TaLBD30 protein and its coding gene in regulating wheat plant type

Abstract

The application discloses TaLBD30 protein and an application of a coding gene thereof in regulating a wheat plant type, and belongs to the technical field of biotechnology, and particularly relates to the application of the TaLBD30 protein and the coding gene thereof in regulating the wheat plant type. The protein is any one of the following: A1) a protein with an amino acid sequence as shown in SEQ ID No:1; A2) a protein obtained by substitution, deletion and / or addition of amino acid residues on the protein of A1), the protein being identical to the protein shown in A1) by more than 80% and having the same function; and A3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of A1) or A2). The TaLBD30 is a transcription factor with transcription inhibition activity, and when overexpressed TaLBD30 , the wheat plant height and ear length are significantly reduced compared with a wild type. TaLBD30 ​

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 Technology

[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:

[0008] A1) A protein with the amino acid sequence shown in SEQ ID No:1;

[0009] A2) a protein having 75% or more identity to the protein shown in A1) and having a plant plant type regulating function, which is obtained by substitution, deletion and / or addition of amino acid residues of the protein of A1); for example, one skilled in the art can obtain a protein mutant having the same function as the amino acid sequence shown in SEQ ID No: 1 by substitution, deletion and / or addition of one or more amino acids without affecting its activity, according to conventional technical means in the art such as the amino acid sequence shown in SEQ ID No: 1 and conservative substitution of amino acids;

[0010] A3) a fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of the protein of A1) or A2).

[0011] The protein of A1) above is named TaLBD30.

[0012] In order to facilitate purification or detection of the protein in A1), a tag protein can be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No: 1 in the sequence listing.

[0013] The above-mentioned protein can be artificially synthesized, or the encoding gene thereof can be synthesized first and then expressed biologically.

[0014] The tag protein includes but is not limited to: a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), an MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, an HA tag protein, a Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein) or an AviTag tag protein.

[0015] One of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein TaLBD30 of the present application using known methods, such as directed evolution or point mutation. Those nucleotides artificially modified, having 75% or more identity to the nucleotide sequence of the protein TaLBD30 isolated from the present application, as long as they encode the protein TaLBD30 and have the function of the protein TaLBD30, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application.

[0016] The above-mentioned 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

[0017] In the present text, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence or a nucleotide sequence can be determined using homology search sites on the Internet, such as the BLAST page of the NCBI home page website. For example, the identity of a pair of amino acid sequences or nucleotide sequences can be calculated by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and performing a search in Advanced BLAST 2.1, and then the value of the identity (%) is obtained.

[0018] In the present text, the identity of more than 80% 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.

[0019] In the present text, the identity of more than 90% can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0020] In the above, the protein is derived from Triticum dicoccum L. (cultivated emmer) Triticum dicoccum Schuebl . ) PI272527.

[0021] The present application also provides biological materials related to the above-mentioned protein, which can be any one of the following:

[0022] B1) a nucleic acid molecule encoding the above-mentioned protein;

[0023] B2) an expression cassette containing the nucleic acid molecule of B1);

[0024] B3) a recombinant vector containing the nucleic acid molecule of B1), or a recombinant vector containing the expression cassette of B2);

[0025] B4) a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the expression cassette of B2), or a recombinant microorganism containing the recombinant vector of B3);

[0026] B5) a transgenic plant cell line containing the nucleic acid molecule of B1), or a transgenic plant cell line containing the expression cassette of B2);

[0027] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0028] B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2);

[0029] C1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the genes encoding the proteins described above;

[0030] C2) expresses the gene encoding the nucleic acid molecule described in C1);

[0031] C3) contains an expression cassette containing the gene encoding described in C2);

[0032] C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3);

[0033] 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);

[0034] 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);

[0035] 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);

[0036] 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).

[0037] In the above-mentioned biological materials, the nucleic acid molecule described in B1) may be a gene as shown in E1) or E2) below:

[0038] E1) The coding sequence is a cDNA molecule of SEQ ID No:2;

[0039] E2) The genome sequence is the DNA molecule of SEQ ID No:3.

