A protein for synergistically regulating flowering and maturation periods of soybean, a coding gene thereof and application thereof

Abstract

This invention discloses a protein that synergistically regulates the flowering and ripening stages of soybean, its encoding gene, and its applications. The protein's amino acid sequence is GmRVE8a, as shown in SEQ ID NO:2. Experiments have demonstrated that gene editing of the GmRVE8a protein encoding gene in the soybean variety Williams 82 yielded the T2 generation. GmRVE8a Homozygous mutant strain. For the T2 generation. Gmrve8a-1 and T2 generation Gmrve8a-2 Phenotypic identification was conducted, and the results showed that, regardless of whether it was long-day, short-day, or naturally long-day, the T2 generation, compared with the soybean variety Williams 82, exhibited [significant differences / performance]. GmRVE8a The flowering and maturity periods of the homozygous mutant were significantly advanced. Therefore, this invention can regulate the flowering and maturity periods of soybeans and has significant application potential.

Description

Technical Field

[0001] This invention belongs to the field of biological breeding, specifically relating to a protein that synergistically regulates the flowering and maturation stages of soybeans, its encoding gene, and its applications. Background Technology

[0002] Timely flowering and maturation are core developmental processes for plants to complete their generational reproduction. The plant's endogenous biological clock, through mediating the spatiotemporal coupling of growth and development rhythms with environmental light and temperature signals, synergistically regulates the initiation of flowering and maturation, and is a core factor determining a plant's environmental adaptability and geographical distribution. Soybean (…) Glycine max (L.) Merr.) is an important crop that can be used for grain, oil, forage and vegetables. As a typical short-day warm-loving crop, its key agronomic traits such as flowering period and maturity period are synergistically regulated by an endogenous molecular regulatory network centered on the biological clock and external light and temperature environmental factors.

[0003] Cultivated soybeans originated in the temperate regions of my country (32°N-40°N). Through long-term domestication and genetic improvement, they are now cultivated globally across ecological zones (53°N-35°S). However, their inherent sensitivity to light and temperature makes them prone to mismatches between flowering and maturity periods and ecological zones when grown across latitudinal boundaries. This can lead to significant yield reductions or even crop failure, severely limiting the widespread application of superior varieties. Especially in high-latitude major producing areas such as Northeast my country, there is still an urgent need for early-maturing, stable-yielding varieties with insensitive photoperiods and synergistic adaptation between flowering and maturity periods.

[0004] Existing research indicates that the geographical adaptability differentiation of soybean varieties depends on the natural variation of genes regulating flowering and maturity stages, as well as the utilization of superior allele combinations. Clock genes are the core hubs of the soybean photoperiod signal transduction and growth rhythm regulation network, directly determining its photoperiodic response characteristics and ecological adaptability. Therefore, identifying and utilizing novel clock genes that can synergistically regulate the adaptability of soybean flowering and maturity stages and enhance the varietal adaptability, and determining new targets for molecular breeding of soybean photoperiod adaptability, is of great significance for using molecular breeding methods to improve varietal adaptability and breed new varieties adapted to specific regions. Summary of the Invention

[0005] The technical problem to be solved by this invention is to regulate the flowering and maturity period of soybeans, and to solve the core problem of mismatch between the flowering and maturity period and the light and temperature conditions of the planting environment in cross-latitude soybean planting, so as to significantly improve the adaptability of soybeans to different light and temperature environments and broaden the suitable planting range of varieties.

[0006] This invention first protects a gene-edited soybean. The gene-edited soybean may be a soybean that does not contain the gene encoding the GmRVE8a protein; The GmRVE8a protein may be a1), a2), a3), or a4. a1) The amino acid sequence of the protein is SEQ ID NO: 2; a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1); a3) Proteins with the same function obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in a1) or a2); Proteins that share more than 70% identity with the amino acid sequence defined by a4) and a1) or a2) and have the same function.

[0007] The aforementioned gene-edited soybeans are specifically soybeans obtained by knocking out the gene encoding the GmRVE8a protein in the soybean to be improved; the soybean to be improved contains the gene encoding the GmRVE8a protein.

[0008] The flowering and / or maturity periods of any of the gene-edited soybeans described above are earlier (i.e. shorter) than those of the soybeans to be improved.

[0009] In any of the gene-edited soybeans described above, the knockout may include performing any of the following operations on the genome of the soybean to be improved: F1) Delete nucleotides 402-405 of SEQ ID NO:3 in the soybean genome to be improved; F2) Delete nucleotides 402-406 of SEQ ID NO:3 in the soybean genome to be improved.

[0010] In any of the gene-edited soybeans described above, the knockout may specifically be performed on the genome of the soybean to be improved by any of the following operations: F1) Delete nucleotides 402-405 of SEQ ID NO:3 in the soybean genome to be improved; F2) Delete nucleotides 402-406 of SEQ ID NO:3 in the soybean genome to be improved.

[0011] This invention also protects the application of biological materials encoding the GmRVE8a protein described above or the gene that has knocked out the GmRVE8a protein described above, which may be at least one of the following A1) to A5): A1) Regulating the flowering period of leguminous plants; A2) Regulating the maturity period of leguminous plants; A3) Cultivating gene-edited legumes with altered flowering periods; A4) Cultivating gene-edited plants with altered maturity periods; A5) Breeding of leguminous plants during flowering and / or maturity.

[0012] In the above applications, the biological material for knocking out the gene encoding the GmRVE8a protein can be any one of D1)-D8): D1) Nucleic acid molecules that target the gene encoding any of the GmRVE8a proteins described above; D2) Cas protein and D1) the nucleic acid molecule described; D3) The nucleic acid molecules and Cas protein-encoding genes described in D1); D4) Vectors, expression cassettes, or recombinant microorganisms containing the nucleic acid molecules described in D1) and the Cas protein-encoding gene; D5) Leguminosae containing the nucleic acid molecules described in D1); D6) Leguminous plants containing Cas protein and the nucleic acid molecules described in D1); D7) Leguminous plants containing the nucleic acid molecules and Cas protein-coding genes described in D1); D8) Leguminosae containing the nucleic acid molecules described in D1) and the Cas protein-encoding gene, vectors, expression cassettes, or recombinant microorganisms.

