Use of pdf9 protein in regulating nitrogen fixation capacity of legume plants

Abstract

The application relates to the field of biotechnology, and discloses application of a PDF9 protein in regulating the nitrogen fixation capacity of leguminous plants. The PDF9 gene is edited by CRISPR / Cas9 technology, the gene coding the PDF9 protein of Medicago truncatula is knocked out, and the plant height and the nitrogen fixation capacity are reduced. The application is very important for studying the efficient nitrogen fixation mechanism of leguminous plants, provides a new idea for improving the biological nitrogen fixation efficiency, and has great application value for plant breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of a PDF9 protein in regulating nitrogen fixation in legumes. Background Technology

[0002] Legumes and rhizobia can form a mutually beneficial symbiotic system. In this system, rhizobia are responsible for converting atmospheric molecular nitrogen into nitrogen that plants can absorb and utilize, while legumes form specialized nitrogen-fixing organs called root nodules, providing the carbon source needed for rhizobia growth. The symbiotic nitrogen fixation between legumes and rhizobia can deliver approximately 50 million tons of nitrogen nutrients to agricultural systems annually. Compared to industrial nitrogen fixation, biological nitrogen fixation is more efficient, economical, and environmentally friendly. Therefore, studying the symbiotic nitrogen fixation mechanism of legumes and rhizobia to improve biological nitrogen fixation efficiency is of great significance for sustainable agricultural development.

[0003] Based on morphology and structure, root nodules can be broadly classified into fixed and amorphous nodules. The main difference lies in their shape: fixed nodules are spherical, lack continuously dividing meristematic tissue, and the rhizobia in the infected cells exhibit a relatively uniform developmental state; in contrast, amorphous nodules are cylindrical or forked, and the rhizobia in the infected cells of amorphous nodules undergo terminal differentiation, forming enlarged, elongated, or spherical bacteriophytes. In amorphous nodules, the differentiation process of these bacteriophytes is highly coupled with their nitrogen-fixing function.

[0004] Current research indicates that plant cells secrete various signaling peptides during amorphous root nodule development, participating in the regulation of rhizobium infection, survival, and differentiation. For example, some members of the NCR peptide family have been shown to participate in the regulation of mycoplasma differentiation and maintenance. However, currently validated plant peptides involved in mycoplasma differentiation regulation are mainly concentrated in a few families (such as NCR), and a systematic understanding of the regulatory network has not yet been formed. A large number of potential plant peptides and their functions remain to be explored. Even for the few known peptides, how they regulate terminal differentiation, metabolic reorganization, and nitrogenase activity activation of mycoplasma through specific signaling pathways, as well as their downstream targets and molecular mechanisms, remain unclear.

[0005] Therefore, discovering and identifying plant polypeptides that can regulate the terminal differentiation of mycoplasma and play a decisive role in the efficiency of symbiotic nitrogen fixation, and elucidating their functions and application potential, has important guiding significance and application value for deepening the research on the regulation of biological nitrogen fixation capacity. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems existing in the prior art and provide an application of the PDF9 protein in regulating nitrogen fixation capacity in legumes. This invention is of great importance for studying the efficient nitrogen fixation mechanism of legumes and provides a new approach to improving biological nitrogen fixation efficiency. This invention also has significant application value for plant breeding.

[0007] To achieve the above objectives, the present invention provides a method for regulating the plant height of leguminous plants.

[0008] A second aspect of the present invention provides a method for regulating the nitrogen fixation capacity of leguminous plants.

[0009] A third aspect of the present invention provides a method for regulating the activity of nitrogenase in legumes.

[0010] The fourth aspect of this invention provides a method for regulating the volume of root nodules in leguminous plants.

[0011] The fifth aspect of this invention provides a method for regulating the length of mycotoxin-like cells in root nodules of leguminous plants.

[0012] The sixth aspect of this invention provides a method for regulating the plant architecture of leguminous plants.

[0013] The seventh aspect of this invention provides a method for regulating the fresh weight of leguminous plants.

[0014] Preferably, the legume is alfalfa.

[0015] Preferably, the gene editing is performed using a CRISPR / Cas9 system.

