Application of wheat tabhlh93 protein or its coding gene in regulating grain development of plants

Abstract

This invention belongs to the field of plant genetic engineering and molecular breeding technology, specifically relating to the application of wheat TabHLH93 protein or its encoding gene in regulating plant grain development. This invention constructs a structure capable of ectopic expression... TabHLH93 A binary expression vector for the gene was genetically transformed into common wheat. TabHLH93 Offspring with high gene expression achieved TabHLH93 The gene was overexpressed and verified, demonstrating that TabHLH93 regulates wheat grain size and plays a significant role in regulating agronomic traits such as plant height, tiller number, spike length, and thousand-grain weight. This provides a promising candidate gene for high-yield genetic improvement of wheat, which is of great importance for ensuring food security.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular breeding technology, specifically involving the application of wheat TabHLH93 protein or its encoding gene in regulating plant grain development. Background Technology

[0002] wheat( Triticum aestivum L. (2n=6x=42, AABBDD), as one of the three staple foods for humans, provides 25% of protein and 20% of carbohydrates. However, with the continuous growth of the global population, urbanization, land degradation leading to a reduction in arable land, and global climate change, food security faces enormous challenges. The yield of major food crops is no longer sufficient to meet the ever-increasing food demand. By 2034, wheat production needs to increase by at least 50% to meet global needs. Therefore, identifying key superior alleles and developing high-yield wheat varieties is of great significance for ensuring my country's food security.

[0003] Wheat yield is mainly determined by three dimensions: number of spikes per unit area, number of grains per spike, and thousand-grain weight. Among these, thousand-grain weight is determined by both grain morphology and filling degree. Grain morphology includes grain length, width, and thickness. Currently, there are six main biological pathways that regulate wheat grain size: the G protein-mediated pathway, the ubiquitin-proteasome pathway, the mitogen-activated protein kinase (MAPK) pathway, the plant hormone regulation pathway, the transcription factor regulation pathway, and the IKU pathway that regulates the endosperm cellification process.

[0004] Brassinosteroids (BRs) are the sixth largest plant hormone and a major plant growth promoter, playing a crucial role in regulating biological processes such as photomorphogenesis, cell elongation, flowering time, root formation, and nutrient signaling. Both BR signaling biosynthesis and signal transduction affect important agronomic traits such as panicle number and grain size. In rice, overexpression of the BR signaling co-receptor OsBAK1 promotes grain enlargement; overexpression of the key kinase OsGSK3 leads to dwarfing and reduced tiller number, while overexpression of the downstream key transcription factor OsBZR1 promotes larger grain morphology. In wheat, the TaGL3-5A-G allele of the OsPPKL1 homolog is significantly associated with longer grains and higher thousand-grain weight, while the TaGL3.3-5B-C allele of the OsPPKL3 homolog promotes wheat yield. The genetic module of ZnF, a positive regulator of the BR signaling pathway, and Rht-B1, a negative regulator of the GA signaling pathway, mediates the balance of BR and GA hormones, which helps to breed short-stalked, lodging-resistant, high-yielding wheat varieties with efficient nitrogen utilization.

[0005] Currently, there is limited research on the functional regulatory molecules in the BR signaling pathway that affect grain size and yield. Therefore, developing key regulatory factors in the BR signaling pathway is of great significance for promoting wheat grain development and cultivating high-yield wheat. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of wheat TabHLH93 protein or its encoding gene in regulating plant grain development.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the invention provides the use of wheat TabHLH93 protein or its encoding gene in regulating plant grain development, wherein the wheat TabHLH93 protein is a protein as shown in (a1) or (a2) below:

[0009] (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;

[0010] (a2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (a1).

[0011] A second aspect of the invention provides the use of wheat TabHLH93 protein in any of the following:

[0012] (c1) Increase plant yield and / or number of tillers and / or ear length and / or ear weight and / or number of grains per ear and / or thousand-grain weight and / or plant height;

[0013] (c2) Plant breeding;

[0014] The wheat TabHLH93 protein is the protein shown in (a1) or (a2) below:

[0015] (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;

[0016] (a2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (a1);

[0017] The plant is selected from at least one of rice, corn, soybean, sunflower, sorghum, wheat, alfalfa, cotton, barley and millet.

[0018] A third aspect of the invention provides the use of a recombinant DNA construct in regulating plant seed development, said recombinant DNA construct comprising a DNA molecule operably linked to at least one heterologous regulatory sequence as shown in (b1) or (b2) below:

[0019] (b1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2;

[0020] (b2) DNA molecules other than (b1) that encode the amino acid sequence shown in SEQ ID NO: 1.

[0021] A fourth aspect of the present invention provides the use of a recombinant DNA construct in any of the following:

[0022] (c1) Increase plant yield and / or number of tillers and / or ear length and / or ear weight and / or number of grains per ear and / or thousand-grain weight and / or plant height;

[0023] (c2) Plant breeding;

[0024] The recombinant DNA construct comprises a DNA molecule operably linked to at least one heterologous regulatory sequence, as shown in (b1) or (b2) below:

[0025] (b1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2;

[0026] (b2) DNA molecules that encode the amino acid sequence shown in SEQ ID NO: 1, except for (b1);

[0027] The plant is selected from at least one of rice, corn, soybean, sunflower, sorghum, wheat, alfalfa, cotton, barley and millet.

[0028] A fifth aspect of the invention provides a method for increasing plant seed size, comprising the steps of: (d1) introducing a recombinant DNA construct into regenerable plant cells, the recombinant DNA construct comprising a DNA molecule operatively linked to at least one regulatory sequence; (d2) regenerating a transgenic plant from the regenerable plant cells following step (d1), wherein the transgenic plant contains the recombinant DNA construct in its genome; and (d3) obtaining progeny plants derived from the transgenic plant of step (d2), wherein the progeny plants contain the recombinant DNA construct in their genome and exhibit increased seed size compared to a control plant;

[0029] The DNA molecule is as shown in (b1) or (b2) below:

[0030] (b1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2;

[0031] (b2) DNA molecules other than (b1) that encode the amino acid sequence shown in SEQ ID NO: 1.

