CN114478728A

CN114478728A - Application of nKCBP protein in regulating and controlling nitrogen fixation capacity of leguminous plants

Info

Publication number: CN114478728A
Application number: CN202011266389.6A
Authority: CN
Inventors: 孔照胜; 张霞霞; 王琪; 黄弋戈; 吴菁霞
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-11-13
Filing date: 2020-11-13
Publication date: 2022-05-13
Anticipated expiration: 2040-11-13
Also published as: CN114478728B

Abstract

The invention discloses application of nKCBP protein in regulating and controlling nitrogen fixation capacity of leguminous plants. The amino acid sequence of the nKCBP protein is shown in SEQ ID No. 2. The invention utilizes CRISPR/Cas9 technology to edit nKCBP gene at fixed points, knocks out the nKCBP gene of medicago truncatula, and reduces plant height and nitrogen fixation capacity. The invention is very important for researching the high-efficiency nitrogen fixation mechanism of leguminous plants, and provides a new idea for improving the biological nitrogen fixation efficiency. The invention has great application value for plant breeding.

Description

Application of nKCBP protein in regulating and controlling nitrogen fixation capacity of leguminous plants

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of nKCBP protein in regulating and controlling nitrogen fixation capacity of leguminous plants.

Background

Leguminous plants are important economic crops and play a role in the production and economic development process of agriculture and animal husbandry, and the leguminous plants and rhizobia can establish a mutual and beneficial symbiotic relationship to realize biological nitrogen fixation, namely a process that the rhizobia converts molecular nitrogen in the atmosphere into nitrogen which can be absorbed and utilized by plants. Symbiotic nitrogen fixation is a biological nitrogen fixation system with highest nitrogen fixation capacity and maximum nitrogen fixation amount in nature, and the effective utilization of the system is important for the sustainable development of agriculture and the nitrogen circulation of biospheres. The development of modern agriculture depends on the application of nitrogen fertilizer to a great extent, which not only causes huge waste of economy and resources, but also destroys the ecological environment and the development of green agriculture. The symbiotic nitrogen fixation of leguminous plants and rhizobia is an extremely complex biological process, and is controlled by a complex gene network. Therefore, by deeply excavating key genes involved in regulating and controlling symbiotic nitrogen fixation, the method has important guiding significance and application value for further improving the biological nitrogen fixation capacity.

Disclosure of Invention

The invention aims to regulate and control the nitrogen fixation capacity of leguminous plants.

The invention firstly protects the application of the nKCBP protein, which can be at least one of S1) -S6):

s1) regulating and controlling the plant height of leguminous plants;

s2) regulating and controlling the nitrogen fixation capacity of the leguminous plants;

s3) regulating and controlling the activity of the nitrogenase of the leguminous plants;

s4) regulating and controlling the plant type of the leguminous plants;

s5) regulating and controlling the volume of the leguminous plant root nodule;

s6) cultivating leguminous plants with changed plant height, nitrogen fixation capacity, nitrogen fixation enzyme activity, plant type and/or root nodule volume;

the nKCBP protein can be a1) or a2) or a3) or a 4):

a1) the amino acid sequence is protein shown as SEQ ID No. 2;

a2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in SEQ ID No. 2;

a3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a1) or a2), is derived from leguminous plants, and is related to nitrogen fixation capacity, azotase activity, plant height, plant type and/or nodule volume;

a4) protein which has 80% or more than 80% of identity with the protein shown in a1) or a2), is derived from leguminous plants, and is related to nitrogen fixation capacity, nitrogen fixation enzyme activity, plant height, plant type and/or nodule volume.

Wherein, SEQ ID No: 2 consists of 1265 amino acid residues.

To facilitate purification of the protein of a1), the protein of SEQ ID No: 2 to the amino terminus or carboxy terminus of the protein shown in table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein according to a3), 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 not more than 10 amino acid residues.

The protein of a3) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a3) above can be obtained by converting the amino acid sequence of SEQ ID No: 1, and/or by missense mutation of one or more base pairs, and/or by attaching to its 5 'and/or 3' end a coding sequence for the tag shown in table 1 above.

The invention also protects the application of the nucleic acid molecule for coding the nKCBP protein, which can be at least one of S1) -S6):

s1) regulating and controlling the plant height of leguminous plants;

s4) regulating and controlling the plant type of the leguminous plants;

s5) regulating and controlling the volume of the leguminous plant root nodule;

s6) cultivating leguminous plants with changed plant height, nitrogen fixation capacity, nitrogen fixation enzyme activity, plant type and/or root nodule volume.

Any one of the nucleic acid molecules encoding the nKCBP protein can be a DNA molecule shown as b1), b2), b3) or b4) as follows:

b1) the coding region is SEQ ID No: 1;

b2) the nucleotide sequence is SEQ ID No: 1;

b3) a DNA molecule having 80% or more 80% identity to the nucleotide sequence defined by b1) or b2) and encoding the nKCBP protein;

b4) a DNA molecule which hybridizes with the nucleotide sequence defined by b1) or b2) under strict conditions and codes the nKCBP protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, SEQ ID No: 1 consists of 3798 nucleotides, SEQ ID No: 1 encodes the nucleotide sequence of SEQ ID No: 2, or a pharmaceutically acceptable salt thereof.

The nucleotide sequence encoding the nKCBP protein of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 80% or more identity to the nucleotide sequence of the nKCBP protein isolated in the present invention, as long as the nKCBP protein is encoded, are derived from and identical to the nucleotide sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes the identity to the nucleotide sequence of the present invention encoding SEQ ID No: 2 has a nucleotide sequence of 80% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the nKCBP protein. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The nucleic acid molecule for coding the nKCBP protein can be specifically a gene for coding the nKCBP protein and is named as an nKCBP gene.

In any of the above applications, the regulating the plant height of the leguminous plant may be reducing the plant height of the leguminous plant or increasing the plant height of the leguminous plant.

In any of the above applications, the regulating and controlling the nitrogen fixation capacity of the leguminous plant may be reducing the nitrogen fixation capacity of the leguminous plant or improving the nitrogen fixation capacity of the leguminous plant.

In any of the above applications, the regulating and controlling the activity of the leguminous plant nitrogenase may be reducing the activity of the leguminous plant nitrogenase or improving the activity of the leguminous plant nitrogenase.

In any of the above applications, the regulating the plant type of the leguminous plant may be a reduction in the plant type of the leguminous plant or an increase in the plant type of the leguminous plant.

In any of the above applications, the regulating leguminous plant root nodule volume may be a reduction in leguminous plant root nodule volume or an increase in leguminous plant root nodule volume.

