Genetically modified soybean plant

Abstract

The present invention provides a genetically modified soybean plant and a plant cell or plant part thereof. Compared with the control plant which is not genetically modified, the activity and / or level of at least two endogenous polypeptides in the genetically modified soybean plant selected from polypeptides having amino acid sequences of SEQ ID NOs: 3, 6, 9, and 12 is reduced or eliminated. The present invention also provides a method for preparing the genetically modified soybean plant and the plant cell or plant part thereof.

Description

Genetically modified soybean plants Technical Field

[0001] The present invention relates to plant molecular biology. In particular, the present invention relates to leguminous plants that have been modified such that the activity and / or level of endogenous polypeptides are reduced or eliminated. Background Art

[0002] Soybean (Glycine max [L.]) is the world's largest source of protein for food and animal feed, and the second largest source of vegetable oil. In order to provide sufficient protein and oil sources to meet the needs of the world's growing population, it is predicted that by 2050, global soybean production will need to increase by 140% based on 2000 levels (www.fao.org). However, since seed protein content is negatively correlated with yield and oil content, breeding high-yield, high-protein soybeans has long been difficult (Patil, G. et al. Theoretical and applied genetics. 130, 1975-1991, (2017); and Mahmoud, AA et al. J Agric Food Chem 54, 3916-3922, (2006)).

[0003] Leguminous plants, including soybeans, can fix nitrogen biologically through symbiotic rhizobia, partially meeting their nitrogen needs. Symbiotic nitrogen fixation is beneficial to the environment, but it is a carbon-demanding process. The host plant strictly controls nodulation to maintain a balance between the plant's nitrogen demand and the carbon consumption of nodules (Searle, IR et al. Science 299, 109-112, (2003); and Nishida, H. & Suzaki, T. Plant Cell Physiol 59, 1733-1738, (2018)). Aerial receptors (called NARKs in soybean) sense rhizobium-induced root-derived signals, allowing the host plant to tightly control the number of nodules (Ferguson, Brett J. et al. Plant, cell & environment 42, 41-51, (2019); Mortier, V. et al. Plant Physiol 153, 222-237, (2010); and Reid, DE, et al. Molecular Plant-Microbe 24, 606-618, (2011)). Mutations in NARK lead to "super-nodulation", forming too many nodules. Super-nodulation mutants, first identified in the 1980s, were initially considered a potential resource for soybean breeding (Reid, DE, et al. Mol Plant Microbe In 24, 606-618, (2011) and Kennedy, P., et al. Biological Nitrogen Fixation, 1071-1076 (2015)). However, the improvement in N acquisition in super-nodulation mutants is offset by the increased energy required for a large number of nodules, which ultimately impairs plant growth and leads to a decrease in yield (Ferguson, Brett J. et al. (2019) above; Zipfel, C. Nature 543, 328-336, (2017)).

[0004] There is still a need for the development of soybean varieties with higher yield and higher protein content. Summary of the Invention

[0005] Symbiotic nitrogen fixation in legumes requires a significant energy investment from the host plant, resulting in soybean hyper-nodulation mutants exhibiting stunted growth and yield losses. Due to a lack of success in improving yield, enhancing soybean nodulation has been neglected in breeding programs for decades.

[0006] The inventors have surprisingly discovered that genetic modification to reduce or eliminate the levels and / or activity of specific endogenous polypeptides in leguminous plants can produce plants with improved nodulation, balanced carbon partitioning, and enhanced carbon and nitrogen acquisition. In multi-year, multi-site field trials, the genetically modified plants demonstrated increased yield and protein content, while maintaining stable oil content, providing a new solution to address the need in the field for developing high-yield, high-protein soybean varieties.

[0007] Therefore, in a first aspect, the present invention provides a genetically modified legume plant, wherein the activity and / or level of at least two endogenous polypeptides selected from the group consisting of polypeptides comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12 is reduced or eliminated in the genetically modified legume plant compared to a control plant that has not been genetically modified.

[0008] In some embodiments, the activity and / or level of two or three, e.g., two, endogenous polypeptides selected from the group consisting of polypeptides comprising the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12 is reduced or eliminated in the genetically modified soybean plant compared to a non-genetically modified control plant.

[0009] In some embodiments, the leguminous plant is a Glycine max plant, such as a soybean plant.

[0010] The present invention also provides the genetically modified plant cells or plant parts of the leguminous plant.

[0011] In some embodiments, the plant part is a seed.

[0012] In a second aspect, the present invention provides a method for increasing yield and / or protein content in a leguminous plant, the method comprising: reducing or eliminating in the plant the activity and / or level of at least two endogenous polypeptides selected from the group consisting of polypeptides comprising the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12. In some embodiments, the method comprises reducing or eliminating in the plant the activity and / or level of two or three, for example, two, endogenous polypeptides selected from the group consisting of polypeptides comprising the amino acid sequence of SEQ ID NO: 3, 6, 9, or 12.

[0013] In some embodiments, the leguminous plant is a Glycine max plant, such as a soybean plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 shows the expression of target genes in each mutant detected by RT-qPCR.

[0015] Figure 2 shows the number of nodules (Figure 2A) and the dry weight of the whole plant (Figure 2B) of soybean plants. Points represent measurements of individual plants (n ≥ 10). t-test p values ​​are marked as: *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed t-test, compared with HC-6); ns: not significantly different.

[0016] Figure 3 shows 13 C distribution between nodules and aboveground parts of plants (Fig. 3A); the proportion of nitrogen from the environment (% Ndfa, Fig. 3B), representing the contribution of symbiotic nitrogen fixation in mutant and HC-6 plants; total nitrogen content in HC-6 and mutant plants (Fig. 3C); chlorophyll content in HC-6 and mutant plants (Fig. 3D); K 15 Absolute isotopes of HC-6 and mutant He5-1 plants grown with or without rhizobium inoculation in NO3 labeling analysis 15 N abundance (Figure 3E) and atomic % 15 N (Fig. 3F) (n≥14); in HC-6 and mutant plants 13 C fixation rate (μg 13 C / g dw plant / 12 h) (Figure 3G); and total carbon content in HC-6 and mutant plants (Figure 3H). Points represent measurements from individual plants (n ≥ 10). t-test p values ​​are marked as: *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed t-test, compared with HC-6); ns: not significantly different.

[0017] Figure 4A shows violin plots of the yield distribution of HC-6 and mutant 5 planted in Fuzhou and Shijiazhuang in 2021 (n≥14); Figure 4B shows the plot yield of HC-6 and mutants in Fuzhou (6.75 m2) and Shijiazhuang (7 m2) in 2022 (n=3); and Figure 4C shows the plot yield of HC-6 and mutants in Yangzhong in 2023 (50 m2). 2 ) (n=3). Each point represents an independent measurement / plant. t-test P values ​​are marked as *P<0.05, **P<0.01, ***P<0.001 (two-tailed t-test, compared with HC-6).

[0018] Figure 5 shows plant height (Figure 5A), branch number (Figure 5B), pod number (Figure 5C), seed number (Figure 5D), and 100-grain weight (Figure 5E) of HC-6 and mutants in trials conducted in Fuzhou and Shijiazhuang in 2022 (n ≥ 14); and the number of nodules of HC-6 and mutants in the Fuzhou field during flowering in 2022 (Figure 5F). Each point represents an independent measurement per plant. T-test P values ​​are marked as *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test, compared with HC-6).

[0019] Figure 6 shows soybean protein content (Figure 6A) and oil content (Figure 6B) across all trials, as well as calculated protein (Figure 6C) and oil (Figure 6D) yield per plant (2021) or per plot (2022 and 2023) at all sites. Each dot represents an independent measurement per plant. T-test P values ​​are marked as *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test, compared to HC-6).

