Application of NLP4 in improving symbiotic nitrogen fixation efficiency of soybeans

Abstract

The invention provides application of NLP4 in improving symbiotic nitrogen fixation efficiency of soybeans. The invention finds that the NLP4 gene participates in the nodulation process of nitrate regulation and control. The NLP4 gene is subjected to gene editing, and a stable NLP4 mutant transgenic soybean plant is obtained. Compared with a wild type, the NLP4 mutant plant shows a nitrogen-resistant nodulation phenotype and a lower expression level of nitrate-related genes. In addition, NLP4a can respond to nitrate induction nucleation. Therefore, gene editing is carried out aiming at the NLP4 gene, the symbiotic nitrogen fixation efficiency of the soybeans can be improved, and the NLP4 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 NLP4 in improving the efficiency of soybean symbiotic nitrogen fixation. BACKGROUND

[0002] Nitrogen is an important life element and a key factor limiting crop production. 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, finding the genes in soybean that perceive and transmit nitrogen signals to adapt to the nitrogen level in the environment to regulate nodule symbiosis, elucidating its molecular mechanism, and genetically modifying it, have important agricultural production significance for cultivating soybeans with excellent performance. SUMMARY

[0005] The application aims at the application of NLP4 in improving the efficiency of symbiotic nitrogen fixation of soybean. The application finds that NLP4 gene is involved in the noduleation process regulated by nitrate. By gene editing of NLP4 gene, a stable NLP4 mutant transgenic soybean plant is obtained. Compared with the wild type, the NLP4 mutant plant shows a more nitrogen-tolerant noduleation phenotype and a lower expression level of nitrate-related genes. In addition, NLP4a can be induced into the nucleus in response to nitrate. Therefore, gene editing of NLP4 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 NLP4 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 of root nodule, increasing the activity of nitrogen fixation enzyme, reducing the expression level of nitrate-related gene, and / or inducing into the nucleus in response to nitrate.

[0007] In the second aspect of the application, the application of a reagent for reducing the expression of NLP4 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 of root nodule, increasing the activity of nitrogen fixation enzyme, reducing the expression level of nitrate-related gene, and / or inducing into the nucleus in response to nitrate.

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

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

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

[0011] In one or more embodiments, the reduction of the expression of NLP4 is realized by targeting NLP4a and / or NLP4b through gene editing technology.

[0012] In one or more embodiments, the gene editing technology is CRISPR / Cas9 technology.

[0013] In one or more embodiments, the reagent comprises sgRNA and Cas9 enzyme for NLP4a and / or NLP4b.

[0014] In one or more embodiments, the reduction of expression of NLP4 is achieved by disrupting exon 2 of NLP4a, or disrupting exon 2 of NLP4b, or disrupting exon 2 of both NLP4a and NLP4b using CRISPR / Cas9 technology.

[0015] In one or more embodiments, the CRISPR / Cas9 technology comprises targeting exon 2 of NLP4a, exon 2 of NLP4b, or both exon 2 of NLP4a and NLP4b with a specific sgRNA.

[0016] In one or more embodiments, the sequence of the sgRNA is set forth in SEQ ID NO: 9.

[0017] 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 transcription and / or translation of the NLP4 protein.

[0018] In one or more embodiments, the targeted site comprises position 1560 of SEQ ID NO: 5, position 847 of SEQ ID NO: 6, position 1555 and / or 1556 of SEQ ID NO: 7, position 840 and / or 841 of SEQ ID NO: 8, position 1559 and / or 1560 of SEQ ID NO: 5, position 846 and / or 847 of SEQ ID NO: 6, position 1555 and / or 1556 of SEQ ID NO: 7, position 840 and / or 841 of SEQ ID NO: 8.

[0019] In a third aspect of the present application, a method of making a NLP4 gene edited soybean plant is provided, comprising the steps of:

[0020] (a) introducing into a soybean plant cell, tissue, or other explant a plasmid for reducing expression of the NLP4 gene containing a coding sequence for a specific sgRNA and a Cas9 enzyme;

[0021] (b) regenerating the soybean cell, tissue, or other explant into a genetically altered soybean plantlet;

[0022] (c) growing the NLP4 gene edited plantlet into a soybean plant having improved symbiotic nitrogen fixation efficiency as compared to an untransformed wild type soybean plant;

[0023] The improved symbiotic nitrogen fixation efficiency comprises: improved tolerance to high content of nitrogen element, increased number of nodule nodulation, increased nitrogenase activity, reduced expression level of nitrate-related gene, and / or induced into nucleus in response to nitrate.

[0024] In one or more embodiments, the method further comprises: screening or selecting the soybean cell, tissue or other explant prior to step (b); screening or selecting the soybean plantlet between steps (b) and (c); or screening or selecting the soybean after step (c) to identify successful editing of the NLP4 gene.

[0025] In one or more embodiments, the method of transformation comprises: particle bombardment, Agrobacterium-mediated transformation, Rhizobium-mediated transformation or protoplast transfection or transformation.

[0026] In one or more embodiments, the sequence of the sgRNA is set forth in SEQ ID NO: 9.