[0040] 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.

[0041] The nucleotide sequence shown in SEQ ID No: 2 is the nucleotide sequence of a gene (CDS) encoding the protein TaLBD30.

[0042] The gene described in the present application TaLBD30 The gene can be any nucleotide sequence capable of encoding the protein TaLBD30. Considering the degeneracy of codons and the preference of codons in different species, a person skilled in the art can use codons suitable for expression in a specific species as needed.

[0043] B1) The nucleic acid molecule can also include a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No: 2.

[0044] B1) The nucleic acid molecule can also include a nucleic acid molecule having more than 95% identity with the nucleotide sequence shown in SEQ ID No: 2 and derived from the same species.

[0045] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0046] The vectors described herein are well known to those skilled in the art, including but not limited to: plasmids, bacteriophages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e. cosmids), Ti plasmids or viral vectors. Specifically, the vector pCambia3301 can be used to construct a transformation vector; pQBV3 can be used to construct a gateway reaction vector; pAN580 can be used to construct a subcellular localization vector; pGADT7 and pGBKT7 can be used to construct a self-activation activity analysis vector; P2GW7, GAL4-LUC and pRLC can be used to construct a luciferase transient transcription experiment vector; pEASY-Blunt Zero cloning vector can be mainly used for gene cloning and sequencing analysis.

[0047] A recombinant expression vector containing the gene can be constructed using existing plant expression vectors. TaLBD30 The plant expression vectors include but are not limited to binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. The plant expression vectors can also contain a 3' untranslated region of the foreign gene, i.e. a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as the 3' untranslated region of the Agrobacterium crown gall inducing (Ti) plasmid gene (such as the Nos gene of nopaline synthase), the plant gene (such as the soybean storage protein gene) all have similar functions.

[0048] The useTaLBD30 When 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] The microorganisms mentioned in this article may be yeast, bacteria, algae, or fungi. Among them, bacteria may originate from the genus *Escherichia* (…).Escherichia ), Erwinia (E. Erwinia ), Agrobacterium (A. Agrobacterium ), Flavobacterium (F. Flavobacterium ), Alcaligenes (A. Alcaligenes ), Pseudomonas (P. Pseudomonas ), Bacillus (B. Bacillus ), etc. Specifically, it can be Agrobacterium tumefaciens EHA105 and Trans-T1 Escherichia coli competence.

[0053] The vector pCambia3301- TaLBD30 expresses the TaLBD30 protein shown in SEQ ID No: 1 in the sequence listing.

[0054] In a specific embodiment, the recombinant microorganism can be specifically a recombinant Agrobacterium EHA105 / pCambia3301- TaLBD30 .

[0055] The recombinant Agrobacterium EHA105 / pCambia3301- TaLBD30 is a recombinant bacterium obtained by introducing the recombinant vector pCambia3301- TaLBD30 into Agrobacterium tumefaciens EHA105.

[0056] The plant obtained by the above method can be a transgenic plant, or a plant obtained by conventional breeding techniques such as hybridization. In the above method, the transgenic plant is understood to include not only the first generation to the second generation transgenic plant, but also its offspring. For the transgenic plant, the gene can be propagated in the species, or the gene can be transferred into other varieties of the same species using conventional breeding techniques, especially including commercial varieties.

[0057] The transgenic plant includes seeds, callus, whole plants, and cells.

[0058] The present application also provides the use of the protein TaLBD30 or the expression substance of the regulatory gene described above, or the substance that regulates the activity or content of the protein, in any of the following:

[0059] U1) in regulating plant architecture;

[0060] U2) in preparing a product for regulating plant architecture;

[0061] U3) in cultivating a plant with plant architecture;

[0062] U4) in preparing a product for cultivating a plant with architecture;

[0063] U5) in plant breeding.

[0064] In the present application, the substance that regulates the activity and / or content of the protein can be a substance that regulates the expression of a gene encoding the protein TaLBD30.

[0065] In the above applications, the substance that regulates the expression of a gene or the substance that regulates the activity or content of the protein can be a biological material related to the protein, which can be the biological material described above.

