CN113429467A

CN113429467A - Application of NPF7.6 protein in regulation and control of nitrogen tolerance of leguminous plant root nodule

Info

Publication number: CN113429467A
Application number: CN202010199885.8A
Authority: CN
Inventors: 孔照胜; 王琪; 张霞霞; 黄弋戈; 任静
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2021-09-24
Anticipated expiration: 2040-03-20
Also published as: CN113429467B

Abstract

The invention discloses an application of NPF7.6 protein in regulation and control of nitrogen tolerance of leguminous plant nodules, wherein the amino acid sequence of the NPF7.6 protein is shown in SEQ ID No. 2. The invention provides a method for improving nitrogen resistance of leguminous plant nodules, which is characterized in that a CRISPR/Cas9 technology is used for editing a gene encoding NPF7.6 protein at a fixed point, namely the NPF7.6 gene of medicago truncatula is knocked out, so that a new germplasm with obviously improved nitrogen resistance of nodules is obtained; the improvement of the nitrogen tolerance of the root nodule is reflected by the increase of the biomass of the root nodule and the increase of the length of the root nodule. The invention has great application value for plant breeding.

Description

Application of NPF7.6 protein in regulation and control of nitrogen tolerance of leguminous plant root nodule

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of NPF7.6 protein in regulation and control of nitrogen tolerance of leguminous plant nodules.

Background

Biological nitrogen fixation can be realized between leguminous plants and rhizobia through establishing a mutualistic symbiotic relationship, namely, the rhizobia converts nitrogen in the air into a form which can be utilized by plants to provide nutrition for the plants, and the plants provide necessary energy and carbon sources for the rhizobia. According to statistics, about 5000 ten thousand tons of nitrogen nutrition is provided for agricultural production by biological nitrogen fixation in nature. However, nitrogen fertilizer residues input in modern agricultural production are extremely unfavorable for biological nitrogen fixation, and due to the excessive application of nitrogen fertilizer, the nitrogen concentration in soil is increased, so that the utilization and supply of the efficient and clean nitrogen source of biological nitrogen fixation are greatly damaged. Therefore, by searching key genes participating in the nitrogen transport process, the creation of symbiosis-related mutants capable of tolerating high nitrogen stress has important significance for improving the nitrogen fixation efficiency of leguminous plants.

In recent years, the creation of mutants by using a CRISPR/Cas9 technology-mediated plant gene editing technology and the acquisition of functional plant materials become a new idea for crop variety improvement.

Disclosure of Invention

The invention aims to improve the nitrogen resistance of leguminous plant nodules.

The invention firstly protects the application of NPF7.6 protein, which can be at least one of S1) -S3):

s1) regulating and controlling nitrogen absorption of leguminous plants;

s2) regulating and controlling the nitrogen resistance of the leguminous plant root nodule;

s3) cultivating leguminous plants with altered nitrogen uptake and/or altered nodule nitrogen tolerance;

the NPF7.6 protein may 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 absorption and/or nodule nitrogen resistance;

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 absorption and/or nitrogen tolerance of root nodules.

Wherein, SEQ ID No: 2 consists of 589 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 NPF7.6 protein, which can be at least one of S1) -S3):

s1) regulating and controlling nitrogen absorption of leguminous plants;

s3) cultivating leguminous plants with altered nitrogen uptake and/or altered root nodule nitrogen tolerance.

Any one of the above nucleic acid molecules encoding NPF7.6 protein may be DNA molecule as shown in b1) or b2) or b3) or b 4):

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 than 80% identity with the nucleotide sequence defined by b1) or b2) and encoding the NPF7.6 protein;

b4) a DNA molecule which hybridizes with the nucleotide sequence defined by b1) or b2) under strict conditions and codes for the NPF7.6 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 1770 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 NPF7.6 protein of the invention can be readily mutated by one 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 NPF7.6 protein isolated according to the present invention, as long as they encode the NPF7.6 protein, 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, or 85% or more, or 90% or more, or 95% or more, of the nucleotide sequence of the NPF7.6 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 encoding the NPF7.6 protein may specifically be a gene encoding the NPF7.6 protein, designated NPF7.6 gene.

In any of the above applications, the regulating nitrogen absorption by the legume may be promoting nitrogen absorption by the legume or inhibiting nitrogen absorption by the legume.

In any of the above applications, the regulating the nitrogen tolerance of the legume root nodule may be to increase the nitrogen tolerance of the legume root nodule or decrease the nitrogen tolerance of the legume root nodule.

