Application of Gm-1i-Q23 protein and Gm-1i-Q63 protein in regulation and control of symbiotic nitrogen fixation capacity and yield of plants

Abstract

The invention discloses an application of a Gm-1i-Q63 protein and a Gm-1i-Q23 protein in regulation and control of symbiotic nitrogen fixation capability and yield of plants. Statistics of symbiotic nitrogen fixation and yield related traits of Gm-1i-Q63 / Gm-1i-Q23 gene edited double mutants show that knockout of the Gm-1i-Q63 and Gm-1i-Q23 genes can significantly increase the nodule number and the root nodule dry weight, and significantly increase the plant height, the single plant grain number, the single plant grain weight, the hundred-grain weight and the protein content. Therefore, the Gm-1i-Q63 protein and the Gm-1i-Q23 protein can prolong the nodulation and nitrogen fixation time by increasing the nodulation number and the root nodule weight, so that the nitrogen fixation efficiency is improved, and the yield is improved. The invention provides a gene resource for obtaining a new soybean variety with high nitrogen fixation capacity or high yield, and has a good application prospect in the aspects of research on an efficient nitrogen fixation mechanism of leguminous crops and improvement of soybean productivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to the application of Gm-1i-Q23 protein and Gm-1i-Q63 protein in regulating the symbiotic nitrogen fixation ability and yield of plants. Background Art

[0002] Symbiotic nitrogen fixation between legumes and rhizobia is an important natural nitrogen source in agricultural ecosystems and is the most economical and environmentally friendly way of nitrogen fixation. Soybeans are the most important oil crops and livestock feed raw materials in the world, and are also excellent rotation and intercropping crops in agricultural production. Soybeans require a large amount of nitrogen source during cultivation. In the case of not excessive nitrogen fertilizer application, it is estimated that 58-68% of the nitrogen in soybeans comes from symbiotic nitrogen fixation. After harvest, 30-60% of the nitrogen in the roots and nodules of the plants underground can be replenished in the surrounding soil. Therefore, giving full play to the symbiotic nitrogen fixation of soybeans is of great significance to weight loss and efficiency and the sustainable development of green agriculture. In addition, soybean symbiotic nitrogen fixation is also an energy-intensive enzyme-catalyzed process. The carbohydrates fixed by the photosynthesis of the plant itself are the main carbon source and energy source of symbiotic nitrogen fixation. Cultivating new soybean varieties that can prolong the nodulation and nitrogen fixation time can provide a theoretical basis and material basis for increasing soybean yield and the application of symbiotic nitrogen fixation in agricultural weight loss and efficiency, and is an effective way to breed high-efficiency nitrogen-fixing and high-yield organisms. Summary of the invention

[0003] The technical problem to be solved by the present invention is how to regulate the symbiotic nitrogen fixation ability of plants, prolong the nodulation and nitrogen fixation time of plants, and increase plant yield. The technical problem to be solved is not limited to the technical subject matter described, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0004] In order to solve the above technical problems, the present invention first provides a new use of protein or a substance for regulating the content and / or activity of the protein.

[0005] The present invention provides the use of a protein or a substance for regulating the content and / or activity of the protein in any of the following A1)-A6):

[0006] A1) Regulate the symbiotic nitrogen fixation ability of plants;

[0007] A2) regulating plant yield;

[0008] A3) regulating plant height;

[0009] A4) regulating the content of plant protein;

[0010] A5) cultivating transgenic plants with altered symbiotic nitrogen fixation ability and / or yield and / or plant height and / or protein content;

[0011] A6) Plant breeding;

[0012] The proteins are Gm-1i-Q63 protein and Gm-1i-Q23 protein;

[0013] The Gm-1i-Q63 protein is any one of the following B1)-B4):

[0014] B1) The amino acid sequence is the protein shown in SEQ ID NO: 3;

[0015] B2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 3;

[0016] B3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 3;

[0017] B4) a protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 3 and having the same function;

[0018] The Gm-1i-Q23 protein is any one of the following C1)-C4):

[0019] C1) The amino acid sequence is the protein shown in SEQ ID NO: 6;

[0020] C2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 6;

[0021] C3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 6;

[0022] C4) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 6 and having the same function.

[0023] In the protein described in B2) or C2) above, the tag refers to a polypeptide or protein that is fused and expressed together with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0024] In the protein described in B3) or C3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is substitution and / or deletion and / or addition of no more than 10 amino acid residues, or substitution and / or deletion and / or addition of no more than 9 amino acid residues, or substitution and / or deletion and / or addition of no more than 8 amino acid residues, or substitution and / or deletion and / or addition of no more than 7 amino acid residues, or substitution and / or deletion and / or addition of no more than 6 amino acid residues, or substitution and / or deletion and / or addition of no more than 5 amino acid residues, or substitution and / or deletion and / or addition of no more than 4 amino acid residues, or substitution and / or deletion and / or addition of no more than 3 amino acid residues, or substitution and / or deletion and / or addition of no more than 2 amino acid residues, or substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0025] In the protein described in B4) or C4), the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gapexistence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be obtained. The identity includes amino acid sequences having 80% or higher, or 85% or higher, or 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity with the amino acid sequence shown in Sequence 3 or Sequence 6 of the present invention.

[0026] The protein described in B1) or B2) or B3) or B4) or C1) or C2) or C3) or C4) can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0027] In the above application, the substance that regulates the protein content and / or activity includes a substance that regulates the Gm-1i-Q63 protein content and / or activity and a substance that regulates the Gm-1i-Q23 protein content and / or activity.

[0028] Among them, the substances that regulate the content and / or activity of the Gm-1i-Q63 protein include substances that increase the content and / or activity of the Gm-1i-Q63 protein and substances that reduce the content and / or activity of the Gm-1i-Q63 protein.

[0029] Substances that regulate the content and / or activity of the Gm-1i-Q23 protein include substances that increase the content and / or activity of the Gm-1i-Q23 protein and substances that decrease the content and / or activity of the Gm-1i-Q23 protein.

[0030] Further, the substance that increases the activity of Gm-1i-Q63 protein may be a protein, polypeptide or small molecule compound that enhances or promotes the function of Gm-1i-Q63 protein. The substance that increases the content of Gm-1i-Q63 protein may be a substance that promotes the synthesis of Gm-1i-Q63 protein or inhibits the degradation of Gm-1i-Q63 protein or overexpresses Gm-1i-Q63 protein.

