Biological clock protein related to flowering period and mature period of soybean as well as coding gene and application of biological clock protein

Abstract

The invention discloses a biological clock protein related to a soybean flowering period and a soybean maturation period as well as a coding gene and application of the biological clock protein. Statistical analysis on the flowering period and the mature period of the soybean homozygous mutant plant with the Gmrve4d gene knocked out shows that compared with wild soybeans, the flowering period and the mature period of the soybean homozygous mutant plant with the Gmrve4d gene knocked out are remarkably shortened under the conditions of long sunlight, short sunlight and natural light. The invention discovers that the Gmrve4d protein can regulate and control the flowering period and the mature period of the plant for the first time, and has important significance on plant variety adaptability improvement and breeding of new plant varieties capable of adapting to specific regions by using a molecular breeding means.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a biological clock protein related to the flowering and ripening stages of soybeans, its encoding gene, and its applications. Background Technology

[0002] Timely flowering and ripening are crucial for plant reproduction. The biological clock promotes the spatiotemporal synchronization of plant growth and development with environmental light and temperature signals, playing a central role in regulating flowering and ripening, and enhancing the plant's environmental adaptability. Soybean (Glycine max (L.) Merr.) is a typical short-day, warm-season crop, highly sensitive to light and temperature. Various developmental traits, such as flowering and ripening periods, as well as yield, are comprehensively regulated by the internal gene network and light and temperature factors.

[0003] It is generally believed that soybeans originated in the temperate regions of China (32°N-40°N), and then expanded northward to cold, high-latitude regions and southward to tropical, low-latitude regions. Today, they are widely distributed throughout the world (53°N-35°S). However, there is still an urgent need in high-latitude regions for a large number of ultra-early maturing varieties with insensitive photoperiods.

[0004] Previous studies have found that the widespread distribution of soybean varieties is due to rich natural variation and different combinations of genes regulating maturity traits. Clock-related genes, as core components of the soybean photothermal molecular regulatory pathway, play a crucial role in soybean photothermal response and the formation of its genetic diversity. Therefore, discovering and utilizing novel clock-related genes that regulate soybean flowering and maturity traits is of great significance for improving variety adaptability and breeding new varieties adapted to specific regions using molecular breeding methods.

[0005] In summary, finding new targets for molecular breeding of soybeans with light and temperature adaptability is a technical problem that researchers in this field need to solve. Summary of the Invention

[0006] The technical problem this invention aims to solve is how to regulate the flowering and / or maturity period of plants, improve their adaptability to different light and temperature environments, and broaden the suitable planting range of varieties. The technical problem to be solved is not limited to the described technical subject matter; those skilled in the art will clearly understand other technical subjects not mentioned herein through the following description.

[0007] To address the aforementioned technical problems, the present invention first provides the application of Gmrve4d protein or a substance regulating the content and / or activity of said Gmrve4d protein in any of the following A1)-A5):

[0008] A1) Regulating plant flowering period;

[0009] A2) Regulating plant maturity;

[0010] A3) Cultivate plants with altered flowering and / or maturity periods;

[0011] A4) Prepare products from plants that have altered flowering and / or maturity periods;

[0012] A5) Plant breeding;

[0013] The Gmrve4d protein is any one of the following B1)-B4):

[0014] B1) The amino acid sequence is the protein shown in sequence 2;

[0015] B2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2;

[0016] B3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 2.

[0017] B4) is a protein that has 80% or more of the same amino acid sequence as shown in Sequence 2 and has the same function.

[0018] In the protein described in B2) above, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracking, and / or purification of the target protein. The tag includes, but is 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.

[0019] In the protein described in B3) above, the substitution and / or deletion and / or addition of one or more amino acid residues is as follows: substitution and / or deletion and / or addition of no more than 10 amino acid residues, or substitution and / or deletion and / or addition of no more than 9 amino acid residues, or substitution and / or deletion and / or addition of no more than 8 amino acid residues, or substitution and / or deletion and / or addition of no more than 7 amino acid residues, or substitution and / or deletion and / or addition of no more than 6 amino acid residues, or substitution and / or deletion and / or addition of no more than 5 amino acid residues, or substitution and / or deletion and / or addition of no more than 4 amino acid residues, or substitution and / or deletion and / or addition of no more than 3 amino acid residues, or substitution and / or deletion and / or addition of no more than 2 amino acid residues, or substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0020] In the protein described in B4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. The identity includes amino acid sequences that have 80% or higher, or 85% or higher, or 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity with the amino acid sequence shown in Sequence 2 of this invention.

[0021] The proteins described in B1), B2), B3), or B4) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0022] In the above applications, substances that regulate the content and / or activity of the Gmrve4d protein include substances that increase the content and / or activity of the Gmrve4d protein or substances that decrease the content and / or activity of the Gmrve4d protein.

[0023] Furthermore, the substance that enhances the activity of the Gmrve4d protein can be a protein, polypeptide, or small molecule compound that enhances or promotes the function of the Gmrve4d protein.

[0024] The substance that increases the content of Gmrve4d protein may be a substance that promotes the synthesis of Gmrve4d protein, inhibits the degradation of Gmrve4d protein, or overexpresses Gmrve4d protein.