[0016] This invention utilizes CRISPR / Cas9 technology to specifically edit the PDF9 gene, knocking out the gene encoding the PDF9 protein in alfalfa, thereby reducing plant height and nitrogen fixation capacity. This invention is crucial for studying the efficient nitrogen fixation mechanisms of leguminous plants and provides a new approach to improving biological nitrogen fixation efficiency. This invention also has significant application value for plant breeding. Attached Figure Description

[0017] Figure 1 The transgenic alfalfa obtained in Example 1 of this invention was inoculated with Sinorhizobium meliloti Images of tissue expression analysis after 2011, where (a) is a photo taken 7 days after vaccination, (b) is a photo taken 10 days after vaccination, and (c) is a photo taken 21 days after vaccination; Figure 2 This is a schematic diagram showing the location of the target sequence on the PDF9 gene in this invention, where (A) is the mutant. pdf9 - 1 The diagram shows (B) as the mutant. pdf9 - 2 A schematic diagram; Figure 3 These are photographs of the growth status of the alfalfa plant in this invention. Figure 4 This is a photograph showing the development of root nodules in *Alfalfa tribulus* as described in this invention. Figure 5 This is a bar chart showing the plant height of Alfalfa tribulus in this invention; Figure 6 This is a bar chart of the fresh weight of alfalfa in this invention; Figure 7 This is a bar chart showing the reduction of acetylene to ethylene by root nodules of alfalfa in this invention. Figure 8 This is a transmission electron microscope image of the root nodules of Alfalfa Tribulus terrestris in this invention. Detailed Implementation

[0018] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0019] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] The purpose of this invention is to regulate the nitrogen fixation capacity of leguminous plants.

[0021] This invention first protects the application of the PDF9 protein, which may be at least one of (S1)-(S7): (S1) Regulates plant height in leguminous plants; (S2) Regulates the nitrogen fixation capacity of leguminous plants; (S3) Regulates nitrogenase activity in legumes; (S4) Regulates the volume of root nodules in leguminous plants; (S5) Regulates the length of mycoplasma-like cells in root nodules of leguminous plants; (S6) Regulates the plant architecture of leguminous plants; (S7) Regulates the fresh weight of leguminous plants; The PDF9 protein may be (a1), (a2), (a3), or (a4): (a1) is the protein whose amino acid sequence is shown in SEQ ID No:2; (a2) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 2; (a3) is a protein derived from legumes and related to nitrogen fixation capacity, nitrogenase activity, plant height and / or root nodule volume, obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in (a1) or (a2). (a4) is a protein that has 80% or more identity with the protein shown in (a1) or (a2), is derived from a legume and is associated with nitrogen fixation capacity, nitrogenase activity, plant height, plant type and / or root nodule volume.

[0022] Of these, SEQ ID No.2 consists of 82 amino acid residues.

[0023] The protein in (a3) ​​above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0024] The proteins mentioned in (a3) ​​above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0025] The gene encoding the protein in (a3) ​​above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in SEQ ID No:1, and / or by performing a missense mutation on one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0026] The present invention also protects the application of nucleic acid molecules encoding the PDF9 protein, which may be at least one of (S1)-(S7): (S1) Regulates plant height in leguminous plants; (S2) Regulates the nitrogen fixation capacity of leguminous plants; (S3) Regulates nitrogenase activity in legumes; (S4) Regulates the volume of root nodules in leguminous plants; (S5) Regulates the length of mycoplasma-like cells in root nodules of leguminous plants; (S6) Regulates the plant architecture of leguminous plants; (S7) Regulates the fresh weight of leguminous plants; The nucleic acid molecule encoding the PDF9 protein described above may be a DNA molecule as shown in (b1), (b2), (b3), or (b4) below: (b1) The coding region is the DNA molecule shown in SEQ ID No:1; (b2) The nucleotide sequence is the DNA molecule shown in SEQ ID No:1; (b3) has 80% or more identity with the nucleotide sequence defined by (b1) or (b2) and is a DNA molecule encoding the PDF9 protein; (b4) Hybridizes under stringent conditions to a nucleotide sequence defined by (b1) or (b2) and to a DNA molecule encoding the PDF9 protein.

[0027] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0028] Of these, SEQ ID No:1 consists of 351 nucleotides, and the nucleotides of SEQ ID No:1 encode the amino acid sequence shown in SEQ ID No:2.

