Proteins derived from wheat and related biological materials and applications

Abstract

The application discloses a protein derived from wheat and related biological materials and application thereof, and belongs to the field of genetic engineering.The technical problem to be solved by the application is how to improve the yield of plants.The protein provided by the application is TaNRK-4D and / or TaNRK-4AB, wherein TaNRK-4AB is a protein with the amino acid sequence of SEQ ID NO:2, and TaNRK-4D is a protein with the amino acid sequence of SEQ ID NO:8.The application can be used for improving the yield of wheat.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to proteins derived from wheat and related biomaterials and applications. Background Technology

[0002] wheat( Triticum aestivum Nitrogen is one of the world's most important food crops, and ensuring its high and stable yields is crucial for maintaining global food security. Nitrogen is one of the most important mineral nutrients for plant growth and development, and the availability of nitrogen in the soil directly determines crop yield and quality.

[0003] Most nitrogen fertilizer applied in agricultural production can be directly absorbed and utilized by crops. It is estimated that cereal crops absorb and utilize 30%-50% of the total nitrogen applied. Nitrogen fertilizer not absorbed and utilized by plants is converted into N2O and emitted into the atmosphere, contributing to climate change as a greenhouse gas. Some seeps into groundwater or flows into rivers or oceans with rainwater, causing groundwater pollution and eutrophication of rivers and nearshore waters. Long-term application of large amounts of chemical fertilizers can also directly or indirectly lead to increased nitrogen (H2O) in the soil. + Excessive accumulation of nitrogen fertilizer leads to soil acidification. Furthermore, nitrogen fertilizer production consumes large amounts of fossil fuels, further exacerbating the damage to the ecological environment. Excessive nitrogen fertilizer increases the environmental burden, causing a series of environmental problems such as soil acidification, eutrophication of water bodies, air pollution, and the greenhouse effect. Therefore, studying the molecular mechanisms of nitrogen absorption and utilization by crops is crucial to providing a theoretical and material basis for achieving the breeding goal of reducing fertilizer use without reducing yield. The continuous input of chemical fertilizers does not necessarily lead to a sustained increase in crop yield; under high nitrogen fertilizer input, crop yield growth gradually slows down or even stagnates, indicating that the relationship between nitrogen fertilizer input and crop yield is not absolutely linear.

[0004] The three key factors determining wheat yield are the number of effective spikes, the number of grains per spike, and the thousand-grain weight. The number of effective spikes, also known as the number of effective tillers, directly determines crop yield. Therefore, studying the tillering regulation mechanism is crucial for high-yield crop breeding. Tillering is one of the important yield-related traits in wheat, and its growth process includes the formation of tiller primordia and the elongation of tiller buds. Factors influencing wheat tillering mainly include genetic factors and the growth environment. Applying nitrogen fertilizer can significantly increase the number of wheat tillers, thereby increasing yield. Therefore, understanding the patterns of wheat tillering and the molecular mechanisms by which nitrogen fertilizer affects tillering is of great significance for achieving high-yield wheat production with low fertilizer consumption. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to increase the yield and / or number of ears of plants (such as wheat).

[0006] To address the aforementioned problems, this invention provides applications of proteins, wherein the proteins may be TaNRK-4D and / or TaNRK-4AB.

[0007] The TaNRK-4AB can be any of the following: A1) The amino acid sequence of this protein is SEQ ID NO: 2. A2) A protein with more than 70% amino acid sequence identity and the same function as A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence of A1). A3) A fusion protein obtained by attaching a tag to the end of the protein in A1); The TaNRK-4D can be any of the following: D1) The amino acid sequence of this protein is that of SEQ ID NO: 8. D2) A protein with more than 70% amino acid identity and the same function as the amino acid sequence of D1) obtained by substituting and / or deleting and / or adding amino acid residues. D3) is a fusion protein obtained by attaching a tag to the end of the protein in D1); The application can be any of the following: Application of F1 in regulating plant agronomic traits; Application of F2 in the preparation of products that regulate plant agronomic traits; Application of F3 in the cultivation of plants with altered agronomic traits; F4) Application in the preparation of products from plants with altered agronomic traits; Application of F5 in plant breeding.

[0008] In the above applications, the protein may be derived from wheat.

[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 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, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0012] In this invention, the 70% or more of identity can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0013] Both SEQ ID NO: 2 and SEQ ID NO: 8 consist of 369 amino acid residues. SEQ ID NO: 2 is as follows: MDGAPVAEFRPTMTHGGRFLLYNIFGNQFEITAKYQPPIMPIGRGAYGIVCSVMNFETREMVAIKKIANAFDNNMDAKRTLREIKLLRHLDHENIVGLRDVIPPAIPQSFNDVYIATELMDTDLHHIIRSNQELSEEHCQYFLYQLLRGLKYIHSANVIHRDLKPSNLLLNANCDLKICDFGLAR PSSESDMMTEYVVTRWYRAPELLLNSTDYSAAIDVWSVGCIFMELINRAPLFPGRDHMHQMRLITEVIGTPTDDDLGFIRNEDARRYMRHLPQFPRRSFPGQFPKVQPAALDLIERMLTFNPLQRITVEEALEHPYLERLHDVADEPICTDPFSFDFEQHPLTEDQMKQLIFNEALELNPNFRY.

[0014] SEQ ID NO: 8 is as follows: MDGAPVAEFRPTMTHGGRFLLYNIFGNQFEITAKYQPPIMPIGRGAYGIVCSVMNFETREMVAIKKIANAFDNNMDAKRTLREIKLLRHLDHENIVGLRDVIPPATPQSFNDVYIATELMDTDLHHIIRSNQELSEEHCQYFLYQLLRGLKYIHSANVIHRDLKPSNLLLNANCDLKICDFGLAR PSSESDMMTEYVVTRWYRAPELLLNSTDYSAAIDVWSVGCIFMELINRAPLFPGRDHMHQMRLITEVIGTPTDDDLGFIRNEDARRYMRHLPQFPRRSFPGQFPKVQPAALDLIERMLTFNPLQRITVEEALEHPYLERLHDVADEPICTDPFSFDFEQHPLTEDQMKQLIFNEALELNPNFRY.

