Genetically modified soybean plants

Abstract

The present invention provides a genetically modified leguminous plant and a plant cell or plant part thereof 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 in the genetically modified leguminous plant is reduced or eliminated as compared to an ungenetically modified control plant. The invention also provides methods of making the genetically modified leguminous plants and plant cells or plant parts thereof.

Description

Technical Field

[0001] The present invention relates to plant molecular biology. In particular, the present invention relates to leguminous plants modified to reduce or eliminate the activity and / or level of an endogenous polypeptide. Background Art

[0002] Soybean (Glycine max [L.]) is the world's largest source of food and animal feed protein and the second largest source of vegetable oil. To provide sufficient sources of protein and oil to meet the needs of the growing world population, it is predicted that by 2050, global soybean production will need to increase by 140% based on the 2000 level (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, A. A. et al. J Agric Food Chem 54, 3916 - 3922, (2006)).

[0003] Leguminous plants, including soybean, can fix biological nitrogen through symbiotic rhizobia, partially meeting their nitrogen requirements. Symbiotic nitrogen fixation is beneficial to the environment, but it is a process that requires carbon. The host plant strictly controls nodulation to maintain a balance between the plant's nitrogen requirements and the carbon consumption of nodulation (Searle, I. R. et al. Science 299, 109 - 112, (2003); and Nishida, H. & Suzaki, T. Plant Cell Physiol 59, 1733 - 1738, (2018)). The receptor located in the above-ground part (known as NARK in soybean) senses the root-derived signal induced by rhizobia, enabling the host plant to strictly 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, D. E., et al. Molecular Plant-Microbe 24,606 - 618, (2011)). Mutations in NARK result in "hyper - nodulation", forming an excessive number of root nodules. The hyper - nodulation mutants first identified in the 1980s were initially considered potential resources for soybean breeding (Reid, D.E., 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 hyper - nodulation mutants is offset by the increased energy required for a large number of root 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 high yield and high protein content. Summary of the Invention

[0005] Symbiotic nitrogen fixation in leguminous plants requires a large amount of energy input from the host plant. Therefore, soybean hyper - nodulation mutants show retarded development and yield loss. Due to the lack of success in increasing yield, the improvement of soybean nodulation rate has been ignored in breeding programs for decades.

[0006] The inventors unexpectedly found that by genetically modifying to reduce or eliminate the level and / or activity of specific endogenous polypeptides in leguminous plants, plants with increased nodulation ability, balanced carbon allocation, and enhanced carbon and nitrogen acquisition can be produced. In multi - year and multi - location field trials, the genetically modified plants showed increased yield, protein content, and stable oil content, providing a new solution to the need in the art for the development of soybean varieties with high yield and high protein content.

[0007] Accordingly, in a first aspect, the present invention provides 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 are reduced or eliminated as compared to an un - genetically modified control plant.

[0008] In some embodiments, the activity and / or level of two or three, such as 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 soybean plant are reduced or eliminated as compared to an un - genetically modified control plant.

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

[0010] The present invention also provides a plant cell or a plant part of the genetically modified 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 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 having the amino acid sequences set forth in SEQ ID NO: 3, 6, 9 or 12 in the plant. In some embodiments, the method comprises reducing or eliminating the activity and / or level of two or three, such as two, endogenous polypeptides selected from the polypeptides having the amino acid sequences set forth in SEQ ID NO: 3, 6, 9 or 12 in the plant.

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

[0014] Figure 1 Showing the expression of the target gene in each mutant detected by RT–qPCR.

[0015] Figure 2 Showing the number of nodules Figure 2 A) and the dry weight of the whole plant Figure 2 B) of soybean plants. Dots represent measurements of individual plants (n≥10). t-test p-values are labeled as: *p<0.05, **p<0.01, ***p<0.001 (two-tailed t-test, compared with HC-6), ns: no significant difference.

[0016] Figure 3 Showing 13 C partitioning in nodules and the above-ground part of the plant Figure 3 A); the proportion of nitrogen from the environment (%Ndfa, Figure 3 B), representing the contribution of symbiotic nitrogen fixation in mutant and HC-6 plants; the total nitrogen content Figure 3 C) in HC-6 and mutant plants; the chlorophyll content Figure 3 D) in HC-6 and mutant plants; K 15 absolute isotope 15 N abundances Figure 3 E) and atom % 15 N Figure 3 F) (n≥14) of HC-6 and ric1a / 2a-1 plants grown with or without rhizobia inoculation in the K 13 NO3 labeling assay; the 13 C fixation rate (μg Figure 3G); and total carbon content in HC-6 and mutant plants ( Figure 3 H). Dots 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 to HC-6), ns: no significant difference.

[0017] Figure 4 A shows a violin plot of the yield per plant distribution of HC-6 and ric1a / 2a mutants planted in Fuzhou and Shijiazhuang in 2021 (n≥14); Figure 4 B shows the plot yield of HC-6 and mutants in Fuzhou (6.75 square meters) and Shijiazhuang (7 square meters) in 2022 (n = 3); and Figure 4 C shows the plot yield of HC-6 and mutants in Yangzhong in 2023 (50m 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 to HC-6).

[0018] Figure 5 shows plant height of HC-6 and mutants in the Fuzhou and Shijiazhuang trials in 2022 ( Figure 5 A), number of branches ( Figure 5 B), number of pods ( Figure 5 C), number of seeds ( Figure 5 D), and 100-seed weight ( Figure 5 E) (n≥14); and number of nodules of HC-6 and mutants at the flowering stage in the Fuzhou field in 2022 ( Figure 5 F). 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 to HC-6).

[0019] Figure 6 shows soybean protein content ( Figure 6 A) and oil content ( Figure 6 B) in all trials; and protein ( Figure 6 C) and oil ( Figure 6 D) yield calculated values per plant (2021) or per plot (2022 and 2023) at all locations. 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 to HC-6).

[0020] Figure 7 shows N content in seeds of HC-6 and mutant plants (N≥15) grown in the field ( Figure 7A); Determination of %Ndfa in mutant and HC-6 plant seeds grown in the field in Fuzhou in 2022 using the natural abundance method Figure 7 B); 15 Natural abundance % of N atoms Figure 7 C) and 15 N / 14 N ratio Figure 7 D), n = 15. 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).

[0021] Figure 8 Net photosynthetic rate ( Figure 8 A) and stomatal conductance ( Figure 8 B) of plants grown in the field in Fuzhou in 2022 during the flowering period; carbon content in leaves Figure 8 C) and seeds Figure 8 D) of mutant and HC-6 plants (n = 8) grown in the field in Fuzhou in 2022. 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).

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

[0023] Figure 10 Shows the composition of the insert in plasmid pGES702. DETAILED DESCRIPTION OF THE INVENTION

[0025] I. Definitions

[0026] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures used herein are all widely used terms and conventional procedures in the respective 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 comprehensively described in the following literature: Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (abbreviated as "Sambrook"). Meanwhile, for a better understanding of the present invention, the definitions and explanations of relevant terms are provided below.

[0027] As used herein, the term "and / or" encompasses all combinations of the items connected by this term, and should be regarded as each combination having been separately 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".

[0028] As used herein, the term "root nodule" is a structure formed on the root system of a plant by the symbiosis of bacteria and the root tissue of the plant. Such bacteria are called "rhizobia". Rhizobia can convert free nitrogen in the air into nitrogen-containing compounds that can be utilized by plants, i.e., nitrogen fixation. Leguminous plants and some green manure plants, lawn grasses, etc. have root nodules. Plants capable of nitrogen fixation can reduce the amount of nitrogen fertilizer used, increase soil organic matter, and improve the physical properties of the soil. However, hypernodulation can also lead to a significant increase in carbon consumption, thus affecting plant growth.

[0029] As used herein, the term "hypernodulation" refers to excessive nodulation in leguminous plants. A "hypernodulation mutant" refers to a mutant that exhibits excessive nodulation due to certain gene mutations.

[0030] The "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 of the plant such as roots, stems, leaves, flowers, fruits, and / or seeds or any tissue.

[0031] As used herein, the term "genetic modification" involves modifying the genetic material of an organism, such as DNA, to obtain altered traits.

[0032] As used herein, "genome" encompasses not only chromosomal DNA present in the cell nucleus, but also organelle DNA present in subcellular components of the cell such as mitochondria and plastids.

[0033] "Genetically modified plant" or "genetically modified plant cell" means a plant organism or plant cell that contains within its genome an exogenous polynucleotide or a modified gene or an expression regulatory sequence. For example, the exogenous polynucleotide can be stably integrated into the genome of the organism or cell and inherited through successive generations. 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 in the genome of the organism or cell contains single or multiple deoxynucleotide substitutions, deletions, and additions.

[0034] "Exogenous" with respect to a sequence means a sequence from a foreign species, or if from the same species, a sequence that has been substantially altered in composition and / or locus from its natural form through deliberate human intervention.