[0029] Those skilled in the art can readily mutate the nucleotide sequence encoding the PDF9 protein of this invention using known methods, such as directed evolution and point mutation. Any artificially modified nucleotides having 80% or higher identity with the nucleotide sequence of the PDF9 protein isolated according to this invention, as long as they encode the PDF9 protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0030] The term "identity" as used in this invention refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence of the PDF9 protein encoding the amino acid sequence shown in SEQ ID No:2 of this invention. Identity can be evaluated visually or using 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.

[0031] The nucleic acid molecule encoding the PDF9 protein may specifically be a gene encoding the PDF9 protein, named the PDF9 gene.

[0032] In any of the above applications, the regulation of legume plant height can be achieved by decreasing or increasing the plant height.

[0033] In any of the above applications, regulating the nitrogen fixation capacity of legumes can be done by reducing or increasing the nitrogen fixation capacity of legumes.

[0034] In any of the above applications, the regulation of nitrogenase activity in legumes can be described as either reducing or increasing nitrogenase activity in legumes.

[0035] In any of the above applications, regulating the volume of leguminous root nodules can be done by decreasing or increasing the volume of leguminous root nodules.

[0036] In any of the above applications, regulating the length of mycoplasma in leguminous root nodules can be done by decreasing or increasing the length of mycoplasma in leguminous root nodules.

[0037] The present invention also protects two specific sgRNAs whose guide sequences are identical to those shown in SEQ ID NO:1 at positions 208-227 or 249-268 from the 5' end, respectively, wherein the guide sequence is the RNA sequence obtained by replacing thymine (T) with uracil (U) in the 20 nt DNA sequence.

[0038] The present invention also protects specific recombinant plasmids; said specific recombinant plasmids contain the coding gene for the Cas9 protein and the coding gene for any of said specific sgRNAs.

[0039] This invention also protects the application of any of the above-described specific sgRNAs or any of the above-described specific recombinant plasmids in legume breeding; the purpose of the legume breeding may be to reduce the plant height of legumes, reduce the nitrogen fixation capacity of legumes, reduce the volume of root nodules of legumes, and reduce the length of mycoplasma-like cells in the root nodules of legumes.

[0040] This invention also protects a method for preparing gene-edited legumes, which may include the following steps: (1) The coding gene of any of the above-mentioned specific sgRNA and the coding gene of Cas9 protein are introduced into legumes to obtain transgenic legumes; (2) Screening gene-edited legumes from the transgenic legumes; Compared to wild-type or untransformed recipient plants, gene-edited plants exhibited reduced plant height, decreased nitrogen fixation capacity, reduced nitrogenase activity, smaller root nodule volume, and reduced length of mycotoxin-like cells in leguminous root nodules.

[0041] In the above method, the encoding gene of any of the specific sgRNAs and the encoding gene of the Cas9 protein are specifically introduced into legumes via the recombinant plasmid.

[0042] In the above method, the screening of gene-edited legumes from transgenic legumes can specifically refer to screening transgenic legumes that have undergone gene editing and whose PDF9 gene expression is suppressed.

[0043] The "transgenic legumes that have undergone gene editing and whose PDF9 gene expression is suppressed" mentioned above can specifically refer to transgenic legumes in which the target region has been mutated and all of them are homozygous mutants.

[0044] The present invention also protects any one of (Q1)-(Q7).

[0045] (Q1) A method for regulating the plant height of a legume, the method comprising: gene editing of a gene encoding a PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding the PDF9 protein is shown in SEQ ID No:1.

[0046] (Q2) A method for regulating the nitrogen fixation capacity of legumes, the method comprising: gene editing of a gene encoding PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding PDF9 protein is shown in SEQ ID No:1.

[0047] (Q3) A method for regulating nitrogenase activity in legumes, the method comprising: gene editing of a gene encoding PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding PDF9 protein is shown in SEQ ID No:1.

[0048] (Q4) A method for regulating the volume of root nodules in legumes, the method comprising: gene editing of a gene encoding PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding PDF9 protein is shown in SEQ ID No:1.

[0049] (Q5) A method for regulating the length of mycotoxin-like cells in root nodules of leguminous plants, the method comprising: gene editing of a gene encoding PDF9 protein in a leguminous plant, wherein the nucleotide sequence of the gene encoding PDF9 protein is shown in SEQ ID No:1.

[0050] (Q6) A method for regulating the plant architecture of a legume, the method comprising: gene editing of a gene encoding a PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding the PDF9 protein is shown in SEQ ID No:1.