[0031] The substance that reduces the activity of Gm-1i-Q63 protein may be a protein, polypeptide or small molecule compound that inhibits the function of Gm-1i-Q63 protein. The substance that reduces the content of Gm-1i-Q63 protein may be a substance that inhibits the synthesis of Gm-1i-Q63 protein or promotes the degradation of Gm-1i-Q63 protein or knocks down (knock-down) or knocks out the gene encoding Gm-1i-Q63 protein.

[0032] The substance that increases the activity of the Gm-1i-Q23 protein may be a protein, polypeptide or small molecule compound that enhances or promotes the function of the Gm-1i-Q23 protein. The substance that increases the content of the Gm-1i-Q23 protein may be a substance that promotes the synthesis of the Gm-1i-Q23 protein or inhibits the degradation of the Gm-1i-Q23 protein or overexpresses the Gm-1i-Q23 protein.

[0033] The substance that reduces the activity of the Gm-1i-Q23 protein may be a protein, polypeptide or small molecule compound that inhibits the function of the Gm-1i-Q23 protein. The substance that reduces the content of the Gm-1i-Q23 protein may be a substance that inhibits the synthesis of the Gm-1i-Q23 protein or promotes the degradation of the Gm-1i-Q23 protein or knocks down (knocks down) or knocks out the Gm-1i-Q23 protein encoding gene.

[0034] Furthermore, the substance that knocks down (knocks down) the Gm-1i-Q63 protein encoding gene can be any nucleic acid molecule that can inhibit the expression of the above-mentioned Gm-1i-Q63 protein encoding gene, such as gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0035] The substance for knocking out the Gm-1i-Q63 protein encoding gene can be a substance that achieves the host cell not producing the functional protein product of the Gm-1i-Q63 gene in any way, such as removing all or part of the encoding gene sequence, introducing mutations so that no functional protein is produced, removing or changing regulatory components (such as promoter editing) so that the encoding gene sequence is not transcribed, and preventing translation by binding to mRNA, etc. Usually, the knockout is performed at the genomic DNA level, so that the offspring of the cell also permanently carry the knockout.

[0036] The substance for knocking down (knocking down) the Gm-1i-Q23 protein encoding gene can be any nucleic acid molecule that can inhibit the expression of the above-mentioned Gm-1i-Q23 protein encoding gene, such as gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA, antisense RNA, etc.

[0037] The substance for knocking out the Gm-1i-Q23 protein encoding gene can be a substance that achieves the host cell not producing the functional protein product of the Gm-1i-Q23 gene in any way, such as removing all or part of the encoding gene sequence, introducing mutations so that no functional protein is produced, removing or changing regulatory components (such as promoter editing) so that the encoding gene sequence is not transcribed, and preventing translation by binding to mRNA, etc. Usually, the knockout is performed at the genomic DNA level, so that the offspring of the cell also permanently carry the knockout.

[0038] In some specific embodiments, the material for knocking out the Gm-1i-Q63 protein-coding gene and the material for knocking out the Gm-1i-Q23 protein-coding gene are CRISPR / Cas9 vectors for knocking out the Gm-1i-Q63 protein-coding gene and the Gm-1i-Q23 protein-coding gene; the CRISPR / Cas9 vector expresses sgRNA and Cas9 protein targeting the Gm-1i-Q63 protein-coding gene and the Gm-1i-Q23 protein-coding gene.

[0039] In some preferred embodiments, the target sequence of the sgRNA includes the DNA molecule described in sequence 7, the DNA molecule described in sequence 8, and the DNA molecule shown in sequence 9.

[0040] In order to solve the above technical problems, the present invention also provides new uses of biomaterials related to the above Gm-1i-Q63 protein and biomaterials related to the above Gm-1i-Q23 protein.

[0041] The present invention provides the use of the biomaterial related to the above-mentioned Gm-1i-Q63 protein and the biomaterial related to the above-mentioned Gm-1i-Q23 protein in any one of the following A1)-A6):

[0042] A1) Regulate the symbiotic nitrogen fixation ability of plants;

[0043] A2) regulating plant yield;

[0044] A3) regulating plant height;

[0045] A4) regulating the content of plant protein;

[0046] A5) cultivating transgenic plants with altered symbiotic nitrogen fixation ability and / or yield and / or plant height and / or protein content;

[0047] A6) Plant breeding;

[0048] The biological material related to the Gm-1i-Q63 protein is any one of the following D1) to D5):

[0049] D1) a nucleic acid molecule encoding the Gm-1i-Q63 protein;

[0050] D2) knocking down or knocking out a nucleic acid molecule of the Gm-1i-Q63 protein encoding gene;

[0051] D3) an expression cassette containing the nucleic acid molecule described in D1) or D2);

[0052] D4) a recombinant vector containing the nucleic acid molecule described in D1) or D2);

[0053] D5) A recombinant microorganism containing the nucleic acid molecule described in D1) or D2);

[0054] The biological material related to the Gm-1i-Q23 protein is any one of the following E1) to E5):

[0055] E1) a nucleic acid molecule encoding the Gm-1i-Q23 protein;

[0056] E2) knocking down or knocking out a nucleic acid molecule of the Gm-1i-Q23 protein encoding gene;

[0057] E3) an expression cassette containing the nucleic acid molecule described in E1) or E2);

[0058] E4) a recombinant vector containing the nucleic acid molecule described in E1) or E2);

[0059] E5) A recombinant microorganism containing the nucleic acid molecule described in E1) or E2).

[0060] In the above application, the nucleic acid molecule in D1) is any one of the following:

[0061] F1) The DNA molecule shown in Sequence 1 or Sequence 2;

[0062] F2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in F1) and encodes the Gm-1i-Q63 protein.

[0063] E1) The nucleic acid molecule is any one of the following:

[0064] G1) The DNA molecule shown in Sequence 4 or Sequence 5;

[0065] G2) is a DNA molecule that has 75% or more identity with the nucleotide sequence defined in G1) and encodes the Gm-1i-Q23 protein.

[0066] A person skilled in the art can easily mutate the nucleotide sequence encoding the Gm-1i-Q63 protein or Gm-1i-Q23 protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the Gm-1i-Q63 or Gm-1i-Q23 nucleotide sequence isolated from the present invention, as long as they encode the Gm-1i-Q63 protein or Gm-1i-Q23 protein and have the same function, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention. The identity refers to the sequence similarity with the natural nucleic acid sequence, including nucleotide sequences having 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence of the protein composed of the amino acid sequence shown in Sequence 3 or Sequence 6 of the present invention. The identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0067] The nucleic acid molecule described in D2) or E2) above may be gRNA (such as sgRNA), mRNA, siRNA, dsRNA, shRNA, miRNA or antisense RNA.