[0066] In the above, the substance that regulates the expression of a gene can be a substance that regulates at least one of the following six regulations:

[0067] 1) regulation at the transcription level of the gene;

[0068] 2) regulation after the transcription of the gene (i.e. regulation of splicing or processing of the primary transcript of the gene);

[0069] 3) regulation of RNA transport of the gene (i.e. regulation of mRNA transport from the nucleus to the cytoplasm of the gene);

[0070] 4) regulation of translation of the gene;

[0071] 5) regulation of mRNA degradation of the gene;

[0072] 6) post-translational regulation of the gene (i.e. regulation of the activity of the protein translated from the gene).

[0073] The present application also provides a method for regulating the plant architecture, which comprises regulating the activity and / or content of the protein described above or the expression of the gene encoding the protein in a plant of interest to regulate the plant architecture.

[0074] In the above method, the regulation of the activity and / or content of the protein TaLBD30 or the expression of the gene encoding the protein in a plant of interest comprises introducing the gene encoding the protein TaLBD30 into a plant of interest. TaLBD30 to change the plant architecture; the TaLBD30 gene encodes the protein TaLBD30.

[0075] The introduction refers to introduction by recombination means, including but not limited to Agrobacterium-mediated transformation, biolistic method, electroporation, in planta technique, etc.

[0076] In the above applications and methods, the regulation can be enhancement, increase or up-regulation.

[0077] In the above-mentioned applications and methods, the regulation can be inhibition, reduction or silencing.

[0078] The present application also provides a method for breeding a plant with altered plant type, comprising:

[0079] 1) increasing, enhancing and / or up-regulating the expression level of the gene encoding the protein described above in a plant of interest, or / and increasing, enhancing and / or up-regulating the activity and / or content of the gene encoding the protein described above, to obtain a plant with reduced plant height and ear length;

[0080] 2) inhibiting or reducing or silencing the expression level of the gene encoding the protein described above in a plant of interest, or / and inhibiting or reducing or silencing the activity and / or content of the gene encoding the protein described above, to obtain a plant with increased plant height and ear length.

[0081] As an embodiment of the present application, the method for breeding a plant with reduced plant height and ear length comprises the following steps:

[0082] (1) constructing an expression vector containing the gene sequence as shown in SEQ ID No: 4; TaLBD30

[0083] (2) introducing the expression vector constructed in step (1) into a plant;

[0084] (3) obtaining a plant with reduced plant height and ear length through screening and identification.

[0085] In the present application, the purpose of plant breeding includes breeding a plant with increased / reduced plant type.

[0086] In the present application, the plant type includes plant height and ear morphology.

[0087] In the above-mentioned applications or methods, the plant is any one of the following:

[0088] N1) a monocotyledonous plant;

[0089] N2) a plant of the order Poales;

[0090] N3) a plant of the family Poaceae;

[0091] N4) a plant of the genus Triticum;

[0092] N5) wheat.

[0093] The present application studies the TaLBD30 gene in wheat TaLBD30 The present application studies the TaLBD30 gene in wheat TaLBD30 It is found that, compared with the wild type, overexpression of the TaLBD30 gene​TaLBD30 After that, plant height and spike length were significantly reduced. BRIEF DESCRIPTION OF DRAWINGS

[0094] TaLBD30 The gene structure of TaLBD30. The gene has two exons and one intron. Figure 1

[0095] Subcellular localization of TaLBD30 in wheat protoplasts. TaLBD30 is localized in the nucleus. TaLBD30

[0096] Transcription factor TaLBD30 self-activation activity analysis. SD-Trp-Leu represents a two-lacking yeast medium lacking tryptophan (Trp) and leucine (Leu); SD-Trp-Leu-His-Ade represents a four-lacking yeast medium lacking tryptophan (Trp), leucine (Leu), histidine (His), and adenine (Ade). Figure 2