[0032] A sixth aspect of the invention provides a method for increasing plant yield and / or tiller number and / or ear length and / or ear weight and / or ear grain number and / or thousand-grain weight and / or plant height, comprising the following steps: (e1) introducing a recombinant DNA construct into regenerable plant cells, the recombinant DNA construct comprising a DNA molecule operatively linked to at least one regulatory sequence; (e2) regenerating a transgenic plant from the regenerable plant cells following step (e1), wherein the transgenic plant contains the recombinant DNA construct in its genome; and (e3) obtaining progeny plants derived from the transgenic plant of step (e2), wherein the progeny plants contain the recombinant DNA construct in their genome and exhibit increased yield and / or tiller number and / or ear length and / or ear weight and / or ear grain number and / or thousand-grain weight and / or plant height compared to a control plant;

[0033] The DNA molecule is as shown in (b1) or (b2) below:

[0034] (b1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2;

[0035] (b2) DNA molecules other than (b1) that encode the amino acid sequence shown in SEQ ID NO: 1.

[0036] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0037] This invention is the first to discover that TabHLH93 is a key transcription factor downstream of the BR signaling pathway, which regulates wheat grain size and plays an important regulatory role in agronomic traits such as wheat plant height, tiller number, spike length, and thousand-grain weight. Through the construction of a structure that can express this transcription factor ectopically... TabHLH93 A binary expression vector for the gene was genetically transformed into common wheat. TabHLH93 The offspring with high gene expression achieved overexpression of the TabHLH93 gene, and this was verified. TabHLH93 High expression of this compound can promote increased wheat plant height and spike length, increase the number of tillers and thousand-grain weight, and ultimately improve wheat yield. It provides a practical method for promoting wheat grain development and increasing wheat yield, possessing significant breeding application value and broad market application prospects. Attached Figure Description

[0038] Figure 1 As described in the embodiments of the present invention TabHLH93 Gene amplification electrophoresis image. In the image, lane M is the Transgen 2K marker, and lane 1... # and 2 # For the purpose of the strip;

[0039] Figure 2 As described in the embodiments of the present inventionTabHLH93 Gene-linked entry cloning vector pENTRY The PCR identification results are shown in the figure. Lane M is the Transgen 2K plus marker, and lane 1 is... # Up to 24 # Electrophoresis results of PCR for single-clone bacterial cultures;

[0040] Figure 3 As described in the embodiments of the present invention TabHLH93 Genes are ligated into binary expression vectors via the LR response. pLGY02-YFP In the image, lane M is the Transgen 2K plus marker, and lane 1... # Up to 11 # for TabHLH93 Successful connection pLGY02- YFP Positive clones of the vector;

[0041] Figure 4 As described in the embodiments of the present invention OE-TabHLH93-1 # PCR identification of transgenic offspring, lane M is the Transgen2K marker, lane 1 # up to 14 # The results of the target band detection for the transgenic strain;

[0042] Figure 5 Overexpression in the embodiments of the present invention TabHLH93 The expression levels of transgenic offspring were identified, and the different letters above the bar chart indicated statistically significant differences between groups (Owo-way ANOVA followed by uncorrected Fisher's LSD multiple comparisons test, p<0.05).

[0043] Figure 6 As described in the embodiments of the present invention TabHLH93 Comparison of seed size and epidermal cell length and width in overexpression lines, where A represents... TabHLH93 Comparison of seed size between overexpression lines and wild-type plants, B is... TabHLH93 A bar chart comparing the seed length of overexpression lines and wild-type plants, where C represents... TabHLH93 A bar chart comparing the grain width of overexpression lines and wild-type plants, where D represents... TabHLH93 Comparison of seed epidermal cell length between overexpression lines and wild-type plants, scale bar: 20 μm, E: TabHLH93 A bar chart comparing the epidermal cell length of seeds from overexpression lines and wild-type plants, where F represents... TabHLH93A bar chart comparing the length of epidermal cells in seeds from overexpression lines and wild-type plants; different letters above the bars indicate statistically significant differences between groups (Owo-way ANOVA followed by uncorrected Fisher's LSD multiple comparisons test). p <0.05);

[0044] Figure 7 As described in the embodiments of the present invention OE-TabHLH93 Comparison of agronomic traits of overexpression lines, where A is... OE- TabHLH93 Comparison of phenotypes of overexpressing transgenic lines and wild-type plants, B is... OE-TabHLH93 A bar chart comparing the plant height of overexpressing transgenic lines and wild-type plants, where C represents... OE-TabHLH93 A bar chart comparing the number of tillers in overexpressing transgenic lines and wild-type plants, where D represents... OE-TabHLH93 A bar chart comparing the spike length of overexpressing transgenic lines with that of wild-type plants, where E represents... OE-TabHLH93 A bar chart comparing the number of spikelets in overexpressing transgenic lines and wild-type plants, where F represents... OE-TabHLH93 A bar chart comparing the thousand-grain weight of overexpressing transgenic lines and wild-type plants; different letters above the bars indicate statistically significant differences between groups (Owo-way ANOVA followed by uncorrected Fisher's LSD multiple comparisons test). p <0.05); Scale bar = 10 cm. Detailed Implementation

[0045] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0046] As used herein, the singular forms “a / species” and “described” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “plant” includes multiple such plants; reference to “cell” includes one or more cells and their equivalents known to those skilled in the art, and so on.

[0047] As used in this article:

[0048] The terms "monocot" and "monocotyledonous plant" are used interchangeably herein. The monocots disclosed herein include plants of the Poaceae family.

[0049] The terms "dicot" and "dicotyledonous plant" are used interchangeably herein. The dicotyledonous plants disclosed herein include the following families: Brassicaceae, Fabaceae, and Solanaceae.

[0050] The terms “full complement” and “full-length complement” are used interchangeably in this document and refer to the complementary sequence of a given nucleotide sequence, wherein the complementary sequence and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary. An “expressed sequence tag” (“EST”) is a DNA sequence derived from a cDNA library and therefore represents a transcribed sequence. ESTs are typically obtained by single-pass sequencing of the cDNA insert. The sequence of the entire cDNA insert is called the “full-length insert”. A “contig” sequence is a sequence assembled from two or more sequences, optionally from, but not limited to, ESTs, FISs, and PCR sequences. A sequence encoding a whole or functional protein is called a “complete gene sequence” and can be derived from FISs or contigs.

[0051] The term "trait" refers to the physiological, morphological, biochemical, or physical characteristics of a plant or a particular plant material or cell. In some cases, this trait is visible to the human eye, such as seed or plant size, or it can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seeds or leaves, or by observing metabolic or physiological processes, such as by measuring tolerance to water deprivation or specific salt, sugar, or nitrogen concentrations, or by observing the expression levels of one or more genes.