[0025] The substance that reduces the activity of Gmrve4d protein can be a protein, polypeptide, or small molecule compound that inhibits the function of Gmrve4d protein.

[0026] The substance that reduces Gmrve4d protein content may be a substance that inhibits Gmrve4d protein synthesis, promotes Gmrve4d protein degradation, or knocks down (reduces) or eliminates the Gmrve4d protein encoding gene.

[0027] Furthermore, the substance that knocks down (reduces) the Gmrve4d protein-coding gene can be any substance that can inhibit or interfere with the expression of the Gmrve4d protein-coding gene, such as gRNA (e.g., sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0028] The substance that knocks out the Gmrve4d protein-coding gene can be any substance that prevents the host cell from producing the functional protein product of the Gmrve4d gene. Specific methods include removing all or part of the coding gene sequence, introducing mutations to prevent the production of the functional protein, removing or altering regulatory components (e.g., promoter editing) to prevent transcription of the coding gene sequence, or blocking translation by binding to mRNA. Typically, knockout occurs at the genomic DNA level, ensuring that the cell's offspring permanently carry the knockout.

[0029] Furthermore, the substance that knocks out the Gmrve4d protein-coding gene can be a substance that causes the Gmrve4d gene in plants to mutate (the mutation can be a deletion mutation and / or an insertion mutation and / or a base substitution) and thus lose its activity. The mutation can be caused by any method known in the art, such as the zinc finger protein ZFN gene editing system, the TALENs gene editing system, the CRISPR / Cas9 gene editing system, T-DNA insertion, etc.

[0030] In some embodiments, the substance for knocking out the Gmrve4d protein-coding gene is a CRISPR / Cas9 gene-editing vector that knocks out the Gmrve4d protein-coding gene. The CRISPR / Cas9 gene-editing vector expresses sgRNA and Cas9 protein targeting the Gmrve4d protein-coding gene. Preferably, the target sequence of the sgRNA is shown at positions 344-363 of Sequence 3.

[0031] To address the aforementioned technical problems, this invention also provides novel uses for biomaterials related to the Gmrve4d protein.

[0032] This invention provides the use of biomaterials related to the Gmrve4d protein in any of the following A1)-A5):

[0033] A1) Regulating plant flowering period;

[0034] A2) Regulating plant maturity;

[0035] A3) Cultivate plants with altered flowering and / or maturity periods;

[0036] A4) Prepare products from plants that have altered flowering and / or maturity periods;

[0037] A5) Plant breeding;

[0038] The biomaterial is any one of the following E1) to E5):

[0039] E1) The nucleic acid molecule that encodes the Gmrve4d protein mentioned above;

[0040] E2) Knock down or knock out the nucleic acid molecules encoding the Gmrve4d protein;

[0041] E3) An expression cassette containing the nucleic acid molecules described in E1) or E2);

[0042] E4) A recombinant vector containing the nucleic acid molecules described in E1) or E2), or a recombinant vector containing the expression cassette described in E3);

[0043] E5) Recombinant microorganisms containing the nucleic acid molecules described in E1) or E2), or recombinant microorganisms containing the expression cassette described in E3), or recombinant microorganisms containing the recombinant vector described in E4).

[0044] In the above applications, the nucleic acid molecule described in E1) is any of the following:

[0045] F1) DNA molecules shown in sequence 1 or sequence 3;

[0046] The nucleotide sequences defined by F2) and F1) have 75% or more identity and encode the DNA molecule of the Gmrve4d protein described above.

[0047] Those skilled in the art can readily mutate the nucleotide sequence encoding the Gmrve4d protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides having 75% or higher identity with the Gmrve4d nucleotide sequence isolated according to this invention, provided they encode the Gmrve4d protein and have the same function, are nucleotide sequences derived from and equivalent to those of this invention. This identity refers to sequence similarity to natural nucleic acid sequences, including nucleotide sequences having 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence of a protein composed of the amino acid sequence shown in Sequence 2 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0048] The nucleic acid molecule mentioned in E2 above can be gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, or antisense RNA.

[0049] Any of the nucleic acid molecules mentioned above can be DNA, such as cDNA, genomic DNA, or recombinant DNA.

[0050] Any of the nucleic acid molecules mentioned above can be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0051] The expression cassette described above may include a promoter, the nucleic acid molecule described in E1) or E2) above, and a terminator. Promoters that can be used in this invention include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Furthermore, the expression cassette may also include an enhancer sequence.

[0052] The vector mentioned above refers to a vector capable of carrying the nucleic acid molecules described in E1) or E2) into the host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage, etc.), granules (i.e., Cos plasmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