In any of the above applications, the leguminous plant with the altered nitrogen fixation capacity, nitrogenase activity, plant height, plant type and/or nodule volume may be "leguminous plant with reduced nitrogen fixation capacity, reduced nitrogenase activity, reduced plant height, reduced plant type and/or reduced nodule volume" or "leguminous plant with improved nitrogen fixation capacity, improved nitrogenase activity, increased plant height, increased plant type and/or increased nodule volume".

The invention also protects the specific sgRNA; the target sequence of the specific sgRNA can be shown as SEQ ID No: 1 at positions 118-137 from the 5' end.

The invention also protects the specific recombinant plasmid; the specific recombinant plasmid contains a coding gene of Cas9 protein and a coding gene of the specific sgRNA.

The invention also protects the application of any one of the specific sgrnas or any one of the specific recombinant plasmids in leguminous plant breeding; the breeding purpose of the leguminous plants can be to reduce the plant height of the leguminous plants, reduce the nitrogen fixation capacity of the leguminous plants, reduce the nitrogen fixation enzyme activity of the leguminous plants, reduce the plant type of the leguminous plants and/or reduce the root nodule volume of the leguminous plants.

The invention also provides a method for preparing a gene-edited leguminous plant, which comprises the following steps:

(1) introducing the coding gene of any one of the specific sgRNAs and the coding gene of the Cas9 protein into a leguminous plant to obtain a transgenic leguminous plant;

(2) screening said transgenic legume for gene-editing legumes;

the gene-editing plant has a reduced plant height, reduced nitrogen fixation capacity, reduced nitrogenase activity, reduced plant type, and/or reduced nodule volume as compared to the recipient plant.

In the above method, the gene encoding any one of the specific sgrnas and the gene encoding Cas9 protein are specifically introduced into the leguminous plant by the recombinant plasmid.

In the above method, the screening of the transgenic leguminous plant for gene editing may specifically be screening of the transgenic leguminous plant for gene editing and inhibition of expression of the nKCBP gene.

The "transgenic leguminous plant in which gene editing occurs and expression of the nKCBP gene is suppressed" as described above may specifically be a transgenic leguminous plant in which the target region is mutated and all of which are homozygous mutant types.

The invention also protects any one of the methods Q1) -Q5).

Q1) A method for reducing the plant height of leguminous plants, comprising the steps of: carrying out gene editing on the gene for coding the nKCBP protein or reducing the activity and/or expression quantity of the nKCBP protein in leguminous plants to obtain the leguminous plants with reduced plant height.

Q2) A method for reducing the nitrogen fixation capacity of leguminous plants, comprising the steps of: carrying out gene editing on the gene for coding the nKCBP protein or reducing the activity and/or expression quantity of the nKCBP protein in leguminous plants to obtain the leguminous plants with reduced nitrogen fixation capacity.

Q3) A method for reducing the nitrogenase activity of leguminous plants, comprising the steps of: carrying out gene editing on the gene for coding the nKCBP protein or reducing the activity and/or expression quantity of the nKCBP protein in leguminous plants to obtain the leguminous plants with reduced nitrogen-fixing enzyme activity.

Q4) a method for reducing the plant type of leguminous plants, comprising the steps of: carrying out gene editing on the gene for coding the nKCBP protein or reducing the activity and/or expression quantity of the nKCBP protein in leguminous plants to obtain the leguminous plants with reduced plant types.

Q5) a method for reducing the volume of legume nodules, comprising the steps of: gene editing of a gene encoding an nKCBP protein or "reduction of the activity and/or expression level of an nKCBP protein in leguminous plants" is carried out to obtain leguminous plants with reduced nodule volume.

In the above method, the "reducing the activity and/or expression level of the nKCBP protein in the leguminous plants" can be achieved by the methods known in the art, such as RNA interference, homologous recombination, gene site-directed editing and the like, so as to achieve the purpose of reducing the activity and/or expression level of the nKCBP protein in the leguminous plants.

In the above method, the "reducing the activity and/or expression level of the nKCBP protein in the leguminous plant" can be specifically realized by knocking out or silencing the nKCBP gene. The knockout includes the knockout of the entire gene, as well as the knockout of a partial segment of the gene.

In the above method, the "reduction of the activity and/or expression level of the nKCBP protein in the leguminous plant" can be specifically achieved by gene editing of the nKCBP gene.

Any one of the above gene editing is realized by means of a CRISPR/Cas9 system.

In the CRISPR/Cas9 system, the target sequence of sgRNA can be as set forth in SEQ ID No: 1 at positions 118-137 from the 5' end.

The gene editing is realized by introducing a specific DNA molecule containing a coding gene of Cas9 protein and a coding gene of the sgRNA into leguminous plants. The gene editing is realized by introducing the recombinant plasmid containing the specific DNA molecule into leguminous plants.

The gene editing is realized by respectively introducing a DNA molecule containing a coding gene of Cas9 protein and a DNA molecule containing a coding gene of sgRNA into leguminous plants.

Any one of the specific recombinant plasmids or the recombinant plasmid can be specifically a recombinant plasmid pCambia1300-Cas 9-nKCBP. The construction process of the recombinant plasmid pCambia1300-Cas9-nKCBP is as follows:

1. and inserting a target sequence into a recognition site of a restriction enzyme BsaI of the vector AtU6-26-sgRNA-SK to obtain an intermediate vector.

2. After the step 1 is completed, the intermediate vector is taken and cut by restriction enzymes NheI and SpeI, and a DNA fragment of about 642bp, namely sgRNA cassette, is recovered.

3. The pCambia1300-pUBQ10: Cas9 plasmid was digested with the restriction enzyme SpeI to give a linearized plasmid.

4. And (3) connecting the linearized plasmid with sgRNA cassette to obtain a recombinant plasmid pCambia1300-Cas 9-nKCBP.

In the present example, when the leguminous plant is medicago truncatula R108, the gene-edited leguminous plant obtained by the above method may be an nkcbp-1 mutant or an nkcbp-2 mutant.

Any one of the above leguminous plants may be Medicago truncatula.

Any one of the medicago truncatula can be medicago truncatula R108 or medicago truncatula A17.

The invention utilizes CRISPR/Cas9 technology to edit nKCBP gene at fixed points, knocks out the nKCBP gene of medicago truncatula, and reduces plant height and nitrogen fixation capacity. The invention is very important for researching the high-efficiency nitrogen fixation mechanism of leguminous plants, and provides a new idea for improving the biological nitrogen fixation efficiency. The invention has great application value.

Drawings

FIG. 1 is a phylogenetic tree analysis of KCBP protein.

FIG. 2 is a tissue expression analysis of the nKCBP gene.