In any of the above applications, the legume having altered nutrient nitrogen uptake and/or altered nodule nitrogen tolerance may be a "legume having increased nutrient nitrogen uptake and/or reduced nodule nitrogen tolerance" or a "legume having decreased nutrient nitrogen uptake and/or increased nodule nitrogen tolerance".

The invention also provides a method for promoting nitrogen absorption, which comprises the following steps: increasing the activity and/or expression level of NPF7.6 protein in cells, tissues or individuals.

In the above method, the "increasing activity and/or expression level of NPF7.6 protein in cells, tissues or individuals" may be achieved by increasing expression level and/or activity of NPF7.6 protein by means of multiple copies, by means of altering promoters, regulatory factors, transgenes, etc., which are well known in the art.

In the above method, the "increasing activity and/or expression level of NPF7.6 protein in a cell, tissue or subject" may be achieved by introducing any of the nucleic acid molecules encoding NPF7.6 protein described above into the cell, tissue or subject.

The "introducing a nucleic acid molecule encoding the NPF7.6 protein into a cell, tissue or individual" may be specifically effected by introducing a recombinant vector comprising any of the nucleic acid molecules described above into a cell, tissue or individual. The "introduction of a recombinant vector containing a nucleic acid molecule" described above may be specifically a method in which a recombinant vector containing the nucleic acid molecule is linearized and then introduced. Linearization can be achieved by a single enzyme cut (e.g., the ApaI endonuclease).

Any one of the above recombinant vectors containing nucleic acid molecules may be a vector obtained by inserting a nucleotide sequence of SEQ ID NO: 1 in the sequence listing.

Any one of the above recombinant vectors containing nucleic acid molecules may specifically be a vector in which the nucleotide sequence of SEQ ID NO: 1 to obtain the recombinant plasmid.

Any of the above cells may be animal cells or plant cells. The animal cell can be Xenopus laevis oocyte (stage V-VI).

Any of the above tissues may be animal or plant tissues.

Any of the above individuals may be animal individuals or plant individuals.

Any of the above plants may be a leguminous plant.

Any of the above promoting nitrogen uptake may be promoting nitrogen uptake under low nitrogen (0.2mM) or high nitrogen (10mM) conditions.

The invention also provides a method for improving the nitrogen resistance of leguminous plant root nodules, which comprises the following steps: gene editing of a gene encoding NPF7.6 protein (i.e., NPF7.6 gene) or "reduction of NPF7.6 protein activity and/or expression level in leguminous plants" is performed.

In the above method, the "reducing the activity and/or expression level of NPF7.6 protein in leguminous plants" can be achieved by RNA interference, homologous recombination, gene site-directed editing, and other methods known in the art.

In the above method, the "reducing activity and/or expression level of NPF7.6 protein in leguminous plants" may be specifically achieved by knocking out or silencing NPF7.6 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 NPF7.6 protein in leguminous plants" may be specifically achieved by gene editing of NPF7.6 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 from the 5' end, positions 28-47.

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.

The invention also protects the specific sgRNA; the target sequence of the specific sgRNA can be shown as SEQ ID No: 1 from the 5' end, positions 28-47.

The invention also protects the specific recombinant plasmid; the specific recombinant plasmid can contain a gene encoding Cas9 protein and a gene encoding 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 of leguminous plants may aim at improving nitrogen tolerance and/or inhibiting nitrogen uptake of nodules.

The invention also provides a method for preparing the 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;

compared to leguminous plants, the gene-edited leguminous plants have improved nitrogen tolerance of nodules.

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 transgenic leguminous plants for gene editing may specifically be screening of transgenic leguminous plants in which gene editing occurs and expression of NPF7.6 gene is suppressed from the transgenic leguminous plants.

The "transgenic leguminous plant in which gene editing occurs and expression of NPF7.6 gene is suppressed" described above may specifically be a transgenic leguminous plant in which a target region is mutated and which is all homozygous mutant.

Any one of the specific recombinant plasmids or the recombinant plasmid can be specifically a recombinant plasmid pCAMBIA 1300-sgRNA. The construction process of the recombinant plasmid pCAMBIA1300-sgRNA is as follows:

1. the recognition site of restriction enzyme BsaI of vector AtU6-26-sgRNA-SK is inserted with the sequence shown in SEQ ID No: 1 from position 28 to 47 from the 5' end 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-pUBQ 10-Cas 9 plasmid was digested with the restriction enzyme SpeI to obtain a linearized plasmid.

