Application of NLP1 in improving symbiotic nitrogen fixation efficiency of soybeans

Abstract

The invention provides application of NLP1 in improving symbiotic nitrogen fixation efficiency of soybeans. The invention finds that the NLP1 gene participates in the nodulation process of nitrate regulation and control. The NLP1 gene is subjected to gene editing, and a stable NLP1 mutant transgenic soybean plant is obtained. Compared with a wild type, the NLP1 mutant plant shows a nitrogen-resistant nodulation phenotype and a lower expression level of nitrate-related genes. NLP1b can respond to nitrate induction nucleation. In addition, the content of photosynthetic metabolites such as malic acid, alpha ketoglutaric acid and cane sugar in the NLP1a or NLP1b overexpression transgenic plant is increased. Therefore, gene editing is carried out aiming at the NLP1 gene, the symbiotic nitrogen fixation efficiency of the soybeans can be improved, and the NLP1 gene can be applied to improvement of soybean varieties.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant molecular biology and plant genetic engineering, and more particularly, the present application relates to the application of NLP1 in improving the efficiency of symbiotic nitrogen fixation of soybean. BACKGROUND

[0002] Nitrogen is an important life element and a key factor limiting crop yield. Plants mainly absorb and utilize inorganic nitrogen sources (nitrate and ammonia) in the soil to maintain their series of life activities. The content of nitrogen in the air is very high, but it cannot be directly absorbed and utilized by plants. At present, it is mainly converted into nitrogen that can be directly absorbed and utilized by plants through chemical nitrogen fixation or biological nitrogen fixation. Although the application of nitrogen fertilizer produced by chemical nitrogen fixation has greatly improved the yield of crops. However, due to the low absorption efficiency of plants to nitrogen in the soil, the current nitrogen fertilizer consumption is far more than the need for high yield of crops, which has caused serious damage to the soil and aquatic ecosystems, and caused water eutrophication. At the same time, the production of nitrogen fertilizer also consumes a large amount of non-renewable energy. Therefore, in the face of energy shortage and environmental deterioration today, improving the utilization efficiency of nitrogen in the soil by plants is of great significance to maintain crop yield and reduce environmental pollution.

[0003] In addition to chemical nitrogen fixation, biological nitrogen fixation (including autotrophic, symbiotic and associative nitrogen fixation) provides more than 2 / 3 of the nitrogen in the earth's ecosystem. The symbiotic nitrogen fixation between soybean and rhizobium is the most efficient and most in-depth researched system in biological nitrogen fixation. The symbiotic nitrogen fixation of soybean-rhizobium starts from the complex molecular signal exchange between the two, and finally induces the formation of nodule organ. The bacteroids in the nodule can convert nitrogen in the air into ammonia, while the plant provides carbon fixed by photosynthesis to the rhizobium for its growth, which is a mutually beneficial relationship. This symbiotic way greatly reduces the use of industrial fertilizers and is a sustainable development way of nitrogen fixation. However, since symbiotic nitrogen fixation is very energy-consuming, too much nitrate nitrogen in the soil environment will affect the establishment of symbiotic relationship between the two, including rhizobium infection, nodule initiation, formation and nitrogen fixation, and other key processes.

[0004] Therefore, it is of great agricultural production significance to find the genes in soybean that perceive and transmit nitrogen signals to adapt to the nitrogen level in the environment to regulate nodule symbiosis, elucidate its molecular mechanism, and genetically modify it to cultivate soybean with excellent performance. SUMMARY

[0005] The application aims at the application of NLP1 in improving the efficiency of symbiotic nitrogen fixation of soybean. The application finds that NLP1 gene is involved in the noduleation process regulated by nitrate. By gene editing of NLP1 gene, stable NLP1 mutant transgenic soybean plants are obtained. Compared with wild type, NLP1 mutant plants show more nitrogen-tolerant noduleation phenotype and lower expression level of nitrate-related genes. NLP1b can be induced into nucleus in response to nitrate. Moreover, the content of photosynthetic metabolites such as malate, alpha-ketoglutarate and sucrose in NLP1a or NLP1b overexpression transgenic plants is increased. Therefore, gene editing of NLP1 gene can improve the efficiency of symbiotic nitrogen fixation of soybean and can be applied to realize the improvement of soybean varieties.

[0006] In the first aspect of the application, a method for improving the efficiency of symbiotic nitrogen fixation of soybean is provided, comprising: reducing the expression of NLP1 in soybean; wherein the improvement of the efficiency of symbiotic nitrogen fixation of soybean comprises: improving the tolerance to high content of nitrogen element, increasing the number of noduleation, increasing the activity of nitrogen fixation enzyme, reducing the expression level of nitrate-related genes, inducing into nucleus in response to nitrate, and / or affecting the content of plant photosynthetic metabolites.

[0007] In the second aspect of the application, the application of a reagent for reducing the expression of NLP1 in improving the efficiency of symbiotic nitrogen fixation of soybean is provided; wherein the improvement of the efficiency of symbiotic nitrogen fixation of soybean comprises: improving the tolerance to high content of nitrogen element, increasing the number of noduleation, increasing the activity of nitrogen fixation enzyme, reducing the expression level of nitrate-related genes, inducing into nucleus in response to nitrate, and / or affecting the content of plant photosynthetic metabolites.

[0008] In one or more embodiments, the content of nitrogen element is represented by the concentration of nitrate, and the high content of nitrogen element is nitrate concentration ≥ 5 mM, preferably ≥ 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 14 mM, 15 mM, 18 mM or 20 mM.

[0009] In one or more embodiments, the nitrate is potassium nitrate or sodium nitrate.

[0010] In one or more embodiments, the nitrate-related genes include: NR1, NR2, NiR, NRT2.1a, NRT2.1b, NRT2.1c, NRT2.1d.

[0011] In one or more embodiments, the photosynthetic metabolites include: malate, alpha-ketoglutarate, sucrose.

[0012] In one or more embodiments, the reduction of NLP1 expression is achieved by targeting NLP1a and / or NLP1b through gene editing technology.

[0013] In one or more embodiments, the gene editing technology is CRISPR / Cas9 technology.

[0014] In one or more embodiments, the reagent comprises sgRNA and Cas9 enzyme directed against NLP1a and / or NLP1b.

[0015] In one or more embodiments, the reduction of NLP1 expression is achieved by disrupting exon 1 of NLP1a, or disrupting exon 1 of NLP1b, or disrupting exon 3 of both NLP1a and NLP1b using CRISPR / Cas9 technology.

[0016] In one or more embodiments, the CRISPR / Cas9 technology comprises: targeting exon 1 of NLP1a, or exon 1 of NLP1b, or exon 3 of both NLP1a and NLP1b with a specific sgRNA.

[0017] In one or more embodiments, the sequence of the sgRNA is set forth in SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 23.

[0018] In one or more embodiments, the disruption comprises: introducing at least one base insertion, deletion, substitution, or a combination thereof into the targeted region, such that the codon at the targeted site is edited to a stop codon, resulting in premature termination of the transcription and / or translation of the NLP1 protein.

[0019] In one or more embodiments, the targeted site comprises: position 38 of SEQ ID NO: 1 or SEQ ID NO: 2, position 809 and / or 810 of SEQ ID NO: 3 or SEQ ID NO: 4, position 1627 and / or 1628 of SEQ ID NO: 1, position 1108 and / or 1109 of SEQ ID NO: 2, position 1650 and / or 1651 of SEQ ID NO: 3, position 1114 and / or 1115 of SEQ ID NO: 4.

[0020] In a third aspect of the present application, a method of making a NLP1 gene edited soybean plant is provided, comprising the steps of:

[0021] (a) introducing into a soybean plant cell, tissue, or other explant a plasmid containing coding sequences for a specific sgRNA and Cas9 enzyme for reducing expression of the NLP1 gene;

[0022] (b) regenerating the soybean cell, tissue, or other explant into a genetically altered soybean plantlet;

[0023] (c) growing the NLP1 gene-edited small soybean plant into a soybean plant with improved symbiotic nitrogen fixation efficiency as compared to an untransformed wild-type soybean plant;

[0024] wherein the improved symbiotic nitrogen fixation efficiency comprises: improved tolerance to high content of nitrogen element, increased nodule number, increased nitrogenase activity, reduced expression level of nitrate-related genes, induced into nucleus in response to nitrate, and / or affected content of photosynthetic metabolites.