FIG. 3 shows the construction of nKCBP gene knockout vector and the acquisition of nKCBP gene knockout mutant.

FIG. 4 shows that the knockout of nKCBP gene can reduce the plant height, root length, biomass and nitrogen fixation capacity of Medicago truncatula.

FIG. 5 is a graph showing the effect of knock-out of the nKCBP gene on Medicago truncatula symbiont and vacuolar development.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 evolution analysis of KCBP protein and tissue expression analysis of nKCBP protein

Evolutionary tree analysis of KCBP protein

The inventors of the present invention performed gene copy number and evolutionary tree analysis on KCBP proteins of 26 species (including alfalfa of 14 families) using the amino acid sequence of the KCBP protein from Arabidopsis thaliana.

The results are shown in FIG. 1. The result shows that the KCBP protein can perform gene amplification with 7 species of rhizobium symbiotic nodulation in leguminous plants, the gene copy number is increased, and the gene replication phenomenon is generated.

II, nKCBP protein is enriched and expressed in alfalfa root nodule of tribulus

The medicago truncatula has two homologous genes for coding KCBP protein, namely Medtr5g025100 and Medtr8g 072430. The Medtr8g072430 was named as nKCBP gene (Nodulation-specific Kinesin-like regulatory-Binding Protein). The nucleotide sequence of the nKCBP gene is shown as SEQ ID NO: 1, encoding the amino acid sequence shown in SEQ ID NO: 2, and (3) the nKCBP protein shown in the figure.

1. Respectively collecting tissues of the medicago truncatula A17, including roots, leaves, flowers and fruit pods, and root nodules at 4 th, 7 th, 14 th and 21 st days after rhizobium inoculation, and freezing with liquid nitrogen for later use.

2. Total RNA of the tissues collected in step 1 was extracted using a plant RNA rapid extraction kit from Bomader EASYspin. The extraction method refers to kit instruction.

3. Total RNA from each tissue was collected and applied to Invitrogen reverse transcriptase (SuperScript)^TMIII Reverse Transcriptase) to synthesize first strand cDNA.

4. First strand cDNA of the tissue is respectively taken and subjected to real-time quantitative PCR amplification by adopting a CFX96(Bio-Rad) reactor and a Realtime PCR Master Mix (TOYOBO) to obtain the relative expression level of the nKCBP gene (the reference gene is an Actin11 gene).

Primers for detecting the nKCBP gene are 5'-GTATCTCCAGTAGAATCAAGC-3' and 5'-CTACATTGAATGGCGGCCA-3'.

Primers for detecting the Actin11 gene are 5'-ACGAGCGTTTCAGATG-3' and 5'-ACCTCCGATCCAGACA-3'.

The results are shown in FIG. 2A. The result shows that the nKCBP gene is enriched and expressed in the alfalfa root nodule of the tribulus (namely, the nKCBP protein is enriched and expressed in the alfalfa root nodule of the tribulus), and the expression level is relatively low in other tissues.

In addition, the protein coded by Medtr5g025100 is expressed in different tissues of medicago truncatula and has no tissue specificity through detection of the inventor.

Expression of protein nKCBP in different regions of rhizobium organ of medicago truncatula

1. The DNA small fragment between the restriction enzymes SalI and PstI of the pCambia1381 vector was replaced with SEQ ID NO: 3 to obtain recombinant plasmid; the recombinant plasmid was named pCambia 1381-pnKCBP.

SEQ ID NO: 3 is upstream 2185bp of the initiation codon of the nKCBP gene in Medicago truncatula A17.

2. After step 1 is completed, the recombinant plasmid pCambia1381-pnKCBP is transferred into the Agrobacterium rhizogenes MSU440 to obtain the recombinant Agrobacterium rhizogenes.

3. After completion of step 2, the transgenic Tribulus Terrestris medica is obtained by transforming the recombinant Agrobacterium rhizogenes into Tribulus Terrestris medica A17 using Agrobacterium-mediated rhizogenesis transformation method (described in Boisson-Dernier A, Chabaud M, Garcia F, Becard G, Rosenberg C, Barker DG. Agrobacterium rhizogenes-transformed roots of medical trunula for the study of nitrogen-transforming and endomycorrhizal systematic associations. mol. plant Microbe interaction. 200614(6): 695. 700).

4. After the step 3 is completed, inoculating the transgenic medicago truncatula with the Sinorhizobium Sm2011 strain (OD)₆₀₀0.01, the inoculation amount is 1 mL/plant), and root nodules at 4 days, 7 days, 14 days and 21 days after infection are respectively taken for GUS staining and observed.

The results of GUS staining are shown in B of FIG. 2.

The result shows that the protein nKCBP is enriched and expressed in different areas of the rhizobium organ of the medicago truncatula and participates in the regulation and control of rhizobium development.

Example 2 obtaining of nKCBP Gene knockout mutant and application of nKCBP protein in reducing Nitrogen fixation ability of Medicago truncatula

Construction of nKCBP gene knockout vector

An nKCBP gene knockout vector, namely, a recombinant plasmid pCambia1300-Cas9-nKCBP is constructed by adopting a CRISPR/Cas9 system. The target sequence is 5'-GTGATGGATATGACAGTGA-3' (i.e., position 118-137 from the 5 ' end of SEQ ID No: 2). The vector construction methods are referenced in the following documents: yan L, Wei S, Wu Y, Hu R, Li H, Yang W, Xie Q.high-Efficiency Genome Editing in Arabidopsis Using YAO Promoter-drive CRISPR/Cas9 System.mol.plant.20158 (12):1820-1823.

The construction process of the knock-out vector pCambia1300-Cas9-nKCBP is as follows:

1. construction of sgRNA cassette

And inserting a target sequence into a recognition site of a restriction enzyme BsaI of the vector AtU6-26-sgRNA-SK to obtain an intermediate vector.

The position of the target sequence on the nKCBP gene is shown as A in FIG. 3.

II, obtaining of nKCBP Gene knockout mutant (i.e., nKCBP-1 mutant and nKCBP-2 mutant)

1. Transforming the recombinant plasmid pCambia1300-Cas9-nKCBP into agrobacterium tumefaciens EHA105 to obtain recombinant agrobacterium tumefaciens; then, the recombinant agrobacterium is transformed into the Medicago truncatula R108 by using the Medicago truncatula genetic transformation method (described in Medicago Handbook: https:// www.noble.org/Medicago-Handbook /), so as to obtain T₀The transgenic medicago truncatula is simulated.