[0015] This invention also provides the application of biomaterials, which may be biomaterial B or biomaterial C; biomaterial B may be any of the following: B1) Nucleic acid molecules that target the genes encoding the proteins described above; B2) Cas protein and B1) the nucleic acid molecule described; B3) Gene, which is the gene encoding the nucleic acid molecule described in B1) and the gene encoding the Cas protein; B4) Expression cassettes, vectors, recombinant microorganisms, or plants containing the genes described in B3); The biomaterial C may be any of the following: C1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the genes encoding the proteins described above; C2) is the gene encoding the nucleic acid molecule described in C1); C3) Expression cassettes, recombinant vectors, recombinant microorganisms, or plants containing the gene described in C2); The application can be any of the following: E1) Application in regulating plant agronomic traits; E2) Application in the preparation of products that regulate plant agronomic traits; E3) Application in the cultivation of plants with altered agronomic traits; E4) Application in the preparation of products from plants with altered agronomic traits; Application of E5 in plant breeding.

[0016] In this application, Cas protein is an "RNA-directed nuclease," which refers to an RNA-directed DNA endonuclease associated with the CRISPR system. Unrestricted examples of RNA-directed nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, their homologs or modified forms thereof. In one implementation, the RNA-directed nuclease is Cas9 or nCas9 (D10A).

[0017] In some embodiments of the present invention, the nucleic acid molecule described in B1) may be a gRNA targeting the protein-coding gene. In some embodiments, the gRNA targets a double-stranded DNA whose nucleotide sequence is SEQ ID NO: 1 at positions 446-468 and / or a double-stranded DNA whose nucleotide sequence is SEQ ID NO: 1 at positions 703-725.

[0018] The term "sgRNA (single-guide RNA)" is a component of the CRISPR-Cas system, responsible for guiding the Cas protein to recognize and cleave target nucleic acid molecules. In practical gene editing applications, sgRNA can be synthesized directly or obtained through plasmid expression or in vitro transcription. In this field, "gRNA" and "sgRNA" are often used interchangeably. In this document, "gRNA" and "sgRNA" are also used interchangeably. sgRNA generally refers to a single RNA structure formed by artificially modifying the crRNA / tracrRNA complex (gRNA) with a dual RNA structure, directly (or through a linker) linking the crRNA and tracrRNA. sgRNA is a short RNA containing a recognition region and a framework region.

[0019] The term "recognition region," also known as a guide sequence, is typically an RNA sequence (referred to herein as the "guide sequence") that is identical to or complementary to the target sequence or target site within the target RNA (sgRNA or gRNA). The guide sequence is generally sufficiently complementary to the target sequence to hybridize with the target site and guide the CRISPR / Cas complex to bind specifically to the target. Perfect complementarity between the guide sequence and the target sequence is preferred, but some mismatch (e.g., 1-6 nucleotide mismatch) is permissible as long as it still results in gene knockout. The complementarity between the guide sequence and its corresponding target sequence is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Methods for determining the complementarity of two nucleic acid sequences are within the capabilities of those skilled in the art.

[0020] The term "scaffold" generally refers to the structural or scaffold RNA sequence that guides the binding or interaction of RNA with RNA-directed nucleases and / or other RNA molecules (e.g., tracrRNA) into RNA (sgRNA or gRNA), and can also be called the backbone sequence of sgRNA. The scaffold can be conventionally selected by those skilled in the art; for example, it can be the backbone sequence of the sgRNA corresponding to Cas9, or it can be a mutant constructed based on this sequence that still retains the function of binding the corresponding Cas9.

[0021] In the above applications, the introduction of the gene described in B3) or C2) into the recipient plant can specifically be achieved by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrocoagulation, or 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.

[0022] In one embodiment of the present invention, the plant gene editing vector is pYLCRISPR / Cas9. The plant gene editing vector may be a pYLCRISPR / Cas9-TaNRK vector.

[0023] As a specific embodiment, the recombinant vector may be the recombinant vector pYLCRISPR / Cas9-TaNRK. The recombinant vector is a recombinant expression vector in which the DNA fragment (SEQ ID NO: 10) shown in the TaNRK-sgRNA expression cassette is inserted into the sequence between the BsaI restriction sites of the pYLCRISPR / Cas9 plasmid, while keeping other nucleotide sequences unchanged.

[0024] Those skilled in the art can readily mutate the nucleotide sequences encoding the proteins TaNRK-4AB and / or TaNRK-4D of this invention using known methods, such as directed evolution or point mutation. Artificially modified nucleotides that possess 75% or more of the nucleotide sequence identity with the proteins TaNRK-4AB and / or TaNRK-4D isolated in this invention, provided they encode and function as proteins TaNRK-4AB and / or TaNRK-4D, are derived from and equivalent to the nucleotide sequences of this invention.

[0025] The vectors described herein are known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cos plasmids), Ti plasmids, or viral vectors.

[0026] Recombinant expression vectors containing the TaNRK-4AB and / or TaNRK-4D encoding genes can be constructed using existing plant expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthase) and plant genes (such as the soybean storage protein gene).

[0027] When constructing recombinant plant expression vectors using the TaNRK-4AB and / or TaNRK-4D coding genes, any enhancing or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0028] In the above applications, the microorganism described in B4) or C3) may be yeast, bacteria, algae, or fungi. In some specific embodiments, the recombinant microorganism may be EHA105-pYLCRISPR / Cas9-TaNRK.

