Application of TaLBD30 protein and coding gene thereof in regulation and control of wheat plant type

Abstract

The invention discloses application of a TaLBD30 protein and a coding gene thereof in regulating and controlling a wheat plant type. The invention belongs to the technical field of biology, and particularly relates to application of TaLBD30 protein and a coding gene thereof to regulation and control of wheat plant types. The protein is any one of the following proteins: A1) a protein with an amino acid sequence as shown in SEQ ID No: 1; a2) a protein which is obtained by substitution and / or deletion and / or addition of amino acid residues on the protein of A1), has 80% or more of identity with the protein of A1) and has the same function as the protein of A1); a3) a fusion protein obtained by connecting a protein tag to the N terminal or / and the C terminal of A1) or A2). TaLBD30 is a transcription factor with transcription inhibition activity, and compared with a wild type, the TaLBD30 gene overexpression shows that after TaLBD30 overexpression, the wheat plant height and ear length are remarkably reduced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of TaLBD30 protein and its encoding gene in regulating wheat plant architecture. Background Art

[0002] wheat( Triticum aestivum Wheat (L.) is the most widely cultivated food crop, providing approximately one-fifth of the world's dietary calories. It is projected that by 2050, the growing global population will increase its demand for wheat by 60%, while wheat production will inevitably be affected by extreme weather and the depletion of natural resources. Increasing food production is an urgent task for achieving global food and nutrition security. Plant architecture is an important agronomic trait closely related to wheat yield; therefore, research on wheat plant architecture-related genes has significant practical implications.

[0003] Crop plant architecture, mainly including plant height, number of tillers, tiller angle, and ear morphology, is an important agronomic trait that determines crop yield. Rht-B1b and Rht-D1b The use of [specific gene] reduces wheat plant height and enhances lodging resistance, which is crucial for increasing wheat yield. Wheat spike traits, including spike length, number of spikelets, number of grains per spike, and thousand-grain weight, are important agronomic traits that determine wheat yield. Therefore, identifying wheat plant architecture-related genes and analyzing their regulatory networks will provide a theoretical basis for the ultimate goal of breeding new wheat varieties and improving plant architecture.

[0004] LBD ( LATERAL ORGAN BOUNDARIES DOMAIN Genes are a class of plant-specific transcription factors that are highly conserved. LOB ( LATERAL ORGAN BOUNDARIES The LBD transcription factor plays a crucial role in regulating metabolic processes such as lateral organ development, pollen development, plant regeneration, photomorphogenesis, and pathogen response in higher plants. Currently, research on the role of LBD transcription factors in regulating wheat plant architecture is still very limited. Therefore, further exploration of the effects of LBD transcription factors on wheat plant architecture regulation has significant practical application value. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to regulate the plant shape.

[0006] To address the problems existing in the prior art, the present invention provides a protein.

[0007] The protein provided by this invention may be any of the following: A1) A protein with the amino acid sequence shown in SEQ ID No:1; A2) A protein that has more than 75% identity with the protein shown in A1) and has the function of regulating plant plant architecture, obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1); For example, those skilled in the art can obtain protein mutants with the same function as the amino acid sequence shown in SEQ ID No:1 by substituting, deleting and / or adding one or more amino acids, without affecting its activity, based on the amino acid sequence shown in SEQ ID No:1 and other conventional techniques in the art such as the conserved substitution of amino acids. A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0008] The protein described in A1 above is named TaLBD30.

[0009] To facilitate the purification or detection of the protein in A1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID No:1 in the sequence listing.

[0010] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0011] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0012] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein TaLBD30 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 protein TaLBD30 isolated in this invention, as long as they encode and function as protein TaLBD30, are derived from and equivalent to the nucleotide sequence of this invention.

[0013] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0014] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid or nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences or nucleotide sequences, then the identity value (%) can be obtained.

[0015] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0016] In this document, the above 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0017] The protein mentioned above is derived from cultivated emmer wheat ( Triticum dicoccum Schuebl . PI272527.

[0018] The present invention also provides biomaterials related to the above-mentioned proteins, said biomaterials may be any of the following: B1) Nucleic acid molecules that encode the proteins described above; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2); C1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the genes encoding the proteins described above; C2) expresses the gene encoding the nucleic acid molecule described in C1); C3) contains an expression cassette containing the gene encoding described in C2); C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3); C5) A recombinant microorganism containing the encoding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4); C6) A transgenic plant cell line containing the encoding gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4); C7) Transgenic plant tissue containing the encoding gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4); C8) A transgenic plant organ containing the encoding gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

[0019] In the above-mentioned biological materials, the nucleic acid molecule described in B1) may be a gene as shown in E1) or E2) below: E1) The coding sequence is a cDNA molecule of SEQ ID No:2; E2) The genome sequence is the DNA molecule of SEQ ID No:3.