[0025] In one or more embodiments, the method further comprises: screening or selecting soybean cells, tissues or other explants prior to step (b); screening or selecting soybean small plants between steps (b) and (c); or screening or selecting soybean plants after step (c) to identify successful editing of the NLP1 gene.

[0026] In one or more embodiments, the method of transformation comprises: particle bombardment, Agrobacterium-mediated transformation, Rhizobium-mediated transformation, or protoplast transfection or transformation.

[0027] In one or more embodiments, the sequence of the sgRNA is as set forth in SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 23.

[0028] In a fourth aspect of the present application, there is provided a NLP1 gene-edited soybean plant, wherein the NLP1 is silenced in the soybean plant, and the soybean plant has improved symbiotic nitrogen fixation efficiency; wherein the improved symbiotic nitrogen fixation efficiency comprises: improved tolerance to high content of nitrogen element, increased nodule number, increased nitrogenase activity, reduced expression level of nitrate-related genes, induced into nucleus in response to nitrate, and / or affected content of photosynthetic metabolites.

[0029] In one or more embodiments, the NLP1 gene-edited soybean plant is prepared by the method described in any of the embodiments of the present application.

[0030] In one or more embodiments, the content of nitrogen element is represented by the concentration of nitrate, and the high content of nitrogen element is a concentration of nitrate ≥ 5 mM, preferably ≥ 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 14 mM, 15 mM, 18 mM, or 20 mM.

[0031] In one or more embodiments, the nitrate is potassium nitrate or sodium nitrate.

[0032] In one or more embodiments, the nitrate-related genes include: NR1, NR2, NiR, NRT2.1a, NRT2.1b, NRT2.1c, NRT2.1d.

[0033] In one or more embodiments, the photosynthetic metabolites include: malate, alpha-ketoglutarate, sucrose.

[0034] Other aspects of the application will be apparent to those skilled in the art from consideration of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Figure 6, Multiple mutation analysis of Gmnlp1s mutants of CRISPR-Cas9 construct. (a-c) Target sites of DNA sequence mutations in Gmnlp1a, Gmnlp1b and Gmnlp1a nlp1b mutants and protein structure prediction. Arrow indicates the frameshift mutation position in the amino acid sequence.

[0036] Figure 2 Figure 8, Nodulation phenotype and infection events of Gmnlp1s mutants. (a-f) Nodulation phenotype of wild type (Huachun 6) and Gmnlp1b mutant plants treated with 0.25 mM, 10 mM, 20 mM nitrate (potassium nitrate, KNO3). Scale bar is 5 mm. (g) Nodule number of wild type and Gmnlp1s mutant plants under 0.25 mM, 10 mM, 20 mM nitrate (potassium nitrate, KNO3) concentration. (h) Infection events of wild type and Gmnlp1b mutant plants under 10 mM nitrate (potassium nitrate, KNO3) concentration. Numbers represent the percentage of infection events reduction. Error bars represent standard deviation. Foci represent infection points, IT represents infection lines. (i) Nitrogenase activity of nodules of wild type and Gmnlp1b mutant plants under 10 mM nitrate (potassium nitrate, KNO3) concentration. Different letters represent statistical differences between each experimental group, one way ANOVA (multiple comparisons).

[0037] Figure 3 Figure 10, Expression analysis of nitrate-related genes in Gmnlp1b mutants. (a-g) Real-time quantitative PCR detection of the relative expression level of nitrate-related genes NR1, NR2, NiR, NRT2.1a, NRT2.1b, NRT2.1c, NRT2.1d in wild type and Gmnlp1b mutant plants under 0 mM, 0.5 mM, 5 mM, 10 mM nitrate (potassium nitrate, KNO3) concentration treatment. Error bars represent standard deviation.

[0038] Figure 4Figure 6. Nitrate promotes GmNLP1b accumulation in the nucleus. (a-b) Localization of p35S:GFP, p35S:GFP-GmNLP1b in tobacco leaves after nitrogen starvation and 10 mM KNO3 treatment for 30 min. (c-d) Localization of p35S:GFP, p35S:GFP-GmNLP1b in soybean roots after nitrogen starvation (c) and 10 mM KNO3 treatment for 1 h (d). Pictures a-d were taken using confocal fluorescence microscopy, scale bar = 50 μm. Experimental results were performed in triplicate.

[0039] Figure 5 Figure 7. GmNLP1a and GmNLP1b overexpression plants have higher contents of photosynthetic metabolites malate, alpha-ketoglutarate and sucrose in leaves. LN: low nitrogen treatment, KNO3 was 0.25 mM; HN: high nitrogen treatment, KNO3 was 10 mM. Bar = 1 cm. Error bars represent standard deviation. Different letters indicate statistical differences between individual experimental groups, one way ANOVA (multiple comparisons). DETAILED DESCRIPTION

[0040] The inventors have found that NLP1 gene in soybean is involved in the nodule process regulated by nitrate. Through CRISPR-Cas9 technology, the NLP1 gene is edited to obtain a stable NLP1 mutant transgenic plant. Compared with the wild type, the NLP1 mutant plant shows a more nitrogen-tolerant nodule phenotype and a lower expression level of nitrate-related genes. NLP1b can be induced into the nucleus in response to nitrate. Moreover, the content of photosynthetic metabolites malate, alpha-ketoglutarate and sucrose in NLP1a or NLP1b overexpression transgenic plants is increased. Therefore, gene editing of NLP1 gene can improve the efficiency of symbiotic nitrogen fixation in soybean, and can be applied to improve soybean varieties.

[0041] As used herein, the "soybean" includes soybeans having a root nodule symbiotic system and expressing NLP1. Preferably, the "soybean" is Glycine max. As used herein, the "symbiotic nitrogen fixation" refers to the process by which soybeans utilize and fix nitrogen in the environment through a mycorrhizal symbiotic system. The nitrogen content in the environment affects the efficiency of symbiotic nitrogen fixation in soybeans. When the nitrogen content in the environment is too high, the efficiency of symbiotic nitrogen fixation in soybeans will decrease, inhibiting the formation of nodules (for example, a decrease in the number of nodules) and the activity of nitrogenase (ARA), which is called the "nitrogen repression" phenomenon. Nitrogen in the environment usually exists in the form of nitrates (such as potassium nitrate or sodium nitrate). Therefore, the concentration of nitrates can reflect the content of nitrogen in the environment. Herein, a nitrate concentration ≥5 mM, such as ≥6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 14 mM, 15 mM, 18 mM or 20 mM, is considered to have a high nitrogen content; a nitrate concentration ≤1 mM, such as ≤0.9 mM, 0.8 mM, 0.7 mM, 0.6 mM, 0.5 mM, 0.4 mM, 0.3 mM, 0.25 mM, 0.2 mM or 0.1 mM, is considered to have a low nitrogen content.

[0042] The function of the NLP1 gene, its relationship with photosynthetic metabolism, and its correlation with the efficiency of symbiotic nitrogen fixation in soybeans remain largely unresolved. In the present invention, the inventors, after in-depth research, discovered for the first time that gene editing of the NLP1 gene improves the efficiency of symbiotic nitrogen fixation in soybeans.