[0027] In a fourth aspect of the present application, there is provided a NLP4 gene edited soybean plant, wherein the NLP4 is silenced in the soybean plant and the soybean has improved symbiotic nitrogen fixation efficiency; wherein the improved symbiotic nitrogen fixation efficiency comprises: improved tolerance to high content of nitrogen element, increased number of nodule nodulation, increased nitrogenase activity, reduced expression level of nitrate-related gene, and / or induced into nucleus in response to nitrate.

[0028] In one or more embodiments, the NLP4 gene edited soybean plant is prepared by the method described in any of the embodiments of the present application.

[0029] In one or more embodiments, the content of the nitrogen element is represented by the concentration of nitrate, and the high content of nitrogen element is a nitrate concentration of ≥5mM, preferably ≥6mM, 7mM, 8mM, 9mM, 10mM, 12mM, 14mM, 15mM, 18mM or 20mM.

[0030] In one or more embodiments, the nitrate is potassium nitrate or sodium nitrate.

[0031] In one or more embodiments, the nitrate-related gene comprises: NR1, NR2, NiR, NRT2.1a, NRT2.1b, NRT2.1c, NRT2.1d.

[0032] Other aspects of the present application will be apparent to those skilled in the art from consideration of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1Figure 6. Multiple mutation analysis of Gmnlp4s mutant constructed by CRISPR-Cas9, and target sites of DNA sequence mutation in Gmnlp4a, Gmnlp4b, Gmnlp4a nlp4b mutants and protein structure prediction. The arrow indicates the frameshift mutation position in the amino acid sequence.

[0034] Figure 2 Figure 7. Nodulation phenotype and infection events statistics of Gmnlp4s mutant. (a-f) Nodulation phenotype of wild type (Huachun 6) and Gmnlp4a mutant plants treated with 0.25 mM, 10 mM, 20 mM nitrate (potassium nitrate KNO3). Scale bar is 5 mm. (g) Nodule number statistics of wild type and Gmnlp4s mutant plants under 0.25 mM, 10 mM, 20 mM nitrate (potassium nitrate KNO3) concentration. (h) Infection events statistics of wild type, Gmnlp4a 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, Gmnlp4a mutant plants under 10 mM nitrate (potassium nitrate KNO3) concentration. Different letters represent statistical differences between each experimental group, one way ANOVA (multiple comparisons).

[0035] Figure 3 Figure 8. Expression analysis of nitrate-related genes in Gmnlp4a mutant. (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 Gmnlp4a mutant plants under 0 mM, 0.5 mM, 5 mM, 10 mM nitrate (potassium nitrate KNO3) concentration treatment. Error bars represent standard deviation.

[0036] Figure 4 Figure 9. Nitrate promotes GmNLP4a accumulation in the nucleus. (a-b) Localization of p35S:GFP and p35S:GFP-GmNLP4a in tobacco leaves after 30 min nitrogen starvation and 10 mM KNO3 treatment. (c-d) Localization of p35S:GFP and p35S:GFP-GmNLP4a in soybean roots after 1 h nitrogen starvation and 10 mM KNO3 treatment. Pictures a-d were taken using confocal fluorescence microscopy, scale bar is 50 μm. Experimental results were repeated three times biologically. DETAILED DESCRIPTION

[0037] The inventors have found that NLP4 gene in soybean is involved in the nodule formation process regulated by nitrate. By CRISPR-Cas9 technology, the inventors have edited the NLP4 gene and obtained stable NLP4 mutant transgenic plants. Compared with wild type, the NLP4 mutant plants show more nitrogen-tolerant nodule phenotype and lower expression level of nitrate-related genes. In addition, NLP4a can be induced into the nucleus in response to nitrate. Therefore, editing the NLP4 gene can improve the efficiency of symbiotic nitrogen fixation in soybean and can be applied to improve soybean varieties.

[0038] As used herein, the "soybean" includes soybean with nodule symbiotic system and expressing NLP4. Preferably, the "soybean" is Glycine max. As used herein, the "symbiotic nitrogen fixation" refers to the process by which soybean utilizes and fixes nitrogen elements in the environment through the mycorrhizal symbiotic system. The content of nitrogen elements in the environment affects the efficiency of symbiotic nitrogen fixation in soybean. When the content of nitrogen elements in the environment is too high, the efficiency of symbiotic nitrogen fixation in soybean will be reduced, inhibiting the formation of root nodules (e.g. reducing the number of nodule nodulation) and nitrogenase (ARA) activity, which is known as "nitrogen repression" phenomenon. Nitrogen elements in the environment usually exist in the form of nitrate (e.g. potassium nitrate or sodium nitrate), therefore, the concentration of nitrate can reflect the content of nitrogen elements in the environment. In this paper, when the concentration of nitrate is ≥5mM, such as ≥6mM, 7mM, 8mM, 9mM, 10mM, 12mM, 14mM, 15mM, 18mM or 20mM, it is considered that the content of nitrogen elements is high; when the concentration of nitrate is ≤1mM, such as ≤0.9mM, 0.8mM, 0.7mM, 0.6mM, 0.5mM, 0.4mM, 0.3mM, 0.25mM, 0.2mM or 0.1mM, it is considered that the content of nitrogen elements is low.