[0020] The DNA molecule shown in SEQ ID No:3 (which regulates plant architecture) TaLBD30 The gene encodes the protein TaLBD30, whose amino acid sequence is SEQ ID No:1.

[0021] The nucleotide sequence shown in SEQ ID No:2 is the nucleotide sequence of the gene encoding the protein TaLBD30 (CDS).

[0022] The present invention TaLBD30 Genes can be any nucleotide sequence that encodes the protein TaLBD30. Considering codon degeneracy and the codon preferences of different species, those skilled in the art can use codons suitable for expression in a specific species as needed.

[0023] B1) The nucleic acid molecule may also include a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No:2.

[0024] B1) The nucleic acid molecule may also include nucleic acid molecules that have a nucleotide sequence identity of more than 95% with the nucleotide sequence shown in SEQ ID No:2 and originate from the same species.

[0025] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0026] The vectors described herein are well-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., Cosmids), Ti plasmids, or viral vectors. Specifically, they may be vectors such as pCambia3301 for constructing transformation vectors; pQBV3 for constructing entry vectors in Gateway reactions; pAN580 for constructing subcellular localization vectors; pGADT7 and pGBKT7 for constructing self-activation activity analysis vectors; P2GW7, GAL4-LUC, and pRLC for constructing luciferase transient transcription experiment vectors; and pEASY-Blunt Zero cloning vectors, primarily used for gene cloning and sequencing analysis.

[0027] Existing plant expression vectors can be used to construct structures containing... TaLBD30 Recombinant gene 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 genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0028] use TaLBD30When constructing recombinant plant expression vectors, any type of enhancing promoter 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.

[0029] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0030] Using any vector capable of guiding the expression of exogenous genes in plants, the present invention can be used to... TaLBD30 Introducing genes or gene fragments into plant cells or recipient plants can yield transgenic cell lines and transgenic plants with altered plant morphology. (Carrying...) TaLBD30 Gene expression vectors can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0031] In one specific embodiment, the recombinant vector may be pCambia3301- TaLBD30 pCambia3301- TaLBD30 The structure is described as follows: a recombinant vector is obtained by inserting a DNA fragment with sequence SEQ ID No:4 between 5'-cagctatgacatgattacgaattc-3' and 5'-gctggtcacctgtaattcacacgtg-3' of the vector pCambia3301, while keeping the other sequences of the vector pCambia3301 unchanged.

[0032] The microorganisms mentioned in this article may be yeast, bacteria, algae, or fungi. Among them, bacteria may originate from the genus *Escherichia* (…). EscherichiaErwinia ( Erwinia Agrobacterium tumefaciens ( ), Agrobacterium tumefaciens Agrobacterium Flavobacterium ( Flavobacterium Alcaligenes ( ) Alcaligenes ), Pseudomonas ( Pseudomonas ), Bacillus spp. ( Bacillus Specifically, these could be Agrobacterium tumefaciens EHA105 and Trans-T1 Escherichia coli competent cells.

[0033] vector pCambia3301- TaLBD30 The TaLBD30 protein shown in SEQ ID No:1 of the sequence listing is expressed.

[0034] In one specific embodiment, the recombinant microorganism may be recombinant Agrobacterium EHA105 / pCambia3301- TaLBD30 .

[0035] The recombinant Agrobacterium EHA105 / pCambia3301- TaLBD30 The recombinant vector pCambia3301- TaLBD30 Recombinant bacteria obtained by introducing Agrobacterium tumefaciens EHA105.

[0036] The plants obtained by the above methods can be transgenic plants or plants obtained through conventional breeding techniques such as hybridization. In the above methods, the transgenic plants are understood to include not only first- and second-generation transgenic plants, but also their progeny. For transgenic plants, the gene can be propagated within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties.

[0037] The genetically modified plant includes seeds, callus tissue, complete plant, and cells.

[0038] The present invention also provides the use of the protein TaLBD30 described above, or an expression substance of the gene regulating it, or a substance regulating the activity or content of the protein, in any of the following: U1) Application in regulating plant architecture; U2) Applications in the preparation of products that regulate plant architecture; U3) Applications in plants for cultivating plant shape; U4) Application in the preparation of products using cultivated plant varieties; U5) Applications in plant breeding.

[0039] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein TaLBD30.