[0043] As used herein, "NLP1" derived from soybean (Glycine max) generally comprises the nucleotide sequence of the NLP1a gene coding region set forth in SEQ ID NO:2, or the amino acid sequence of the NLP1a protein set forth in SEQ ID NO:5, or the nucleotide sequence of the NLP1b gene coding region set forth in SEQ ID NO:4, or the amino acid sequence of the NLP1b protein set forth in SEQ ID NO:6. Any polypeptide / gene with a high homology (e.g., 80% or greater homology; preferably 90% or greater homology, such as 95%, 98%, or 99%) to the NLP1 specifically cited herein and having the same function as the NLP1 is also encompassed by the present invention. In the present invention, the NLP1 also includes its homologs, i.e., polypeptides / genes present in species other than soybean that have homology to the sequences described above and have the same function as the NLP1 of the present invention, or polypeptides / genes that play the same or similar role in the same or similar signaling pathways. Since NLP1 is conserved in multiple species, it should be understood that although the NLP1 gene proposed in the examples of the present invention is preferred, the present invention is not limited to the genes specifically listed in the examples.

[0044] In the present application, the "genetic modification" or "gene editing" includes insertion, deletion or substitution of one or more (e.g. 1, 2, 3, 4, 5, 10, 15 or more) nucleotides. In some preferred embodiments, after insertion, deletion or substitution of one or more nucleotides, the corresponding codon at these sites is modified or edited into a stop codon, resulting in premature termination of transcription and translation of NLP1 protein.

[0045] As an embodiment of the present application, targeted gene editing technology is used for genetic modification of NLP1, for example, CRISPR / Cas (such as Cas9) system can be used. The method comprises: co-transferring (the co-transferring can be transferring an expression vector containing sgRNA and an expression vector containing Cas9 mRNA separately, sequentially or simultaneously, or transferring an expression vector containing both sgRNA and Cas9 mRNA at the same time) sgRNA or nucleic acid capable of forming the sgRNA, Cas9 mRNA or nucleic acid capable of forming the Cas9 mRNA that target bind to the specific sites into the target region or target cells. Then, the sgRNA and Cas9 are introduced into the target cells. For example, the nucleic acid capable of forming the sgRNA can be amplified and constructed into an expression vector (such as pGel-201) containing Cas9 mRNA, and the expression vector is introduced into the target cells, so as to form active sgRNA and Cas9 mRNA in the cells. After repeated screening, the present inventors screened some sgRNA sequences, and the positions targeted by these sequences are very suitable, which can efficiently guide the Cas9 enzyme to break the DNA double strand and induce NLP1 to be silenced.

[0046] The present application provides a method for reducing the expression of NLP1 based on gene editing technology, which is achieved by using CRISPR / Cas9 technology to destroy the 1st exon of NLP1a, or destroy the 1st exon of NLP1b, or destroy the 3rd exon of NLP1a and NLP1b at the same time. Preferably, the sequence of the specific sgRNA is shown in SEQ ID NO: 16 (targeting NLP1a), SEQ ID NO: 19 (targeting NLP1a and NLP1b) or SEQ ID NO: 23 (targeting NLP1a and NLP1b).

[0047] The CRISPR / Cas9 technology of the specific sgRNA can introduce random insertion or deletion or insertion and deletion at the 1st or 3rd exon of NLP1a, or the 1st or 3rd exon of NLP1b.

[0048] In some preferred embodiments, for the NLP1a gene, a deletion mutation can be introduced at exon 1 (e.g., corresponding to position 38 of SEQ ID NO: 1 or SEQ ID NO: 2);

[0049] For the NLP1b gene, a deletion mutation can be introduced at exon 1 (e.g., corresponding to positions 809 and / or 810 of SEQ ID NO: 3 or SEQ ID NO: 4);

[0050] For the NLP1a gene, one or more insertion mutations, e.g., insertion of G, can be introduced at exon 3 (e.g., between positions 1627 and 1628 of SEQ ID NO: 1, or between positions 1108 and 1109 of SEQ ID NO: 2).

[0051] For the NLP1b gene, one or more insertion mutations, e.g., insertion of G, can be introduced at exon 3 (e.g., between positions 1650 and 1651 of SEQ ID NO: 3, or between positions 1114 and 1115 of SEQ ID NO: 4).

[0052] In some more preferred embodiments, the sgRNAs can be amplified using PCR techniques, e.g., using primers as shown in Table 2.

[0053] In some more specific embodiments, the sgRNAs are amplified using the five sets of primer pairs of SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 21, SEQ ID NO: 24 and SEQ ID NO: 25, and SEQ ID NO: 26 and SEQ ID NO: 27.

[0054] After gene editing, an NLP1 gene edited vector, host cell, or soybean plant can be obtained. The NLP1 gene edited vector, host cell, or soybean plant has improved symbiotic nitrogen fixation efficiency compared to wild type. The improved symbiotic nitrogen fixation efficiency of soybean includes: improved tolerance to high nitrogen content, increased number of nodule nodulation, increased nitrogenase activity, reduced expression level of nitrate-related genes, induced into the nucleus in response to nitrate, and / or affecting the content of photosynthetic metabolites of the plant.

[0055] In some embodiments, the nitrate-related genes include, but are not limited to: NR1, NR2, NiR, NRT2.1a, NRT2.1b, NRT2.1c, NRT2.1d.

[0056] In some embodiments, the photosynthetic metabolites include, but are not limited to, malate, alpha-ketoglutarate, and sucrose.

[0057] Therefore, the present application also provides the use of a reagent for reducing the expression of NLP1 protein based on gene editing technology to improve the efficiency of symbiotic nitrogen fixation of soybean. The related application is achieved by destroying the 1st exon of NLP1a or the 1st exon of NLP1b or the 3rd exon of NLP1a and NLP1b based on CRISPR / Cas9 technology. The related application includes specific sgRNA sequences of gene editing, gene editing reagents, gene editing plasmids, gene editing soybean plant cells and their preparation methods, gene editing soybean plant preparation methods, and gene editing soybean plants.

[0058] The gene editing reagent comprises a specific sgRNA, as described in any embodiment of the present application.

[0059] The gene editing plasmid is an expression plasmid containing a specific sgRNA and a Cas9 enzyme coding sequence.

[0060] The gene editing soybean plant cell preparation method is to introduce an expression plasmid containing a specific sgRNA and a Cas9 enzyme coding sequence into a soybean plant cell to express, thereby obtaining a soybean plant cell in which the NLP1 gene is edited and the NLP1 protein is silenced. The gene editing soybean plant cell is a soybean plant cell in which the NLP1 gene is edited and the NLP1 protein is silenced, obtained by the gene editing cell preparation method.

[0061] The gene editing soybean plant preparation method is a method of introducing an expression plasmid containing a specific sgRNA and a Cas9 enzyme coding sequence into a soybean plant cell, tissue or other explant to express, silencing the NLP1 protein in the soybean plant cell, tissue or other explant, and then regenerating the soybean plant from the soybean plant cell, tissue or other explant.

[0062] The gene editing soybean plant includes a soybean plant, plant part or plant cell that is edited by any embodiment of the present application, for example, edited for the NLP1 gene. The plant part includes but is not limited to leaves, stems, roots, tubers, flowers, seeds, kernels, grains, fruits.

[0063] The present application also provides a method for preparing a NLP1 gene edited soybean plant, comprising the following steps:

[0064] (a) introducing an expression plasmid for reducing the expression of NLP1 gene containing a specific sgRNA and a Cas9 enzyme coding sequence into a soybean plant cell, tissue or other explant;

[0065] (b) regenerating the soybean cells, tissues, or other explants into genetically altered soybean plantlets;

[0066] (c) growing the NLP1 gene-edited plantlets into soybean plants with improved symbiotic nitrogen fixation efficiency as compared to untransformed wild-type soybean plants;

[0067] wherein the improved symbiotic nitrogen fixation efficiency comprises: increased tolerance to high levels of nitrogen elements, increased nodule number, increased nitrogenase activity, decreased expression level of nitrate-related genes, and / or nitrate-induced nuclear import.

[0068] In one or more embodiments, the method further comprises: screening or selecting the soybean cells, tissues, or other explants prior to step (b); screening or selecting the soybean plantlets between steps (b) and (c); or screening or selecting the soybean plants after step (c) to identify successful editing of the NLP1 gene.