[0029] In the above applications, the plant described in B4) or C3) may be the whole plant or a part of it (cells, tissues and / or organs).

[0030] In this invention, the plant may be any of the following: G1) dicotyledonous plants, G2) monocotyledonous plants, G3) grasses, G4) grasses, G5) wheat, G6) wheat.

[0031] In this invention, the plant agronomic traits may be plant yield and / or number of spikes, and the regulation of plant agronomic traits may be to increase the number of spikes and / or yield; the yield may be yield per plant.

[0032] In this invention, the regulation of plant agronomic traits can be accomplished by regulating the genes encoding the proteins TaNRK-4AB and / or TaNRK-4D.

[0033] In the above applications, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the gene transcription level; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of gene translation; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0034] The present invention also provides a method, which may be M1, M2, M3 or M4: M1. A method for increasing wheat yield and / or number of ears, the method comprising knocking out the coding gene of a wheat to be improved containing the protein described above to obtain gene knockout wheat, wherein the yield and / or number of ears of the gene knockout wheat is higher than that of the wheat to be improved. M2. A breeding method for producing wheat with increased yield and / or number of spikes, the method comprising knocking out the coding gene of the wheat to be improved containing the protein described above to obtain wheat with increased number of spikes and / or yield, wherein the yield and / or number of spikes of the wheat with increased number of spikes and / or yield is higher than that of the wheat to be improved. M3. A method for increasing wheat yield and / or number of ears, the method comprising reducing the activity and / or content of the proteins described above in the wheat to be improved, and / or reducing the expression level of the gene encoding the proteins described above, to increase the yield and / or number of ears of the wheat to be improved. M4. A breeding method for producing wheat with increased yield and / or number of ears, the method comprising reducing the activity and / or content of the proteins described above in the wheat to be improved, and / or reducing the expression level of the gene encoding the proteins described above, to obtain improved wheat, wherein the yield and / or number of ears of the improved wheat is higher than that of the wheat to be improved.

[0035] The “yield and / or number of spikes” are comparisons made under comparable conditions. “Comparable conditions” refer to the same or similar environmental conditions and agronomic practices used for meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices significantly contribute to or explain any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water, humidity, soil, and nutrients (e.g., nitrogen and phosphorus). Experiments have demonstrated that the yield and / or number of spikes in the gene-knockout wheat are higher than those in the wheat to be improved, both under nitrogen-free and nitrogen-fertilized nutrient conditions.

[0036] The methods described above, M1 and M2, include the step of introducing the substance described above that knocks out the gene encoding the protein into the wheat to be improved.

[0037] Specifically, in methods M1 and M2, the knockout includes performing any of the following operations on the genome of the wheat to be improved: H1) Delete positions 893-1164 of SEQ ID NO:7 in the wheat genome to be improved, causing a frameshift in the translation of amino acids after the editing site, premature termination of the protein sequence, and thus knocking out the gene; H2) The T base at position 62 and 22 bases at positions 1190-1211 in the wheat genome to be improved are deleted; a T base is inserted between positions 989-990 in SEQ ID NO:4, and 3 bases are deleted between positions 1233-1235; an A base is inserted between positions 922-923 and a T base is inserted between positions 1164-1165 in SEQ ID NO:7, resulting in a frameshift mutation that causes premature termination of translation.

[0038] H3) Delete nucleotides from positions 942-1194 of SEQ ID NO:1 in the wheat genome to be improved, insert 253 bases between positions 454 and 455 of SEQ ID NO:4 (TGCGGGATCGCCGGCGGGATCACATCTCGGAGGCCTACTATCTGCGTGTGATGTCCGTCGATACAGCGTATCAGGTTAGCGCCTGAATTGCCGAATTCAGTCAGATACAAACATCTGAAGCGGAAGAGAAAGATGTTGATTACGTTCTCGTGGTCGAGGTGCCTGAGGAGCTTGATCTCCCGGAGCGTGCGCTTGGCGTCCATGTTGTTGTCGAAGGCGTTTGCGATCTTCTTGATTGCCACCATCTCCCTCG), and delete 252 bases from positions 451-692 of SEQ ID NO:7, resulting in a frameshift mutation and premature termination of translation.

[0039] The present invention also provides a substance, which may be the protein or biological material described above.

[0040] The present invention also provides gene-edited wheat, wherein the gene-edited wheat is wheat that does not contain the coding gene for the protein described above.

[0041] The aforementioned gene-edited wheat can be gene-knockout wheat obtained by knocking out the coding gene of the wheat to be improved, which contains the coding gene of the protein described above.

[0042] The number of spikes and / or yield of the gene-edited wheat is higher than that of the wheat to be improved.

[0043] The yield mentioned can be the yield per plant.

[0044] The gene-edited wheat refers to the whole wheat plant or a part of it (cells, tissues, and / or organs).

[0045] The cells include original knockout cells (T0 generation), cells regenerated or developed from T0 generation cells, cells from any progeny or descendant of T0, including seed or embryo cells, or cultured cells, callus cells, etc.

[0046] The tissues and / or organs may be meristems, bud organs / structures (e.g., leaves, stems, or nodes), roots, flowers or floral organs / structures (e.g., flowers, bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (e.g., embryos, endosperm, and seed coats), fruits (e.g., mature ovaries), propagules, or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue).

[0047] In some specific embodiments of the present invention, the wheat to be improved may be the wheat variety Kenong 199 (KN199).