[0068] Any of the above-mentioned nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA.

[0069] Any of the above-mentioned nucleic acid molecules can be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0070] Any of the above-mentioned expression cassettes may include a promoter, the above-mentioned nucleic acid molecule D1) or D2) or E1) or E2) and a terminator. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development-specific promoters and inducible promoters. Further, the expression cassette may also include an enhancer sequence.

[0071] Any of the above-mentioned vectors refers to a vector capable of carrying the nucleic acid molecule described in D1) or D2) or E1) or E2) into a host cell for amplification and expression. The vector may be a cloning vector or an expression vector, including but not limited to: a plasmid, a phage (such as lambda phage or M13 filamentous phage, etc.), a cosmid (i.e., a cosmid), a Ti plasmid, a viral vector (such as a retrovirus (including a lentivirus), adenovirus, adeno-associated virus, etc.).

[0072] Any of the above-mentioned recombinant vectors refers to a recombinant DNA molecule constructed by connecting the nucleic acid molecule described in D1) or D2) or E1) or E2) with the vector in vitro. The recombinant vector containing the nucleic acid molecule described in E1) or E2) can be constructed using an existing plant expression vector. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector may also contain the 3'-end non-translated region of the exogenous gene, i.e., a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the non-translated region transcribed at the 3' end of the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos) and the plant gene (such as the soybean storage protein gene) all have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation start region can come from the transcription start region or the structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene that confers resistance to kanamycin and related antibiotics, bar gene that confers resistance to the herbicide phosphinothricin, hph gene that confers resistance to the antibiotic hygromycin, dhfr gene that confers resistance to methotrexate, EPSPS gene that confers resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and transformed plants can be directly screened by adversity.

[0073] Any of the above-mentioned microorganisms may be bacteria, fungi, actinomycetes, protozoa, algae or viruses. Wherein, the bacteria may be from Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but not limited thereto, for example, the bacteria may be Escherichia coli, Bacillus subtilis or Bacillus pumilus. The fungus may be yeast, and the yeast may be from the genus Saccharomyces (such as Saccharomyces cerevisiae), the genus Kluyveromyces (such as Kluyveromyces lactis), the genus Pichia (such as Pichia pastoris), the genus Schizosaccharomyces (such as Schizosaccharomyces pombe), the genus Hansenula (such as Hansenula polymorpha), etc., but not limited thereto. The fungus may also be from the genus Fusarium (Fusarium sp.), the genus Rhizoctonia (Rhizoctonia sp.), the genus Verticillium (Verticillium sp.), the genus Penicillium (Penicillium sp.), the genus Aspergillus (Aspergillus sp.), the genus Cephalosporium (Cephalosporium sp.), etc., but not limited thereto. The actinomycetes may be from Streptomyces sp., Nocardia sp., Micromonospora sp., Streptosporangium sp., Actinoplanes sp., Thermoactinomyces sp., etc., but not limited thereto. The algae may be from Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., Boekelovia sp., etc., but not limited thereto. The virus may be rotavirus, herpes virus, influenza virus, adenovirus, etc., but not limited thereto.

[0074] Any of the above-mentioned recombinant microorganisms refers to a recombinant microorganism with a changed function obtained by operating and modifying the genes of the target microorganism. For example, a recombinant microorganism obtained by introducing the above-mentioned recombinant vector into the target microorganism. The recombinant microorganism can be understood to refer not only to a specific recombinant microorganism, but also to the offspring of such a cell, and due to natural, accidental or intentional mutations and / or changes, the offspring may not be completely identical to the original parent cell, but is still included in the scope of the recombinant microorganism.

[0075] In any of the above applications, the regulation of the symbiotic nitrogen fixation ability of the plant is embodied in any one of the following M1)-M4):

[0076] M1) Regulate the number of plant nodules;

[0077] M2) regulates plant nodule dry weight;

[0078] M3) regulating the time of plant nodulation and / or nitrogen fixation;

[0079] M4) regulates the efficiency of plant nitrogen fixation.

[0080] The regulation is negative regulation, that is, when the content and / or activity of Gm-1i-Q63 protein and Gm-1i-Q23 protein in the plant increases, the symbiotic nitrogen fixation ability of the plant decreases; when the content and / or activity of Gm-1i-Q63 protein and Gm-1i-Q23 protein in the plant decreases or is absent, the symbiotic nitrogen fixation ability of the plant increases.

[0081] In some embodiments, when the genes encoding Gm-1i-Q63 protein and Gm-1i-Q23 protein are knocked out in a plant, the number of nodules in the plant increases, the dry weight of nodules increases, the nodulation time is prolonged, the nitrogen fixation time is prolonged, and the nitrogen fixation efficiency is improved.

[0082] In any of the above applications, the regulation of plant yield is embodied in any of the following N1)-N3):

[0083] N1) Regulate the number of seeds per plant;

[0084] N2) regulates the grain weight per plant;

[0085] N3) regulates the 100-grain weight of plants.

[0086] The regulation is negative regulation, that is, when the content and / or activity of Gm-1i-Q63 protein and Gm-1i-Q23 protein in the plant is increased, the yield of the plant is reduced; when the content and / or activity of Gm-1i-Q63 protein and Gm-1i-Q23 protein in the plant is reduced or absent, the yield of the plant is increased.

[0087] In some embodiments, when the genes encoding Gm-1i-Q63 protein and Gm-1i-Q23 protein are knocked out in a plant, the number of grains per plant, the grain weight per plant, and the 100-grain weight of the plant are increased.

[0088] In any of the above applications, regulating the plant height is to increase the plant height or to reduce the plant height.

[0089] The regulation is negative regulation, that is, when the content and / or activity of Gm-1i-Q63 protein and Gm-1i-Q23 protein in the plant is increased, the plant height is reduced; when the content and / or activity of Gm-1i-Q63 protein and Gm-1i-Q23 protein in the plant is reduced or absent, the plant height is increased.

[0090] In some embodiments, when the genes encoding Gm-1i-Q63 protein and Gm-1i-Q23 protein are knocked out in a plant, the plant height is increased.