[0040] In the above applications, the substance that regulates gene expression or the substance that regulates the activity or content of the protein can be a biological material related to the protein, and the biological material can be the biological material described above.

[0041] In the above text, the substance that regulates gene expression can be a substance that performs at least one of the following six types of regulation: 1) Regulation occurring at the transcriptional level of the aforementioned gene; 2) Regulation that occurs after the gene is transcribed (i.e., regulation of the splicing or processing of the primary transcript of the gene). 3) Regulation of RNA transport of the gene (that is, regulation of the transport of mRNA of the gene from the nucleus to the cytoplasm). 4) Regulation of the translation of the aforementioned genes; 5) Regulation of mRNA degradation of the aforementioned gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0042] The present invention also provides a method for regulating plant architecture, including regulating the activity and / or content of the proteins described above in the target plant, and / or the expression level of the genes encoding the proteins, to regulate plant architecture.

[0043] In the above method, regulating the activity and / or content of the protein TaLBD30 in the target plant, and / or the expression level of the gene encoding the protein, includes introducing the gene encoding the protein into the recipient plant. TaLBD30 This alters the plant's shape; TaLBD30 The gene encodes the protein TaLBD30.

[0044] The importation refers to the importation through recombination methods, including but not limited to Agrobacterium-mediated transformation, bio-projectile methods, electroporation, in-planta technology, and so on.

[0045] In the above applications and methods, the regulation can be to increase, enhance, or upregulate.

[0046] In the above applications and methods, the regulation can be suppression, reduction, or silencing.

[0047] The present invention also provides a method for cultivating plants with altered plant form, comprising: 1) Increase, enhance and / or upregulate the expression level of the coding genes of the proteins mentioned above in the target plant, or / and increase, enhance and / or upregulate the activity and / or content of the coding genes of the proteins mentioned above, to obtain plants with reduced plant height and spike length. 2) Inhibit, reduce, or silence the expression level of the coding genes of the proteins mentioned above in the target plant, and / or inhibit, reduce, or silence the activity and / or content of the coding genes of the proteins mentioned above, to obtain plants with increased plant height and spike length.

[0048] As one embodiment of the present invention, the method for cultivating plants with reduced plant height and spike length includes the following steps: (1) Construct a structure containing the following as shown in SEQ ID No:4 TaLBD30 Gene sequence expression vectors; (2) Introduce the expression vector constructed in step (1) into plants; (3) Plants with reduced plant height and spike length were obtained through screening and identification.

[0049] In this invention, the purpose of plant breeding includes cultivating plants with improved / reduced plant type.

[0050] In this invention, the plant type includes plant height and ear morphology.

[0051] In the above applications or methods, the plant is any one of the following: N1) Monocotyledons; N2) Plants of the order Poales; N3) Gramineae plants; N4) Plants of the Triticum genus; N5) Wheat.

[0052] This study focuses on wheat. TaLBD30 Genetic studies were conducted to preliminarily investigate the function of TaLBD30 in wheat, including the creation of transgenic materials, phenotypic identification, expression pattern analysis, subcellular localization, and verification of yeast self-activation. Overexpression was performed using winter wheat 'Ke Nong 199' as the receptor. TaLBD30 Genetic discovery revealed that, compared to the wild type, it was overexpressed TaLBD30 Afterwards, plant height and ear length decreased significantly. Attached Figure Description

[0053] Figure 1 for TaLBD30 The gene structure. This gene has two exons and one intron.

[0054] Figure 2 Subcellular localization of TaLBD30 in wheat protoplasts. TaLBD30 is localized in the nucleus.

[0055] Figure 3This study analyzed the self-activation activity of transcription factor TaLBD30. SD-Trp-Leu represents a two-deficient yeast culture medium lacking tryptophan (Trp) and leucine (Leu); SD-Trp-Leu-His-Ade represents a four-deficient yeast culture medium lacking tryptophan (Trp), leucine (Leu), histidine (His), and adenine (Ade).

[0056] Figure 4 This is an analysis of TaLBD30 transcriptional activity. Where a: plasmids used to transform protoplasts; control group plasmids include... 35s::GAL4-TATA-LUC (GAL4-LUC), 35s::GAL4-BD (P2GW7 - GAL4-BD), 35s::REN (pRLC) is abbreviated as CK group; the experimental group transformation plasmids include 35s::GAL4-TATA-LUC (GAL4-LUC), 35s::GAL4-BD-TaLBD30 (P2GW7 - GAL4-BD -TaLBD30 ), 35s::REN (pRLC), abbreviated as TaLBD30 group, the ratio of plasmid transformation in CK group and TaLBD30 group is 1:6:1; b: relative fluorescence value of control group and experimental group, n = 8. Error bar is expressed as ±SD (***P<0.001, ****P<0.0001, standard t test).