[0039] In the art, the function of NLP4 gene and whether NLP4 gene is related to the efficiency of symbiotic nitrogen fixation in soybean have not been studied. In the present invention, the inventors have found for the first time that by editing the NLP4 gene, the efficiency of symbiotic nitrogen fixation in soybean is improved.

[0040] As used herein, "NLP4" from Glycine max typically includes the nucleotide sequence of the NLP4a gene coding region set forth in SEQ ID NO: 6, or the nucleotide sequence of the NLP4b gene coding region set forth in SEQ ID NO: 8. Any polypeptide / gene that is highly homologous (e.g., 80% or more homologous; preferably 90% or more homologous, such as 95%, 98%, or 99% homologous) to the NLP4 specifically recited herein and has the same function as the NLP4 is also included in the present application. In the present application, the NLP4 also includes its homologs, i.e., polypeptides / genes that exist in other species other than soybean, are homologous to the sequences recited above in the present application, and have the same function as the NLP4 in the present application, or polypeptides / genes that play the same or similar role in the same or similar signaling pathways. Since NLP4 is conserved in multiple species, it should be understood that, although the NLP4 gene recited in the embodiments of the present application is preferred, the present application is not limited to only the gene specifically recited in the embodiments.

[0041] In the present application, "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 to be a stop codon, thereby causing premature termination of transcription and translation of the NLP4 protein.

[0042] As an embodiment of the present application, targeted gene editing technology is used to genetically modify NLP4, for example, the CRISPR / Cas (e.g., Cas9) system can be used. The method includes co-transferring (the co-transferring can be separately, sequentially, or simultaneously transferring an expression vector containing sgRNA and an expression vector containing Cas9 mRNA, or can be transferring one expression vector containing both sgRNA and Cas9 mRNA) 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 site into the target region or target cell. Then, the sgRNA and Cas9 are introduced into the target cell. For example, the nucleic acid capable of forming the sgRNA can be amplified and constructed into an expression vector (e.g., pGel-201) containing Cas9 mRNA, and the expression vector is introduced into the target cell, so that active sgRNA and Cas9 mRNA are formed in the cell. After repeated screening, the present inventors screened some sgRNA sequences, and the positions targeted by these sequences are very suitable for efficiently guiding the Cas9 enzyme to break the DNA double strand and trigger the silent expression of NLP4.

[0043] The present application provides a method for reducing the expression of NLP4 based on gene editing technology, which is achieved by disrupting the 2nd exon of NLP4a, or disrupting the 2nd exon of NLP4b, or disrupting the 2nd exon of both NLP4a and NLP4b using CRISPR / Cas9 technology. Preferably, the sequence of a specific sgRNA is shown in SEQ ID NO: 28 (targeting NLP4a and NLP4b).

[0044] The CRISPR / Cas9 technology of a specific sgRNA can introduce random insertion or deletion or insertion and deletion at the 2nd exon of NLP4a, or the 2nd exon of NLP4b.

[0045] In some preferred embodiments, for the NLP4a gene, a deletion mutation can be introduced at the 2nd exon (e.g., corresponding to position 1560 of SEQ ID NO: 5, or corresponding to position 847 of SEQ ID NO: 6);

[0046] For the NLP4b gene, one or more insertion mutations, e.g., insertion of A, can be introduced at the 2nd exon (e.g., between positions 1555 and 1556 of SEQ ID NO: 7, or between positions 840 and 841 of SEQ ID NO: 8);

[0047] For the NLP4a gene, one or more insertion mutations, e.g., insertion of T, can be introduced at the 2nd exon (e.g., between positions 1559 and 1560 of SEQ ID NO: 5, or between positions 846 and 847 of SEQ ID NO: 6);

[0048] For the NLP4b gene, one or more insertion mutations, e.g., insertion of T, can be introduced at the 2nd exon (e.g., between positions 1555 and 1556 of SEQ ID NO: 7, or between positions 840 and 841 of SEQ ID NO: 8).

[0049] In some more preferred embodiments, the sgRNA can be obtained by amplification using PCR technology, e.g., using primers as shown in Table 2.

[0050] In some more specific embodiments, the sgRNA is obtained by amplification using two sets of primer pairs of SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

[0051] After gene editing, a NLP4 gene edited vector, host cell or soybean plant can be obtained. The NLP4 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, and / or response to nitrate induction into the nucleus.

[0052] In some embodiments, the nitrate-related genes include but are not limited to: NR1, NR2, NiR, NRT2.1a, NRT2.1b, NRT2.1c, NRT2.1d.

[0053] Therefore, the present application also provides the use of a reagent for reducing the expression of NLP4 protein based on gene editing technology to improve the efficiency of symbiotic nitrogen fixation of soybean. The related application is achieved based on the CRISPR / Cas9 technology to destroy the 2nd exon of NLP4a, or destroy the 2nd exon of NLP4b, or destroy the 2nd exon of NLP4a and NLP4b at the same time. The related application includes specific sgRNA sequence of gene editing, gene editing reagent, gene editing plasmid, gene editing soybean plant cell and its preparation method, gene editing soybean plant preparation method, gene editing soybean plant.

