Application of protein WPA1 and related biological materials in regulating low temperature stress response

Abstract

The application discloses application of a protein WPA1 and related biological materials thereof in regulating low-temperature stress response, and belongs to the field of variation or genetic engineering. WPA1 The amino acid sequence of the protein WPA1 is shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. Experiments prove that editing the gene of the protein WPA1 in a WT can reduce the survival rate of wheat under low-temperature stress and shorten the flowering time. Therefore, the protein WPA1 has important application value in regulating low-temperature stress response and flowering time of wheat, and has a broad 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 protein WPA1 and related biomaterials in regulating low-temperature stress response. 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 gene 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-resistance breeding. This invention explores the low-temperature phenotype of gene-edited materials and provides new genes for wheat low-temperature response. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a new use for the protein WPA1. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: The technical problem to be solved by this invention is how to use gene editing to achieve [the following]: WPA1 Gene knockout regulates wheat's low-temperature response.

[0005] To address the aforementioned technical problems, the present invention first provides the application of proteins or biological materials related to said proteins.

[0006] The application of the protein or related biomaterials provided by this invention can be any of the following: E1) The use of proteins or biomaterials related to said proteins in regulating the flowering time of plants; E2) The use of proteins or biomaterials related to said proteins in the preparation of products that regulate the flowering time of plants; E3) The application of proteins or biomaterials associated with said proteins in regulating low-temperature responses. E4) The use of proteins or biomaterials related to said proteins in the preparation of products that regulate plant cold tolerance. E5) The use of proteins or biomaterials related to said proteins in plant breeding and / or the preparation of plant breeding products; E6) regulates plant survival rate under low temperature; E7) Prepare products that regulate plant survival rates under low temperatures; E8) Plant breeding; The biological material is a substance that regulates the expression of the gene encoding the protein or a substance that regulates the content of the protein. The protein is WPA1, and is any of the following: B1) The amino acid sequence of the protein is shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. B2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in B1), which has more than 80% identity with the protein shown in B1) and can regulate the plant's low-temperature response. B3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2.

[0007] In the above applications, the indicators for plant breeding may include the low-temperature phenotype. The purpose of plant breeding may include regulating the low-temperature phenotype of plants.

[0008] In the above applications, the protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Poly-Arg tag, Strep-tag II tag, Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0009] In the above applications, identity refers to the identity of amino acid 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 Lambda ratio 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.

[0010] In the above applications, the 80% or more of identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0011] In the above applications, the protein may be derived from wheat.

[0012] In the above applications, the substance can regulate the expression of the protein-coding gene or regulate the content of the protein in at least one of the following six aspects: 1) regulation at the transcriptional level of the coding gene; 2) post-transcriptional regulation of the coding gene (i.e., regulation of splicing or processing of the primary transcript of the coding gene); 3) regulation of RNA transport of the coding gene (i.e., regulation of mRNA transport of the coding gene from the nucleus to the cytoplasm); 4) regulation of translation of the coding gene; 5) regulation of mRNA degradation of the coding gene; 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the coding gene).

[0013] In the above applications, the substance may specifically be any of the following: C1) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein, or nucleic acid molecules that knock out the gene encoding the aforementioned protein; C2) expresses the gene encoding the nucleic acid molecule described in C1); C3) contains 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) 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).

[0014] The transgenic plant is a plant obtained through biological methods such as recombinant DNA technology in genetic engineering.

[0015] In one specific embodiment of the present invention, the regulation of the flowering time of the plant is to advance the flowering time of the plant.

[0016] In some embodiments of the present invention, in the biological material, C1) the nucleic acid molecule may specifically be a gRNA that targets the protein-coding gene.

[0017] In one specific embodiment of the present invention, the protein-coding gene in the biological material is SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0018] The target sequence of the gRNA may be nucleotides 193 to 212 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0019] In some embodiments of the present invention, the recombinant microorganism in the biological material (C5) may specifically be yeast, bacteria, algae, and fungi.

[0020] In some embodiments of the present invention, the recombinant microorganism may be Agrobacterium. The Agrobacterium may be Agrobacterium tumefaciens.

[0021] In some embodiments of the present invention, the plant tissue in the biological material (C7) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and / or anthers.