4. And connecting the linearized plasmid with the sgRNA cassette to obtain the recombinant plasmid pCAMBIA 1300-sgRNA.

In the examples of the present invention, when the leguminous plant is medicago truncatula R108, the gene-edited leguminous plant obtained by the above method may be the npf 7.6.6-1 mutant or the npf 7.6.6-2 mutant.

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

Any one of the medicago truncatula can be specifically medicago truncatula R108.

Any of the above-described nodule biomass may be a fresh weight of nodules and/or a dry weight of nodules.

Any of the above nitrogen absorptions may specifically be nitrate absorptions.

Any of the above-described improvements in nitrogen tolerance may be manifested as an increase in nodule biomass and/or nodule length.

Any of the above-mentioned nitrogen resistance may be nitrogen resistance under a nitrogen concentration of 20mM or less.

The inventor utilizes CRISPR/Cas9 technology to edit NPF7.6 gene at fixed points, knocks out Zygophylli alfalfa NPF7.6 gene, and obtains a new germplasm with obviously improved nitrogen resistance of root nodule; the improvement of the nitrogen tolerance of the root nodule is reflected by the increase of the biomass of the root nodule and the increase of the length of the root nodule. The present invention utilizes clover as material to create leguminous plant material insensitive to high nitrogen (20mM) concentration and to provide a new method for improving the variety of leguminous crops. The invention has great application value.

Drawings

FIG. 1 is a tissue-specific expression analysis of NPF7.6 gene.

FIG. 2 shows the nitrate transport assay of NPF7.6 protein.

FIG. 3 shows that the knockout of NPF7.6 gene can improve the nitrogen tolerance of alfalfa root nodules of Tribulus terrestris.

FIG. 4 shows that the knockout of NPF7.6 gene has no effect on the fresh weight, main root length and lateral root number of Medicago truncatula.

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 a conventional biochemical reagent store unless otherwise specified.

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

Example 1 tissue-specific expression analysis of NPF7.6 protein

First, NPF7.6 protein is specifically expressed in R108 root nodule organ of medicago truncatula

The inventor of the invention carries out Blast analysis on 97 NPFs gene sequences of medicago truncatula R108 on an MtGEA website (the website is https:// MtGEA. noble. org/v3/), and determines the NPF genes with high expression and specific expression in a root nodule organ according to the expression mode of each NPF gene.

The expression pattern of the NPF7.6 gene in Medicago truncatula R108 is shown in FIG. 1A.

The results show that the NPF7.6 gene (the nucleotide sequence is shown as SEQ ID NO: 1) is specifically expressed in R108 root nodule organs of Medicago truncatula.

Secondly, the promoter of the NPF7.6 gene specifically promotes the expression of GUS gene in the root nodule vascular bundle

1. The DNA fragment between restriction enzymes PstI and BamHI of pCambia1391z vector was replaced with SEQ ID NO: 3 to obtain a recombinant plasmid pCambia1391 z-pNPF7.6.

SEQ ID NO: 3 is about 1.9kb upstream of the initiation codon of the NPF7.6 gene in Medicago truncatula R108.

2. After completion of step 1, the recombinant plasmid pCambia1391z-pNPF7.6 was transformed into Agrobacterium rhizogenes msu440 to obtain recombinant Agrobacterium rhizogenes.

3. After step 2 is completed, the recombinant Agrobacterium rhizogenes is transformed into Tribulus terrestris R108 by the hairy root transformation method (described in Boisson-Derner A, Chabaud M, Garcia F, Becard G, Rosenberg C, Barker DG. Agrobacterium rhizogenes-transformed roots of medical truncus for the student of nitrogen-transforming and endomorphic systematic associations. mol Plant Microbe interaction.2001; 14(6): 695-700. doi:10.1094/MPMI.2001.14.6.695) to obtain transgenic Tribulus terrestris alfalfa.

4. After the step 3 is completed, inoculating the transgenic medicago truncatula with the Sinorhizobium Sm2011 strain (OD)₆₀₀Is 0.01Inoculation amount is 1 mL/plant), after 3 weeks, mature root nodules are taken and are respectively subjected to GUS staining and 'resin section preparation and ruthenium red staining', and observation is carried out.

The results of GUS staining are shown in FIG. 1B.

The results of resin section preparation and ruthenium red staining are shown in FIG. 1C.