[0054] The gene editing reagent comprises a specific sgRNA, as described in any embodiment of the present application.

[0055] The gene editing plasmid is an expression plasmid containing a specific sgRNA and a Cas9 enzyme coding sequence.

[0056] 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, and obtain a soybean plant cell in which the expression of NLP4 protein is silenced after the NLP4 gene is edited. The gene editing soybean plant cell is a soybean plant cell in which the expression of NLP4 protein is silenced after the NLP4 gene is edited by the gene editing cell preparation method.

[0057] The gene editing soybean plant preparation method is to introduce an expression plasmid containing a specific sgRNA and a Cas9 enzyme coding sequence into a soybean plant cell, tissue or other explant to express, silence the expression of NLP4 protein in the soybean plant cell, tissue or other explant, and then regenerate the soybean plant from the soybean plant cell, tissue or other explant.

[0058] In some embodiments, the genetically edited soybean plant includes a soybean plant, plant part, or plant cell that has been genetically edited using any of the embodiments described herein (e.g., edited for the NLP4 gene). The plant part includes, but is not limited to, a leaf, stem, root, tuber, flower, seed, kernel, grain, fruit.

[0059] The present disclosure also provides a method of making a NLP4 genetically edited soybean plant, comprising the steps of:

[0060] (a) introducing into a soybean plant cell, tissue, or other explant a plasmid for reducing expression of the NLP4 gene containing a coding sequence for a specific sgRNA and a Cas9 enzyme;

[0061] (b) regenerating the soybean cell, tissue, or other explant into a genetically altered soybean plantlet;

[0062] (c) growing the NLP4 gene-edited plantlet into a soybean plant having increased symbiotic nitrogen fixation efficiency as compared to a non-transformed wild-type soybean plant;

[0063] In some embodiments, the increased symbiotic nitrogen fixation efficiency includes increased tolerance to high levels of nitrogen, increased nodule number, increased nitrogenase activity, decreased expression level of nitrate-related genes, and / or induction into the nucleus in response to nitrate.

[0064] In one or more embodiments, the method further comprises identifying successful editing of the NLP4 gene by screening or selecting the soybean cell, tissue, or other explant prior to step (b), the soybean plantlet between steps (b) and (c), or the soybean plant after step (c).

[0065] In one or more specific embodiments, the method of transformation includes, but is not limited to, particle bombardment (i.e., biolistics, gene gun), Agrobacterium-mediated transformation, Rhizobium-mediated transformation, or protoplast transfection or transformation.

[0066] Compared to other gene silencing techniques such as antisense nucleotide technology, CRISPR / Cas9-mediated gene editing can disrupt the NLP4 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, increased nodule number, increased ARA activity, decreased expression level of nitrate-related genes, and induction into the nucleus in response to nitrate. 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.

[0067] The application will be further described in connection with the following specific examples. It should be understood that these examples are only used to illustrate but not limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers.

[0068] Materials and methods

[0069] 1. Plant materials and growth conditions

[0070] Soybean (Glycine max) (Huachun 6) :

[0071] In the artificial climate chamber: planted in a moist environment of perlite and vermiculite (1:1), the temperature was 22℃ / 18℃ (day / night), the light cycle was 16h / 8h (day / night), the light intensity was 250 μmol·m -2 -1 , and the relative humidity was 70%;

[0072] In the natural environment: the optimal temperature was maintained at 28-30℃, and the natural light cycle and intensity were maintained for 5-6 months for growth.

[0073] 2. Strains and their growth conditions

[0074] (1) Escherichia coli (E. coli) : DH5α 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.

[0075] (2) Agrobacterium : A. rhizogenes K599 was used for the rooting transformation experiment of soybean, A. tumefaciens EHA105 and GV3101 were used for the transient transformation experiment of tobacco and the CRISPR-Cas9 stable transformation mutant transformation experiment of soybean; all were cultured in LB medium, solid in a 28℃ constant temperature incubator, and liquid in a 28℃ constant temperature shaker.

[0076] (3) Rhizobium : Bradyrhizobium japonicum USDA110-GUS was used for inoculating soybean. Bradyrhizobium japonicum was grown in HM medium, solid in a 28℃ constant temperature incubator, and liquid in a 28℃ constant temperature shaker.

[0077] 3. GmNLP4a / b gene

[0078] ​The genomic sequence of GmNLP4a (SEQ ID NO: 5, wherein the bold part is the exon part, in order, exon 1, exon 2, exon 3, exon 4 and exon 5, and the underlined part is the GmNLP4a / 4b sgRNA targeting sequence):

[0079]

[0080]

[0081] The nucleotide sequence of the coding region of GmNLP4a (SEQ ID NO: 6):

[0082]

[0083] GmNLP4b genomic sequence (SEQ ID NO: 7, wherein the bolded parts are exon parts, in order are exon 1, exon 2, exon 3, exon 4 and exon 5, respectively, and the underlined part is the GmNLP4a / 4b sgRNA targeting sequence):

[0084]

[0085]

[0086]

[0087] GmNLP4b coding region nucleotide sequence (SEQ ID NO: 8):

[0088]

[0089] PCR amplification of the target fragment (high-fidelity DNA polymerase Max, amplification system and program refer to the instruction) using primers under appropriate conditions, with soybean (wild type Hua Chun 6) cDNA as template; run gel electrophoresis and recover the target fragment with gel recovery kit, or directly recover the target fragment with PCR product recovery kit.

