMAFSEHSLPKPIAGHPQPQPSPPSSPSERPAPRGRRRAQEPGRFLGVRRRPWGRYAAAEIRDPTTKERHWLGTFDTAQEAALAYDRAALSMKGAQARTNFVYAHAAYNNYPPFLAPFHAQQQPAAYASSTMPYAGQQHAA PHIGSSYHHGHGHGGLGYHQQGPGAGAGECSMPVPNAADHGASSPMDVRGSSGHDFLFPSADDNSGYLSSVVPESCLRPRGDLQDARRYSVSDADAYGLGLREDVDDLASMVAGFWGGADAAYGGFAPANGGGHDMVASSQGSDNGYSPFSFLSH.

[0051] 1.2 Primer design and amplification Target gene TaWCR1-A and its homologous genes TaWCR1-D The conserved sequences were used to design gene editing targets. The targets are as follows: T1: 5′-CCTCGCGCCGTTCCACGCGCAG-3′ (corresponding to nucleotides 381 to 402 of SEQ ID NO: 1, nucleotides 390 to 411 of SEQ ID NO: 3); T2: 5′-GCTACCACCAGCAGGGCCCGGG-3′ (corresponding to nucleotides 512 to 533 of SEQ ID NO: 1 and nucleotides 521 to 542 of SEQ ID NO: 3).

[0052] The forward primer F: 5'-ccgaggtctcgggcgCCTCGCGCCGTTCCACGCGCAGgtttcagagctatgctggaaac -3' and the reverse primer R: 5'-acctcggtctccaaacCCCGGGCCCTGCTGGTGGTAGCcaagtctgatgcagcaagc -3' were designed and synthesized. PCR amplification was performed using the intermediate vector pMETaU6.1 as a template. The PCR amplification system is shown in Table 1, and the PCR amplification procedure is shown in Table 2. The PCR product of the target band of 812 bp was recovered. The obtained PCR product contained two target sequences and the TaU6.1 promoter sequence.

[0053] The PCR product contains an sgRNA expression cassette for T1 and an sgRNA expression cassette for T2 and is named sgRNA1-TaU6.1-snRNA2-sgRNA2. The nucleotide sequence of the PCR product is SEQ ID NO: 5, wherein bp 1-15 are a BsaI restriction site and its protective bases, bp 16-37 are a T1 sequence, bp 38-62 are a regulatory sequence, bp 63-127 are a gRNA scaffold, bp 128-412 are a regulatory sequence, bp 413-774 are a wheat U6 promoter, bp 775-796 are a T2 sequence, and bp 797-812 are a BsaI restriction site and its protective bases.

[0054] The specific sequence of SEQ ID NO: 5 (812 bp) is: 5’-CCGAGGTCTCGGGCGCCTCGCGCCGTTCCACGCGCAGGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTTTCGTTTTGCATTGAGTTTTCTCCGTCGCATGTTTGCAGTTTTATTTTCCGTTTTGCATTGAAATTTCTCCGTCTCATGTTTGCAGCGTGTTCAAAAAGTACGCAGCTGTATTTCACTTATTTACGGCGCCACATTTTCATGCCGTTTGTGCCAACTATCCCGAGCTAGTGAATACAGCTTGGCTTCACACAACACTGGTGACCCGCTGACCTGCTCGTACCTCGTACCGTCGTACGGCACAGCATTTGGAATTAAAGGGTGTGATCGATACTGCTTGCTGCTACCAAGCCCGTTATTCTGACAGTTCTGGTGCTCAACACATTTATATTTATCAAGGAGCACATTGTTACTCACTGCTAGGAGGGAATCGAACTAGGAATATTGATCAGAGGAACTACGAGAGAGCTGAAGATAACTGCCCTCTAGCTCTCACTGATCTGGGTCGCATAGTGAGATGCAGCCCACGTGAGTTCAGCAACGGTCTAGCGCTGGGCTTTTAGGCCCGCATGATCGGGCTTTTGTCGGGTGGTCGACGTGTTCACGATTGGGGAGAGCAACGCAGCAGTTCCTCTTAGTTTAGTCCCACCTCGCCTGTCCAGCAGAGTTCTGACCGGTTTATAAACTCGCTTGCTGCATCAGACTGCTACCACCAGCAGGGCCCGGGGTTTGGAGACCGAGGT-3’。

[0055] 表1、PCR扩增体系

[0056] 表2、PCR扩增程序

[0057] 3、PCR产物与基因编辑载体的连接 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 by gel excision. The PCR product was ligated with the aforementioned enzyme digestion to obtain the recombinant plasmid pLGY-E003- TaWCR1- sgRNA. The ligation reaction conditions were as follows: 37°C for 5 min, 16°C for 10 min, for a total of 60 cycles; then 16°C for 1 h. The ligation reaction system is shown in Table 3. The recombinant vector pLGY-E003- TaWCR1- sgRNA can produce Cas9 and sgRNA targeting T1 and sgRNA targeting T2.

[0058] The nucleotide sequence of the sgRNA targeting T1 is as follows: 5′-CCUCGCGCCGUUCCACGCGCAG-3′.