[0022] In some embodiments of the present invention, the biological material, C8), the transgenic plant organs may be the roots, stems, leaves, flowers, fruits and seeds of the transgenic plant.

[0023] The present invention also provides a method for regulating the low-temperature stress response of plants, the method comprising regulating the agronomic traits of the target plant through step S.

[0024] The present invention also provides a method for cultivating plants with altered phenotypes under low-temperature stress, including obtaining plants with altered low-temperature responses through step S.

[0025] Step S includes knocking out, inhibiting, reducing, or downregulating the expression of the gene encoding the protein in the target plant, and / or the activity and / or content of the protein; the target plant contains the gene encoding the protein.

[0026] Step S can be achieved through gene knockout or gene silencing.

[0027] The present invention also provides a method for regulating the survival rate of plants under low temperature, comprising the following steps: the method includes introducing a substance into a target plant that inhibits, reduces, or downregulates the expression of the gene encoding the aforementioned protein or inhibits, reduces, or downregulates the activity and / or content of the aforementioned protein, thereby changing the survival rate of the target plant under low temperature, wherein the target plant contains the gene encoding the aforementioned protein.

[0028] The present invention also provides a method for cultivating plants with altered survival rates under low temperatures, comprising the following steps: the method includes introducing substances into a target plant that inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein, or inhibit, reduce, or downregulate the activity and / or content of the aforementioned protein, to obtain a plant with altered survival rates under low temperatures, wherein the target plant contains the gene encoding the aforementioned protein.

[0029] 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.

[0030] 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.

[0031] Step S includes introducing a gene knockout vector into the target plant targeting nucleotides 193-212 of sequence 1, 2, or 3 in the sequence listing.

[0032] The target plant mentioned above may be wheat.

[0033] The encoding gene can be a DNA molecule whose nucleotide sequence is sequence 1, 2, or 3 in the sequence listing.

[0034] Specifically, step S can be performed as follows: in the first case, 20 nucleotides from positions 195 to 214 of SEQ ID NO.1 are deleted, thymine deoxyribonucleotide T is inserted between positions 208 and 209 of SEQ ID NO.2, and 4 nucleotides from positions 209 to 212 of SEQ ID NO.3 are deleted; in the second case, thymine deoxyribonucleotide T is inserted between positions 208 and 209 of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0035] The regulation of wheat's response to low-temperature stress mentioned above mainly includes regulating the survival rate of plants under low temperatures.

[0036] The present invention also provides a method for altering the flowering time of a plant, comprising the following steps: regulating the expression of the encoding gene of the aforementioned protein in the target plant or regulating the activity and / or content of the protein, or knocking out the encoding gene of the aforementioned protein in the target plant, thereby altering the flowering time of the target plant, wherein the target plant contains the encoding gene of the aforementioned protein.

[0037] The regulation includes at least the following aspects: regulating the expression of the gene encoding the protein or regulating the content of the protein: 1) regulation at the transcriptional level of the gene encoding; 2) post-transcriptional regulation of the gene encoding (i.e., regulation of splicing or processing of the primary transcript of the gene encoding); 3) regulation of RNA transport of the gene encoding (i.e., regulation of the transport of mRNA of the gene encoding from the nucleus to the cytoplasm); 4) regulation of translation of the gene encoding; 5) regulation of mRNA degradation of the gene encoding; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene encoding).

[0038] In some specific embodiments of the present invention, regulating the expression of the encoding gene of the aforementioned protein in the recipient plant or regulating the activity and / or content of the protein may specifically be inhibiting, reducing or downregulating the expression of the encoding gene of the aforementioned protein in the recipient plant or inhibiting, reducing or downregulating the activity and / or content of the protein.

[0039] In some specific embodiments of the present invention, the inhibition, reduction, or downregulation of the expression of the gene encoding the aforementioned protein in the recipient plant, or the inhibition, reduction, or downregulation of the activity and / or content of the aforementioned protein, includes introducing a substance into the target plant that inhibits, reduces, or downregulates the expression of the gene encoding the aforementioned protein, or the inhibition, reduction, or downregulation of the activity and / or content of the aforementioned protein.