[0051] (Q7) A method for regulating the fresh weight of a legume, the method comprising: gene editing of a gene encoding a PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding the PDF9 protein is shown in SEQ ID No:1.

[0052] The gene editing methods described above include well-known methods in the art such as RNA interference, homologous recombination, and site-specific gene editing, in order to reduce the activity and / or expression level of PDF proteins in legumes.

[0053] In the above method, the gene editing can be specifically achieved by knocking out or silencing the PDF9 gene. Knockout includes knocking out the entire gene or knocking out a portion of the gene.

[0054] The gene editing described above was achieved using the CRISPR / Cas9 system.

[0055] The gene editing is achieved by introducing a specific DNA molecule containing the gene encoding the Cas9 protein and the gene encoding the sgRNA into a legume. The gene editing is also achieved by introducing a recombinant plasmid containing the specific DNA molecule into the legume.

[0056] The gene editing was achieved by introducing DNA molecules containing the gene encoding the Cas9 protein and DNA molecules containing the gene encoding sgRNA into legumes, respectively.

[0057] In an embodiment of the present invention, when the legume is alfalfa R108, the gene-edited legume obtained by the above method can be... pdf9-1 mutant or pdf9-2 Mutant.

[0058] Any of the legumes mentioned above may be alfalfa.

[0059] The aforementioned alfalfa may specifically be alfalfa R108 or alfalfa A17.

[0060] SEQ ID Number 1 TGTAGATGAAGAAGCAAAAATTTGTAAAATTTTGCTTAGTTCAACAATGAACCATGGTTGTGAAGAAATCAAGTGTAACGAAGGCTGCATCAACGAGGAGAATGCAAATTCCGGTGAGTGTATGGCTACAGGCATCGTTGTTTTTTTTTGCTACTGCTTCTACGAATGTGAGCAGTGA.

[0061] Positions 77-178 are introns: GTACAAACTAACTAAACAACCTATATTTATTCTCTCATACTTCAATTCTAATCCTTCTTCATGTTCAATTTTTTATTTTTTATTGTTTATTTTTATGTGTAG.

[0062] sgRNA1:CUUAGUUCAACAAUGAACCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC。

[0063] sgRNA2:UAACGAAGGCUGCAUCAACGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC。

[0064] SEQ ID No:2:MTFSASKFYIIFMYLYLAIFINSAKDEEAKICKILLSSTMNHGCEEIKCNEGCINEENANSGECMATGIVVFFCYCFYECEQ。

[0065]

[0066] The following examples further illustrate the application of the PDF9 protein described in this invention in regulating nitrogen fixation in legumes. These examples are implemented based on the technical solution of this invention, providing detailed implementation methods and specific operating procedures; however, the scope of protection of this invention is not limited to the following examples.

[0067] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available.

[0068] Example 1 PDF9 protein is expressed in different regions of the root nodule organs of alfalfa. (1) The pCambia1391 vector was digested with the restriction endonuclease PstI and inserted into the DNA molecule shown in SEQ ID NO:3 to obtain the recombinant plasmid; the recombinant plasmid was named pCambia1391-pPDF9. The DNA molecule shown in SEQ ID NO:3 is 2185 bp upstream of the start codon of the PDF9 gene in Alfalfa R108.

[0069] (2) After completing step (1), the recombinant plasmid pCambia1381-pPDF9 was transferred into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium tumefaciens.

[0070] (3) The recombinant plasmid pCambia1391-pPDF9 was transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium; then, the recombinant Agrobacterium was transformed into alfalfa R108 using the alfalfa genetic transformation method (described in Medicago Handbook: https: / / www.noble.org / medicago-handbook / ) to obtain T0 generation pseudo-transgenic alfalfa.

[0071] (4) After completing step (3), inoculate the transgenic alfalfa. Sinorhizobium meliloti In 2011 (OD600 = 0.01, inoculum size 1 mL / plant), root nodules were collected at 7, 10, and 21 days post-infection for GUS staining and observation. The results of GUS staining are shown below. Figure 1 As shown in (A).

