Use of mutant gmln protein and gene encoding same in regulation of soybean yield

Abstract

The present disclosure pertains to the field of genetic engineering of plants. Specifically, the present disclosure relates to use of a mutant GmLn protein and a gene encoding same in the regulation of soybean yield, particularly to, but not limited to, a method for obtaining a soybean tolerant of dense planting, a method for detecting a soybean tolerant of dense planting, and a related primer and kit, and further to a soybean plant tolerant of dense planting and a method for improving soybean planting density. The methods provided by the present disclosure involve expressing a gene editing tool in a soybean to genetically edit a target sequence in the soybean, resulting in a soybean tolerant of dense planting with increased yield.

Description

Mutated GmLn protein and its encoding gene for regulating soybean yield

[0001] Priority and Related Applications

[0002] The present disclosure claims priority to Chinese Patent Application No. 202410544468.0, filed on April 30, 2024, entitled “Mutated GmLn Protein and Its Encoding Gene for Regulating Soybean Yield,” the entire contents of which, including appendices, are incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure belongs to the field of plant genetic engineering, and relates to a mutated GmLn protein and its encoding gene for regulating soybean yield, in particular to, but not limited to, a method for obtaining high-density-plantable soybean, a method for detecting high-density-plantable soybean, and related primers and kits, and further relates to high-density-plantable soybean plants and a method for increasing soybean planting density. BACKGROUND

[0004] Crops are the basic guarantee for human survival and reproduction, and therefore scientists have been continuously domesticating better crop varieties by selecting seeds with excellent traits such as high yield for planting and breeding, in order to meet the basic needs for crops.

[0005] Soybean is an important food and oil crop. As a food, soybean seeds are rich in protein and oil. The composition ratio of its oil, protein, carbohydrates, and crude fiber is very close to that of meat food. Among all cereal and legume crops, soybean seeds have the highest protein content, about 40%, which is 2 to 4 times higher than that of wheat, corn, and rice, etc. Soybean seeds contain about 20% oil, which is the second highest among all cereal and legume crops. Soybean contains 8 essential amino acids for the human body, among which lysine and tryptophan (their contents are 2.3% and 0.5%, respectively) cannot be synthesized by the human body and are easily absorbed by the human body, and therefore soybean is known as “vegetable meat”.

[0006] Therefore, how to improve soybean yield is the focus of researchers. It has been reported that single base substitution mutation of soybean GmJaGGED1-2 may produce phenotypic differences in soybean plants (Jeong N, Suh S J, Kim M H, et al. Ln is a key regulator of leaflet shape and number of seeds per pod in soybean [J]. The Plant Cell, 2012, 24(12): 4807-4818.). It has been reported that a high-density soybean mutant library (1 mutation per 74 kb) is designed to screen mutant systems with target phenotypes (Tsuda M, Kaga A, Anai T, et al. Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing [J]. BMC genomics, 2015, 16: 1-18.). In breeding research, it has been reported that two gRNAs targeting GmJAG1 and GmJAG2 are designed to edit multiple target gene sites of the two genes at the same time, so as to obtain soybean varieties with yield improvement (Cai Z, Xian P, Cheng Y, et al. CRISPR / Cas9-mediated gene editing of GmJAGGED1 increased yield in the low-latitude soybean variety Huachun 6 [J]. Plant biotechnology journal, 2021, 19(10): 1898.). However, high-density mutation or multi-target mutation in the prior art may produce unexpected mutations, which may mask the expected phenotype or affect other traits. It is of great significance and social and economic value to achieve site-directed gene editing on the target sequence of the genome and to create high-yield and safe soybean materials. SUMMARY

[0007] To solve the above technical problems, the present disclosure designs a site-directed gene editing tool construct, which realizes site-directed editing of target genes in soybean plants by using the gene editing tool, and screens soybean germplasm with high-density planting and high-yield traits.

[0008] Specifically, in a first aspect, the present disclosure provides a genetically engineered non-propagating material soybean plant cell, comprising a mutated GmLn gene, wherein the mutated GmLn gene comprises: a nucleotide sequence obtained by insertion or deletion mutation at nucleotides 345-372 in the nucleotide sequence as set forth in SEQ ID NO: 15.

[0009] The mutated GmLn gene is knocked down or knocked out compared with the unmutated GmLn gene.

[0010] Exemplarily, the "nucleotide sequence obtained by insertion or deletion mutation at nucleotides 345-372 in the nucleotide sequence as set forth in SEQ ID NO: 15" comprises a nucleotide sequence obtained by insertion or deletion mutation at any position of nucleotides 345-372 in the nucleotide sequence as set forth in SEQ ID NO: 15, which can comprise:

[0011] insertion of one or more nucleotides between any two of nucleotides 345-372 in the nucleotide sequence as set forth in SEQ ID NO: 15;

[0012] deletion of one or more nucleotides in nucleotides 345-372 in the nucleotide sequence as set forth in SEQ ID NO: 15; or

[0013] deletion of one or more nucleotides in nucleotides 345-372 in the nucleotide sequence as set forth in SEQ ID NO: 15, and insertion of one or more nucleotides between any two of nucleotides 345-372 in the nucleotide sequence as set forth in SEQ ID NO: 15.

[0014] In an exemplary embodiment provided by the present disclosure, the mutated GmLn gene comprises: a nucleotide sequence obtained by insertion or deletion mutation at nucleotides 368-372 in the nucleotide sequence as set forth in SEQ ID NO: 15.

[0015] In an exemplary embodiment provided by the present disclosure, the mutated GmLn gene comprises: insertion of a nucleotide between nucleotide 370 and nucleotide 371 in the nucleotide sequence as set forth in SEQ ID NO: 15.

[0016] In an exemplary embodiment provided by the present disclosure, the stop codon of the mutated GmLn gene is located between nucleotides 368-388 in the nucleotide sequence as set forth in SEQ ID NO: 15.

[0017] In an exemplary embodiment provided in this disclosure, the soybean plant cells contain a mutated GmLn protein;

[0018] The amino acid sequence of the mutated GmLn protein is shown in SEQ ID NO:4.

[0019] In an exemplary embodiment provided in this disclosure, the soybean plant cell includes a nucleotide sequence encoding the mutated GmLn protein.