[0040] The substance may specifically be any of the following: C1) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the gene encoding the aforementioned protein, or nucleic acid molecules that knock out the gene encoding the aforementioned protein; C2) expresses the gene encoding the nucleic acid molecule described in C1); C3) contains 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) 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).

[0041] In some specific embodiments of the present invention, the nucleic acid molecule described in C1) may specifically be a gRNA that targets the protein-coding gene.

[0042] In some specific embodiments of the present invention, the protein-coding gene is SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0043] The target sequence of the gRNA may be nucleotides 193 to 212 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some specific embodiments of the present invention, the recombinant microorganism in C5) of the method may specifically be yeast, bacteria, algae and fungi.

[0044] In some specific embodiments of the present invention, the recombinant microorganism in the method may be Agrobacterium. The Agrobacterium may be Agrobacterium tumefaciens.

[0045] In some specific embodiments of the present invention, the plant tissue in C7) of the method may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and / or anthers.

[0046] In some specific embodiments of the present invention, the transgenic plant organs in C8) of the method may be the roots, stems, leaves, flowers, fruits and seeds of the transgenic plant.

[0047] In one specific embodiment of the present invention, the flowering time is changed to the flowering time being advanced.

[0048] In one specific embodiment of the present invention, knocking out the gene encoding the protein in the target plant includes introducing a gene knockout vector into the target plant with nucleotides 494-510 of sequence 1 in the sequence listing as the target site.

[0049] A “target site” refers to a location within a plant genome containing a polynucleotide sequence that is bound by a genome-modifying enzyme to introduce modifications into the nucleic acid backbone of the polynucleotide sequence and / or its complementary DNA strand within the plant genome. A target site may contain at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, or 30 consecutive nucleotides. A target site may contain the complementary strand of a double-stranded nucleic acid (DNA) molecule at the target site or a chromosomal sequence. The site-specific nuclease Cas9 can bind to the target site, for example, via a non-coding guide nucleic acid (e.g., but not limited to, CRISPR RNA (crRNA) or single guide RNA (sgRNA) as further described herein). The non-coding guide nucleic acids provided herein may be complementary to the target site (e.g., complementary to either strand of a double-stranded nucleic acid molecule or to the chromosome at the target site). It should be understood that the binding or hybridization of non-coding guide nucleic acids to target sites may not require perfect identity or complementarity. For example, there may be at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight mismatches (or more) between the target site and the guide nucleic acid. "Target site" also refers to the location of a polynucleotide sequence within the plant genome that may be bound and cleaved by any other site-specific nuclease that is not guided by the guide nucleic acid, such as broad-spectrum nucleases, zinc finger nucleases (ZFNs), transcription activators such as effector nucleases (TALENs), to introduce double-strand breaks (or single-strand cuts) into the polynucleotide sequence and / or its complementary DNA strand.

[0050] In one specific embodiment of the present invention, the knockout of the coding gene in the target plant is achieved by mutating the coding gene in the target plant as follows: deleting 20 nucleotides from positions 195 to 214 of SEQ ID NO.1, inserting thymine deoxyribonucleotide T between positions 208 and 209 of SEQ ID NO.2, and deleting 4 nucleotides from positions 209 to 212 of SEQ ID NO.3.

[0051] In another specific embodiment of the present invention, the knockout of the coding gene in the target plant is achieved by mutating the coding gene in the target plant as follows: inserting thymine deoxyribonucleotide T between positions 208-209 of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

[0052] In the applications or methods described above, the plant is any of the following: G1) Dicotyledons; G2) Monocotyledons; G3) Gramineae plants; G4) Wheat; G5 wheat varieties.

[0053] The wheat may be K35.

[0054] Experiments have shown that gene editing in WT... WPA1 This protein, WPA1, can reduce the survival rate of wheat under low-temperature stress and cause it to flower earlier. Therefore, WPA1 has important application value in regulating the low-temperature stress response and flowering time of wheat, and has broad prospects in the breeding of wheat varieties. Attached Figure Description

[0055] Picture 1 for WPA1 Genotypic and phenotypic analysis of gene knockout mutants. (a) WPA1 Genotypic analysis of gene knockout mutants, knockout lines KO#1 and KO#2 Compared to the control K35, in terms of genes WPA1 / WPA1-7A / WPA1-4A Gene editing occurred in all of them, including base insertion and deletion; b is... WPA1 Phenotypic analysis of gene knockout mutants: After wheat seedlings at the three-leaf stage were treated at -6℃ for 10 hours, the mutant lines... KO#1 and KO#2 Compared to the control K35, it is more sensitive to low temperatures; the scale bar is 5 cm; c is... WPA1 Survival statistics of gene knockout mutants, mutant strains KO#1 and KO#2Compared to the control K35, the survival rate was lower under low-temperature treatment. Statistical analysis was performed using a one-way ANOVA test. P <0.01 (**) indicates a significant difference.