[0097] Transcriptional activity analysis of TaLBD30. a: plasmid for transforming protoplasts, the control group transformed plasmid includes Figure 3 (GAL4-LUC), Figure 4 (P2GW7 35s::GAL4-TATA-LUC GAL4-BD), - (pRLC), abbreviated as CK group; the experimental group transformed plasmid includes 35s::GAL4-BD (GAL4-LUC), 35s::REN (P2GW7 35s::GAL4-TATA-LUC GAL4-BD - ), 35s::GAL4-BD-TaLBD30 (pRLC), abbreviated as TaLBD30 group, the ratio of the transformed plasmid of CK group and TaLBD30 group is 1:6:1; b: relative fluorescence value of the control group and the experimental group, n = 8. Error bars represent ± SD (***P < 0.001, ****P < 0.0001, standard t test). 35s::REN

[0098] Phenotype identification of transgenic materials. a: KN199 and transgenic lines Figure 5 TaLBD30 pro::TaLBD30- 3 TaLBD30 pro::TaLBD30-4 and TaLBD30 pro::TaLBD30-5 phenotype in the grain filling stage, the scale is 10 cm; b: data statistics of plant height and spike length of KN199 and transgenic lines, n = 10; c: relative expression of KN199 and transgenic lines of stem below ear and rachis TaLBD30 gene, n = 3. Error bars represent ± SD (***P < 0.001, ****P < 0.0001, standard t test).​​ Detailed Implementation

[0099] 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.

[0100] 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.

[0101] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0102] 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.

[0103] 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.

[0104] The reagents and formulations required for subcellular localization in the following examples are detailed in Table 1 below.

[0105] Table 1. Reagents and formulations required for subcellular localization experiments

[0106]

[0107] Winter wheat ‘KN199’ in the following examples is a national registered wheat variety bred by the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and has been described in: Li Junming, et al. High-yield and widely adaptable new wheat variety—KN199. Journal of Triticeae Crops 27.2 (2007): 1. The biological material is available from the applicant for the purpose of repeating the experiments of the present invention only, and cannot be used for other purposes.

[0108] 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 to fusarium head blight in synthetic hexaploid wheat derived from Aegilops tauschii and diverse Triticum turgidum subspecies. Front Plant Sci. 2018, 9: 1829. doi: 10.3389 / fpls.2018.01829. The biological material is available from the applicant for the purpose of repeating the experiments of the present invention only, and cannot be used for other purposes.

[0109] pEASY-Blunt Zero cloning vector in the following examples was purchased from Beijing Zixingjin Biotechnology Co., Ltd., item number: CB501-01.

[0110] Various restriction enzymes in the following examples were purchased from New England Biolabs.

[0111] pGBKT7, pQBV3, P2GW7, GAL4-LUC and pRLC in the following examples have been described in: Liu Pan. Molecular genetic regulation mechanism of key genes Q in wheat spike development[D]. Chinese Academy of Agricultural Sciences, 2019. The biological material is available from the applicant for the purpose of repeating the experiments of the present invention only, and cannot be used for other purposes.

[0112] pCambia3301 and pAN580 in the following examples are described in Dong C, et al. Tiller Number1encodes an ankyrin repeat protein that controls tillering in bread wheat. Nat Commun. 2023, 14(1): 836. doi: 10.1038 / s41467-023-36271-z. The biological material is available from the applicant for the purpose of repeating the experiments of the present application only and cannot be used for other purposes.

[0113] Example 1, Obtaining of TaLBD30 gene

[0114] TaLBD30 The gene structure is shown in Figure 1 , the open reading frame (ORF) of the 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 the genomic sequence is SEQ ID No: 3.

[0115] 1. Localization study of TaLBD30 protein

[0116] (1) Design specific primers (TaLBD30-ORF-F2: 5'-GATCGATCTAGCACAGCAATGA-3'; (TaLBD30-ORF-R3: 5'-CGAAAACCGGCCAATGCTTC-3', amplified from the cDNA of the above tissues TaLBD30 The full-length CDS of the gene was connected to the pEASY-Blunt Zero cloning vector, and after sequencing, the plasmid was extracted for use (plasmid extraction reagent kit purchased from Beijing Zison Biotechnology Co., Ltd., item number: EM101-02).