[0013] In the above applications, the nucleic acid molecule described in D1) may be a gRNA encoding a gene that targets any of the GmRVE8a proteins described above.

[0014] In some embodiments, the gRNA targets the double-stranded DNA whose nucleotide sequence is SEQ ID NO: 3, positions 389-408.

[0015] The term "sgRNA (single-guide RNA)" is a component of the CRISPR-Cas system, responsible for guiding the Cas protein to recognize and cleave target nucleic acid molecules. In practical gene editing applications, sgRNA can be synthesized directly or obtained through plasmid expression or in vitro transcription. In this field, "gRNA" and "sgRNA" are often used interchangeably. In this document, "gRNA" and "sgRNA" are also used interchangeably. sgRNA generally refers to a single RNA structure formed by artificially modifying the crRNA / tracrRNA complex (gRNA) with a dual RNA structure, directly (or through a linker) linking the crRNA and tracrRNA. sgRNA is a short RNA containing a recognition region and a framework region.

[0016] The term "recognition region," also known as a guide sequence, is typically an RNA sequence (referred to herein as the "guide sequence") that is identical to or complementary to the target sequence or target site within the target RNA (sgRNA or gRNA). The guide sequence is generally sufficiently complementary to the target sequence to hybridize with the target site and guide the CRISPR / Cas complex to bind specifically to the target. Perfect complementarity between the guide sequence and the target sequence is preferred, but some mismatch (e.g., a mismatch of 1-6 nucleotides) is permissible, as long as it still results in gene knockout. The complementarity between the guide sequence and its corresponding target sequence is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Methods for determining the complementarity of two nucleic acid sequences are within the capabilities of those skilled in the art.

[0017] The term "scaffold" generally refers to the structural or scaffold RNA sequence that guides the binding or interaction of RNA with RNA-directed nucleases and / or other RNA molecules (e.g., tracrRNA) into RNA (sgRNA or gRNA), and can also be called the backbone sequence of sgRNA. The scaffold can be conventionally selected by those skilled in the art; for example, it can be the backbone sequence of the sgRNA corresponding to Cas9, or it can be a mutant constructed based on this sequence that still retains the function of binding the corresponding Cas9.

[0018] The legumes mentioned above can specifically refer to plants of the genus *Glycine*.

[0019] This invention also protects methods for cultivating soybeans with advanced flowering and / or maturity periods.

[0020] The method for cultivating soybeans with earlier flowering and / or maturity periods protected by this invention may specifically be K1). K1) may involve knocking out the gene encoding the GmRVE8a protein in the soybean to be improved containing any of the aforementioned GmRVE8a protein encoding genes, thereby obtaining gene-knockout soybeans with earlier flowering and / or maturity periods than the soybean to be improved.

[0021] The method for cultivating soybeans with earlier flowering and / or maturity periods protected by this invention may specifically be K2). K2 may be to reduce the content of any of the aforementioned GmRVE8a proteins in the soybean to be improved and / or reduce the expression level of the gene encoding the GmRVE8a protein in the soybean to be improved, thereby obtaining improved soybeans; the flowering and / or maturity periods of the improved soybeans are earlier than those of the soybeans to be improved.

[0022] This invention also protects methods for advancing the flowering and / or maturity of soybeans.

[0023] The method for advancing the flowering and / or maturity period of soybeans protected by this invention may specifically be S1). S1) may involve knocking out the gene encoding the GmRVE8a protein in the soybean to be improved, which contains any of the aforementioned GmRVE8a protein encoding genes, to obtain gene knockout soybeans; the flowering and / or maturity period of the gene knockout soybeans is advanced compared to the soybean to be improved.

[0024] The method for advancing the flowering and / or maturity period of soybeans protected by this invention may specifically be S2). S2) may be to advance the flowering and / or maturity period of soybeans by reducing the content of any of the GmRVE8a proteins mentioned above in the soybeans to be improved and / or reducing the expression level of the gene encoding the GmRVE8a protein in the soybeans to be improved.

[0025] In K1) or S1), the step of "knocking out the gene encoding the GmRVE8a protein in the soybean to be improved containing any of the above-mentioned gene encoding the GmRVE8a protein" may include the step of introducing biological material containing any of the above-mentioned gene encoding the GmRVE8a protein that has been knocked out into the soybean to be improved.

[0026] In K1) or S1), the knockout may include performing any of the following operations on the genome of the soybean to be improved: F1) Delete nucleotides 402-405 of SEQ ID NO:3 in the soybean genome to be improved; F2) Delete nucleotides 402-406 of SEQ ID NO:3 in the soybean genome to be improved.

[0027] In some specific embodiments, the knockout is achieved via a CRISPR-Cas system. The CRISPR-Cas system includes an RNA-directed nuclease (Cas protein) and sgRNA.

[0028] In this application, Cas protein is an "RNA-directed nuclease," which refers to an RNA-directed DNA endonuclease associated with the CRISPR system. Non-restricted examples of RNA-directed nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, their homologs or modified forms thereof. In one implementation, the RNA-directed nuclease is Cas9 or nCas9 (D10A).

[0029] In some embodiments, the legume and the gene-edited soybean are the whole soybean plant or other parts (cells, tissues, and / or organs).

[0030] The cells include original knockout cells (T0 generation cells), cells regenerated or developed from T0 generation cells, cells from any progeny or descendant of T0, including seed or embryo cells, or cultured cells, callus cells, etc.

[0031] The tissues and / or organs may be original knockout T0 generation, meristematic tissues regenerated or developed from T0 generation, bud organs / structures (e.g., leaves, stems, or nodes), roots, flowers or floral organs / structures (e.g., flowers, bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (e.g., embryos, endosperm, and seed coats), fruits (e.g., mature ovaries), propagules, or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue). The tissues and / or organs may also be tissues and / or organs from any progeny or descendant of T0.

[0032] In this application, the flowering period is the time from sowing to the opening of a flower at any node on the main stem. The maturity period is the time from sowing to the time when any normal pod on the main stem reaches the color of a mature pod. In some embodiments, regulating the flowering period of legumes is to advance flowering (the opening of a flower at any node on the main stem). In some embodiments, regulating the maturity period of legumes is to advance maturity (the time when any normal pod on the main stem reaches the color of a mature pod).