[0056] Picture 2 for WPA1 Flowering phenotype of gene knockout mutant. a is... WPA1 Growth phenotype of gene knockout mutants, knockout lines KO#1 and KO#2 All of them headed earlier than the control K35; b is WPA1 Flowering time statistics of gene knockout mutants, mutant lines KO#1 and KO#2 Compared to the control K35, the flowering time was shorter. The statistical analysis was performed using a one-way ANOVA test. P <0.001 (***) indicates a highly significant difference. Detailed Implementation

[0057] 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.

[0058] 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.

[0059] pBUE411 plasmid: described in the non-patent literature "Jihu Li, Shujuan Zhang, Rongzhi Zhang, Jie Gao, Yiping Qi 4 5, Guoqi Song, Wei Li, Yulian Li, Genying Li. Efficient multiplex genome editing by CRISPR / Cas9 in common wheat. Plant Biotechnol J .2021 Mar;19(3):427-429. doi: 10.1111 / pbi.13508. Epub 2020 Nov 30.", is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only for repeating the relevant experiments of this invention and cannot be used for other purposes.

[0060] 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 <0.01 (**) indicates a significant difference.

[0061] Example 1: Knockout of wheat WPA1 Genes influence wheat's response to low temperature stress 1. Wheat WPA1 Genetic information wheat WPA1 The gene is located on chromosome 7D, and its homologous genes are located on chromosomes 7A and 4A. Therefore, gene knockout is used in this invention. WPA1 This means that WPA1 - 7D / - 7A / - 4A Knock it out at the same time. WPA1 - 7D / - 7A / - 4A The corresponding genome sequences do not contain introns, therefore their CDS sequences are identical to their genome sequences.

[0062] wheat WPA1-7D The coding sequence (CDS) of the gene and the nucleic acid sequence of the genome are both sequence 1 (SEQ ID NO: 1, 1575 bp) in the sequence listing. Wheat WPA1-7A The coding sequence (CDS) of the gene and the nucleic acid sequence of the genome are both sequence 2 (SEQ ID NO:2, 1575bp) in the sequence listing. Wheat WPA1-4A The coding sequence (CDS) of the gene and the nucleic acid sequence of the genome are both sequence 3 (SEQ ID NO:3, 1575bp) in the sequence listing. Wheat WPA1-7D The amino acid sequence of the protein WPA1-7D encoded by the gene is sequence 4 in the sequence listing (SEQ ID NO:4, 524 aa). Wheat WPA1-7A The amino acid sequence of the protein WPA1-7A encoded by the gene is sequence 5 in the sequence listing (SEQ ID NO: 5, 524 aa). Wheat WPA1-4A The amino acid sequence of the protein WPA1-4A encoded by the gene is sequence 6 in the sequence listing (SEQ ID NO: 6, 524 aa).

[0063] 2. Primer design and amplification For target genes WPA1-7D and its homologous genes WPA1-7A / -4AGene editing targets were designed based on conserved sequences, with the selected targets being T1 (SgRNA Target1) and T2 (SgRNA Target2). The T1 sequence is 5'-GACGTGAGCGGCAGCATGCA-3' (corresponding to nucleotides 193-212 of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3), and the T2 sequence is 5'-GAGGACATCGTGCTGAACGA-3' (corresponding to nucleotides 1171-1190 of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3). T1 and T2... WPA1-7D / - 7A / -4A Location in genes such as Picture 1 As shown.