2. Respectively taking the T obtained in the step 1₀Replacing leaves of a transgenic medicago truncatula plant, and extracting genome DNA; then, taking the genome DNA of each plant as a template, and adopting a primer F: 5'-GTCAACATAGCTCAAATTGTG-3' and primer R: 5'-GAACCAAACATACCCTCAGAG-3' to obtain the corresponding PCR amplification product.

3. Sequencing each PCR amplification product. The sequencing result is compared with the nucleotide sequence of the nKCBP gene.

Co-detection of T₀A transgenic medicago truncatula 10 is generated, wherein one of the plants is a heterozygous mutant (the heterozygous mutant refers to that nKCBP genes of two homologous chromosomes of the plant are mutated but have different mutation forms), specifically, one homologous chromosome is subjected to deletion of 1 nucleotide A at the target position of the nKCBP gene to cause frame shift and early termination, and the other homologous chromosome is subjected to deletion of 2 nucleotide AG at the target position of the nKCBP gene to cause frame shift and early termination, so that the function of the nKCBP protein is lost.

4. Selfing the heterozygous mutant obtained in the step 3 to obtain T₁Transgenic medicago truncatula.

5. According to the above step 2-3, adding T₀Replacement of transgenic Medicago truncatula with T₁The transgenic medicago truncatula is generated, and other steps are not changed.

Two homozygous mutants, an nkcbp-1 mutant and an nkcbp-2 mutant, were finally obtained. Homozygous mutant means that the nKCBP genes of two homologous chromosomes of the plant have the same mutation.

The mutant forms of the nkcbp-1 mutant and the nkcbp-2 mutant at the target site are shown in FIG. 3B. The nKCBP-1 mutant has 1 nucleotide deletion of 'A' in the nKCBP gene, thereby causing frame shift and early termination, and causing the functional deletion of nKCBP protein. The nKCBP-2 mutant has 'AG' 2 nucleotide deletion in nKCBP gene to cause frame shift and early termination, resulting in nKCBP protein function deletion.

Thirdly, knocking out the nKCBP gene can reduce the plant height, the plant type, the root nodule volume and the nitrogen fixation capacity of the medicago truncatula

1. Taking seeds of medicago truncatula (nkcbp-1 mutant, nkcbp-2 mutant or R108 medicago truncatula) to be detected with basically the same size, firstly treating the seeds with concentrated sulfuric acid for 5min, and washing the seeds with clear water for 5 to 10 times; treating with 6% (m/v) sodium hypochlorite solution for 10min, and washing with sterile water for 5-10 times; and then placing the mixture on a shaking table for shaking culture for 24 hours to obtain the treated medicago truncatula seeds to be detected.

2. And (3) after the step 1 is finished, placing the treated alfalfa seeds to be detected in an FM solid culture medium, and carrying out inverted culture at the temperature of 22 ℃ for 12 h.

3. After the step 2 is finished, 20 germinated seeds are planted in vermiculite potted pots containing FM liquid medium (2 seeds in each pot), and after 5 days, the alfalfa rhizobium Sm2011 strain (OD) is inoculated₆₀₀0.01, inoculum size 1mL per strain).

4. After the completion of the step 3, the cultivation is carried out for 4 weeks, and the plant growth state and the root nodule development condition are observed.

The growth status of the plant parts is shown in A in FIG. 4.

The development of part of the nodules is shown in B in FIG. 4.

Results show that compared with alfalfa R108, the nkcbp-1 mutant and the nkcbp-2 mutant have obviously reduced plant heights and plant types; the nodules of the nkcbp-1 and nkcbp-2 mutants were pink in color, but were somewhat smaller in volume and relatively lighter in color than those of alfalfa R108.

5. After completion of step 3, the resulting nodules were cultured for 4 weeks, taken separately and tested for their azotase activity, i.e., the amount of acetylene-reduced ethylene per hour per plant root in. mu.M/h/plant, by acetylene reduction (described in Wych RD, and rain DW. Simultaneous root measurement of nitrogen implantation by acetyl-ethylene assay and nitrate adsorption by sobeybeans. plant Physiol.197862 (3): 443. mu.448.). The test results were averaged by group.

The results are shown in FIG. 4C. Results show that compared with alfalfa R108, the nitrogenase activity of the nkcbp-1 mutant and the nkcbp-2 mutant is remarkably reduced, namely the nitrogenase activity of the nkcbp-1 mutant and the nkcbp-2 mutant is remarkably reduced.

6. After completion of step 3, the cells were cultured for 21 days, and the nodules were collected and observed by transmission electron microscopy.

Partial results are shown in FIG. 5. The result shows that in the root nodule of the medicago truncatula R108, the infected cells of the nitrogen fixation area have obvious central large vacuole, and the thallus like bodies in the cells are normal in development and are in regular long rod shapes; in nodules of the nkcbp-1 mutant and the nkcbp-2 mutant, large central vacuoles cannot be formed in infected cells, a plurality of small vacuoles with irregular shapes and different sizes exist at the edges of the cells, some symbionts are slow in development and cannot form long-rod-shaped normal symbionts, and the symbiont membranes are locally thickened and abnormally developed.

<110> institute of microbiology of Chinese academy of sciences

Application of <120> nKCBP protein in regulation and control of nitrogen fixation capacity of leguminous plants