[0033] In this application, the flowering and maturity periods are compared under comparable conditions. "Comparable conditions" refer to the same or similar environmental conditions and agronomic practices used to make meaningful comparisons between two or more plant genotypes, such that neither the environmental conditions nor the agronomic practices significantly contribute to or explain any differences observed between the two or more plant genotypes. Environmental conditions include, for example, light, temperature, water, humidity, soil, and nutrients (e.g., nitrogen and phosphorus).

[0034] Experiments have shown that this invention, through gene editing in the soybean variety Williams 82, yielded the T2 generation. GmRVE8a homozygous mutant strain—T2 generation Gmrve8a-1 and T2 generation Gmrve8a-2 Testing revealed that, compared to the Williams 82, the T2 generation... Gmrve8a- 1 The only difference in the genome is: T2 generation Gmrve8a-1 Two homologous chromosomes GmRVE8a The same mutation occurred in the genes, specifically on two homologous chromosomes. GmRVE8a All genes underwent a 4-nucleotide deletion—CAAT (i.e., deletion at positions 402-405 of SEQ ID NO: 3 (corresponding to positions 36-39 of SEQ ID NO: 1)), resulting in a frameshift and premature termination of GmRVE8a protein translation, thus knocking out the gene encoding GmRVE8a. Compared to Williams 82, the T2 generation... Gmrve8a-2 The only difference in the genome is: T2 generation Gmrve8a-2 Two homologous chromosomes GmRVE8a The same mutation occurred in the genes, specifically on two homologous chromosomes. GmRVE8a All genes underwent a 5-nucleotide deletion—CAATG (i.e., deletion at positions 402-406 of SEQ ID NO: 3 (corresponding to positions 36-40 of SEQ ID NO: 1)), resulting in a frameshift and premature termination of GmRVE8a protein translation, thus knocking out the gene encoding GmRVE8a. For the T2 generation... Gmrve8a-1 and T2 generation Gmrve8a-2 Phenotypic identification was conducted, and the results showed that, regardless of whether it was long-day, short-day, or naturally long-day, the T2 generation, compared with the soybean variety Williams 82, exhibited [significant differences / performance]. GmRVE8a homozygous mutants (such as T2 generation) Gmrve8a-1 T2 generation Gmrve8a-2 The flowering and maturity periods of soybeans are significantly advanced (i.e. significantly shortened). This invention can regulate the flowering and maturity periods of soybeans and has important application prospects.

[0035] The terms used in this application are explained as follows: Examples of resources describing many of the molecular biology-related terms used in this paper can be found in the following literature: Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, Genes IX, Oxford University Press: New York, 2007.

[0036] Any references cited in this article, including, for example, all patents, published patent applications and non-patent publications, are incorporated in their entirety by reference.

[0037] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0038] The term "identity" generally refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is usually expressed as a percentage. The identity described herein can refer to the identity of an amino acid sequence or a nucleotide sequence. Two copies having completely identical sequences have 100% identity. Those skilled in the art will recognize that the identity of an amino acid sequence or nucleotide sequence can be determined using identity search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, the identity of an amino acid sequence 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 the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), and performing a search, thus obtaining the identity value (%). Alternatively, sequence analysis software such as CLC Main Workbench and MegAlign can be used. TM ) to perform the measurement, for example using the computer program BLAST with default parameters, especially BLASTP or TBLASTN.

[0039] Specifically, the 70% or higher degree of similarity can be 75% or higher. Specifically, the 75% or higher degree of similarity can be 80% or higher. Specifically, the 80% or higher degree of similarity can be 85% or higher. Specifically, the 85% or higher degree of similarity can be 90% or higher. Specifically, the 90% or higher degree of similarity can be 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher. More specifically, the 70% or more of identity can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0040] When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0041] As used in this article, "plant" includes explants, plant parts, seedlings, plantlets, or whole plants at any stage of regeneration or development.

[0042] Plant cells are biological cells of plants, derived from plants or derived from cultures obtained by culturing cells taken from plants. As used herein, “transgenic plant cell” means any plant cell transformed with a stably integrated recombinant DNA molecule, construct, expression cassette, or sequence. Transgenic plant cells can include original transformed plant cells, transgenic plant cells regenerated or developed from R0 generation transgenic plant cells, transgenic plant cells cultured from another transgenic plant cell, or transgenic plant cells from any progeny or offspring of a transformed R0 generation plant, including cells of plant seeds or embryos, or cultured plant cells, callus cells, etc.

[0043] As is commonly understood in the art, the term "promoter" generally refers to DNA containing an RNA polymerase binding site, a transcription start site, and / or a TATA box that assists or promotes the transcription of transcribed DNA. Promoters can be artificially synthesized, modified, or derived from known or naturally occurring promoters. Promoters can also include chimeric promoters comprising combinations of two or more heterologous sequences. Therefore, the promoters of this application may include variants of promoter sequences that are compositionally similar but not identical to other promoter sequences provided herein.

[0044] Promoters can be classified according to various criteria related to the expression patterns of the associated coding or transcribed sequences or genes (including transgenes) operably linked to them, such as constitutive, developmental, tissue-specific, and inducible promoters. A promoter that drives expression in all or most tissues of a plant is called a "constitutive" promoter. A promoter that drives expression at certain times or stages of development is called a "developmental" promoter. A promoter that drives enhanced expression in certain tissues of a plant relative to other tissues is called a "tissue-enhancing" or "tissue-preferred" promoter. Therefore, a "tissue-preferred" promoter elicits relatively high or preferential expression in a specific tissue of the plant, but lower expression levels in other tissues. A promoter that is expressed in a specific tissue of the plant but rarely or not expressed in other tissues is called a "tissue-specific" promoter. An "inducible" promoter is a promoter that initiates transcription in response to environmental stimuli (e.g., cold, drought, or light) or other stimuli (e.g., injury or chemical application). Promoters can also be classified according to their origin, such as heterologous, homologous, chimeric, synthetic, etc.