[0020] In the exemplary embodiments provided in this disclosure, the nucleotide sequence encoding the mutated GmLn protein is selected from the nucleotide sequence shown in SEQ ID NO:16 (or the CDS sequence of the mutated GmLn as shown in SEQ ID NO:3).

[0021] Secondly, this disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding the aforementioned mutated GmLn protein.

[0022] In an exemplary embodiment provided in this disclosure, the nucleic acid molecule comprises a nucleotide sequence encoding a mutated GmLn protein with an amino acid sequence as shown in SEQ ID NO:4.

[0023] In the exemplary embodiments provided in this disclosure, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:16 (or the CDS sequence of the mutated GmLn is shown in SEQ ID NO:3).

[0024] Thirdly, this disclosure provides a method for improving the length-to-width ratio of soybean leaves, the method comprising: the soybean expressing the above-mentioned mutated GmLn protein, or the soybean including the above-mentioned nucleic acid molecule.

[0025] In the exemplary embodiments provided in this disclosure, the endogenous GmLn gene of soybean is mutated to encode the aforementioned mutated GmLn protein and / or the endogenous GmLn gene of soybean is mutated to the aforementioned nucleic acid molecule.

[0026] Fourthly, this disclosure provides a method for manufacturing or obtaining soybean plants, wherein all or part of the cells of the soybean plant are modified by gene editing, physical mutagenesis and / or chemical mutagenesis to express the above-mentioned mutated GmLn protein (e.g., expressing the mutated GmLn protein with an amino acid sequence as shown in SEQ ID NO:4), wherein the endogenous GmLn gene of the soybean plant is mutated into the above-mentioned nucleic acid molecule (e.g., encoding the mutated GmLn protein with an amino acid sequence as shown in SEQ ID NO:4 or the nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO:16).

[0027] In the embodiments exemplarily provided in the present disclosure, the tool adopted for the gene editing is CRISPR / Cas nuclease or its derivative technology, zinc finger nuclease or its derivative technology (ZFN), or transcription activator-like effector (TALE) or its derivative technology.

[0028] In the embodiments exemplarily provided in the present disclosure, the CRISPR / Cas nuclease comprises TraC effector protein, preferably eTraC protein, the amino acid sequence of which is shown in SEQ ID NO: 14.

[0029] In the embodiments exemplarily provided in the present disclosure, the DNA target sequence recognized by the DNA recognition region of the gRNA molecule comprises at least SEQ ID NO: 5 or its complementary sequence.

[0030] In the embodiments exemplarily provided in the present disclosure, the DNA target sequence is located in the GmLn gene.

[0031] In the embodiments exemplarily provided in the present disclosure, the DNA target sequence recognized by the DNA recognition region of the gRNA molecule comprises at least SEQ ID NO: 5 or its complementary sequence.

[0032] In the embodiments exemplarily provided in the present disclosure, the DNA target sequence is located in the GmLn gene.

[0033] In the embodiments exemplarily provided in the present disclosure, the composition is used for reducing the expression of GmLn gene.

[0034] In the embodiments exemplarily provided in the present disclosure, the reduction of the expression of GmLn gene leads to the expression of the protein with the amino acid sequence shown in SEQ ID NO: 4.

[0035] In the embodiments exemplarily provided in the present disclosure, the DNA target sequence recognized by the DNA recognition region of the gRNA molecule comprises at least SEQ ID NO: 5 or its complementary sequence.

[0036] In the embodiments exemplarily provided in the present disclosure, the DNA target sequence is located in the GmLn gene.

[0037] In the embodiments exemplarily provided in the present disclosure, the composition is used for reducing the expression of GmLn gene.

[0038] In a tenth aspect, the Cas effector protein in the gRNA molecule, the composition, the vector, the ribonucleoprotein complex, or the method for reducing expression of a GmLn gene described above, wherein the Cas effector protein is a transposon and CRISPR-Cas12 intermediate (TraC) effector protein.

[0039] In an eleventh aspect, the present disclosure provides a soybean plant cell of a non-propagating material, comprising: the gRNA molecule, the composition, the vector, and / or the ribonucleoprotein complex described above.

[0040] The soybean plant cell further comprises: reduced expression of a GmLn gene.

[0041] In an exemplary embodiment of the present disclosure, the reduced expression of the GmLn gene results in expression of a protein shown in SEQ ID NO: 4.

[0042] In an exemplary embodiment of the present disclosure, the leaf shape of a soybean plant comprising the soybean plant cell changes from an oval shape to a pointed leaf shape compared to a soybean plant in which the expression of the GmLn gene is not reduced.

[0043] In a twelfth aspect, the present disclosure provides a method for detecting a soybean with a mutation in a GmLn gene, the method comprising: contacting a sample to be detected with at least two primers for amplifying a target amplification product in a nucleic acid amplification reaction;

[0044] performing a nucleic acid amplification reaction;

[0045] detecting the presence of the target amplification product;

[0046] The target amplification product comprises a nucleic acid sequence shown in SEQ ID NO: 3.

[0047] In an exemplary embodiment provided by the present disclosure, the two primers comprise primers shown in SEQ ID NO: 11 and SEQ ID NO: 12.

[0048] In a thirteenth aspect, the present disclosure provides a kit for detecting a soybean with reduced expression of a GmLn gene, the kit comprising primers shown in SEQ ID NO: 11 and SEQ ID NO: 12.

[0049] In a fourteenth aspect, the present disclosure provides a method for reducing leaf area of soybean, the method comprising: causing a soybean plant cell to express the mutant GmLn protein described above (e.g., a mutant GmLn protein having an amino acid sequence as set forth in SEQ ID NO: 4) and / or mutating the endogenous GmLn gene in the soybean plant into the nucleic acid molecule described above (e.g., a nucleic acid molecule encoding a mutant GmLn protein having an amino acid sequence as set forth in SEQ ID NO: 4 or a nucleotide sequence as set forth in SEQ ID NO: 16).