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 3798

<212> DNA

<213> Medicago truncatula

<400> 1

atgatatttg atatgccacc atccggcgcc caaagtgtga ggacaaacag atcttcattt 60

ggttctatca atggcagtga gggtacacct ttacatagct atgctactgt tagcaatggt 120

gatggatatg acagtgatgg ctccaatttc gcaccaccca caccaacaac tctatcaaca 180

gctattccag cagaacttgc tggagcagca cctttgattg acagatttca ggtggaaggt 240

tttttaaagt tgatgcaaaa gcaaattcac tctgctggga agcgaggatt tttttctaaa 300

cgatctgtgg gaccccaagt tcgagagaaa cttacaattg aagacatgct ttgcttccaa 360

aaggatccaa taccaacatc attgcttaag ctgaatggcg atttggcaag ccgggcaaca 420

aagttgttcc aaatgatttt gaagtatatg ggtgttgatt cgtcttcact aagcttagag 480

gaccgggttg agcttgtagg taaactatac aagcaaagtt tgaagcgttc ggaactccga 540

gatgaacttt ttgtccagat atcaaaacaa acgagaaaca atccagagag ggaatacttg 600

atgaaatcat gggagctaat gtatttatgt gtgtcgtgca tgcctcctag caaagacatt 660

ggaggatatc tgtcagaata tacccataac gtggcgcatg gtgtggctgt tgattctggg 720

atccgaggtg ttgcattgaa cactttaaat gctttgaagc actctctcaa ggctggtcct 780

aggcatataa tacctggacc tgctgagatc gaagctcgga tgactgggaa aaagctcaca 840

actatagtct tctttctgga tgaaacattt gaagaaatta catatgacat gtcaacaacc 900

gtagcggatg ctgtcgagga acttgcagga atcattaaac tgccaacata ctctagcttt 960

ggcttgtacg aatgccgtaa agttgtcaca agcgctaaat catctgattc tggcaatgag 1020

gagtacattg gtttggacga caataaacat atcggggatt tactagcgga atttaaggca 1080

gtaaaagaac gaagtaaggg agaagttttg cactgcaaac tggtcttcaa gaaaaagtta 1140

tttcgtgaat cggatgaagc cgtgacagat cctatgtttt ggcagttgtc ctatgtacaa 1200

ttgcagcatg attatatttt gggtaattat cctattggaa gggaagatgc atcaaagctt 1260

tctgcattgc aaatcttggc tgagattgga tttgttagaa gacctgaatc atgccctgat 1320

tggaattcat ttctcgagcg atttcttcca aggcaaatag caatgactag agctagacgc 1380

gaatgggagt tggacattct ttcttgctat cattcactgg agaatgtcac gaaagatgat 1440

gcaagacaac agtttctgca tataataaga acacttcctt atggcttttc tgtattcttt 1500

aatgttcgca aaattgatga tcctattgga ctcctacctg gacgaataat actagcaatt 1560

aacaagagag gggttcattt ttttcgtccc attccaaagg aatatttgca ctcagctgaa 1620

ttgagagaca taatgcaatt tggaagcagt aacactgctg tattttttaa aatgcgagtt 1680

gccggtgttc ttcacatatt ccagtttgaa accaagcagg gagaagaaat ttgtgtagcc 1740

cttcagacac acataaatga tgtaatgctg cgccgatatt ccaaaacacg atcttctcct 1800

acaggttctt tggataaaga aatttctact gaattcaagt ctccgaattt ggaatcgtac 1860

gagaaacgtg ttcaagattt aacaaaagct gttgaagatt ctcaaaggac ggctgatcaa 1920

ttgatggaaa aattgcatga gaagcaatta caagaagagg agactctcca cgaattagaa 1980

ggcttgaaag aatctttaga aacttccaag aatagtcttg cagaagttac aaatgaccgt 2040

gataggctta catcactatg cagtgaaaaa gataatgaac ttcaggctaa aatgatagaa 2100

aaaaggagct tggaagcaaa gatagccaag ttgaatagtt tgatgataga ggaagttacc 2160

aataaggact ctagtggagc taacgaccga gcgttgcaaa agcttgaaga tgacttgaga 2220

ctttgtagag acgagcttat gttagctgaa gagaccatca aatgcttgac aaatgagaag 2280

ttagttttga gacaaaagat atctgagctt gagaagaaga atactgaaga gattaattat 2340

cttcaacgaa aacttgaaca agaacgcaaa gctctgaaca ctcaagtaca cggccttgaa 2400

agaaaactag atgtgcttaa acaagaatta gttatggctg aatctacact tttggcgaag 2460

gacactgaat tggctgtgtt gaagaacaat ttaaaagaac tagaagattt gagagaaatg 2520

aaagaggaca ttgacagaaa gaatgaacaa acagcttcca tactgaggat gcaagcggca 2580

caactagctg acatggaatc actttataag gaggagcagg ttctcagaaa gcgatatttc 2640

aataccatag aagatatgaa aggaaaaata cgagtttact gtcgtctaag accccttggt 2700

gaaaaagaga ttgccgtgaa agaaagaaaa gttcttactt atgtagacga gtttactgtt 2760

gagcatccat ggaaagatga taaagcaaag caacacatat atgatcgcgt gtttaacggc 2820

aatgcaactc aagaagatgt atttgaagat acaaggtact tggtgcaatc tgctgtagac 2880

ggttataatg tttgcgtatt tgcctatggt caaactggct ctggaaagac gtttacaata 2940

tatggatccg aaaacaatcc cggtcttacg ccacgtgcta ctacagagct ttttagaatt 3000

ttaaggagag atggtaacaa gttttctttt tccttgaagg cgtacatgtt agaattatat 3060

caagatacac ttgtagatct cttgttgccg aagaatgcaa agcgattaaa attggatatt 3120

aagaaggatt caaagggaat ggtaatggta gaaaatgcaa ccactgtgtc gatttccacc 3180

atggaggaat tgaatagaat aatacaaagg ggttcagaac ggcggcatac agcagggaca 3240

caaatgaatg aagaaagctc aagatctcat cttatactat caattgtaat cgaaagtgtc 3300

aatcttcaaa gccaatcaac tgcaagagga aagctgagtt ttgtggatct tgctggctct 3360

gaaagaatta aaaagtcagg ctctgagggt agtcaattga aagaagctca aagtataaac 3420

aaatcattat cagctcttgg agatgttatt agtgctttat cttcaggggg tcaacacata 3480

ccttacagaa atcacaagtt aactatgttg atgagtgatt cacttggagg caacgccaaa 3540

actctcatgt ttgtgaatgt atctccagta gaatcaagct tggatgagac acataactct 3600

cttatgtatg catcgcgtgt gaggtcaatt gtgaatgatc caagcaaaaa catatcttcg 3660

aaagagatcg ctcgactgaa gaaattagtt gcttattgga aggagcaagc aggtaggaaa 3720