[0069] In one or more specific embodiments, the transformation method comprises, but is not limited to: particle bombardment (i.e., biolistics, gene gun), Agrobacterium-mediated transformation, Rhizobium-mediated transformation, or protoplast transfection or transformation.

[0070] Compared to other gene silencing techniques such as antisense nucleotide technology, CRISPR / Cas9-mediated gene editing can disrupt the NLP1 gene at the DNA level with higher silencing efficiency, and can improve the efficiency of soybean symbiotic nitrogen fixation, including increased tolerance to high levels of nitrogen elements, increased nodule number, increased ARA activity, decreased expression level of nitrate-related genes, and nitrate-induced nuclear import. Using this method for gene editing, it is expected to breed soybeans with excellent symbiotic nitrogen fixation efficiency, thereby maintaining high crop yields and reducing environmental pollution.

[0071] The present application is further described in conjunction with the specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are generally carried out under conventional conditions, or under the conditions recommended by the manufacturer.

[0072] Materials and Methods

[0073] 1. Plant materials and growth conditions

[0074] Soybean Glycine max (Huachun 6):

[0075] In a natural environment: maintain the optimum temperature at 28-30℃, maintain the natural light period and intensity for 5-6 months. -2 ·s -1 , relative humidity is 70%;

[0076] In a natural environment: maintain the optimum temperature at 28-30℃, maintain the natural light period and intensity for 5-6 months.

[0077] 2. Strains and their growth conditions

[0078] (1) Escherichia coli (E. coli): DH5a was cultured in LB medium, solid LB medium was cultured in a 37℃ constant temperature incubator, and liquid LB medium was cultured in a 37℃ constant temperature shaker.

[0079] (2) Agrobacterium: A. rhizogenes K599 was used for root transformation experiment of soybean, A. tumefaciens EHA105 and GV3101 were used for tobacco transient transformation experiment and CRISPR-Cas9 stable mutant transformation experiment of soybean; all were cultured in LB medium, solid in 28℃ constant temperature incubator, liquid in 28℃ constant temperature shaker.

[0080] (3) Rhizobium: Bradyrhizobium japonicum USDA110-GUS was used for inoculation of soybean. Bradyrhizobium japonicum was grown in HM medium, solid in 28℃ constant temperature incubator, liquid in 28℃ constant temperature shaker.

[0081] 3. GmNLP1a / b gene

[0082] GmNLP1a genomic sequence (SEQ ID No. 1, wherein the bold part is the exon part, in order are exon 1, exon 2, exon 3 and exon 4, respectively, the underlined part is the GmNLP1a sgRNA targeting sequence, and the italic part is the GmNLP1a / 1b sgRNA targeting sequence):

[0083]

[0084]

[0085] GmNLP1a coding region nucleotide sequence (SEQ ID NO: 2):

[0086]

[0087] GmNLP1b genomic sequence (SEQ ID NO: 3, wherein the bold part is the exon part, in order are exon 1, exon 2, exon 3 and exon 4, respectively, the underlined part is the GmNLP1b sgRNA targeting sequence, and the italic part is the GmNLP1a / 1b sgRNA targeting sequence):

[0088]

[0089]

[0090]

[0091] GmNLP1b coding region nucleotide sequence (SEQ ID NO: 4):

[0092]

[0093] NLP1a amino acid sequence (SEQ ID NO: 5):

[0094] MGDSGVTSSAATMMEAPPPDGTTTTATSMDFDYMGEFFLDGCWLEASADVSDFLLQSPSFSNPLFDPSLSWPALETNHNKSQDAAFGTQQESHNNIVSVVAGGGYSQQFQSETHSVEGVSEGVRRWWFAPSPIPSPGPGPSIMEKLIRALMWIKDYNRNKDMLIQIWVPIHKEGRPILAADDLLFSLESKSLNLAKYREISVTYEFSAEESDSKELARGLPGRVFRYKVPEWTPDVRFFRSDEYPRVDHAQEYDVRGTVAVPIFEQGSKTCLGVIEVVMTTQQINYGPELESVCKALEAVDLRSSKQLSIQNVKQACNRTYEAALPEIYEVLRSACEMHRLPLAQTWVPCVQQGKEGCRHSEDNYLLCISPVEHACYVGDPSIRSFHEACTEHHLLKGEGVAGGAFMTNQPCFSDDITSLSKKDYPLSHYARLFGLHAAVAIRLRSIYNSTDDFVLEFFLPVDCNDSEEQRKMLTALSIIIQRVCRSLRVIRDKELEEANLSVDEVIALADSGFARNAIFSEPQYKGMVASLDAEEKSSETMGRKFSDLRQQQESPILKGNLDCVKECSTSVEGNLSSLGTNKTGERRRAKAEKTITLQVLRQYFAGSLKDAAKNIGVCTTTLKRICRQHGIKRWPSRKIKKVGHSLQKLQLVINSVQGASGAFQIGSFYSNFPDLASPNLSGTGFFSTLNQSDYPNSTSTQPDHGSLSPEGASKSPSSSCSQSSISSHSCSSMSELQQHRTANGAGNKVSTTVSEDSAGVVLKRISSEAELKSLSQDRAKLLPRSQSQETLGEHPKTQYQQPLLKTSSSKVDSHRVKVAYGDEKTRFRMPKSWGYEDLLQEIARRFNVSDMSKFDVKYLDDDCEWVLLTCDADLEECIDVCQSSESGTIKLSLQPSSHSMRSSLEFR

[0095] NLP1b amino acid sequence (SEQ ID NO: 6):

[0096] MMGDGGVISSAATMMEAPPPDGTTTTSMDFDYMGELFLDGCWMEASADGSDFLLQSPSFSNTLFDPSFSWPALETNHNESQVAAFGSQQESHNNNMVSVVAGGDYSQQFQSETHSVEGASEGIRRWWFAPTHTPTPSPGPGPSIMEKLIRALMWIKDYNRNKDMLIQIWVPVHKEGRPILAADDLLFSLESKSLNLAKYREISVTYKFSAEESDSKELAWGLPGRVFRDKVPEWTPDVRFFKIDEYPRVDHAQEYDVRGTLAVPIFEQGSKTCLGVIEVVMTTQQINYGPELESVCKALEAVDLRSSKQLSIQNVKQACNRSYEAALPEIHEVLRSACEMHKLPLAQTWVPCVQQGKEGCRHSEDNYLLCISPVEHACYVGDPSIRSFHEACTEHHLLKGEGVAGGAFMTNQPCFSDDITSLSKKDYPMSHHARLFGLRAAVAIRLRSIYNSTDDFVLEFFLPVDCNDIEEQRKMLTSLSNIIQRVCRSLRVIREKELEEANLSVNEVIALADSGFTRDEICSEPQQKGMVASLDTEEKSSETMGRKFSEPRQQQESPILKGNLDCVRECSTSVEGNLSSPGTSKTGERRRVKAEKTITLQVLRQYFAGSLKDAAKNIGVCTTTLKRICRQHGIKRWPSRKIKKVGHSLQKLQLVIDSVQGASGAFQINSFYSNFPDLASPNLSGTGFFSTLNQSDNPNSTSTQPEHGSLSPEGASKSPSSSSSQSSISSHSCSSMSELQQQHTTNIASDKDPATVGEYSADVVLKLIRNEAKLKSLSQDRAKLLPRSLSQETLGEHPKTQYQLPLLKTSSSKVDSHRVKVTYGDEKTRFRMLKNWVYEDLLQEIGRKFNVSDMSKFDVKYLDDDCEWILLTCDADLEECIDVCQSSESGTIKLSLQPSSHSVRSSLEFR

[0097] The soybean (wild type Huachun 6) cDNA was used as a template, and the target fragments were amplified by PCR using primers under appropriate conditions (high-fidelity DNA polymerase Max, amplification system and procedure reference); gel electrophoresis was performed, and the target fragments were recovered using a gel recovery kit or directly recovered using a PCR product recovery kit.