[0053] The recombinant vector described above refers to a recombinant DNA molecule constructed by in vitro ligation of the nucleic acid molecule described in E1) or E2) with the vector. Recombinant vectors containing the nucleic acid molecule described in E1) or E2) can be constructed using existing plant expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA). The plant expression vector may also contain the 3′ untranslated region of the exogenous gene, i.e., containing the polyadenylated signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated acid to the 3′ end of the mRNA precursor. Similar functions exist in the untranslated regions transcribed at the 3′ end of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the nosine synthase gene) and plant genes (such as the soybean storage protein gene). 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 translation control signal and start codons are widely available and can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed. This can involve adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic marker genes (such as the nptII gene for resistance to kanamycin and related antibiotics, the bar gene for resistance to the herbicide phosphinic acid, the hph gene for resistance to the antibiotic hygromycin, the dhfr gene for resistance to methotrexate, and the EPSPS gene for resistance to glyphosate), or chemical reagent resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0054] The microorganisms mentioned above can be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. The bacteria may originate from, but are not limited to, species such as *Escherichia sp.*, *Erwinia sp.*, *Agrobacterium sp.*, *Flavobacterium sp.*, *Alcaligenes sp.*, *Pseudomonas sp.*, and *Bacillus sp.*. For example, the bacteria may be *Escherichia coli*, *Bacillus subtilis*, or *Bacillus pumilus*. The fungus may be a yeast, and the yeast may originate from genera such as *Saccharomyces cerevisiae*, *Kluyveromyces* (e.g., *Kluyveromyces lactis*), *Pichia pastoris* (e.g., *Pichia pastoris*), *Schizosaccharomyces pombe* (e.g., *Schizosaccharomyces pombe*), and *Hansenula* (e.g., *Hansenula polymorpha*), but is not limited thereto. The fungus may also originate from genera such as *Fusarium* sp., *Rhizoctonia* sp., *Verticillium* sp., *Penicillium* sp., *Aspergillus* sp., and *Cephalosporium* sp., but is not limited thereto. The actinomycetes may originate from genera such as *Streptomycess*, *Nocardia*, *Micromonospora*, *Streptosporangium*, *Actinoplanes*, and *Thermoactinomyces*, but are not limited to these. The algae may originate from genera such as *Fucus*, *Achnanthes*, *Amphiprora*, *Amphora*, *Ankistrodesmus*, *Asteromonas*, and *Boekelovia*, but are not limited to these. The viruses may be rotavirus, herpesvirus, influenza virus, adenovirus, etc., but are not limited to these.

[0055] The recombinant microorganisms mentioned above refer to those obtained by manipulating and modifying the genes of a target microorganism, thereby altering its function. For example, recombinant microorganisms obtained after introducing the aforementioned recombinant vector into a target microorganism. The term "recombinant microorganism" can be understood not only to a specific recombinant microorganism but also to the offspring of such cells. Due to natural, accidental, or intentional mutations and / or alterations, the offspring may not necessarily be completely identical to the original parent cell, but are still included within the scope of recombinant microorganisms.

[0056] In the above applications, the regulation of plant flowering period refers to shortening the plant flowering period (making the plant flower earlier) or prolonging the plant flowering period (making the plant flower later), specifically manifested as: increasing the content and / or activity of Gmrve4d protein in the plant, prolonging the plant flowering period (delaying the flowering time), and decreasing or eliminating the content and / or activity of Gmrve4d protein in the plant, shortening the plant flowering period (making the flowering time earlier).

[0057] In some implementations, when a mutation in the Gmrve4d protein-coding gene in a plant leads to a decrease in the expression level of the Gmrve4d protein-coding gene, the flowering period of the plant is shortened (flowering time is advanced).

[0058] In the above applications, the regulation of plant maturity period refers to shortening the plant maturity period (making the plant mature earlier) or prolonging the plant maturity period (making the plant mature later). Specifically, this is manifested as follows: when the content and / or activity of Gmrve4d protein in the plant increases, the plant maturity period is prolonged (maturity time is delayed); when the content and / or activity of Gmrve4d protein in the plant decreases or is absent, the plant maturity period is shortened (maturity time is advanced).

[0059] In some implementations, when mutations in the Gmrve4d protein-coding gene in plants lead to a decrease in the expression level of the Gmrve4d protein-coding gene, the plant's maturation period is shortened (maturation time is advanced).

[0060] In the above applications, the purpose of plant breeding is to cultivate early-maturing or late-maturing plant varieties.

[0061] To address the aforementioned technical problems, the present invention also provides a method for cultivating transgenic plants with shortened flowering and / or maturity periods.

[0062] The method for cultivating transgenic plants with shortened flowering and / or maturity periods provided by the present invention includes the following steps: reducing the content and / or activity of Gmrve4d protein in the target plant to obtain a transgenic plant; wherein the flowering and / or maturity periods of the transgenic plant are shorter than those of the target plant.

[0063] In the above method, the method for reducing the content and / or activity of Gmrve4d protein in the target plant is to introduce the substance that knocks out the Gmrve4d protein encoding gene into the target plant.

[0064] In the above method, the flowering period and / or maturity period of the transgenic plant is shorter than that of the target plant, as manifested in any one of the following X1)-X3):

[0065] X1) Under short-day conditions, the flowering period and / or maturity period of the transgenic plant is shorter than that of the target plant;

[0066] X2) Under long-day conditions, the flowering and / or maturity period of the transgenic plant is shorter than that of the target plant;

[0067] X3) Under natural light conditions, the flowering and / or maturity period of the transgenic plant is shorter than that of the target plant.

[0068] To address the aforementioned technical problems, the present invention ultimately provides a method for cultivating transgenic plants with shortened flowering and / or maturity periods.