[0020] Figure 7 shows the N content in seeds of HC-6 and mutant plants (N≥15) grown in the field (Figure 7A); the % Ndfa in seeds of mutant and HC-6 plants grown in the field in Fuzhou in 2022 was determined using the natural abundance method (Figure 7B); the % Ndfa in seeds of HC-6 and mutant plants harvested in Fuzhou in 2022 was determined using the natural abundance method (Figure 7B). 15 The natural abundance of N atoms (Figure 7C) and 15 N / 14 N ratio (Figure 7D), n = 15. Each point represents an independent measurement per plant. t-test P values ​​are marked as *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test, compared with HC-6).

[0021] Figure 8 shows the net photosynthetic rate (Figure 8A) and stomatal conductance (Figure 8B) of plants grown in the field in Fuzhou in 2022 (n ≥ 15) during the flowering period; and carbon content in leaves (Figure 8C) and seeds (Figure 8D) of mutant and HC-6 plants (n = 8) grown in the field in Fuzhou in 2022. Each point represents an independent measurement per plant. T-test P values ​​are marked as *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test, compared with HC-6).

[0022] Figure 9 shows the composition of the insert in plasmid pGES701.

[0023] Figure 10 shows the composition of the insert in plasmid pGES702.

[0024] Figure 11 shows the grain weight per plant of plants grown in the Yangzhong field in 2024.

[0025] Detailed Description of the Invention

[0026] 1. Definition

[0027] In the present invention, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and routine procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following literature: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (referred to as "Sambrook"). At the same time, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0028] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0029] As used herein, the term "root nodule" refers to structures formed on plant roots by bacteria that coexist with the root tissue of a plant. These bacteria are called "rhizobia." Rhizobia can convert free nitrogen in the air into nitrogenous compounds that plants can use, a process known as nitrogen fixation. Leguminous plants, some green manure plants, and lawn grasses have root nodules. Plants that can fix nitrogen can reduce the use of nitrogen fertilizers, increase soil organic matter, and improve the physical properties of the soil. However, excessive nodulation can also lead to significantly increased carbon consumption, thereby affecting plant growth.

[0030] As used herein, the term "hyper-nodulation" refers to excessive nodulation in legumes. A "hyper-nodulation mutant" refers to a mutant that causes excessive nodulation due to certain gene mutations, which are called "hyper-nodulation mutations."

[0031] "Yield" or "protein content" of a leguminous plant refers to the yield or protein content of the plant part of interest, including the yield or protein content of any organ such as roots, stems, leaves, flowers, fruits and / or seeds or any tissue of the plant.

[0032] As used herein, the term "genetic modification" refers to the modification of an organism's genetic material, such as DNA, to obtain an altered trait.

[0033] As used herein, "genome" encompasses not only the chromosomal DNA present in the cell nucleus, but also the organelle DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0034] "Genetically modified plants" or "genetically modified plant cells" refer to plant organisms or plant cells that contain exogenous polynucleotides or modified genes or expression control sequences within their genome. For example, the exogenous polynucleotides are capable of stably integrating into the genome of the organism or cell and being inherited through successive generations. The exogenous polynucleotides can be integrated into the genome alone or as part of a recombinant DNA construct. A modified gene or expression control sequence is one in which the sequence contains single or multiple deoxynucleotide substitutions, deletions, and additions in the genome of the organism or cell. It will be understood by those skilled in the art that genetic modification in the present invention does not include super-nodulation mutations, i.e., the genetically modified plants of the present invention are not super-nodulation mutants.

[0035] "Exogenous" with respect to a sequence refers to a sequence that is from a foreign species, or, if from the same species, has been significantly altered from its native form in composition and / or locus through deliberate human intervention.

[0036] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded polymers of RNA or DNA that optionally contain synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single-letter names as follows: "A" is adenosine or deoxyadenosine (RNA or DNA, respectively), "C" is cytidine or deoxycytidine, "G" is guanosine or deoxyguanosine, "U" is uridine, "T" is deoxythymidine, "R" is a purine (A or G), "Y" is a pyrimidine (C or T), "K" is G or T, "H" is A or C or T, "I" is inosine, and "N" is any nucleotide.

[0037] As used herein, the term "encoding" refers to a polynucleotide that directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or other start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG and TGA. The coding sequence can be a DNA, cDNA or recombinant nucleotide sequence.

[0038] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" may also include modified forms including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0039] An "endogenous" polypeptide refers to a polypeptide that occurs naturally in an organism, ie, is encoded by the organism's native genome and is expressed within the organism.

[0040] In the context of peptides, the terms "amino acid," "residue," and "amino acid residue" are used interchangeably to include both naturally occurring amino acids and non-natural amino acids found in proteins. The single-letter and three-letter nomenclature for naturally occurring amino acids in proteins follows the common nomenclature used in the art, as described in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0041] As used herein, "percent identity" refers to a comparison of the amino acids of two polypeptides or the nucleotides of two nucleic acid molecules, which, when optimally aligned, have approximately the specified percentage of identical amino acids or nucleotides. For example, "95% amino acid / nucleotide identity" means a comparison of the amino acids of two polypeptides or the nucleotides of two nucleic acid molecules, which, when optimally aligned, have 95% of the identical amino acids / nucleotides.

[0042] As used herein, an "expression construct" refers to a vector, such as a recombinant vector, suitable for expressing a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to produce mRNA or functional RNA) and / or translation of RNA into a precursor or mature protein.

[0043] The "expression construct" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be an RNA (such as mRNA) that can be translated.

[0044] An "expression construct" of the present invention may comprise regulatory sequences and a nucleotide sequence of interest from different sources, or regulatory sequences and a nucleotide sequence of interest from the same source but arranged in a manner different from that normally found in nature.

[0045] "Regulatory sequence" and "regulatory element" are used interchangeably to refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0046] "Promoter" refers to a nucleic acid fragment that is capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the present invention, a promoter is a promoter that is capable of controlling the transcription of a gene in a cell, whether or not it is derived from the cell. A promoter can be a constitutive promoter, a tissue-specific promoter, a developmentally regulated promoter, or an inducible promoter.

[0047] "Constitutive promoter" refers to a promoter that will generally cause a gene to be expressed in most cell types under most circumstances. "Tissue-specific promoter" and "tissue-preferred promoter" are used interchangeably and refer to a promoter that is expressed primarily, but not necessarily exclusively, in one tissue or organ, and may also be expressed in one specific cell or cell type. "Developmentally regulated promoter" refers to a promoter whose activity is determined by developmental events. "Inducible promoter" selectively expresses an operably linked DNA sequence in response to endogenous or exogenous stimuli (environmental, hormonal, chemical signals, etc.).

[0048] As used herein, the term "operably linked" refers to the connection of a regulatory element (e.g., but not limited to, a promoter sequence, a transcription termination sequence, etc.) to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.

[0049] "Introducing" a nucleic acid molecule (e.g., a plasmid, a linear nucleic acid fragment, RNA, etc.) or a protein into an organism refers to transforming the cells of the organism with the nucleic acid or protein so that the nucleic acid or protein can function in the cell. "Transformation" as used in the present invention includes stable transformation and transient transformation. "Stable transformation" refers to the introduction of an exogenous nucleotide sequence into the genome, resulting in the stable inheritance of the exogenous gene. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof. "Transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell to perform a function without the stable inheritance of the exogenous gene. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.

[0050] As used herein, "antisense nucleic acid" refers to a nucleic acid molecule that has a complementary sequence to a target nucleic acid (e.g., mRNA) and participates in gene expression regulation by base pairing with the target nucleic acid. Antisense nucleic acids include RNA or DNA molecules that precisely complement a specific mRNA and specifically block its translation.