[0091] In any of the above applications, regulating the plant protein content is to increase the plant protein content or reduce the plant protein content.

[0092] The regulation is negative regulation, that is, when the content and / or activity of Gm-1i-Q63 protein and Gm-1i-Q23 protein in the plant is increased, the protein content in the plant grain is reduced; when the content and / or activity of Gm-1i-Q63 protein and Gm-1i-Q23 protein in the plant is reduced or absent, the protein content in the plant grain is increased.

[0093] In some embodiments, when the genes encoding Gm-1i-Q63 protein and Gm-1i-Q23 protein are knocked out in a plant, the protein content in the grain of the plant is increased.

[0094] In any of the above applications, the purpose of the breeding is to cultivate plant varieties with high nitrogen fixation ability or high yield plant varieties.

[0095] In order to solve the above technical problems, the present invention also provides a method for cultivating transgenic plants with improved symbiotic nitrogen fixation ability and / or improved yield and / or increased plant height and / or increased protein content.

[0096] The method for cultivating transgenic plants with improved symbiotic nitrogen fixation ability and / or improved yield and / or increased plant height and / or increased protein content provided by the present invention comprises the following steps: reducing the content and / or activity of Gm-1i-Q63 protein and Gm-1i-Q23 protein in the target plant to obtain a transgenic plant; the symbiotic nitrogen fixation ability and / or yield and / or plant height and / or protein content of the transgenic plant are higher than those of the target plant.

[0097] In the above method, the method for reducing the content and / or activity of Gm-1i-Q63 protein and Gm-1i-Q23 protein in the target plant is to introduce the above-mentioned substance for knocking out the Gm-1i-Q63 protein encoding gene and the above-mentioned substance for knocking out the Gm-1i-Q23 protein encoding gene into the target plant.

[0098] In the above method, the symbiotic nitrogen fixation ability of the transgenic plant is higher than that of the target plant, which is embodied in any one of the following X1)-X4):

[0099] X1) after inoculation with rhizobia, the number of nodules of the transgenic plant is higher than that of the target plant;

[0100] X2) after inoculation with rhizobia, the dry weight of nodules of the transgenic plant is higher than that of the target plant;

[0101] X3) after inoculation with rhizobia, the nodulation time and / or nitrogen fixation time of the transgenic plant are longer than those of the target plant;

[0102] X4) After inoculation with rhizobia, the nitrogen fixation efficiency of the transgenic plant is higher than that of the target plant.

[0103] Furthermore, the rhizobium is rhizobium USDA110.

[0104] The yield of the transgenic plant is higher than that of the target plant, which is embodied in any one of the following Y1)-Y3):

[0105] Y1) the number of grains per plant of the transgenic plant is higher than that of the target plant;

[0106] Y2) the grain weight per plant of the transgenic plant is higher than that of the target plant;

[0107] Y3) The 100-grain weight of the transgenic plant is higher than that of the target plant.

[0108] In any of the above-mentioned uses or methods, the plant is any one of the following Z1)-Z4):

[0109] Z1) dicotyledonous plants or monocotyledonous plants;

[0110] Z2) Leguminosae;

[0111] Z3) soybean plants;

[0112] Z4) Soybean (such as Tianlong No. 1).

[0113] The present invention constructs a gm-1i-q63 / gm-1i-q23 gene editing double mutant based on CRISPR / Cas9 gene editing technology, and statistically analyzes the symbiotic nitrogen fixation and yield-related traits of the gm-1i-q63 / gm-1i-q23 gene editing double mutant, and finds that knocking out the Gm-1i-Q63 and Gm-1i-Q23 genes in soybeans can significantly increase the number of soybean nodules and nodule dry weight, prolong the nodulation and nitrogen fixation time of soybeans, especially in the late development period, the newly formed vigorous nodules on the root system are significantly increased, and the plant height, the number of grains per plant, the grain weight per plant, the 100-grain weight, and the protein content of the plant are significantly increased. It is shown that Gm-1i-Q63 protein and Gm-1i-Q23 protein can increase the number of nodules and the weight of nodules, prolong the nodulation and nitrogen fixation time, and then improve the nitrogen fixation efficiency, thereby achieving an increase in yield. The present invention provides genetic resources for obtaining new soybean varieties with high nitrogen fixation ability or high yield, which is not only of great significance in agriculture and ecology, but also has good application prospects in the study of efficient nitrogen fixation mechanism of leguminous crops and in agricultural production and soybean production capacity improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1 Construct a map for the pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 vector.

[0115] Figure 2 PCR was used to detect the expression of gRNA, Cas9 and Bar genes in soybeans transformed with pYLCRISPR / Cas9P35S-B-Gm-1i-Q63 / 1i-Q23.

[0116] Figure 3 Editing type detection for gm-1i-q63 / gm-1i-q23 gene editing double mutant.

[0117] Figure 4 Off-target site analysis for gm-1i-q63 / gm-1i-q23 gene-edited double mutants.

[0118] Figure 5 This is a comparison of the growth conditions of the gm-1i-q63 / gm-1i-q23 gene-edited double mutant and the control plants 23 days and 45 days after inoculation.

[0119] Figure 6 Comparison of root nodulation phenotypes between gm-1i-q63 / gm-1i-q23 gene-edited double mutants and control plants 23 and 45 days after inoculation. Red circles represent newly formed nodules.

[0120] Figure 7Schematic diagram of the statistical analysis and comparison of plant height, root nodule number and nodule dry weight of the gm-1i-q63 / gm-1i-q23 gene-edited double mutant and the control plants 23 days and 45 days after inoculation.

[0121] Figure 8 Schematic diagram of the phenotypic comparison between the gm-1i-q63 / gm-1i-q23 gene-edited double mutant and the control plants in the field.

[0122] Fig. 9 This is a schematic diagram of the statistical analysis and comparison of plant height, number of grains per plant, grain weight per plant, 100-grain weight, protein content, etc. between the gm-1i-q63 / gm-1i-q23 gene-edited double mutant and the control plants. DETAILED DESCRIPTION

[0123] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0124] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0125] The culture medium and reagent formulas involved in the following examples are as follows:

[0126] MS medium: MS basal salt mixture (Sigma) 4.3 g, MS vitamin powder (Sigma) 0.103 g, 0.7-0.8% (mass volume ratio) agar powder, ddH2O to 1000 mL, adjusted to pH 5.8.