The results show that the promoter of the NPF7.6 gene specifically promotes the expression of GUS gene in the root nodule vascular bundle.

The NPF7.6 gene encodes SEQ ID NO: 2, namely the NPF7.6 protein is specifically expressed in R108 root nodule organs of medicago truncatula.

Example 2 nitrate transport assay of NPF7.6 protein

1. The restriction enzymes EcoRI and XbaI of the pGEMHE vector were inserted between SEQ ID NO: 1 to obtain a recombinant plasmid pGEMHE-NPF 7.6. The recombinant plasmid pGEMHE-NPF7.6 expresses the nucleotide sequence shown in SEQ ID NO: 2, NPF7.6 protein.

2. And (3) after the step 1 is finished, taking the recombinant plasmid pGEMHE-NPF7.6, and carrying out enzyme digestion by using ApaI endonuclease to obtain a linearized recombinant plasmid.

3. After completion of step 2, the linearized recombinant plasmid was injected into Xenopus laevis oocytes using a syringe (stage V-VI), and then treated at ND96 Buffer for 24h at 18 ℃.

The solute of ND96 Buffer and its concentration are 96mM NaCl, 2mM KCl, 1mM MgCl₂，1.8mM CaCl₂5mM HEPES, 50mg/mL gentamicin sulfate; the solvent is water; the pH was 7.4.

4. After step 3 is completed, the nitrate transport capacity of the NPF7.6 protein is detected by using low-affinity and high-affinity nitrate transport experiments. The method comprises the following specific steps: by means of stable isotopes¹⁵N-labelled K¹⁵NO₃Processing Xenopus laevis oocytes, and determining with isotope ratio mass spectrometer (ThermoFinnigan Delta PlusXP; Flash EA1112)¹⁵And (4) N content. Low affinity and high affinity nitrate transport assays¹⁵NO₃Concentrations of 0.2mM and 10mM, respectively.

The linearized recombinant plasmid was replaced with an equal volume of water as a negative control according to the procedure described above.

According to the above steps, SEQ ID NO: 1 was replaced with the nucleotide sequence of the coding region of the AtCHL1 gene as a positive control. The AtCHL1 gene encodes a southwestern nitrate transporter.

The results of the low affinity nitrate transport experiments are shown in FIG. 2A (H)₂0 is negative control, NPF7.6 is experimental group, CHL1 is positive control).

The results of the high affinity nitrate transport experiments are shown in FIG. 2B (H)₂0 is negative control, NPF7.6 is experimental group, CHL1 is positive control).

The results show that the NPF7.6 protein has nitrate uptake capacity under both low nitrogen (0.2mM) and high nitrogen (10mM) treatments. Therefore, the NPF7.6 protein can regulate and control the absorption of plant nitrate, namely improve the absorption of plant nitrogen.

5. The nitrate transport kinetics analysis of NPF7.6 protein mainly included 10 nitrate concentrations (0.05mM, 0.1mM, 0.2mM, 0.5mM, 1mM, 2mM, 5mM, 10mM, 20mM and 30mM, pH5.5), and the nitrate uptake values were obtained after fitting using the Michaelis-Menten equation.

The results of the nitrate transport kinetic analysis of the NPF7.6 protein are shown in FIG. 2C.

Thus, the NPF7.6 protein is a high-affinity nitrate transporter.

Example 3 use of the NPF7.6 Gene in regulating the number, length and fresh weight of nodules of Medicago sativa L.Tribuli I. NPF 7.6.6-1 mutant and NPF 7.6.6-2 mutant

The target sequence is 5'-GAGGTTGTTATGGAAGAAAG-3' (i.e., positions 28-47 from the 5 ' end of SEQ ID No: 1).

The primer pair is composed of a primer NPF 7.6-sgRNA-F: 5'-ATTGGAGGTTGTTATGGAAGAAAG-3' and primer NPF 7.6-sgRNA-R: 5'-AAACCTTTCTTCCATAACAACCTC-3', for amplifying the target.

1. The target sequence was inserted into the recognition site of the restriction enzyme BsaI of vector AtU 6-26-sgRNA-SK. An intermediate vector is obtained.

The position of the target sequence on the NPF7.6 gene is shown in FIG. 3A.

3. The pCAMBIA1300-pUBQ10: Cas9 plasmid (described in Yan L, Wei S, Wu Y, et al, high-Efficiency Genome Editing in Arabidopsis Using YAO Promoter-Driven CRISPR/Cas9 System. mol plant.2015; 8(12) 1820-1823. doi:10.1016/j. mol p.2015.10.004) was digested with the restriction enzyme SpeI to give a linearized plasmid.