[0048] This invention involves editing the TaNRK gene on a homologous chromosome of wheat and knocking out the TaNRK-4D and / or TaNRK-4A and TaNRK-4B genes using a CRISPR / Cas9 vector to obtain homozygous mutants. The knockout wheat plants KO-1, KO-6, and KO-10 mutants showed significantly higher spike number and yield per plant under both normal and low nitrogen conditions compared to the control KN199 plant. Statistical analysis of these traits indicates that the TaNRK gene negatively regulates wheat spike number and yield, and reducing TaNRK expression significantly increases wheat yield. Attached Figure Description

[0049] Figure 1 The bar charts show the expression levels of the TaNRK4D gene in wheat (Kenong 199) under hydroponic and field conditions. A represents the hydroponic treatment results, with bars from left to right showing the TaNRK4D gene expression levels in high-nitrogen aboveground parts, low-nitrogen aboveground parts, high-nitrogen roots, and low-nitrogen roots, respectively. B represents the field treatment results, with bars from left to right showing the TaNRK4D gene expression levels in high-nitrogen flag leaves and low-nitrogen flag leaves, respectively.

[0050] Figure 2 The expression levels of the TaNRK4D and TaNRT2.1 genes in nitrate-induced Kenong 199 wheat seedlings at different time points are shown.

[0051] Figure 3 This represents the agronomic traits of the TaNRK knockout lines KO-1, KO-6, and KO-10 and the wild-type Kenong 199 (nitrogen control) under normal and low nitrogen conditions. A represents the number of spikes under normal and low nitrogen conditions; B represents the yield per plant under normal and low nitrogen conditions.

[0052] Figure 4 The expression levels of primary nitrate response genes TaNRT2.1, TaNR, and TaGOGAT in the flag leaves of TaNRK knockout strain KO-10 and wild-type Kenong 199 (KN199) are shown. Detailed Implementation

[0053] 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.

[0054] 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.

[0055] The following examples demonstrate the data processing. Experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, and P < 0.05 was considered satisfactory. () indicates a significant difference, P < 0.01. () indicates a highly significant difference, P < 0.001. () indicates a highly significant difference.

[0056] KN199 is a common commercially available wheat variety. It was approved by the National Variety Approval Committee in 2006, with the approval number being National Approval Wheat 2006017.

[0057] The pYLCRISPR / Cas9 in the following examples is the vector named "pYLCRISPR / Cas9Pubi-H" in the following literature, which is publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of this invention and should not be used for other purposes: Hong Yu et al., A route to de novodomestication of wild allotetraploid rice, Cell, Volume 184, Issue 5, 2021, Pages 1156-1170.e14, ISSN 0092-8674.

[0058] Example 1 TaNRK Acquisition of gene knockout materials 1. Construction of CRISPR / Cas9 expression vector 1.1 gRNA Design RNA was extracted from wheat Kenong 199, cDNA was prepared, and the TaNRK gene was cloned from the cDNA. The primer sequences for cloning the gene were: TaNRK4-F: 5'-TCCTCAGCTGGGCCGAGG-3' (SEQ ID NO: 5), TaNRK4-R: 5'-TTTTTATTCGCACAGCCG-3' (SEQ ID NO: 11). Wheat Kenong 199 contains three TaNRK genes: the TaNRK gene in the wheat A genome, also known as TaNRK-4A; the TaNRK gene in the wheat B genome, also known as TaNRK-4B; and the TaNRK gene in the wheat D genome, also known as TaNRK-4D.

[0059] The nucleotide sequence of the TaNRK-4A genome is SEQ ID NO: 1 (2454 bp), as follows: positions 1-152, 443-572, 668-805, 904-1420, and 2282-2454 are exons, and the rest are introns. SEQ ID NO: 1 is as follows:

[0060] The amino acid sequence encoded by the wheat TaNRK-4A gene is the protein TaNRK-4A, sequence 2 (SEQ ID NO: 2,369aa) in the sequence listing, as follows: MDGAPVAEFRPTMTHGGRFLLYNIFGNQFEITAKYQPPIMPIGRGAYGIVCSVMNFETREMVAIKKIANAFDNNMDAKRTLREIKLLRHLDHENIVGLRDVIPPAIPQSFNDVYIATELMDTDLHHIIRSNQELSEEHCQYFLYQLLRGLKYIHSANVIHRDLKPSNLLLNANCDLKICDFGLAR PSSESDMMTEYVVTRWYRAPELLLNSTDYSAAIDVWSVGCIFMELINRAPLFPGRDHMHQMRLITEVIGTPTDDDLGFIRNEDARRYMRHLPQFPRRSFPGQFPKVQPAALDLIERMLTFNPLQRITVEEALEHPYLERLHDVADEPICTDPFSFDFEQHPLTEDQMKQLIFNEALELNPNFRY.

[0061] The coding sequence (CDS) for protein TaNRK-4A is SEQ ID NO: 3 (1110bp), as follows:

[0062] The nucleotide sequence of the TaNRK-4B genome is SEQ ID NO: 4 (2343bp), where positions 1-152, 315-564, 667-804, 947-1463, and 2171-2343 are exons, and the rest are introns; its specific nucleotide sequence is as follows:

[0063] The amino acid sequence encoded by the wheat TaNRK-4B gene is identical to that of the protein TaNRK-4A, both being SEQ ID NO: 2, and can be referred to as TaNRK-4AB or protein TaNRK-4AB.