[0052] The term "agronomic traits" refers to measurable parameters, including but not limited to grain size, grain yield, growth rate, total biomass or accumulation rate, fresh weight at maturity, dry weight at maturity, fruit yield, grain yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in vegetative tissues, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in vegetative tissues, total plant protein content, fruit protein content, seed protein content, protein content in vegetative tissues, drought tolerance, nitrogen uptake, root lodging, harvest index, stem lodging, plant height, ear height, ear length, salt tolerance, number of tillers, panicle size, early seedling vigor, and emergence under low temperature stress.

[0053] The increase in biomass can be measured, for example, as an increase in plant grain size, plant height, plant ear length, number of ears, number of tillers, total leaf area, or plant fresh or dry weight compared to control plants. Increases in grain size, ear length, number of ears, and number of tillers can be particularly significant and can be used to increase yield.

[0054] The term “transgenic” refers to any cell, cell line, callus, tissue, plant part, or plant whose genome has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct. As used herein, the term “transgenic” includes those initial transgenic events and those resulting from those events through sexual hybridization or asexual reproduction, and does not cover genomic (chromosomal or extrachromosomal) alterations made through conventional plant breeding methods or through naturally occurring events such as random cross-fertilization, infection with a non-recombinant virus, transformation by a non-recombinant bacteria, non-recombinant transposition, or spontaneous mutation.

[0055] The terms "control" or "control plant" or "control plant cell" provide a reference point for measuring phenotypic changes in a subject plant or plant cell in which a genetic alteration has been achieved, such as through transformation, to target a gene for a specific purpose, and which has been affected. The subject plant or plant cell may be inherited from a plant or cell that has undergone such alteration and will contain that alteration.

[0056] Control plants or plant cells may include, for example: (a) wild-type plants or cells, i.e., plants or cells having the same genotype as the starting material used to perform the genetic alteration that would result in the subject plant or cell; (b) plants or plant cells having the same genotype as the starting material but transformed with a null construct (i.e., a construct that does not have a known effect on the target trait, such as a construct containing a marker gene); (c) plants or plant cells that are non-transformed segregants in the progeny of the subject plant or plant cell; (d) plants or plant cells that are genetically identical to the subject plant or plant cell but are not exposed to conditions or stimuli that would induce the expression of the target gene; or (e) the subject plant or plant cell itself under conditions in which the target gene is not expressed.

[0057] When the genome is applied to plant cells, it not only covers chromosomal DNA present in the cell nucleus, but also organelle DNA present in subcellular components of the cell (such as mitochondria and plastids).

[0058] "Plant" includes indexing of whole plants, plant organs, plant tissues, seeds, and plant cells, as well as their progeny. Plant cells include, but are not limited to, cells derived from seeds, suspension cultures, plumules, meristematic regions, callus, leaves, roots, seedlings, gametophytes, sporophytes, pollen, and microspores.

[0059] "Offspring" includes any subsequent generations of a plant.

[0060] "Transgenic plants" refers to plants that contain heteropolynucleotides within their genome. For example, a heteropolynucleotide is stably integrated into the genome, allowing it to be passed down through successive generations. Heteropolynucleotides can be integrated into the genome alone or as part of a recombinant DNA construct. T0 plants are recovered directly from transformation and regeneration. The offspring of T0 plants are called T1 (first generation), T2 (second generation), etc.

[0061] The term "heterogeneous" in relation to a sequence refers to a sequence that originates from an alien species, or, if it originates from the same species, a sequence that has been substantially modified in its natural form in terms of composition and / or loci through deliberate human intervention.

[0062] "Recombinant" refers to an artificial combination of two originally separate sequence segments, achieved, for example, through chemical synthesis or by manipulating isolated nucleic acid segments using genetic engineering techniques. "Recombinant" also includes cells or vectors that have been modified by introducing heterologous nucleic acids, or derived from such modified cells, but does not cover changes to cells or vectors caused by naturally occurring events (e.g., spontaneous mutations, natural transformation / transduction / transposition), such as those that occur without intentional human intervention.

[0063] A "recombinant DNA construct" refers to a combination of nucleic acid fragments that do not normally exist together in nature. Therefore, recombinant DNA constructs can contain regulatory and coding sequences from different sources, or regulatory and coding sequences from the same source but arranged in a manner different from those found in nature.

[0064] The terms “introductory clone” and “introductory vector” are used interchangeably in this article.

[0065] "Regulatory sequence" refers to a nucleotide sequence located upstream (5′ non-coding sequence), inside, or downstream (3′ non-coding sequence) of a coding sequence that affects the transcription, RNA processing, or stability of the associated coding sequence, or translation. Regulatory sequences may include, but are not limited to, promoters, translational leader sequences, introns, and polyadenylation recognition sequences. The terms "regulatory sequence" and "regulatory element" are used interchangeably in this document.

[0066] A promoter is a nucleic acid fragment that controls the transcription of another nucleic acid fragment.

[0067] "A functional promoter in a plant" refers to a promoter that can control gene transcription in plant cells, regardless of whether it originates from a plant cell.

[0068] "Tissue-specific promoters" and "tissue-preferred promoters" can refer to promoters that are primarily, but not necessarily, expressed in a single tissue or organ, but can also be expressed in a specific cell or cell type.

[0069] "Developmental regulator promoters" are promoters whose activity is determined by developmental events.

[0070] "Operationally ligated" refers to the linking of nucleic acid fragments into a single fragment, such that the function of one nucleic acid fragment is regulated by the other. For example, when a promoter can regulate the transcription of a nucleic acid fragment, the promoter is operably ligated to that nucleic acid fragment.

[0071] "Expression" refers to the production of a functional product. For example, the expression of a nucleic acid fragment can refer to the transcription of the nucleic acid fragment (e.g., the transcription of mRNA or functional RNA) and / or the translation of mRNA into a precursor or mature protein.

[0072] "Phenomenon" refers to the detectable characteristics of a cell or organism.

[0073] In the context of inserting nucleic acid fragments (e.g., recombinant DNA constructs) into cells, “introduction” means “transfection” or “conversion” or “transduction”, and includes the incorporation of nucleic acid fragments into eukaryotic or prokaryotic cells, wherein the nucleic acid fragments can be incorporated into the cell’s genome (e.g., chromosome, plasmid, plasmid, or mitochondrial DNA), transformed into autonomous replicons, or transiently expressed (e.g., transfected mRNA).