4. The linearized plasmid and the sgRNA cassette were ligated with T4 DNA ligase (Takara) to obtain the recombinant plasmid pCAMBIA 1300-sgRNA.

5. After step 4 is completed, the recombinant plasmid pCAMBIA1300-sgRNA is transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium.

6. After step 5 is completed, the recombinant Agrobacterium is transformed into Medicago truncatula R108 by the method of genetic transformation of Medicago truncatula (described in Song Y, Nolan KE, Rose RJ. Stable transformation of medical truncatalacv. Jelong for gene analysis using Agrobacterium tumefaciens. MethodsMol biol. 2013; 1069: 203-214. doi:10.1007/978-1-62703-₀The transgenic medicago truncatula is simulated.

7. T obtained in step 6 respectively₀Using genome DNA of the medicago truncatula as a template, and performing PCR amplification by using a primer pair to obtain a corresponding PCR amplification product; sequencing each PCR amplification product. The sequencing result was compared with the nucleotide sequence of the NPF7.6 gene.

Since medicago truncatula is a diploid plant, when Cas9 functions to cut a particular gene, it is possible that both alleles on both homologous chromosomes in the same cell can be edited, resulting in the same type or different types of mutation, so that two alleles in one plant are considered to be two gene editing events. Homozygous mutant refers to the plant having the same mutation in the NPF7.6 genes on two homologous chromosomes. A biallelic mutant refers to a plant in which the NPF7.6 genes of both homologous chromosomes are mutated but in different forms. The heterozygous mutant number means that the NPF7.6 gene of one of the two homologous chromosomes of the plant is mutated, and the NPF7.6 gene of the other homologous chromosome is not mutated. Wild type means that the NPF7.6 gene is not mutated in both homologous chromosomes of the plant. In this example, homozygous mutations are designated as mutants.

Co-detection of T₀The generation stable transgenic medicago truncatula has at least 10 strains, wherein the number of homozygous mutants is 2. 2 of these homozygous mutants were designated npf 7.6.6-1 mutant and npf 7.6.6-2 mutant.

The sequences of the npf 7.6.6-1 mutant and the npf 7.6.6-2 mutant at the target site are shown in FIG. 3B. The NPF 7.6.6-1 mutant has 2-nucleotide AG deletion in NPF7.6 gene to cause frame shift and early termination, resulting in loss of function of NPF7.6 protein. The NPF7.6-2 mutant has 9-nucleotide deletion in NPF7.6 gene, resulting in 3 amino acids deletion in the N-terminal of NPF7.6 protein.

Secondly, the NPF7.6 gene is knocked out to improve the nitrogen tolerance of alfalfa root nodules of caltrops

The experiment was repeated three times to obtain an average, and the procedure for each repetition was as follows:

1. taking seeds of medicago truncatula (npf 7.6.6-1 mutant or R108) with basically the same size to be detected, treating the seeds with concentrated sulfuric acid for 5min, and washing the seeds with clear water for 6 times; treating with 6% (m/v) sodium hypochlorite solution for 6min, and washing with sterile water for 6 times; and then placing the mixture on a shaking table for shaking culture for 12 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, the germinated seeds are planted in vermiculite potted pots (4 seeds per pot), 100mL of liquid culture medium is added into each pot, and then the Chinese alfalfa rhizobium Sm2011 strain (OD) is inoculated₆₀₀0.01, inoculum size 1mL per plant), cultured for 3 weeks. During the culture period, 50mL of liquid medium was added to each pot every 10 days.

The liquid medium was an FM liquid medium, an FM liquid medium containing 0.2mM potassium nitrate, an FM liquid medium containing 20mM potassium nitrate or an FM liquid medium containing 50mM potassium nitrate.

4. And (4) after the step 3 is completed, observing the state of the nodules and counting the number, the length and the fresh weight of the nodules.

The nodule status is shown in fig. 3C.

The statistics of the fresh weight of the nodules are shown in FIG. 3D.

The statistics of nodule length are shown in fig. 3E.

The statistics of the number of nodules are shown in fig. 3F.

The results show that the npf 7.6.6-1 mutant has significantly increased fresh root nodule weight, root nodule length, and root nodule number compared to medicago truncatula R108 when the potassium nitrate concentration is 20 mM. Therefore, the NPF7.6 gene knockout can improve the nitrogen tolerance of alfalfa root nodules of the caltrops.