[0064] Design and synthesize forward primers. WPA1 -CRISPR-F and reverse primers WPA1 CRISPR-R was used with the intermediate vector pMETaU6.1 as a template for PCR amplification. The PCR amplification system is shown in Table 1, and the PCR amplification program is shown in Table 2. The PCR product containing approximately 800 bp of the target band was recovered. This PCR product contains sgRNA expression cassettes targeting T1 and T2, and is named sgRNA1-TaU6 snRNA-sgRNA2. The nucleotide sequence of this PCR product is SEQ ID NO:7, where bp 1-15 of SEQ ID NO:7 is... Bsa The I restriction site and its protective bases: 16-35 bp is the T1 sequence, 36-62 bp is the regulatory sequence, 63-127 bp is the gRNA scaffold, 128-412 bp is the regulatory sequence, 413-774 bp is the wheat U6 promoter, 775-794 bp is the T2 sequence, and 795-810 bp is... Bsa I. Enzyme cleavage site and its protective base.

[0065] The primer sequences are as follows: WPA1 -CRISPR-F: 5'-ccgaggtctcgggcgGACGTGAGCGGCAGCATGCAgtttcagagctatgctggaaac-3'; WPA1-CRISPR-R:5'-acctcggtctccaaacTCGTTCAGCACGATGTCCTCcaagtctgatgcagcaagc-3'.

[0066] Table 1. PCR amplification system (50 μL)

[0067] Table 2 PCR amplification program

[0068] 3. Ligation of PCR products with gene editing vectors use Bsa I. Digest the gene editing backbone vector pBUE411 with a single enzyme, and then recover the vector via gel extraction. Mix the PCR product with the gene editing backbone vector pBUE411 (… Bsa The vector recovered after single enzyme digestion was ligated to obtain the recombinant plasmid pBUE411-WPA1-sgRNA. The ligation reaction was performed using a PCR instrument under the following conditions: 37℃ for 5 min, 16℃ for 10 min, for a total of 60 cycles; then 16℃ for 1 h. pBUE411-WPA1-sgRNA can generate Cas9 and sgRNAs targeting T1 and T2.

[0069] Table 3 Reaction system (50 μL)

[0070] 4. Agrobacterium-mediated transformation The recombinant plasmid pBUE411-WPA1-sgRNA was transformed into the Agrobacterium tumefaciens strain EHA105 to obtain recombinant Agrobacterium EHA105 / pBUE411-WPA1-sgRNA containing the recombinant plasmid pBUE411-WPA1-sgRNA.

[0071] Wheat K35 (recipient) embryonic scutellaria was infected with recombinant Agrobacterium EHA105 / pBUE411-WPA1-sgRNA. The embryos were then transferred to an induction medium and cultured at 22-23°C in the dark to induce embryogenic callus. The embryogenic callus was then transferred to a rooting medium and cultured at 22-23°C for 3-4 weeks under 12h light / 12h dark conditions to obtain T0 generation regenerated seedlings with good rhizome growth, i.e., T0 generation transgenic wheat.

[0072] The formulations of the relevant culture media are as follows: Callus induction medium (1 L): 100 mL MS macro salts (×10), 1 mL L7 micro salts (×1000), 10 mL FeNaEDTA (×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 Picloram, and 80 mg Timentin.

[0073] Regeneration medium (1 L): 100 mL L7 macro salts (×10), 1 mL L7 micro salts (×1000), 10 mL ferric sodium EDTA (FeNaEDTA) (×100), 5 mL vitamins / 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 Timentin, and 2.5 mg Zeatin. The screening medium is the regeneration medium with 1.25 mg glufosinate added.

[0074] MS Macro 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.

[0075] L7 Macro 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.

[0076] L7 Micro 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.

[0077] 5. Identification of positive plants T0 generation transgenic wheat was selected for PCR detection. The specific steps are as follows: 1) Total DNA was extracted from the leaves of T0 generation transgenic wheat seedlings using the CTAB genomic DNA extraction method. The DNA content in the leaves of each T0 generation transgenic wheat seedling was approximately 200 ng / μL.