[0045] The term "transcribed DNA" refers to DNA that can be transcribed into RNA molecules.

[0046] The term "operationally ligated" can refer to a functional connection between a promoter and transcribed DNA, enabling the promoter to function and initiate transcription of the transcribed DNA. The term "operationally ligated" can also refer to a functional connection between other regulatory elements and a target gene to regulate the transcription and / or expression of the target gene.

[0047] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by linking a foreign target gene to a vector in vitro. It can be constructed in any suitable way, as long as the constructed recombinant vector can carry the foreign target gene into the recipient cell and provide the foreign target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0048] The term "linkage" generally refers to the association of two or more molecules. Linkages can be covalent or non-covalent. The linkages described herein can be direct peptide bonds or linkages via linkers (connectors).

[0049] The term "comprising" is not intended to be restrictive, but rather inclusive and implies the presence of other elements besides those listed, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "substantially composed of". In this document, the terms "comprising" and "including" are used interchangeably.

[0050] The term "construct" refers to any recombinant DNA or recombinant RNA molecule. Recombinant DNA molecules can be plasmids, granules, viruses, bacteriophages, or linear or circular DNA. Constructs typically include one or more expression cassettes.

[0051] The term "expression cassette" generally refers to a nucleic acid construct containing sufficient nucleic acid elements to express a target gene. A typical expression cassette includes a promoter, a multiple cloning site (MCS), and / or a terminator. Expression cassettes may also include the target gene, a marker gene, a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly(A) signal sequence, and / or an mRNA splicing signal sequence. Elements within an expression cassette can be directly linked or indirectly linked via adapters. An "expression cassette" contains at least transcribed DNA operatively linked to one or more regulatory elements, typically at least a promoter and a 3' UTR (such as a terminator).

[0052] The term "tag" includes, but is not limited to: GST (glutathione thioredoxin) tagged protein, Trx (thioredoxin) tagged protein, nitrogen utilization substrate A (NusA) tagged protein, His-tag protein, MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA (influenza hemagglutinin) tagged protein, Myc tagged protein, LacZ tagged protein, CBD (cellulose-binding domain) tagged protein, phage T7 protein kinase (T7PK) tagged protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein. Those skilled in the art know how to select appropriate tagged proteins according to the desired purpose. The use of tags does not alter the function of the target protein; its purpose is to separate, purify, detect, or trace it. Therefore, the tagged proteins applicable to this invention are not limited to a specific type. Tags can be separated from the target protein by chemical cleavage methods or enzymatic methods (such as introducing protease cleavage sites to remove the tag using TEV protease).

[0053] The term "vector" generally refers to a vector capable of delivering exogenous DNA or a target gene into a host cell for amplification and / or expression. This vector can be a cloning vector or an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be amplified and / or expressed within the host cell. Those skilled in the art can select appropriate vectors based on the purpose of genetic engineering and the properties of the recipient cells. These vectors include, but are not limited to: plasmids, phages (such as λ phage or M13 phage), cosmids (i.e., Cosmids), phagemids, shuttle vectors (such as the E. coli-Agrobacterium dual-host shuttle vector), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 phage artificial chromosomes (PAC), or Ti-derived artificial chromosomes (TAC)), and viral vectors (such as baculovirus vectors, tobacco mosaic virus (TMV), potato virus, etc.). A vector may contain multiple elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site.

[0054] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasmas, chlamydiae, spirochetes, algae, etc. For example, the bacteria mentioned may originate from the genus *Escherichia* (…). Escherichia sp. (such as Escherichia coli), Erwinia spp. Erwinia sp. ), Agrobacterium ( Agrobacterium sp. (such as Agrobacterium tumefaciens), Flavobacterium ( Flavobacterium sp. ), Alcaligenes ( Alcaligenes sp. ), Pseudomonas spp. Pseudomonas sp. ) and Bacillus spp. ( Bacillus sp. (e.g., Bacillus). The viruses may include baculoviruses, retroviruses, etc. The fungi may be derived from the genus *Saccharomyces* (e.g., *Bacillus*). Saccharomyces sp. (such as Saccharomyces cerevisiae, Saccharomyces methylbenzene, Pichia pastoris), Fusarium genus ( Fusarium sp. ), Rhizoctonia spp. Rhizoctonia sp. Verticillium ( Verticillium sp. ), Penicillium ( Penicillium sp. Aspergillus ( ) Aspergillus sp. ) and Cephalosporin ( Cephalosporium sp. The actinomycetes may originate from the genus Streptomyces (…). Streptomyces sp. (e.g., Streptomyces). The algae may originate from the phylum Cyanophyta (e.g., cyanobacteria), genus Fucus (e.g., *Fucus*). Fucus sp. ), genus *Cyclocarya* ( Achnanthes sp. ), genus *Codonopsis* ( Amphiprora sp. ), genus Dipterocarpa (Amphora sp. ), Fiber Algae ( Ankistrodesmus sp. ), genus *Stellaria* ( Asteromonas sp. ) and the genus *Golden Color Algae* ( Boekelovia sp. (and others, but not limited to these.)

[0055] The recombinant microorganisms (or recombinant host cells) described herein refer to recombinant microorganisms (or recombinant host cells) whose functions have been altered by manipulating and modifying the genes of a target microorganism (or target host cell). This includes recombinant microorganisms (or recombinant host cells) obtained by introducing exogenous target genes or recombinant vectors into the target microorganism (or target host cell), or recombinant microorganisms (or recombinant host cells) obtained by directly editing the endogenous genes of the target microorganism (or target host cell). The term "recombinant microorganism" (or recombinant host cell) can be understood to refer not only to a specific recombinant microorganism (or recombinant host cell), but also to its offspring. 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 (or recombinant host cells). Attached Figure Description

[0056] Figure 1 for GmRVE8a Gene structure diagram, target location, and T1 generation Gmrve8a-1 and T1 generation Gmrve8a- 2 The sequencing results and mutation types.

[0057] Figure 2 To test the T2 generation GmRVE8a homozygous mutants GmRVE8a Relative gene expression levels. Different lowercase letters indicate the expression level of genes. P＜ There was a significant difference at the 0.05 level.