[0050] In a fifteenth aspect, the present disclosure provides a method for increasing planting density of soybean, the method comprising: causing a soybean plant cell to express the mutant GmLn protein described above (e.g., a mutant GmLn protein having an amino acid sequence as set forth in SEQ ID NO: 4) and / or mutating the endogenous GmLn gene in the soybean plant into the nucleic acid molecule described above (e.g., a nucleic acid molecule encoding a mutant GmLn protein having an amino acid sequence as set forth in SEQ ID NO: 4 or a nucleotide sequence as set forth in SEQ ID NO: 16).

[0051] Technical effects

[0052] The present disclosure provides a mutant soybean GmLn protein and its encoding gene. The soybean plant with the mutation has an increased number of soybean plants per mu under high planting density, and the soybean plant with the mutation has no change in soybean nutritional components under high-density planting (i.e., dense planting) conditions, and the number of pods per plant, the number of grains per plant, and the number of grains per pod are significantly increased, thereby increasing the yield of soybean.

[0053] The technical solution provided by the present disclosure only needs to edit one target sequence in the soybean GmLn gene to obtain a homozygous mutant genotype, thereby significantly reducing the high off-target risk that may exist when multiple target sequences are edited.

[0054] Definitions

[0055] As used herein, unless otherwise indicated, the scientific and technical terms used herein have the meanings that would be understood by one of ordinary skill in the art. Also, as used herein, the terms "protein" and "nucleic acid chemistry," "molecular biology," "cell culture," "microbiology," "immunology" are related terms and laboratory procedures steps used in connection with the practice of the respective art, and such terms are understood by those of ordinary skill in the art. For example, standard recombinant DNA and molecular cloning techniques used in the disclosure are well known and described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Sambrook"). Also, for better understanding of the disclosure, the definitions and explanations of the relevant terms are provided below.

[0056] As used herein, the term "and / or" encompasses all combinations of the items linked by the term "and / or". For example, "A and / or B" covers the possibilities of "A", "A and B", and "B". For example, "A, B, and / or C" covers the possibilities of "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0057] As used herein, the terms "comprises", "comprising", and "comprised of" should be understood to mean that the item(s) listed after the terms are at least included in the composition, mixture, reaction medium, process, method, or article of manufacture, but not that the item(s) listed after the terms are the only item(s) included in the composition, mixture, reaction medium, process, method, or article of manufacture.

[0058] The term "plant" as used herein includes immature or mature whole soybean plants, including plants that have been removed of seed, grain, or anthers. Any seed or embryo that is capable of producing a plant is also considered a soybean plant.

[0059] As used herein, the terms "gene", "genome" encompass not only chromosomal DNA present in the nucleus of a cell, but also organelle DNA present in subcellular components of a cell, such as mitochondria, plastids.

[0060] A "genetically modified organism" or "genetically modified cell" means an organism or cell that comprises within its genome an exogenous polynucleotide or a modified gene or expression regulatory sequence. The exogenous polynucleotide is capable of stably integrating into the genome of the organism or cell and being inherited through successive generations, for example. The exogenous polynucleotide can be integrated into the genome either alone or as part of a recombinant DNA construct. The modified gene or expression regulatory sequence is one in which the sequence comprises a single or multiple deoxynucleotide substitutions, deletions and additions in the genome of the organism or cell.

[0061] As used herein, "polynucleotide," "nucleic acid sequence," "nucleotide sequence," "DNA sequence," or "nucleic acid fragment" are used interchangeably and are single- or double-stranded RNA or DNA polymers, optionally can contain synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single letter designation as follows: "A" is adenine or deoxyadenosine (corresponding to RNA or DNA, respectively), "C" denotes cytosine or deoxycytosine, "G" denotes guanine or deoxyguanosine, "U" denotes uridine, "T" denotes deoxythymidine, "R" denotes purine (A or G), "Y" denotes pyrimidine (C or T), "K" denotes G or T, "H" denotes A or C or T, "I" denotes inosine, and "N" denotes any nucleotide.

[0062] As used herein, a polynucleotide is an isolated polynucleotide. Also provided are biological samples and soybean byproducts comprising any of the above polynucleotides. In certain embodiments, the byproducts are processed products made from soybean plants or seeds thereof having a mutated Ln gene, including: (a) soybean meal (defatted or non-defatted); (b) extracted soy protein, oil, sugar, syrup, and starch; (c) soybean fermentation products; (d) soybean-based animal feed or human food (e.g., feed and food comprising soybean meal), (e) pharmaceuticals; (1) unprocessed or processed biomass (e.g., cellulosic and / or lignocellulosic material; silage); and (g) various industrial products.

[0063] As used herein, "polypeptide," "peptide," "amino acid sequence," and "protein" are used interchangeably in the present disclosure to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" can also include modified forms, including but not limited to glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0064] "Identity" in the context of a sequence is intended to mean the percentage of residues that are the same in two sequences, when aligned for maximum correspondence. Sequence identity can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide, or along a region of the molecule. (See, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are a number of methods for measuring sequence identity between two polynucleotides or polypeptides, the term "identity" is well known to one of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0065] In peptides or proteins, suitable conservative amino acid substitutions are known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. In general, those of skill in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide will not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. co., p. 224).

[0066] As used herein, "promoter" refers to a nucleic acid segment capable of controlling the transcription of another nucleic acid segment. In some embodiments of the disclosure, the promoter is a promoter capable of controlling the transcription of a gene in a cell, whether or not it is derived from the cell. The promoter can be a constitutive promoter or a tissue-specific promoter or a developmentally-regulated promoter or an inducible promoter.

[0067] As used herein, "sample" or "specimen" refers to whole or non- whole soybean plant tissue (e.g., ground soybean seed or soybean plant tissue, chopped soybean plant tissue, or lyophilized tissue, etc.). It can also be an extract comprising whole or non- whole seed or soybean plant tissue. The biological specimen can include all or a portion of meal, meal, syrup, oil, starch, and grain containing soybean plant byproducts. In certain embodiments, the biological specimen is "non-regenerable" (i.e., cannot be regenerated into a soybean plant or a portion of a soybean plant).

[0068] As used herein, "knockdown" refers to the down-regulation of the expression and / or activity of a gene of interest (typically an endogenous gene of interest) in a soybean plant or cell relative to a wild-type plant not undergoing the same manipulation by artificial means (e.g., genetic engineering). Expression can be at the level of transcription or at the level of translation. Knockdown of a gene of interest can result in a reduction of its function. Knockdown of a gene of interest, for example, also encompasses mutating (e.g., point mutations) its encoded product to result in a reduction of activity, e.g., biological activity.