[0127] YMA medium: mannitol (or sucrose) 10.0 g, Yeast Extract 0.4 g, K2HPO4 0.5 g, MgSO4·7H2O 0.2 g, CaCl2·6H2O 0.1 g, NaCl 0.1 g, Rh trace element solution 4 mL, dilute to 1000 mL with ddH2O, adjust to pH 6.8-7.0, and sterilize at 115°C for 20 min.

[0128] Rh trace element solution: H3BO3 5.0g, Na2MoO4 5.0g, ddH2O, make up to 1000mL.

[0129] Fahraeus nitrogen-free nutrient solution: CaCl2·2H2O 0.10g, MgSO4·7H2O 0.12g, KH2PO4 0.10g, Na2HPO4·12H2O 0.15g, Gibson trace element solution 1mL, ferric citrate 5mg, and ddH2O to make up to 1000mL.

[0130] Gibson trace element solution: H3BO3 2.86g, ZnSO4·7H2O 0.22g, MnSO4·4H2O 2.03g, Na2MoO4·2H2O 0.13g, CuSO4·5H2O 0.08g, make up to 1000mL with ddH2O.

[0131] The soybean variety Tianlong No. 1 (hereinafter referred to as Tianlong No. 1) in the following embodiments is recorded in the document "Wang Ruizhen, Yang Zhonglu, Zhou Xin'an, Zhao Xianwei, Zhao Chaosen, Xiong Wenhua, Peng Yang. Introduction and promotion and application of high-yield and high-quality spring soybean Tianlong No. 1. Soybean Technology. Issue 05, 2015." The public can obtain it from the applicant. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0132] The gene editing vector pYLCRISPR / Cas9P35S-B in the following embodiment is recorded in the document “Zhang YX, Guo W, Chen LM et al. CRISPR / Cas9-Mediated Targeted Mutagenesis of GmUGT Enhanced Soybean Resistance Against Leaf-Chewing Insects Through Flavonoids Biosynthesis.” and is available to the public from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0133] The gRNA vector in the following embodiments is recorded in the document “Zhang YX, Guo W, Chen LM et al. CRISPR / Cas9-Mediated Targeted Mutagenesis of GmUGT Enhanced Soybean Resistance Against Leaf-Chewing Insects Through Flavonoids Biosynthesis.”, which is available to the public from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0134] The Agrobacterium tumefaciens EHA105 in the following examples is recorded in the document "Duan X, Zheng L et al. Co-culturing on dry filter paper significantly increased the efficiency of Agrobacterium-mediated transformations of maize immature embryos. Physiol Mol Biol Plants. 2019, 25(2): 549-560", which is available to the public from the applicant. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0135] The soybean rhizobium USDA110 in the following embodiment is recorded in the document "Quentin N, Florian L et al. Bradyrhizobium diazoefficiens USDA110 nodulation of Aeschynomene afrasperais associated with a typical terminal bacteroid differentiation and suboptimal symbiotic efficiency. mSystems. 2021, 6(3): e01237-20", which is available to the public from the applicant. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0136] The CDS sequence of Gm-1i-Q63 in the following examples is shown in Sequence 1, the genome sequence of Gm-1i-Q63 is shown in Sequence 2, and the amino acid sequence of Gm-1i-Q63 protein is shown in Sequence 3.

[0137] The CDS sequence of Gm-1i-Q23 in the following examples is shown in Sequence 4, the genome sequence of Gm-1i-Q23 is shown in Sequence 5, and the amino acid sequence of Gm-1i-Q23 protein is shown in Sequence 6.

[0138] Example 1. Preparation of gm-1i-q63 / gm-1i-q23 gene-edited double mutant material

[0139] In this example, CRISPR / Cas9 technology was used to knock out the Gm-1i-Q63 and Gm-1i-Q23 genes in wild-type soybeans to obtain gm-1i-q63 / gm-1i-q23 gene-edited double mutant materials. The specific steps are as follows:

[0140] 1. Construction of pYLCRISPR / Cas9P35S-B-Gm-1i-Q63 / 1i-Q23 knockout vector and recombinant Agrobacterium

[0141] 1. Design of target sequence

[0142] Referring to the gene sequences of Gm-1i-Q63 and Gm-1i-Q23 in the soybean genome database, three target sites were set, which were recorded as T1, T2 and T3. The target site sequences are as follows:

[0143] T1: 5′-TTCTCAAATGCACAGGCCCT-3′ (SEQ ID NO: 7);

[0144] T2: 5′-TGGAGCTCAGATGATCAGGC-3′ (SEQ ID NO: 8);

[0145] T3: 5'-TGGAGCCCAGATAATCAAGG-3' (sequence 9).

[0146] Among them, target T1 is shared by both genes, target T3 is specific to the Gm-1i-Q63 gene, and target T2 is specific to the Gm-1i-Q23 gene.

[0147] 2. Preparation of target gRNA expression cassette

[0148] Anneal the adapter primers corresponding to each target to obtain three target adapters; then use T4 DNA enzyme to connect each target adapter to the gRNA vector digested by BsaI to obtain connection products; then perform two rounds of PCR amplification on the connection products to obtain three target gRNA expression cassettes.

[0149] The adapter primer sequences corresponding to the T1 target are as follows:

[0150] AtU3bT T1F: 5'-gtcaTTCTCAAATGCACAGGCCCT-3'.

[0151] AtU3bT T1R: 5'-aaacAGGGCCTGTGCATTTGAGAA-3'.

[0152] The adapter primer sequences corresponding to the T2 target are as follows:

[0153] AtU6-1 T2F: 5'-attgTGGAGCTCAGATGATCAGGC-3'.

[0154] AtU6-1 T2R: 5'-aaacGCCTGATCATCTGAGCTCCA-3'.

[0155] The adapter primer sequences corresponding to the T3 target are as follows:

[0156] AtU6-29 T3F: 5'-attgTGGAGCCCAGATAATCAAGG-3'.

[0157] AtU6-29 T3R: 5'-aaacCCTTGATTATCTGGGCTCCA-3'.

[0158] The first round of PCR amplification primer sequences are as follows:

[0159] UF: 5'-CTCCGTTTTACCTGTGGAATCG-3'.

[0160] gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3'.

[0161] The primer sequences for the second round of PCR amplification of the T1 target site are as follows:

[0162] Uctcg-B1': 5'-TTCAGAggtctcTctcgACTAGTGGAATCGGCAGCAAAGG-3'.