[0058] Figure 3 For T2 generation GmRVE8a Flowering and maturity phenotypes of homozygous mutants. The scale bar for flowering is 0.5 cm, and the scale bar for maturity is 1 cm.

[0059] Figure 4 For T2 generation GmRVE8a Statistical results of flowering and maturity times for homozygous mutants. Different lowercase letters indicate the flowering and maturity periods. P＜ There was a significant difference at the 0.05 level. Detailed Implementation

[0060] 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.

[0061] 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.

[0062] In the quantitative experiments described below, three replicate experiments were performed.

[0063] The following examples used SPSS 23.0 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, and different lowercase letters indicate significant differences. P <0.05).

[0064] The soybean variety Williams 82 is a common soybean variety, recorded on the China Crop Germplasm Information Network, and serves as the model material for soybean genome research. This biological material can be obtained from the applicant (National Southern Breeding Research Institute, Chinese Academy of Agricultural Sciences, Sanya). This biological material is solely for the purpose of replicating the relevant experiments of this invention and may not be used for any other purpose. In the following text, the soybean variety Williams 82 will be referred to as Williams 82.

[0065] Agrobacterium tumefaciens EHA105 is a common Agrobacterium widely used in plant transgenic research. This biological material can be obtained from the applicant (National Southern Breeding Research Institute, Chinese Academy of Agricultural Sciences, Sanya). This biological material is intended solely for repeating the experiments related to this invention and may not be used for any other purpose.

[0066] MS Salt, MS Organic, B5 Organic, and B5 Salt are all products of Phyto Tech, with catalog numbers M524, M533, G219, and G768, respectively.

[0067] The Fe salt (200×) was obtained by diluting 100× Iron Sulfate / Chelate Solution (a product of Phyto Tech, catalog number F318) with ultrapure water at a volume ratio of 1:1.

[0068] The culture media involved in the following examples are as follows: The solutes and their concentrations in the liquid culture medium were 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. The solvent was water, and the pH was 5.4.

[0069] The solutes and their concentrations in the co-culture medium were 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. The solvent was water, and the pH was 5.4.

[0070] The solutes and their concentrations in the recovery culture medium were 3.1 g / L B5 salt, 1 mL / L B5 organic, 30 g / L sucrose, 150 mg / L cephalosporin, 150 mg / L ticarcillin (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. The solvent was water, and the pH was 5.4.

[0071] The solutes and their concentrations in the screening medium were 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. The solvent was water, and the pH was 5.4.

[0072] The solutes and their concentrations in the elongation medium were 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 ticarcillin, 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, with water as the solvent and a pH of 5.6.

[0073] Example: Knocking out the gene encoding the GmRVE8a protein in the soybean to be improved yielded gene-edited soybeans with earlier flowering and maturity periods. In this embodiment, the soybean variety Williams 82 is used as the soybean to be improved. The nuclear genome of the soybean variety Williams 82 is the most commonly used reference genome for soybean gene function research, and the genes (such as...) can be obtained through the Phytozome v14 database (https: / / phytozome-next.jgi.doe.gov / info / Gmax_Wm82_a2_v1). GmRVE8a The nucleotide sequence of the gene. The genome of soybean variety Williams 82 contains the nucleotide sequence shown in SEQ ID NO: 3. GmRVE8a Gene, GmRVE8a The coding sequence of the gene (i.e., the CDS) is shown in SEQ ID NO: 1. GmRVE8a The gene encodes the GmRVE8a protein. The amino acid sequence of the GmRVE8a protein is shown in SEQ ID NO: 2.

[0074] one, GmRVE8a Gene editing vectors—recombinant vectors GmRVE8a Construction of -gRNA 1. Target acquisition Suitable targets were screened using CRISPR-P v2.0 (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR) for experimental purposes. The target sites are located at... GmRVE8a The exon region of the gene, the target sequence is: 5'-TGTCAATCAATGGCAATGGT-3' (i.e., positions 389-408 of SEQ ID NO: 3).

[0075] 2. Recombinant vector GmRVE8a Construction of -gRNA Recombinant vector GmRVE8a - The gRNA was constructed according to the instructions of the Plant Cas9 / gRNA Plasmid Construction Kit (product of Beijing Weishang Lide Biotechnology Co., Ltd., catalog number VK005-15). The specific steps are as follows: (1) Based on the target, the sense strand 5'-TTGTGTCAATCAATGGCAATGGT-3' (SEQ ID NO: 10) and the antisense strand 5'-AACACCATTGCCATTGATTGACA-3' (SEQ ID NO: 11) were designed and synthesized artificially. Then, the sense strand and the antisense strand were diluted with deionized water to obtain a 10 μM solution of the sense strand and a 10 μM solution of the antisense strand.

[0076] (2) After completing step (1), prepare the Oligo mixture system. Then take the Oligo mixture system, treat it at 95°C for 3 min, then slowly cool it to 25°C at room temperature, and keep it at 16°C for 5 min to obtain Oligo dimer.

[0077] The Oligo mixture consists of 25 μL of 5 μL of positive chain diluent, 5 μL of negative chain diluent, and 15 μL of ddH2O.

[0078] (3) After completing step (2), prepare the Oligo ligation system. Then, take the Oligo ligation system and react it at 16°C for 2 hours to obtain the recombinant vector. GmRVE8a -gRNA.

[0079] The Oligo ligation system consisted of 10 μL of Oligo dimer obtained in step (2), 1 μL of gRNA / Cas9 vector, 1 μL of solution 1, 1 μL of solution 2, and 6 μL of ddH2O. The gRNA / Cas9 vector, solution 1, and solution 2 were all components of the plant Cas9 / gRNA plasmid construction kit.

[0080] Recombinant vector GmRVE8a Sequencing was performed on the -gRNA. Sequencing results showed that the recombinant vector... GmRVE8a -gRNA is a recombinant plasmid obtained by replacing the blank gRNA insertion site downstream of the atU6 promoter in the gRNA / Cas9 vector with the DNA molecule shown in positions 389-408 of SEQ ID NO: 3.