[0098] 4. Construction of the vector

[0099] Gateway method for constructing the localization vector:

[0100] According to the GmNLP1b gene, a primer was designed, and then an attB long linker was added to both ends of the GmNLP1b gene primer. The primer is shown in Table 1. The GmNLP1b gene fragment was amplified using the GmNLP1b-attb-F / R primer pair and soybean cDNA as a template, and then mixed with the intermediate vector pDONR-207. After adding Gateway BP recombinase, the mixture was incubated at 22°C overnight. The reaction product was heat-shocked in a 42°C water bath for 30s, and then 1mL of antibiotic-free liquid LB was added. After incubation at 37°C for 50min, the mixture was uniformly coated on LB solid medium containing gentamicin resistance, and then incubated at 37°C in an incubator overnight. The next day, positive clones were identified by PCR, and then the plasmid was extracted and sequenced. The correctly sequenced plasmid was mixed with the target vector pK7WGF2 (p35S:GFP), and then Gateway LR recombinase was added. After incubation at 22°C overnight, the mixture was heat-shocked, and finally the correctly sequenced plasmid p35S:GFP-GmNLP1b vector was obtained.

[0101] In order to transform the hairy roots into soybeans, a marker for the transformed root marker was screened. The 35S-GFP-GmNLP1b fragment in the vector p35S:GFP-GmNLP1b was amplified using the GmNLP1b-MssI-F primer and the GmNLP1b-AscI-R primer, and then recombined into the vector pUB:3flag-mCherry (the promoter UB was cut off, and only the 3flag-mCherry skeleton remained) digested with MssI / AscI enzymes to obtain the final localization vector, i.e. p35S-GFP-GmNLP1b-3flag, mCherry (GFP-GmNLP1b-3flag, mCherry was expressed alone after the 35S promoter).

[0102] The final localization vector was electroporated into Agrobacterium, and then antibiotic-free LB was added and incubated at 28°C for 1h. The mixture was coated on the corresponding LB plate, and then incubated at 28°C for 48h. Colony PCR was performed using vector primers, and then the correct clones were picked and used in the soybean hairy root experiment and the tobacco transient transformation experiment. The subcellular localization of the GmNLP1b protein was observed under a laser confocal microscope.

[0103] Table 1

[0104]

[0105] Note: The underlined part in Table 1 is the enzyme cutting site.

[0106] Enzymatic recombination method to construct CRISPR-Cas9 vector:

[0107] Using the complete genomic DNA sequence of the target gene GmNLP1a and GmNLP1b, specific, normal GC content and high editing efficiency small-guide RNAs (sgRNAs) were screened in the soybean database of Guangzhou University Guanyuefeng Research Group. According to the sgRNAs, primers were designed (as shown in Table 2), and PCR reaction was carried out under the PCR reaction conditions to amplify sgRNAs against GmNLP1a and GmNLP1b, respectively. At the same time, the target vector pGel-201 was subjected to enzyme cutting reaction using CRISPR-Cas9 enzyme cutting system, and the enzyme cutting was carried out at 37°C for 2h. The target fragments amplified by PCR and the vector fragments after enzyme cutting were recovered using a gel recovery kit, mixed, and then subjected to recombination reaction using a recombination enzyme, and then subjected to heat shock transformation and sequencing identification. The plasmid verified by sequencing was transformed into Agrobacterium GV3101 by root induction, and the correct clones were picked for the construction of stable transformation plants of soybean (stable transformation plants were purchased from Guangzhou University Guanyuefeng Research Group).

[0108] Table 2

[0109]

[0110]

[0111] 5. Sterilization treatment and germination of soybean seeds

[0112] Soybean seeds were placed in a clean glass culture dish and placed in a glass vacuum pump; a beaker containing 100mL of 100% sodium hypochlorite solution was placed in the middle of the vacuum pump; after sealing the cover of the vacuum pump with vaseline, 5-10mL of concentrated hydrochloric acid was added to the funnel, and the piston was rotated to slowly drip into the sodium hypochlorite solution, and the funnel was closed after the addition was completed. After standing for 12-16h, the cover of the vacuum pump was opened, and the seeds were transferred to a clean bench (>10h), and the seeds were constantly shaken during the period, and then dried and collected for germination. The sterilized soybean seeds were buried in a planting tray containing perlite and vermiculite (1:1), with the bean navel facing down, buried to a depth of 1-2cm, and then poured with an appropriate amount of tap water, and covered with a transparent cover. After growing for 7-10d, the cotyledons were opened and young leaves grew (water was constantly supplemented during the period to keep it moist).

[0113] ​6. Extraction of soybean genomic DNA

[0114] After the soybean seeds germinate and grow leaves, take the appropriate amount of leaves into 2 mL EP tube, add steel beads and 300 μL of DNA extraction solution (formula as shown in Table 3), put into the grinder to break, 50HZ / 3min; put the ground sample into the 65°C oven for 30min, then centrifuge at 12000rpm for 10min; take the supernatant into a 1.5mL EP tube, add 100 μL of phenol-chloroform-isoamyl alcohol (25:24:1) in the fume hood, mix well by inverting, centrifuge at 12000rpm for 10min; take the supernatant into a new 1.5mL EP tube, add equal volume of isopropanol and one-tenth volume of 3M NaAc, mix well by inverting, centrifuge at 12000rpm for 10min; discard the supernatant, add 500 μL of 75% ethanol, wash the precipitate by blowing, centrifuge at 12000rpm for 5min; aspirate the ethanol, dry at room temperature, then add 50 μL of double distilled water until the precipitate is completely dissolved.

[0115] Table 3

[0116]

[0117]

[0118] 7. GUS staining and phenotype observation of rhizobium inoculation and infection of soybean

[0119] 7.1 Inoculation of rhizobium

[0120] The seeds of the pure and stable transgenic mutants were sterilized and germinated, and after one week, they were inoculated with rhizobium. At the same time, the frozen USDA110-GUS strain was taken out from the -80°C ultra-low temperature freezer, spread on HM solid medium containing kanamycin, and cultured in an inverted incubator at 28°C for 3-5 days; the activated bacteria were picked into 3-5 mL of HM liquid medium containing kanamycin, and cultured at 28°C for 3-5 days; the bacteria were transferred into new 50-100 mL of HM liquid medium at a ratio of 1:50, and cultured at 28°C until the OD600 was about 1.0, about 3 days; the bacteria were centrifuged at 3000rpm for 15min at 4°C, the supernatant was discarded, and the bacteria were resuspended by adding an appropriate amount of sterile water and blowing gently, and then diluted with sterile water to OD600 of 0.08, and then inoculated into the stable transgenic mutants of soybean with an inoculation flask.

[0121] 7.2 GUS staining and observation of infection events

[0122] After 7 days of inoculation with USDA110-GUS, the soybean seedlings were pulled out and the roots were washed clean, then fixed in fixative (formula as shown in Table 4) on ice for 45 min under vacuum; the fixative was discarded and the roots were washed three times (formula as shown in Table 5) with 0.1M phosphate buffer for 10 min each time; the root material was transferred to GUS staining solution and vacuum-stained for 45 min; after vacuum was removed, the GUS staining solution was replenished (formula as shown in Table 6) and the material was placed in a 37℃ incubator for overnight staining; the next day, the GUS staining solution was discarded and the material was washed three times with 0.1M phosphate buffer for 10 min each time; the material was placed under a microscope for observation and statistics of infection events.