[0069] As used herein, "knockout" refers to the substantial lack of expression of a gene of interest (typically an endogenous gene of interest) in a soybean plant or cell relative to a wild-type plant not undergoing the same manipulation by artificial means (e.g., genetic engineering), i.e., substantially no functional expression product is produced and / or expression of a product that is substantially non-functional. Expression can be at the level of transcription or at the level of translation. Knockout of a gene of interest can result in a loss of function. Knockout of a gene of interest, for example, also encompasses mutating (e.g., point mutations) its encoded product to result in a loss of activity, e.g., biological activity.

[0070] As used herein, a soybean Ln gene, referred to simply as GmLn gene or Ln gene, is a class of transcription factors with single-stranded C2H2 zinc finger structure (Jeong N, Suh S J, Kim M H, et al. Ln is a key regulator of leaflet shape and number of seeds per pod in soybean [J]. The Plant Cell, 2012, 24(12): 4807-4818.). Exemplary wild-type GmLn gene is shown in SEQ ID NO: 15 and its homologs, and the CDS sequence of wild-type GmLn is shown in SEQ ID NO: 1.

[0071] As used herein, “reducing GmLn gene expression” refers to a mutation that results in loss or reduction of gene function as compared to a non-mutated or wild-type allele.

[0072] As used herein, a “mutation” can be an increase of 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 15 bp, 20 bp, 25 bp, or more in the position of DNA double-strand break in the genome of GmLn as compared to wild-type; or a “mutation” can be a decrease of 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 15 bp, 20 bp, 25 bp, or more in the position of DNA double-strand break in the genome of GmLn as compared to wild-type.

[0073] As used herein, “mutated” or “edited” refers to comprising a knockdown or knockout of an endogenous soybean Ln gene, i.e., reducing GmLn gene expression. In certain embodiments, such knockdown or knockout mutation results in GmLn gene expressing a protein as shown in SEQ ID NO: 4. “Mutated” can also refer to a mutation to a CRISPR / Cas nuclease to achieve higher editing efficiency against a target sequence, or other beneficial effects related to CRISPR / Cas nuclease.

[0074] In certain embodiments, the insertion shown in SEQ ID NO: 3 is a GmLn null mutation, which comprises at least an insertion of 1 base A between the 24th and 25th nucleotides of SEQ ID NO: 1.

[0075] As used herein, “plant” or “planting” includes a whole soybean plant and any progeny, cells, tissues, parts, or components thereof. Thus, the term “plant” or “planting” includes a whole soybean plant, whether immature or mature, including plants or plantings that have had seeds, grains, or anthers removed.

[0076] As used herein, a "plant part" includes any part of a plant, such as, but not limited to, a seed (including a mature seed and an immature seed); a grain; a stover; a plant section; a plant cell; a plant cell culture; or a plant organ (such as pollen, an embryo, a pod; a flower, a fruit, a shoot, a leaf, a root, a stem, and an explant). A plant tissue or plant organ can be a seed, a protoplast, a callus, or any other group of plant cells organized into a structural or functional unit. A plant cell or tissue culture can regenerate a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained and regenerate a plant that is substantially identical to the plant from which the cell or tissue was obtained. Regenerable cells in a plant cell or tissue culture can be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, or stems. Conversely, some plant cells cannot regenerate a whole plant, referred to herein as "non- propagating" plant cells.

[0077] In certain embodiments, soybean plants with a knocked out or knocked down GmLn gene have a higher planting density with an increased or unchanged yield per soybean plant compared to a corresponding wild type soybean plant. The planting density can be increased by up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0078] As used herein, a "trait" refers to a physiological, morphological, biochemical, or physical characteristic of a cell or organism.

[0079] As used herein, an "agronomic trait" refers specifically to a measurable indicator parameter of a crop plant, including but not limited to, leaf greenness, grain yield, growth rate, total biomass or rate of accumulation, fresh weight at maturity, dry weight at maturity, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content of vegetative plant tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content of vegetative plant tissue, total plant protein content, fruit protein content, seed protein content, protein content of vegetative plant tissue, herbicide resistance, drought resistance, nitrogen uptake, root lodging, harvest index, stalk lodging, plant height, ear height, ear length, disease resistance, cold resistance, salt resistance, and tiller number.

[0080] As used herein, a "nuclease" refers to an RNA-dependent DNA endonuclease herein. In some embodiments, the nucleases of the present disclosure can be selected from transposons and CRISPR-Cas12 intermediate (TraC) effector proteins.

[0081] The transposon and CRISPR-Cas intermediate (TraC) effector protein refers to a class of CRISPR effector proteins that have the target cleavage activity of the TnpB system and the CRISPR system, that is, they can target and bind to the target DNA under the guidance of reRNA, and can target and bind to the target DNA under the guidance of a guide RNA such as sgRNA composed of tracrRNA and / or crRNA, as disclosed in Chinese Patent CN117187213A, which is incorporated herein by reference.

[0082] In some preferred embodiments, the nuclease of the present disclosure is an eTraC effector protein, the amino acid sequence of which is shown in SEQ ID NO: 14. The structure of the gRNA corresponding to the TraC effector protein includes a scaffold. The TraC effector protein and its corresponding gRNA can be described and selected in more detail with reference to Chinese Patent CN117187213A.

[0083] In this context, CRISPR / Cas nuclease has the same meaning as Cas effector protein, which refers to the core functional protein directly responsible for target recognition and cleavage in the CRISPR-Cas system.

[0084] In this context, soybean coverage refers to the planting coverage of soybeans on a certain land area, usually expressed in percentage. It can reflect the planting scale and distribution of soybeans in a specific area. Soybean coverage = (soybean planting area ÷ total land area) x 100%.

[0085] In this context, the breeding coefficient refers to the ratio of the number of offspring obtained by breeding means to the number of parents within a certain period of time.

[0086] In this context, shattering refers to the property of crop seeds or fruits naturally falling off the mother plant after maturation.