[0081] II. Recombinant Agrobacterium EHA- GmRVE8a Preparation of -gRNA The recombinant vector constructed in step one was prepared using a freeze-thaw method. GmRVE8a Agrobacterium tumefaciens EHA105 was transformed with -gRNA, and plasmids were extracted and sequenced for verification. The recombinant Agrobacterium that was correctly sequenced was named EHA- GmRVE8a -gRNA. EHA- GmRVE8a -gRNA contains recombinant vector GmRVE8a -gRNA.

[0082] III. Obtaining T0 generation gene knockout soybeans and detecting mutation types Using Agrobacterium-mediated transformation to deliver EHA- GmRVE8a Transforming soybean variety Williams 82 with -gRNA yielded T0 generation pseudogene knockout soybeans. The specific steps are as follows: 1. Seed sterilization (1) Take seeds of the Williams 82 soybean variety that are free from disease, pests and spots, plump, uniform and dry, spread them evenly in a petri dish, and then put the petri dish into a desiccator.

[0083] (2) After completing step (1), place a 100mL beaker in the desiccator, pour 80mL of sodium hypochlorite aqueous solution with a concentration of 12mol / L into the beaker, then slowly add 4mL of concentrated hydrochloric acid, then quickly cover the desiccator, seal it with petroleum jelly, and place it for 16h for chlorine sterilization.

[0084] 2. Obtaining T0 generation gene knockout soybeans (1) Cultivate the EHA- prepared in step one at 28℃ GmRVE8a -gRNA, then resuspended in liquid culture medium to obtain OD. 600nm Infected bacterial solution with a value of 0.6.

[0085] (2) Place the sterilized seeds obtained in step 1 into a laminar flow hood, peel off the seed coat under a microscope, separate the two cotyledons along the long axis, retain the cotyledons with complete hypocotyls, and make scratches at the junction of the hypocotyl and cotyledons (generally 3-5 scratches per cotyledon); then add the invading bacterial solution and soak at 28°C for 2 hours.

[0086] (3) After completing step (2), place the cotyledons with the inner surface (smooth surface) facing up on the co-culture medium that has been covered with sterile filter paper, and incubate in the dark at 22°C for 5 days.

[0087] (4) After completing step (3), wait until the hypocotyl elongates to 2cm, then cut off part of the hypocotyl, leaving 0.5cm. Then place the treated explants in recovery medium and culture for 7 days at 28℃ under 16h light / 8h dark conditions.

[0088] (5) After completing step (4), remove the explant from the recovery medium, remove the new shoots, cut off part of the hypocotyl, retain 0.5 cm of the hypocotyl, and then transfer the trimmed explant into the screening medium and culture for 21 days at 28℃ under 16h light / 8h dark conditions.

[0089] (6) After the screening and induction in step (5), the explant produces a large number of adventitious buds. The cotyledons and brown leaves are removed, and the remaining part is transferred to the elongation medium and cultured at 28°C under 16h light / 8h darkness until 5-8cm young stems emerge.

[0090] (7) After completing step (6), remove all the unfolded leaves of the regenerated bud in the elongation medium, and retain the apical vegetative bud and the 2-4cm main stem below it. According to the number of nodes of the regenerated bud, divide it into sections to ensure that each scion has a 2-3cm main stem and an apical vegetative bud or axillary vegetative bud on it.

[0091] (8) Four to six days after soybean emergence, cotyledonary nodes are created by removing all tissues above the cotyledonary node and the vegetative buds in the cotyledonary axils. Using the cotyledonary node as rootstock and the regenerated bud as scion, the rootstock and scion are grafted using the cleft grafting method. The graft union is fixed with a sealing film and covered with plastic film to retain moisture for hardening-off treatment. When the first new trifoliate compound leaf has fully unfolded, the plastic film is removed and the plant is transplanted into a pot. The resulting plant is the T0 generation of gene knockout soybean.

[0092] 3. Mutation type detection Because soybeans are diploid plants, when Cas9 begins to cleave specific genes, both alleles on the two homologous chromosomes within the same cell can potentially be edited, resulting in the same or different types of mutations. Therefore, two alleles in a single plant are considered two gene editing events. A homozygous mutant refers to a plant whose two homologous chromosomes... Gmrve8a The genes have undergone the same mutation. A biallelic mutant refers to a plant whose two homologous chromosomes have undergone the same mutation. Gmrve8a All genes underwent mutations, but the forms of mutation differed. The number of heterozygous mutant strains refers to the number of homozygous chromosomes on one of the plant's two homozygous chromosomes. Gmrve8a A gene mutation occurred, on another homologous chromosome Gmrve8a No gene mutation occurred. Wild type refers to the type of chromosome between the two homologous chromosomes of this plant. Gmrve8a No gene mutations were observed.

[0093] Genomic DNA from leaves of several T0 generation soybeans with gene knockout or Williams 82 (as a control) was used as templates for PCR amplification of the target region, yielding PCR amplification products. The primers used for PCR amplification were... GmRVE8a - CRISPR-F : 5'-ACGGCTTGATAATGGGTG-3' (SEQ ID NO: 12) and GmRVE8a-CRISPR-R :5'-CAACATAATGGCATAACAGA-3' (SEQ ID NO: 13).

[0094] The PCR amplification products were sequenced. The sequencing data were compared using Chromas software. The presence of overlapping peaks (two or more peaks) near the target site identified heterozygous mutants or biallelic mutants, named generation T0. GmRVE8a Gene-edited soybeans.

[0095] IV. T1 Generation GmRVE8a Obtaining homozygous mutants and detecting mutation types 1. Take the T0 generation obtained in step three respectively. GmRVE8aGenetically edited soybeans are self-pollinated to produce seeds that are T1 generation seeds, and plants grown from T1 generation seeds are T1 generation plants.

[0096] A total of 8 T1 generation plants were obtained.

[0097] 2. Using genomic DNA from leaves of T1 generation plants as templates, respectively, GmRVE8a - CRISPR-F and GmRVE8a- CRISPR-R The primer pairs were used for PCR amplification to obtain PCR amplification products. The PCR amplification products were then sequenced. The sequencing results were compared with... GmRVE8a The Cas9 target sequence of the gene (SEQ ID NO: 3) was compared, and the mutation types were counted.