[0123] Table 4

[0124] Fixing solution 50 mL 10% paraformaldehyde 2.5 mL 0.1 M phosphate buffer 47.5 mL

[0125] Table 5

[0126] 0.1 M potassium phosphate buffer (pH 7.0) 1L KH2PO4 5.3g K2HPO4 13.9g

[0127] Table 6

[0128] GUS staining solution (pH 7.0) 200L NaH2PO4.2H2O 1.17g Na2HPO4·12H2O 4.47g K3[Fe(CN)]6 0.066g K4[Fe(CN)]6.3H2O 0.084g 0.5 M EDTA 2 mL Triton X-100 0.2 mL 25 g / ml X-Gluc (in DMF) 4 mL

[0129] 7.3 Detection of nitrogenase activity

[0130] After 3 weeks of inoculation with USDA110-GUS, the soybean seedlings were pulled out and the roots were washed clean, and the Gmnlp1b mutant was detected for nitrogenase activity. The specific steps are as follows: 2 root nodules were removed from each plant and placed in 10 mL glass tubes sealed well, 15 samples were set for each experimental group with 7-8 plants; 1 mL of air was first drawn from the glass tube with a syringe, and then 1 mL of acetylene gas was injected into the glass tube; after 2 h of reaction at 28℃, the reaction was terminated with 1 mL of 1M NaOH and waited for determination; at the same time, the gas chromatograph was opened, 0.3 mL of gas sample in the glass tube was sucked with a sample needle and injected into the gas chromatograph, the acetylene and ethylene peak values of the sample were recorded, the nodule weight was weighed and counted, and the nitrogenase activity (nodule acetylene reduction activity, ARA activity) was calculated. The calculation formula of nitrogenase activity is as follows:

[0131] Total formula:

[0132]

[0133] In the formula, FW refers to the fresh weight of the nodule which can be weighed in advance, X refers to the ethylene peak area measured by the gas chromatograph, and 2h refers to two hours.

[0134] 7.4 Observation of nodule formation events

[0135] Three weeks after inoculation of USDA110-GUS, the soybean seedlings were pulled out and the roots were washed clean. The nodule phenotype of Gmnlp1s mutant was observed by naked eye and stereomicroscope.

[0136] 8. Extraction of total RNA from plant roots (kit) and real-time fluorescent quantitative PCR (RT-qPCR)

[0137] The roots of Gmnlp1b mutant were cut, 50-100 mg per tube, steel beads were added, and then the roots were quickly placed into a pre-cooled grinder after being rapidly frozen in liquid nitrogen, 50 HZ / 3 min; 500 μL of lysis solution SL (5% β-mercaptoethanol was added before use) was added, and then the mixture was vortexed and centrifuged at 12000 rpm for 2 min; the supernatant was transferred to a filter column CS using a pipette, and then centrifuged at 12000 rpm for 2 min, and the supernatant in the collection tube was transferred to a new RNase-Free centrifuge tube; 0.4 times the volume of anhydrous ethanol was added, and then the mixture was inverted to mix, and then the obtained solution and precipitate were transferred to an adsorption column CR3, which was centrifuged at 12000 rpm for 1 min, and then the waste liquid was discarded; 350 μL of deproteinization solution RW1 was added to the adsorption column CR3, which was centrifuged at 12000 rpm for 1 min, and then the waste liquid was discarded; 500 μL of de-rinsing solution RW (anhydrous ethanol was added before use) was added to the adsorption column CR3, which was centrifuged at 12000 rpm for 1 min, and then the waste liquid was discarded, and the operation was repeated once; the adsorption column CR3 was placed in a new RNase-Free centrifuge tube, 30-50 μL of RNase-Free ddH2O was added, and then the mixture was allowed to stand at room temperature for 2 min, and then centrifuged at 12000 rpm for 1 min to obtain an RNA solution; the RNA concentration was determined using an instrument, and then the solution was labeled.

[0138] The reverse transcription kit One-Step gDNA Removal and cDNA SynthesisSuperMix was used for reverse transcription. Primers (Table 7) were designed, and the transcription levels of nitrate-related genes NR1, NR2, NiR, NRT2.1a, NRT2.1b, NRT2.1c, and NRT2.1d were detected by real-time fluorescent quantitative PCR.

[0139] Table 7

[0140]

[0141]

[0142] 9. Tobacco transient transformation

[0143] Take the correct identification of the 35S-GFP-NLP1b containing Agrobacterium EHA105 from the ultra-low temperature freezer, spread on LB solid medium containing spectinomycin and rifampicin antibiotics, 28°C overnight; pick a single colony with a sterile gun head in 4 ml LB liquid medium containing spectinomycin and rifampicin antibiotics, 28°C overnight; centrifuge at 4000 rpm for 5 min to collect the bacteria, resuspend the bacteria with tobacco transformation solution; centrifuge at 4000 rpm for 5 min, discard the supernatant, resuspend the bacteria with tobacco transformation solution again, wash 3 times; add an appropriate amount of tobacco transformation solution to resuspend the bacteria, measure the OD 600 value, and dilute to a certain concentration; mix the bacteria samples in equal volume, cultivate at 28°C in the dark for 2-4 h; inject the bacteria solution into the back of the tobacco leaf with a 1 ml syringe, irrigate the tobacco with an appropriate amount of tap water, and cultivate in the greenhouse for 2-3 days; use scissors to cut the leaves of an appropriate size, and use a confocal laser microscope to observe the subcellular localization of GmNLP1b under nitrogen starvation and 10 mM nitrate conditions.

[0144] Table 8

[0145] Tobacco transformation solution components Final concentration MgCl2 10 mM MES 10 mM Acetosyringone 0.2 mM

[0146] 10. Rooting transformation of soybean seeds

[0147] At the same time of germination of soybean seeds, activate the correct identification of the 35S-GFP-NLP1b containing Agrobacterium K599, pick a single colony into LB liquid medium containing spectinomycin, rifampicin and streptomycin resistance, cultivate overnight at 28°C on a shaker; evenly spread 200 μl of bacteria solution on LB solid medium containing the corresponding resistance, and cultivate in an inverted culture in a 28°C incubator for about 20 h. Use a blade to smoothly cut 3-5 cm below the cotyledon of the soybean seedling, and insert the seedling into a 96-well plate containing sterile water. At the same time, collect the activated bacteria, and dip the cut of the seedling with an appropriate amount of bacteria, and place it in a bag containing FP solid medium (about 30 mL FP solid medium per bag, and additionally supplement with 1 / 1000 KNO3 and CaCl2). Place the bag vertically in a 22°C light incubator for cultivation, and constantly supplement with liquid FP medium (containing 1 / 1000 KNO3 and CaCl2); remove the non-transformed roots that grow above the cut of the seedling at 1-2 weeks, and place the seedling under a fluorescence microscope to screen for transformed roots with fluorescent markers at 3-4 weeks, remove the non-transformed roots, and use the screened plants to observe the subcellular localization of GmNLP1b protein in soybean roots.

[0148] 11. Construction of overexpression vector and obtaining of stable transgenic plants:

[0149] According to the GmNLP1a and GmNLP1b gene design primer, then add homologous arm for homologous recombination at both ends of GmNLP1a and GmNLP1b gene primer, the primer sequence is shown in Table 9, the reaction system of PCR is shown in Table 10, and the PCR reaction program is shown in Table 11; using GmNLP1a-infu-F / R primer pair and GmNLP1b-infu-F / R primer pair, taking GmNLP1a and GmNLP1b gene as a template, the target gene fragments of GmNLP1a and GmNLP1b are amplified respectively, and the recovered PCR product is mixed with the linearized vector pFGC5941 after being digested with AscI and BamHI, 1 μL Exnase II recombinase is added, and then the reaction is carried out at 37℃ for 30 min; the reaction product is heat shocked in a 42℃ water bath for 30 s, 1 mL of liquid LB without antibiotics is added, and then the mixture is incubated at 37℃ for 50 min, and then uniformly coated on LB solid medium containing kanamycin, and then cultured in a 37℃ incubator overnight; the next day, the positive clones are identified by PCR, and then the bacteria are shaken and the plasmid is extracted for sequencing. The plasmid with correct sequencing is heat shocked into EHA105 strain, and then the overexpression vector GmNLP1a and GmNLP1b gene is transferred into soybean callus by using the method of agrobacterium-mediated genetic transformation, and then the transgenic soybean with overexpressed GmNLP1a and GmNLP1b gene is obtained by culture. The genomic DNA of T3 generation transgenic soybean is extracted, and then the overexpression positive is detected by PCR using Bar-F / R primer, which is the GmNLP1a and GmNLP1b overexpression stable transgenic plant.