[0051] As used herein, "interfering nucleic acid" refers to a nucleic acid molecule encoding an RNA molecule for RNA interference (RNAi), including siRNA, shRNA, miRNA, etc.

[0052] "Gene editing", also known as genome editing, uses engineered nucleases or "molecular scissors" to insert, delete or replace DNA in the genome of an organism. Gene editing causes site-specific double-strand breaks (DSBs) at the desired location in the genome and then introduces the desired DNA insertion, deletion or substitution during the repair of the DSB. Gene editing typically uses large-range nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR systems.

[0053] Meganucleases are a class of endonucleases with large recognition sites (12-40 bp of double-stranded DNA sequences) that typically occur only once in any given genome. For example, the 18 bp sequence recognized by the I-SceI meganuclease would need to occur only once, on average, in a genome 20 times larger than the human genome.

[0054] Zinc finger nucleases are artificial restriction enzymes made by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger DNA binding domain of a single ZFN typically contains 3-6 individual zinc finger repeats, each of which can recognize, for example, 3 bp.

[0055] "Transcription activator-like effector nucleases" are restriction enzymes that can be engineered to cleave specific DNA sequences. They are typically made by fusing the DNA-binding domain of a transcription activator-like effector (TALE) to a DNA-cleavage domain. TALEs can be engineered to bind to virtually any desired DNA sequence.

[0056] Clustered regularly interspaced short palindromic repeats (CRISPR) are prokaryotic DNA segments containing short, repetitive sequences. The CRISPR system is a prokaryotic immune system that confers resistance to foreign genetic elements, such as those found in plasmids and bacteriophages, providing acquired immunity. In this system, Cas proteins or similar proteins cleave foreign nucleic acids under the guidance of RNA.

[0057] The CRISPR / Cas system refers to clustered regularly interspaced short palindromic repeats and their related systems, in which CRISPR nucleases can cut DNA chains at specific locations under the guidance of guide RNA.

[0058] As used herein, the term "CRISPR nuclease" generally refers to nucleases present in naturally occurring CRISPR systems, as well as modified forms thereof, variants thereof, catalytically active fragments thereof, etc. The term encompasses any effector protein based on the CRISPR system that is capable of achieving gene targeting (e.g., gene editing, gene targeting regulation, etc.) in cells.

[0059] Examples of "CRISPR nucleases" include Cas9 nucleases or variants thereof. The Cas9 nucleases can be Cas9 nucleases from different species, such as spCas9 from Streptococcus pyogenes (S. pyogenes) or SaCas9 derived from Staphylococcus aureus (S. aureus). "Cas9 nuclease" and "Cas9" are used interchangeably herein to refer to RNA-guided nucleases comprising Cas9 proteins or fragments thereof (e.g., proteins comprising the active DNA cleavage domain of Cas9 and / or the gRNA binding domain of Cas9). Cas9 is a component of the CRISPR / Cas genome editing system that can target and cut a DNA target sequence to form a DNA double-strand break (DSB) under the guidance of a guide RNA.

[0060] Examples of "CRISPR nucleases" may also include Cpf1 nucleases or variants thereof, such as highly specific variants. The Cpf1 nucleases may be Cpf1 nucleases from different species, such as Cpf1 nucleases from Francisella novicida U112, Acidaminococcus sp. BV3L6, and Lachnospiraceae bacterium ND2006.

[0061] As used herein, "gRNA" and "guide RNA" are used interchangeably and refer to an RNA molecule that can form a complex with a CRISPR nuclease and can target the complex to a target sequence due to having a certain complementarity with the target sequence. For example, in a gene editing system based on Cas9, gRNA is generally composed of crRNA and tracrRNA molecules that partially complement each other to form a complex, wherein crRNA includes a sequence that has sufficient complementarity with the target sequence to hybridize with the target sequence and guide the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to the target sequence sequence. However, it is known in the art that single guide RNA (sgRNA) can be designed, which includes the features of crRNA and tracrRNA at the same time. In a genome editing system based on Cpf1, gRNA is generally composed of only mature crRNA molecules, wherein the sequence included in crRNA has sufficient homology to the target sequence to hybridize with the complementary sequence of the target sequence and guide the complex (Cpf1+crRNA) to specifically bind to the target sequence sequence. It is within the capabilities of those skilled in the art to design a suitable gRNA sequence based on the CRISPR nuclease used and the target sequence to be edited.

[0062] The CRISPR-dCas system involves modifying Cas to inactivate its nuclease activity, thereby preventing it from cutting DNA chains, while simultaneously connecting it to a transcriptional activation domain to achieve site-specific transcriptional activation.

[0063] Similarly, the CRISPR-dCas system can also be used to regulate gene expression in the region of interest. For example, CRISPR-dCas can be connected to a transcriptional activation or repression domain to activate or inhibit gene expression in the region of interest. Transcription can also be regulated by adjusting the methylation level of the region of interest. Specifically, dCas can be connected to a sequence that promotes methylation, such as an amino acid sequence with methyltransferase activity (e.g., CRISPR-dCas-SunTag-DNMT3A), to increase the methylation level of the region of interest, thereby reducing transcription in the region of interest. dCas can also be connected to a sequence that inhibits methylation, such as the Tet (ten-eleven translocation) catalytic domain (CRISPR-dCas-Tet) to reduce the methylation level of the region of interest, thereby increasing transcription in the region of interest.

[0064] 2. Modified Leguminous Plants

[0065] The inventors unexpectedly obtained genetically modified soybean plants in which the expression and / or activity of certain endogenous polypeptides is reduced or eliminated, resulting in more nodules than unmodified plants, thereby improving the plants' nitrogen fixation ability. Simultaneously, the modified plants have increased photosynthesis, i.e., carbon fixation capacity, resulting in higher biomass and enabling higher yields. Furthermore, the modified soybean plant seeds have increased protein content while maintaining the same oil content. This provides significant production advantages compared to existing technologies, where increased protein content results in decreased oil content.

[0066] Thus, the present invention provides a modified Fabaceae plant, wherein the activity and / or level of at least two endogenous polypeptides in the genetically modified Fabaceae plant is reduced or eliminated compared to a control plant that has not been genetically modified.

[0067] The present invention also provides a genetically modified plant cell of a legume plant, wherein the activity and / or level of at least two endogenous polypeptides in the genetically modified plant cell is reduced or eliminated compared to a control plant cell that has not been genetically modified.

[0068] In some embodiments, the at least two endogenous polypeptides are selected from polypeptides comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12, or a polypeptide comprising an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 6, 9 or 12.

[0069] In some embodiments, the two endogenous polypeptides are two or three endogenous polypeptides selected from a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 6, 9 or 12.

[0070] In some embodiments, the two endogenous polypeptides are two endogenous polypeptides selected from a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 6, 9 or 12.

[0071] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; and a second polypeptide comprising SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6.

[0072] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; and a second polypeptide comprising SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.

[0073] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; and a second polypeptide comprising SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0074] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6; and a second polypeptide comprising SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.

[0075] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6; and a second polypeptide comprising SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0076] In some embodiments, the at least two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9; and a second polypeptide comprising SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0077] In some embodiments, the at least two endogenous polypeptides are three endogenous polypeptides selected from a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12, or a polypeptide comprising an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 6, 9 or 12.

[0078] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; a second polypeptide comprising SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6; and a third polypeptide comprising SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.

[0079] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; a second polypeptide comprising SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6; and a third polypeptide comprising SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0080] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; a second polypeptide comprising SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9; and a third polypeptide comprising SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0081] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6; a second polypeptide comprising SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9; and a third polypeptide comprising SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0082] In some embodiments, the activity and / or level of four endogenous polypeptides comprising the amino acid sequences of SEQ ID NOs: 3, 6, 9, and 12, respectively, or comprising an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 3, 6, 9, or 12, is reduced or eliminated in the genetically modified legume plant compared to a control plant that has not been genetically modified.