[0035] "Polynucleotide", "nucleic acid sequence", "nucleotide sequence", or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers, optionally containing synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single letter designations: "A" is adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

[0036] As used herein, the term "encoding" means that a polynucleotide directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame that typically begins with an ATG start codon or an alternative start codon such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence can be DNA, cDNA, or a recombinant nucleotide sequence.

[0037] "Polypeptide", "peptide", and "protein" are used interchangeably in the present invention and refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence", and "protein" also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0038] An "endogenous" polypeptide refers to a polypeptide that naturally exists in an organism, that is, a polypeptide encoded by the natural genome of the organism and expressed in the organism.

[0039] In the context of peptides, the terms "amino acid", "residue", and "amino acid residue" are used interchangeably and include both naturally occurring amino acids and non-natural amino acids in proteins. The single-letter and three-letter nomenclatures of naturally occurring amino acids in proteins are those commonly used in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0040] As used herein, "percent identity" refers to comparing the amino acids of two polypeptides or the nucleotides of two nucleic acid molecules, and when optimally aligned, the two polypeptides or two nucleic acid molecules have approximately the specified percentage of identical amino acids or nucleotides. For example, "95% amino acid / nucleotide identity" means comparing the amino acids of two polypeptides or the nucleotides of two nucleic acid molecules, and when optimally aligned, the two polypeptides have 95% identical amino acids / nucleotides.

[0041] As used in the present invention, 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, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (such as transcription to generate mRNA or functional RNA) and / or the translation of the RNA into a precursor or mature protein.

[0042] 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 translatable RNA (such as mRNA).

[0043] The "expression construct" of the present invention can 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 which is normally found in nature.

[0044] The terms "regulatory sequence" and "regulatory element" are used interchangeably and refer to nucleotide sequences that are located upstream (5' non-coding sequence), in the middle, or downstream (3' non-coding sequence) of a coding sequence and affect the transcription, RNA processing or stability, or translation of the relevant coding sequence. Regulatory sequences can include but are not limited to promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0045] "Promoter" refers to a nucleic acid fragment that can control the transcription of another nucleic acid fragment. In some embodiments of the present invention, the promoter is a promoter capable of controlling gene transcription 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.

[0046] "Constitutive promoter" refers to a promoter that generally causes 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 mainly but not necessarily exclusively in one tissue or organ and can also be expressed in a 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 (environment, hormone, chemical signal, etc.).

[0047] As used herein, the term "operably linked" refers to the connection of a regulatory element (such as, but not limited to, a promoter sequence, a transcription termination sequence, etc.) to a nucleic acid sequence (such as a coding sequence or an open reading frame) such that the transcription of the nucleotide sequence is controlled and regulated by the transcription regulatory element. Techniques for operably linking a regulatory element region to a nucleic acid molecule are known in the art.

[0048] "Introducing" a nucleic acid molecule (such as a plasmid, a linear nucleic acid fragment, RNA, etc.) or a protein into an organism means transforming a cell of the organism with the nucleic acid or protein such 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 a 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.

[0049] As used herein, "antisense nucleic acid" refers to a nucleic acid molecule having a complementary sequence to a target nucleic acid (such as mRNA) and participating in the regulation of gene expression by binding to the target nucleic acid through base pairing. Antisense nucleic acids include RNA or DNA molecules that can be precisely complementary to a specific mRNA and specifically block its translation.

[0050] As used herein, "interfering nucleic acid" refers to a nucleic acid molecule encoding an RNA molecule for RNA interference (RNAi), including nucleic acid molecules such as siRNA, shRNA, miRNA, etc.

[0051] "Gene editing", also known as genome editing, uses engineered nucleases or "molecular scissors" to perform DNA insertion, deletion, or replacement in the genome of an organism. Gene editing causes site-specific double-strand breaks (DSBs) at desired locations in the genome and then introduces the desired DNA insertions, deletions, or substitutions during the repair of the DSBs. Gene editing commonly uses meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR systems.

[0052] "Meganucleases" are a class of endodeoxyribonucleases with large recognition sites (double-stranded DNA sequences of 12 - 40 bp), and their recognition sites typically occur only once in any given genome. For example, the 18 bp sequence recognized by the I-SceI meganuclease would on average occur by chance only once in a genome 20 times larger than the human genome.

[0053] "Zinc finger nucleases" are artificial restriction enzymes prepared by fusing zinc finger DNA-binding domains with DNA cleavage domains. The zinc finger DNA-binding domain of a single ZFN typically contains 3 - 6 individual zinc finger repeats, and each zinc finger repeat can recognize, for example, 3 bp.

[0054] "Transcription activator-like effector nucleases" are restriction enzymes that can be engineered to cut specific DNA sequences, typically prepared by fusing the DNA-binding domain of a transcription activator-like effector (TALE) with a DNA cleavage domain. TALEs can be engineered to bind to almost any desired DNA sequence.

[0055] "Clustered regularly interspaced short palindromic repeats (CRISPR)" are prokaryotic DNA segments containing short repeat sequences. The CRISPR system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present in plasmids and phages, and this resistance provides acquired immunity. In this system, Cas proteins or similar proteins cut foreign nucleic acids under the guidance of RNA.

[0056] The CRISPR / Cas system refers to clustered regularly interspaced short palindromic repeats and their associated systems, in which CRISPR nucleases can cut DNA strands at specific locations under the guidance of guide RNA.

[0057] As used herein, the term "CRISPR nuclease" generally refers to nucleases present in naturally occurring CRISPR systems, as well as their modified forms, their variants, their catalytically active fragments, etc. This term encompasses any effector protein based on the CRISPR system that can achieve gene targeting (such as gene editing, gene targeting regulation, etc.) within a cell.

[0058] Examples of “CRISPR nuclease” include Cas9 nuclease or its variants. The Cas9 nuclease can be a Cas9 nuclease 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 and refer to an RNA-guided nuclease comprising a Cas9 protein or a fragment thereof (such as a protein containing 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 and can target and cleave a DNA target sequence under the guidance of a guide RNA to form a DNA double-strand break (DSB).

[0059] Examples of “CRISPR nuclease” can also include Cpf1 nuclease or its variants such as highly specific variants. The Cpf1 nuclease can be a Cpf1 nuclease from different species, such as Cpf1 nucleases from Francisella novicida U112, Acidaminococcus sp. BV3L6, and Lachnospiraceae bacterium ND2006.

[0060] 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 Cas9-based gene editing system, the gRNA is typically composed of a crRNA and a tracrRNA molecule that partially complement to form a complex, where the crRNA contains a sequence with sufficient complementarity to the target sequence to hybridize with the target sequence and direct the CRISPR complex (Cas9 + crRNA + tracrRNA) to specifically bind to the target sequence. However, it is known in the art that single-guide RNA (sgRNA) can be designed, which contains the characteristics of both crRNA and tracrRNA. In a Cpf1-based genome editing system, the gRNA is typically composed of only a mature crRNA molecule, where the crRNA contains a sequence with sufficient identity to the target sequence to hybridize with the complementary sequence of the target sequence and direct the complex (Cpf1 + crRNA) to specifically bind to the target sequence. Designing a suitable gRNA sequence based on the CRISPR nuclease used and the target sequence to be edited is within the capabilities of those skilled in the art.

[0061] The CRISPR-dCas system involves modifying to cause the loss of nuclease activity of Cas, so that it cannot cleave DNA strands, while connecting it with a transcriptional activation domain to achieve the effect of site-specific transcriptional activation.

[0062] Similarly, the CRISPR-dCas system can also be used to regulate gene expression in a region of interest. For example, CRISPR-dCas can be connected with a transcriptional activation or repression domain to activate or repress gene expression in the region of interest. Transcription can also be regulated by modulating the methylation level in the region of interest. Specifically, dCas can be connected with 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 in the region of interest, thereby reducing the transcription in the region of interest. dCas can also be connected with a sequence that inhibits methylation, such as the Tet (ten-eleven translocation) catalytic domain (CRISPR-dCas-Tet), to decrease the methylation level in the region of interest, thereby increasing the transcription in the region of interest.

[0063] II. Modified Leguminous Plants

[0064] The inventors unexpectedly obtained genetically modified soybean plants in which the expression and / or activity of certain endogenous polypeptides was reduced or eliminated, resulting in more root nodules than unmodified plants, thereby enhancing the nitrogen fixation ability of the plants. At the same time, the modified plants had improved photosynthesis, i.e., carbon fixation ability, and thus had higher biomass and could achieve higher yields. In addition, in the seeds of the modified soybean plants, the protein content was increased while the oil content remained unchanged. Compared with the prior art (where increasing the protein content would lead to a decrease in the oil content), it provided significant production advantages.

[0065] Therefore, the present invention provides a modified leguminous (Fabaceae) plant in which the activity and / or level of at least two endogenous polypeptides in the genetically modified leguminous plant is reduced or eliminated compared with an ungenetically modified control plant.

[0066] The present invention also provides a plant cell of a genetically modified leguminous plant in which the activity and / or level of at least two endogenous polypeptides in the genetically modified plant cell is reduced or eliminated compared with an ungenetically modified control plant cell.