Thirdly, the knockout of NPF7.6 gene has no influence on the fresh weight, the length of the main root and the number of lateral roots of the medicago truncatula

3. After completing step 2, the germinated seeds were planted in vermiculite pot wells (4 seeds per pot), 100mL of liquid medium (FM liquid medium or FM liquid medium containing 0.2mM potassium nitrate) was added per pot, cultured for 10 days or 21 days, and the fresh weight, main root length and lateral root number of the plants were counted.

Statistics for 10 days of culture are shown in A, B and C in FIG. 4.

Statistics for 21 days of culture are shown in D, E and F in FIG. 4.

The results show that the fresh weight, the length of the main root and the number of the lateral roots of the npf 7.6.6-1 mutant and the medicago truncatula R108 have no significant difference no matter the culture is carried out for 10 days or 21 days, and no potassium nitrate is added or added.

<110> institute of microbiology of Chinese academy of sciences

Application of <120> NPF7.6 protein in regulation and control of nitrogen tolerance of leguminous plant root nodules

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 1770

<212> DNA

<213> Medicago truncatula of Tribulus terrestris

<400> 1

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ataaaaagtt ttgggagttc actttgtatg gcttcaattt ctcttggaaa ctatgtgagt 1500

agcatgcttg ttcacgtggt catgaaaatc actgcaagag ggaatgacaa aggttggatt 1560

ccagagaacc taaacaaagg gcacatggat aggtttttct ttcttctagc agggctagtt 1620

gtttttgatt ttgtgattta cttgttctgt gctaagtggt acaagagtat caatgttcaa 1680

ggtgaccaag aggagttgga tgatgatgat gctcaagtta ttataattaa gtcggcatcc 1740

aaagaccaac ctttaacacc acaagaataa 1770

<210> 2

<211> 589

<212> PRT

<213> Medicago truncatula of Tribulus terrestris

<400> 2

Met Glu Gly Asn Glu Asn Tyr Arg Glu Glu Val Val Met Glu Glu Ser

1 5 10 15

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

20 25 30

Leu Leu Val Asn Gln Ala Leu Ala Thr Leu Ala Phe Phe Gly Val Gly

35 40 45

Val Asn Leu Val Leu Phe Leu Thr Arg Val Leu Gly Gln Asp Asn Ala

50 55 60

Glu Ala Ala Asn Asn Val Ser Lys Trp Thr Gly Thr Val Tyr Met Phe

65 70 75 80

Ser Leu Val Gly Ala Phe Leu Ser Asp Ser Tyr Trp Gly Arg Tyr Leu

85 90 95

Thr Cys Thr Ile Phe Gln Leu Phe Phe Val Leu Gly Leu Ala Leu Ser

100 105 110

Cys Leu Ser Ser Trp Arg Phe Leu Ile Asn Pro Ser Gly Cys Gly Asn

115 120 125

Gly His Ile Pro Cys Lys Pro Ser Ser Ile Gly Val Ser Ile Phe Tyr

130 135 140

Phe Ser Ile Tyr Leu Val Ala Phe Gly Tyr Gly Gly His Gln Pro Thr

145 150 155 160

Leu Ala Thr Phe Gly Ala Asp Gln Tyr Asp Glu Arg Asn Pro Lys Glu

165 170 175

Arg Ser Leu Lys Val Ala Phe Phe Cys Tyr Phe Tyr Phe Ser Leu Asn

180 185 190

Val Gly Ser Leu Phe Ser Asn Thr Val Leu Val Tyr Tyr Glu Asp Thr

195 200 205

Gly Lys Trp Thr Met Gly Phe Phe Ile Ser Leu Ile Ser Ala Ile Ile

210 215 220

Ala Leu Leu Thr Phe Leu Ser Gly Ser Pro Lys Tyr Arg Tyr Leu Lys

225 230 235 240

Pro Ser Gly Asn Pro Val Val Arg Val Ala Gln Val Phe Thr Ala Ala

245 250 255

Ala Arg Lys Trp Asp Val Ala Pro Ala Lys Ala Asp Lys Leu Phe Glu

260 265 270

Val Leu Gly Ser Arg Ser Ala Ile Lys Gly Cys Arg Lys Ile Leu His

275 280 285

Ser Asp Asp Phe Arg Leu Met Asp Lys Ala Ala Thr Ile Thr Lys Asn

290 295 300

Asp Asp Glu Gln Ile Gly Asn Asn Pro Trp Lys Leu Cys Thr Val Thr

305 310 315 320

Gln Val Glu Glu Thr Lys Cys Val Leu Arg Met Leu Pro Ile Trp Leu

325 330 335