[0057] Figure 5 Phenotypic identification of transgenic materials. Among them, a: KN199 and transgenic lines. TaLBD30 pro::TaLBD30- 3 , TaLBD30pro::TaLBD30-4 and TaLBD30pro::TaLBD30-5 Phenotypic data during the grain-filling stage, scale bar: 10 cm; b: Statistical data on plant height and ear length of KN199 and transgenic lines, n = 10; c: Data on the lower stem and rachis of KN199 and transgenic lines. TaLBD30 Relative gene expression levels, n = 3. Error bars represent ±SD (***P<0.001, ****P<0.0001, standard t-test). DETAILED DESCRIPTION

[0058] 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.

[0059] 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.

[0060] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0061] RNA extraction in the following examples was performed using the TRIZOL extraction method (detailed steps can be found in: Liu, Pan. Study on the molecular genetic regulatory mechanism of Q, a key gene for wheat spike development [D]. Chinese Academy of Agricultural Sciences, 2019.). RNA reverse transcription was performed using a 5×All-In-One RT MasterMix kit, purchased from abm.

[0062] The PCR amplification reaction system for the target fragment in the following examples is as follows: KOD-Fx-Neo 0.4 μL, 2× PCR Buffer for KOD-Fx 10.0 μL, dNTPs (2 mM) 4.0 μL, Primer (2 µM) 2.0 μL, cDNA 2.0 μL, and ddH2O to a final volume of 20.0 μL. The PCR reaction program is as follows: 94℃ pre-denaturation for 5 min; 98℃ denaturation for 30 s, 62℃ annealing for 30 s, 68℃ extension for 30 s-3 min, 33 cycles; 68℃ extension for 10 min, and incubation at 12℃. High-fidelity KOD FX Neo was purchased from TOYOBO.

[0063] The reagents and formulations required for subcellular localization in the following examples are detailed in Table 1 below.

[0064] Table 1. Reagents and formulations required for subcellular localization experiments

[0065] The winter wheat 'KN199' in the following examples is a nationally approved wheat variety bred by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and has been recorded in: Li Junming et al. High-yielding and widely adaptable new wheat variety - KN199. Journal of Triticeae Crops 27.2 (2007):1. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of this invention and cannot be used for other purposes.

[0066] The PI272527 material in the following examples has the unified number MY020988 in the "Catalogue of Chinese Wheat Genetic Resources" and has been described in: Szabo-Hever A, et al. Genetic diversity and resistance tofusarium head blight in synthetic hexaploid wheat derived from Aegilopstauschii and diverse Triticum turgidum subspecies. Front Plant Sci. 2018, 9:1829. doi: 10.3389 / fpls.2018.01829. This biological material is available to the public from the applicant and is only for the purpose of repeating the experiments of this invention and shall not be used for other purposes.

[0067] The pEASY-Blunt Zero cloning vector used in the following examples was purchased from Beijing TransGen Biotech Co., Ltd., catalog number: CB501-01.

[0068] All restriction endonucleases used in the following examples were purchased from New England Biolabs.

[0069] The pGBKT7, pQBV3, P2GW7, GAL4-LUC, and pRLC mentioned in the following examples are described in: Liu, Pan. Study on the molecular genetic regulation mechanism of Q, a key gene for wheat spike development [D]. Chinese Academy of Agricultural Sciences, 2019. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0070] The pCambia3301 and pAN580 in the following examples are described in: Dong C, et al. Tiller Number1 encodes an ankyrin repeat protein that controls tillering in bread wheat. NatCommun. 2023,14(1):836. doi: 10.1038 / s41467-023-36271-z. This biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of this invention and may not be used for any other purpose.

[0071] Example 1: Obtaining the TaLBD30 gene TaLBD30 See gene structure Figure 1The open reading frame (ORF) of this gene is 789 bp (nucleotide sequence as shown in SEQ ID No:2), encoding a protein containing 262 amino acids (amino acid sequence as shown in SEQ ID No:1). The gene contains 2 exons and 1 intron, and its genome sequence is SEQ ID No:3.

[0072] 1. TaLBD30 protein localization study (1) Design specific primers (TaLBD30-ORF-F2:5'-GATCGATCTAGCACAGCAATGA-3'; (TaLBD30-ORF-R3:5'-CGAAAACCGGCCAATGCTTC-3') to amplify cDNA from the above tissues. TaLBD30 The full-length CDS of the gene was ligated into the pEASY-Blunt Zero cloning vector. After successful sequencing, the plasmid was extracted for use (the plasmid miniprep kit was purchased from Beijing TransGen Biotech Co., Ltd., catalog number: EM101-02).