[0090] 4. Construction of the vector

[0091] Construction of the localization vector by Gateway method:

[0092] According to the GmNLP4a gene design primers, then add attB long adapter to both ends of the GmNLP4a gene primers, the primers are shown in Table 1; use GmNLP4a-attB-F / R primers to amplify the target gene fragment of GmNLP4a with soybean cDNA as template, mix it with intermediate vector pDONR-207, add Gateway BP recombinase, and react overnight at 22℃; heat shock the reaction product in 42℃ water bath for 30s, add 1mL of antibiotic-free liquid LB; after 37℃ incubation for 50min, evenly spread it on LB solid medium containing gentamicin resistance, and incubate in 37℃ incubator overnight; the next day, PCR identify positive clones, shake the bacteria and extract plasmid for sequencing; mix the correctly sequenced plasmid with the target vector pK7WGF2 (p35S:GFP), add Gateway LR recombinase, and react overnight in 22℃ incubator, then heat shock the transformation, and finally obtain the correctly sequenced plasmid p35S:GFP-GmNLP4a vector.

[0093] In order to transform it into soybean, it is necessary to screen the transformation root marker marker. Use GmNLP4a-MssI-F primer and GmNLP4a-AscI-R to amplify the 35S-GFP-GmNLP4a fragment in the vector p35S:GFP-GmNLP4a, and recombine it into the vector pUB:3flag-mCherry (cut off the promoter UB, only the 3flag-mCherry skeleton is left) digested with MssI / AscI enzyme, to obtain the final localization vector, i.e. p35S-GFP-GmNLP4a-3flag, mCherry (GFP-GmNLP4a-3flag, mCherry is expressed alone after 35S promoter expression).

[0094] The final positioning vector was electroporated into Agrobacterium, added to LB without antibiotics, and incubated at 28°C for 1 h. The mixture was then plated on LB plates with the corresponding antibiotic and incubated at 28°C for 48 h. Colony PCR was performed using vector primers to identify the correct clones. The GmNLP4a protein subcellular localization was observed under a laser confocal microscope using a root hair experiment in soybean and a tobacco transient transformation experiment.

[0095] Table 1

[0096]

[0097] Note: The underlined part in Table 1 is the enzyme digestion site.

[0098] The CRISPR-Cas9 vector was constructed by enzyme digestion:

[0099] Using the complete genomic DNA sequence of the target genes GmNLP4a and GmNLP4b, specific, normal GC content, and high editing efficiency small-guide RNAs (sgRNAs) were screened from the soybean database of the Guanyuefeng research group at Guangzhou University. Primers were designed according to the sgRNAs (as shown in Table 2) and PCR amplification was performed under the PCR reaction conditions. The sgRNAs for GmNLP4a and GmNLP4b were obtained, respectively. At the same time, the target vector pGel-201 was subjected to enzyme digestion reaction using the CRISPR-Cas9 enzyme digestion system at 37°C for 2 h. The PCR amplified target fragments and the vector fragments after enzyme digestion were recovered using a gel recovery kit, mixed, and then subjected to recombination reaction using a recombinase at 37°C for 30 min. The mixture was then subjected to heat shock transformation and sequencing identification. The plasmid verified by sequencing was transformed into Agrobacterium GV3101 by root hair, and the correct clones were selected for the construction of stable transformation plants in soybean (stable transformation plants were purchased from the Guanyuefeng research group at Guangzhou University).

[0100] Table 2

[0101]

[0102] 5. Soybean seed sterilization and germination

[0103] 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 rotated 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 then an appropriate 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 were opened, and young leaves grew (water was constantly supplemented during the period to keep the soil moist).

[0104] 6. Extraction of soybean genomic DNA

[0105] After the soybean seeds germinated and grew leaves, an appropriate amount of leaves was cut and placed in a 2 mL EP tube, steel balls and 300 μL of DNA extraction solution (the formula is shown in Table 3) were added, and the tube was placed in a grinder for grinding and crushing at 50 HZ / 3 min; after the ground sample was placed in a 65°C oven for 30 min, it was centrifuged at 12000 rpm for 10 min; the supernatant was taken into a 1.5 mL EP tube, 100 μL of phenol-chloroform-isoamyl alcohol (25:24:1) was added in a fume hood, and the mixture was inverted and mixed, and then centrifuged at 12000 rpm for 10 min; the supernatant was taken into a new 1.5 mL EP tube, and an equal volume of isopropanol and one-tenth volume of 3M NaAc were added, and the mixture was inverted and mixed, and then centrifuged at 12000 rpm for 10 min; the supernatant was discarded, 500 μL of 75% ethanol was added, and the precipitate was washed by blowing, and then centrifuged at 12000 rpm for 5 min; the ethanol was aspirated, and after air-drying at room temperature, 50 μL of double-distilled water was added until the precipitate was completely dissolved.