[0069] The present invention provides a method for cultivating transgenic plants with shortened flowering and / or maturity periods, comprising the following steps: replacing the DNA molecule shown at positions 344-363 of sequence 3 in the plant Gmrve4d gene with the DNA molecule shown in sequence 4 or sequence 5 to obtain a transgenic plant; wherein the flowering and / or maturity period of the transgenic plant is shorter than that of the target plant.

[0070] In the above method, the substitution is a homozygous substitution, that is, the same substitution occurs in homologous chromosomes.

[0071] In any of the above applications or methods, the flowering period refers to the number of days from the emergence of cotyledons until the first flower appears at any node on the main stem of the plant.

[0072] The flowering time refers to the time when the first flower appears at any node on the main stem of the plant.

[0073] The maturity period refers to the number of days from the emergence of the cotyledons until any normal pod on the main stem of the plant reaches the color of a mature pod.

[0074] The maturity time refers to the point in time when any normal pod on the main stem of the plant reaches the color of a mature pod.

[0075] In any of the above applications or methods, the plant may be a dicotyledonous plant or a monocotyledonous plant.

[0076] Furthermore, the plant is a dicotyledonous plant.

[0077] Furthermore, the dicotyledonous plant is soybean (such as wild-type soybean Jack).

[0078] This invention utilizes CRISPR / Cas9 gene editing technology to knock out the Gmrve4d gene in wild-type soybean Jack, obtaining two homozygous plants with different Gmrve4d gene knockout mutations, Gmrve4d-1 and Gmrve4d-2. Statistical analysis of the flowering and maturity periods of Gmrve4d-1 and Gmrve4d-2 revealed that, compared to wild-type soybean Jack, the flowering and maturity periods of both mutant lines Gmrve4d-1 and Gmrve4d-2 were significantly shortened under long-day, short-day, and natural light conditions. Under short-day conditions, the flowering time of the two different mutant lines, Gmrve4d-1 and Gmrve4d-2, was significantly earlier than that of wild-type soybean Jack by 2.5 and 1.6 days, respectively, and their maturity time was significantly earlier by 8.6 and 9.9 days, respectively. Under long-day conditions, the flowering time of the two different mutant lines, Gmrve4d-1 and Gmrve4d-2, was significantly earlier than that of wild-type soybean Jack by 3.5 and 6.5 days, respectively, and their maturity time was significantly earlier by 10.6 days. Under natural light... Under summer sowing conditions in Beijing, the flowering time of two different mutant lines, Gmrve4d-1 and Gmrve4d-2, was significantly earlier than that of wild-type soybean Jack by 5.0 days and 4.9 days, respectively, and their maturity time was significantly earlier by 5.4 days and 6.3 days, respectively. Under natural light conditions (summer sowing in Xinxiang), both mutant lines Gmrve4d-1 and Gmrve4d-2 showed a significantly earlier flowering time than wild-type soybean Jack by 2.7 days, and a significantly earlier maturity time by 4.7 days and 4.0 days, respectively. These results indicate that the Gmrve4d protein can regulate the flowering and maturity periods of plants. This invention is of great significance for improving the adaptability of varieties and breeding new varieties that can adapt to specific regions using molecular breeding methods. Attached Figure Description

[0079] Figure 1 This is the mutation type of the Gmrve4d mutant.

[0080] Figure 2 The expression level of Gmrve4d in the Gmrve4d mutant.

[0081] Figure 3 The flowering and maturation phenotypes of the Gmrve4d mutant.

[0082] Figure 4 Statistical results for flowering and maturity of the Gmrve4d mutant. Different lowercase letters indicate highly significant differences compared to the control (Jack) (P < 0.01). Detailed Implementation

[0083] 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.

[0084] Unless otherwise specified, the experimental methods used in the following embodiments 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 embodiments are commercially available. Unless otherwise specified, the quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged.

[0085] The data in the following examples were processed using SPSS 23.0 statistical software. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, and different lowercase letters indicate highly significant differences (P < 0.01).

[0086] The wild-type soybean Jack in the following examples is described in the following literature: Effects of exogenous sulfate and methionine on expression of soybean cystathionine-γ-synthase gene GmCGS, which is available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

[0087] The culture media and their formulations involved in the following examples are as follows:

[0088] YEP solid medium (pH 7.0) consists of a solvent and a solute; the solvent is water, and the solutes and their concentrations are as follows: 5 g / L NaCl, 5 g / L yeast extract, 10 g / L tryptone, and 15 g / L agar.

[0089] The germination medium (pH 5.8) consists of a solvent and a solute; the solute is water, and the concentrations of each solute are as follows: 3.12 g / L B5 salt, 1 ml / L B5 organic, 20 g / L sucrose, and 7.5 g / L agar.

[0090] The liquid culture medium (pH 5.4) consists of a solvent and a solute; the solute is water, and the concentrations of each solute are as follows: 0.43 g / L MS salt, 1 ml / L B5 organic, 40 mg / L acetylsuccinone, 150 mg / L dithiothreitol, 100 mg / L L-cysteine, 30 g / L sucrose, and 3.9 mg / L 2-morpholinoethanesulfonic acid.

[0091] The co-culture medium (pH 5.4) consisted of a solvent and a solute; the solvent was water, and the solutes and their concentrations were as follows: 0.43 g / L MS salt, 1 ml / L B5 organic, 40 mg / L acetylsuccinone, 150 mg / L dithiothreitol, 100 mg / L L-cysteine, 30 g / L sucrose, 7.5 g / L agar, and 3.9 mg / L 2-morpholinoethanesulfonic acid.