[0073] (2) The homologous recombination adapter designed in the laboratory based on the pAN580 vector sequence was added to the target gene-specific primers to amplify the CDS sequence. The gel recovery yielded a fragment of about 800 bp. The homologous recombination adapter primers were pAN580-LBD-F: 5'-agcccagatcactagtATGAGCTCGGGCGGCGGC-3' and pAN580-LBD-R: 5'-tgctcaccatggatccTCTTGGATGCGGCGGCGGT-3'.

[0074] (3) Use the pAN580 stored in the laboratory as an empty load. Spe I-HF and BamH I-HF was used for double digestion, and the specific system is shown in Table 2.

[0075] Table 2. Double enzyme digestion reaction system

[0076] (4) The fragment recovered by double enzyme digestion is mixed with the fragment recovered by gel in step (2) at a ratio of 1:3, and then ligated and transformed using In-Fusion seamless cloning enzyme.

[0077] (5) After transformation, the correct bacterial culture and plasmid were obtained by bacterial culture PCR and sequencing and then put into use.

[0078] (6) Extract high-quality, high-concentration plasmids according to the instructions of the Wieglas Large Plasmid Extraction Kit (Catalog No.: N001) for later use.

[0079] (7) Isolation of wheat protoplasts and plasmid transfection, detailed steps can be found in (Dong C, et al. TillerNumber1 encodes an ankyrin repeat protein that controls tillering in breadwheat. Nat Commun. 2023,14(1):836. doi: 10.1038 / s41467-023-36271-z.).

[0080] (8) GFP detection: Using a flattened pipette tip, 20 μL of transfection solution was dropped onto a clean glass slide, and a coverslip was gently placed on top to avoid air bubbles. The expression of wheat protoplasts was detected and photographed using a laser confocal scanning microscope (Zeiss Lsm700, Germany).

[0081] To determine the specific location of the TaLBD30 protein in the cell and to provide foundational information for subsequent gene function studies, a [construction / engineering] was performed. TaLBD30 The CDS sequence of the CDS is fused with eGFP in the expression vector pAN580-TaLBD30-eGFP. The structure of the fusion expression vector pAN580-TaLBD30-eGFP is described as follows: It is expressed in the pAN580 vector... Spe I and BamH I. A DNA fragment with the sequence SEQ ID No:2 is inserted between the two restriction enzyme sites, while keeping the other sequences of the pAN580 vector unchanged, to obtain the recombinant vector. The pAN580-TaLBD30-eGFP vector can express the TaLBD30 protein, the amino acid sequence of which is SEQ ID No:1.

[0082] Simultaneously, NLS-mCherry, which is expressed by fusing a marker gene with known location information with mCherry, was constructed.

[0083] The structure of the fusion expression vector NLS-mCherry is described below: it originates from the starting vector pAN580. Spe I and Bgl II. A DNA fragment with the sequence SEQ ID No:7 was inserted between the two restriction sites, while keeping the other sequences of the starting vector pAN580 unchanged to obtain the recombinant vector.

[0084] Plasmids pAN580-eGFP and pAN580-TaLBD30-eGFP were mixed in equal amounts with the nuclear localization signal plasmid NLS-mCherry and co-transformed into wheat protoplasts for subcellular localization. Laser confocal microscopy revealed that TaLBD30 was localized in the cell nucleus. Figure 2 ).

[0085] 2. Verify the self-activation activity of TaLBD30 protein. To verify whether the TaLBD30 protein has self-activation activity, TaLBD30 The full-length CDS sequence of the gene was cloned into the pGBKT7 vector of the yeast two-hybrid system and then transformed into yeast. The specific steps are as follows: (1) The homologous recombination adapter designed in the laboratory based on the pGBKT7 vector sequence was added to the target gene-specific primers to amplify the CDS sequence. The fragment of about 800 bp was obtained by gel recovery. The homologous recombination adapter primers are as follows: pGBKT7-LBD-F: 5'-atctcagaggaggacctgcatatgATGAGCTCGGGCGGCGGC-3'; pGBKT7-LBD-R: 5'-aggtcgacggatccccgggaattcTCTTGGATGCGGCGGCGT-3'.

[0086] (2) Use the pGBKT7 stored in the laboratory without load. BamH I-HF and EcoR I-HF was used for double digestion, and the specific system is shown in Table 3.