[0106] Table 3

[0107]

[0108]

[0109] 7. Inoculation of rhizobium, GUS staining of infected soybean and phenotype observation

[0110] 7.1 Inoculation of rhizobium

[0111] The seeds of the pure and stable transgenic mutants are sterilized and germinated well, and after one week, rhizobium inoculation is waited. At the same time, the frozen USDA110-GUS strain is taken out from the ultra-low temperature freezer at -80℃, spread on the HM solid medium containing kanamycin, and cultured in an inverted incubator at 28℃ for 3-5 days; the activated bacteria are picked into 3-5 mL HM liquid medium containing kanamycin, and cultured in a shaker at 28℃ for 3-5 days; the bacteria are transferred into new 50-100 mL HM liquid medium at a ratio of 1:50, and cultured at 28℃ until the OD600 is about 1.0, about 3 days; the bacteria liquid is dispensed into 50 mL centrifuge tubes, centrifuged at 3000 rpm for 15 min at 4℃; the supernatant is discarded, and the bacteria are resuspended by adding an appropriate amount of sterile water and gently blowing, and then diluted to OD600 of 0.08, and then the soybean stable transgenic mutants are inoculated with the inoculation pot.

[0112] 7.2 GUS staining and observation of infection events

[0113] After 7 days of inoculation with USDA110-GUS, the soybean seedlings are pulled out, and the roots are washed clean, and then fixed liquid (the formula is shown in Table 4) is added, and vacuumed on ice for 45 min; the fixed liquid is discarded, and the roots are washed with 0.1M phosphate buffer three times (the formula is shown in Table 5), 10 min each time; the root material is transferred into GUS staining liquid, and vacuumed for 45 min; after vacuuming, the GUS staining liquid is supplemented (the formula is shown in Table 6), and the material is placed in a 37℃ incubator for overnight staining; the next day, the GUS staining liquid is discarded, and the material is washed with 0.1M phosphate buffer three times, 10 min each time; the material is placed under a microscope for observation and statistics of infection events.

[0114] Table 4

[0115] Fixative 50 mL 10% paraformaldehyde 2.5 mL 0.1 M phosphate buffer 47.5 mL

[0116] Table 5

[0117] 0.1 M potassium phosphate buffer (pH 7.0) 1L KH2PO4 5.3g K2HPO4 13.9g

[0118] Table 6

[0119] 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

[0120] 7.3 Detection of nitrogenase activity

[0121] After 3 weeks of inoculation with USDA110-GUS, the soybean seedlings are pulled out, and the roots are washed clean, and then the nitrogenase activity of the Gmnlp4a mutant is detected. The specific steps are as follows: 2 root nodules are removed from each plant, and placed in 10 mL glass tubes which are tightly sealed, 7-8 plants are set for 15 samples in each experimental group; 1 mL air is first drawn from the glass tube with a syringe, and then 1m L Acetylene gas was injected into the glass tube; after 2h reaction at 28°C, the reaction was terminated with 1 m L 1M NaOH was added to terminate the reaction and wait for the measurement; meanwhile, the gas chromatograph was turned on and 0.3 m L The gas sample in the glass tube was injected into the gas chromatograph, the acetylene and ethylene peak values were recorded, the nodule weight was measured, and the nodule acetylene reduction activity (ARA activity) was calculated. The formula for calculating the nodule acetylene reduction activity is as follows:

[0122] Total formula:

[0123]

[0124] In the formula, FW refers to the fresh weight of the nodule, which can be measured in advance, X refers to the ethylene peak area measured by the gas chromatograph, and 2h refers to two hours.

[0125] 7.4 Observation of nodule formation events

[0126] After 3 weeks of inoculation with USDA110-GUS, the soybean seedlings were pulled out and the roots were washed clean. The nodule phenotype of the Gmnlp4s mutant was observed and counted by naked eye and stereomicroscope.

[0127] 8. Extraction of total RNA from plant roots (kit) and real-time fluorescent quantitative PCR (RT-qPCR)

[0128] The roots of the Gmnlp4a mutant were cut, 50-100mg per tube, steel balls were added, and after quick freezing in liquid nitrogen, they were quickly placed in a pre-cooled grinder, 50HZ / 3min; 500μL of lysis solution SL (5% β-mercaptoethanol was added before use) was added, vortexed to mix, and then centrifuged at 12000rpm for 2min; the supernatant was transferred to a filter column CS with a pipette, centrifuged at 12000rpm for 2 min min, and the supernatant in the collection tube was transferred to a new RNase e -Fre eThe centrifuge tube was placed in a centrifuge at 12000 rpm for 1 min, and the supernatant was discarded. 350 μL of deproteinization solution RW1 was added to the centrifugal column CR3, which was centrifuged at 12000 rpm for 1 min, and the waste liquid was discarded. 500 μL of deproteinization solution RW (anhydrous ethanol was added before use) was added to the centrifugal column CR3, which was centrifuged at 12000 rpm for 1 min, and the waste liquid was discarded. The centrifugal column CR3 was placed in a new RNase-Free centrifuge tube, 30-50 μL of RNase-Free ddH2O was added, and the mixture was allowed to stand at room temperature for 2 min. The mixture was centrifuged at 12000 rpm for 1 min, and the RNA solution was obtained. The concentration of the RNA solution was determined by an instrument.