[0083] Examples of legumes include, but are not limited to, soybean, faba bean, pea, mung bean, adzuki bean, cowpea, common bean, lentil, pigeon pea, peanut, lupine, redbud plant, catechu, cassia, licorice, sophora flavescens, milk vetch, alfalfa, broad bean, schisandra chinensis, albizzia julibrissin, dalbergia paniculata, honey loquat, red bean, locust, indigo plant, sappanwood, acacia, astragalus, twin-leaved bean, Indian hemp, and kudzu.

[0084] In some embodiments, the leguminous plant is a soybean plant. In some embodiments, the leguminous plant is a soybean plant.

[0085] The present invention also provides a plant part of a genetically modified legume plant of the present invention, or a plant part comprising a plant cell of the present invention. Examples of plant parts include, but are not limited to, plant organs, tissues, and any combination thereof. Examples of plant organs include roots, stems, leaves, flowers, fruits, and seeds. In some embodiments, the plant part is a seed. In some embodiments, the plant part is a soybean seed.

[0086] 3. Methods to increase the yield of legumes

[0087] The present invention provides a method for increasing the yield and / or protein content of leguminous plants, the method comprising: reducing or eliminating the activity and / or level of at least two endogenous polypeptides in the plant.

[0088] In some embodiments, the at least two endogenous polypeptides are selected from polypeptides comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12, or a polypeptide comprising an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 6, 9 or 12.

[0089] In some embodiments, the two endogenous polypeptides are two or three endogenous polypeptides selected from a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 6, 9 or 12.

[0090] In some embodiments, the two endogenous polypeptides are two endogenous polypeptides selected from a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12, or an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 6, 9 or 12.

[0091] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; and a second polypeptide comprising SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6.

[0092] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; and a second polypeptide comprising SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.

[0093] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; and a second polypeptide comprising SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0094] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6; and a second polypeptide comprising SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.

[0095] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6; and a second polypeptide comprising SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0096] In some embodiments, the at least two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9; and a second polypeptide comprising SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0097] In some embodiments, the at least two endogenous polypeptides are three endogenous polypeptides selected from a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12, or a polypeptide comprising an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 6, 9 or 12.

[0098] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; a second polypeptide comprising SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6; and a third polypeptide comprising SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.

[0099] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; a second polypeptide comprising SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6; and a third polypeptide comprising SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0100] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3; a second polypeptide comprising SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9; and a third polypeptide comprising SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0101] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6; a second polypeptide comprising SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9; and a third polypeptide comprising SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0102] In some embodiments, the method comprises reducing or eliminating the activity and / or level of four endogenous polypeptides in the plant, wherein the four endogenous polypeptides comprise the amino acid sequences of SEQ ID NOs: 3, 6, 9, and 12, respectively, or comprise an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 3, 6, 9, or 12.

[0103] The present invention provides a method for increasing the yield and / or protein content of legumes, the method comprising:

[0104] a) providing a first leguminous plant having reduced or eliminated activity and / or level of a first endogenous polypeptide;

[0105] b) providing a second legume having reduced or eliminated activity and / or levels of a second endogenous polypeptide; and

[0106] c) crossing the first and second legume plants to produce a first progeny plant having reduced or eliminated activity and / or level of the first and second endogenous polypeptides.

[0107] In some embodiments, the first and second legumes are species of the same genus, preferably the same species. In some embodiments, the legume is a Glycine max plant. In some embodiments, the legume is a soybean plant.

[0108] In some embodiments, the first and second endogenous polypeptides are selected from polypeptides comprising the amino acid sequence of SEQ ID NO: 3, 6, 9 or 12, or a polypeptide comprising an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3, 6, 9 or 12.

[0109] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, and the second endogenous polypeptide comprises SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6.

[0110] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, and the second endogenous polypeptide comprises SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.

[0111] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, and the second endogenous polypeptide comprises SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0112] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6, and the second endogenous polypeptide comprises SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.

[0113] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6, and the second endogenous polypeptide comprises SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0114] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9, and the second endogenous polypeptide comprises SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0115] The present invention provides a method for increasing the yield and / or protein content of legumes, the method comprising:

[0116] a) providing a first leguminous plant having reduced or eliminated activity and / or level of a first endogenous polypeptide;

[0117] b) providing a second legume having reduced or eliminated activity and / or levels of a second endogenous polypeptide;

[0118] c) providing a third legume having reduced or eliminated activity and / or level of a third endogenous polypeptide;

[0119] d) crossing the first and second legume plants to produce a first progeny plant having reduced or eliminated activity and / or level of the first and second endogenous polypeptides; and

[0120] e) crossing said first progeny plant with said third legume plant to produce a second progeny plant having reduced or eliminated activity and / or levels of the first, second and third endogenous polypeptides.

[0121] Those skilled in the art will appreciate that the second offspring plant may also be produced by crossing the offspring of the hybridization of the second and third legume plants with the first legume plant.

[0122] In some embodiments, the first, second, and third legumes are species of the same genus, preferably the same species. In some embodiments, the legume is a Glycine max plant. In some embodiments, the legume is a soybean plant.

[0123] In some embodiments, the first, second and third endogenous polypeptides are selected from polypeptides comprising the amino acid sequence of SEQ ID NO:3, 6, 9 or 12, or a polypeptide comprising an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3, 6, 9 or 12.

[0124] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, the second endogenous polypeptide comprises SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6, and the third endogenous polypeptide comprises SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9.

[0125] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, the second endogenous polypeptide comprises SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6, and the third endogenous polypeptide comprises SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0126] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:3, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, the second endogenous polypeptide comprises SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9, and the third endogenous polypeptide comprises SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0127] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:6, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:6, the second endogenous polypeptide comprises SEQ ID NO:9, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9, and the third endogenous polypeptide comprises SEQ ID NO:12, or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:12.

[0128] The present invention provides a method for increasing the yield and / or protein content of legumes, the method comprising:

[0129] a) providing a first leguminous plant (plant 1) having reduced or eliminated activity and / or level of a first endogenous polypeptide;

[0130] b) providing a second leguminous plant (plant 2) having reduced or eliminated activity and / or levels of a second endogenous polypeptide;

[0131] c) providing a third leguminous plant (Plant 3) having reduced or eliminated activity and / or level of a third endogenous polypeptide;

[0132] d) providing a fourth leguminous plant (plant 4) having reduced or eliminated activity and / or level of a fourth endogenous polypeptide

[0133] e) crossing the first and second legume plants (plant 1 x plant 2) to produce a first progeny plant having reduced or eliminated activity and / or level of the first and second endogenous polypeptides;

[0134] f) crossing said first progeny plant with said third legume plant ((plant 1 x plant 2) x plant 3) to produce a second progeny plant having reduced or eliminated activity and / or levels of the first, second and third endogenous polypeptides; and

[0135] g) crossing said first progeny plant with said fourth legume plant (((plant 1 x plant 2) x plant 3) x plant 4) to produce a third progeny plant having reduced or eliminated activity and / or levels of the first, second, third and fourth endogenous polypeptides.

[0136] Those skilled in the art will understand that the third offspring plants can also be produced by different hybridization strategies, such as (plant 1×plant 2)×(plant 3×plant 4), (plant 1×plant 3)×(plant 2×plant 4), (plant 1×plant 4)×(plant 2×plant 3), ((plant 2×plant 3)×plant 1)×plant 4, ((plant 1×plant 3)×plant 2)×plant 4, ((plant 1×plant 2)×plant 4)×plant 3, ((plant 2×plant 4)×plant 1)×plant 3, ((plant 1×plant 4)×plant 2)×plant 3, ((plant 1×plant 3)×plant 4)×plant 2, ((plant 1×plant 4)×plant 3)×plant 2, ((plant 3×plant 4)×plant 2)×plant 2, ((plant 2×plant 3)×plant 4)×plant 1, ((plant 2×plant 4)×plant 3)×plant 1 and ((plant 3×plant 4)×plant 2)×plant 1.