[0078] 2) PCR technology was used to obtain fragment sequences, including gene editing target sites, from each T0 generation of transgenic wheat. This included primers for identifying whether WPA1-7D and its homologous genes WPA1-7A / 4A had mutated. The sequences are as follows: WPA1-7D -seq-F:5'-TGCTTGGTGTGTGAGTGTGA-3'; WPA1-7D -seq-R: 5'-AAATGGAGTGATGAAACAAACG-3'; WPA1-7A -seq-F:5'-TGCTCTGCTAGCTCATTGGA-3'; WPA1-7A -seq-R: 5'-GCCAGGTTACAACCATGAAGA-3'; WPA1-4A -seq-F:5'-ACACGTACGCCAGACACAGA-3'; WPA1-4A -seq-R: 5'-GGAAAACGTAGCAGTAAATGGA-3'.

[0079] The reaction system consisted of 20 μL of 10 μL 2× Phanta Max Master Mix (Dye Plus), 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 1 μL of T0 generation transgenic wheat genomic DNA, and 8.0 μL ddH2O.

[0080] Reaction program: 94℃ pre-denaturation for 3 min; 95℃ denaturation for 3 sec; 60℃ annealing for 30 sec; 35 cycles.

[0081] 3) Sequencing method for detecting mutants The PCR products were sequenced, and the mutation type was detected by comparing the sequencing results with the corresponding nucleotide sequences of the recipient plants. The detected T0 generation mutant plants were then self-crossed for two consecutive generations to obtain the T2 generation. WPA1 -7D / 7A / 4A gene knockout wheat, i.e., homozygous mutant wpa1 In KO#1, compared to wheat variety K35, WPA1 The sequences corresponding to the -7D / 7A / 4A genes have undergone the following changes: 20 nucleotides from positions 195-214 of SEQ ID NO.1 have been deleted; thymine deoxyribonucleotide T has been inserted between positions 208-209 of SEQ ID NO.2; and 4 nucleotides from positions 209-212 of SEQ ID NO.3 have been deleted. In KO#2, thymine deoxyribonucleotide T has been inserted between positions 208-209 of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 (see...). Picture 1 ).

[0082] 6. T2 generation WPA1 Phenotypic identification of gene knockout wheat under low temperature stress Wild-type control and mutant wheat seeds were sown in soil and placed in a light incubator at 16°C with 16 hours of light followed by 8 hours of darkness. Five replicates were used for each line, with six wheat seeds per replicate. After approximately 30 days of cultivation, when the seedlings reached the three-leaf stage, uniformly growing seedlings were selected for low-temperature treatment. After testing at different temperatures (-4°C, -6°C, -8°C, -10°C) and for different durations (2 h, 4 h, 6 h, 8 h, 10 h), -6°C for 10 h was determined to be the most suitable temperature for phenotypic identification of the experimental materials in this invention. Therefore, after 10 h of treatment at -6°C, the seedlings were then restored to 16°C for 3 days. Thirty wild-type and mutant seedlings were treated each time, for a total of three treatments. Photos were taken before and after treatment to record the survival rate.

[0083] The phenotype of the low-temperature stress experiment described above was as follows: compared with the control K35, the knockout mutant... wpa1 KO#1 and KO#2 The survival rate of all decreased ( Picture 1 ).

[0084] The results show that WPA1 Gene knockout lines have reduced survival rates at low temperatures and exhibit a phenotype that is more sensitive to low temperatures.

[0085] 7. T2 generation WPA1 Identification of flowering phenotype in gene knockout wheat Wild-type control and mutant wheat seeds were sown separately in soil and placed in a light incubator at 16℃ with 16 h light / 8 h darkness. Each line was replicated in 5 pots, with 3 wheat seeds per pot. The plants were cultured until flowering, and the flowering time of each line was recorded, with a total of 15 plants per line. Flowering time refers to the time it takes for half of the flag leaf to emerge from the ear of the main tiller of each wheat plant.

[0086] The results are as follows Picture 2 As shown, WPA1 The flowering time of the gene knockout line was significantly earlier than that of the wild-type K35. The flowering time of the wild-type K35 was 43±1.5 days, the flowering time of ko#1 was shortened to 35±2 days, and the flowering time of ko#2 could reach 36±1 days.