[0150] Table 9

[0151]

[0152] Table 10 PCR reaction system

[0153]

[0154] Table 11 PCR reaction program

[0155]

[0156] 12, Sterilization and germination of soybean seeds

[0157] Soybean seeds were placed in a clean glass Petri dish and put in a glass vacuum pump; a beaker containing 100 mL of 100% sodium hypochlorite solution was placed in the middle of the vacuum pump; after the lid of the vacuum pump was sealed with vaseline, 5-10 mL of concentrated hydrochloric acid was added to the funnel, and the plunger was turned to slowly drip into the sodium hypochlorite solution, and the funnel was closed after the addition was completed; after standing for 12-16 h, the lid of the vacuum pump was opened, and the seeds were transferred to a clean bench (> 10 h), and the seeds were constantly shaken during the period, and then the seeds were collected and germinated after being dried. The sterilized soybean seeds were buried in a planting tray containing perlite and vermiculite (1:1), with the bean navel facing down, buried to a depth of 1-2 cm, and a suitable amount of tap water was poured into the tray, and a transparent cover was placed on the tray; after 7-10 days of growth, the cotyledons opened and young leaves grew (water was constantly supplemented during the period to keep the soil moist).

[0158] 13. Nodulation with rhizobia

[0159] The seeds of the pure and stable transgenic mutants were sterilized and germinated, and after one week, the rhizobia were inoculated. At the same time, the frozen USDA110 strain was taken out from the -80°C ultra-low temperature freezer, spread on YMA solid medium, and cultured in an inverted incubator at 28°C for 3-5 days; the activated bacteria were picked into 3-5 mL of YMA liquid medium, and cultured in a 28°C shaker for 3-5 days; the bacteria were transferred to new 50-100 mL of YMA liquid medium at a ratio of 1:50, and cultured at 28°C until the OD600 was about 1.0, which took about 3 days; the bacterial solution was aliquoted into 50 mL centrifuge tubes, centrifuged at 4°C and 3000 rpm for 15 min; the supernatant was discarded, and an appropriate amount of sterile water was added to gently blow the bacteria to resuspend them, and the bacteria were diluted with sterile water to an OD600 of 0.08, and then the stable transgenic soybean plants were inoculated with the inoculation flask. The rhizobia were inoculated for 21 days.

[0160] 14. Detection of the content of photosynthetic metabolites in the leaves of overexpressing plants

[0161] Take the third three out of the expression of soybean plants, liquid nitrogen grinding, weighing about 50 mg sample quickly into a 2 ml EP tube, add 1.4 mL methanol, shake well, add 100 μL inositol (4 μg / mL), as an internal standard. 70℃ shock 15 min, 14000g centrifugation 3 min. The supernatant was removed into a 15 mL tube, add 1.4 mL ddH2O, add 1 mL chloroform, shake. 6000 rpm, centrifugation 15 min. Take the polar phase (upper) 500 μL to 1.5 mL EP tube, dry in nitrogen blowing instrument, concentrated in a centrifugal dryer for 2 h. After complete drying, add 50 μL BSTFA + TMCS and 100 μL pyridine, 70℃ reaction 30 min. Take 100 μL into the sample bottle. GC-TOF-MS injection volume 1 μL, with helium as the carrier gas, through the gas flow rate of 2 mL / min. From 80℃, isothermal 2 min, then 10℃ / min to 300℃, isothermal 6 min. The transfer line temperature is 275℃. Using 70V bombardment ionization, ion source temperature is 250℃. Ion source filament energy setting is 70eV. Solvent delay 240 s after opening the filament, in full scan mode to collect mass spectrum, mass resolution R = 600, m / z = 45-500, 20 spectra per second. Detector voltage is 1460V, record 30 min. Mass spectrometry results in NIST database in search of library identification material.

[0162] Example 1, obtaining of Gmnlp1s mutant

[0163] In this embodiment, the GmNLP1 gene was edited by CRIPSR-CAS9 technology. First, the sgRNA specific to GmNLP1 gene was designed, and the specific primers were designed according to the sequence of sgRNA. The sgRNA sequence and primer sequence obtained are shown in Table 2. After annealing, double-stranded was formed, and was connected into CRISPR-Cas9 vector, which was transformed into wild type Huachun 6 soybean variety by Agrobacterium-mediated genetic transformation method, and several homozygous mutant soybean plants were obtained by PCR identification. The base mutation information and amino acid coding of T2 generation Gmnlp1s are shown in Table 11 and Table 12. Figure 1

[0164] Table 12

[0165]

[0166] Example 2, Gmnlp1s mutant presents more nitrogen-tolerant nodule phenotype

[0167] ​The seeds of wild type (WT) soybean Huachun 6 and homozygous soybean mutants obtained in Example 1 were inoculated with rhizobium USDA110-GUS, respectively, and treated by pouring 0.25 mM, 10 mM, 20 mM potassium nitrate solution into the soil. After 7 days of inoculation with USDA110-GUS rhizobium, GUS staining was performed, and the infection events were observed under a microscope. After 3 weeks of inoculation with USDA110-GUS rhizobium, the soybean seedlings were pulled out and the roots were washed clean. The nodule phenotype of Gmnlp1s mutants was observed and counted by naked eye and body microscope, and the nitrogenase activity of Gmnlp1b mutants was detected.

[0168] The nodule pictures of wild type and Gmnlp1b mutant plants treated by 0.25 mM, 10 mM, 20 mM potassium nitrate solution poured into the soil are shown in a-f, and the counting results of the number of nodules on each plant are shown in g. The results show that, compared with the wild type (Huachun 6), after GmNLP1 mutation, the number of nodules is significantly reduced under the treatment of 10 mM or 20 mM potassium nitrate, and only white nodules can be formed under 20 mM. However, in Gmnlp1a, Gmnlp1b and Gmnlp1a nlp1b, more red nodules can still be formed, and the total number of nodules is also more than that of the wild type. Figure 2 a-f, and the counting results of the number of nodules on each plant are shown in g. The results show that, compared with the wild type (Huachun 6), after GmNLP1 mutation, the number of nodules is significantly reduced under the treatment of 10 mM or 20 mM potassium nitrate, and only white nodules can be formed under 20 mM. However, in Gmnlp1a, Gmnlp1b and Gmnlp1a nlp1b, more red nodules can still be formed, and the total number of nodules is also more than that of the wild type. Figure 2 g. The results show that, compared with the wild type (Huachun 6), after GmNLP1 mutation, the number of nodules is significantly reduced under the treatment of 10 mM or 20 mM potassium nitrate, and only white nodules can be formed under 20 mM. However, in Gmnlp1a, Gmnlp1b and Gmnlp1a nlp1b, more red nodules can still be formed, and the total number of nodules is also more than that of the wild type.

[0169] The results of infection events show that, under nitrogen starvation and 10 mM nitrate treatment, the inhibition efficiency of Gmnlp1b infection events is significantly reduced compared with the wild type. Figure 2 h).

[0170] The detection results of nitrogenase activity show that, under the condition of 10 mM nitrate, the ARA activity in the wild type (Huachun 6) plant is significantly reduced, while the ARA activity in the Gmnlp1b mutant is not affected. Figure 2 i).

[0171] The above results show that, compared with the wild type, the stable transgenic lines Gmnlp1s mutants all exhibit a more nitrogen-tolerant nodule phenotype.

[0172] Example 3, Expression level of nitrate-related genes in Gmnlp1s mutants is reduced

[0173] Using the primers in Table 7, the transcription levels of nitrate-related genes NR1, NR2, NiR, NRT2.1a, NRT2.1b, NRT2.1c, NRT2.1d in wild type (WT) and homozygous soybean mutants Gmnlp1b obtained in Example 1 were detected by real-time fluorescent quantitative PCR.

[0174] The results show that, compared with the wild type, the expression levels of NR1, NR2, NiR, NRT2.1a, NRT2.1b, NRT2.1c, and NRT2.1d genes in Gmnlp1b are significantly reduced Figure 3 ) compared with the wild type.