Cys Thr Ile Cys Tyr Ser Val Val Phe Thr Gln Met Ala Ser Leu Phe

340 345 350

Val Glu Gln Gly Asp Val Met Asn Ser Asn Ile Gly Glu Phe His Leu

355 360 365

Pro Ala Ala Ser Met Ser Val Phe Asp Ile Cys Ser Val Leu Val Cys

370 375 380

Thr Val Ile Tyr Arg Thr Ile Leu Val Pro Leu Val Gly Arg Leu Ile

385 390 395 400

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

405 410 415

Ile Ile Ala Met Leu Ser Met Val Ala Ser Gly Met Thr Glu Met Val

420 425 430

Arg Leu Arg Asn Ile Ile Pro Gly Gln Lys Arg Ser Ser Met Ser Ile

435 440 445

Phe Tyr Gln Ile Pro Gln Tyr Val Leu Ile Gly Ala Ser Glu Val Phe

450 455 460

Met Tyr Val Gly Gln Leu Glu Phe Phe Asn Gly Gln Ala Pro Asp Gly

465 470 475 480

Ile Lys Ser Phe Gly Ser Ser Leu Cys Met Ala Ser Ile Ser Leu Gly

485 490 495

Asn Tyr Val Ser Ser Met Leu Val His Val Val Met Lys Ile Thr Ala

500 505 510

Arg Gly Asn Asp Lys Gly Trp Ile Pro Glu Asn Leu Asn Lys Gly His

515 520 525

Met Asp Arg Phe Phe Phe Leu Leu Ala Gly Leu Val Val Phe Asp Phe

530 535 540

Val Ile Tyr Leu Phe Cys Ala Lys Trp Tyr Lys Ser Ile Asn Val Gln

545 550 555 560

Gly Asp Gln Glu Glu Leu Asp Asp Asp Asp Ala Gln Val Ile Ile Ile

565 570 575

Lys Ser Ala Ser Lys Asp Gln Pro Leu Thr Pro Gln Glu

580 585

<210> 3

<211> 1932

<212> DNA

<213> Medicago truncatula of Tribulus terrestris

<400> 3

atattggtga taatctctac tttttgtgga tgaaataatg ctgatattag aatatattga 60

aggattataa attgcactgg ggctatctct ttgtaaccac cttgtacgtt agaagacact 120

gatgtattat gcgtcatgtg ttcgataact gtttttgtta taaccctgcg ctcatctggg 180

ttccatcaaa ttgtaaaaat gatctgtgaa ttgcagagac cattcaaata ttttactaat 240

tttttaatct gaacagatgg tttatatgta tgttacgtta gtttaatatt atatgctttc 300

atctattatg cagcacctct ggatgtccat ttggcgaggg atgccatttc ctgcattatg 360

ttcctggggg ctttaaagct gtctatcaga tgatcaatgt tggtagcagt cctgctattc 420

caccaattgg tagaaatcca aatgttccac aatccttccc agacggttct tctccaccag 480

ttgctaagac ccggttgtgc aacaagttca atacagctga aggttgcaaa tttggggata 540

aatgtcactt tgcccatggt gagtgggagc ttggcaggcc aacggtccct gcatatgaag 600

atactcgcgc catgggacaa atgcaaagca gcagtgttgg tgggagaatt gagcccccac 660

ctccagctca tggagctgct gccggttttg gagtctcagc cactgcaaca gttagtatta 720

atgctaccct tgctggagct atcattggaa aaaatgatgt taactccaag caaatctgtc 780

acataacagg tgctaaactt tccattaggg aacatgattc agatcctaac cttagaaata 840

ttgagcttga aggcagtttt gatcagatca aacaagccag tgccatggtc catgacctta 900

ttctaaatgt gagttcggtc tctggacctc caggaaaaaa cattacttca cagacttctg 960

cccctgcaaa caacttcaag accaagctgt gtgaaaactt taccaaagga tcctgcactt 1020

ttggggaaag gtgccacttt gcgcatggaa cagatgaatt acgcaagcct ggaatgtgat 1080

agaaatttta gtttgcattg tttttagtga ggattgaact gtgcagaatt ttgtagttat 1140

ctataatttt gccaatcact ttttctattt tgcaatatgg taggtaacta atgggagagt 1200

tttaccccag cttcaaaagg tcaattgctt tcctttgtca ccattttttt aatatttatt 1260

gtttctgaag tatcgagcca agatgtggtt tcagttctga tttttatgtt tttgttattt 1320

ttgtattttt agtttgaata tattggttgt tcgccaacat gattaatgca agagtctcat 1380

gtctatatat aagggtggga tttgaggttg cacaattcat aaattcaaat ggaaagtgaa 1440

attaaaaata aataaaaaaa taaaaaagct agcttctatt aactaacttg agaagaatat 1500

ttataaataa ataaatgaaa aatggacgat taagaatgaa cgaaggagaa tattttttat 1560

gatagtgagt agaatattga ctccaaagct ctggtctagt tgcaaaaata cagctttgaa 1620

tctaaaagat aattacaccc ctcataacaa tggcagggca gcccatttga cattgcctat 1680

gagtcatact caactattat aaaaaaatca aaaaatagtt ttgaatgtaa ttacgtatct 1740

ccatttagct gcaccgtaaa catttcgcat caagtagcat ttttataata aaattaattt 1800