[0092] The recovery medium (pH 5.4) consisted of a solvent and a solute; the solvent was water, and the solutes and their concentrations were as follows: 3.1 g / L B5 salt, 1 ml / L B5 organic, 30 g / L sucrose, 150 mg / L cephalosporin, 150 mg / L termethin, 1 mg / L 6-BA, 0.98 g / L 2-morpholinoethanesulfonic acid, 7.5 g / L agar, 4 ml / L Fe salt (200×), 50 mg / L L-asparagine, and 50 mg / L L-glutamine.

[0093] The screening medium (pH 5.4) consisted of a solvent and a solute; the solute was water, and the concentrations of each solute were as follows: 3.1 g / L B5 salt, 1 ml / L B5 organic, 0.98 g / L 2-morpholinoethanesulfonic acid, 30 g / L sucrose, 150 mg / L cephalosporin, 150 mg / L termethin, 1 mg / L 6-BA, 6 mg / L glufosinate, 7.5 g / L agar, 4 ml / L Fe salt (200×), 50 mg / L L-asparagine, and 50 mg / L L-glutamine.

[0094] The elongation medium (pH 5.6) consisted of a solvent and a solute; the solute was water, and the concentrations of each solute were as follows: 4.0 g / L MS salt, 1 ml / L B5 organic, 0.6 g / L 2-morpholinoethanesulfonic acid, 30 g / L sucrose, 150 mg / L cephalosporin, 150 mg / L termethin, 0.1 mg / L IAA, 0.5 mg / L GA, 1 mg / L 6-BA, 6 mg / L glufosinate, 7.5 g / L agar, 4 ml / L Fe salt (200×), 50 mg / L L-asparagine, and 50 mg / L L-glutamine.

[0095] The rooting medium (pH 5.7) consists of a solvent and a solute; the solute is water, and the concentrations of each solute are as follows: 2.165 g / L MS salt, 1 ml / L B5 organic, 0.6 g / L 2-morpholinoethanesulfonic acid, 20 g / L sucrose, 7.5 g / L agar, 50 mg / L L-asparagine, and 50 mg / L L-glutamine.

[0096] The MS salt mentioned above is a product of Phyto Tech, catalog number M524; MS organic is a product of Phyto Tech, catalog number M533; B5 organic is a product of Phyto Tech, catalog number G219; B5 salt is a product of Phyto Tech, catalog number G768; Fe salt (200×) is a product of Phyto Tech, catalog number F629.

[0097] The CDS sequence of the Gmrve4d gene in the following examples is shown in Sequence 1, the amino acid sequence of the Gmrve4d protein it encodes is shown in Sequence 2, and the genomic sequence of the Gmrve4d gene is shown in Sequence 3.

[0098] Example 1: Obtaining the Gmrve4d mutant

[0099] I. Construction of the Gmrve4d gene editing CRISPR vector

[0100] 1. Obtaining gRNA

[0101] Based on the Gmrve4d genome sequence, suitable sgRNA target sequences were designed and screened. The final target sequence is as follows: 5'-TCAACTTCCACAAGTGGGTC-3' (corresponding to positions 344-363 of sequence 3).

[0102] 2. Construction of the recombinant vector Gmrve4d-sgRNA

[0103] The Gmrve4d gene-editing CRISPR vector was constructed using the Cas9 / gRNA plasmid construction kit (Beijing Weishang Lide Biotechnology Co., Ltd., catalog number: VK005-15) according to the kit instructions. The specific steps are as follows:

[0104] 1) Synthesize the sense and antisense strand sequences. After synthesis, dilute to 10 μM and prepare the Oligo mixture according to the following formulation: 5 μL sense (10 μM), 5 μL antisense (10 μM), and 15 μL ddH2O. The sense and antisense strand sequences are as follows:

[0105] Chain of Justice: 5'-TTGGACCCACTTGTGGAAGTTGA-3';

[0106] Antonym chain: 5'-AACTCAACTTCCACAAGTGGGTC-3'.

[0107] 2) After the Oligo mixture is mixed evenly, it is treated in a metal bath at 95°C for 3 min, then slowly cooled to 25°C at room temperature, and held at 16°C for 5 min to obtain oligo dimer.

[0108] 3) Prepare the oligo ligation system according to the following formula: 1 μL oligo dimer, 1 μL sgRNA / Cas9 vector (from Cas9 / gRNA plasmid construction kit), 1 μL solution 1 (from Cas9 / gRNA plasmid construction kit), 1 μL solution 2 (from Cas9 / gRNA plasmid construction kit), and 6 μL ddH2O.

[0109] 4) The oligo ligation system was incubated at 16°C for 2 hours to obtain the recombinant vector Gmrve4d-sgRNA. The recombinant vector Gmrve4d-sgRNA expresses Cas9 protein and sgRNA targeting the Gmrve4d gene. The target sequence of the sgRNA targeting the Gmrve4d gene is 5'-TCAACTTCCACAAGTGGGTC-3'.