[0059] The nucleotide sequence of the sgRNA targeting T2 is as follows: 5′-GCUACCACCAGCAGGGCCCGGG-3′.

[0060] Table 3. Ligation reaction system

[0061] 4. Agrobacterium transformation The recombinant plasmid pLGY-E003- TaWCR1- sgRNA was transformed into Agrobacterium tumefaciens strain EHA105 to obtain the recombinant plasmid pLGY-E003- TaWCR1- sgRNA recombinant Agrobacterium EHA105 / pLGY-E003- TaWCR1- sgRNA.

[0062] Recombinant Agrobacterium EHA105 / pLGY-E003- TaWCR1- sgRNA was used to infect wheat Fielder (recipient), and the T0 generation of transgenic wheat was obtained by following the following operation: Agrobacterium-mediated genetic transformation was used to transform the recombinant bacteria EHA105 / pLGYE-3- TaWCR1- sgRNA was transferred into the scutellum of immature embryos of wild-type wheat variety Fielder and tissue cultured.

[0063] The formula of the relevant culture medium involved is as follows: Callus induction medium (1 L): 100 mL MS macronutrient salts (×10), 1 mL L7 trace element salts (×1000), 10 mL sodium ferric 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, adjust the pH to 5.7, add 2 g of plant gelatin, and sterilize at high temperature at 121°C for 15 min. After the culture medium cools to 55°C, add filter-sterilized reagents according to the mother liquor ratio, including 0.25 mg 2,4-D, 1 mg picloram, and 80 mg timentin.

[0064] Regeneration medium (1 L): 100 mL L7 macronutrient salts (×10), 1 mL L7 trace element salts (×1000), 10 mL sodium ferric EDTA (×100), 5 mL vitamin / inositol mixture (×200), 100 mg inositol, 30 g maltose. Mix thoroughly, adjust the pH to 5.7, add 2 g of plant gelatin, and sterilize at high temperature. After the medium cools to 55°C, add filter-sterilized reagents according to the stock solution ratio, including 0.05 mg 2,4-D, 80 mg timentin, and 2.5 mg zeatin. The screening medium is the regeneration medium with 1.25 mg glufosinate added.

[0065] 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°C for 15 min, and store at 4°C.

[0066] 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°C for 15 min, and store at 4°C.

[0067] 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 sterilize and store at 4°C. Vitamin / inositol mixture (×200): 40.0 g / l inositol, 2.0 g / l thiamin hydrochloride, 0.2 g / l pyridoxine hydrochloride, 0.2 g / l niacin, 0.2 g / l calcium pantothenate, 0.2 g / l vitamin C, filter sterilize, store at 4°C.

[0068] 5. Identification of positive plants Select 20 T0 generation plants TaWCR1 DNA was extracted from the transgenic wheat seedlings. The genomic DNA of the T0 generation seedlings was used as a template. PCR amplification was performed on the plants using the upstream identification primer: 5'-CTAGGCTGGAGCAGTAGTATAGTAG -3' and the downstream identification primer: 5'-TCTCCAAACAAACACTACTACCTAG-3'. The PCR products were sequenced by Sanger sequencing to identify the mutation of the target site in the T0 generation plants. The sequencing results were compared with those of the recipient plants. TaWCR1 Compare nucleotide sequences and detect mutation types of mutants 。 The specific steps are as follows: 1) Total DNA was extracted from leaves of 20 T0-generation transgenic wheat plants at the seedling stage using the CTAB genomic DNA extraction method. The DNA content in the leaves of each T0-generation transgenic wheat plant was approximately 200 ng / μL.

[0069] 2) Use PCR technology to obtain the fragment sequence including the gene editing target site in each T0 generation of transgenic wheat.

[0070] The reaction system is 20 μL, consisting of 10 μL SYBR ® PremixExTaq TM , 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 1 μL of T0 generation TaFZP The mixture consisted of wheat genomic DNA and 8.0 μL nuclease-free water.

[0071] Reaction procedure: pre-denaturation at 94°C for 3 min; denaturation at 95°C for 3 sec, annealing at 55°C for 30 sec, 35 cycles.

[0072] 3) Detect mutants by Sanger sequencing.

[0073] The PCR products were subjected to Sanger sequencing to detect their gene editing status and the mutants were named tawcr1-ad#1 and tawcr1-ad#2 , and harvested their seeds as the T1 generation for subsequent experiments. tawcr1-ad#1 and tawcr1-ad#2 The A and D genomes TaWCR1 Gene sequence changes图1 shown.

[0074] Compared with wild-type Fielder, tawcr1-ad#1 In the genome TaWCR1 The region corresponding to the gene has undergone the following changes: On chromosome 2A in the A genome TaWCR1-A The 140bp deoxyribonucleotides at positions 399-538 of the gene (SEQ ID NO: 1) were deleted, and the 2D chromosome in the D genome TaWCR1-D The 139 bp deoxyribonucleotide sequence from position 399 to 537 of the gene was deleted.