[0074] "Transformed cells" are any cells in which nucleic acid fragments (such as recombinant DNA constructs) have been introduced.

[0075] The term "transformation" used in this article refers to both stable transformation and instantaneous transformation.

[0076] "Stable transformation" refers to the introduction of a nucleic acid fragment into the genome of a host organism, resulting in its genetic stability. Once stable transformation occurs, the nucleic acid fragment is stably integrated into the genome of the host organism and in any subsequent generations.

[0077] "Transient conversion" refers to the introduction of nucleic acid fragments into the cell nucleus or DNA-containing organelles of a host organism, resulting in the expression of genes that are not genetically stable.

[0078] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations of the present invention.

[0079] One specific embodiment of the present invention provides the application of wheat TabHLH93 protein or its encoding gene in regulating plant grain development, wherein the wheat TabHLH93 protein is a protein as shown in (a1) or (a2) or (a3) ​​below:

[0080] (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;

[0081] (a2) A protein having an amino acid sequence that is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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% sequence identity with SEQ ID NO: 1;

[0082] (a3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (a1) and (a2).

[0083] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0084] In some embodiments, the gene encoding the wheat TabHLH93 protein is a DNA molecule as shown in (b1) or (b2) or (b3) below:

[0085] (b1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2;

[0086] (b2) A DNA molecule or its full-length complementary sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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% sequence identity with SEQ ID NO: 2;

[0087] (b3) A DNA molecule that encodes the amino acid sequence shown in SEQ ID NO: 1, except for (b1) and (b2).

[0088] In some embodiments, the regulation of plant grain development specifically involves upregulating or enhancing or increasing the expression of the gene encoding the wheat TabHLH93 protein, or upregulating or enhancing or increasing the protein activity or content, to increase the length of plant grains and the length of the epidermal cells of the grain outer skin.

[0089] Another specific embodiment of the present invention provides the use of wheat TabHLH93 protein in any of the following:

[0090] (c1) Increase plant yield and / or number of tillers and / or ear length and / or ear weight and / or number of grains per ear and / or thousand-grain weight and / or plant height;

[0091] (c2) Plant breeding;

[0092] The wheat TabHLH93 protein is the protein shown in (a1) or (a2) or (a3) ​​below:

[0093] (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;

[0094] (a2) A protein having an amino acid sequence that is at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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% sequence identity with SEQ ID NO: 4;

[0095] (a3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (a1) and (a2).

[0096] This invention has determined TabHLH93 It is a transcription factor downstream of the BR signaling pathway, which regulates wheat grain size and plays an important regulatory role in agronomic traits such as wheat plant height, tiller number, spike length, and thousand-grain weight. Experiments have clarified the role of wheat... TabHLH93 The relationship between genes and wheat plant height, tiller number, spike length, and thousand-grain weight was verified. TabHLH93 High expression of this substance can promote increased wheat plant height and ear length, increase the number of tillers and thousand-grain weight, and ultimately increase wheat yield.

[0097] Another specific embodiment of the present invention provides the application of a recombinant DNA construct in regulating plant seed development, said recombinant DNA construct comprising a DNA molecule operably linked to at least one heterologous regulatory sequence as shown in (b1) or (b2) or (b3) below:

[0098] (b1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2;

[0099] (b2) A DNA molecule or its full-length complementary sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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% sequence identity with SEQ ID NO: 2;

[0100] (b3) A DNA molecule that encodes the amino acid sequence shown in SEQ ID NO: 1, except for (b1) and (b2).

[0101] Another specific embodiment of the present invention provides the use of the recombinant DNA construct in any of the following:

[0102] (c1) Increase plant yield and / or number of tillers and / or ear length and / or ear weight and / or number of grains per ear and / or thousand-grain weight and / or plant height;

[0103] (c2) Plant breeding;

[0104] The recombinant DNA construct comprises a DNA molecule operably linked to at least one heterologous regulatory sequence, as shown in (b1) or (b2) or (b3) below:

[0105] (b1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2;

[0106] (b2) A DNA molecule or its full-length complementary sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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% sequence identity with SEQ ID NO: 2;

[0107] (b3) A DNA molecule that encodes the amino acid sequence shown in SEQ ID NO: 1, except for (b1) and (b2).

[0108] It should be understood that those skilled in the art will recognize that this invention is not limited to specific exemplary sequences. Changes to nucleic acid fragments that produce chemically equivalent amino acids at a given site without affecting the functional properties of the encoded polypeptide are well known in the art. For example, the codon for the hydrophobic amino acid alanine may be replaced by a codon encoding another less hydrophobic residue such as glycine, or a more hydrophobic residue such as valine, leucine, or isoleucine. Similarly, it is contemplated that changes such as replacing one negatively charged residue with another, such as aspartic acid with glutamic acid, or replacing one positively charged residue with another, such as lysine with arginine, will produce functionally equivalent products. It is also contemplated that nucleotide changes altering the N-terminal and C-terminal portions of a polypeptide molecule will not change the activity of the polypeptide. Each of the recommended changes is within the conventional art, as is the preservation of the biological activity of the encoded product.

[0109] In some implementations, the regulatory sequence may be a promoter.

[0110] A variety of promoters can be used in the recombinant DNA constructs of this invention. Promoters can be selected based on desired results and may include constitutive promoters, tissue-specific promoters, inducible promoters, or other promoters for expression in a host organism. Promoters may be entirely derived from natural genes, or may consist of different elements derived from different naturally occurring promoters, or may even contain synthetic DNA segments.

[0111] In some embodiments, the recombinant DNA construct may also include other regulatory sequences, including but not limited to pre-translational sequences, introns, and polyadenylation recognition sequences. In some embodiments, the recombinant DNA construct may also include enhancers or silencers.

[0112] Another specific embodiment of the present invention provides a method for increasing plant seed size, comprising the following steps: (d1) introducing a recombinant DNA construct into regenerable plant cells, the recombinant DNA construct comprising a DNA molecule operatively linked to at least one regulatory sequence; (d2) regenerating a transgenic plant from the regenerable plant cells following step (d1), wherein the transgenic plant contains the recombinant DNA construct in its genome; and (d3) obtaining progeny plants derived from the transgenic plant of step (d2), wherein the progeny plants contain the recombinant DNA construct in their genome and exhibit increased seed size compared to a control plant;

[0113] The DNA molecule is as shown in (b1), (b2), or (b3) below:

[0114] (b1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2;

[0115] (b2) A DNA molecule or its full-length complementary sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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% sequence identity with SEQ ID NO: 2;

[0116] (b3) A DNA molecule that encodes the amino acid sequence shown in SEQ ID NO: 1, except for (b1) and (b2).