[0110] II. Obtaining and Identifying the Gmrve4d Mutant

[0111] 1. Preparation of recombinant bacteria

[0112] The recombinant vector Gmrve4d-sgRNA was transformed into Agrobacterium tumefaciens EHA105, and the plasmid was extracted and sequenced for verification. The recombinant strain that was correctly sequenced was named EHA-Gmrve4d-sgRNA (containing the recombinant vector Gmrve4d-sgRNA).

[0113] 2. Agrobacterium-mediated transformation

[0114] Recombinant bacteria EHA-Gmrve4d-sgRNA was transformed into soybean variety Jack (hereinafter referred to as wild-type soybean or wild soybean Jack) using Agrobacterium-mediated transformation to obtain T0 generation transformed soybeans. The specific steps are as follows:

[0115] 1) Seed sterilization

[0116] 1-1) Take healthy, plump, uniform, and dry wild-type soybean Jack seeds that are free from pests, diseases, and spots, spread them evenly in a petri dish, and then place the petri dish in a desiccator.

[0117] 1-2) After completing step 1-1), place a 100mL beaker in the desiccator, pour 80mL of 12M sodium hypochlorite aqueous solution into the beaker, then slowly add 4mL of concentrated hydrochloric acid, and then quickly cover the desiccator, seal it with petroleum jelly, and place it for 16 hours for chlorine sterilization to obtain sterilized seeds.

[0118] 2) Infection

[0119] 2-1) Recombinant bacteria EHA-Gmrve4d-sgRNA was cultured at 28℃, resuspended in liquid culture medium, and OD was obtained.600nm =0.6% of the infecting bacterial solution.

[0120] 2-2) Place the sterilized seeds in a laminar flow hood and, under a microscope, peel off the seed coat and separate the two cotyledons along the long axis, keeping the cotyledon with the intact hypocotyl. Make 3-5 cuts at the junction of the hypocotyl and cotyledon. Then, incubate the seeds in a 28°C incubator with an infecting bacterial solution for 2 hours.

[0121] 2-3) After infection, place the cotyledons with the inner surface (smooth side) facing up on a co-culture medium that has been covered with sterile filter paper, and incubate in the dark at 22°C for 5 days.

[0122] 2-4) After co-culturing for 5 days, the hypocotyl of the explants elongated to 2 cm. Part of the hypocotyl was cut off, leaving 0.5 cm. The treated explants were then placed in recovery medium and cultured for 7 days at 28°C under 16h light / 8h dark conditions.

[0123] 2-5) Remove the explants from the recovery medium, remove the new shoots, cut off part of the hypocotyl, leaving 0.5 cm of the hypocotyl, and then transfer the trimmed explants into the selection medium and culture them for 21 days at 28℃ under 16h light / 8h dark conditions.

[0124] 2-6) After 21 days of selection and induction, the explants produced a large number of adventitious buds. The cotyledons and brown leaves were removed, and the remaining parts were transferred to elongation medium for culture at 28°C under 16h light / 8h dark conditions.

[0125] 2-7) In the elongation medium, when the clustered buds produce 5-8 cm young stems, cut them off from the base of the adventitious buds; dip the stem base in 1 mg / L IBA solution for 1 min, and then transfer it to the rooting medium for culture. Culture for one week at 28℃ under 16 h light / 8 h dark conditions. After a large number of roots are produced at the base of the stem, transplant them into pots. The resulting plants are T0 generation transformed soybeans.

[0126] 3. Molecular detection of edited plants

[0127] DNA was extracted from leaves of T0 generation transformed soybean and wild-type soybean Jack as templates. PCR amplification was performed using primers Gmrve4d-CRISPR-F and Gmrve4d-CRISPR-R. The PCR products were obtained and sequenced to detect editing near the Gmrve4d gene target site. Primer sequences are as follows:

[0128] Gmrve4d-CRISPR-F: 5′-AACACGCCCTCCAAGCATA-3′;

[0129] Gmrve4d-CRISPR-R: 5′-TGAACCAGCAAGATGACCAAC-3′.

[0130] The PCR reaction system (total volume 25 μL) is as follows: 2×KOD One™ PCR Master Mix-Blue 12.5 μL, DNA (200 ng / μL) 2 μL, Gmrve4d-CRISPR-F (10 pmol / μL) 1 μL, Gmrve4d-CRISPR-R (10 pmol / μL) 1 μL, ddH2O 8.5 μL.

[0131] The PCR reaction system was as follows: 98℃ for 3 min; 98℃ for 10 sec, 58℃ for 10 sec, 72℃ for 10 sec, 35 cycles; 72℃ for 5 min.

[0132] The plants exhibiting overlapping peaks near the target site were heterozygous edited plants, named T0 generation Gmrve4d gene-edited soybeans.

[0133] T0 generation Gmrve4d gene-edited soybeans were sown and self-pollinated to harvest T1 generation Gmrve4d gene-edited soybean seeds. After sowing, T1 generation Gmrve4d gene-edited soybean plants were obtained, totaling 10 plants, which were named T1-4d-1, T1-4d-2, T1-4d-3, T1-4d-4, T1-4d-6, T1-4d-8, T1-4d-9, T1-4d-12, T1-4d-13 and T1-4d-14, respectively.