[0067] In some embodiments, the at least two endogenous polypeptides are selected from the group consisting of the amino acid sequences comprising SEQ ID NO: 3, 6, 9, or 12, or polypeptides 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.

[0068] In some embodiments, the two endogenous polypeptides are two or three endogenous polypeptides selected from the group consisting of the amino acid sequences comprising SEQ ID NO: 3, 6, 9, or 12, or polypeptides 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 endogenous polypeptides selected from the group consisting of the amino acid sequences comprising SEQ ID NO: 3, 6, 9, or 12, or polypeptides 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.

[0070] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO: 3 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, and a second polypeptide comprising SEQ ID NO: 6 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: 6.

[0071] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO: 3 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, and a second polypeptide comprising SEQ ID NO: 9 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: 9.

[0072] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3 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, and a second polypeptide comprising SEQ ID NO: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:12.

[0073] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:6 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:6, and a second polypeptide comprising SEQ ID NO:9 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:9.

[0074] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:6 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:6, and a second polypeptide comprising SEQ ID NO: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:12.

[0075] In some embodiments, the at least two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:9 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:9, and a second polypeptide comprising SEQ ID NO: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:12.

[0076] In some embodiments, the at least two endogenous polypeptides are three endogenous polypeptides selected from the group consisting of the amino acid sequences comprising SEQ ID NO: 3, 6, 9, or 12, or polypeptides comprising amino acid sequences that are 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.

[0077] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO: 3 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; a second polypeptide comprising SEQ ID NO: 6 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: 6; and a third polypeptide comprising SEQ ID NO: 9 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: 9.

[0078] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO: 3 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; a second polypeptide comprising SEQ ID NO: 6 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: 6; and a third polypeptide comprising SEQ ID NO: 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: 12.

[0079] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3 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; a second polypeptide comprising SEQ ID NO:9 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:9; and a third polypeptide comprising SEQ ID NO: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:12.

[0080] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:6 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:6; a second polypeptide comprising SEQ ID NO:9 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:9; and a third polypeptide comprising SEQ ID NO: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:12.

[0081] In some embodiments, compared to an unmodified control plant, the activity and / or level of four endogenous polypeptides in the genetically modified leguminous plant is reduced or eliminated, the four endogenous polypeptides comprising the amino acid sequences of SEQ ID NOs: 3, 6, 9, and 12, 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.

[0082] Examples of leguminous plants include, but are not limited to: soybean, broad bean, pea, mung bean, adzuki bean, cowpea, kidney bean, hyacinth bean, pigeon pea, peanut, lupin, Cercideae plants, catechu, cassia, liquorice, sophora flavescens, milk vetch, alfalfa, broad bean, lesser galangal, albizia, rosewood, Chinese honey locust, Indian indigo, sappanwood, acacia, woody milk vetch, twinleaf bean, Indian hemp, and kudzu vine.

[0083] In some embodiments, the leguminous plant is a Glycine plant. In some embodiments, the leguminous plant is a soybean plant.

[0084] The present invention also provides a plant part of the genetically modified leguminous plant of the present invention, or a plant part comprising the plant cells 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.

[0085] III. Method for increasing the yield of leguminous plants

[0086] The present invention provides 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 in the plant.

[0087] In some embodiments, the at least two endogenous polypeptides are selected from the group consisting of amino acid sequences comprising SEQ ID NO: 3, 6, 9, or 12, or polypeptides 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.

[0088] In some embodiments, the two endogenous polypeptides are two or three endogenous polypeptides selected from the group consisting of amino acid sequences comprising SEQ ID NO: 3, 6, 9, or 12, or polypeptides 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 endogenous polypeptides selected from the group consisting of amino acid sequences comprising SEQ ID NO: 3, 6, 9, or 12, or polypeptides 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.

[0090] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3 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, and a second polypeptide comprising SEQ ID NO:6 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:6.

[0091] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3 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, and a second polypeptide comprising SEQ ID NO:9 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:9.

[0092] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3 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, and a second polypeptide comprising SEQ ID NO: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:12.

[0093] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:6 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:6, and a second polypeptide comprising SEQ ID NO:9 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:9.

[0094] In some embodiments, the two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:6 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:6, and a second polypeptide comprising SEQ ID NO: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:12.

[0095] In some embodiments, the at least two endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:9 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:9, and a second polypeptide comprising SEQ ID NO: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:12.

[0096] In some embodiments, the at least two endogenous polypeptides are three endogenous polypeptides selected from the group consisting of the amino acid sequences comprising SEQ ID NO:3, 6, 9, or 12, or polypeptides 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.

[0097] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3 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, a second polypeptide comprising SEQ ID NO:6 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:6, and a third polypeptide comprising SEQ ID NO:9 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:9.

[0098] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3 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, a second polypeptide comprising SEQ ID NO:6 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:6, and a third polypeptide comprising SEQ ID NO: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:12.

[0099] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:3 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, a second polypeptide comprising SEQ ID NO:9 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:9, and a third polypeptide comprising SEQ ID NO: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:12.

[0100] In some embodiments, the three endogenous polypeptides include: a first polypeptide comprising SEQ ID NO:6 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:6, a second polypeptide comprising SEQ ID NO:9 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:9, and a third polypeptide comprising SEQ ID NO: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:12.

[0101] In some embodiments, the method comprises reducing or eliminating the activity and / or level of four endogenous polypeptides in a plant, the four endogenous polypeptides comprising the amino acid sequences of SEQ ID NO: 3, 6, 9, and 12, respectively, or comprising amino acid sequences that are 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.

[0102] The present invention provides a method for increasing the yield and / or protein content of leguminous plants, the method comprising:

[0103] a) providing a first leguminous plant having reduced or eliminated activity and / or level of a first endogenous polypeptide;

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

[0105] c) crossing the first and second leguminous plants to produce a first progeny plant having reduced or eliminated activity and / or level of the first and second endogenous polypeptides.

[0106] In some embodiments, the first and second leguminous plants are species of the same genus, preferably the same species. In some embodiments, the leguminous plant is a plant of the genus Glycine. In some embodiments, the leguminous plant is a soybean plant.

[0107] In some embodiments, the first and second endogenous polypeptides are selected from polypeptides comprising the amino acid sequences of SEQ ID NO: 3, 6, 9, or 12, or polypeptides comprising amino acid sequences that are 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.

[0108] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO: 3 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, and the second endogenous polypeptide comprises SEQ ID NO: 6 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: 6.

[0109] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:3 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, and the second endogenous polypeptide comprises SEQ ID NO:9 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:9.

[0110] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:3 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, and the second endogenous polypeptide comprises SEQ ID NO: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:12.

[0111] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:6 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:6, and the second endogenous polypeptide comprises SEQ ID NO:9 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:9.

[0112] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:6 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:6, and the second endogenous polypeptide comprises SEQ ID NO: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:12.

[0113] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:9 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:9, and the second endogenous polypeptide comprises SEQ ID NO: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:12.

[0114] The present invention provides a method for increasing the yield and / or protein content of leguminous plants, the method comprising:

[0115] a) providing a first leguminous plant having reduced or eliminated activity and / or level of a first endogenous polypeptide;

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

[0117] c) providing a third leguminous plant having reduced or eliminated activity and / or level of a third endogenous polypeptide;

[0118] d) crossing the first and second leguminous plants to produce a first progeny plant having reduced or eliminated activity and / or level of the first and second endogenous polypeptides; and

[0119] e) crossing the first progeny plant with the third leguminous plant to produce a second progeny plant having reduced or eliminated activity and / or level of the first, second, and third endogenous polypeptides.

[0120] Those skilled in the art will understand that the second progeny plant can also be produced by crossing the progeny of the second and third leguminous plants with the first leguminous plant.

[0121] In some embodiments, the first, second, and third leguminous plants are species of the same genus, preferably the same species. In some embodiments, the leguminous plant is a plant of the genus Glycine. In some embodiments, the leguminous plant is a soybean plant.

[0122] In some embodiments, the first, second, and third endogenous polypeptides are selected from the group consisting of the amino acid sequences comprising SEQ ID NO:3, 6, 9, or 12, or polypeptides 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.

[0123] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:3 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, the second endogenous polypeptide comprises SEQ ID NO:6 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:6, and the third endogenous polypeptide comprises SEQ ID NO:9 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:9.

[0124] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:3 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, the second endogenous polypeptide comprises SEQ ID NO:6 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:6, and the third endogenous polypeptide comprises SEQ ID NO: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:12.

[0125] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:3 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, the second endogenous polypeptide comprises SEQ ID NO:9 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:9, and the third endogenous polypeptide comprises SEQ ID NO: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:12.

[0126] In some embodiments, the first endogenous polypeptide comprises SEQ ID NO:6 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:6, the second endogenous polypeptide comprises SEQ ID NO:9 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:9, and the third endogenous polypeptide comprises SEQ ID NO: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:12.