[0087] The sequences involved in the above embodiments are: SEQ ID NO:1 (1575bp)

[0088] SEQ ID NO:2(1575bp)

[0089] SEQ ID NO:3(1575bp):

[0090] SEQ ID NO:4(524 aa): MAFNDDEKPPASNAGNTKGLVTITEPKFSKDEAALSADEVTAVVELKATSSTAVREGLDLVAVLDVSGSMQGDKLQSMKMAMQFVIMKLTPVDRLSVVSFSGSATRHCPLRSVTQQAQADLKAIVDGLVANGGTNIKAGLDTALAIVAGRATTKARTPNVFLMSDGQQSDGDARQVDPGNVAVYTFGFGKDADHALLSDVARKSPGGTFNSVPDGGNVTAPFSQLLGGLLTIVAQDVQLTLTPKAEDPSAPDLDTMTVAPGTDYTQTTDGGTGVITIKFGTLFSGETRKVAINFKLLESTLTTPYDGLVAEAQHSYNVQGSPQGQTPQDVVIPRSPDAPGEEAVSVKARGVLAEMARRQHAGAIGEARQMADGKNLEEARYKLADAQNALEDIVLNDGEKLVGMLRAELQQLLDLMETQELYEAEGRPYALASETSHGRQRYAARGGDMDAVRLFATPRMDTYLEQAKKFEEDPTAPLPSADEDAKEEMAANPLAAISAPIAFYIKVAIQALQEIEKLVAPPTK。

[0091] SEQ ID NO:5(524 aa): MAFNDDEKPPASNVGSTKGLVTISTPTYSKDAAALTADAVTAVVELNATSSTAVREGLDLVAVLDVSGSMQGDKLQSMKRAMQFVIMKLTPVDRLSVISFASSANRHCPLRSVTQQAQTDLKSIVDGLVANGGTNIKAGLDTALAVIAGRATTKARTPNIFLMSDGQQSDGDARQVDPGNVAVYTFGFGKDADHALLSDVAKKSPGGTFNSVPDGGNVSAPFSQLLGGLLTIVAQDVQLTLTPKAEDPSAPDLDTMTVAPGTDYTQTTDGNTGVITIKFGTLFSGETRKVAINLKLLESTLTTAYDGLVAEAQHSYTVQGSPQGQTPQDIVIPRSPDAPADPPTSGKAQAVLAEMARRQHAGAIGEARQMADGKNLEEARYKLADAQNALEDIVLNDGEKLVGMLRAELQQLLDLMETQELYEAEGRPYALASETSHGRQRYAARGGDMDAVRLFATPRMDTYLEQAKKFEEDPTAPLPSADDDAKEEMAANPLAAISAPIAFYIKVAIQALQEIEKLVAPPTK。

[0092] SEQ ID NO:6(524 aa): MAFNDDEKPPASNAGNTKGLVTITEPKFSKDAAALSADEVTAVVELKATSSTAVREGLDLVAVLDVSGSMQGDKLQSMKMAMQFVIMKLTPVDRLSVVSFSGSATRHCPLRSVTQQAQADLKAIVDGLVANGGTNIKAGLDTALAIVAGRATTKARTPNVFLMSDGQQSDGDARQVDPGNVAVYTFGFGKDADHALLSDVAKKSPGGTFNSVPDGGNVSAPFSQLLGGLLTIVAQDVQLTLTPKAEDPTAPDLDTMTVAPGTDYTQTTDGDTGVITIKFGTLFSGETRKVAINFKLLESTLTTAYDGLVAEAQHSYTVQGSLQGQTPQDVVIPRSPDAPADPPTSGKAQAVLAEMARRQHAGAIGEARQMADGKNLEEARYKLADAQNALEDIVLNDGEKLVGMLRAELQQLLDLMETQELYEAEGRPYALASETSHGRQRYAARGGDMDAVRLFATPRMDTYLEQAKKFEEDPTAPLPSADDDAKEEMAANPLAAISAPIAFYIKVAIQALQEIEKLVAPPTK。