[0129] The reverse transcription kit was used to synthesize cDNA. One-Step gDNA Removal and cDNA SynthesisSuperMix was used for reverse transcription. Primers were designed (Table 7), 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.

[0130] Table 7

[0131]

[0132]

[0133] 9. Tobacco transient transformation

[0134] The correct Agrobacterium EHA105 containing 35S-GFP-NLP4a was taken out from the ultra-low temperature freezer at -80°C, and was coated on LB solid medium containing spectinomycin and rifampicin antibiotics, and was incubated at 28°C overnight. A sterile gun head was used to pick a single colony of bacteria in 4 ml of LB liquid medium containing spectinomycin and rifampicin antibiotics, and was incubated at 28°C overnight. The bacteria were collected by centrifugation at 4000 rpm for 5 min, and were resuspended in tobacco transformation solution. The bacteria were centrifuged at 4000 rpm for 5 min, and the supernatant was discarded. The bacteria were resuspended in tobacco transformation solution again, and were washed three times. An appropriate amount of tobacco transformation solution was added to resuspend the bacteria, and the OD 600 value was measured. The bacteria were diluted to a certain concentration, and were mixed in equal volumes. The mixture was incubated at 28°C in the dark for 2-4 h. The bacteria were injected into the back of tobacco leaves using a 1 ml syringe, and the tobacco was watered with an appropriate amount of tap water. The tobacco was cultured in a greenhouse for 2-3 days. The leaves were cut to an appropriate size using scissors, and the subcellular localization of GmNLP4a under nitrogen starvation and the application of 10 mM nitrate was observed using a confocal laser microscope.

[0135] Table 8

[0136] Tobacco transformation solution components Final concentration MgCl2 10 mM MES 10 mM Acetosyringone 0.2 mM

[0137] 10. Rooted transformation of soybean seeds

[0138] At the same time of germination of soybean seeds, the correct Agrobacterium K599 containing 35S-GFP-NLP4a construct was activated and identified, and single colonies were picked into LB liquid medium containing spectinomycin, rifampicin and streptomycin resistance and incubated at 28°C overnight on a shaker; 200 μl of bacterial solution was evenly spread on LB solid medium containing corresponding resistance and incubated in a 28°C incubator for about 20 h. A blade was used to smoothly cut the soybean seedling hypocotyl 3-5 cm below, and the seedling was inserted into a 96-well plate containing sterile water. At the same time, the activated bacteria were collected, and the seedling incision was dipped in an appropriate amount of bacteria, and placed in a bag containing FP solid medium (about 30 mL of FP solid medium per bag and additional 0.1% KNO3 and CaCl2). The bag was placed vertically in a 22°C light incubator for incubation, and the liquid FP medium (containing 0.1% KNO3 and CaCl2) was supplemented continuously during the incubation; the non-transformed roots growing above the seedling incision were pulled out at 1-2 weeks, and the seedling was placed under a fluorescence microscope to screen for transformed roots with fluorescent markers at 3-4 weeks, and the non-transformed roots were pulled out, and the screened plants were used to observe the subcellular localization of GmNLP4a protein in soybean roots.

[0139] Example 1, obtaining of Gmnlp4s mutant

[0140] In this example, the GmNLP4 gene was edited by CRIPSR-CAS9 technology. First, the sgRNA specific to the GmNLP4 gene was designed, and specific primers were designed according to the sequence of the sgRNA. The sgRNA sequence and primer sequence obtained are shown in Table 2. After annealing, a double-stranded was formed and connected into a CRISPR-Cas9 vector, which was transformed into wild-type Huachun 6 soybean varieties 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 Gmnlp4s are shown in Table 9. Figure 1 and Table 9.

[0141] Table 9

[0142]

[0143] Example 2, Gmnlp4s mutant shows more nitrogen-tolerant nodule phenotype

[0144] 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 rhizobium USDA110-GUS, GUS staining was performed, and the infection events were observed under a microscope. After 3 weeks of inoculation with rhizobium USDA110-GUS, the soybean seedlings were pulled out and the roots were washed clean. The nodule phenotype of the Gmnlp4s mutant was observed and counted by naked eye and body microscope, and the nitrogenase activity of the Gmnlp4a mutant was detected.

[0145] The nodule pictures of wild type and Gmnlp4a mutant plants treated by 0.25 mM, 10 mM, 20 mM potassium nitrate solution poured into the soil are shown in Figure 2 a-f, and the counting results of the number of nodules on each plant are shown in Figure 2 g. The results show that, compared with the wild type (Huachun 6), after the GmNLP4 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 Gmnlp4a, Gmnlp4b and Gmnlp4a nlp4b, more red nodules can still be formed, and the total number of nodules is also more than that of the wild type.

[0146] The results of infection events show that, under nitrogen starvation and 10 mM nitrate treatment, the number of Gmnlp4a infection events is significantly reduced compared with the wild type, and the inhibition efficiency is obviously reduced Figure 2 h).