[0117] Another specific embodiment of the present invention provides a method for increasing plant yield and / or tiller number and / or ear length and / or ear weight and / or ear grain number and / or thousand-grain weight and / or plant height, comprising the following steps: (e1) introducing a recombinant DNA construct into regenerable plant cells, the recombinant DNA construct comprising a DNA molecule operably linked to at least one regulatory sequence; (e2) regenerating a transgenic plant from the regenerable plant cells after step (e1), wherein the transgenic plant contains the recombinant DNA construct in its genome; and (e3) obtaining progeny plants derived from the transgenic plant of step (e2), wherein the progeny plants contain the recombinant DNA construct in their genome and exhibit increased yield and / or tiller number and / or ear length and / or ear weight and / or ear grain number and / or thousand-grain weight and / or plant height compared to a control plant;

[0118] The DNA molecule is as shown in (b1), (b2), or (b3) below:

[0119] (b1) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 2;

[0120] (b2) A DNA molecule or its full-length complementary sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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% sequence identity with SEQ ID NO: 2;

[0121] (b3) A DNA molecule that encodes the amino acid sequence shown in SEQ ID NO: 1, except for (b1) and (b2).

[0122] In any or any other embodiment of the foregoing method, during the introduction step, the regenerative plant cells may include callus cells, embryogenic callus cells, gamete cells, meristematic cells, or cells of immature embryos.

[0123] In any or any other embodiment of the foregoing methods, there are alternative methods for introducing a recombinant DNA construct comprising a polynucleotide operatively linked to at least one regulatory sequence into a regenerative plant cell. For example, a regulatory sequence (such as one or more enhancers, optionally as part of a transposon element) may be introduced into a regenerative plant cell, followed by screening for events in which the regulatory sequence is operatively linked to an endogenous gene encoding the TabHLH93 protein disclosed herein.

[0124] The recombinant DNA constructs disclosed herein can be introduced into plants using any suitable technique, including but not limited to direct DNA uptake, chemical treatment, electroporation, microinjection, cell fusion, infection, vector-mediated DNA transfer, bombardment, or Agrobacterium-mediated transformation.

[0125] Furthermore, methods for modifying or altering the host's endogenous genomic DNA are available. This includes altering the host's natural DNA sequence or a pre-existing transgenic sequence, which includes regulatory elements, coding and non-coding sequences. These methods are also used to enable nucleic acids to target pre-engineered target recognition sequences in the genome.

[0126] The development or regeneration of plants containing isolated, foreign nucleic acid fragments encoding proteins of interest is well known in the art. Regenerated plants can be self-pollinated to provide homozygous transgenic plants. Alternatively, pollen obtained from the regenerated plants can be hybridized with seed-derived plants from agronomically important lines. In turn, the regenerated plants are pollinated with pollen from these important lines. Transgenic plants containing the desired TabHLH93 protein are then cultured using methods well known to those skilled in the art.

[0127] Those skilled in the art will also understand that changes can be introduced through mutations in the nucleic acid sequence, thereby causing alterations in the expression of the encoded mRNA or the amino acid sequence of the encoded polypeptide, resulting in changes in the biological activity of the mRNA or protein, or both, respectively. Therefore, variant nucleic acid molecules can be generated by introducing one or more nucleotide substitutions, additions, and / or deletions into the corresponding nucleic acid sequence or surrounding sequences disclosed herein. Such variant nucleic acid sequences are also covered by this invention.

[0128] Variant nucleic acid sequences can be prepared by randomly introducing sequence changes along all or part of a gene region, including but not limited to chemical or radiation mutagenesis and oligonucleotide-mediated mutagenesis (OMM). Alternatively or otherwise, sequence changes can be introduced using double-strand break techniques at specific selected sites, such as ZNF, custom homing endonucleases, TALEN, CRISPR / CAS (also known as guide RNA / Cas endonuclease systems), or other protein and / or nucleic acid-based mutagenesis techniques. The resulting variants can be screened for altered activity. It should be understood that the techniques are generally not mutually exclusive. In fact, the various methods can be used alone or in combination, in parallel or sequentially, to produce or obtain a variety of sequence variants.

[0129] In this invention, the plant can be a monocotyledonous or dicotyledonous plant. The plant is selected from at least one of rice, corn, soybean, sunflower, sorghum, wheat, alfalfa, cotton, barley, and millet. Wheat is preferred. The wheat includes, but is not limited to, JW1, Yannong 15, Lumai 15, Jimai 38, Zhengmai 379, Jimai 44, Chuanmai 104, Xinong 511, Zhongmai 578, Weilong 169, Zhengmai 1860, Bainong 207, Luyuan 502, Huaimai 33, Bainong 4199, Xinmai 26, Malan 1, Yangmai 25, Yumai 14 (superior variety), Xinmai 19, Fengdecunmai 20, Zhongxinmai 998, Zhengmai 136, Bainong 307, Yannong 1212, Zhoumai 16, Zhoumai 22, or Fielder.

[0130] The test materials used in the examples and their sources are as follows:

[0131] The high-fidelity enzyme required for PCR amplification was KOD-FX Neo (Toyobo); the vector used for the LR reaction was Gateway entry-level cloning vector. pENTRY TM / SD / D-TOPO TM (Abbreviation: pENTRY The plasmid vector and target gene were purchased from Thermo Fisher Scientific, Inc., USA, for constructing recombination between the plasmid vector and the target gene; the 2×PCR master mix was purchased from Beijing Polymer Biotechnology Co., Ltd.; and the PerfectStart real-time qPCR kit was used. TM Green qPCR SuperMix was purchased from Beijing TransGen Biotech Co., Ltd.; XcmI restriction endonuclease was purchased from NEB; T4 ligase was purchased from Ise-Hisashi Biosciences; LR Clonase for homologous recombination, gel extraction kits for enzyme fragment recovery, plasmid extraction kits, and antibodies for Western blotting were all purchased from Thermo Fisher Scientific. Inorganic salts required for culture medium preparation were purchased from Sinopharm Group, and vitamins and antibiotics were purchased from Sigma-Aldrich.