[0163] gRaaga-B3: 5'-AGCGTGggtctcGtcttGGTCCATCCACTCCAAGCTC-3'.

[0164] The primer sequences for the second round of PCR amplification of the T2 target site are as follows:

[0165] Uaaga-B3':5'-TTCAGAggtctcTaagaCACTGGAATCGGCAGCAAAGG-3'.

[0166] gRgact-B4: 5'-AGCGTGggtctcGagtcGGTCCATCCACTCCAAGCTC-3'.

[0167] The primer sequences for the second round of PCR amplification of the T3 target site are as follows:

[0168] Ugact-B4':5'-TTCAGAggtctcTgactCACTGGAATCGGCAGCAAAGG-3'.

[0169] gRcggt-BL: 5'-AGCGTGggtctcGaccgACGCGTCCATCCACTCCAAGCTC-3'.

[0170] 3. Preparation of knockout vector pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23

[0171] The three target gRNA expression cassettes and the pYLCRISPR / Cas9P35s-B vector were digested with BsaI, and then the large vector fragment obtained by BsaI digestion was connected with the three target gRNA expression cassettes digested with BsaI using T4 DNA ligase to obtain the knockout vector pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 and sequenced for verification. The results showed that the three target gRNA expression cassettes had been successfully connected to the pYLCRISPR / Cas9P35s-B vector ( Figure 1 ).

[0172] The knockout vector pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 expresses three sgRNAs targeting the Gm-1i-Q63 and / or Gm-1i-Q23 genes (the targets of the three sgRNAs are T1, T2 and T3 mentioned above, respectively).

[0173] 4. Preparation of recombinant bacteria EHA105 / pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23

[0174] The knockout vector pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 was introduced into Agrobacterium tumefaciens EHA105. After identification, Agrobacterium tumefaciens EHA105 containing the knockout vector pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 was obtained and named as the recombinant bacterium EHA105 / pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23.

[0175] 2. Obtaining and Molecular Detection of Soybeans Transfected with pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 1. Obtaining T0 Generation Soybeans Transfected with pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23

[0176] The recombinant bacteria EHA105 / pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 obtained in step 1 was transformed into Tianlong No. 1 by Agrobacterium-mediated soybean cotyledonary node genetic transformation method (herbicides were used for screening during the transformation process). The specific steps were referred to the method in the literature "Paz M, Martinez JC, Kalvig AB et al. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Reports, 2006, 25: 206-213" to obtain T0 generation pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 soybean. The seeds of T0 generation soybeans to be transfected with pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 were harvested by self-pollination to obtain T1 generation soybean plants to be transfected with pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23.

[0177] 2. Molecular detection of T1 generation soybean plants to be transformed with pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23

[0178] PCR detection of target genes and marker genes in T1 generation pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 soybean plants was performed. The specific steps were as follows: DNA was extracted from leaves of wild-type soybean Tianlong No. 1 and T1 generation pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 soybean plants, with wild-type soybean Tianlong No. 1 plant DNA as a negative control and pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 plasmid as a positive control. The gRNA expression cassette was detected using T1 target-specific primers and vector primers (T1-F: 5'-TTCTCAAATGCACAGGCCCT-3'; gRNA-R1: 5'-CCCGACATAGATGCAATAACTTC-3'), and Cas9-specific primers (Cas9-F: 5'-GAAGTACTCCATCGGCCTCG-3'; Cas9-R: 5'-GCGCTTGATCATGGAAGCAG-3') and bar gene-specific primers (bar-F: 5'-GTGCCACCGAGGCGGACAT-3'; bar-R: 5'-TCAGATCTCGGTGACGGGCAG-3') were used to detect Cas9 and bar genes. The PCR reaction program was as follows: 94°C for 5 min; 94°C for 30 s, 56°C for 30 s, 72°C for 2 min, 34 cycles; 72°C for 10 min.

[0179] The results are as follows Figure 2 The results showed that the plasmid positive control and the three T1 generation soybean plants that were intended to be transformed with pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 amplified a band of 1184 bp (gRNA expression box), a band of 950 bp (Cas9 gene), and a band of 514 bp (bar gene).

[0180] 3. Detection and analysis of gene editing types in T3 transfected soybeans with pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23

[0181] 1. Detection of editing types

[0182] The T1 generation transgenic pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 soybean obtained in step 2 was propagated and multiplied to obtain T3 generation transgenic pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 soybean plants, and then the editing type was detected. The specific steps are as follows: According to the Gm-1i-Q63 and Gm-1i-Q23 sequences, specific primers were designed (Gm-1i-Q63-F: 5'-CGTAGATGCGTAGCTTCTGTAG-3', Gm-1i-Q63-R: 5'-ACTTGTGGCCTTGATTGAG-3'; Gm-1i-Q23-F: 5'-CTCAGCCAATCTGTGTTGACC-3', Gm-1i-Q23-R : 5'-TCTTTGTGAGGAAAACTGAATGG-3'), Gm-1i-Q63 and Gm-1i-Q23 were amplified from the genome of wild-type soybean Tianlong No. 1 and T3 generation pYLCRISPR / Cas9P35s-B-Gm-1i-Q63 / 1i-Q23 soybean plants and sequenced to compare whether the target sequence had changed. Finally, three homozygous Gm-1i-Q63 and Gm-1i-Q23 gene-edited double mutant materials were obtained, and they were named gm-1i-q63 / gm-1i-q23-1(-9 / -4), gm-1i-q63 / gm-1i-q23-2(+1 / -4) and gm-1i-q63 / gm-1i-q23-3(+1 / +1), respectively.

[0183] The only difference between the gm-1i-q63 / gm-1i-q23-1(-9 / -4) gene-edited double mutant and the wild-type soybean Tianlong No. 1 genome sequence is that a fragment deletion (CAGGCCCTG) occurs in the gene encoding the Gm-1i-Q63 protein (sequence 2), and the deleted fragment is located at positions 348-356 of sequence 2, and a fragment deletion (AGGC) occurs in the gene encoding the Gm-1i-Q23 protein (sequence 5), and the deleted fragment is located at positions 2251-2254 of sequence 5.

[0184] The only difference between the gm-1i-q63 / gm-1i-q23-2 (+1 / -4) gene-edited double mutant and the wild-type soybean Tianlong No. 1 genome sequence is that a base T is inserted in the gene encoding the Gm-1i-Q63 protein (sequence 2), and the insertion position of the base T is located between positions 352 and 353 of sequence 2, and a fragment deletion (AGGC) occurs in the gene encoding the Gm-1i-Q23 protein (sequence 5), and the deleted fragment is located at positions 2251-2254 of sequence 5.