[0098] Ultimately obtained 2 T1 generation GmRVE8a Homozygous mutants were named T1 generation. Gmrve8a-1 and T1 generation Gmrve8a- 2 .

[0099] T1 generation Gmrve8a-1 Two homologous chromosomes GmRVE8a The same mutation occurred in the genes, specifically on two homologous chromosomes. GmRVE8a All genes underwent a 4-nucleotide deletion—CAAT (i.e., deletion at positions 402-405 of SEQ ID NO: 3 (corresponding to positions 36-39 of SEQ ID NO: 1)), resulting in a frameshift and premature termination of GmRVE8a protein translation, thus knocking out the gene encoding GmRVE8a protein. This is the T1 generation. Gmrve8a-1 middle, GmRVE8a Gene mutation GmRVE8a / 4 Gene: Gmrve8a-1 In the genome, GmRVE8a / 4 The nucleotide sequence of the gene is shown in SEQ ID NO: 7; GmRVE8a / 4 The nucleotide sequence of the gene's coding sequence (i.e., the CDS) is shown in SEQ ID NO: 6. GmRVE8a / 4 The gene encodes the GmRVE8a / 4 protein. The amino acid sequence of the GmRVE8a / 4 protein is shown in SEQ ID NO: 9.

[0100] T1 generation Gmrve8a-2 Two homologous chromosomes GmRVE8a The same mutation occurred in the genes, specifically on two homologous chromosomes. GmRVE8aAll genes underwent a 5-nucleotide deletion—CAATG (i.e., deletion at positions 402-406 of SEQ ID NO: 3 (corresponding to positions 36-40 of SEQ ID NO: 1)), resulting in a frameshift and premature termination of GmRVE8a protein translation, thus knocking out the gene encoding GmRVE8a protein. This is the T1 generation. Gmrve8a-2 middle, GmRVE8a Gene mutation GmRVE8a / 5 Gene: Gmrve8a-2 In the genome, GmRVE8a / 5 The nucleotide sequence of the gene is shown in SEQ ID NO: 5; GmRVE8a / 5 The nucleotide sequence of the gene's coding sequence (i.e., the CDS) is shown in SEQ ID NO: 4. GmRVE8a / 5 The gene encodes the GmRVE8a / 5 protein. The amino acid sequence of the GmRVE8a / 5 protein is shown in SEQ ID NO: 8.

[0101] GmRVE8a Gene structure diagram, target location, and T1 generation Gmrve8a-1 and T1 generation Gmrve8a-2 See sequencing results and mutation types. Figure 1 .

[0102] V. T2 Generation GmRVE8a Obtaining homozygous mutants T1 generation respectively Gmrve8a-1 and T1 generation Gmrve8a-2 Propagation and generation were carried out, and strains without transgenic elements were selected and named T2 generation. Gmrve8a-1 and T2 generation Gmrve8a-2 .

[0103] Testing revealed that, compared to the Williams 82, the T2 generation... Gmrve8a-1 The only difference in the genome is the placement of the two homologous chromosomes of Williams82. GmRVE8a Gene mutation GmRVE8a / 4 Gene.

[0104] Testing revealed that, compared to the Williams 82, the T2 generation... Gmrve8a-2 The only difference in the genome is the placement of the two homologous chromosomes of Williams82. GmRVE8a Gene mutation GmRVE8a / 5 Gene.

[0105] VI. Real-time quantitative PCR detection of T2 generation GmRVE8ahomozygous mutants (such as T2 generation) Gmrve8a-1 T2 generation Gmrve8a-2 )middle GmRVE8a Relative expression level of genes The soybeans tested were Williams 82 and T2 generation. Gmrve8a-1 and T2 generation Gmrve8a-2 .

[0106] (1) Total RNA was extracted from the soybean to be tested, and then reverse transcriptase was used to reverse transcribe the cDNA of the soybean to be tested.

[0107] (2) Real-time quantitative PCR detection of cDNA in soybeans to be tested GmRVE8a Relative gene expression levels (in terms of) GmActin Gene( Glyma.18G290800 (This is an internal reference gene).

[0108] Used for detection GmActin The primers for the gene are 5'-CGGTGGTTCTATCTTGGCATC-3' (SEQ ID NO: 14) and 5'-GTCTTTCGCTTCAATAACCCTA-3' (SEQ ID NO: 15).

[0109] Used for detection GmRVE8a The primers for the gene are 5'-GCTCGCCTATCTTCTTGG-3' (SEQ ID NO: 16) and 5'-GCCTGTTTCCCTTGTTTT-3' (SEQ ID NO: 17).

[0110] Williams 82 GmRVE8a The relative expression level of the gene was taken as 1, and the T2 generation was statistically analyzed separately. Gmrve8a-1 and T2 generation Gmrve8a-2 middle GmRVE8a The relative expression level of genes.

[0111] Test results are shown Figure 2 The results show that, compared to Williams 82, the T2 generation... Gmrve8a-1 and T2 generation Gmrve8a-2 middle GmRVE8a The relative expression levels of all genes were significantly reduced.

[0112] VII. Phenotypic Identification The soybeans tested were Williams 82 and T2 generation. Gmrve8a-1 and T2 generation Gmrve8a-2 .

[0113] Soybeans were planted under natural long-day, short-day, and long-day conditions. Each line consisted of 30 plants. During the planting period, individual plants were recorded according to the soybean growth stage classification standards (see the following literature: Fehr, WR and Caviness, CE (1977). Stages of soybean development. Agriculture and Home Economics Experiment Station, Iowa State University of Science and Technology.): Initial flowering period (R1): the time from sowing to the opening of a flower at any node on the main stem; Physiological maturity period (R7): the time from sowing to the time when any normal pod on the main stem reaches the color of a mature pod.

[0114] Both long-day and short-day cultures were incubated in an artificial climate chamber. Long-day conditions were: 26°C, with alternating light and dark cycles of 16 hours light / 8 hours darkness. Short-day conditions were: 26°C, with alternating light and dark cycles of 12 hours light / 12 hours darkness.