[0132] In this invention, the plasmid pLGY02-YFP and the wheat variety JW1 were obtained from the Crop Research Institute of Shandong Academy of Agricultural Sciences. The wheat variety JW1 is a new germplasm with good tissue culture ability bred by the Crop Research Institute of Shandong Academy of Agricultural Sciences. It can be obtained by the public from the Crop Research Institute of Shandong Academy of Agricultural Sciences. The above-mentioned biological materials are only used to repeat the relevant experiments of this invention and cannot be used for other purposes.

[0133] The Escherichia coli strain used in this invention is E.coli Transgen5α was purchased from Beijing Quanshijin Company; Agrobacterium tumefaciens. EHA105 Competent cells were purchased from Beijing Huayueyang Company, and the primers used were synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd. The relevant primer sequences are shown in Table 1.

[0134] Table 1 Primer Sequences

[0135]

[0136] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0137] Example 1: Construction of expression vector

[0138] 1. Cloning of the TabHLH93 coding sequence:

[0139] To obtain the coding sequence of the TabHLH93 protein, this experiment amplified cDNA from the wheat variety Chinese spring (CS). TabHLH93 The gene sequence was obtained using TabHLH93-IF (SEQ ID NO: 3) and TabHLH93-IR (SEQ ID NO: 4) primers. The PCR reaction system was as follows: KOD Fx Neo buffer: 15 μL, dNTPs (2mM): 6 μL, TabHLH93-IF (10 μM): 0.9 μL, TabHLH93-IR (10 μM): 0.9 μL, CS cDNA: 1 μL, KOD Fx Neo: 0.5 μL, and ddH2O to a final volume of 30 μL. The PCR reaction program was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 25 s, 58°C annealing for 25 s, 72°C extension for 1 min, 35 cycles, 72°C annealing for 5 min, and storage at 4°C.

[0140] PCR results as follows Figure 1As shown, lane 1 is the Transgen 2K marker, and lanes 2 and 3 are... TabHLH93 Target band.

[0141] After PCR product extraction using the GeneJET Gel Extraction Kit from Thermo Scientific, the samples were sent to Qingdao Qingke Biotechnology Co., Ltd. for sequencing. The reference sequence was from IWGSC RefSeq v1.1 (WheatOmics 1.0, http: / / 202.194.139.32). TabHLH93 The gene (TraesCS7B02G152800) sequence. The full-length coding sequence of this gene is 1155 bp and contains 8 exons. Its nucleotide sequence is shown in SEQ ID NO: 2.

[0142] 2. The product of TabHLH93 reacts with A:

[0143] The PCR products recovered from the gel were subjected to an A-addition reaction. The reaction mixture consisted of: 10 × Buffer: 2 μL, 10 mM dNTPs: 2 μL, PCR products: 15.7 μL, and Taq DNA polymerase: 0.3 μL. The A-addition reaction program was: 72°C for 40 min; storage at 4°C.

[0144] The product from the addition of A was recovered using the GeneJET Gel Extraction Kit from Thermo Scientific, and its concentration was measured.

[0145] 3. pENTRY Carrier linearization:

[0146] Extraction using Thermo Scientific's GeneJET Plasmid Miniprep Kit pENTRY The vector plasmid was linearized using XcmI restriction endonuclease. The digestion reaction system was 10 × NE Buffer. TM r2.1: 5 μL, XcmI: 0.5 μL, vector: 5 μg, ddH2O: up to 50 μL. Incubate at 37°C for 1 h 30 min. The enzyme digestion products were recovered using the GeneJET Gel Extraction Kit from Thermo Scientific, and their concentrations were measured.

[0147] 4. T4 connection:

[0148] The PCR products recovered from the gel were ligated to... pENTRY In the support, the T4 linkage reaction system is linearized. pENTRY Vector fragment: 56 ng, insert fragment: 46 ng, 10 × T4 DNA Ligase Buffer: 2 μL, T4 DNA Ligase: 1 U (0.2 μL), Nuclease-Free Water: up to 20 μL. Mix thoroughly and briefly incubate at 16°C overnight. Transform the reaction product into Trans5α competent cells and incubate upside down on LB agar plates (containing kanamycin) for 1 day. Single colonies are picked for colony PCR identification using primers M13F (SEQ ID NO: 5) and M13R (SEQ ID NO: 6). Electrophoresis results of the PCR products are shown below. Figure 2 As shown, the selected positive recombinant strains were sent to Qingdao Ruiboxingke Biotechnology Co., Ltd. for sequencing, which indicated that the TabHLH93 gene had been ligated into the entry vector. pENTRY In this context, the recombinant vector is named... pENTRY-TabHLH93 .

[0149] 5. LR reaction:

[0150] Will pENTRY-TabHLH93 plasmids and pLGY02-YFP The carrier undergoes the LR reaction, and the specific reaction system is as follows: pENTRY-TabHLH93 Plasmid (50 ng / μL): 1 μL, pLGY02pYFP Plasmid (50 ng / μL): 1 μL, LRclonase: 0.5 μL. The reaction system was incubated at 25°C for 1 h, and then the reaction product was transformed into Escherichia coli Transgen 5α and cultured at 37°C for 16 h on LB solid medium containing kanamycin antibiotic. Single clones were picked for identification and sequencing by colony-linked PCR using primers LGY-dF (SEQ ID NO: 7) and TabHLH93-IR (SEQ ID NO: 4).

[0151] Electrophoresis results as follows Figure 3 As shown, clones with the target band are carried by binary vectors. pLGY02-TabHLH93-YFP Positive clones. Example 2: Obtaining and identifying transgenic progeny.

[0152] 1. Obtaining the offspring of the TabHLH93 transgenic strain:

[0153] The binary expression vector constructed in Example 1 pLGY02-TabHLH93-YFP Transformed into Agrobacterium EHA105 competent cells, specifically: 1 μL of recombinant binary expression vector was added. pLGY02-TabHLH93-YFPThe plasmid (500 ng / μL) was added to Agrobacterium EHA105 competent cells, incubated on ice for 5 min, rapidly transferred to liquid nitrogen for 5 min, heat-shocked at 37°C for 5 min, incubated on ice for 2 min, and then 700 μL of antibiotic-free liquid YEP medium was added. The cells were then placed in a shaker at 28°C for 2 h to recover. The cells were then spread on YEP solid medium containing rifampicin, gentamicin, and kanamycin and incubated upside down at 28°C for 2 days.