ggggaggatg aagatttaga agaaattcaa gataaacgac caactaaaga gcggagcgat 3780

ggccgccatt caatgtag 3798

<210> 2

<211> 1265

<212> PRT

<213>Medicago truncatula

<400> 2

Met Ile Phe Asp Met Pro Pro Ser Gly Ala Gln Ser Val Arg Thr Asn

1 5 10 15

Arg Ser Ser Phe Gly Ser Ile Asn Gly Ser Glu Gly Thr Pro Leu His

20 25 30

Ser Tyr Ala Thr Val Ser Asn Gly Asp Gly Tyr Asp Ser Asp Gly Ser

35 40 45

Asn Phe Ala Pro Pro Thr Pro Thr Thr Leu Ser Thr Ala Ile Pro Ala

50 55 60

Glu Leu Ala Gly Ala Ala Pro Leu Ile Asp Arg Phe Gln Val Glu Gly

65 70 75 80

Phe Leu Lys Leu Met Gln Lys Gln Ile His Ser Ala Gly Lys Arg Gly

85 90 95

Phe Phe Ser Lys Arg Ser Val Gly Pro Gln Val Arg Glu Lys Leu Thr

100 105 110

Ile Glu Asp Met Leu Cys Phe Gln Lys Asp Pro Ile Pro Thr Ser Leu

115 120 125

Leu Lys Leu Asn Gly Asp Leu Ala Ser Arg Ala Thr Lys Leu Phe Gln

130 135 140

Met Ile Leu Lys Tyr Met Gly Val Asp Ser Ser Ser Leu Ser Leu Glu

145 150 155 160

Asp Arg Val Glu Leu Val Gly Lys Leu Tyr Lys Gln Ser Leu Lys Arg

165 170 175

Ser Glu Leu Arg Asp Glu Leu Phe Val Gln Ile Ser Lys Gln Thr Arg

180 185 190

Asn Asn Pro Glu Arg Glu Tyr Leu Met Lys Ser Trp Glu Leu Met Tyr

195 200 205

Leu Cys Val Ser Cys Met Pro Pro Ser Lys Asp Ile Gly Gly Tyr Leu

210 215 220

Ser Glu Tyr Thr His Asn Val Ala His Gly Val Ala Val Asp Ser Gly

225 230 235 240

Ile Arg Gly Val Ala Leu Asn Thr Leu Asn Ala Leu Lys His Ser Leu

245 250 255

Lys Ala Gly Pro Arg His Ile Ile Pro Gly Pro Ala Glu Ile Glu Ala

260 265 270

Arg Met Thr Gly Lys Lys Leu Thr Thr Ile Val Phe Phe Leu Asp Glu

275 280 285

Thr Phe Glu Glu Ile Thr Tyr Asp Met Ser Thr Thr Val Ala Asp Ala

290 295 300

Val Glu Glu Leu Ala Gly Ile Ile Lys Leu Pro Thr Tyr Ser Ser Phe

305 310 315 320

Gly Leu Tyr Glu Cys Arg Lys Val Val Thr Ser Ala Lys Ser Ser Asp

325 330 335

Ser Gly Asn Glu Glu Tyr Ile Gly Leu Asp Asp Asn Lys His Ile Gly

340 345 350

Asp Leu Leu Ala Glu Phe Lys Ala Val Lys Glu Arg Ser Lys Gly Glu

355 360 365

Val Leu His Cys Lys Leu Val Phe Lys Lys Lys Leu Phe Arg Glu Ser

370 375 380

Asp Glu Ala Val Thr Asp Pro Met Phe Trp Gln Leu Ser Tyr Val Gln

385 390 395 400

Leu Gln His Asp Tyr Ile Leu Gly Asn Tyr Pro Ile Gly Arg Glu Asp

405 410 415

Ala Ser Lys Leu Ser Ala Leu Gln Ile Leu Ala Glu Ile Gly Phe Val

420 425 430

Arg Arg Pro Glu Ser Cys Pro Asp Trp Asn Ser Phe Leu Glu Arg Phe

435 440 445

Leu Pro Arg Gln Ile Ala Met Thr Arg Ala Arg Arg Glu Trp Glu Leu

450 455 460

Asp Ile Leu Ser Cys Tyr His Ser Leu Glu Asn Val Thr Lys Asp Asp

465 470 475 480

Ala Arg Gln Gln Phe Leu His Ile Ile Arg Thr Leu Pro Tyr Gly Phe

485 490 495

Ser Val Phe Phe Asn Val Arg Lys Ile Asp Asp Pro Ile Gly Leu Leu

500 505 510

Pro Gly Arg Ile Ile Leu Ala Ile Asn Lys Arg Gly Val His Phe Phe

515 520 525

Arg Pro Ile Pro Lys Glu Tyr Leu His Ser Ala Glu Leu Arg Asp Ile

530 535 540

Met Gln Phe Gly Ser Ser Asn Thr Ala Val Phe Phe Lys Met Arg Val

545 550 555 560

Ala Gly Val Leu His Ile Phe Gln Phe Glu Thr Lys Gln Gly Glu Glu

565 570 575

Ile Cys Val Ala Leu Gln Thr His Ile Asn Asp Val Met Leu Arg Arg

580 585 590

Tyr Ser Lys Thr Arg Ser Ser Pro Thr Gly Ser Leu Asp Lys Glu Ile

595 600 605

Ser Thr Glu Phe Lys Ser Pro Asn Leu Glu Ser Tyr Glu Lys Arg Val

610 615 620

Gln Asp Leu Thr Lys Ala Val Glu Asp Ser Gln Arg Thr Ala Asp Gln

625 630 635 640

Leu Met Glu Lys Leu His Glu Lys Gln Leu Gln Glu Glu Glu Thr Leu

645 650 655

His Glu Leu Glu Gly Leu Lys Glu Ser Leu Glu Thr Ser Lys Asn Ser

660 665 670

Leu Ala Glu Val Thr Asn Asp Arg Asp Arg Leu Thr Ser Leu Cys Ser

675 680 685

Glu Lys Asp Asn Glu Leu Gln Ala Lys Met Ile Glu Lys Arg Ser Leu

690 695 700

Glu Ala Lys Ile Ala Lys Leu Asn Ser Leu Met Ile Glu Glu Val Thr

705 710 715 720

Asn Lys Asp Ser Ser Gly Ala Asn Asp Arg Ala Leu Gln Lys Leu Glu

725 730 735

Asp Asp Leu Arg Leu Cys Arg Asp Glu Leu Met Leu Ala Glu Glu Thr

740 745 750

Ile Lys Cys Leu Thr Asn Glu Lys Leu Val Leu Arg Gln Lys Ile Ser

755 760 765

Glu Leu Glu Lys Lys Asn Thr Glu Glu Ile Asn Tyr Leu Gln Arg Lys

770 775 780

Leu Glu Gln Glu Arg Lys Ala Leu Asn Thr Gln Val His Gly Leu Glu

785 790 795 800

Arg Lys Leu Asp Val Leu Lys Gln Glu Leu Val Met Ala Glu Ser Thr

805 810 815

Leu Leu Ala Lys Asp Thr Glu Leu Ala Val Leu Lys Asn Asn Leu Lys

820 825 830

Glu Leu Glu Asp Leu Arg Glu Met Lys Glu Asp Ile Asp Arg Lys Asn

835 840 845

Glu Gln Thr Ala Ser Ile Leu Arg Met Gln Ala Ala Gln Leu Ala Asp

850 855 860

Met Glu Ser Leu Tyr Lys Glu Glu Gln Val Leu Arg Lys Arg Tyr Phe

865 870 875 880

Asn Thr Ile Glu Asp Met Lys Gly Lys Ile Arg Val Tyr Cys Arg Leu

885 890 895

Arg Pro Leu Gly Glu Lys Glu Ile Ala Val Lys Glu Arg Lys Val Leu