[0087] If any of the foregoing definitions is inconsistent with the definition in any patent or non-patent reference cited herein, any patent or non-patent reference cited herein, or any patent or non-patent reference elsewhere, it is understood that the foregoing definition will be used herein. BRIEF DESCRIPTION OF DRAWINGS

[0088] Figure 1 is a vector map of Example 1.

[0089] Figure 2 is a schematic diagram of the T-DNA insertion region of the vector of Example 1.

[0090] Figure 3 is a comparison of the phenotype of T1 generation genome editing homozygous soybean plant (QH64111) numbered 1 in Table 3 and wild type soybean.

[0091] Figure 4 is a peak plot (excerpts) and alignment analysis of the detection results of the editing homozygote QH64111 numbered 1, which shows that 1 bp (A) is inserted in the target region of the GmLn gene, and no editing occurs in the potential off-target region, which is the same as the wild type.

[0092] Figures 5A and 5B are peak plots (excerpts) and alignment analysis of the detection results of the T2 generation genome editing homozygotes QH64111-T2-1 to QH64111-T2-3 numbered 1. It can be seen that the plant numbered 1 inserts 1 bp (single base A) in the target region (target sequence, SEQ ID NO: 5) of the GmLn gene, which ultimately leads to premature termination of protein translation, and the amino acid sequence is truncated from 256 aa to 10 aa. Figure 5A is the amino acid alignment result before and after editing; Figure 5B is a peak plot (excerpts) and alignment analysis of the detection results of the T2 generation genome editing homozygotes QH64111-T2-1 to QH64111-T2-3 numbered 1.

[0093] Figure 6 is an exemplary photograph of investigating the leaf type of the middle canopy of soybeans at the grain filling stage. The right side is a leaf photograph of QH64112 soybeans, which is a pointed leaf type; the left side is a leaf photograph of the wild type control Zhonghuang 13, which is an oval type, and the two have obvious differences. DETAILED DESCRIPTION

[0094] The present disclosure will now be described with reference to the following examples, which are intended to illustrate the present disclosure (but not to limit the present disclosure).

[0095] The experimental methods in the following examples are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0096] In the following examples, unless otherwise specified, T1 generation and each number of its offspring are randomly mixed into 1 sample for detection or analysis, and the maximum number is taken if the number is less than 10.

[0097] In the following examples, the regeneration frequency = explants with regenerative buds / total number of explants x 100%. The transformation efficiency = number of transgenic seedlings / total number of explants x 100%. The editing efficiency = number of mutants / number of transgenic seedlings x 100%.

[0098] In the following examples, the soybean sprouts resistant to glufosinate ammonium show normal green color similar to the wild type soybean sprouts without glufosinate ammonium treatment, while the wild type soybean sprouts treated with the same amount of glufosinate ammonium will not show normal green color but yellow color. This indicates that the presence of the glufosinate ammonium resistance gene (i.e. marker gene) makes the soybean sprouts resistant to glufosinate ammonium.

[0099] In the following examples, the soybean variety used is Zhonghuang 13, which was approved by the National Crop Variety Approval Committee in 2001, with the approval number: Guoshoudou 2001008; and was approved by Henan Province in 2011, with the introduction number: YuyinDou 2011003.

[0100] In the following examples, the yield increase or yield improvement in the field test is calculated by the following formula: (the yield of the edited soybean - the yield of the wild type) / the yield of the wild type x 100%; the yield of the edited soybean or the yield of the wild type is obtained based on the actual weight of the planting plot after conversion of the standard moisture content of 13.5% (i.e. actual weight x (1-harvest moisture %) / 86.5%).

[0101] In the field test, each planting plot has an area of 40m 2 , 8 rows are set, the row length is 10m, the ridge bottom width is 100cm, the ridge surface width is 50cm, the ridge row spacing is 40cm, and the ridge spacing is 60cm.

[0102] In the following examples, the host cells (recombinant cells) containing the vector are Agrobacterium rhizogenes and / or Agrobacterium tumefaciens, the Agrobacterium rhizogenes is preferably a wild type or a derivative strain selected from one or more of A4, Arqua1, MSU440, C58C1, Ar1193, K599, ATCC15834 and NCPPB1855; the Agrobacterium tumefaciens is preferably a wild type or a derivative or disarmed strain C58 selected from one or more of EHA101, EHA105, LBA4404, GV3101 and AGL1.

[0103] In the following examples, the identification test step of gene editing is a conventional experimental step in the art, when detecting the genome editing site, the reference genome of Zhonghuang 13 is used as a primer design template, the sequence shown in SEQ ID NO: 15 is the GmLn genome sequence (SoyZH13_2e0G103500, https: / / ngdc.cncb.ac.cn / gwh / Assembly / 125 / show), and SEQ ID NO: 13 is a selection of SEQ ID NO: 15. A pair of primers (i.e. SEQ ID NO: 11 and SEQ ID NO: 12) are designed on both sides of the editing site as shown in SEQ ID NO: 13, and the editing of the target site is amplified. The primer for site-specific PCR test of genome editing is shown in Table 1:

[0104] Table 1: Primer for site-specific PCR test of genome editing

[0105] Example 1: Construction of gene editing vector

[0106] Construction of vector: This example uses the backbone vector pCAMBIA (Hellens et al, Trends in Plant Science (2000) 5, 446-451), which is a binary vector commonly used in plant gene editing process, derived from E. coli, and no pathogenicity and possible evolution into pathogenicity has been reported. The replication origin site ori of the vector (numbered p010-0068, as shown in Figure 1) in this example is derived from the natural plasmid of E. coli. The vector contains the coding sequence of nuclease (eTraC effector protein, SEQ ID NO: 14), gRNA (LaTraC-sgRNA, also referred to as sgRNA in this specification) which guides the anchoring of eTraC effector protein to the target gene, the backbone sequence of which is shown in SEQ ID NO: 19, and the selection marker Bar gene. The vector map is shown in Figure 1 and Figure 2.

[0107] Genetic transformation of soybean was completed using methods well known in the art, which can use the method described in Paz M M, Martinez J C, Kalvig A B, et al. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation [J]. Plant cell reports, 2006, 25: 206-213. to transform soybean explants using Agrobacterium-mediated transformation.