attttccctt cttttacttt aaaagtttta agacatggtg aaaaggcaaa tgattacaaa 1860

tatgtagcaa aatgttaagt atgcatgtag taaataatat agatttgctt tataaacaaa 1920

taggtaggta aa 1932

Claims

The application of NPF7.6 protein, which is at least one of S1) -S3):

s1) regulating and controlling nitrogen absorption of leguminous plants;

s2) regulating and controlling the nitrogen resistance of the leguminous plant root nodule;

s3) cultivating leguminous plants with altered nitrogen uptake and/or altered nodule nitrogen tolerance;

the NPF7.6 protein is 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 absorption and/or nodule nitrogen resistance;

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 absorption and/or nitrogen tolerance of root nodules.
2. Use of a nucleic acid molecule encoding the NPF7.6 protein of claim 1, at least one of S1) -S3):

s1) regulating and controlling nitrogen absorption of leguminous plants;

s2) regulating and controlling the nitrogen resistance of the leguminous plant root nodule;

s3) cultivating leguminous plants with altered nitrogen uptake and/or altered root nodule nitrogen tolerance.
3. Use according to claim 1 or 2, characterized in that:

the regulation and control of the nitrogen absorption of the leguminous plants are to promote the nitrogen absorption of the leguminous plants or inhibit the nitrogen absorption of the leguminous plants;

the regulation and control of the nitrogen tolerance of the leguminous plant root nodule are to improve the nitrogen tolerance of the leguminous plant root nodule or reduce the nitrogen tolerance of the leguminous plant root nodule;

the leguminous plant with altered nutrient nitrogen uptake and/or altered nodule nitrogen tolerance is a "leguminous plant with increased nutrient nitrogen uptake and/or reduced nodule nitrogen tolerance" or a "leguminous plant with reduced nutrient nitrogen uptake and/or increased nodule nitrogen tolerance".
4. A method of promoting nitrogen absorption comprising the steps of: increasing the activity and/or expression level of NPF7.6 protein in cells, tissues or individuals.
5. A method for improving nitrogen resistance of leguminous plant root nodules comprises the following steps: gene editing of the gene encoding NPF7.6 protein or "reduction of NPF7.6 protein activity and/or expression level" in leguminous plants.
6. The method of claim 5, 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 from the 5' end, positions 28-47.
7. A specific sgRNA or a specific recombinant plasmid;

the target sequence of the specific sgRNA is shown as SEQ ID No: 1 is shown at positions 28-47 from the 5' end;

the specific recombinant plasmid contains a coding gene of Cas9 protein and a coding gene of the specific sgRNA.
8. Use of the specific sgRNA or the specific recombinant plasmid of claim 7 in leguminous plant breeding; the purpose of the leguminous plant breeding is to improve nitrogen tolerance and/or inhibit nitrogen uptake of nodules.
9. A method of preparing a gene-edited leguminous plant comprising the steps of:

(1) introducing a gene encoding the specific sgRNA of claim 7 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;

compared to leguminous plants, the gene-edited leguminous plants have improved nitrogen tolerance of nodules.
10. The use of claim 1, 2, 3 or 8, or the method of claim 5 or 9, wherein: the leguminous plant is medicago truncatula.