[0154] JW1 wheat seeds, approximately 15 days post-pollination, were harvested, and embryos were extracted. 1 mL of Agrobacterium tumefaciens culture was placed in a 1.5 mL centrifuge tube, and 1.4 μL of 0.1 M acetylsylcholine was added and mixed thoroughly to prepare an Agrobacterium tumefaciens suspension. The embryos were added to the Agrobacterium tumefaciens suspension for 5 min, then placed on a co-culture medium and incubated in the dark at 23°C for 3 days. After co-culture, the culture was placed on a resting medium and incubated in the dark at 25°C for 5 days. Callus tissue was transferred to selection medium 1, the culture dishes were sealed with sealing film, and incubated in the dark at 25.5°C for 2 weeks. After cutting and selecting the callus for 2 weeks, resistant callus exhibiting green buds was transferred to a regeneration medium. The culture dishes were sealed and incubated in a 25°C incubator under light / dark conditions (16 h / 8 h) for 2 weeks. Two weeks after regeneration, the healthy seedlings were transferred to new resistance regeneration boxes. Once the seedlings reached a certain size, samples were taken for testing.

[0155] The formulations of the co-culture medium, rest medium, selection medium 1, selection medium 2 and regeneration medium involved in the above-mentioned wheat genetic transformation are shown in Table 2.

[0156] Table 2 Culture medium formulation

[0157]

[0158] 2. Identification of TabHLH93 transgenic offspring:

[0159] gDNA was extracted from young leaves of T0 generation wheat plants using the CTAB method. Using gDNA as a template, PCR identification of transgenic wheat was performed using LGY-dF (SEQ ID NO: 7) and TabHLH93-IR (SEQ ID NO: 4) as primers. The reaction system was: 10 μL 2× PCR master mix, 0.5 μL 10 μM LGY-dF, 0.5 μL 10 μM LGY-dR, 0.5 μL gDNA (50 ng / μL), and ddH2O to a final volume of 20 μL. PCR conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 25 s, annealing at 58°C for 25 s, extension at 72°C for 1 min, for a total of 35 cycles; 72°C for 5 min; storage at 4°C. Agarose gel electrophoresis results are shown below. Figure 4 As shown.

[0160] 3. Using RT-qPCR experiments to detect positive transgenic lines OE-TabHLH93 -1 # , OE-TabHLH93 -3 # Expression level identification was performed. The main experimental procedures were as follows: Total RNA was extracted from wheat leaves using the TransZol method (Beijing TransGen Biotech Co., Ltd.). The RNA was then reverse transcribed into cDNA using reverse transcriptase (5 × All-in-One RT Mastermix, Abcam). The reaction mixture consisted of 4 μL of 5 × All-in-One RT Mastermix, 2 μL of total RNA (500 ng / μL), and Nuclease-free H2O to a final volume of 20 μL. The reverse transcription PCR program was 37°C for 15 min; 55°C for 30 min; and 95°C for 3 min. The obtained cDNA was used for subsequent RT-qPCR experiments. The reaction system was as follows: 5 μL 2 × M5 Hiper Realtime PCR mix (SYBR green), 0.25 μL 10 μM TabHLH93-qF (SEQ ID NO: 8), 0.25 μL 10 μM TabHLH93-qR (SEQ ID NO: 9), 2.5 μL cDNA (10× dilution), and 2 μL ddH2O. The PCR program was 95°C, 3 min; 95°C, 20 s, 60°C, 20 s, 72°C, 15 s, 45 cycles; signal acquisition at 70°C - 95°C, 0.2°C / 5 s, 72°C, 5 min, and storage at 4°C. The results were analyzed using relative quantification methods, and the results are shown below. Figure 5 As shown, positive transgenic lines OE-TabHLH93 -1# and OE- TabHLH93 -3 # It has a high expression level.

[0161] Example 3 Wheat overexpression TabHLH93 Genetic representation identification

[0162] Wild-type recipient variety JW1 and overexpression-positive transgenic lines were used. OE-TabHLH93 -1 # and OE-TabHLH93 -3 # Cultivated in the artificial climate chamber of Shandong University Qingdao Campus, the cultivation conditions were: 16 hours of light, 8 hours of darkness, daytime temperature of 22°C, nighttime temperature of 16°C, humidity of 40% - 50%, and CO2 concentration of 500 ppm - 700 ppm.

[0163] For JW1, OE-TabHLH93 -1 # and OE-TabHLH93 -3 # The length and width of the grains, as well as the length and width of the outer epidermal cells of the grains, were analyzed.

[0164] like Figure 6 As shown in A, B, and C, compared to the wild-type plant JW1, OE-TabHLH93 Overexpressing transgenic lines resulted in increased grain length and decreased grain width; for example... Figure 6 As shown in D, E, and F, compared to the wild-type plant JW1, OE-TabHLH93 The transgenic lines overexpressing this gene showed increased length and decreased width of the outer epidermal cells in their grains. This indicates... TabHLH93 It can significantly promote wheat grain elongation.

[0165] Further research on JW1, OE-TabHLH93 -1 # and OE-TabHLH93 -3 # The agronomic traits such as plant height, number of tillers, panicle length, number of grains per panicle, and thousand-grain weight were analyzed.

[0166] The results are as follows Figure 7 As shown in A, B, C, D, E, and F, compared to the wild-type plant JW1, OE-TabHLH93 The overexpression transgenic lines showed a significant increase in tiller number, panicle length, panicle grain number, and thousand-grain weight, with a slight increase in plant height, indicating that... TabHLH93 It can promote wheat growth.

[0167] This invention constructs TabHLH93An overexpression vector was used to transfer the Ubi::TabHLH93 element into wheat through Agrobacterium infection of wheat embryo callus. The results demonstrated that TabHLH93 promotes wheat cell elongation, wheat growth, and grain development, thereby increasing wheat yield. This not only provides new insights into the regulatory mechanisms of wheat growth and development but also offers a promising candidate gene for high-yield genetic improvement of wheat, which is of great significance for ensuring food security.

[0168] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a wheat TabHLH93 protein or a gene encoding the same in regulating grain development in a plant, characterized in that, The wheat TabHLH93 protein is a protein as shown in (a1) or (a2) below: (a1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing; (a2) a fusion protein obtained by connecting a protein tag to the N terminus and / or C terminus of the protein defined in (a1); The regulation of plant kernel development refers to promoting wheat kernel elongation by overexpressing the TabHLH93 gene; The TabHLH93 gene is a DNA molecule as shown in (b1) or (b2) below: (b1) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 2; (b2) a DNA molecule other than (b1) that encodes the amino acid sequence shown in SEQ ID NO: 1; The plant is wheat.