[0127] The present invention provides a method for increasing the yield and / or protein content of leguminous plants, the method comprising:

[0128] a) providing a first leguminous plant (Plant 1) having reduced or eliminated activity and / or level of a first endogenous polypeptide;

[0129] b) providing a second leguminous plant (Plant 2) having reduced or eliminated activity and / or level of a second endogenous polypeptide;

[0130] c) providing a third leguminous plant (Plant 3) having reduced or eliminated activity and / or level of a third endogenous polypeptide;

[0131] d) providing a fourth leguminous plant (Plant 4) having reduced or eliminated activity and / or level of a fourth endogenous polypeptide

[0132] e) crossing the first and second leguminous plants (Plant 1×Plant 2) to produce a first progeny plant having reduced or eliminated activity and / or level of the first and second endogenous polypeptides;

[0133] f) crossing the first progeny plant with the third leguminous plant ((Plant 1×Plant 2)×Plant 3) to produce a second progeny plant having reduced or eliminated activity and / or level of the first, second and third endogenous polypeptides; and

[0134] g) crossing the first progeny plant with the fourth leguminous plant (((Plant 1×Plant 2)×Plant 3)×Plant 4) to produce a third progeny plant having reduced or eliminated activity and / or level of the first, second, third and fourth endogenous polypeptides.

[0135] Those skilled in the art will understand that the third progeny plant 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.

[0136] In some embodiments, the first, second, third, and fourth leguminous plants are species of the same genus, preferably the same species. In some embodiments, the leguminous plant is a plant of the genus Glycine. In some embodiments, the leguminous plant is a soybean plant.

[0137] In some embodiments, the first, second, third, and fourth endogenous polypeptides are selected from the group consisting of amino acid sequences comprising SEQ ID NO: 3, 6, 9, or 12, or polypeptides comprising amino acid sequences that are 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 that is 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 that is 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 that is at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, and the fourth endogenous polypeptide comprises SEQ ID NO: 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: 12.

[0138] Techniques for reducing or eliminating the activity and / or expression of endogenous plant polypeptides are known to those skilled in the art. Examples of such techniques include, but are not limited to, antisense nucleic acid techniques, RNA interference (RNAi) techniques, and gene editing techniques. Examples of gene editing techniques include, but are not limited to, meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR systems. Plants with reduced or eliminated activity and / or expression of endogenous polypeptides can also be obtained by TILLING technology.

[0139] In some embodiments, the activity or expression of endogenous plant polypeptides is reduced or eliminated by a CRISPR system. In some embodiments, the 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, the expression of the gene encoding the endogenous plant polypeptide is inhibited by a CRISPR / dCas system.

[0140] 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. Exemplary sequences of the targeting portion are shown in Table 1.

[0141] Table 1. Sequences of the targeting portion of sgRNA and primers for testing genome editing

[0142]

[0143]

[0144] Beneficial effects

[0145] The protein content of soybeans is negatively correlated with yield and oil content, which has historically meant that breeding for 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 the oil content, a significant contribution can be made to increasing soybean yield and protein content in modern crop production practices. This application provides an accurate, rapid, durable, low-cost, and eco-friendly method that can be widely applied to soybean breeding programs. Assuming that, through the method provided in this application, a 10% increase in yield and a 1% increase in protein could result in an increase in global protein production of more than 30 million tons (an increase of approximately 210 kg of protein per hectare x 136 million hectares (as of 2020)), and make a significant contribution to filling the projected protein shortage of 60 million to 70 million tons by 2050. Examples

[0146] Example 1, Materials and Methods

[0147] 1.1. Construction of Plant Materials

[0148] 1.1.1. Plasmids and Strains

[0149] In the examples of this application, Escherichia coli DH5α was used to construct transgenic vectors. 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) backbone, plasmid vectors pGES701 / pGES702 ( Figure 9 and 10 ) were constructed, and Agrobacterium tumefaciens GV3101 was used for soybean transformation. Bradyrhizobium japonicum BXYD3 (see CN101182476A) was used to inoculate soybean plants to form root nodules.

[0150] 1.1.2. Gene Editing System

[0151] In the high-throughput CRISPR / Cas9 system, the pGES701 vector contains a nucleotide sequence encoding sgRNA operably linked to the GmU6 promoter, and a nucleotide sequence encoding spCas9 optimized for soybean codons operably linked to the soybean endogenous promoter pM4 ( Figure 9 ); the single transcript pGES702 vector contains a tandem nucleotide sequence encoding Cas9 and encoding multiple sgRNAs operably linked to the GmpM4 promoter ( Figure 10 ).

[0152] The pGES701 / pGES702 vectors utilize the characteristic that the BsaI restriction nuclease has different recognition sites and cleavage sites to clone sgRNA. The single transcript vector pGES702 is characterized in that both ends of the sgRNA+sgRNA Scaffold structure are tRNAs, and the tRNAs can be cleaved by RNase in vivo, separating Cas9 and sgRNA after transcription into mRNA.

[0153] 1.1.3. Transgenic Method

[0154] In a dedicated laminar flow hood for plant tissue culture, use a scalpel to excise the cotyledon part of sodium hypochlorite-sterilized soybean seeds. Vertically cut the seeds along the direction of the hypocotyl of the cotyledon and remove the germ. Then, dip the scalpel in the colony and make 7-8 scratches near the hypocotyl of the cotyledon node. Transfer the explants to a square Petri dish containing double-layer filter paper moistened with sterile water and seal it. Incubate at 25 °C in a light incubator for 4-5 days, resume growth for 7 days, induce buds and screen for resistance for 21 days, elongate buds and screen for resistance for 21 days, and culture on a rooting medium for 30 days to obtain transgenic 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).

[0155] 1.1.4. Detection of gene knockout

[0156] Remove the transgenic soybean seedlings from the rooting medium, use clear water to remove the excess medium and browning tissue from the roots of the seedlings, transfer the transgenic seedlings to a black square box with a ratio of vermiculite: nutrient soil: perlite of 1:1:1, cover it with plastic wrap to keep it moist, uncover it along the periphery of the plastic wrap for ventilation after one week, and completely uncover the plastic wrap after 14 days. Sample and identify the transgenic seedlings.

[0157] 1.1.4.1. Cultivation and identification of T0 generation transgenic soybean seedlings

[0158] Spray the T0 generation transgenic soybean seedlings with a 1000-fold diluted Bar (glufosinate-ammonium aqueous solution, 10% solution, Sangon Biotech (Shanghai) Co., Ltd., A614229) herbicide for preliminary screening. Observe the changes in the leaves of the transgenic seedlings after one week, remove the non-positive seedlings, number the surviving seedlings and take leaf samples. Extract the leaf DNA using the rapid plant DNA extraction method and perform PCR detection for the Bar gene (primers: forward 5’-CTGCACCATCGTCAACCACTA-3’; reverse 5’-CTGAAGTCCAGCTGCCAGAAA-3’). Identify the positive plants according to the electrophoresis results of the PCR products (1% agarose gel electrophoresis). Perform PCR amplification with the primers in Table 1, and sequence the PCR

[0159] products using Hi–TOM. Statistically analyze the gene editing types and vector editing efficiency based on the sequencing results. Finally, transfer the positive plants to a cultivation pot with a ratio of nutrient soil: vermiculite: perlite of 1:1:1 for planting and harvesting seeds.

[0160] 1.1.4.2. Screening and Genotype Identification of Homozygous Mutant Plants of T1 Generation Soybeans

[0161] Plant the seeds of the edited plants among the T0 generation positive plants, and screen for plants with homozygous editing types that do not contain vector sequences in the offspring. Specifically, at the seedling stage (about one week after seed germination, when true leaves emerge), apply Basta liquid diluted 1000 times to one end of the true leaves and mark them. Observe the changes in the leaves three days later (withered leaves indicate that the plants have no Bar resistance). After numbering, extract leaf DNA to further confirm whether it contains vector sequences. Perform PCR amplification using the primers in Table 1, sequence the products by Sanger sequencing, analyze the editing types of each plant through the sequencing results, and screen out plants that do not contain vector sequences and are homozygous mutants.

[0162] 1.1.4.3. Phenotypic Analysis of Homozygous Mutants of T2 Generation Soybeans

[0163] Plant the seeds of the homozygous mutant plants screened in the T1 generation, perform PCR amplification using the primers in Table 1, and confirm whether the plants are homozygous mutants by sequencing. The T2 generation plants are planted in pots, and agronomic traits are analyzed. Among them, at the mature stage, plant height, number of branches, and number of pods are counted for the plants, and the number of seeds per plant, seed weight per plant, and 100-seed weight are counted after harvesting the seeds. Wild-type plants (Huachun 6, HC-6) are used as controls.

[0164] 1.1.4.4. Detection of Gene Editing Types by Sanger Sequencing

[0165] Sample preparation: First, obtain the target DNA fragment for Sanger sequencing through PCR amplification reaction, send its PCR product to a sequencing company for sequencing, and either the forward primer or the reverse primer of the editing primer can be used as the sequencing primer (there should be no poly structure or repeat sequence within the range of the forward and reverse primers from the sgRNA). Sequencing result analysis: Gene editing types can be divided into four types: no gene editing, heterozygous editing, biallelic editing, and homozygous editing.