900 905 910

Thr Tyr Val Asp Glu Phe Thr Val Glu His Pro Trp Lys Asp Asp Lys

915 920 925

Ala Lys Gln His Ile Tyr Asp Arg Val Phe Asn Gly Asn Ala Thr Gln

930 935 940

Glu Asp Val Phe Glu Asp Thr Arg Tyr Leu Val Gln Ser Ala Val Asp

945 950 955 960

Gly Tyr Asn Val Cys Val Phe Ala Tyr Gly Gln Thr Gly Ser Gly Lys

965 970 975

Thr Phe Thr Ile Tyr Gly Ser Glu Asn Asn Pro Gly Leu Thr Pro Arg

980 985 990

Ala Thr Thr Glu Leu Phe Arg Ile Leu Arg Arg Asp Gly Asn Lys Phe

995 1000 1005

Ser Phe Ser Leu Lys Ala Tyr Met Leu Glu Leu Tyr Gln Asp Thr

1010 1015 1020

Leu Val Asp Leu Leu Leu Pro Lys Asn Ala Lys Arg Leu Lys Leu

1025 1030 1035

Asp Ile Lys Lys Asp Ser Lys Gly Met Val Met Val Glu Asn Ala

1040 1045 1050

Thr Thr Val Ser Ile Ser Thr Met Glu Glu Leu Asn Arg Ile Ile

1055 1060 1065

Gln Arg Gly Ser Glu Arg Arg His Thr Ala Gly Thr Gln Met Asn

1070 1075 1080

Glu Glu Ser Ser Arg Ser His Leu Ile Leu Ser Ile Val Ile Glu

1085 1090 1095

Ser Val Asn Leu Gln Ser Gln Ser Thr Ala Arg Gly Lys Leu Ser

1100 1105 1110

Phe Val Asp Leu Ala Gly Ser Glu Arg Ile Lys Lys Ser Gly Ser

1115 1120 1125

Glu Gly Ser Gln Leu Lys Glu Ala Gln Ser Ile Asn Lys Ser Leu

1130 1135 1140

Ser Ala Leu Gly Asp Val Ile Ser Ala Leu Ser Ser Gly Gly Gln

1145 1150 1155

His Ile Pro Tyr Arg Asn His Lys Leu Thr Met Leu Met Ser Asp

1160 1165 1170

Ser Leu Gly Gly Asn Ala Lys Thr Leu Met Phe Val Asn Val Ser

1175 1180 1185

Pro Val Glu Ser Ser Leu Asp Glu Thr His Asn Ser Leu Met Tyr

1190 1195 1200

Ala Ser Arg Val Arg Ser Ile Val Asn Asp Pro Ser Lys Asn Ile

1205 1210 1215

Ser Ser Lys Glu Ile Ala Arg Leu Lys Lys Leu Val Ala Tyr Trp

1220 1225 1230

Lys Glu Gln Ala Gly Arg Lys Gly Glu Asp Glu Asp Leu Glu Glu

1235 1240 1245

Ile Gln Asp Lys Arg Pro Thr Lys Glu Arg Ser Asp Gly Arg His

1250 1255 1260

Ser Met

　　1265

<210> 3

<211> 2185

<212> DNA

<213> Medicago truncatula

<400> 3

gacggatgga tgggtctaat cccatcatcc attacgatga tcaatttttc tctataaaac 60

aacgatagac gactaatttg aggggtgtaa tcccatcatc cattacgaag atccatttaa 120

agtcaaatca taatagtacc gtatgaattt atcccaccga aattgatgct taaagaaatc 180

gaaattgaaa ctttaagaac acattctaaa atcttaaacc aatcattaaa tcaaccctaa 240

acaaattaaa attagtttct tattagctta aactttcttc tcgaaaaatg aaaaattaat 300

ccccaacctt gctaataggc gtcaacaatt tttttaacta ttccgtaaaa aaaagtttta 360

aactgtttcc ccccaatttt aaagtaaaat atacattttt aattatacaa ttaaattgga 420

aaaccctatg agctggaaaa tgaagggtgg gtgacgacta agcaagaatt ttgggtggcc 480

tcttcccgcc ggaggcagac agtgacaaat tttatacaaa gattgtgggg tccactacga 540

ccttttcaaa ttatcatgaa ttattattaa attatatttt tatattacgc cacatatcta 600

tgacaaatat atttattcaa aaataaaaag acaaagtgta aacaaaaaat ggcgaaattt 660

ttatcattaa tctcatctgt ctatatttga caaagtttag ataattacat attttttatg 720

aaacaatgaa tttaatttta accgaagatg tcacatttat ttaaatacat tttttattga 780

aagtatgtga cattacacta acacgaaatg acataattgt agagtgtgtg aaaaaccaaa 840

agatagtaac acaaacaaaa aatatgtaca ttaatatcat atgcatttaa taaataaatt 900

agggtcttgc taaccaatgt cctcaaggta atagttaagt aaactaaaaa tagtatattt 960

atattgaatt cagcaatatt ttgactttta aaaagttaaa ttcttcactt ttcacccttt 1020

tttcaataca tgtttttatt tttatcatct taaccaatgc ttcaagggta cttttagttt 1080

ttcctaataa atgaagggaa gaagagaaag agactcacat ttatgtaaac caaagtattt 1140

ggccgcataa tcactagatt atgagaatct tttttcggtt gaatagttat aaatcatcaa 1200

ttactatcaa ttattattaa ctccaaagac cattcatatt ttagattttt tctaaacaaa 1260

acactcaaac aaacatagtc ttatgccaaa tggaaatgca agatttaaaa aataaaaact 1320

atataatata atataatgtt acttaaatga caatgaagtt tatgtttatg tatatttagt 1380

tgtgtaacaa gaagaatctt gtttatctgt gttttctttc atgtcaatct ttatttttga 1440

gtgaagaaga agaaagtgta aaagtagttt aagttcccca aaagcctcat tgtttaataa 1500

tgttctcgtg actttctctc acctgttttt tttttgtcca ctggtaacaa tttcagcctc 1560

aaaaatcata acttcaatta tttttctttc atttttcaac caaatctatt cttaatttgc 1620

ttcatcttca ctttctcact ggtccagatc tctctattgc actgcacacc attgtttgtt 1680

ccattgctgc tacaaaggtt gtttttttcc cttcatctta agctcattct tcatgttttg 1740

gatcctcatg tgttttttag tggaaatgca aaagtttctt agttttgtta accttgtact 1800

tgctagttta atgttgtagc attttggttt ctgggtctgt ttctttcttt gtccttcata 1860

attgttatgt tttatttttt gttcatgaaa ttcatgaatt gttaaaaaaa ttgtattttt 1920

gttaatttga tgtatttttg tttgtttgtt tctgttctgt tctatttatg atatggtttt 1980

gaatttatac ctgacttgtg ttttatgcgg caatttacaa actaacattt ttatttaagt 2040

caacatagct caaattgtgt gtgtgtgtgt gtgttgtatt gtttgaactt atgtttaggt 2100

ttttggtgat gcagtggatg agcttgaaga agaacaagct ttgttgcttt gactagtttc 2160

aatttttgtt tgttggcaaa agaat 2185

Claims

The application of the nKCBP protein is at least one of S1) -S6):