[0064] The coding sequence (CDS) corresponding to the amino acid sequence encoded by the wheat TaNRK-4B gene is SEQ ID NO: 6 (1110bp), as follows:

[0065] The nucleotide sequence of the TaNRK-4D genome is SEQ ID NO: 7 (2559 bp), where positions 1-152, 431-560, 652-789, 893-1409, and 2387-2559 are exons, and the rest are introns. Its specific nucleotide sequence is as follows:

[0066] The amino acid sequence encoded by the wheat TaNRK-4D gene is the protein TaNRK-4D, which is sequence 8 (SEQ ID NO: 8,369aa) in the sequence listing, as follows: MDGAPVAEFRPTMTHGGRFLLYNIFGNQFEITAKYQPPIMPIGRGAYGIVCSVMNFETREMVAIKKIANAFDNNMDAKRTLREIKLLRHLDHENIVGLRDVIPPATPQSFNDVYIATELMDTDLHHIIRSNQELSEEHCQYFLYQLLRGLKYIHSANVIHRDLKPSNLLLNANCDLKICDFGLAR PSSESDMMTEYVVTRWYRAPELLLNSTDYSAAIDVWSVGCIFMELINRAPLFPGRDHMHQMRLITEVIGTPTDDDLGFIRNEDARRYMRHLPQFPRRSFPGQFPKVQPAALDLIERMLTFNPLQRITVEEALEHPYLERLHDVADEPICTDPFSFDFEQHPLTEDQMKQLIFNEALELNPNFRY.

[0067] The coding sequence (CDS) for the protein TaNRK-4D is SEQ ID NO: 9 (1110bp), as follows:

[0068] Based on the TaNRK genome gene in wheat genomes A, B, and D, gene editing gRNA sequences were designed: sgRNA1: 5'-GGTGATGAACTTCGAGACGAGGG-3' (SEQ ID NO: 12) targets positions 446-468 of SEQ ID NO: 1, positions 438-460 of SEQ ID NO: 4, and positions 434-456 of SEQ ID NO: 7; sgRNA2: 5'-CCCGCAGTCCTTCAACGACGTCT-3' (SEQ ID NO: 13) targets positions 703-725 of SEQ ID NO: 1, positions 702-724 of SEQ ID NO: 4, and positions 687-709 of SEQ ID NO: 7.

[0069] 1.2 Low nitrogen levels inhibit TaNRK4D expression 1.2.1 Hydroponic treatment Hydroponics was conducted in the laboratory hydroponic room in January 2025. The hydroponic treatment involved selecting uniformly sized and plump wheat seeds, soaking them overnight in a 1% hydrogen peroxide solution, and rinsing them with distilled water to remove residual hydrogen peroxide. The seeds were then neatly arranged on a seedbed saturated with calcium sulfate. Once the seeds germinated and grew to approximately 10 cm in height, the uniformly growing seedlings were selected, residual endosperm was removed, and they were transplanted into a solution containing 2 mM NO3. - (High nitrogen treatment) and containing 0.2 mM NO3 - Samples were taken after two weeks of growth in the (low-nitrogen treatment) nutrient solution. Each hydroponic container had 24 cells, with 2 plants per cell, representing one replicate per cell, and 4-6 replicates per line. Growth conditions were set at 20°C. 2°C, relative humidity 50%-60%, light intensity 150-200 μmol·cm -2 ·s -1 The seedlings were exposed to 16 hours of light and 8 hours of darkness, with the nutrient solution changed every two days. All seedling nutrient solution culture experiments in this study used a uniform nutrient solution formula (Table 1).

[0070] Table 1. Solute formulation in nutrient solution during wheat seedling stage

[0071] 1.2.2 Field treatment During the 2024-2025 wheat growing season, the Zhaoxian Experimental Station of the Shijiazhuang Academy of Agricultural and Forestry Sciences in Hebei Province established high-nitrogen and low-nitrogen treatments (field treatments). The high-nitrogen treatment was based on 12 g N / m³. 2 Apply base fertilizer, and during the jointing stage, apply 6g N / m 2Topdressing was applied; no nitrogen fertilizer was applied for the low-nitrogen treatment. KN199 was sown in autumn in 2024 with a row length of 2 m, a row spacing of 23 cm, and a plant spacing of 10 cm. Three biological replicates were set up for each material, and four rows were sown for each replicate.

[0072] 1.2.3 Detection of TaNRK4D gene expression level At maturity, aboveground samples (1.2.1) and root system samples (1.2.2) were collected for RT-qPCR analysis, with the TaActin gene used as an internal reference gene.

[0073] The primers for amplifying the TaNRK4D gene are: RT-TaNRK4D-F: 5'-CGGGAGATCAAGCTCCTG-3' (SEQ ID NO: 14); RT-TaNRK4D-R: 5'-CGTCATCATGTCGCTCTCG-3' (SEQ ID NO: 15); The primers for amplifying the TaActin gene are: RT-TaActin-F: 5'-ACCTTCAGTTGCCCAGCAAT-3' (SEQ ID NO: 16); RT-TaActin-R: 5'-8-3' (SEQ ID NO: 17).

[0074] Experimental results are as follows Figure 1 As shown, whether it is hydroponic treatment ( Figure 1 (A) or field treatment ( Figure 1 (B) In low-nitrogen environments, whether in the aboveground parts or the root system TaNRK4D Their expressions were suppressed.

[0075] 2. Nitrates can induce TaNRK4D expression Select wheat seedlings with uniform growth, remove the endosperm, and pre-culture them in a semi-nutrient solution for 2 days. Then, transfer them to a full nutrient solution for 2 days. Finally, transplant the seedlings into a 2 mM NH4+ solution. + They were cultured in a nutrient solution for 3 days, and then transferred to an environment with nitrate nitrogen as the sole nitrogen source (2 mM NJO3). - In the nutrient solution, samples were taken at time points (0 h, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, and 72 h), with three biological replicates at each time point. The expression level of the TaNRK4D gene was detected using method 1.2.3, and the expression level of the TaNRT2.1 gene was detected using method 3.4. The results showed that at 5 min of nitrate induction... TaNRK4DThe expression level of [the gene] was significantly downregulated by 4-fold, then reached its lowest level within 15 minutes, and rapidly upregulated by 2-fold after 2 hours, followed by a slow increase back to the initial level; the expression level of the TaNRT2.1 gene increased after nitrate induction, peaked at 12 hours, and then decreased. Figure 2 ).