[0166] 1. No gene editing: When the sequencing result sequence is exactly the same as the reference genome sequence and the sequencing peak map is a single peak, it means no gene editing has occurred.

[0167] 2. Homozygous editing: Since soybeans are ancient tetraploid evolved diploids, gene editing (insertion, substitution, or deletion) occurs at the sgRNA site on one chromosome, and the homologous chromosome also has the same gene editing type. When there are base deletions, insertions, or substitutions at the sgRNA site in the sequencing result sequence compared with the reference genome sequence, and the sequencing peak map is a single peak, it is homozygous gene editing.

[0168] 3. Heterozygous editing: When double peaks start to appear in the sequencing peak map near the sgRNA site, and the sequencing sequence may or may not be identical to the reference genome, it is heterozygous editing.

[0169] 4. Biallelic editing: When the sequencing result sequence starts to be inconsistent with the reference genome sequence near the sgRNA, and at the same time double peaks start to appear in the sequencing peak map near the sgRNA site, the gene editing type may be biallelic editing or heterozygous editing.

[0170] 1.2 Growth conditions of plant materials

[0171] Using the soybean (Glycine max) cultivar Huachun 6 (HC-6) as the material, physiological and agronomic experiments were carried out. The hydroponic culture of soybean plants was as described in the article by Yang et al. in 2017 (doi: 10.3389 / fpls.2017.02091). The nutrient solution contained Ca(NO3)2 0.12 mM, KNO3 0.19 mM, MgCl2 2.5 μM, MgSO4 0.5 mM, K2SO4 1 mM, MnSO4 0.5 μM, ZnSO4 1.5 μM, CuSO4 0.5 μM, (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, the culture medium was changed once a week, and the biomass was measured after about 45 days of culture. Soybean plants were grown in a growth chamber with a light intensity of 450 μmol photons m -2 s -1 , a photoperiod of 14 hours of light (28 °C) / 10 hours of dark (24 °C), and a humidity of 65%.

[0172] For the field experiment, plants of different genotypes (HC-6 and mutants) were planted in a randomized block design from 2021 to 2023, with three replicates for each genotype, and were planted in experimental fields in Fuzhou, Fujian Province, China (26.08 N, 119.24 E) and Yangzhong (26.28 N, 118.49 E, only in 2023) and Shijiazhuang, Hebei Province (38.03 N, 114.48 E).

[0173] In 2021, two rows of each genotype were planted, with a row length of 3.5 m and a row spacing of 0.5 m. The plot design in Fuzhou in 2022 was three rows per plot (~6.75 square meters), each row being 4.5 m long, and in Shijiazhuang in 2022, four rows per plot (~7 square meters), each row being 3.5 m long. In 2023, the plots in Yangzhong were designed to have six rows per plot (about 50 square meters), each row being 25 m long. In 2022 and 2023, the growth density per plot was calculated to be 140,000–150,000 plants / ha. 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 fertilizers. In Shijiazhuang, all plots were fertilized with 375 kg / ha of compound fertilizer (N:P2O5:K2O = 15:15:15) as basal fertilizer. The available nitrogen content in the field soil was 53–107 mg / kg.

[0174] 1.3. Plant field performance

[0175] At the flowering stage, at least 8 representative plants of each genotype were taken to record the flowering time. After full maturity (growth stage R8), in 2021 and 2022, about 15 randomly selected individual plants per row of each plot were harvested. Five agronomic traits (plant height, number of branches per plant, 100-seed weight, number of pods per plant, and seed yield per plant) were scored. The seed protein and oil contents were determined using a MATRIX-I Fourier transform near-infrared reflectance spectrometer (FT-NIRS) (Bruker Optics, Bremen, Germany). The estimated protein and oil contents per plant were calculated by multiplying the seed yield of each plant by the seed protein content and the seed oil content, respectively.

[0176] 1.4. Real-time quantitative PCR

[0177] Total RNA was extracted from plant tissues using reagents (OMEGA Bio-Tek, Norcross, GA, United States).

[0178] First-strand cDNA was synthesized from the extracted total RNA using a PrimeScript RT kit (RR037A, TaKaRa) according to the manufacturer's instructions, and SYBR1 Premix Ex Taq was used TM(RR820A, TaKaRa) RT-qPCR amplification was performed using the iQ5 real-time PCR detection system (BioRad). The amplification was carried out in a 20 μL reaction volume (2x SYBR mixture, 10 μL; primer-F / R (10 μmol, primer sequences are shown in Table 2) 1 μL each, H2O 4 μL, template cDNA 4 μL). 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, K. J. & Schmittgen, T. D. Methods 25, 402-408, (2001)).

[0179] Table 2. Primers for real-time quantitative PCR

[0180]

[0181] 1.5. C / N content and isotope labeling determination

[0182] For 15 N isotope labeling determination, 21 days after seed germination, soybean hydroponic plants (inoculated and non-inoculated with Bradyrhizobium japonicum BXYD3) were transferred to a hydroponic culture containing 26.5 μM K 15 NO3 to absorb 15 N. The whole seedlings were harvested on the 49th day to measure 15 N content.

[0183] For 13 C isotope labeling determination, soybean plants grown in soil at the filling stage were placed in a transparent sealed chamber to maintain photosynthesis. They were placed at room temperature for 12 hours at a light intensity of 450 μmol photons m -2 s -1 to fix the 13 CO2 released from the reaction of barium carbonate (Ba 13 CO3, 0.5 g) and lactic acid (10 mL). After two labeling treatments (with a two-day interval), the plants were transferred to the outdoor environment and harvested when the seeds matured.

[0184] For isotope measurement, the 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). The N and C concentrations were determined by comparing the peak areas of the samples with those of three standard samples, and calculations were made based on the concentrations and dry weights. The 15 N / 14 N ratio (for N) or 13 C / 12 C ratio (for C), and then compared with the international standard material to calculate δ 15 N and δ 13 C values (‰, PDB). The N fixation rate of the labeled samples grown in hydroponic culture is shown in Equation (1), and the N fixation rate of natural seeds is shown in Equation (2):

[0185]

[0186]

[0187] δ 15 N uninoculated : Without inoculation with rhizobia 15 δ of the N-labeled material 15 N

[0188] δ 15 N inoculated : Inoculated with rhizobia 15 δ of the N-labeled material 15 Nδ 15 N natrue : Under natural growth conditions ( 15 N unlabeled) δ of the control material 15 N value

[0189] δ 15 N reference : δ of the seeds of the control plants (non-nodulating soybeans) 15 N

[0190] δ 15 N legume : δ of the seeds of leguminous plants (here referring to soybeans) 15 NB: Constant (B = -2.1).

[0191] 1.6. Net photosynthetic rate and stomatal conductance

[0192] The net photosynthetic rate (Pn) and stomatal permeability of the leaves of field-grown soybeans were measured using a LICOR-6400XT instrument (LI-COR Biosciences, USA) at the flowering stage. The measurements were carried out in the ambient air between 10:00 and 11:00 am in Fuzhou in summer.

[0193] 1.7. Statistical analysis

[0194] 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. A P-value less than 0.05 was 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 each legend.

[0195] Example 2. Mutants with altered nodule number and their growth

[0196] It is known in the art that although an increase in nodule number can improve nitrogen acquisition ability, it causes huge carbon consumption, thereby affecting plant growth. Mutants with a large increase in nodule number known in the prior art show a poor growth phenotype.

[0197] The inventors unexpectedly found that reducing the expression of a specific endogenous polypeptide can balance nitrogen fixation and carbon consumption while increasing nodule number and achieving altered nitrogen fixation ability, thus still being beneficial to the growth of soybean plants and increasing the yield of soybean plants.

[0198] Specifically, as shown in Example 1, using a multiplex CRISPR–Cas9 mutagenesis system, mutants as shown in Table 3 were obtained in the background 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 lines, mutant 5-1 and mutant 5-2, with a 1-nucleotide insertion and a 4-nucleotide deletion introduced into the coding sequence (SEQ ID NO: 1) of SEQ ID NO: 3, respectively.

[0199] Table 3. Prepared mutants and their nodules and growth

[0200]

[0201] All mutants carried loss-of-function mutations at their respective target sites, and no off-target editing was observed at predicted potential off-target sites. RT–qPCR showed that the mRNA levels of the target proteins encoded in the mutants were significantly reduced compared with HC-6 ( Figure 1 ).