[0137] In some embodiments, the first, second, third, and fourth legumes are species of the same genus, preferably the same species. In some embodiments, the legume is a Glycine max plant. In some embodiments, the legume is a soybean plant.

[0138] In some embodiments, the first, second, third and fourth endogenous polypeptides are selected from polypeptides comprising the amino acid sequence of SEQ ID NO:3, 6, 9 or 12, or a polypeptide comprising an amino acid sequence that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3, 6, 9 or 12. For example, the first endogenous polypeptide comprises SEQ ID NO:3 or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3, the second endogenous polypeptide comprises SEQ ID NO:6 or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:6, the third endogenous polypeptide comprises SEQ ID NO:9 or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9, the fourth endogenous polypeptide comprises SEQ ID NO:12 or an amino acid sequence at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1 NO:12 is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence.

[0139] The technology of reducing or eliminating the activity and / or expression of plant endogenous polypeptides is known to those skilled in the art, and examples of such technology include but are not limited to antisense nucleic acid technology, RNA interference (RNAi) technology and gene editing technology. Examples of gene editing technology include but are not limited to large-scale nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR systems. Plants in which the activity and / or expression of endogenous polypeptides are reduced or eliminated can also be obtained by TILLING technology.

[0140] In some embodiments, the activity or expression of an endogenous plant polypeptide is reduced or eliminated by a CRISPR system. In some embodiments, a gene encoding the endogenous plant polypeptide is knocked out or knocked down by a CRISPR / Cas system comprising a Cas protein with nuclease activity. In some embodiments, expression of a gene encoding the endogenous plant polypeptide is inhibited by a CRISPR / dCas system.

[0141] In some embodiments, the CRISPR / Cas system comprises a gRNA targeting the gene. The gRNA can be a single guide RNA (sgRNA). The sgRNA can comprise a backbone portion (e.g., SEQ ID NO: 19) and a targeting portion. The sequences of exemplary targeting portions are shown in Table 1.

[0142] Table 1. Sequences of the sgRNA targeting portion and primers tested for genome editing Beneficial effects

[0143] Soybean protein content is generally negatively correlated with yield and oil content, which historically means that breeding towards high yield, high protein and high oil has been difficult. This application demonstrates that by reducing the expression of specific endogenous polypeptides, improving nitrogen and carbon assimilation, increasing the yield and protein content of soybean plants, and maintaining oil content, a significant contribution can be made to increasing soybean yield and protein content in modern crop production practices. This application provides a precise, rapid, durable, low-cost and eco-friendly method that can be widely used in soybean breeding programs. Hypothetically, through the method provided by this application, a 10% yield and a 1% protein increment could increase global protein production by more than 30 million tons (approximately 210 kg of protein per hectare x 136 million hectares (as of 2020)), and make a significant contribution to filling the 60 to 70 million tons of protein shortage expected to occur by 2050. Example

[0144] Example 1. Materials and methods

[0145] 1.1. Plant material construction

[0146] Plasmids and strains

[0147] In the examples of the present application, Escherichia coli DH5α was used for transgenic vector construction. Based on the pGES (see Bai et al., Generation of a multiplex mutagenesis population via pooled CRISPR-Cas9 in soya bean. Plant Biotechnol J, 2020, 18 (3): 721-731) skeleton, plasmid vectors pGES701 / pGES702 (Figures 9 and 10) were constructed, and soybean transformation was performed using Agrobacterium tumefaciens GV3101. Bradyrhizobium BXYD3 (see CN101182476A) was used to inoculate soybean plants to form nodules.

[0148] 1.1.2. Gene Editing System

[0149] In the high-throughput CRISPR / Cas9 system, the pGES701 vector contains a nucleotide sequence encoding sgRNA operably linked to the GmU6 promoter, and a soybean codon-optimized nucleotide sequence encoding spCas9 operably linked to the soybean endogenous promoter pM4 ( FIG. 9 ); the single transcript pGES702 vector contains a tandem nucleotide sequence encoding Cas9 and encoding multiple sgRNAs operably linked to the GmpM4 promoter ( FIG. 10 ).

[0150] The pGES701 / pGES702 vectors utilize the different recognition and cleavage sites of the BsaI restriction enzyme to clone sgRNA. The single-transcript vector pGES702 is characterized by tRNAs at both ends of the sgRNA+sgRNA Scaffold structure. In vivo, tRNAs can be cleaved by RNase, separating Cas9 and sgRNA after transcription into mRNA.

[0151] 1.1.3. Transgenic methods

[0152] In a clean bench dedicated to plant tissue culture, the cotyledons of soybean seeds sterilized with sodium hypochlorite were removed with a scalpel. The seeds were then cut vertically along the hypocotyl of the cotyledons to remove the embryo. The scalpel was then used to dip the colony into 7-8 wounds near the hypocotyl of the cotyledonary node. The explants were transferred to a square culture dish containing a double layer of filter paper soaked in sterile water and sealed. The culture was carried out in a light incubator at 25°C for 4-5 days, followed by 7 days of growth recovery, 21 days of bud induction and resistance screening, 21 days of bud elongation and resistance screening, and 30 days of rooting medium culture to obtain transformed soybean seedlings (see Song S. et al., (2013) Soybean seeds expressing feedback-insensitive cystathionine r-synthase exhibit a higher content of methionine., Journal of Experimental Botany, 64(7):1917-1926).

[0153] 1.1.4. Detection of gene knockout

[0154] The transformed soybean seedlings were removed from the rooting medium, and the excess medium and browned tissue at the roots of the seedlings were removed with clean water. The transformed seedlings were moved to a black square box with a ratio of vermiculite: nutrient soil: perlite of 1:1:1. The transformed seedlings were covered with plastic wrap to keep them moist. After one week, the plastic wrap was peeled off along the edge to allow for ventilation. After 14 days, the plastic wrap was completely peeled off and the transformed seedlings were sampled and identified.

[0155] 1.1.4.1. Cultivation and identification of T0 generation soybean transformed seedlings

[0156] T0-generation soybean seedlings were initially screened by spraying them with a 1000-fold dilution of Bar (10% solution of glufosinate ammonium aqueous solution, Sangon Biotech (Shanghai) Co., Ltd., A614229). One week later, changes in the leaves of the transformed seedlings were observed, and non-positive seedlings were removed. Surviving seedlings were numbered and leaf samples were taken. Leaf DNA was extracted using a rapid plant DNA extraction method, and PCR was performed for the Bar gene (primers: forward 5'-CTGCACCATCGTCAACCACTA-3'; reverse 5'-CTGAAGTCCAGCTGCCAGAAA-3'). Positive plants were identified based on electrophoresis of the PCR products (1% agarose gel electrophoresis). PCR amplification was performed using the primers listed in Table 1, and the PCR products were sequenced by Hi-TOM sequencing. The gene editing type and vector editing efficiency were analyzed based on the sequencing results. Positive plants were then transferred to culture pots in a 1:1:1 ratio of nutrient soil:vermiculite:perlite, and planted and harvested.

[0157] 1.1.4.2. Screening and genotyping of homozygous mutant soybean plants in the T1 generation

[0158] Plant the seeds of the plants that have been edited in the T0 generation of positive plants, and screen the plants of homozygous editing type and no vector sequence in the offspring. Specifically, in the seedling stage (about one week after seed germination, when true leaves grow), apply 1000 times diluted Basta liquid to one end of the true leaf and mark it. After three days, observe the changes in the leaves (withered leaves represent that the plant has no Bar resistance), extract the leaf DNA after numbering, and further confirm whether it contains the vector sequence. PCR amplification was performed using the primers in Table 1, and the products were subjected to Sanger sequencing. The editing type of each plant was analyzed and counted by sequencing results, and plants that do not contain vector sequences and are homozygous mutants were screened out.