[0115] Natural long-day conditions are determined in the fields, specifically divided into Beijing summer sowing natural long-day conditions and Xinxiang summer sowing natural long-day conditions. The Beijing summer sowing natural long-day conditions are: planting location in Beijing, sowing on June 15, 2025, emergence on June 22, 2025, with a maximum day length of 15 hours during the growing season. The Xinxiang summer sowing natural long-day conditions are: planting location in Xinxiang, Henan, sowing on June 16, 2025, emergence on June 22, 2025, with a maximum day length of 14.5 hours during the growing season.

[0116] T2 generation Gmrve8a-1 and T2 generation Gmrve8a-2 See flowering and maturity phenotypes. Figure 3 .

[0117] T2 generation Gmrve8a-1 and T2 generation Gmrve8a-2 The statistical results of the flowering and ripening periods are shown in the figure. Figure 4 .

[0118] The results showed that under short-day conditions, the T2 generation Gmrve8a-1 and T2 generation Gmrve8a-2 The flowering period was significantly earlier than that of Williams 82 by an average of 4.6 days (i.e., flowering occurred 4.6 days earlier). P <0.05%, the average maturity period was significantly advanced by 6.4 days ( P <0.05); Under long-day conditions, T2 generation Gmrve8a-1 and T2 generation Gmrve8a-2The flowering period was significantly earlier than that of Williams 82 by 7.1 days on average (i.e., flowering was 7.1 days earlier). P <0.05), and the average maturity period was significantly advanced by 9.4 days ( P <0.05); Under the natural long-day conditions of summer sowing in Beijing, T2 generation Gmrve8a-1 and T2 generation Gmrve8a-2 The flowering period was significantly earlier than Williams 82 by an average of 3.2 days (i.e., flowering was 3.2 days earlier), and the maturity period was significantly earlier by an average of 4.8 days. P <0.05); Under the long-day conditions of summer sowing in Xinxiang, the T2 generation Gmrve8a-1 and T2 generation Gmrve8a-2 The flowering time was significantly earlier than that of Williams 82 by an average of 3.1 days (i.e., flowering was 3.1 days earlier). P <0.05%, the average maturity period was significantly advanced by 4.6 days ( P <0.05). This shows that, regardless of whether it's long-day, short-day, or naturally long-day conditions, compared to the soybean variety Williams 82, the T2 generation... GmRVE8a homozygous mutants (such as T2 generation) Gmrve8a-1 T2 generation Gmrve8a-2 The flowering and maturity periods of ) were significantly advanced (significantly shortened).

[0119] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Gene-edited soybeans, characterized by: The gene-edited soybean is obtained by knocking out the gene encoding the GmRVE8a protein in the soybean to be improved; the soybean to be improved contains the gene encoding the GmRVE8a protein, and the nucleotide sequence of the gene encoding the GmRVE8a protein is shown in SEQ ID NO: 3; the gene-edited soybean is a soybean that does not contain the gene encoding the GmRVE8a protein. The GmRVE8a protein is either a1 or a2). a1) The amino acid sequence of the protein is SEQ ID NO: 2; a2) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a1); The flowering and / or maturity period of the gene-edited soybeans is earlier than that of the soybeans to be improved.

2. The gene-edited soybean according to claim 1, characterized in that: The knockout includes performing any of the following operations on the genome of the soybean to be improved: F1) Delete nucleotides 402-405 of SEQ ID NO:3 in the soybean genome to be improved; F2) Delete nucleotides 402-406 of SEQ ID NO:3 in the soybean genome to be improved.

3. The application of biological materials that have the gene encoding the GmRVE8a protein of claim 1 knocked out, comprising at least one of the following A1) to A5): A1) It advances the flowering period of soybeans; A2) It advances the maturity period of soybeans; A3) Developing gene-edited soybeans with earlier flowering periods; A4) Developing gene-edited soybeans that mature earlier; A5) Soybean breeding; the purpose of the breeding is to advance the flowering and / or maturity period of soybeans.

4. The application according to claim 3, characterized in that: The biological material from which the gene encoding the GmRVE8a protein is knocked out is any one of D1)-D8): D1) A nucleic acid molecule that targets the gene encoding the GmRVE8a protein as described in claim 1; the nucleic acid molecule is a gRNA that targets the gene encoding the GmRVE8a protein. D2) Cas protein and D1) the nucleic acid molecule described; D3) The nucleic acid molecules and Cas protein-encoding genes described in D1); D4) Vectors, expression cassettes, or recombinant microorganisms containing the nucleic acid molecules described in D1) and the Cas protein-encoding gene; D5) Soybeans containing the nucleic acid molecules described in D1); D6) Soybeans containing Cas protein and the nucleic acid molecules described in D1); D7) Soybeans containing the nucleic acid molecules and Cas protein-encoding genes described in D1); D8) Soybeans containing the nucleic acid molecules described in D1) and the Cas protein encoding gene, vectors, expression cassettes, or recombinant microorganisms.

5. A method for cultivating soybeans with earlier flowering and / or maturity periods, comprising knocking out the gene encoding the GmRVE8a protein in the soybean to be improved containing the gene encoding the GmRVE8a protein as described in claim 1, thereby obtaining gene-knockout soybeans with earlier flowering and / or maturity periods than the soybean to be improved.

6. The method according to claim 5, characterized in that: The knockout includes performing any of the following operations on the genome of the soybean to be improved: F1) Delete nucleotides 402-405 of SEQ ID NO:3 in the soybean genome to be improved; F2) Delete nucleotides 402-406 of SEQ ID NO:3 in the soybean genome to be improved.

7. A method for advancing the flowering and / or maturity period of soybeans, comprising knocking out the gene encoding the GmRVE8a protein in soybeans to be improved containing the gene encoding the GmRVE8a protein as described in claim 1, thereby obtaining gene knockout soybeans; the flowering and / or maturity period of the gene knockout soybeans is advanced compared to that of the soybeans to be improved.

8. The method according to claim 7, characterized in that: The knockout includes performing any of the following operations on the genome of the soybean to be improved: F1) Delete nucleotides 402-405 of SEQ ID NO:3 in the soybean genome to be improved; F2) Delete nucleotides 402-406 of SEQ ID NO:3 in the soybean genome to be improved.

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