[0202] As Figure 2As shown, plants were inoculated with Bradyrhizobium japonicum strain BXYD3 (OD600 = 0.1) under laboratory conditions, and the number of nodules was counted at the flowering stage. The results showed that nin-4m did not form nodules, while the number of nodules in nark was six times that of HC-6 (nark was 617.7 ± 85.9, and HC-6 was 103.8 ± 11.8). As expected according to the prior art, the growth of both mutants (nin-4m and nark) was hindered due to insufficient nitrogen supply and imbalanced nitrogen and carbon supply (the dry weight of the whole plant 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). The number of nodules formed by mutant 8 (322.5 ± 24.1) was three times that of HC-6, and the biomass decreased (4.62 ± 0.39 g), indicating that mutant 8 experienced the same C / N trade-off as the hypernodulating mutant nark, although to a lesser extent. The number of nodules in mutant 6 was approximately 20% more than that of HC-6 (121.3 ± 14.4), and its biomass increased slightly but significantly (5.74 ± 0.57 g). Interestingly, in mutant 5-1, the number of nodules was twice that of HC-6 (202.4 ± 18.0), and the shoot biomass increased by 31.6% (6.78 ± 0.50 g, while it was 5.08 ± 0.43 g in HC-6).

[0203] The results showed that reducing the expression of two of SEQ ID NO: 3, 6, 9, and 12 in soybean plants unexpectedly increased the number of soybean nodules while improving soybean growth.

[0204] Example 3. Carbon / Nitrogen Balance in Soybean Mutant Plants

[0205] The purpose of this example was to determine the metabolic fluxes of carbon and nitrogen in the mutants.

[0206] Isotope labeling analysis was performed as described in Example 1.4.

[0207] 13 CO2 tracer analysis showed that in mutants 8 and nark, the assimilated 13 C was overly distributed to nodules and not to the shoot( Figure 3 A). In contrast, the distribution of the assimilated 13 C in plants of mutants 5-1 and 6 was relatively higher compared to HC-6. The balanced distribution of C in mutant 5-1 led to an improvement in the C / N status in the plant.

[0208] As Figure 3As shown in Figure B, the increase in nodules in mutant 5-1 led to a higher total nitrogen fixation capacity, indicating a higher nitrogen fixation contribution in mutant 5-1 compared to HC-6 (71.24% for mutant 5-1 and 65.02% for HC-6).

[0209] The inventors also observed that the nitrogen and chlorophyll contents in the leaves of mutant 5-1 were higher ( Figure 3 C - 3D), and at the same time, the nitrogen content also increased significantly ( Figure 3 E - 3F). In the leaves of mutant 5-1, 13 the C assimilation rate increased significantly statistically ( Figure 3 G). This confirmed the enhanced photosynthesis of the mutant. Due to the enhanced photosynthesis and increased biomass, the total carbon content of mutant 5-1 plants increased ( Figure 3 H). Therefore, the increase in nodules in mutant 5-1 unexpectedly led to a balance in carbon allocation, while enhancing carbon and nitrogen assimilation and improving plant growth.

[0210] Example 4. Field Experiments on Soybean Mutants

[0211] The purpose of this example is to test the practical application potential of soybean mutants.

[0212] For this purpose, as described in Example 1.1, field experiments were conducted with mutants 5-1 and 5-2 at three locations from 2021 to 2023, with HC-6 as the reference plant. The field experiments were carried out following typical production practices in China, without inoculating the seeds with additional Bradyrhizobium, but relying on the Bradyrhizobium in the local environment. As described in Example 1.2, the agronomic traits of the plants were analyzed.

[0213] It is worth noting that in all experiments, compared with HC-6, the yields of the two mutants increased significantly statistically. In 2021, small-scale experiments were conducted in Shijiazhuang, Hebei Province and Fuzhou, Fujian Province. As Figure 4 shown in Figure A, the average yield per plant 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.

[0214] In 2022, small-plot experiments were conducted in Shijiazhuang and Fuzhou. As Figure 4 shown in Figure B, in the experiment in Shijiazhuang, the yields of mutants 5-1 and 5-2 increased by 21.67% and 25.02% respectively (7 square meters per small plot). In the experiment in Fuzhou in 2022, heavy summer rains caused waterlogging, affecting the yields of all plants. Nevertheless, mutants 5-1 and 5-2 still showed statistically significant yield increases of 27.93% and 31% (6.75 m per plot2 )。

[0215] In 2023, large-scale field trials were conducted in the ocean. As Figure 4 shown in C, compared with HC-6, the yields of mutants 5-1 and 5-2 increased (by 11.99% and 16.17% respectively), which was consistent with the previous test results.

[0216] The results also showed that the substantial increase in mutant yield was accompanied by an increase in plant height, as well as an increase in the number of branches and pods per plant. As Figure 5 shown, there was no statistically significant difference in 100-seed weight between the mutants and HC-6. The number of nodules of the mutants in the Fuzhou field was twice that of HC-6 ( Figure 5 F), indicating that Bradyrhizobium in the local environment was sufficient to support the increased nodulation in the mutants.

[0217] Notably, in all tests, although the protein content of HC-6 was very high (41.75–46.90% in different field trials), in all tests, the seed protein content of the mutants increased significantly statistically (increased by 1.77–4.42%, Figure 6 A). At the same time, there was no statistically significant change in the oil content of the mutant plants ( Figure 6 B). Therefore, the protein and oil yields per plant or plot calculated by multiplying the protein / oil content by the grain yield both increased (protein yield increased by 13.96–33.61%, oil yield increased by 10.67–31.69%, Figure 6 C and D).

[0218] As described in Examples 1.4 and 1.5, the seeds harvested from the field were tested. The results showed that the total nitrogen content of mutant 5-1 was higher in the seeds harvested from the field ( Figure 7 A). This may be attributed to the increased supply of fixed nitrogen in the mutant seeds, as shown by the 15N natural abundance analysis ( Figure 7 B-D). Therefore, the enhanced nitrogen fixation in the ric1a / 2a mutants provided an additional nitrogen supply for the seeds.

[0219] The net photosynthetic rate (Pn) and stomatal conductance (gs) of the mutant plants grown in the field were higher than those of the HC-6 control ( Figure 8 A-B). This was consistent with the results observed in the laboratory, that is, mutant 5-1 showed a higher C assimilation rate in the leaves ( Figure 3 E). Related to the improvement of photosynthesis, the C content in the leaves and seeds of the mutants grown in the field was higher than that of HC-6 ( Figure 8 C-D). Therefore, the negative correlation between protein content and yield / oil content may be eliminated due to the simultaneous increase in nitrogen and carbon supply in the mutant plants.

[0220] Since different combinations of mutants of the four genes SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7, and SEQ ID NO.10 produce different numbers of root nodules, we detected the agronomic trait performances of these mutant materials with different degrees of nodulation in the field. We focused on analyzing the yield traits of mutant materials with different degrees of increased root nodule numbers, and determined the traits of multiple knockout mutants obtained by knocking out the genes of SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7, and SEQ ID NO.10 or their combinations using multiplex editing technology. It was found that there were no significant differences in plant height, branching, and number of nodes among the different combinations of mutants of the four genes SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7, and SEQ ID NO.10 compared with the control material (WT: HC-6). However, mutant materials with moderately increased root nodule numbers, such as the mutant combinations of 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 hypernodulating mutant (mutant 8) showed significant differences in root nodules compared with the control, but the pod number, grain number, and yield per plant were significantly reduced, as shown in Table 4. From the above results, it can be seen that obtaining mutants with optimal control of root nodule numbers by different combinations of genes can significantly increase soybean yield.

[0221] Table 4

[0222]

[0223] Sequence

[0224] SEQ ID NO.1

[0225]

[0226] SEQ ID NO.2

[0227] ATGGCAAATGCAACAATGGCTACTCGAGTGTCTATACTAATTGCACTAATCATCCTCTCCACCTTCTTCATGACTTTGCAGGCTAGTAATCTCCATGGCCATCCCTTCATTCGTGAAAATAATATTGCAGATAGCCATCACTTTCTTCACAAATATTTAGACGACCTATCAAAGCATATACATGTTCAAGATGCTGATGATGCCCCTCACAAAGATGGAAATACGCATAGGCTCGCACCAGAGGGACCAGATCCTCATCATAATTTTGCAACACCACCAAGAAACTAA