[0108] Example 2: Obtain gene editing plants

[0109] The target sequence for eTraC effector protein is shown in Table 2 as follows:

[0110] Table 2: Gene editing target sequence statistics

[0111] According to the target sequence of Table 2, the gRNA (for example, the DNA recognition region of the gRNA molecule is shown in SEQ ID NO: 10) in the gene editing system was designed, and the plasmid containing the gene editing tool was delivered into the soybean explant for genetic transformation, and glufosinate was used as a selection marker to identify the explant with regenerated shoots, i.e. sprouts. The appearance of the sprouts was observed, and the tolerance of the sprouts was recorded. Glufosinate-tolerant soybean sprouts were obtained.

[0112] The glufosinate-resistant soybean sprouts were transferred to rooting medium, and the rooted soybean plants were transferred to a greenhouse to obtain rooted seedlings. Whether the vector described in Example 1 was integrated into the soybean genome was identified, and the integrated seedlings were recorded as transgenic seedlings.

[0113] The transgenic seedlings were subjected to glufosinate resistance screening again. The transgenic seedlings subjected to glufosinate resistance screening were subjected to second-generation sequencing, and it was analyzed that there were plants that had undergone gene editing and plants that had not undergone gene editing, and a total of 10 plants that had undergone gene editing were selected.

[0114] Example 3: Phenotype analysis, off-target and genetic stability analysis

[0115] The transgenic seedlings subjected to glufosinate screening were transferred to a greenhouse for growth, and it was found through observation that, compared with the wild type control, there were mutant plants in which the leaf shape changed from oval to lanceolate. The leaves of the transgenic seedlings subjected to glufosinate screening were measured, and multiple flower bud differentiation period leaves were randomly selected during the measurement. The average value of the leaf size was measured and the average value of the leaf length-width ratio was calculated, as shown in Table 3 below.

[0116] Table 3

[0117] The leaves of soybean No. 1 and No. 2 changed significantly from oval to lanceolate. Figure 3 is a comparison of the plant No. 1 with the wild type. In Figure 3, the left plant is the wild type, and the right plant is the edited plant. It can be seen that the leaf size is significantly smaller than the wild type, and the leaf length-width ratio is significantly larger than the wild type, that is, the narrow leaf phenotype is obtained.

[0118] The plants No. 1 and No. 2 in Table 3 were propagated, and T1 and T2 generation seeds were harvested. Analysis found that the three-pod and four-pod numbers of the progeny of No. 1 and No. 2 had no statistical difference with the wild type. The soybean yield of the plants No. 1 and No. 2 in Table 3 was basically the same as that of the wild type, and there was no statistical difference.

[0119] Genome analysis of the plants No. 1 and No. 2 in Table 3 and their progeny with narrow leaf phenotype found that the Ln gene of each of them had a mutation as shown in SEQ ID NO: 3.

[0120] Genomic DNA was extracted from each of the multiple T1 generation plants No. 1 and No. 2 in Table 3, and a plurality of primers were used to detect whether there was a vector residue in the T1 generation plants. The target gene editing type and potential off-target sites were verified by targeted PCR and sequencing, and finally the strain in which the target site was homozygous mutation, there was no exogenous vector sequence residue, and the potential target site and other sites were not edited was selected for self-pollination, and T2 generation seeds were harvested.

[0121] Potential off-target sites were verified using the crop genome editing target site off-target prediction software Cas-OFFinder (v2.4, Sangsu et al., 2014) (http: / / github.com / snugel / cas-offinder) to predict potential off-target sites of sgRNA targeting the target sequence shown in SEQ ID NO: 5 in the soybean genome (Williams82_v2.1), as shown in Table 4. By analyzing whether the predicted potential off-targets exist in the genetically edited transgenic seedlings by second-generation sequencing, the conclusion is shown in Figure 4, i.e. no editing occurs at the potential off-target sites and no non-target editing occurs.

[0122] Table 4: Information statistics table of predictable off-target regions of sgRNA in this example

[0123] Since the genome editing site is located inside the target gene, the T1, T2 generations of plants numbered 1 and 2 in Table 3 were identified and tracked according to the primers described in Table 1. The results show that in the edited material of the T1 generation selected in this example, the genome editing site can be stably inherited, and does not contain editing elements, is off-target free, and the editing site is homozygous, and the T2 generation plants also have similar effects. As shown in the peak chart of Figure 5B (Figure 5B selects QH64111-T2-1, QH64111-T2-2, QH64111-T2-3 of No. 1 as an exemplary illustration of T2 generation plants).

[0124] It is identified that the plants numbered 1 and 2 in Table 3 correspond to the insertion of 1 bp (single base A) into the Target region (target sequence, SEQ ID NO: 5) of GmLn gene in yellow 13 in the recipient soybean, which ultimately leads to premature termination of protein translation, and the amino acid sequence is truncated from 256 aa (SEQ ID NO: 2) to 10 aa (SEQ ID NO: 4). Through phenotype analysis and genome analysis, it can be concluded that this mutation can significantly increase the length-width ratio of soybean leaves and reduce leaf area.

[0125] Example 4: Field test

[0126] The T5 generation (hereinafter referred to as QH64112) of soybean plants which are completely identical to the GmLn genotype mutated in Table 3, off-target free, do not contain editing elements, the editing site is homozygous, and no other modifications are made, and its wild type yellow 13 (hereinafter referred to as control) are selected for field planting to analyze the effect on soybean growth under high planting density.

[0127] The experimental site is located in Yizhou District, Sanya City, Hainan Province, which belongs to tropical marine monsoon climate. The average annual temperature is 25.5℃, the average temperature in June is the highest, reaching 28.5℃, and the average temperature in January is the lowest, reaching 20.9℃. The annual rainfall is abundant, with an average annual rainfall of 1279 mm in recent years, the maximum annual rainfall is 1871 mm, and the minimum annual rainfall is 747 mm. The average annual sunshine hours is 2588 hours, there is no frost throughout the year, and the accumulated temperature of ≥10℃ is 9300.7℃.