[0075] Compared with wild-type Fielder, tawcr1-ad#2 In the genome TaWCR1 The region corresponding to the gene has undergone the following changes: On chromosome 2A in the A genome TaWCR1-A The 140bp deoxyribonucleotides at positions 399-538 of the gene (SEQ ID NO: 1) were deleted, and the 2D chromosome in the D genome TaWCR1-D The 140 bp deoxyribonucleotides at positions 399-538 of the gene were deleted.

[0076] Example 2 T1 generation TaWCR1 Phenotypic identification of gene knockout wheat under low temperature stress Harvest the mutant in Example 1 tawcr1-ad#1 、 tawcr1-ad#2 Wheat seeds and wild-type control Fielder seeds were planted in the soil, placed in a light incubator, and cultured at 16°C, 16 h light / 8 h dark. After about 30 days of culture, when the seeds grew to the three-leaf stage, wheat seedlings with consistent growth were selected for low-temperature treatment. After testing and treating at different temperatures (-4°C, -6°C, -8°C, -10°C) and different times (3 h, 6 h, 9 h, 12 h), it was finally determined that -6°C, 12 h were more suitable for the phenotypic identification of the experimental materials in the present invention. Therefore, after being treated at -6°C for 12 h, the culture was restored at 16°C for 3 days, and 30 wild-type and mutant seedlings were treated each time, for a total of 3 times. Photos were taken before and after treatment, and the survival rate was counted. The photos of wheat before and after recovery are as follows: 图2 In a, the statistical results are 图2 Middle b.

[0077] The phenotype of the low temperature phenotype in the above low temperature stress experiment is: compared with the control Fielder, the knockout line tawcr1- ad#1 and tawcr1-ad#2 The survival rates of the mutants were improved, Fielder's survival rate was 33.3%, tawcr1-ad#1The survival rate of the mutant was 87.78%, and the survival rate of the mutant tawcr1-ad#2 The survival rate of the mutant was 88.89%.

[0078] The results show that, TaWCR1 The survival rate of the gene knockout strain at low temperature is increased, and the phenotype is more resistant to low temperature.

[0079] The above has carried out the detailed description to the present application. For the person skilled in the art, in the case where not departing from the purpose and range of the present application, and without unnecessary experiments, the present application can be implemented in a wide range under the equivalent parameters, concentration and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In short, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the present application.

Claims

1. Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein, characterized in that: The application is any one of A1) to A5): A1) Application in regulating plant cold tolerance, A2) Application in the preparation of products for regulating plant cold tolerance, A3) Application in cultivating plants with altered cold tolerance, A4) use in the preparation of products for cultivating plants with altered cold tolerance, A5) Application in plant breeding; The protein is TaWCR1 protein, which is any one of the following B1)-B5): B1) a protein having an amino acid sequence of SEQ ID NO: 2, B2) a protein having an amino acid sequence of SEQ ID NO: 4, B3) A protein having the same function as the amino acid sequence shown in B1) or B2) by substitution and / or deletion and / or addition of one or more amino acid residues, B4) A protein having an amino acid sequence identity of 80% or more to any of B1) to B3) and having the same function, B5) A fusion protein obtained by ligating a tag to the end of any of the proteins defined in B1) to B4).

2. The use according to claim 1, characterized in that The TaWCR1 protein is derived from wheat.

3. The use 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) a nucleic acid molecule that inhibits, reduces or downregulates the expression of a gene encoding the protein according to claim 1 or 2, C2) expressing a gene encoding the nucleic acid molecule described in C1), C3) an expression cassette containing 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) a recombinant microorganism containing the gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism 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) a transgenic plant tissue containing the gene described in C2), or a transgenic plant tissue containing the expression cassette described in C3), or a 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 use according to claim 3, characterized in that C1) The nucleic acid molecule is a gRNA targeting the protein encoding gene according to claim 1.

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

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

7. The method according to claim 5 or 6, wherein: The step M comprises introducing into the target plant a gene knockout vector targeting nucleotides 381 to 402 of sequence 1 in the sequence list and / or nucleotides 512 to 533 of sequence 1 in the sequence list.

8. The method according to claim 5 or 6, wherein: The genome of the target plant contains a DNA molecule whose nucleotide sequence is sequence 1 and / or sequence 3 in the sequence list, and the gene knockout comprises performing any of the following operations on the genome of the target plant: D1) deleting 140 bp of deoxyribonucleotides from positions 399 to 538 of sequence 1 in the sequence listing in the genome of the target plant, and deleting 139 bp of deoxyribonucleotides from positions 399 to 537 of sequence 3 in the sequence listing in the genome of the target plant; D2) deleting 140 bp of deoxyribonucleotides from positions 399 to 538 of sequence 1 in the sequence listing in the genome of the target plant, and deleting 140 bp of deoxyribonucleotides from positions 399 to 538 of sequence 3 in the sequence listing in the genome of the target plant.

9. The use according to any one of claims 1 to 4 or the method according to any one of claims 5 to 8, characterized in that: The plant is any one of the following E1) to E4): E1) Dicotyledons, E2) Monocots, E3) Grasses, E4) 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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