[0076] 3. TaNRK Biological function identification To further study the function of TaNRK, a CRISPR / Cas9-mediated TaNRK knockout line of KN199 was created.

[0077] 3.1 TaNRK Knockout System Material Creation 3.1.1 Carrier Construction The recombinant expression vector pYLCRISPR / Cas9-TaNRK is obtained by inserting the DNA fragment (SEQ ID NO: 10, 933bp) shown in the TaNRK-sgRNA expression cassette between the BsaI restriction sites of the pYLCRISPR / Cas9 plasmid, while keeping other nucleotide sequences unchanged. pYLCRISPR / Cas9-TaNRK expression targets the sgRNAs (sgRNA1 and sgRNA2) of the wheat TaNRK gene and the TaNRK gene knockout vector of the Cas9 gene. In SEQ ID NO: 10, positions 1-363 represent the promoter TaU6, positions 364-382 represent the sgRNA1 gene, positions 383-458 represent gRNAscaffold, positions 459-468 represent polyT, positions 469-831 represent a repeat of the promoter TaU6, positions 832-850 represent the sgRNA2 gene, and positions 851-926 represent a repeat of gRNAscaffold, as detailed below: 5' -GACCAAGCCCGTTATTCTGACAGTTCTGGTGCTCAACACATTTATATTTATCAAGGAGCACATTGTTACTCACTGCTAGGAGGGAATCGAACTAGGAATATTGATCAGAGGAACTACGAGAGAGCTGAAGATAACTGCCCTCTAGCTCTCACTGATCTGGGTCGCATAGTGAGATGCAGCCCACGTGAGTTCAGCAACGGTCTAGCGCTGGGCTTTTAGGCCCGCATGATCGGGCTTTTGTCGGGTGGTCGACGTGTTCACGATTGGGGAGAGCAACGCAGCAGTTCCTCTTAGTTTAGTCCCACCTCGCCTGTCCAGCAGAGTTCTGACCGGTTTATAAACTCGCTTGCTGCATCAGACTTGGTGATGAACTTCGAGACGAgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttTCCCGACCAAGCCCGTTATTCTGACAGTTCTGGTGCTCAACACATTTATATTTATCAAGGAGCACATTGTTACTCACTGCTAGGAGGGAATCGAACTAGGAATATTGATCAGAGGAACTACGAGAGAGCTGAAGATAACTGCCCTCTAGCTCTCACTGATCTGGGTCGCATAGTGAGATGCAGCCCACGTGAGTTCAGCAACGGTCTAGCGCTGGGCTTTTAGGCCCGCATGATCGGGCTTTTGTCGGGTGGTCGACGTGTTCACGATTGGGGAGAGCAACGCAGCAGTTCCTCTTAGTTTAGTCCCACCTCGCCTGTCCAGCAGAGTTCTGACCGGTTTATAAACTCGCTTGCTGCATCAGACTTGGCAGTCCTTCAACGACGTCgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc-3'.

[0078] 3.1.2 Construction of gene knockout plants pYLCRISPR / Cas9-TaNRK was transformed into Agrobacterium tumefaciens EHA105 competent cells to obtain recombinant Agrobacterium EHA105-pYLCRISPR / Cas9-TaNRK. Wheat variety KN199 was infected with EHA105-pYLCRISPR / Cas9-TaNRK to obtain wheat plants with the pseudo-TaNRK gene edited. Seeds from the T0 generation plants were harvested (T0 generation seeds). These T0 generation seeds were then planted and self-pollinated to obtain T1 generation seeds. T1 generation seedlings were then obtained and tested.

[0079] 3.1.3 Detection of gene knockout plants The plants obtained in step 2 were tested using the following method: Genomic DNA was extracted from the plants using the CTAB method, and PCR was performed using the following primers to identify the mutation status of genomes A, B, and D, respectively: TaNRK-4A-F-cr: 5'-GTAGTCGGTGGAGTTGAGCA-3' (SEQ ID NO: 18); TaNRK-4A-R-cr: 5'- ACGCACAGAAAAGAGCCAGA-3' (SEQ ID NO: 19); TaNRK-4B-F-cr: 5'- CCATCGATTTCCTGCCAAGC-3' (SEQ ID NO: 20); TaNRK-4B-R-cr: 5'- CAAGAACACCACGGCACATG-3' (SEQ ID NO: 21); TaNRK-4D-F-cr: 5'-CAAGAACACCACGTCGCATG-3' (SEQ ID NO: 22); TaNRK-4D-R-cr: 5'-CGTTCCCGCCCGTCTTAATTT-3' (SEQ ID NO: 23); The T1 generation homozygous gene knockout plants KO-1, KO-6, and KO-10 were identified, and the gene editing details are as follows: Compared to wild-type wheat Kenong 199, the KO-1 genome has a deletion of 271 nucleotides in positions 893-1164 of SEQ ID NO:7 (corresponding to positions 421-692 of SEQ ID NO:9). This deletion causes a frameshift in the amino acid translation after the edited site, resulting in premature termination of the protein sequence and thus knocking out the TaNRK-4D gene. The A and B genomes of KO-1 remain unchanged.

[0080] Compared to wild-type wheat Kenong 199, KO-6 has the following differences: The A genome lacks nucleotide T at position 62 of SEQ ID NO:1 and nucleotides 1190-1211 of SEQ ID NO:1 (corresponding to positions 62 and 707-728 of SEQ ID NO:3); the B genome has an insertion of a T base between positions 989-990 of SEQ ID NO:4 and a deletion of 3 TCT bases at positions 1233-1235 (corresponding to positions 463-464 and 707-709 of SEQ ID NO:6); the D genome has an insertion of A between positions 922-923 of SEQ ID NO:7 (corresponding to positions 450-451 of SEQ ID NO:9) and a deletion of nucleotides 1164-1165 (corresponding to positions 62 and 707-728 of SEQ ID NO:3). A T base is inserted between positions 692-693 of IDNO:9, resulting in a frameshift mutation that causes premature termination of translation, thereby knocking out the TaNRK-4A, TaNRK-4B, and TaNRK-4D genes.