[0128] The experimental design is a completely randomized block design with 3 replicates. The area of each planting plot is 40m 2 , 8 rows are set, the row length is 10m, the ridge bottom width is 100cm, the ridge surface width is 50cm, the ridge row spacing is 40cm, and the ridge spacing is 60cm. Three density gradients are set, which are 14,000 plants / acre, 18,000 plants / acre, and 22,000 plants / acre. The cultivation method is artificial dibbling. The ridge distance is 40cm, and the plant distance is 9.52cm, 7.41cm, and 6.06cm according to the seeding density. The fertilization method is to apply all the fertilizer at one time, and no additional fertilizer is applied during the growth stage of soybeans. Other management, weeding and pest control are the same as local field production.

[0129] The leaf type of the middle canopy of soybeans during the podding stage was investigated, as shown in Figure 6: the leaf blades of QH64112 soybeans are sharp leaf type, and the leaf blades of wild type control Zhonghuang 13 are oval type, both of which have obvious differences. Further analysis of soybean yield was conducted, as shown in Tables 5 to 9, wherein the letters after the numerical values indicate the significance of the difference between the gene edited soybeans and the corresponding recipient soybeans at the level of α=0.05, the same lowercase letters indicate no significant difference, and different lowercase letters indicate significant difference. All data in Tables 5 to 9 were analyzed by one-way ANOVA using SPSS system to calculate the significance at the level of α=0.05. I, II, and III in Tables 5 to 9 represent the average values obtained by sampling and measuring different multiple soybean plants at the same period in the three groups.

[0130] (1) Pod number per plant

[0131] The results are shown in Table 5, as the seeding density increases, the pod number per plant of both the gene edited soybeans QH64112 and the control decreases. At all densities, the pod number per plant of the gene edited soybeans QH64112 is higher than that of the control Zhonghuang 13, but there is no significant difference.

[0132] Table 5: Comparison of pod number per plant of soybeans

[0133] (2) Grain number per plant

[0134] The results are shown in Table 6, at all seeding densities, the grain number per plant of the gene edited soybeans QH64112 is higher than that of the control Zhonghuang 13, and the difference in grain number per plant between the two is significant at the density of 22,000 plants / acre.

[0135] Table 6: Comparison of soybean plant number

[0136] (3) Pod number

[0137] The results are shown in Table 7. At all seeding densities, the pod number of the gene edited soybean QH64112 was higher than that of the control Zhonghuang 13, but the difference was not significant.

[0138] Table 7: Comparison of soybean pod number

[0139] (4) 100-seed weight

[0140] The results are shown in Table 8. At all seeding densities, there was no significant difference in the 100-seed weight of QH64112 soybean and its control.

[0141] Table 8: Comparison of soybean 100-seed weight

[0142] (5) Yield

[0143] The yield per mu of the gene edited soybean QH64112 and its control Zhonghuang 13 at different seeding densities was determined at the mature stage, and the data of all the soybean yield component factors were summarized. The results are shown in Table 9. With the increase of seeding density, the yield of QH64112 soybean increased more obviously than that of the control Zhonghuang 13. At the seeding densities of 14,000 plants per mu, 18,000 plants per mu and 22,000 plants per mu, the yield of QH64112 soybean was higher than that of the control, and the yield increase effects were 1.4%, 3.47% and 4.95%, respectively.

[0144] Table 9: Comparison of soybean yield

[0145] For the coverage rate of the cultivation land, at the podding stage and the mature stage, the coverage rates of the gene edited soybean QH64112 and its control Zhonghuang 13 at all seeding densities were 100%, and there was no significant difference between them. For the reproductive coefficient, at the seeding densities of 14,000 plants per mu and 18,000 plants per mu, there was no difference in the reproductive coefficient between the gene edited soybean QH64112 and its control. However, at the seeding density of 22,000 plants per mu, the reproductive coefficient of QH64112 soybean was significantly higher than that of the control Zhonghuang 13. For the seed shattering, at all seeding densities, the seeds of the gene edited soybean QH64112 and its corresponding receptor soybean Zhonghuang 13 had a certain seed shattering after 21 days of maturity, but there was no significant difference in the seed shattering between them. For the seed germination rate, there was no significant difference in the seed germination rate between the harvested gene edited soybean QH64112 and its corresponding receptor soybean Zhonghuang 13 at all seeding densities.

[0146] According to the detection standards of various nutritional components in the "National Food Safety Standard", the total nutritional components in the genetically edited soybean QH64112 and the control seeds were determined. The results showed that there was no significant difference in the contents of main nutritional components (including water, ash, protein, total fat, starch, dietary fiber, crude fiber, carbohydrate, energy), 11 kinds of fatty acids (C14:0, C16:0, C16:1, C17:0, C18:0, C18:1, C18:2, C18:3, C20:0, C22:0, C22:1), 16 kinds of amino acids (Asp, Thr, Ser, Glu, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg), 10 kinds of minerals (Na, Mg, K, Ca, Mn, Fe, Cu, Zn, Se), 4 kinds of vitamins (vitamin A, vitamin E, vitamin B1, vitamin B2), anti-nutritional factors (phytic acid, trypsin inhibitor, soybean lectin, soybean isoflavones (including soybean glycoside, soybean glycoside, genistin, genistein, daidzein, daidzein glycoside)) and endogenous allergens (glycinin and β-conglycinin) in genetically edited soybean QH64112 and the control seeds.

[0147] In summary: from the yield index, under high density conditions, the number of pods per plant of genetically edited soybean QH64112 was significantly higher than that of the control Zhonghuang 13; under all seeding densities, the number of grains per pod of genetically edited soybean QH64112 was higher than that of the control but did not reach a significant difference, but there was no significant difference in the hundred-grain weight of the two. With the increase of seeding density, the yield of QH64112 soybean increased more obviously than that of the control, and under the seeding densities of 14,000 plants per mu, 18,000 plants per mu and 22,000 plants per mu, the yield of QH64112 soybean was higher than that of the control, and the yield-increasing effects reached 1.4%, 3.47% and 4.95%, respectively. From the survival competition ability test results, the cultivation coverage, seed drop rate and seed germination rate of genetically edited soybean QH64112 were not significantly different from those of the control Zhonghuang 13. Under high density (22,000 plants per mu) seeding conditions, the breeding coefficient of genetically edited soybean QH64112 was significantly higher than that of the control soybean Zhonghuang 13.