[0159] 1.1.4.3. Phenotypic Analysis of T2 Generation Homozygous Soybean Mutants

[0160] Seeds from T1 generation plants that were homozygous mutants were planted and amplified using the primers listed in Table 1. Sequencing confirmed the plants as homozygous mutants. T2 generation plants were potted and analyzed for agronomic traits. Mature plants were analyzed for plant height, branch number, and pod number. Seeds were harvested and analyzed for number of grains per plant, grain weight, and 100-grain weight. Wild-type plants (Huachun 6, HC-6) were used as controls.

[0161] 1.1.4.4. Sanger sequencing to detect gene editing type

[0162] Sample Preparation: First, a PCR amplification reaction is performed to obtain the target DNA fragment for Sanger sequencing. The PCR product is then sent to a sequencing company for sequencing. Either the front primer or the back primer of the editing primer can be used as a sequencing primer (no polysaccharides or repetitive sequences should exist within the distance from the sgRNA to the front primer). Sequencing Results Analysis: Gene editing can be categorized into four types: no gene editing, heterozygous editing, biallelic editing, and homozygous editing.

[0163] 1. No gene editing has occurred: When the sequencing result sequence is exactly the same as the reference genome sequence and the sequencing peak graph is a single peak, no gene editing has occurred.

[0164] 2. Homozygous editing: Since soybean evolved from a paleotetraploid to a diploid, gene editing (insertion, substitution, or deletion) occurred at the sgRNA site on one of the chromosomes, and the same type of gene editing also occurred on the homologous chromosome. When the sequencing result sequence and the reference genome sequence have base deletions, insertions, or substitutions at the sgRNA site, and the sequencing peak graph is a single peak, it is homozygous gene editing.

[0165] 3. Heterozygous editing: When the sequencing peak graph begins to show double peaks near the sgRNA site, and the sequencing sequence may be completely consistent or inconsistent with the reference genome, it is a heterozygous editing.

[0166] 4. Biallelic editing: The sequencing result sequence begins to be inconsistent with the reference genome sequence near the sgRNA. At the same time, the sequencing peak graph begins to show double peaks near the sgRNA site. The gene editing type may be biallelic editing or heterozygous editing.

[0167] 1.2 Plant material growth conditions

[0168] Physiological and agronomic experiments were conducted using the soybean variety Huachun 6 (HC-6). Soybean plants were hydroponically cultured as described by Yang et al. (2017) (Metabolomics Reveals Distinct Carbon and Nitrogen Metabolic Responses to Magnesium Deficiency in Leaves and Roots of Soybean [Glycine max (Linn.) Merr.], Frontier in Plant Science, 8, 2091). The nutrient solution contained 0.12 mM Ca(NO3)2, 0.19 mM KNO3, 2.5 μM MgCl2, 0.5 mM MgSO4, 1 mM K2SO4, 0.5 μM MnSO4, 1.5 μM ZnSO4, 0.5 μM CuSO4, (NH4)·Mo7O 24 0.15 μM, KH2PO4 0.25 mM, NaB4O7 0.25 μM, Fe·EDTA 0.04 mM, (NH4)2SO4 0.05 mM, and CaCl2 1.2 mM. The pH was adjusted to 5.8 with 1 M KOH every 2 days, and the medium was changed weekly. Biomass was measured after approximately 45 days of cultivation. Soybean plants were grown in a growth chamber with a light intensity of 450 μmol photons m -2 s -1 , the photoperiod was 14 h light (28°C) / 10 h dark (24°C), and the humidity was 65%.

[0169] For the field experiment, plants of different genotypes (HC-6 and mutants 1–8) were planted in a randomized plot design from 2021 to 2023, with three replicates per genotype, in experimental fields in Fuzhou (26.08°N, 119.24°E) and Yangzhong (26.28°N, 118.49°E, 2023) in Fujian Province, China, and Shijiazhuang (38.03°N, 114.48°E) in Hebei Province, China.

[0170] In 2021, each genotype was planted in two rows, each 3.5 meters long, with a spacing of 0.5 meters between rows. The 2022 plot design in Fuzhou was three rows per plot (~6.75 square meters), each 4.5 meters long. In 2022, the Shijiazhuang plot design was four rows per plot (~7 square meters), each 3.5 meters long. In 2023, the Yangzhong plot design was six rows per plot (approximately 50 square meters), each 25 meters long. The planting density per plot was calculated to be 140,000–150,000 plants / ha in 2022 and 2023. In Fuzhou and Yangzhong, 225 kg / ha of compound fertilizer (N:P2O5:K2O = 15:15:15) and 150 kg / ha of calcium magnesium phosphate fertilizer were applied as basal fertilizer. In Shijiazhuang, all plots were fertilized with 375 kg / ha of compound fertilizer (N:P2O5:K2O=15:15:15) as base fertilizer. The available nitrogen content in the field soil was 53-107 mg / kg.

[0171] In addition, HC-6 and mutants 2, 4 and 6-10 were planted in Yangzhong in 2024. The plant growth density of the planting plot was calculated to be 166,600 plants / hectare. 225 kg / hectare of compound fertilizer (N:P2O5:K2O=15:15:15) and 150 kg / hectare of calcium magnesium phosphate fertilizer were applied as base fertilizer. The available nitrogen content of the field soil was 90-100 mg / kg.

[0172] 1.3. Plant field performance

[0173] During the flowering stage, at least 8 representative plants of each genotype were taken to record the flowering time. After full maturity (growth stage R8), about 15 randomly selected plants per row of each plot were harvested in 2021 and 2022. Five agronomic traits (plant height, number of branches per plant, 100-grain weight, number of pods per plant, and seed yield per plant) were scored. Seed protein and oil content were determined using a MATRIX-I Fourier transform near-infrared reflectance spectrometer (FT-NIRS) (Bruker Optics, Bremen, Germany). The estimated protein and oil content of each plant was calculated by multiplying the seed yield of each plant by the seed protein content and seed oil content, respectively.

[0174] Real-time quantitative PCR

[0175] use Total RNA was extracted from plant tissues using OMEGA Bio-Tek reagent (OMEGA Bio-Tek, Norcross, GA, United States).

[0176] First-strand cDNA was synthesized from the extracted total RNA using the PrimeScript RT reagent kit (RR037A, TaKaRa) according to the manufacturer's instructions and SYBR1 Premix Ex Taq TM RT-qPCR amplification was performed using the iQ5 real-time PCR detection system (BioRad) in a 20 μL reaction volume (2x SYBR mix, 10 μL; 1 μL each of primers F / R (10 μmol, primer sequences see Table 2), 4 μL of HO, and 4 μL of template cDNA). eEF-1α (TefS1, accession number X56856) was used as a reference gene, and the relative expression level of each gene was calculated using the 2-ΔΔCT method (Livak, KJ & Schmittgen, TD Methods 25, 402-408, (2001)).

[0177] Table 2. Primers used for real-time quantitative PCR

[0178] 1.5. C / N Content and Isotope Labeling Determination

[0179] for 15 N isotope labeling assay: 21 days after seed germination, soybean hydroponic plants (inoculated and not inoculated with soybean rhizobium BXYD3) were transferred to a 26.5 μM K 15 In the hydroponic culture of NO3, the 15 N. whole seedlings were harvested on day 49 to measure 15 N content.