2. Use of overexpression of wheat TabHLH93 gene in increasing plant yield, characterized in that, The TabHLH93 gene is a DNA molecule as shown in (b1) or (b2) below: (b1) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 2; (b2) a DNA molecule other than (b1) that encodes the amino acid sequence shown in SEQ ID NO: 1; The plant is wheat.

3. Use of overexpression of TabHLH93 gene in increasing the number of tillers in plants, characterized in that, The TabHLH93 gene is a DNA molecule as shown in (b1) or (b2) below: (b1) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 2; (b2) a DNA molecule other than (b1) that encodes the amino acid sequence shown in SEQ ID NO: 1; The plant is wheat.

4. Use of overexpression of TabHLH93 gene in increasing plant ear length, characterized in that, The TabHLH93 gene is a DNA molecule as shown in (b1) or (b2) below: (b1) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 2; (b2) a DNA molecule other than (b1) that encodes the amino acid sequence shown in SEQ ID NO: 1; The plant is wheat.

5. Use of overexpression of TabHLH93 gene in increasing grain number per panicle of a plant, characterized in that, The TabHLH93 gene is a DNA molecule as shown in (b1) or (b2) below: (b1) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 2; (b2) a DNA molecule other than (b1) that encodes the amino acid sequence shown in SEQ ID NO: 1; The plant is wheat.

6. Use of overexpression of TabHLH93 gene in increasing the thousand seed weight of a plant, characterized in that, The TabHLH93 gene is a DNA molecule as shown in (b1) or (b2) below: (b1) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 2; (b2) a DNA molecule other than (b1) that encodes the amino acid sequence shown in SEQ ID NO: 1; The plant is wheat.

7. Use of overexpression of TabHLH93 gene in increasing plant height, characterized in that, The TabHLH93 gene is a DNA molecule as shown in (b1) or (b2) below: (b1) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 2; (b2) a DNA molecule other than (b1) that encodes the amino acid sequence shown in SEQ ID NO: 1; The plant is wheat.

8. Use of a recombinant DNA construct for modulating grain development in a plant, characterized in that, The recombinant DNA construct comprises a TabHLH93 gene, and the sequence of the TabHLH93 gene is as shown in (b1) or (b2): (b1) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 2; (b2) a DNA molecule other than (b1) that encodes the amino acid sequence shown in SEQ ID NO: 1; (b2) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO: 1 other than (b1); The regulation of plant kernel development refers to increasing the length of plant kernels by overexpressing the TabHLH93 gene; The plant is wheat.

9. Use of a recombinant DNA construct for increasing yield in a plant, characterized in that, The recombinant DNA construct comprises a TabHLH93 gene, and the sequence of the TabHLH93 gene is shown in (b1) or (b2): (b1) a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 2; (b2) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO: 1 other than (b1); The plant is wheat.

10. Use of a recombinant DNA construct for increasing the number of tillers in a plant, characterized in that, The recombinant DNA construct comprises a TabHLH93 gene, and the sequence of the TabHLH93 gene is shown in (b1) or (b2): (b1) a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 2; (b2) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO: 1 other than (b1); The plant is wheat.

11. Use of a recombinant DNA construct for increasing ear length in a plant, characterized in that, The recombinant DNA construct comprises a TabHLH93 gene, and the sequence of the TabHLH93 gene is shown in (b1) or (b2): (b1) a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 2; (b2) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO: 1 other than (b1); The plant is wheat.

12. Use of a recombinant DNA construct for increasing grain number per ear in a plant, characterized in that, The recombinant DNA construct comprises a TabHLH93 gene, and the sequence of the TabHLH93 gene is shown in (b1) or (b2): (b1) a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 2; (b2) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO: 1 other than (b1); The plant is wheat.

13. Use of a recombinant DNA construct for increasing the thousand kernel weight of a plant, characterized in that, The recombinant DNA construct comprises a TabHLH93 gene, and the sequence of the TabHLH93 gene is shown in (b1) or (b2): (b1) a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 2; (b2) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO: 1 other than (b1); The plant is wheat.

14. Use of a recombinant DNA construct for increasing plant height, characterized in that, The recombinant DNA construct comprises a TabHLH93 gene, and the sequence of the TabHLH93 gene is shown in (b1) or (b2): (b1) a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 2; (b2) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO: 1 other than (b1); The plant is wheat. The recombinant DNA construct comprises a TabHLH93 gene, and the sequence of the TabHLH93 gene is shown in (b1) or (b2): (b1) a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 2; (b2) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO: 1 other than (b1); The plant is wheat.

15. A method of increasing grain length in a plant, comprising: comprises the following steps: (d1) introducing into a regenerable plant cell a recombinant DNA construct comprising a TabHLH93 gene; (d2) regenerating a transgenic plant from the regenerable plant cell after step (d1), wherein the transgenic plant comprises in its genome the recombinant DNA construct; and (d3) obtaining a progeny plant derived from the transgenic plant of step (d2), wherein the progeny plant comprises in its genome the recombinant DNA construct and exhibits increased grain length as compared to a control plant; the sequence of the TabHLH93 gene is represented by (b1) or (b2): (b1) a DNA molecule whose nucleotide sequence is represented by SEQ ID NO: 2; (b2) a DNA molecule other than (b1) that encodes the amino acid sequence represented by SEQ ID NO: 1; the plant is wheat.

16. A method of increasing yield and / or tiller number and / or ear length and / or grain number per ear and / or thousand kernel weight and / or plant height of a plant, characterized in that, comprises the following steps: (e1) introducing into a regenerable plant cell a recombinant DNA construct comprising a TabHLH93 gene; (e2) regenerating a transgenic plant from the regenerable plant cell after step (e1), wherein the transgenic plant comprises in its genome the recombinant DNA construct; and (e3) obtaining a progeny plant derived from the transgenic plant of step (e2), wherein the progeny plant comprises in its genome the recombinant DNA construct and exhibits increased yield and / or number of tillers and / or ear length and / or number of grains per ear and / or thousand grain weight and / or plant height as compared to a control plant; the sequence of the TabHLH93 gene is represented by (b1) or (b2): (b1) a DNA molecule whose nucleotide sequence is represented by SEQ ID NO: 2; (b2) a DNA molecule other than (b1) that encodes the amino acid sequence represented by SEQ ID NO: 1; the plant is wheat.

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