[0185] The only difference between the gm-1i-q63 / gm-1i-q23-3 (+1 / +1) gene-edited double mutant and the wild-type soybean Tianlong No. 1 genome sequence is that a base T is inserted in the gene encoding the Gm-1i-Q63 protein (sequence 2), and the insertion position of the base T is located between positions 352 and 353 of sequence 2, and a base T is inserted in the gene encoding the Gm-1i-Q23 protein (sequence 5), and the insertion position of the base T is located between positions 2253 and 2254 of sequence 5.

[0186] The changes in genome and protein sequences after mutation are as follows Figure 3 shown.

[0187] 2. Off-target site analysis

[0188] The website http: / / skl.scau.edu.cn / offtarget / was used to analyze the off-target situation of Gm-1i-Q63 and Gm-1i-Q23 genes. The results showed that except for the corresponding target sites of Gm-1i-Q63 and Gm-1i-Q23 genes, the possibility of off-target at other sites was extremely low ( Figure 4 ).

[0189] Example 2: Analysis of the symbiotic nitrogen fixation ability of the gm-1i-q63 / gm-1i-q23 gene-edited double mutant material

[0190] Test soybean materials: wild-type soybean Tianlong No. 1, gm-1i-q63 / gm-1i-q23 gene-edited double mutant materials gm-1i-q63 / gm-1i-q23-1 (-9 / -4), gm-1i-q63 / gm-1i-q23-2 (+1 / -4) and gm-1i-q63 / gm-1i-q23-3 (+1 / +1) prepared in Example 1.

[0191] Experimental method: Soybean rhizobium USDA110 was activated on YMA plates and then inoculated into liquid YMA medium. After shaking culture at 28°C for 2-3 days, wild-type soybean seedlings and gm-1i-q63 / gm-1i-q23 gene-edited double mutant seedlings that were about 5-7 days old were inoculated. The inoculation volume was 2 mL of rhizobium solution (OD 600nm>0.6) / plant. 23 days and 45 days after inoculation with rhizobium USDA110, gm-1i-q63 / gm-1i-q23 gene-edited double mutant plants and wild-type plants were harvested, cut from the cotyledon node, and the nodulation phenotypes of the aboveground part and the root system were photographed and recorded, the growth of the whole plant and the root nodulation phenotype were observed, and the number of root nodules and the dry weight of nodules of each plant were statistically analyzed. Among them, 10 wild-type plants and 13 gm-1i-q63 / gm-1i-q23 gene-edited double mutant plants (mixed with three double mutant materials) were harvested 23 days after inoculation; 10 wild-type plants and 14 gm-1i-q63 / gm-1i-q23 gene-edited double mutant plants (mixed with three double mutant materials) were harvested 45 days after inoculation.

[0192] The experimental results showed that compared with the wild-type soybean Tianlong No. 1, the senescence of the aboveground part of the gm-1i-q63 / gm-1i-q23 gene-edited double mutant was significantly delayed and the growth period was significantly extended ( Figure 5 ), the number of nodules increased significantly, the number of newly formed and vigorous nodules on the root system increased significantly in the late development stage, and the dry weight of nodules increased significantly ( Figure 6 ). Among them, the average nodule numbers of the wild-type Tianlong No. 1 and the gm-1i-q63 / gm-1i-q23 gene-edited double mutant materials 23 days after inoculation were 31.8 and 39.5, and the average nodule dry weights were 0.039g and 0.045g, respectively; the average nodule numbers of the wild-type Tianlong No. 1 and the gm-1i-q63 / gm-1i-q23 gene-edited double mutant materials 45 days after inoculation were 51.3 and 83.7, and the average nodule dry weights were 0.048g and 0.116g, respectively. Figure 7 ).

[0193] The above results show that after knocking out the Gm-1i-Q63 and Gm-1i-Q23 genes, the plant growth period was extended, the number of nodules increased, the dry weight of nodules increased, and the effective nodulation time and nitrogen fixation time of the root system were extended (many new and vigorous nodules still appeared in the late stage of plant development). This shows that knocking out the Gm-1i-Q63 and Gm-1i-Q23 genes can improve the nitrogen fixation efficiency by increasing the number of nodules and the weight of nodules and extending the nodulation and nitrogen fixation time of the entire root system.

[0194] Example 3. Analysis of yield-related traits of gm-1i-q63 / gm-1i-q23 gene-edited double mutant materials

[0195] Test soybean materials: wild-type soybean Tianlong No. 1, gm-1i-q63 / gm-1i-q23 gene-edited double mutant materials gm-1i-q63 / gm-1i-q23-1 (-9 / -4), gm-1i-q63 / gm-1i-q23-2 (+1 / -4) and gm-1i-q63 / gm-1i-q23-3 (+1 / +1) prepared in Example 1.

[0196] Experimental method: In the Hainan biological breeding area, wild soybean Tianlong No. 1, gm-1i-q63 / gm-1i-q23 gene editing double mutant materials gm-1i-q63 / gm-1i-q23-1 (-9 / -4), gm-1i-q63 / gm-1i-q23-2 (+1 / -4) and gm-1i-q63 / gm-1i-q23-3 (+1 / +1) prepared in Example 1 were planted according to conventional methods. Ten plants were continuously taken from the middle row of the plot planted with each material at maturity for copying, and the field harvest phenotype of the plant was observed and photographed and recorded. Each material was copied for conventional agronomic traits (plant height, number of grains per plant, grain weight per plant and 100 grain weight, protein content (percentage)) in three replicates of about 30 plants, wherein the protein content (percentage) was detected using a portable handheld near-infrared grain analyzer.

[0197] The test results showed that after knocking out the Gm-1i-Q63 and Gm-1i-Q23 genes, the plant height, number of grains per plant, grain weight per plant and 100-grain weight were significantly improved. In addition, the protein content was significantly improved in terms of quality ( Figure 8 , Fig. 9 ). Among them, the average plant heights of the wild-type Tianlong No. 1, gm-1i-q63 / gm-1i-q23 gene-edited double mutant materials gm-1i-q63 / gm-1i-q23-1(-9 / -4), gm-1i-q63 / gm-1i-q23-2(+1 / -4) and gm-1i-q63 / gm-1i-q23-3(+1 / +1) were 25.5cm, 29.6cm, 30.77cm and 31.46cm respectively; the average number of grains per plant were 22, 29, 32 and 32.7 respectively; the average grain weight per plant were 3.39g, 4.87g, 5.76g and 6.03g respectively; the average 100-grain weight were 16.55g, 16.90g, 18.37g and 19.13g respectively; the average protein content (percentage) were 37.1%, 38.4%, 40.4% and 42% respectively.