[0180] for 13 For C isotope labeling, soil-grown soybean plants at the grain filling stage were placed in a transparent sealed box to maintain photosynthesis. -2 s -1 The light intensity was set at room temperature for 12 hours to fix the barium carbonate (Ba 13 CO3, 0.5 g) and lactic acid (10 mL) were reacted to release 13 After two labeling treatments (two days apart), the plants were transferred to an outdoor environment and harvested when the seeds were mature.

[0181] For isotope measurements, samples were dried in an oven at 80°C for 1 week, ground into powder, and combusted into N2 (for N) or CO2 (for C) in an isotope ratio mass spectrometry elemental analyzer (DELTA V Advantage; Thermo Fisher Scientific, Inc., USA). N and C concentrations were determined by comparing the peak areas of the samples with those of three standard samples and calculated based on the concentration and dry weight. 15 N / 14 N ratio (for N) or 13 C / 12 C ratio (for C), which is then compared with international standard materials to calculate δ 15 N and δ 13 C value (‰, PDB). The N fixation rate of the labeled samples grown in hydroponics is shown in formula (1), and the N fixation rate of natural seeds is shown in formula (2):

[0182] δ 15 N uninoculated :Not inoculated with rhizobia 15 N marks the δ of the material 15 N

[0183] δ 15 N inoculated : Inoculation with Rhizobium 15 N marks the δ of the material 15 Nδ 15 N natrue :Under natural growth conditions ( 15 N is not labeled) δ of the control material 15 N value

[0184] δ 15 N reference : δ of control plant (non-nodulating soybean) seeds 15 N

[0185] δ 15 N legume : δ of leguminous plant (soybean in this case) seeds 15 NB: constant (B = -2.1).

[0186] 1.6. Net photosynthetic rate and stomatal conductance

[0187] Net photosynthetic rate (Pn) and stomatal permeability of field-grown soybean leaves were measured at the flowering stage using a LICOR-6400XT instrument (LI-COR Biosciences, USA). Measurements were performed in ambient air between 10:00 and 11:00 am during summer in Fuzhou.

[0188] Statistical analysis

[0189] Statistical analysis was performed using Microsoft Excel, and the traits of interest in HC-6 and mutants were determined by two-tailed Student's t-test. P values ​​less than 0.05 were considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001). Data were generated using GraphPad Prism (version 8.0) and R software (version 3.6.1). Details and numbers of biological replicates are described in the legends for each figure.

[0190] Example 2: Mutants with altered nodule number and their growth

[0191] It is known in the art that although an increase in the number of nodules can improve nitrogen acquisition, it will cause a huge carbon consumption, thereby affecting plant growth. Mutants with a significantly increased number of nodules known in the prior art exhibit a poor growth phenotype.

[0192] The inventors unexpectedly discovered that reducing the expression of a specific endogenous polypeptide can achieve a balance between nitrogen fixation and carbon consumption while increasing the number of nodules and achieving an altered nitrogen fixation capacity, thereby still being beneficial to the growth of soybean plants and increasing the yield of soybean plants.

[0193] Specifically, as shown in Example 1, using the multiple CRISPR-Cas9 mutagenesis system, mutants as shown in Table 3 were obtained in the context of HC-6, and their nodulation ability and growth were compared. The mutants nin1a / 1b / 2a / 2bc (Nodule Inception, hereinafter referred to as nin-4m) and nark (Fu, M., et al., Plant Physiol 188, 477-489, (2022)) known in the prior art were used as references. Mutant 5 includes two strains, mutant 5-1 and mutant 5-2, in which a 1 nucleotide insertion and a 4 nucleotide deletion were introduced into the coding sequence of SEQ ID NO: 3 (SEQ ID NO: 1), respectively.

[0194] Table 3. Prepared mutants and their nodules and growth

[0195] All mutants carried loss-of-function mutations at their respective target sites, and no off-target editing was observed in predicted potential off-target sites. RT-qPCR showed that the mRNA levels encoding the target protein were greatly reduced in the mutants compared to HC-6 (Figure 1).

[0196] As shown in Figure 2, plants were inoculated with the bradyrhizobium strain BXYD3 (OD600 = 0.1) under laboratory conditions, and the number of nodules was counted during the flowering stage. The results showed that nin-4m did not form nodules, while nark had six times the number of nodules as HC-6 (617.7 ± 85.9 for nark and 103.8 ± 11.8 for HC-6). As expected based on prior art, the growth of both mutants (nin-4m and nark) was stunted due to insufficient nitrogen supply and an imbalance in nitrogen and carbon supply (whole plant dry weight was 2.10 ± 0.31 g for nin-4m, 3.66 ± 0.3 g for nark, and 5.08 ± 0.43 g for HC-6). Mutant 8 formed three times more nodules (322.5 ± 24.1) than HC-6, with reduced biomass (4.62 ± 0.39 g), suggesting that it experienced the same C / N trade-off as the hyper-nodulating mutant nark, albeit to a lesser extent. Mutant 6 had approximately 20% more nodules than HC-6 (121.3 ± 14.4) and a slightly but statistically significant increase in biomass (5.74 ± 0.57 g). Interestingly, in mutant 5-1, nodule number was twice that of HC-6 (202.4 ± 18.0), and aboveground biomass was increased by 31.6% (6.78 ± 0.50 g, compared to 5.08 ± 0.43 g in HC-6).

[0197] The results showed that reducing the expression of two of SEQ ID NOs: 3, 6, 9, and 12 in soybean plants unexpectedly increased soybean growth while increasing the number of soybean nodules.

[0198] Example 3. Carbon / Nitrogen Balance in Soybean Mutant Plants

[0199] The purpose of this example was to determine the metabolic flux of carbon and nitrogen in the mutant.

[0200] Isotope labeling analysis was performed as described in Example 1.4.

[0201] 13 CO2 tracer analysis showed that in mutant 8 and nark, the assimilated 13 C was excessively distributed to the nodules and not to the aerial parts (Figure 3A). In contrast, the assimilated 13 The allocation of C was relatively higher. The balanced allocation of C in mutant 5-1 resulted in an improvement in the C / N status in the plants.

[0202] As shown in Figure 3B, the increase in nodules in mutant 5-1 resulted in a higher total nitrogen fixation capacity, indicating that the contribution of nitrogen fixation was higher in mutant 5-1 relative to HC-6 (71.24% in mutant 5-1 and 65.02% in HC-6).

[0203] The inventors also observed that the nitrogen and chlorophyll contents in the leaves of mutant 5-1 were higher (Figures 3C-3D), and the nitrogen content was also significantly increased (Figures 3E-3F). 13 The C assimilation rate was statistically significantly increased (Figure 3G). This confirms that the mutant has enhanced photosynthesis. Due to the enhanced photosynthesis and increased biomass, the total carbon content of mutant 5-1 plants increased (Figure 3H). Therefore, the increase in nodules in mutant 5-1 unexpectedly led to a balance in carbon distribution, simultaneously enhancing carbon and nitrogen assimilation and improving plant growth.

[0204] Example 4: Field Experiments on Soybean Mutants

[0205] The purpose of this example is to test the practical application potential of soybean mutants.

[0206] To this end, as described in Example 1.1, field trials were conducted at three locations from 2021 to 2023 using Mutant 5-1 and Mutant 5-2, with HC-6 serving as a reference plant. The field trials followed typical production practices in China, without inoculating seeds with additional Bradyrhizobium bacteria, relying instead on the presence of Bradyrhizobium in the local environment. Agronomic traits of the plants were analyzed as described in Example 1.2.

[0207] Notably, both mutants demonstrated statistically significant increases in yield compared to HC-6 in all experiments. In 2021, small-scale trials were conducted in Shijiazhuang, Hebei Province, and Fuzhou, Fujian Province. As shown in Figure 4A, the average per-plant yield of mutants 5-1 and 5-2 increased by 19.12% and 20.9%, respectively, in Fuzhou and by 10.45% and 12.37%, respectively, in Shijiazhuang.