[0117] (2) The homologous recombination adapter designed by the laboratory according to the pAN580 vector sequence was added to the specific primers of the target gene to amplify the CDS sequence, and a fragment of about 800 bp was recovered by gel. The homologous recombination adapter primers are pAN580-LBD-F: 5'-agcccagatcactagtATGAGCTCGGGCGGCGGC-3', pAN580-LBD-R: 5'-tgctcaccatggatccTCTTGGATGCGGCGGCGGT-3'.

[0118] (3) The pAN580 empty vector preserved in the laboratory was used Spe I-HF andBamH I-HF was double-digested, and the specific system is shown in Table 2.

[0119] Table 2, double enzyme digestion reaction system

[0120]

[0121] (4) The fragments recovered by double enzyme digestion were mixed with the fragments recovered by step (2) according to a ratio of 1:3, and then In-Fusion seamless cloning enzyme was used for ligation and transformation.

[0122] (5) After transformation, correct bacterial liquid and correct plasmid were obtained by bacterial liquid PCR and sequencing for standby use.

[0123] (6) High-quality and high-concentration plasmids were extracted according to the instructions of the WIGLAS large plasmid extraction kit (catalog number: N001) for standby use.

[0124] (7) Isolation of wheat protoplasts and plasmid transfection, detailed steps can be found in (Dong C, et al. Tiller Number 1 encodes an ankyrin repeat protein that controls tillering in bread wheat. Nat Commun. 2023, 14(1): 836. doi: 10.1038 / s41467-023-36271-z.).

[0125] (8) GFP detection: 20 μL of transfection solution was taken with a flat gun head and dropped on a clean glass slide, and a cover glass was gently covered to avoid air bubbles. Laser confocal scanning microscope (Zeiss Lsm700, Germany) was used to detect the fusion expression of wheat protoplasts and take pictures.

[0126] To determine the specific location of TaLBD30 protein in cells, as basic information for subsequent research on gene function, the vector pAN580-TaLBD30-eGFP for fusion expression of CDS sequence of TaLBD30 TaLBD30 and eGFP was constructed. The structure of the fusion expression vector pAN580-TaLBD30-eGFP is described as follows: the DNA fragment of SEQ ID No: 2 is inserted between the two enzyme digestion sites of pAN580 vector Spe I and BamH I, and the other sequences of pAN580 vector remain unchanged to obtain a recombinant vector. The pAN580-TaLBD30-eGFP vector can express TaLBD30 protein, and the amino acid sequence is SEQ ID No: 1.

[0127] Meanwhile, the Marker gene with known positioning information was fused with mCherry to express NLS-mCherry.

[0128] The structure of the fusion expression vector NLS-mCherry is described as follows: the DNA fragment of SEQ ID No: 7 was inserted between the two enzyme cutting sites of I and II of the starting vector pAN580, and the other sequences of the starting vector pAN580 were kept unchanged to obtain the recombinant vector. Spe I and Bgl II, and the inserted sequence was the DNA fragment of SEQ ID No: 7, and the other sequences of the starting vector pAN580 were kept unchanged to obtain the recombinant vector.

[0129] The plasmid pAN580-eGFP and the plasmid pAN580-TaLBD30-eGFP were mixed with the nuclear localization signal plasmid NLS-mCherry in equal amounts, and were co-transformed into wheat protoplasts for subcellular localization. Laser confocal microscopy observation found that TaLBD30 was located in the nucleus. Figure 2 ).

[0130] 2. Verification of the self-activation activity of TaLBD30 protein

[0131] In order to verify whether the TaLBD30 protein has self-activation activity, the full-length CDS sequence of the gene was cloned into the pGBKT7 vector of the yeast two-hybrid system, and the yeast was transformed. The specific steps are as follows: TaLBD30

[0132] (1) The homologous recombination linker designed by the laboratory according to the sequence of the pGBKT7 vector was added to the specific primers of the target gene to amplify the CDS sequence, and a fragment of about 800 bp was obtained by gel recovery. The homologous recombination linker primers are as follows:

[0133] pGBKT7-LBD-F: 5'-atctcagaggaggacctgcatatgATGAGCTCGGGCGGCGGC-3';

[0134] pGBKT7-LBD-R: 5'-aggtcgacggatccccgggaattcTCTTGGATGCGGCGGCGGT-3'.