[0081] Compared to wild-type wheat Kern 199, KO-10's A genome has a deletion of 253 bases at positions 942-1194 of SEQ ID NO:1 (corresponding to positions 459-711 of SEQ ID NO:3); the B genome has an insertion of 253 bases between positions 454-455 of SEQ ID NO:4 (corresponding to positions 172-173 of SEQ ID NO:6) TGCGGGATCGCCGGCGGGATCACATCTCGGAGGCCTACTATCTGCGTGTGATGTCCGTCGATACAGCGTATCAGGTTAGCGCCTGAATTGCCGAATTCAGTCAGATACAAACATCTGAAGCGGAAGAGAAAGATGTTGATTACGTTCTCGTGGTCGAGGTGCCTGAGGAGCTTGATCTCCCGGAGCGTGCGCTTGGCGTCCATGTTGTTGTCGAAGGCGTTTGCGATCTTCTTGATTGCCACCATCTCCCTCG (SEQ ID NO:24), and the D genome has a deletion of 253 bases at positions 942-1194 of SEQ ID NO:1 (corresponding to positions 459-711 of SEQ ID NO:3); the D genome has a deletion of 253 bases between positions 454-455 of SEQ ID NO:4 (corresponding to positions 172-173 of SEQ ID NO:6) TGCGGGATCGCCGGGAATTCGCCGGGAATTCAGTCGCCGGGAATTCAGTCGCCTCGGGAGCGTGCTTGATCTCCCCGGGAGCGTGCTTGGCGTCCATGTTGTTGTCGAAGGCGTTTGCGATCTTCTTGATTGCCACCATCTCCCTCGGGGAATTCAGTCGCCGGGAATTCAGTCGCCTCGGGAATTCAGTCGCCTCGGGAATTCAGTCGCCTCGGGAATTCAGTCGCCTCGGGAATTCAGTCAGTCGCCTCGGGAATTCAG A frameshift mutation occurs when 252 bases are deleted between positions 451-692 of ID NO:7 (corresponding to positions 173-323 of SEQ ID NO:9), causing premature termination of translation and thus knocking out the TaNRK-4A, TaNRK-4B, and TaNRK-4D genes.

[0082] To clarify TaNRKThe impact on wheat yield at maturity was investigated by harvesting seeds from T1 generation homozygous knockout plants KO-1, KO-6, and KO-10 (T2 generation seeds), resulting in three TaNRK knockout lines: KO-1, KO-6, and KO-10. During the 2024-2025 wheat growing season, a field sowing experiment was conducted at the Zhaoxian Experimental Station of the Shijiazhuang Academy of Agricultural and Forestry Sciences in Hebei Province to compare the yield traits of the KO-1, KO-6, and KO-10 lines with wild-type wheat KN199 under normal and low nitrogen treatments. The normal nitrogen treatment was based on 12 g N / m³. 2 Apply base fertilizer, and during the jointing stage, apply 6g N / m 2 Topdressing was applied; no nitrogen fertilizer was applied in the low-nitrogen treatment, and all other operations were the same. Autumn sowing was carried out in 2024, with a row length of 2 m, a row spacing of 23 cm, and a plant spacing of 10 cm. Each line and each treatment (normal nitrogen treatment and low-nitrogen treatment) was set up with 3 biological replicates, and each replicate was sown in 4 rows.

[0083] The results showed that, regardless of whether the nitrogen application was normal or low, the KO-1, KO-6, and KO-10 lines significantly increased the number of spikes and the yield per plant compared to the wild type, as detailed below: Under normal nitrogen application conditions (normal nitrogen treatment), the KO-1, KO-6, and KO-10 lines (corresponding to...) Figure 3 Normal nitrogen KO-1, normal nitrogen KO-6, and normal nitrogen KO-10) compared to wild type (corresponding to Figure 3 The number of spikes (in the normal nitrogen control) increased from 12.07±1.53 to 13.13±1.06, 13.2±1.21, and 13.27±0.96, respectively, representing increases of 8.84%, 9.39%, and 9.94%. Figure 3 (A)

[0084] Under low nitrogen conditions (low nitrogen treatment), the KO-1, KO-6, and KO-10 lines (corresponding to...) Figure 3 Low-nitrogen KO-1, low-nitrogen KO-6, and low-nitrogen KO-10) are derived from wild-type (corresponding to Figure 3 The number of spikes in the low-nitrogen control increased from 7.27±1.53 to 8.47±0.99, 8.6±0.99, and 8.67±1.11, respectively, representing increases of 16.51%, 18.35%, and 19.27%. Figure 3 (A)

[0085] Under normal nitrogen application conditions (normal nitrogen treatment), the yield per plant of KO-1, KO-6, and KO-10 lines increased from 22.29±2.38 g to 23.85±1.45 g, 24.23±1.76 g, and 24.26±1.44 g, respectively, representing increases of 6.98%, 8.71%, and 8.85% compared to the wild type. Figure 3 (B)

[0086] Under low nitrogen conditions (low nitrogen treatment), the yield per plant of KO-1, KO-6, and KO-10 lines increased from 14.74±1.9 g to 16.63±1.49 g, 17.04±1.68 g, and 17.31±1.94 g, respectively, representing increases of 12.83%, 15.60%, and 17.46% compared to the wild type. Figure 3 (B)