[0087] Table 3. Double enzyme digestion reaction system

[0088] (3) The fragments recovered by double enzyme digestion and the fragments recovered by gel extraction in step (2) were mixed at a ratio of 1:3 and then ligated and transformed using In-Fusion seamless cloning enzyme. After transformation, the correct bacterial culture and the correct plasmid were obtained by bacterial PCR and sequencing. The plasmid was then extracted and set aside for use.

[0089] The structure of the recombinant vector pGBKT7-TaLBD30 is described as follows: It originates from the vector pGBKT7... BamH I and EcoR I. A DNA fragment with the sequence SEQ ID No:2 was inserted between the two restriction sites, and the other sequences of the vector pGBKT7 remained unchanged to obtain the recombinant vector.

[0090] (4) Mix plasmid pGBKT7 and plasmid pGBKT7-TaLBD30 with plasmid pGADT7 in equal amounts, and transform yeast according to the yeast transformation experimental method in the product manual of Y2HGold Chemically Competent Cell of Shanghai Weidi Biotechnology Co., Ltd.

[0091] Pick a piece of yeast from the culture medium where colonies have grown, and dilute it with pre-sterilized ddH2O at different concentration gradients. Take 3-10 μL of the bacterial solution from high concentration to low concentration and spot it onto two-deficient (SD-Trp-Leu) and four-deficient (SD-Trp-Leu-His-Ade) culture media in the corresponding order. Incubate at 29℃ for 48-96 h to observe the growth of yeast colonies and take pictures for transcriptional activity analysis.

[0092] The transcriptional activation activity of TaLBD30 was determined by detecting the growth of yeast on different auxotrophic media, and it was found that the TaLBD30 protein does not have transcriptional autoactivation activity. Figure 3 ).

[0093] 3. TaLBD30 Gene transcriptional activity For research TaLBD30 Gene transcriptional activity, construction of effector plasmids ( Figure 4 (a)

[0094] (1) pGBKT7 constructed as described above - Using the TaLBD30 vector as a template, homologous recombination adapters designed based on the pQBV3 vector sequence were added to the target gene-specific primers to amplify the gene fused with the GAL4-BD binding domain. TaLBD30 The gene CDS sequence was obtained, and a fragment of approximately 1350 bp was recovered by gel extraction. The homologous recombination adapter primers are as follows: pQBV3-BD-LBD-F: 5'-aaaagcaggctcaggggatatcATGAAGCTACTGTCTTCTATCGAACAA-3'; pQBV3-BD-LBD-R: 5'-aagctgggtgcagggcgatTCTTGGATGCGGCGGCGT-3'; (2) Using the pGBKT7 vector as a template, homologous recombination adapters designed based on the pQBV3 vector sequence were added to the target gene-specific primers to amplify the sequence fused with the GAL4-BD binding domain. The fragment of approximately 550 bp was obtained by gel extraction. The homologous recombination adapter primers are as follows: pQBV3-BD-F: 5'-aaaagcaggctcaggggatatcATGAAGCTACTGTCTTCTATCG-3'; pQBV3-BD-R: 5'-aagctgggtgcagggcgatGAATTCGGCCTCCATGGCC-3'.

[0095] (3) Use the pQBV3 stored in the laboratory without load. EcoRV-HF was used for enzyme digestion, and the specific system is shown in Table 4.

[0096] Table 4. Enzyme digestion reaction system

[0097] (4) The fragments recovered by enzyme digestion are mixed with the fragments recovered by gel in steps (1) and (2) at a ratio of 1:3, and then ligated and transformed using In-Fusion seamless cloning enzyme.

[0098] (5) After transformation, the correct bacterial culture and plasmid were obtained through bacterial culture PCR and sequencing. The plasmid was extracted for later use. The plasmid fused with the GAL4-BD binding domain was then used in the LR reaction (kit purchased from Invitrogen). TaLBD30 The gene CDS sequence and the GAL4-BD binding domain sequence were constructed into the P2GW7 vector to form the effect plasmid P2GW7-GAL4-BD. - The reaction system of TaLBD30 and P2GW7-GAL4-BD is shown in Table 5.

[0099] The structure of pQBV3-GAL4-BD-TaLBD30 is described as follows: it is based on the starting carrier pQBV3. EcoR The recombinant vector was obtained by inserting a DNA fragment with the sequence SEQ ID No:5 between the V restriction sites while keeping the other sequences of the vector pQBV3 unchanged.

[0100] The structure of pQBV3-GAL4-BD is described as follows: it is based on the starting carrier pQBV3. EcoR A DNA fragment with the sequence SEQ ID No:6 was inserted between the V restriction sites, and the other sequences of the vector pQBV3 remained unchanged to obtain the recombinant vector.