[0093] The specific sequence of SEQ ID NO:7 is as follows: 5’-CCGAGGTCTCGGGCGGACGTGAGCGGCAGCATGCAGTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTTTCGTTTTGCATTGAGTTTTCTCCGTCGCATGTTTGCAGTTTTATTTTCCGTTTTGCATTGAAATTTCTCCGTCTCATGTTTGCAGCGTGTTCAAAAAGTACGCAGCTGTATTTCACTTATTTACGGCGCCACATTTTCATGCCGTTTGTGCCAACTATCCCGAGCTAGTGAATACAGCTTGGCTTCACACAACACTGGTGACCCGCTGACCTGCTCGTACCTCGTACCGTCGTACGGCACAGCATTTGGAATTAAAGGGTGTGATCGATACTGCTTGCTGCTACCAAGCCCGTTATTCTGACAGTTCTGGTGCTCAACACATTTATATTTATCAAGGAGCACATTGTTACTCACTGCTAGGAGGGAATCGAACTAGGAATATTGATCAGAGGAACTACGAGAGAGCTGAAGATAACTGCCCTCTAGCTCTCACTGATCTGGGTCGCATAGTGAGATGCAGCCCACGTGAGTTCAGCAACGGTCTAGCGCTGGGCTTTTAGGCCCGCATGATCGGGCTTTTGTCGGGTGGTCGACGTGTTCACGATTGGGGAGAGCAACGCAGCAGTTCCTCTTAGTTTAGTCCCACCTCGCCTGTCCAGCAGAGTTCTGACCGGTTTATAAACTCGCTTGCTGCATCAGACTTGTCGTTCAGCACGATGTCCTCGTTTGGAGACCGAGGT-3’。

[0094] 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 application of a protein or a biomaterial related to said protein, characterized in that, The application is any one of the following: E1) Regulates the flowering time of plants; E2) Prepare products that regulate the flowering time of plants; E3) regulates plant survival rate under low temperature; E4) Prepare products that regulate plant survival rate under low temperature; E5) Plant breeding; The biological material is a substance that regulates the expression of the gene encoding the protein or a substance that regulates the content of the protein. The protein is any one of the following: B1) The amino acid sequence is that of the protein shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6; B2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein described in B1) that has more than 80% identity with and the same function as the protein shown in B1). B3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2).

2. The application according to claim 1, characterized in that, The biomaterial is any one of the following: C1) Inhibit or reduce or downregulate the expression of the nucleic acid molecule encoding the protein of claim 1 or knock out the nucleic acid molecule encoding the protein of claim 1; C2) expresses the gene encoding the nucleic acid molecule described in C1); C3) contains 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) 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).

3. The application according to claim 2, characterized in that, The protein-coding gene is SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:

3.

4. A method for regulating the survival rate of plants under low temperature, characterized in that, The method includes the following steps: the method includes introducing a substance into the target plant that inhibits, reduces, or downregulates the expression of the gene encoding the protein of claim 1 or inhibits, reduces, or downregulates the activity and / or content of the protein, thereby changing the survival rate of the target plant at low temperature, wherein the target plant contains the gene encoding the protein of claim 1.

5. A method for altering the flowering time of a plant, characterized in that, The method includes the following steps: regulating the expression of the gene encoding the protein described in claim 1 in the target plant or regulating the activity and / or content of the protein, or knocking out the gene encoding the protein described in claim 1 in the target plant to change the flowering time of the target plant, wherein the target plant contains the gene encoding the protein described in claim 1.

6. The method according to claim 5, characterized in that, The change in flowering time is now interpreted as an earlier flowering time.

7. The method according to claim 5, characterized in that, The gene encoding the protein in the target plant to be knocked out includes introducing a gene knockout vector into the target plant with nucleotides 193 to 212 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 as the target site.

8. The method according to claim 7, characterized in that, The knockout of the coding gene in the target plant is achieved by mutating the coding gene in the target plant as follows: deleting 20 nucleotides from positions 195 to 214 of SEQ ID NO.1, inserting thymine deoxyribonucleotide T between positions 208 and 209 of SEQ ID NO.2, and deleting 4 nucleotides from positions 209 to 212 of SEQ ID NO.

3.

9. The method according to claim 7, characterized in that, The knockout of the coding gene in the target plant is achieved by mutating the coding gene in the target plant as follows: inserting thymine deoxyribonucleotide T between positions 208-209 of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.

3.

10. The application according to any one of claims 1-3, or the method according to any one of claims 4-9, characterized in that, The plant is any one of the following: G1) Dicotyledons; G2) Monocotyledons; G3) Gramineae plants; G4) Wheat; G5 wheat varieties.

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