[0072] Example 2 Obtaining PDF9 gene knockout mutants and the application of PDF9 protein in reducing nitrogen fixation capacity of alfalfa. I. Construction of PDF9 gene knockout vector The PDF9 gene knockout vector—recombinant plasmid pCambia1300-Cas9-PDF9—was constructed using the CRISPR / Cas9 system. The target sequences were 5'-CTTAGTTCAACAATGAACCA-3' and 5'-TAACGAAGGCTGCATCAACG-3'. The vector construction method was based on the following literature: Yan L, Wei S, Wu Y, Hu R, Li H, Yang W, Xie Q. High Efficiency Genome Editing in Arabidopsis Using YAO Promoter-Driven CRISPR / Cas9 System. Mol. Plant. 2015 8(12):1820-1823. The construction process of the knockout vector pCambia1300-Cas9-PDF9 is as follows: Construction of sgRNA cassette (1) The target sequence was inserted into the recognition site of the restriction endonuclease BsaI in the vector AtU6-26-sgRNA-SK to obtain the intermediate vector. The location of the target sequence on the PDF9 gene is shown in the figure. Figure 2 As shown in (A).

[0073] (2) After completing step (1), take the intermediate vector and digest it with restriction endonucleases NheI and SpeI to recover a DNA fragment of about 642 bp, namely sgRNA cassette.

[0074] (3) The pCambia1300-pUBQ10:Cas9 plasmid was digested with the restriction endonuclease SpeI to obtain a linearized plasmid.

[0075] (4) The linearized plasmid and two sgRNA cassettes were ligated to obtain the recombinant plasmid pCambia1300-Cas9-PDF9.

[0076] II. PDF9 gene knockout mutant (i.e. pdf9 - 1 mutants and pdf9 - 2 Obtaining mutants (1) The recombinant plasmid pCambia1300-Cas9-PDF9 was transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium; then the recombinant Agrobacterium was transformed into alfalfa R108 using the alfalfa genetic transformation method (described in Medicago Handbook: https: / / www.noble.org / medicago-handbook / ) to obtain T0 generation pseudo-transgenic alfalfa.

[0077] (2) Take leaves of the T0 generation transgenic alfalfa plants obtained in step 1 and extract genomic DNA. Then, using the genomic DNA of each plant as a template, perform PCR amplification using primer pair consisting of primer F: 5'-GAAGTGTCATATCTTGGCTTCTCGAC-3' and primer R: 5'-GTGTAACACAGCATCATTGATCATGTG-3' to obtain the corresponding PCR amplification products.

[0078] (3) Sequencing of each PCR amplification product. The sequencing results were compared with the nucleotide sequence of the PDF9 gene. A total of 10 T0 generation transgenic alfalfa plants were tested, one of which was a heterozygous mutant (a heterozygous mutant is one in which the PDF9 gene of both homologous chromosomes of the plant is mutated but the mutations are different). Specifically, one homologous chromosome had a deletion of 41 nucleotides at the target site of the PDF9 gene, namely “CCATGGTTGTGAAGAAATCAAGTGTAACGAAGGCTGCATCA”, and the other homologous chromosome had the original “GTAAAATTTTGCTTAGTTCAACAATGAACCATGGTTGTGAAGAAATCAAGTGTAACGAAGGCTGCATCAAC” nucleotide sequence changed to “GCAAAAATTTTATTGTTTATTTTCAAGTGTAACGAAGGCTGCATCAC” at the target site of the PDF9 gene.

[0079] (4) Self-cross the heterozygous mutant obtained in step (3) to obtain T1 generation transgenic alfalfa.

[0080] (5) Following steps (2) and (3) above, replace the T0 generation transgenic alfalfa with the T1 generation transgenic alfalfa, keeping all other steps unchanged. Two homozygous mutants are finally obtained— pdf9 - 1 mutants and pdf9 - 2 Mutants. Homozygous mutants are those in which the PDF9 gene on two homologous chromosomes of the plant has the same mutation. pdf9 - 1 mutants and pdf9 - 2 See the mutation patterns at the target site. Figure 2 . pdf9 - 1 The mutant exhibits a 41-nucleotide deletion in the PDF9 gene, namely “CCATGGTTGTGAAGAAATCAAGTGTAACGAAGGCTGCATCA”, resulting in a loss of PDF9 protein function. pdf9 - 2 The mutant altered the original nucleotide sequence of "GTAAAATTTTGCTTAGTTCAACAATGAACCATGGTTGTGAAGAAATCAAGTGTAACGAAGGCTGCATCAAC" in the PDF9 gene to "GCAAAAATTTTATTGTTTATTTTCAAGTGTAACGAAGGCTGCATCAC", resulting in the loss of PDF9 protein function.