[0148] The editing site of the genetically edited soybean provided by the disclosure is located in the GmLn gene, and by only editing the GmLn gene and simultaneously knocking out or weakening the soybean GmLn gene, the leaf area of the soybean can be significantly reduced, the length-width ratio of the soybean leaf can be increased, and the overall yield per mu can be increased without affecting the yield per plant of the soybean.

[0149] SEQUENCE LISTING:

[0150] >SEQ ID NO: 1 GmLn CDS original (771 bp):

[0151] >SEQ ID NO: 2 GmLn amino acid original (256 aa):

[0152] >SEQ ID NO: 3 GmLn CDS edited (772 bp):

[0153] where lower case + underlined shows inserted nucleotides.

[0154] >SEQ ID NO: 4 GmLn amino acid edited (10 aa)

[0155] >SEQ ID NO: 5 site 1

[0156] >SEQ ID NO: 6 site 2

[0157] >SEQ ID NO: 7 site 3

[0158] >SEQ ID NO: 8 site 4

[0159] >SEQ ID NO: 9 site 5

[0160] >SEQ ID NO: 10 DNA recognition region of sgRNA

[0161] >SEQ ID NO: 11 D-Gm-Ln-F56

[0162] >SEQ ID NO: 12 D-Gm-Ln-R56

[0163] >SEQ ID NO: 13 Part of GmLn original genome sequence

[0164] >SEQ ID NO: 14 eTraC effector protein

[0165] SEQ ID NO: 15 GmLn original genome sequences

[0166] SEQ ID NO: 16 GmLn edited genome sequences

[0167] SEQ ID NO: 17

[0168] SEQ ID NO: 18

[0169] SEQ ID NO: 19 sgRNA backbone sequence

[0170] Sequences in the figures:

[0171] Figure 4 GmLn3 gene target site region - WT (SEQ ID NO: 20):

[0172] Figure 4 GmLn3 gene target site region - QH64111 (SEQ ID NO: 21):

[0173] Figure 4 Potential target site region - WT (SEQ ID NO: 22):

[0174] Figure 4 Potential target site region - QH64111 (SEQ ID NO: 22):

[0175] Figure 5A - original (SEQ ID NO: 2):

[0176] Figure 5A - edited (SEQ ID NO: 4):

[0177] Figure 5B - WT (SEQ ID NO: 23):

[0178] Figure 5B - QH64111-T2-1 (SEQ ID NO: 24):

[0179] Figure 5B - QH64111-T2-2 (SEQ ID NO: 24):

[0180] Figure 5B - QH64111-T2-3 (SEQ ID NO: 24):

Claims

1. An engineered non-propagating material soybean plant cell, said soybean plant cell comprising a mutated GmLn gene, said mutated GmLn gene comprising: a nucleotide sequence obtained by insertion or deletion mutation at nucleotides 345 to 372 in the nucleotide sequence represented by SEQ ID NO: 15; the mutated GmLn gene is knocked down or knocked out compared with the unmutated GmLn gene.

2. The soybean plant cell of claim 1, wherein, the mutated GmLn gene includes a nucleotide sequence obtained by insertion or deletion mutation at nucleotides 368 to 372 in the nucleotide sequence represented by SEQ ID NO:

15.

3. The soybean plant cell of claim 1, wherein, the mutated GmLn gene includes insertion of nucleotides between nucleotide 370 and nucleotide 371 in the nucleotide sequence represented by SEQ ID NO:

15.

4. The soybean plant cell of any one of claims 1 to 3, wherein, the stop codon of the mutated GmLn gene is located between nucleotides 368 and 388 in the nucleotide sequence represented by SEQ ID NO:

15.

5. The soybean plant cell according to any one of claims 1 to 3, wherein, the soybean plant cell comprises a mutated GmLn protein; the amino acid sequence of the mutated GmLn protein is represented by SEQ ID NO:

4.

6. The soybean plant cell of any one of claims 1 to 3, wherein, the soybean plant cell comprises a nucleotide sequence encoding the mutated GmLn protein.

7. The soybean plant cell of claim 6, wherein, the nucleotide sequence encoding the mutated GmLn protein is selected from the nucleotide sequence represented by SEQ ID NO:

16.

8. A nucleic acid molecule comprising a nucleotide sequence encoding a mutated GmLn protein having an amino acid sequence represented by SEQ ID NO:

4.

9. The nucleic acid molecule according to claim 8, the nucleotide sequence of which is represented by SEQ ID NO:

16.

10. A method for obtaining a soybean plant, all or part of the cells of which are engineered to express a mutated GmLn protein having an amino acid sequence represented by SEQ ID NO: 4 and / or the endogenous GmLn gene of the soybean plant is mutated to the nucleic acid molecule according to claim 8 or 9, by gene editing, physical mutagenesis and / or chemical mutagenesis.

11. The method of claim 10, wherein, The tool used for the gene editing is a CRISPR / Cas nuclease or a derivative technology thereof, a zinc finger nuclease or a derivative technology thereof (ZFN) or a transcription activator-like effector (TALE) or a derivative technology thereof.

12. The method of claim 11, wherein, The CRISPR / Cas nuclease includes a TraC effector protein, preferably an eTraC effector protein, the amino acid sequence of which is represented by SEQ ID NO:

14.

13. A method of reducing leaf area of soybean, the method comprising: The soybean plant cell is caused to express a mutated GmLn protein having an amino acid sequence represented by SEQ ID NO: 4 and / or the endogenous GmLn gene of the soybean plant is mutated to the nucleic acid molecule according to claim 8 or 9.

14. A method of increasing soybean planting density, the method comprising: The soybean plant cell is caused to express a mutated GmLn protein having an amino acid sequence represented by SEQ ID NO: 4 and / or the endogenous GmLn gene of the soybean plant is mutated to the nucleic acid molecule according to claim 8 or 9.

15. A composition comprising a Cas effector protein and a gRNA molecule, wherein, The gRNA molecule comprises a variable DNA recognition region and a Cas effector protein interaction region, the DNA recognition region of the gRNA molecule recognizes a DNA target sequence comprising at least SEQ ID NO: 5 or its complementary sequence, the DNA target sequence is located in the GmLn gene.

16. The composition of claim 15, for use in reducing GmLn gene expression. Preferably, the reduction of GmLn gene expression results in the expression of a protein having an amino acid sequence as set forth in SEQ ID NO: 4.

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