[0135] (2) The empty pGBKT7 vector preserved in the laboratory was double-digested with I-HF and I-HF, and the specific system is shown in Table 3. BamH I-HF and EcoR I-HF.

[0136] Table 3, double enzyme cutting reaction system

[0137]

[0138] ​(3) The recovered fragments from the double enzyme digestion were mixed with the recovered fragments from the gel in step (2) according to a ratio of 1:3, and then ligated and transformed using In-Fusion Seamless Cloning Enzyme. After transformation, correct bacterial liquid and correct plasmids were obtained through bacterial liquid PCR and sequencing, and the plasmids were extracted for later use.

[0139] The structure of the recombinant vector pGBKT7-TaLBD30 is described as follows: the DNA fragment with the sequence of SEQ ID No: 2 is inserted between the two enzyme digestion sites of I and I of the starting vector pGBKT7, and the other sequences of the vector pGBKT7 remain unchanged to obtain the recombinant vector. BamH I and EcoR I two enzyme digestion sites, and the other sequences of the vector pGBKT7 remain unchanged to obtain the recombinant vector.

[0140] (4) The plasmid pGBKT7 and the plasmid pGBKT7-TaLBD30 were mixed with the same amount of plasmid pGADT7, and yeast transformation was performed according to the yeast transformation experimental method of the Y2HGold Chemically Competent Cell product manual of Shanghai Yidai Biotechnology Co., Ltd.

[0141] A piece of bacteria was picked from the culture medium with a gun head, and different concentration gradients were diluted with sterilized ddH2O. 3-10 μL of bacterial liquid was taken in turn according to the corresponding order and then dropped onto the two-deficient (SD-Trp-Leu) and four-deficient (SD-Trp-Leu-His-Ade) medium, and cultured at 29°C for 48-96 h to observe the growth of yeast colonies and take pictures for transcription activity analysis.

[0142] The transcription activation activity of TaLBD30 was determined by detecting the growth of yeast on different deficient media, and it was found that the TaLBD30 protein did not have transcription self-activation activity. Figure 3 ).

[0143] 3、 TaLBD30 Transcription activity of the gene

[0144] To study TaLBD30 the transcription activity of the gene, the effector plasmid (pGADT7-TaLBD30) was constructed. Figure 4

[0145] (1) The pGBKT7-TaLBD30 vector constructed successfully above was used as a template, a homologous recombination linker designed according to the sequence of the pQBV3 vector was added to the specific primers of the target gene, and the CDS sequence of the gene fused with the GAL4-BD binding domain was amplified to obtain a fragment of about 1350 bp, and the homologous recombination linker primers were as follows: - TaLBD30

[0146] ​​​pQBV3-BD-LBD-F: 5'-aaaagcaggctcaggggatatcATGAAGCTACTGTCTTCTATCGAACAA-3';

[0147] pQBV3-BD-LBD-R: 5'-aagctgggtgcagggcgatTCTTGGATGCGGCGGCGGT-3';

[0148] (2) with pGBKT7 vector as template, according to the homologous recombination linker designed for pQBV3 vector sequence added to the target gene specific primers, amplified the sequence of fusion GAL4-BD binding domain, about 550 bp fragments were obtained by gel recovery, the homologous recombination linker primers are as follows:

[0149] pQBV3-BD-F: 5'-aaaagcaggctcaggggatatcATGAAGCTACTGTCTTCTATCG-3';

[0150] pQBV3-BD-R: 5'-aagctgggtgcagggcgatGAATTCGGCCTCCATGGCC-3'.

[0151] (3) the laboratory preserved pQBV3 empty carrier was digested with V-HF, and the specific system is shown in Table 4. EcoR

[0152] Table 4, enzyme reaction system

[0153]

[0154] (4) the fragments recovered by enzyme digestion were mixed with the fragments recovered by gel in step (1) and step (2) according to the ratio of 1:3, and then the In-Fusion seamless cloning enzyme was used for ligation and transformation.