[0198] The above results show that after knocking out the Gm-1i-Q63 and Gm-1i-Q23 genes, the yield-related agronomic traits plant height, number of grains per plant, grain weight per plant and 100-grain weight were significantly improved, and the protein content in terms of quality was significantly improved. This shows that the gm-1i-q63 / gm-1i-q23 gene-edited double mutant has a good yield-increasing potential, and it mainly improves seed development through efficient nitrogen fixation, which is mainly manifested in the increase in the number of grains per plant, grain weight per plant and 100-grain weight.

[0199] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of a protein or a substance for regulating the content and / or activity of the protein in any of the following A1)-A6): A1) Regulate the symbiotic nitrogen fixation ability of plants; A2) regulating plant yield; A3) regulating plant height; A4) regulating the content of plant protein; A5) cultivating transgenic plants with altered symbiotic nitrogen fixation ability and / or yield and / or plant height and / or protein content; A6) Plant breeding; The proteins are Gm-1i-Q63 protein and Gm-1i-Q23 protein; The Gm-1i-Q63 protein is any one of the following B1)-B4): B1) The amino acid sequence is the protein shown in SEQ ID NO: 3; B2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 3; B3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 3; B4) a protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 3 and having the same function; The Gm-1i-Q23 protein is any one of the following C1)-C4): C1) The amino acid sequence is the protein shown in SEQ ID NO: 6; C2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 6; C3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 6; C4) A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 6 and having the same function.

2. Use of biological materials related to Gm-1i-Q63 protein and biological materials related to Gm-1i-Q23 protein in any of the following A1)-A6): A1) Regulate the symbiotic nitrogen fixation ability of plants; A2) regulating plant yield; A3) regulating plant height; A4) regulating the content of plant protein; A5) cultivating transgenic plants with altered symbiotic nitrogen fixation ability and / or yield and / or plant height and / or protein content; A6) Plant breeding; The biological material related to the Gm-1i-Q63 protein is any one of the following D1) to D5): D1) a nucleic acid molecule encoding the Gm-1i-Q63 protein; D2) knocking down or knocking out a nucleic acid molecule of the Gm-1i-Q63 protein encoding gene; D3) an expression cassette containing the nucleic acid molecule described in D1) or D2); D4) a recombinant vector containing the nucleic acid molecule described in D1) or D2); D5) A recombinant microorganism containing the nucleic acid molecule described in D1) or D2); The biological material related to the Gm-1i-Q23 protein is any one of the following E1) to E5): E1) a nucleic acid molecule encoding the Gm-1i-Q23 protein; E2) knocking down or knocking out a nucleic acid molecule of the Gm-1i-Q23 protein encoding gene; E3) an expression cassette containing the nucleic acid molecule described in E1) or E2); E4) a recombinant vector containing the nucleic acid molecule described in E1) or E2); E5) A recombinant microorganism containing the nucleic acid molecule described in E1) or E2).

3. The use according to claim 2, characterized in that: D1) The nucleic acid molecule is any of the following: F1) The DNA molecule shown in Sequence 1 or Sequence 2; F2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in F1) and encoding the Gm-1i-Q63 protein; E1) The nucleic acid molecule is any one of the following: G1) The DNA molecule shown in Sequence 4 or Sequence 5; G2) is a DNA molecule that has 75% or more identity with the nucleotide sequence defined in G1) and encodes the Gm-1i-Q23 protein.

4. The use according to any one of claims 1 to 3, characterized in that: The symbiotic nitrogen fixation ability of the regulating plant is embodied in any one of the following M1)-M4): M1) Regulate the number of plant nodules; M2) regulates plant nodule dry weight; M3) regulating the time of plant nodulation and / or nitrogen fixation; M4) regulates the efficiency of plant nitrogen fixation.

5. The use according to any one of claims 1 to 4, characterized in that: The regulating plant yield is embodied in any one of the following N1)-N3): N1) Regulate the number of seeds per plant; N2) regulates the grain weight per plant; N3) regulates the 100-grain weight of plants.

6. A method for cultivating transgenic plants with improved symbiotic nitrogen fixation ability and / or improved yield and / or increased plant height and / or increased protein content, comprising the following steps: reducing the content and / or activity of Gm-1i-Q63 protein and Gm-1i-Q23 protein in the target plant to obtain a transgenic plant; the symbiotic nitrogen fixation ability and / or yield and / or plant height and / or protein content of the transgenic plant are higher than those of the target plant.

7. The method according to claim 6, characterized in that The method for reducing the content and / or activity of Gm-1i-Q63 protein and Gm-1i-Q23 protein in the target plant is to introduce a substance for knocking out the Gm-1i-Q63 protein encoding gene and a substance for knocking out the Gm-1i-Q23 protein encoding gene into the target plant.

8. The method according to claim 6 or 7, characterized in that: The symbiotic nitrogen fixation ability of the transgenic plant is higher than that of the target plant, which is embodied in any one of the following X1)-X4): X1) after inoculation with rhizobia, the number of nodules of the transgenic plant is higher than that of the target plant; X2) after inoculation with rhizobia, the dry weight of nodules of the transgenic plant is higher than that of the target plant; X3) after inoculation with rhizobia, the nodulation time and / or nitrogen fixation time of the transgenic plant are longer than those of the target plant; X4) After inoculation with rhizobia, the nitrogen fixation efficiency of the transgenic plant is higher than that of the target plant.

9. The method according to any one of claims 6 to 8, characterized in that: The yield of the transgenic plant is higher than that of the target plant, which is embodied in any one of the following Y1)-Y3): Y1) the number of grains per plant of the transgenic plant is higher than that of the target plant; Y2) the grain weight per plant of the transgenic plant is higher than that of the target plant; Y3) The 100-grain weight of the transgenic plant is higher than that of the target plant.

10. The use according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9, characterized in that: The plant is any one of the following Z1)-Z4): Z1) dicotyledonous plants or monocotyledonous plants; Z2) Leguminosae; Z3) soybean plants; Z4) Soybean.

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