[0081] III. Knocking out the PDF9 gene can reduce the plant height, nitrogen fixation capacity, and length of mycotoxin-like cells in root nodules of alfalfa. (1) Take alfalfa truncatum of similar size to be tested. pdf9 - 1 mutants pdf9 - 2 Seeds of mutant alfalfa (R108) were first treated with concentrated sulfuric acid for 5 min, then rinsed with water 5-10 times; then treated with 6% (m / v) sodium hypochlorite solution for 10 min, then rinsed with sterile water 5-10 times; and finally placed on a shaker for 24 h to obtain the treated alfalfa seeds to be tested.

[0082] (2) After completing step (1), the treated alfalfa seeds to be tested were placed in FM solid medium and incubated upside down at 22°C for 12 hours.

[0083] (3) After completing step (2), plant 20 germinated seeds in vermiculite pots containing FM liquid culture medium (2 seeds per pot). After 5 days, inoculate. Sinorhizobium meliloti 2011 (OD600 was 0.01, inoculation amount was 1 mL per plant).

[0084] (4) After completing step (3), cultivate for 3 weeks and observe the plant growth status and root nodule development. See below for plant growth status. Figure 3 . wild type and pdf9 - 1 See the development of root nodules. Figure 4 The results showed that, compared with alfalfa R108, pdf9 - 1 mutants and pdf9 - 2 The mutant plant height and plant size were significantly reduced; see the detailed statistical results below. Figure 5 and Figure 6 ; pdf9 - 1 The mutant root nodules exhibited a premature aging phenotype, with the base of the nodules turning noticeably green.

[0085] (5) After completing step (3), culture for 3 weeks, collect root nodules and determine the nitrogenase activity of the nodules using the acetylene reduction method (described in the following literature: Wych RD, and Rains DW. Simultaneous measurement of nitrogen fixation estimated by acetylene-ethylene assay and nitrate absorption by soybeans. Plant Physiol. 1978 62(3):443-448.), i.e., the amount of acetylene reduced to ethylene per hour in the roots of each plant, in μM / h / plant. The test results are averaged by group. The test results are shown in the figure. Figure 7 The results showed that, compared with alfalfa R108, pdf9 - 1 mutants and pdf9 - 2 The mutant almost completely lost its nitrogenase activity.

[0086] (6) After completing step (3), culture for 14 days, collect root nodules and observe them using transmission electron microscopy. Some results are shown in [link to results]. Figure 8 (A) The results showed that in the root nodules of Alfalfa R108, the intracellular bacterioids in the nitrogen-fixing zone were normally developed and had a regular, long, rod-shaped morphology. pdf9 - 1 mutants and pdf9 - 2 In the root nodules of mutants, the bacteriophytes within the nitrogen-fixing zone infected cells cannot differentiate normally into long rod-shaped structures.

[0087] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for regulating the plant height of leguminous plants, characterized in that, The method includes: gene editing of a gene encoding the PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding the PDF9 protein is shown in SEQ ID No:

1.

2. A method for regulating the nitrogen fixation capacity of leguminous plants, characterized in that, The method includes: gene editing of a gene encoding the PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding the PDF9 protein is shown in SEQ ID No:

1.

3. A method for regulating the activity of nitrogenase in legumes, characterized in that, The method includes: gene editing of a gene encoding the PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding the PDF9 protein is shown in SEQ ID No:

1.

4. A method for regulating the volume of root nodules in leguminous plants, characterized in that, The method includes: gene editing of a gene encoding the PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding the PDF9 protein is shown in SEQ ID No:

1.

5. A method for regulating the length of mycorrhizal cells in root nodules of leguminous plants, characterized in that, The method includes: gene editing of a gene encoding the PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding the PDF9 protein is shown in SEQ ID No:

1.

6. A method for regulating the plant architecture of leguminous plants, characterized in that, The method includes: gene editing of a gene encoding the PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding the PDF9 protein is shown in SEQ ID No:

1.

7. A method for regulating the fresh weight of leguminous plants, characterized in that, The method includes: gene editing of a gene encoding the PDF9 protein in a legume, wherein the nucleotide sequence of the gene encoding the PDF9 protein is shown in SEQ ID No:

1.

8. The method according to any one of claims 1-7, characterized in that, The legume in question is alfalfa.

9. The method according to any one of claims 1-7, characterized in that, The gene editing was performed using the CRISPR / Cas9 system.

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