[0228] SEQ ID NO.3

[0229] MANATMATRVSILIALIILSTFFMTLQASNLHGHPFIRENNIADSHHFLHKYLDDLSKHIHVQDADDAPHKDGNTHRLAPEGPDPHHNFATPPRN*

[0230] SEQ ID NO.4

[0231] ATTTAGCCTCTAGAGAGAGTTAATTGATTATTTAATAGACTCCACTTCTATTTATCTAAGGCCATTTTTCTTATTTTGCACATATATAAATAATGCTCATAATATGCTTCCACACTACTTTTTCATCAGACTTCTATCCAAGCTCAAGTTTAGTAGTTCCCACTTTTGTAGCACTTTGTTCAATATTCATTCAGAGTTTACATATATTTCATCTCATGGCAAACACAACAATGGTTACTCGAGTATTTATACTTGCACTAATCATCCTCTCCACCTTCTTCATGACTTTGCAGGCTCGTAATCTCCATGGACATCCCTCCATTCGCGAAAATAATGTTGCAGATAACAACCATCGCTTTCTTCACAATTATTTAGACCTATTAAAGCATATACATGTTGATCATCAAGATGCTGATGATGCCCCTCACAAGGATGGAAATACACATAGACTCACACCAGAAGGACCAGATCCTCATCATAATTTTGCAACACCACCAAGAAACTGAAGCTAGCTTTGTTTGGAGCTAGGTTTCAGTTGTGCATGCCTCATACATATCTATATGTTTATCTTCAAATAGAACTTGCAAAAGTTGTGTGAGCTTAGCTGCACGGTTGAGTTTTAGTATCTTTTTCTTCCCTTATTTAAACTTCATGGTATTTTCAAATAAACCTACTTGTTGCTTTGGAAACCCATGCCAGTGCTCTAGAGCATATATATGTAGGTGGAGTGTATGCATTATTTTTGTTTGTGTTATTTGTCAGCAGTAGTGGTGTGGTATCTCTGTTTCTCTCTTTTCTACGTTCTTTTTTCCTCCTTTTTGTGCTGTATTAATTTTTCCCTTTCTAATGTTAGAGAAACTAGAAGGTCAGTGAAGAGGCTAAGTGTCAAAATTTGTCATGTAGTTCTAGCCACATATATGGTAATAATATATGTCGCTTTTCTTACTC

[0232] SEQ ID NO.5

[0233] ATGCTCATAATATGCTTCCACACTACTTTTTCATCAGACTTCTATCCAAGCTCAAGTTTAGTAGTTCCCACTTTTGTAGCACTTTGTTCAATATTCATTCAGAGTTTACATATATTTCATCTCATGGCAAACACAACAATGGTTACTCGAGTATTTATACTTGCACTAATCATCCTCTCCACCTTCTTCATGACTTTGCAGGCTCGTAATCTCCATGGACATCCCTCCATTCGCGAAAATAATGTTGCAGATAACAACCATCGCTTTCTTCACAATTATTTAGACCTATTAAAGCATATACATGTTGATCATCAAGATGCTGATGATGCCCCTCACAAGGATGGAAATACACATAGACTCACACCAGAAGGACCAGATCCTCATCATAATTTTGCAACACCACCAAGAAACTGASEQ ID NO.6

[0234] MLIICFHTTFSSDFYPSSSLVVPTFVALCSIFIQSLHIFHLMANTTMVTRVFILALI ILSTFFMTLQARNLHGHPSIRENNVADNNHRFLHNYLDLLKHIHVDHQDADDAPHKDGNTHRLTPEGPDPHHNFATPPRN*

[0235] SEQ ID NO.7

[0236]

[0237] SEQ ID NO.8

[0238] ATGGGAAATACAAGTGCAACCCTAGTGCCCATACTTGCCCTAATCATGTTCTCTACATTCTTCATGACTTTGCAAGCTCGTAGTCTCCATGGCCACAATCCATTATTCGCTCACAAAAAAGTTGTTGACATCCAAAACTTTCTCCACAAATCGGGTATTCACCTATCAAAGCGTGTGCGTATTCCATTTGGTGATGATCTCCCACTAGCACCTGCAGATAGACTTGCACCAGGAGGACCAGATCCTCAGCATAATGTGAGAGCTCCACCACGCAAGCCATAG

[0239] SEQ ID NO.9

[0240] MGNTSATLVPILALIMFSTFFMTLQARSLHGHNPLFAHKKVVDIQNFLHKSGIHLSKRVRIPFGDDLPLAPADRLAPGGPDPQHNVRAPPRKP

[0241] SEQ ID NO.10

[0242] TTGTGACTAATAACCCAAATCTAAAGGATACCAAAGAAGACTAGAATATGATGCAAATCACAGTTATAGAATTTCTCTCCTTTAGGAGTGCACAAGAAACCCATTGGGTAGGGGAGACTTTCTGGGTTAATTTTTTGTCCTAGGTTAAAAATAATGTATGTCTTCCAATTTATTGAATTAATCTCACTCACAATACATAACTAATTAAGAAATTTTCTAGTATGAAAACAACAAAATAAAGATATACAAATTAACGAGAAAAAATAAAGAAGATGAGAATATTATCAAGTATGTCAAAATTTTAAATTATTAAGTGAGATGTGTAATTATTTTAGATCACATTATTAATACGCCCCCCTATGCAAAAGAACTTCTTGGGACGACAGAAAGGTGGTAACCTAACCACATATAGATAGTGCTTATACCTTCCAAACTACTCCAACTCATTGATCATTCCACTAGCCATCTCAAGCTTATATATATAATCGTTCTCACTTTATAGCACCTTGTTCAATTCTAAGACTTACATCACGCATATGGGGAATACAAGTGCAGCCCTAGTGTCCATACTTGCCCTAATCATGTTCTCTACATTCTTCATGACTTTACAGGCTCGTAGTACTCTCCATGGTCATAATCCATTATTCGCTCACAAAAAAGTTGTTGCCATCGAAAACCTTCTCCACAAATCGGGTATTCACATATCAAAG