[0155] (5) after transformation, correct bacterial liquid and correct plasmid were obtained by bacterial liquid PCR and sequencing, and the plasmid was extracted for use. The CDS sequence of the gene fused with GAL4-BD binding domain and the sequence of GAL4-BD binding domain were constructed into P2GW7 vector respectively to form effector plasmid P2GW7-GAL4-BD TaLBD30 TaLBD30 and P2GW7-GAL4-BD, and the reaction system is shown in Table 5. -

[0156] pQBV3-GAL4-BD-TaLBD30 is described as follows: in the starting vector pQBV3 EcoR ​​A DNA fragment with the sequence SEQ ID No:5 was inserted between the V restriction sites, and the other sequences of the vector pQBV3 were kept unchanged to obtain the recombinant vector.

[0157] 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 were kept unchanged to obtain the recombinant vector.

[0158] 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.

[0159] 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.

[0160] Table 5. LR Reaction System

[0161]

[0162] (6) Extract high-quality, high-concentration plasmids according to the instructions of the Wieglas large plasmid extraction kit.

[0163] (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.

[0164] 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::RENThe relative fluorescence value of TaLBD30 was significantly reduced, indicating that TaLBD30 is a transcription factor with transcriptional repression activity Figure 4 b) in the middle.

[0165] Example 2, verification by transgenic TaLBD30 Regulatory effect of genes on plant type

[0166] The leaves of PI272527 material were selected, and DNA was extracted using the CTAB method. The full-length +2709bp promoter of the gene was amplified by PCR, and was constructed into the pCambia3301 vector to obtain the recombinant vector pCambia3301- TaLBD30 TaLBD30.

[0167] The structure of pCambia3301- TaLBD30 is described as follows: a DNA fragment with SEQ ID No: 4 is inserted between the enzyme cutting sites EcoR I and Pml I of the pCambia3301 vector, and the other sequences of the pCambia3301 vector remain unchanged to obtain the recombinant vector.

[0168] For TaLBD30 The primers used for gene vector construction are shown in Table 6:

[0169] Table 6, TaLBD30 Information of primers used for gene vector construction

[0170]

[0171] Using Agrobacterium-mediated transgenic technology, the specific method is described in the following reference: Ishida Y, et al. Wheat (L.) transformation using immature embryos. Methods Mol Biol. 2015; 1223: 189-98. doi: 10.1007 / 978-1-4939-1695-5_15. PMID: 25300841. The recombinant Agrobacterium EHA105 / pCambia3301- Triticum aestivum is the recombinant bacteria obtained by introducing the recombinant vector pCambia3301- TaLBD30 into Agrobacterium tumefaciens EHA105. TaLBD30

[0172] ​​The transgenic test strip is used for screening positive plants. An appropriate amount of normally growing wheat plant leaves at the three-leaf stage is taken into a 2.0 mL centrifuge tube. After sample preparation, an appropriate amount (300-600 μL) of extraction solution is added to the centrifuge tube, and a PAT / ba rapid test strip (Shanghai Youlong Biological Technology Co., Ltd.) is used for detection. After about 10 min, the results can be read, and one bar indicates a negative plant, and two bars indicate a positive plant.

[0173] The identification results are as follows: a total of 10 positive single plants and 2 negative single plants are identified in the T0 generation. At the time of heading in the T1 generation, the stems below the ears and ear axes of KN199 and the transgenic positive plants TaLBD30 pro::TaLBD30-3 、 TaLBD30 pro::TaLBD30-4 and TaLBD30 pro::TaLBD30-5 are taken for qRT-PCR analysis, TaLBD30 The gene fluorescence quantitative PCR amplification primers are shown in Table 7.