[0134] Using the primers Gmrve4d-CRISPR-F and Gmrve4d-CRISPR-R, the T1 generation of Gmrve4d gene-edited soybean plants were detected according to the above method. Sequencing results showed that both T1 generation Gmrve4d gene-edited soybean plants were soybean mutants with a homozygous mutation in the Gmrve4d gene (the same mutation occurred on two homologous chromosomes), designated as T1 generation Gmrve4d homozygous soybean mutants Gmrve4d-1 and Gmrve4d-2, respectively.

[0135] The T1 generation Gmrve4d homozygous mutant soybean Gmrve4d-1 has a gene mutation type of -1 bp (deletion at position 347 of sequence 3). Its only difference from the wild-type soybean Jack genome sequence is a base deletion in the gene encoding the Gmrve4d protein (sequence 3), located at position 347. This mutation causes premature termination of Gmrve4d protein translation, thus knocking out the Gmrve4d gene. The target sequence in the T1 generation Gmrve4d homozygous mutant soybean Gmrve4d-1 is as follows: 5'-TCACTTCCACAAGTGGGTC-3' (sequence 4).

[0136] The T1 generation Gmrve4d homozygous mutant soybean Gmrve4d-2 has a gene mutation type of -5bp (deletion at positions 347-351 of sequence 3). Its only difference from the wild-type soybean Jack genome sequence is a deletion in the gene encoding the Gmrve4d protein (sequence 3), located at positions 347-351. This mutation causes premature termination of Gmrve4d protein translation, thus knocking out the Gmrve4d gene. The target sequence in the T1 generation Gmrve4d homozygous mutant Gmrve4d-2 is as follows: 5'-TCACACAAGTGGGTC-3' (sequence 5).

[0137] Soybean homozygous mutants without transgenic elements were screened from the T1 generation Gmrve4d soybean homozygous mutants Gmrve4d-1 and Gmrve4d-2. Seeds from these mutants were then planted to obtain the T2 generation Gmrve4d soybean homozygous mutants Gmrve4d-1 and Gmrve4d-2. Sequencing verification of the T2 generation Gmrve4d soybean homozygous mutants Gmrve4d-1 and Gmrve4d-2 showed that the mutation type in Gmrve4d-1 was -1 bp, and the mutation type in Gmrve4d-2 was -5 bp. These mutants were then used for phenotypic analysis.

[0138] The mutation types and sequencing results of the above-mentioned homozygous mutants of soybean Gmrve4d, Gmrve4d-1 and Gmrve4d-2, are as follows: Figure 1 As shown.

[0139] 4. Detection of Gmrve4d expression level

[0140] The expression level of Gmrve4d in wild-type soybean Jack and the T2 generation Gmrve4d homozygous mutants Gmrve4d-1 and Gmrve4d-2 was detected. The specific steps were as follows: Trifoliate leaves just unfolded from simultaneously sown and uniformly grown wild-type soybean Jack and T2 generation Gmrve4d homozygous mutants Gmrve4d-1 and Gmrve4d-2 were harvested, flash-frozen in liquid nitrogen, pulverized in a mortar, and RNA was extracted and reverse transcribed into cDNA. Actin, a housekeeping gene with relatively stable transcription levels at different developmental stages and tissue sites in soybean, was used as an internal control. PCR was performed using an ABI-7600 real-time quantitative PCR instrument. The PCR reaction program was as follows: 50℃, 2 min; 95℃, 10 min; 95℃, 15 sec; 60℃, 1 min; 40 cycles. After the PCR reaction, the expression level was determined according to Formula 2. -△△Ct To compare the relative differences in gene expression, each sample group contained three biological replicates.

[0141] The results are as follows Figure 2 As shown in the results, the expression levels of Gmrve4d in the T2 generation Gmrve4d homozygous mutants Gmrve4d-1 and Gmrve4d-2 were significantly lower than those in wild-type soybean Jack (P < 0.01).

[0142] Example 2: Phenotypic Analysis of the Gmrve4d Mutant

[0143] Test materials: wild-type soybean Jack, T2 generation Gmrve4d homozygous mutant soybean Gmrve4d-1 (hereinafter referred to as Gmrve4d-1) and Gmrve4d-2 (hereinafter referred to as Gmrve4d-2).

[0144] Experimental Methods: From June to October 2023, seeds of the test materials were planted in an artificial climate chamber using conventional methods and cultured to maturity under both short-day (light:dark = 12h:12h, 26℃) and long-day (light:dark = 16h:8h, 26℃) conditions. Flowering and maturity dates were recorded. Simultaneously, from June to October 2023, seeds of the test materials were planted in fields in Beijing and Xinxiang using conventional methods and cultured to maturity under natural light (natural long-day conditions). Flowering and maturity dates were also recorded. Flowering period refers to the number of days (in days) from cotyledon emergence until the first flower appears at any node on the soybean main stem. Maturity period refers to the number of days (in days) from cotyledon emergence until any normal pod on the soybean main stem reaches the color of a mature pod. The experimental results were repeated three times, and the average value was taken.

[0145] The results are shown in Table 1 and Table 2. Figure 3 and Figure 4 As shown.