[0208] In 2022, small plot trials were conducted in Shijiazhuang and Fuzhou. As shown in Figure 4B, in the Shijiazhuang trial, the yield of mutant 5-1 and mutant 5-2 increased by 21.67% and 25.02%, respectively (7 m2 per small plot). In the 2022 Fuzhou trial, heavy summer rains caused waterlogging, which affected the yield of all plants. Despite this, mutant 5-1 and mutant 5-2 still showed statistically significant yield increases of 27.93% and 31% (6.75 m2 per plot). 2 ).

[0209] A large-scale field trial was conducted in Yangzhong in 2023. As shown in Figure 4C, the yield of mutants 5-1 and 5-2 was increased compared with HC-6 (11.99% and 16.17%, respectively), which is consistent with the results of previous trials.

[0210] The results also showed that the mutant's significant yield increase was accompanied by an increase in plant height, as well as an increase in branches and pods per plant. As shown in Figure 5, the 100-grain weight of the mutant was not statistically significantly different from that of HC-6. The number of nodules in the mutant grown in the Fuzhou field was twice that of HC-6 (Figure 5F), indicating that the local Bradyrhizobium environment was sufficient to support the increased nodulation in the mutant.

[0211] Notably, despite the very high protein content of HC-6 (41.75–46.90% across the different field trials), the mutants showed statistically significant increases in seed protein content across all trials (1.77–4.42% increase, Figure 6A). Conversely, the mutants showed no statistically significant changes in oil content (Figure 6B). Consequently, both protein and oil yield per plant or plot, calculated by multiplying protein / oil content by grain yield, were increased (13.96–33.61% increase in protein yield and 10.67–31.69% increase in oil yield, Figures 6C and D).

[0212] As described in Examples 1.4 and 1.5, field-harvested seeds were tested. The results showed that the total nitrogen content of mutant 5-1 was higher in field-harvested seeds ( FIG. 7A ). This may be attributed to the increased fixed nitrogen supply in the mutant seeds, as shown in FIG. 15 As shown by N natural abundance analysis (Figure 7B-D), the enhanced nitrogen fixation in mutant 5 provides an additional nitrogen supply to the seeds.

[0213] Field-grown mutant plants exhibited higher net photosynthetic rates (Pn) and stomatal permeability (gs) than the HC-6 control (Figures 8A-B). This is consistent with laboratory observations that mutant 5-1 exhibited a higher rate of carbon assimilation in leaves (Figure 3E). In line with improved photosynthesis, field-grown mutants exhibited higher carbon content in leaves and seeds than HC-6 (Figures 8C-D). Therefore, the negative correlation between protein content and yield / oil production may be eliminated by the simultaneous increase in nitrogen and carbon supply to the mutant plants.

[0214] Since different combination mutants of the four genes SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 10 produced different numbers of nodules, we tested the agronomic performance of these mutant materials with different degrees of nodulation in the field. The yield traits of mutant materials with varying degrees of increased nodule number were analyzed. The traits of multiple knockout mutants obtained by knocking out genes or combinations of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, and SEQ ID NO:10 using multiple editing techniques were determined. It was found that mutants with different combinations of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, and SEQ ID NO:10 showed no significant differences in plant height, branching, or node number compared to the control material (WT:HC-6). However, mutant materials with moderately increased nodule number, such as SEQ ID NO:1+SEQ ID NO:7, SEQ ID NO:4+SEQ ID NO:10, and SEQ ID NO:7+SEQ ID NO:10, showed significant increases in agronomic traits such as pod number, grain number, and yield per plant. The super-nodulation mutant (Mutant 8), while showing significant differences in nodules, showed significant decreases in pod number, grain number, and yield per plant compared to the control, as shown in Table 4. From the above results, it can be seen that using different combinations of genes to obtain mutants that optimally control the number of nodules can significantly increase soybean yield.

[0215] Table 4

[0216] *Significantly different from HC-6.

[0217] Example 5. Preparation and field trials of soybean mutants

[0218] The purpose of this example is to further screen soybean mutants and test their application potential.

[0219] Mutants were obtained in the HC-6 background using the multiplex CRISPR-Cas9 mutagenesis system as described in Example 1. The plants in Table 5 were tested for yield per plant in 2024 in Yangzhong.

[0220] Table 5. Tested plants

[0221] As shown in Figure 11, the order of average single-plant grain weight from small to large is: Mutant 8 (8.88 g) < HC6 (9.42 g) < Mutant 10 (9.53 g) < Mutant 4 (9.71 g) < Mutant 2 (9.76 g) < Mutant 6 (10.71 g) < Mutant 9 (10.99 g) < Mutant 7 (11.97 g).

[0222] The results of the above examples demonstrate that mutations (knockouts) in SEQ ID NOs: 3, 6, 9, and 12 all contribute to improved soybean production. Compared to the base (HC-6) plants, the single and triple mutants showed an increased grain weight per plant, but without a dominant difference. Plants with more gene mutations (Mutant 8) even showed a decreased grain weight per plant, likely due to excessive nodules depleting the plant's energy supply, leading to decreased yield per plant. The double mutant, on the other hand, exhibited a significantly increased yield.

[0223] It can be seen that deletion of one or more, preferably two, proteins of SEQ ID NOs: 3, 6, 9 and 12 in soybean plants can achieve improved soybean plant production.

[0224] sequence

Claims

1. A genetically modified leguminous plant, wherein the activity and / or level of at least two endogenous polypeptides selected from the polypeptides comprising the amino acid sequences of SEQ ID NO: 3, 6, 9 or 12 in the genetically modified leguminous plant is reduced or eliminated as compared to an unmodified control plant.

2. The genetically modified leguminous plant according to claim 1, wherein the activity and / or level of two or three endogenous polypeptides selected from the polypeptides comprising the amino acid sequences of SEQ ID NO: 3, 6, 9 or 12 in the genetically modified soybean plant is reduced or eliminated as compared to an unmodified control plant.

3. The genetically modified leguminous plant according to claim 1 or 2, wherein the activity and / or level of two endogenous polypeptides selected from the polypeptides comprising the amino acid sequences of SEQ ID NO: 3, 6, 9 or 12 in the genetically modified leguminous plant is reduced or eliminated as compared to an unmodified control plant.

4. The genetically modified leguminous plant according to any one of claims 1 - 3, wherein the leguminous plant is a plant of the genus Glycine.

5. The genetically modified leguminous plant according to any one of claims 1 - 4, wherein the leguminous plant is a soybean (Glycine max) plant.

6. A plant cell of the genetically modified leguminous plant according to any one of claims 1 - 5.

7. A plant part of the genetically modified leguminous plant according to any one of claims 1 - 5.

8. The plant part according to claim 7, which is a seed.

9. A method for increasing the yield and / or protein content of a leguminous plant, the method comprising: reducing or eliminating the activity and / or level of at least two endogenous polypeptides selected from the polypeptides comprising the amino acid sequences of SEQ ID NO: 3, 6, 9 or 12 in the plant.

10. The method according to claim 9, wherein the method comprises: reducing or eliminating the activity and / or level of two or three endogenous polypeptides selected from the polypeptides comprising the amino acid sequences of SEQ ID NO: 3, 6, 9 or 12 in the plant.

11. The method according to claim 9 or 10, wherein the method comprises: reducing or eliminating the activity and / or level of two endogenous polypeptides selected from the polypeptides comprising the amino acid sequences of SEQ ID NO: 3, 6, 9 or 12 in the plant.

12. The method according to any one of claims 9 - 11, wherein the leguminous plant is a plant of the genus Glycine.

13. The method according to any one of claims 9 - 12, wherein the leguminous plant is a soybean plant.

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