[0101] Effect plasmid P2GW7-GAL4-BD - The structure of TaLBD30 is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence SEQ ID No:5 between 5'-tttgtacaaaaaagcaggct-3' and 5'-tttgtacaagaaagctgggt-3' of the starting vector P2GW7 via an LR reaction, while keeping the other sequences of the starting vector P2GW7 unchanged.

[0102] The structure of the effect plasmid P2GW7-GAL4-BD is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with sequence SEQ ID No:6 between 5'-tttgtacaaaaaagcaggct-3' and 5'-tttgtacaagaaagctgggt-3' of the starting vector P2GW7 via an LR reaction, while keeping the other sequences of the starting vector P2GW7 unchanged.

[0103] Table 5. LR Reaction System

[0104] (6) Extract high-quality, high-concentration plasmids according to the instructions of the Wieglas large plasmid extraction kit.

[0105] (7) After transfecting tobacco protoplasts with the reporter plasmid and the effect plasmid (detailed steps can be found in: Liu Pan. Study on the molecular genetic regulation mechanism of Q, a key gene for wheat spike development [D]. Chinese Academy of Agricultural Sciences, 2019.), the enzyme activity values ​​of LUC and REN were measured sequentially on an ELISA reader using the dual-luciferase reporter gene detection system of Promega. The relative luciferase value was calculated by dividing the LUC value by the REN value.

[0106] The results showed that: compared with the CK group ( 35s::GAL4-TATA-LUC , 35s::GAL4-BD , 35s::REN Compared to the TaLBD30 group () 35s::GAL4-TATA-LUC , 35s::GAL4-BD-TaLBD30 , 35s::REN The relative fluorescein value of TaLBD30 was significantly reduced, indicating that TaLBD30 is a transcription factor with transcriptional repressive activity. Figure 4 (b)

[0107] Example 2: Verification using transgenic technology TaLBD30 The regulatory role of genes on plant architecture Leaves of PI272527 material were selected, DNA was extracted using the CTAB method, and then amplified by PCR. TaLBD30 The full-length gene plus a 2709bp promoter was used to construct the recombinant vector pCambia3301 into the pCambia3301-. TaLBD30.

[0108] pCambia3301- TaLBD30 The structure is described as follows: it is located at the restriction enzyme site of the pCambia3301 vector. EcoR I and Pml A recombinant vector was obtained by inserting a DNA fragment with the sequence SEQ ID No:4 between I and II, while keeping the other sequences of the pCambia3301 vector unchanged.

[0109] For TaLBD30 The primers used for gene vector construction are shown in Table 6 below: Table 6 TaLBD30 Primer information used in gene vector construction

[0110] The Agrobacterium-mediated transgenic technology was utilized; for specific methods, please refer to the following literature: Ishida Y, et al. Wheat ( Triticum aestivum L.) transformation using immature embryos. Methods Mol Biol. 2015;1223:189-98. doi: 10.1007 / 978-1-4939-1695-5_15. PMID:25300841. Recombinant Agrobacterium EHA105 / pCambia3301- TaLBD30 The recombinant vector pCambia3301- TaLBD30 Recombinant bacteria obtained by introducing Agrobacterium tumefaciens EHA105.

[0111] Positive plants were screened using transgenic test strips. A suitable amount of leaves from normally grown wheat plants at the three-leaf stage were placed in a 2.0 mL centrifuge tube. After sampling, an appropriate amount (300-600 μL) of extraction solution was added to the centrifuge tube, and the samples were tested using a PAT / ba rapid test strip (Shanghai Youlong Biotechnology Co., Ltd.). Results were read after approximately 10 minutes; one line indicated a negative plant, and two lines indicated a positive plant.

[0112] The identification results are as follows: A total of 10 positive plants and 2 negative plants were identified in the T0 generation. At the heading stage of the T1 generation, KN199 and transgenic positive plants were collected. TaLBD30 pro::TaLBD30-3 , TaLBD30pro::TaLBD30-4 and TaLBD30pro::TaLBD30-5 The lower stalks and rachis of the ear were analyzed by qRT-PCR. TaLBD30 The primers for gene fluorescence quantitative PCR amplification are shown in Table 7 below.

[0113] The relative expression level of the TaLBD30 gene was analyzed using Roche quantitative PCR. TaGAPDH The reaction system, used as an internal reference gene, is shown in Table 8. The reaction program was: 95℃, 10 s; 95℃, 5 s; 60℃, 30 s; 72℃, 10 s; 40 cycles. Each experiment used 3 biological samples, and each biological sample had 3 technical replicates. Data are presented as the mean ± standard error of the 3 biological replicates. Relative expression levels were calculated using a 2-1... -ΔΔCTMethods: Experimental data were analyzed using Graphpad Prism software. Results showed that the Ct values ​​were relatively stable, with good repeatability, and the amplification curves were normal, indicating that the experimental data were reliable.