[0146] Table 1

[0147]

[0148] Table 2

[0149]

[0150] The results showed that compared with wild-type soybean Jack, the flowering and maturity periods of the two mutant lines Gmrve4d-1 and Gmrve4d-2 were significantly shortened under long-day, short-day, and natural light conditions. Specifically, under short-day conditions, the flowering time of the two mutant lines Gmrve4d-1 and Gmrve4d-2 was significantly earlier than that of wild-type soybean Jack by 2.5 days and 1.6 days, respectively, and their maturity time was significantly earlier by 8.6 days and 9.9 days, respectively. Under long-day conditions, the flowering time of the two mutant lines Gmrve4d-1 and Gmrve4d-2 was significantly earlier than that of wild-type soybean Jack by 3.5 days and 6.5 days, respectively, and their maturity time was significantly earlier by 10.6 days. Under natural light... Under normal light conditions (summer sowing in Beijing), the two mutant lines Gmrve4d-1 and Gmrve4d-2 flowered significantly earlier than wild-type soybean Jack by 5.0 days and 4.9 days, respectively, and matured significantly earlier by 5.4 days and 6.3 days, respectively. Under natural light conditions (summer sowing in Xinxiang), the two mutant lines Gmrve4d-1 and Gmrve4d-2 both flowered significantly earlier than wild-type soybean Jack by 2.7 days, and matured significantly earlier by 4.7 days and 4.0 days, respectively.

[0151] The present invention has been described in detail above. For those skilled in the art, 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. Although specific embodiments have been given, 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. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of Gmrve4d protein or a substance regulating the content and / or activity of said Gmrve4d protein in any of the following A1)-A5): A1) Regulating plant flowering period; A2) Regulating plant maturity; A3) Cultivate plants with altered flowering and / or maturity periods; A4) Prepare products from plants that have altered flowering and / or maturity periods; A5) Plant breeding; The Gmrve4d protein is any one of the following B1)-B4): B1) The amino acid sequence of the protein is shown in sequence 2; B2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; B3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 2. B4) is a protein that has 80% or more of the same amino acid sequence as shown in Sequence 2 and has the same function.

2. Use of biomaterials related to the Gmrve4d protein of claim 1 in any of the following A1)-A5): A1) Regulating plant flowering period; A2) Regulating plant maturity; A3) Cultivate plants with altered flowering and / or maturity periods; A4) Prepare products from plants that have altered flowering and / or maturity periods; A5) Plant breeding; The biomaterial is any one of the following E1) to E5): E1) A nucleic acid molecule encoding the Gmrve4d protein as described in claim 1; E2) Knock down or knock out the nucleic acid molecule of the Gmrve4d protein-encoding gene as described in claim 1; E3) An expression cassette containing the nucleic acid molecules described in E1) or E2); E4) A recombinant vector containing the nucleic acid molecules described in E1) or E2), or a recombinant vector containing the expression cassette described in E3); E5) Recombinant microorganisms containing the nucleic acid molecules described in E1) or E2), or recombinant microorganisms containing the expression cassette described in E3), or recombinant microorganisms containing the recombinant vector described in E4); The Gmrve4d protein is any one of the following B1)-B4): B1) The amino acid sequence of the protein is shown in sequence 2; B2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; B3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 2. B4) is a protein that has 80% or more of the same amino acid sequence as shown in Sequence 2 and has the same function.

3. The application according to claim 2, characterized in that: E1) The nucleic acid molecule is any one of the following: F1) DNA molecules shown in sequence 1 or sequence 3; The nucleotide sequences defined by F2) and F1) have 75% or more identity and encode the DNA molecule of the Gmrve4d protein.

4. The application according to any one of claims 1-3, characterized in that: The plant is a dicotyledonous plant or a monocotyledonous plant.

5. A method for cultivating transgenic plants with shortened flowering and / or maturity periods, comprising the following steps: reducing the content and / or activity of Gmrve4d protein in a target plant to obtain a transgenic plant; wherein the flowering and / or maturity period of the transgenic plant is shorter than that of the target plant; The Gmrve4d protein is any one of the following B1)-B4): B1) The amino acid sequence of the protein is shown in sequence 2; B2) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2; B3) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in Sequence 2. B4) is a protein that has 80% or more of the same amino acid sequence as shown in Sequence 2 and has the same function.

6. The method according to claim 5, characterized in that: The method for reducing the content and / or activity of Gmrve4d protein in the target plant is to introduce a substance that knocks down or eliminates the Gmrve4d protein encoding gene into the target plant.

7. The method according to claim 6, characterized in that: The substance used to knock out the Gmrve4d protein-coding gene is a CRISPR / Cas9 gene editing vector that knocks out the Gmrve4d protein-coding gene; the CRISPR / Cas9 gene editing vector expresses sgRNA and Cas9 protein targeting the Gmrve4d protein-coding gene.

8. The method according to claim 7, characterized in that: The target sequence of the sgRNA is shown in positions 344-363 of sequence 3.

9. A method for cultivating transgenic plants with shortened flowering and / or maturity periods, comprising the following steps: replacing the DNA molecule shown at positions 344-363 of sequence 3 in the plant Gmrve4d gene with the DNA molecule shown in sequence 4 or sequence 5 to obtain a transgenic plant; wherein the flowering and / or maturity period of the transgenic plant is shorter than that of the target plant.

10. The application according to any one of claims 5-9, characterized in that: The plant is a dicotyledonous plant or a monocotyledonous plant.

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