s1) regulating and controlling the plant height of leguminous plants;

s2) regulating and controlling the nitrogen fixation capacity of the leguminous plants;

s3) regulating and controlling the activity of the nitrogenase of the leguminous plants;

s4) regulating and controlling the plant type of the leguminous plants;

s5) regulating and controlling the volume of the leguminous plant root nodule;

s6) cultivating leguminous plants with changed plant height, nitrogen fixation capacity, nitrogen fixation enzyme activity, plant type and/or root nodule volume;

the nKCBP protein is a1) or a2) or a3) or a 4):

a1) the amino acid sequence is protein shown in SEQ ID No. 2;

a2) a fusion protein obtained by connecting labels at the N end or/and the C end of the protein shown in SEQ ID No. 2;

a3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a1) or a2), is derived from leguminous plants, and is related to nitrogen fixation capacity, azotase activity, plant height, plant type and/or nodule volume;

a4) protein which has 80% or more than 80% of identity with the protein shown in a1) or a2), is derived from leguminous plants, and is related to nitrogen fixation capacity, nitrogen fixation enzyme activity, plant height, plant type and/or nodule volume.
2. Use of a nucleic acid molecule encoding said nKCBP protein of claim 1, which is at least one of S1) -S6):

s1) regulating and controlling the plant height of leguminous plants;

s2) regulating and controlling the nitrogen fixation capacity of the leguminous plants;

s3) regulating and controlling the activity of the nitrogenase of the leguminous plants;

s4) regulating and controlling the plant type of the leguminous plants;

s5) regulating and controlling the volume of the leguminous plant root nodule;

s6) cultivating leguminous plants with changed plant height, nitrogen fixation capacity, nitrogen fixation enzyme activity, plant type and/or root nodule volume.
3. Use according to claim 1 or 2, characterized in that:

the leguminous plant height is regulated to be reduced or increased;

the regulation and control of the nitrogen fixation capacity of the leguminous plants is to reduce the nitrogen fixation capacity of the leguminous plants or improve the nitrogen fixation capacity of the leguminous plants;

the regulating and controlling of the activity of the nitrogen-fixing enzyme of the leguminous plants is to reduce the activity of the nitrogen-fixing enzyme of the leguminous plants or improve the activity of the nitrogen-fixing enzyme of the leguminous plants;

the plant type of the leguminous plant is regulated to be reduced or increased;

the regulating and controlling the volume of the leguminous plant root nodule is that the volume of the leguminous plant root nodule is reduced or the volume of the leguminous plant root nodule is increased;

the leguminous plant with the nitrogen fixing capacity, the nitrogen fixing enzyme activity, the plant height, the plant type and/or the root nodule volume changed is cultivated to 'the leguminous plant with the reduced nitrogen fixing capacity, the reduced nitrogen fixing enzyme activity, the reduced plant height, the reduced plant type and/or the reduced root nodule volume' or 'the leguminous plant with the improved nitrogen fixing capacity, the improved nitrogen fixing enzyme activity, the increased plant height, the increased plant type and/or the increased root nodule volume'.
4. A specific sgRNA or a specific recombinant plasmid;

the target sequence of the specific sgRNA is shown as SEQ ID No: 1 at positions 118-137 from the 5' end;

the specific recombinant plasmid contains a coding gene of Cas9 protein and a coding gene of the specific sgRNA.
5. Use of the specific sgRNA or the specific recombinant plasmid of claim 4 in leguminous plant breeding; the breeding purpose of the leguminous plants is to reduce the plant height of the leguminous plants, reduce the nitrogen fixation capacity of the leguminous plants, reduce the nitrogen fixation enzyme activity of the leguminous plants, reduce the plant type of the leguminous plants and/or reduce the root nodule volume of the leguminous plants.
6. The use of claim 1, 2, 3 or 5, wherein: the leguminous plant is medicago truncatula.
7. A method of preparing a gene-edited leguminous plant comprising the steps of:

(1) introducing a gene encoding the specific sgRNA of claim 4 and a gene encoding the Cas9 protein into a leguminous plant to obtain a transgenic leguminous plant;

(2) screening said transgenic legume for gene-editing legumes;

the gene-editing plant has reduced plant height, reduced nitrogen fixation capacity, reduced nitrogenase activity, reduced plant type, and/or reduced nodule volume as compared to the recipient plant.
8, Q1) -Q5):

q1) A method for reducing the plant height of leguminous plants, comprising the steps of: carrying out gene editing on a gene for coding nKCBP protein or reducing the activity and/or expression quantity of the nKCBP protein in leguminous plants to obtain leguminous plants with reduced plant height;

q2) A method for reducing the nitrogen fixation capacity of leguminous plants, comprising the steps of: carrying out gene editing on a gene for coding nKCBP protein or reducing the activity and/or expression quantity of the nKCBP protein in leguminous plants to obtain the leguminous plants with reduced nitrogen fixation capacity;

q3) A method for reducing the nitrogenase activity of leguminous plants, comprising the steps of: carrying out gene editing on the gene for coding the nKCBP protein or reducing the activity and/or expression quantity of the nKCBP protein in leguminous plants to obtain leguminous plants with reduced activity of the azotobacter;

q4) A method for reducing the plant type of leguminous plants, comprising the steps of: carrying out gene editing on a gene for coding nKCBP protein or reducing the activity and/or expression quantity of the nKCBP protein in leguminous plants to obtain leguminous plants with reduced plant types;

q5) a method for reducing the volume of legume nodules comprising the steps of: gene editing of a gene encoding an nKCBP protein or "reduction of the activity and/or expression level of an nKCBP protein in leguminous plants" is carried out to obtain leguminous plants with reduced nodule volume.
9. The method of claim 8, wherein: the gene editing is realized by means of a CRISPR/Cas9 system; in the CRISPR/Cas9 system, the target sequence of sgRNA is shown in SEQ ID No: 1 at positions 118-137 from the 5' end.
10. The method of any of claims 7 to 9, wherein: the leguminous plant is medicago truncatula.