[0175] Example 4, GmNLP1b responds to nitrate induction into the nucleus

[0176] Using Agrobacterium EHA105 containing 35S-GFP-NLP1b, tobacco is used for transient transformation, and then soybean hairy root transformation is carried out. The subcellular localization of GmNLP1b under the conditions of nitrogen starvation and the application of 10 mM nitrate is observed by confocal laser microscopy.

[0177] The results show that, under the condition of nitrogen starvation, GmNLP1b is mainly located in the cytoplasm Figure 4 a, 4c), and after 10 mM nitrate treatment, GmNLP1b is obviously aggregated in the nucleus Figure 4 b, 4d).

[0178] The above results show that GmNLP1b can respond to nitrate induction and aggregate from the cytoplasm into the nucleus Figure 4 d). It can be seen that GmNLP1 plays a core role in the inhibition of nodulation by nitrate.

[0179] Example 5, the content of photosynthetic metabolites in GmNLP1a and GmNLP1b overexpression plant leaves is significantly increased

[0180] In this embodiment, transgenic technology is used to overexpress GmNLP1a and GmNLP1b genes. First, the GmNLP1a and GmNLP1b gene overexpression vectors are transformed into wild type Huachun 6 soybean varieties by Agrobacterium-mediated genetic transformation method. Through PCR identification, several homozygous soybean lines are screened. The wild type Huachun 6 soybean (WT) and homozygous soybean overexpression plants are inoculated with rhizobium USDA110, respectively, and 0.25 mM potassium nitrate solution (low nitrogen) and 10 mM potassium nitrate solution (high nitrogen) are used to treat the soil, respectively. After 21 days of inoculation with rhizobium, the leaf is taken to detect the content of photosynthetic metabolites. The results show that the contents of malic acid, alpha-ketoglutaric acid, and sucrose in the leaves of GmNLP1a and GmNLP1b overexpression lines are higher than those of the wild type under low nitrogen (LN) or high nitrogen (HN) treatment Figure 5 ).

[0181] The above embodiments only express several implementation manners of the present application, and the description is relatively specific and detailed, but should not be understood as a limitation on the patent scope of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims. Meanwhile, all the documents mentioned in the present application are cited as references in the present application, just as each document is cited as a separate reference.

Claims

1. A method of increasing the efficiency of symbiotic nitrogen fixation in soybean, comprising: The method for improving the symbiotic nitrogen fixation efficiency of soybean comprises: reducing the expression of NLP1 in soybean; wherein the improving the symbiotic nitrogen fixation efficiency of soybean comprises: improving the tolerance to high content of nitrogen element, increasing the number of nodule nodulation, increasing the nitrogenase activity, reducing the expression level of nitrate-related genes, inducing into nucleus in response to nitrate, and / or affecting the content of photosynthetic metabolites of plants.

2. Use of an agent that reduces expression of NLP1 to increase the efficiency of symbiotic nitrogen fixation in soybean; wherein the increase in the efficiency of symbiotic nitrogen fixation in soybean comprises: The method for improving the symbiotic nitrogen fixation efficiency of soybean comprises: improving the tolerance to high content of nitrogen element, increasing the number of nodule nodulation, increasing the nitrogenase activity, reducing the expression level of nitrate-related genes, inducing into nucleus in response to nitrate, and / or affecting the content of photosynthetic metabolites of plants.

3. The method of claim 1 or 2, wherein, The content of the nitrogen element is represented by the concentration of nitrate, and the high content of nitrogen element is the concentration of nitrate ≥ 5 mM, preferably ≥ 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 14 mM, 15 mM, 18 mM or 20 mM; preferably, the nitrate is potassium nitrate or sodium nitrate; and / or, The nitrate-related genes include: NR1, NR2, NiR, NRT2.1a, NRT2.1b, NRT2.1c, NRT2.1d; and / or, The photosynthetic metabolites include: malic acid, alpha-ketoglutaric acid, sucrose.

4. The method of any one of claims 1 to 3, wherein, The reduction of the expression of NLP1 is achieved by targeting NLP1a and / or NLP1b through a gene editing technology; Preferably, the gene editing technology is CRISPR / Cas9 technology; More preferably, the reagent comprises sgRNA and Cas9 enzyme for NLP1a and / or NLP1b.

5. The method of any one of claims 1-4, wherein, The reduction of the expression of NLP1 is achieved by destroying the 1st exon of NLP1a or the 1st exon of NLP1b or the 3rd exon of NLP1a and NLP1b simultaneously using CRISPR / Cas9 technology.

6. The method of claim 5, wherein, The CRISPR / Cas9 technology comprises: using specific sgRNA to target the 1st exon of NLP1a or the 1st exon of NLP1b or the 3rd exon of NLP1a and NLP1b simultaneously; Preferably, the sequence of the sgRNA is as shown in SEQ ID NO: 16, SEQ ID NO: 19 or SEQ ID NO:

23.

7. The method of claim 5, wherein, The destruction comprises: introducing at least one base insertion, deletion, substitution or combination thereof into the targeted region, so that the codon at the targeted site is edited into a stop codon, thereby causing premature termination of transcription and / or translation of NLP1 protein.

8. The method of claim 7, wherein, The targeted site comprises: 38th of SEQ ID NO: 1 or SEQ ID NO: 2, 809th and / or 810th of SEQ ID NO: 3 or SEQ ID NO: 4, 1627th and / or 1628th of SEQ ID NO: 1, 1108th and / or 1109th of SEQ ID NO: 2, 1650th and / or 1651th of SEQ ID NO: 3, 1114th and / or 1115th of SEQ ID NO:

4.

9. A method for preparing NLP1 gene edited soybean plants, comprising the following steps: (a) introducing into a soybean plant cell, tissue or other explant a plasmid containing a coding sequence for a specific sgRNA and a Cas9 enzyme for reducing expression of the NLP1 gene; (b) regenerating the soybean cell, tissue or other explant into a genetically altered soybean plantlet; (c) growing the NLP1 gene-edited plantlet into a soybean plant having improved symbiotic nitrogen fixation efficiency as compared to a non-transformed wild-type soybean plant; wherein the improved symbiotic nitrogen fixation efficiency comprises: increased tolerance to high levels of nitrogen elements, increased nodule number, increased nitrogenase activity, reduced expression level of nitrate-related genes, and / or nitrate-induced into nucleus; Preferably, the method further comprises: screening or selecting the soybean cell, tissue or other explant before step (b); screening or selecting the soybean plantlet between steps (b) and (c); or screening or selecting the soybean plant after step (c) to identify successful editing of the NLP1 gene; Preferably, the method of transformation comprises: particle bombardment, Agrobacterium-mediated transformation, Rhizobium-mediated transformation or protoplast transfection or transformation; Preferably, the sequence of the sgRNA is as set forth in SEQ ID NO: 16, SEQ ID NO: 19 or SEQ ID NO:

23.

10. A soybean plant edited for NLP1, characterized in that, The NLP1 is silenced in the soybean plant, and the soybean has improved symbiotic nitrogen fixation efficiency; wherein the improved symbiotic nitrogen fixation efficiency comprises: increased tolerance to high levels of nitrogen elements, increased nodule number, increased nitrogenase activity, reduced expression level of nitrate-related genes, nitrate-induced into nucleus, and / or affecting the content of photosynthetic metabolites in the plant; Preferably, the NLP1 gene-edited soybean plant is prepared by the method of claim 9; Preferably, the content of nitrogen elements is represented by the concentration of nitrate, and the high levels of nitrogen elements are nitrate concentrations of ≥5 mM, preferably ≥6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 14 mM, 15 mM, 18 mM or 20 mM; more preferably, the nitrate is potassium nitrate or sodium nitrate; Preferably, the nitrate-related genes comprise: NR1, NR2, NiR, NRT2.1a, NRT2.1b, NRT2.1c, NRT2.1d; Preferably, the photosynthetic metabolites comprise: malate, alpha-ketoglutarate, sucrose.

Images