[0114] The results showed that among the three positive strains... TaLBD30 The expression levels of all three were significantly upregulated. Figure 5 (c)

[0115] Table 7 TaLBD30 Primer information for gene fluorescence quantitative PCR amplification

[0116] Table 8. Real-time quantitative PCR system

[0117] To further determine the function of candidate genes, the plant height and ear length phenotypes of the T1 generation were examined.

[0118] KN199 and three positive lines were planted and treated in a vernalization chamber at 4℃ for 4 weeks. After treatment, they were transferred to an artificial climate chamber for cultivation under the following conditions: 23℃, 16 hours of light, and 8 hours of darkness. Twenty plants were planted for each material, and ten individual plants from each line were selected for statistical analysis.

[0119] The results showed that the plant height and spike length of the three positive lines during the wheat grain-filling stage were significantly lower than those of KN199. Figure 5 (a and b). This shows that overexpression... TaLBD30 It significantly reduced plant height and ear length.

[0120] 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. A protein, wherein the protein is any of the following: A1) A protein with the amino acid sequence shown in SEQ ID No:1; A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1). A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

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

3. A biomaterial relating to the protein of claim 1 or 2, wherein the biomaterial is any one of the following: B1) A nucleic acid molecule encoding the protein described in claim 1 or 2; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2); C1) A nucleic acid molecule that inhibits, reduces, or silences the expression of the gene encoding the protein described in claim 1 or 2; C2) expresses the gene encoding the nucleic acid molecule described in C1); C3) contains an expression cassette containing the gene encoding described in C2); C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3); C5) A recombinant microorganism containing the encoding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4); C6) A transgenic plant cell line containing the encoding gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4); C7) Transgenic plant tissue containing the encoding gene described in C2), or transgenic plant tissue containing the expression cassette described in C3), or transgenic plant tissue containing the recombinant vector described in C4); C8) A transgenic plant organ containing the encoding gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

4. The biomaterial according to claim 3, characterized in that, B1) The nucleic acid molecule described is a gene as shown in E1) or E2) below: E1) The coding sequence is a cDNA molecule of SEQ ID No:2; E2) The genome sequence is the DNA molecule of SEQ ID No:

3.

5. The use of the protein or gene expression regulator or substance regulating the activity or content of said protein as described in claim 1 or 2 in any of the following: U1) Application in regulating plant architecture; U2) Applications in the preparation of products that regulate plant architecture; U3) Applications in plants for cultivating plant shape; U4) Application in the preparation of products using cultivated plant varieties; U5) Applications in plant breeding.

6. The application according to claim 5, characterized in that, The substance regulating gene expression or the substance regulating protein activity or content is a biological material related to the protein, and the biological material is any one of B1) to B7) below: B1) A nucleic acid molecule encoding the protein described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1) or transgenic plant organs containing the expression cassette described in B2).

7. A method for regulating plant architecture, characterized in that, This includes regulating plant architecture by controlling the activity and / or content of the protein described in claim 1 or 2 in the recipient plant, and / or the expression level of the gene encoding the protein described in claim 1 or 2.

8. The method according to claim 7, characterized in that, The regulation of the activity and / or content of the protein described in claim 1 or 2 in the recipient plant, or / and the expression level of the gene encoding the protein described in claim 1 or 2, includes introducing the gene encoding the protein into the recipient plant to obtain a target plant with a plant height and spike length lower than that of the recipient plant; the gene encoding the protein described in claim 1 or 2.

9. Methods for cultivating plants with altered plant shapes, including: 1) Increase, enhance and / or upregulate the expression level of the gene encoding the protein described in claim 1 in the recipient plant, or / and increase, enhance and / or upregulate the activity and / or content of the gene encoding the protein described in claim 1, to obtain plants with reduced plant height and spike length. 2) Inhibit or reduce or silence the expression level of the gene encoding the protein described in claim 1 in the recipient plant, and / or inhibit or reduce or silence the activity and / or content of the gene encoding the protein described in claim 1, to obtain plants with increased plant height and spike length.

10. The method according to any one of claims 7-9, characterized in that, The plant is any one of the following: N1) Monocotyledonous plants; N2) Plants of the order Poales; N3) Gramineae plants; N4) Plants of the Triticum genus; N5) Wheat.

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