[0174] The Roche fluorescence quantitative PCR instrument is used to analyze the relative expression level of the TaLBD30 gene. The TaGAPDH gene is used as an internal reference gene, and the reaction system is shown in Table 8. The reaction program is as follows: 95°C, 10 s; 95°C, 5 s; 60°C, 30 s; 72°C, 10 s; 40 cycles. Each group of experiments sets 3 biological samples, and each biological sample sets 3 technical repeats. The data is the average value ± standard error of 3 biological repeats. The relative expression amount is calculated by the 2 -ΔΔCT method, and the experimental data is analyzed by the Graphpad prism software. The results show that the Ct value is stable, the repeatability is good, and the amplification curve is normal, indicating that the experimental data is reliable.

[0175] The results show that the expression amount of TaLBD30 in the 3 positive lines is significantly up-regulated Figure 5 in c).

[0176] Table 7, TaLBD30 Gene fluorescence quantitative PCR amplification primer information

[0177]

[0178] Table 8, real-time fluorescence quantitative PCR system

[0179]

[0180] In order to further determine the function of the candidate gene, the plant height and ear length phenotype of the T1 generation lines are investigated.

[0181] KN199 and 3 positive lines were planted, and treated in a vernalization room for 4 weeks, and the temperature of the vernalization room was 4℃; after the treatment, the plants were moved into an artificial climate room for culture, and the culture conditions were as follows: temperature 23℃, light 16h, and darkness 8h. 20 plants of each material were planted, and 10 single plants of each line were selected for statistical analysis.

[0182] It was found that the plant height and ear length of the 3 positive lines in the wheat filling stage were significantly lower than those of KN199. Figure 5 a and b). It can be seen that the overexpression of the gene significantly reduces the plant height and ear length. TaLBD30 significantly reduces the plant height and ear length.

[0183] The application has been described in detail above. For those skilled in the art, the application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the application, and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application.

Claims

1. Use of a substance that increases, enhances or up-regulates the expression of a protein-encoding gene, or a substance that increases, enhances or up-regulates the activity or content of the protein, in any of the following: U1) in reducing plant plant height and ear length; U2) in the manufacture of a product for reducing plant plant height and ear length; U3) in breeding a plant with reduced plant height and ear length; U4) in the manufacture of a product for breeding a plant with reduced plant height and ear length; U5) in plant breeding; the protein is a protein with an amino acid sequence as shown in SEQ ID No: 1; the breeding is for breeding a plant variety with reduced plant height and ear length; the plant is wheat.

2. Use according to claim 1, characterized in that, the substance is a biological material related to the protein, and the biological material is any of the following B1) to B7): B1) a nucleic acid molecule encoding the protein as claimed in claim 1; B2) an expression cassette containing the nucleic acid molecule as claimed in B1); B3) a recombinant vector containing the nucleic acid molecule as claimed in B1), or an expression cassette as claimed in B2); B4) a recombinant microorganism containing the nucleic acid molecule as claimed in B1), or an expression cassette as claimed in B2), or a recombinant vector as claimed in B3); B5) a transgenic plant cell line containing the nucleic acid molecule as claimed in B1), or an expression cassette as claimed in B2); B6) a transgenic plant tissue containing the nucleic acid molecule as claimed in B1), or an expression cassette as claimed in B2); B7) a transgenic plant organ containing the nucleic acid molecule as claimed in B1), or an expression cassette as claimed in B2).

3. A method of modulating plant architecture, characterized by, The activity and / or content of the protein as claimed in claim 1, or / and the expression level of the protein-encoding gene as claimed in claim 1, in a recipient plant is increased, enhanced or up-regulated to reduce the plant height and ear length of the plant; the plant is wheat.

4. The method of claim 3, wherein, The activity and / or content of the protein as claimed in claim 1, or / and the expression level of the protein-encoding gene as claimed in claim 1, in a recipient plant is increased, enhanced or up-regulated to reduce the plant height and ear length of the plant; the plant is wheat.

5. A method for breeding a plant with a plant type, comprising: increasing, enhancing and / or up-regulating the expression level of the protein-encoding gene as claimed in claim 1, or / and increasing, enhancing and / or up-regulating the activity and / or content of the protein-encoding gene as claimed in claim 1, in a recipient plant to obtain a plant with reduced plant height and ear length; the plant is wheat.

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