[0243] CATGTACATATTCCAATTGGTAATGATCTCCCACTAGCACCTGCAGATAGACTTGCACCAGGAGGACCTGA

[0244] TCCTCAGCATAATGTAAGAGCACCACCACGCAAGTCATAGTTTATTATGTGTGGAGATCATATATAGTGGC

[0245] TACATTCATGTATGCATGCATGCCTTGTTTTGTTTAGCTACAGTGGTGTGAGCTTAATTTTCAGGTGTGCA

[0246] TGCATGCAGTGGTACCTTCTCCCTAGCTAAGTTGTAAACTTCATAGTGTTCTCAAATTGAATAATGCTATT

[0247] TCTACCCACAATTTTCTACAATATTTTCTCACAACATTTTTTTTCTCTTTGTCTCTTCTCTTATTATTATT

[0248] ACTTTTCTTGTGTTAAACTTCCTTCTCTTCCCCAAAAAATATTGTGAGAGATTGTTGTGGAAATAGCATTA

[0249] CTCTTTCAAATAAAACCCTACATGCTTGGAAAATCTTTGTATACATGTTTTGGAGCCTATGTGGGTGATGT

[0250] AGGGTGGATAGTATATTGTTTCTGTTATTTTGTCTACGGTGATATACCAGTCCTGTTTGTCTTTATCTTTT

[0251] ATCTATGTTTCTTTTGTTTGTTATTTTATGTAACCTTGTTTACTGGAGGTGTTTTAGCAGATCAATGAAGA

[0252] CGAACCAGGGTTTTTTCTCTCTCTCGTAATTTATATTATTTTTAATCATATAACATAATATATAGCTTTAC

[0253] CTACTTATATGGCATAATTTTTTCTTGTCACAAATCAACTATGCCAAAAACTTAATTTCAATAAAAAGAAC

[0254] ATAAATAGTTTTTTATTAAATTTTTATTAATTTCAAAGGATAAAACTTAATTACATATAATTTTATTTAAG

[0255] TTTTTTTAGTATTGATTTTCAATTAAAATTGGAATTTCACCTCAATAATTTATGTTGACTTTTAATATAAA

[0256] TTTTTATTACACGGGTTATTAAGATAGAATTCTAACTATAATTGAAAAACGACACAAAAAATAATTATAAA

[0257] ACTAGTTTTATCCTTTTTTACACTCTTCTCAGTTTATTTCTTCGCCATAAAGGCAAAATTATTTCTACGAA

[0258] AGGTAGTTAAGGAGGGAACTTAATTCATAATTGTACTGATATTCCTCTCAAAACAATGGAAAAAAAGAATA

[0259] AATTAAGGCAAAGTTCGATTCAAGTTTTCACTTTAATTTCCCTTTCCAAAAGTATATTCAAAGAATATATA

[0260] TATATATATATATATATATATATATATATCGTGAACAAACTAAATTAAACTCACAAGAAACGTGTAACAAA

[0261] ACAAACTTACTAGAGACAATATGTAAACCTTCAAATATCTAGTAAACAAAATGTATAATCAGAGATTTTTT

[0262] TTAGCTCAGTTGGGTATAAGTAGAATACTGAGTTGTTATAAATCTTTTATACTTATTTTTTATTCCTACTG

[0263] ATAAAATAACAAAATGTATAATTTGTAGGATACAAATATAATAAGTTTTTAATGCGTATATTCATATTGAT

[0264] CAGTATTAACAATAATTAGCTGTTGGATGACAATCATGCAGAAAAAGGAAACAAAGAGTCGTTGGAAACAG

[0265] ATCCCTATGATTGTGCTACTGATAATGGAAAGCTGTTGGAAGACTAATCTGCTTTAAGTGGCACAAATCAA

[0266] TCTGGCAAATAAACCAGATTGTAGCTAACCATATTTAGGAAACTTATTCTATATTTAGAATTTCCTAATCT

[0267] GCAATCAATTCACATGTAATTACTTTATGCGGAATCATTGTACACTATGCTATATAATTATCCCTTGACTT

[0268] TCTTGTATCGATGCAGAGAGTTCACGTTTCAAAAAGGTTATACAATTTATCAGTTTTCTTCTTGGTATCTA

[0269] GATCCTTTGTGAACACCTTGCTACACCATGACTTCCT

[0270] SEQ ID NO.11

[0271] ATGGGGAATACAAGTGCAGCCCTAGTGTCCATACTTGCCCTAATCATGTTCTCTACATTCTTCATGACTTT

[0272] ACAGGCTCGTAGTACTCTCCATGGTCATAATCCATTATTCGCTCACAAAAAAGTTGTTGCCATCGAAAACC

[0273] TTCTCCACAAATCGGGTATTCACATATCAAAGCATGTACATATTCCAATTGGTAATGATCTCCCACTAGCA

[0274] CCTGCAGATAGACTTGCACCAGGAGGACCTGATCCTCAGCATAATAAAAAGGAAACAAAGAGTCGTTGGAA

[0275] ACAGATCCCTATGATTGTGCTACTGATAATGGAAAGCTGTTGGAAGACTAATCTGCTTTAASEQ IDNO.12

[0276] MGNTSAALVSILALIMFSTFFMTLQARSTLHGHNPLFAHKKVVAIENLLHKSGIHISKHVHIPIGNDLPLAPADRLAPGGPDPQHNKKETKSRWKQIPMIVLLIMESCWKTNLL*

[0277] SEQ ID NO.13

[0278] CTAAGCTTGAAGCCTTGGGGGAAATGGCCAACGCAAAGCAAGTACTATGCCTAATTTTGTTGGTGCTTTTA

[0279] TTTTCCAAGCTTGAAAGTCGTTCTCTTGAGGCATTCATAGAGGGAAAGAAGACCCTAGCCAAAGGTTGTTC

[0280] ACGAGAATTGATTGAAAAATCACAGCTCCTGAAGGCAAGTTTTGCAAAAGCAACAACACGTTTTACAAATC

[0281] CTAGAGTGTCCAAAAGACTAAGCCCTGGAGGACCTGATCAAAAACATCACTAATTCACTTTTCTAGTTTTC

[0282] TTTTAACGTTACATTATTATTTACACACTATTTGTATGCTCTGGTCATTGAAATGTAAATAACTAAGAATC

[0283] AGTAGATACCATACGCACAATATCACCAATCAGGCAATTACCACGTATGCTGATTATCAGTTAATTACATA

[0284] TTAATTATCAGTGTGTGTGTAAAATAGTGTGGGAATATATAGTGTAAACGCGTTAAAGAGATCACTCGATA

[0285] AATAGTACAGAAAATGTTATTTTGTGCATATTTTTAAACCGGTCGATGTGTTAATAACTTGTAATTAGCGT

[0286] GTACTATCTATGCAGACATGTGATCATTAATTATTCACATTATGCATTTACACGTTGATCATTAATTATTT

[0287] ACACATCGATCAATAATAAAGAAACGTGCACAAAATATATAGCATATATGTACATTTTTTGTAGAACGTGT

[0288] TTCTCGTAAATTTTGTGAAAGGGTCTAAATAAAATAGCGTGGTAAAGATGTTCAAGAGTTATGACGTACTT

[0289] GCTGTTTATGTGTGTTGTAGCATGGTTTGGGCAGTACGAATATTGTATTGAGTTGTTTGTATGCGGTTACA

[0290] AATTTCAATTGTTAAACTACTACTGTATTAAAAGAA

[0291] SEQ ID NO.14

[0292] ATGGCCAACGCAAAGCAAGTACTATGCCTAATTTTGTTGGTGCTTTTATTTTCCAAGCTTGAAAGTCGTTC

[0293] TCTTGAGGCATTCATAGAGGGAAAGAAGACCCTAGCCAAAGGTTGTTCACGAGAATTGATTGAAAAATCAC

[0294] AGCTCCTGAAGGCAAGTTTTGCAAAAGCAACAACACGTTTTACAAATCCTAGAGTGTCCAAAAGACTAAGC

[0295] CCTGGAGGACCTGATCAAAAACATCACTAA

[0296] SEQ ID NO.15

[0297] MANAKQVLCLILLVLLFSKLESRSLEAFIEGKKTLAKGCSRELIEKSQLLKASFAKATTRFTNPRVSKRLSPGGPDQKHH*

[0298] SEQ ID NO.16

[0299] TGGGTATATTCATTTGGAATCACGCAACTAAGCTTGAGGCCTTGGGGAAAATGGCCAACGCAAAGCAAGTA

[0300] CTATGCCTAATTCTGTTGGTGCTTTTATTTGCCAAGTTTGAAAGTCGTTCACTTGAGGCATTCATAGAGAG

[0301] GAAGAAGACCCCACCCAAAGGTTGTTCACGTCAATTGATTGAAAAATCACAGCTCCTCAAGGCAAGTTTTG

[0302] CAAAAGCAACTCCTTTTACAAATCCTATCATCTCCAAAAGACTAAGCCCTGGAGGGCCTGATCACAAACAT

[0303] CACTAAGTCACTTTTCTAGTTTTCTTTTAACGTTACATTATTTACACACTATTTGTTTGTGGATATAGTAT

[0304] AGAAAATGTTATTTTGTGCATATTTTTAAACCGGTCGATGTGTTAATAACTTGTAATTAACGTGTACTATC

[0305] TATACAGACATGTGATCACTTTTCACATTATGCATTTACACGTTGATCATTAATTATTTACACATCGATCA

[0306] ATAGTAATAAAGCAACGTGCACAAAATATATAGTATGTTCATTTTTGTAGAACGTGTTCCCCGTAAATTTT

[0307] GTGAAAGGGTCTTAATAAAATTGCGTGGTGGTAAAGATGATCTATTGATGTAAGACTTTTTTTTTTAGGGA

[0308] GATGTTCAAGAGTTATGACGTAGTTATTGCTGTTTAAGAGTTGTAGCATGGTTTGGGCGGTACGAATGGTA

[0309] TTGAGTTGTTTGTATGCGGTTACAGATTTAAATTGTTAAACTACTACTGTATTAAAAGAAATCAGTGTCTA

[0310] TGTTTTTTTTTAAAAAAAATTTAATTCCCCTCTCACCCTTTTGCTTATCCACTTTTATAGTCTAACCAAAG

[0311] ATATCCTGTTTTAACCTACGTACTAATAAACTTTTAAAAAAAATATTATCTAAGAAGTAAATGGGATAAGT

[0312] ATCTAACATTTTTGAAATCTTTTGGGACCACTTCTCCAGGATTAATTAGTTTAGATAAGCCTTATTCTGAA

[0313] CCACAACTGTATGGTATAATCAATTTTGGGAGAATCCGTACACTGATCATATATATAAAACAATAAGTCCA

[0314] GAAAGCTAGATTGGTCTTGCAAAATATTAATAATATTGACAATGACTATTGTTGATCCTTAAGGTGAGGAA

[0315] GAGACAATACTGATGTCATGCTTTCAGACTGATCTACAAGGTCCTTAACCTTAATTAAGCCATGTCTCTTA

[0316] AGTGCTTATAGCTAAGTGTATAGTCAGCATTTACCTCATGGAAGTTGATGGTGTTATTTCTTGATTCGAGA

[0317] TAAATCTGGACGTACGGGACATTTAGAGTTGGACGTACATAACAAGGGTCGATCCCCTTGGGTCAGTGAGT

[0318] CAGTTTTTGGGTATATATATCTTGAAAGTGTTAATGTTGGGATCTTCCTAATTAAGTGATCGATAAGCTGC

[0319] AGGGATCTTTCTCTAATGTATGTACACGTTTGTATATATCTTGCTTGGAAGTTATGCTATTCGCTASEQID NO.17

[0320] ATGGCCAACGCAAAGCAAGTACTATGCCTAATTCTGTTGGTGCTTTTATTTGCCAAGTTTGAAAGTCGTTC

[0321] ACTTGAGGCATTCATAGAGAGGAAGAAGACCCCACCCAAAGGTTGTTCACGTCAATTGATTGAAAAATCAC

[0322] AGCTCCTCAAGGCAAGTTTTGCAAAAGCAACTCCTTTTACAAATCCTATCATCTCCAAAAGACTAAGCCCT

[0323] GGAGGGCCTGATCACAAACATCACTAA

[0324] SEQ ID NO.18

[0325] MANAKQVLCLILLVLLFAKFESRSLEAFIERKKTPPKGCSRQLIEKSQLLKASFAKATPFTNPIISKRLSPGGPDHKHH*

[0326] SEQ ID NO:19 sgRNA scaffold

[0327] guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgaguc

[0328] ggugcuuuuuuu

Claims

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

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

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

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

11. The method of claim 9 or 10, wherein the method comprises: reducing or eliminating the activity and / or level of two endogenous polypeptides selected from 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 Glycine max plant.

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