[0087] 3.2 TaNRK The expression of primary nitrate response genes was suppressed. To clarify TaNRK To investigate how primary nitrate response genes are affected, aboveground samples of the Tanrk tri-mutant homozygous line KO-10 were collected at maturity at the Zhaoxian Experimental Station of the Shijiazhuang Academy of Agricultural and Forestry Sciences in Hebei Province during the 2024-2025 wheat growing season and analyzed by rt-qPCR. The results showed that compared with wild-type KN199, the expression levels of TaNRT2.1, TaNR, and TaGOGAT were significantly upregulated in the tri-mutant homozygous KO-10 line. Figure 4 The primer sequences used are shown below: RT-TaNRT2.1-F: 5'-GTGGTGCTGTATTTGGCGTCG-3' (SEQ ID NO: 25); RT-TaNRT2.1-R: 5'-AAGAAGTTGCGTTAGCCCTGC-3' (SEQ ID NO: 26); RT-TaNR2-F: 5'-AGCAGTTCACCATGTCCGAG-3' (SEQ ID NO: 27); RT-TaNR2-R: 5'-CAGTCGGATCCCGCATTGAT-3' (SEQ ID NO: 28); RT-TaGOGAT-F: 5'-GCGGAAGTGCCATACCAATAC-3' (SEQ ID NO: 29); RT-TaGOGAT-R: 5'-AAAGTTAATGACATGCTCTGGTTCTC-3' (SEQ ID NO: 30).

[0088] 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 proteins, characterized in that, The protein is TaNRK-4D and / or TaNRK-4AB. The TaNRK-4AB is any one of the following: A1) The amino acid sequence of this protein is SEQ ID NO:

2. A2) A protein with more than 70% amino acid sequence identity and the same function as A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence of A1). A3) A fusion protein obtained by attaching a tag to the end of the protein in A1); The TaNRK-4D is any one of the following: D1) The amino acid sequence of this protein is that of SEQ ID NO:

8. D2) A protein with more than 70% amino acid identity and the same function as the amino acid sequence of D1) obtained by substituting and / or deleting and / or adding amino acid residues. D3) is a fusion protein obtained by attaching a tag to the end of the protein in D1); The application is any one of the following: Application of F1 in regulating plant agronomic traits; Application of F2 in the preparation of products that regulate plant agronomic traits; Application of F3 in the cultivation of plants with altered agronomic traits; F4) Application in the preparation of products from plants with altered agronomic traits; Application of F5 in plant breeding.

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

3. The application of biomaterials, characterized in that, The biomaterial is either biomaterial B or biomaterial C; The biomaterial B is any one of the following: B1) A nucleic acid molecule that targets the gene encoding the protein of claim 1 or 2; B2) Cas protein and B1) the nucleic acid molecule described; B3) Gene, which is the gene encoding the nucleic acid molecule described in B1) and the gene encoding the Cas protein; B4) Expression cassettes, vectors, recombinant microorganisms, or plants containing the genes described in B3); The biomaterial C is any one of the following: C1) A nucleic acid molecule that inhibits, reduces, or silences the expression of the gene encoding the protein of claim 1 or 2; C2) is the gene encoding the nucleic acid molecule described in C1); C3) Expression cassettes, recombinant vectors, recombinant microorganisms, or plants containing the gene described in C2); The application is any one of the following: E1) Application in regulating plant agronomic traits; E2) Application in the preparation of products that regulate plant agronomic traits; E3) Application in the cultivation of plants with altered agronomic traits; E4) Application in the preparation of products from plants with altered agronomic traits; Application of E5 in plant breeding.

4. The application according to claim 1 or 3, characterized in that, The agronomic traits are yield and / or number of wheat ears.

5. The application according to claim 4, characterized in that, The plant is any one of the following: G1) Dicotyledons, G2) Monocotyledons, G3) Plants of the order Poales, G4) Gramineae plants, G5) Wheat genus plants, G6) Wheat.

6. The method, characterized in that, The method is M1, M2, M3, or M4: M1. A method for increasing yield and / or number of wheat ears, the method comprising knocking out the coding gene of a wheat to be improved containing the coding gene of the protein of claim 1 to obtain gene knockout wheat, wherein the yield and / or number of ears of the gene knockout wheat is higher than that of the wheat to be improved. M2. A method for cultivating wheat with increased spike number and / or yield, the method comprising knocking out the coding gene of a wheat to be improved containing the coding gene of the protein of claim 1 to obtain wheat with increased spike number and / or yield, wherein the spike number and / or yield of the wheat with increased spike number and / or yield is higher than that of the wheat to be improved. M3. A method for increasing the number of wheat ears and / or yield, the method comprising reducing the activity and / or content of the protein described in claim 1 in the wheat to be improved, and / or reducing the expression level of the gene encoding the protein described in claim 1, to increase the number of wheat ears and / or yield of the wheat to be improved; M4. A method for cultivating wheat with increased spike number and / or yield, the method comprising reducing the activity and / or content of the protein of claim 1 in the wheat to be improved, and / or reducing the expression level of the gene encoding the protein of claim 1, to obtain improved wheat, wherein the improved wheat has a higher spike number and / or yield than the wheat to be improved.

7. The method according to claim 6, characterized in that, M1 and M2 include the step of introducing the biomaterial of claim 3 into the wheat to be improved.

8. Gene-edited wheat, characterized by, The gene-edited wheat is wheat that does not contain the gene encoding the protein described in claim 1.

9. The gene-edited wheat according to claim 8, characterized in that, The gene-edited wheat is gene-knockout wheat obtained by knocking out the coding gene of the wheat to be improved, which contains the coding gene of the protein described in claim 1.

10. The gene-edited wheat according to claim 8 or 9, characterized in that, The yield and / or number of ears of the gene-edited wheat are higher than those of the wheat to be improved.

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