[0147] 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 Gmnlp4a mutant is not affected Figure 2 i).

[0148] The above results show that, compared with the wild type, the stable transgenic lines Gmnlp4s mutants all exhibit a more nitrogen-tolerant nodule phenotype.

[0149] Example 3, Expression level of nitrate-related genes in Gmnlp4s mutants is reduced

[0150] 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 Gmnlp4a obtained in Example 1 were detected by real-time fluorescent quantitative PCR.

[0151] The results show that the expression levels of NR1, NR2, NiR, NRT2.1a, NRT2.1b, NRT2.1c, NRT2.1d genes in Gmnlp4a are significantly lower than those in the wild type Figure 3 ).

[0152] Example 4, GmNLP4a responds to nitrate induction into the nucleus

[0153] Using Agrobacterium EHA105 containing 35S-GFP-NLP4a construct, tobacco is used for transient transformation, and then soybean hairy root transformation is carried out, and the subcellular localization of GmNLP4a under the conditions of nitrogen starvation and application of 10 mM nitrate is observed by confocal laser microscope.

[0154] The results show that under the condition of nitrogen starvation, GmNLP4a is mainly located in the cytoplasm Figure 4 a, 4c), after 10 mM nitrate treatment, GmNLP4a is obviously aggregated in the nucleus Figure 4 b, 4d).

[0155] The above results show that GmNLP4a can respond to nitrate induction and enter the nucleus from the cytoplasm Figure 4 d). It can be seen that GmNLP4 plays a core role in the inhibition of nodule formation by nitrate.

[0156] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which 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. At the same time, all the documents mentioned in the present application are cited as reference in the present application, just as each document is cited as reference individually.

Claims

1. A method of increasing the efficiency of symbiotic nitrogen fixation in soybean, comprising: reducing expression of NLP4 in soybean; wherein the improving symbiotic nitrogen fixation efficiency of soybean comprises: improving tolerance to high content of nitrogen element, increasing nodule number, increasing nitrogenase activity, reducing expression level of nitrate-related genes, and / or inducing into nucleus in response to nitrate.

2. Use of an agent that reduces expression of NLP4 to increase efficiency of symbiotic nitrogen fixation in soybean; wherein the increase in efficiency of symbiotic nitrogen fixation in soybean comprises: improving tolerance to high content of nitrogen element, increasing nodule number, increasing nitrogenase activity, reducing expression level of nitrate-related genes, and / or inducing into nucleus in response to nitrate.

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 refers to 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.

4. The method of any one of claims 1 to 3, wherein, The reduction of NLP4 expression is achieved by targeting NLP4a and / or NLP4b using gene editing technology. Preferably, the gene editing technology is CRISPR / Cas9 technology. More preferably, the reagent includes sgRNA and Cas9 enzyme targeting NLP4a and / or NLP4b.

5. The method of any one of claims 1-4, wherein, The reduction of NLP4 expression is achieved by disrupting exon 2 of NLP4a, or disrupting exon 2 of NLP4b, or disrupting exon 2 of both NLP4a and NLP4b using CRISPR / Cas9 technology.

6. The method of claim 5, wherein, The CRISPR / Cas9 technology includes: using specific sgRNA to target exon 2 of NLP4a, exon 2 of NLP4b, or both exon 2 of NLP4a and NLP4b. Preferably, the sequence of the sgRNA is shown in SEQ ID NO:

9.

7. The method of claim 5, wherein, The disruption includes: introducing at least one base insertion, deletion, substitution, or a combination thereof into the targeted region, so that the codon at the targeted site is edited into a stop codon, resulting in premature termination of transcription and / or translation of NLP4 protein.

8. The method of claim 7, wherein, The targeted site includes: position 1560 of SEQ ID NO: 5, position 847 of SEQ ID NO: 6, position 1555 and / or position 1556 of SEQ ID NO: 7, position 840 and / or position 841 of SEQ ID NO: 8, position 1559 and / or position 1560 of SEQ ID NO: 5, position 846 and / or position 847 of SEQ ID NO: 6, position 1555 and / or position 1556 of SEQ ID NO: 7, position 840 and / or position 841 of SEQ ID NO:

8.

9. A method for preparing NLP4 gene edited soybean plants, comprising the following steps: (a) introducing a plasmid for reducing expression of NLP4 gene containing coding sequences of specific sgRNA and Cas9 enzyme into soybean plant cells, tissues or other explants; (b) regenerating the soybean cells, tissues or other explants into genetically altered soybean plantlets; (c) growing the NLP4 gene-edited plantlets into soybean plants having improved symbiotic nitrogen fixation efficiency as compared to untransformed wild-type soybean plants; wherein the improved symbiotic nitrogen fixation efficiency comprises: improved 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 response; Preferably, the method further comprises: screening or selecting the soybean cells, tissues or other explants before 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 NLP4 gene; Preferably, the transformation method comprises: particle bombardment, Agrobacterium-mediated transformation, Rhizobium-mediated transformation or protoplast transfection or transformation; Preferably, the sgRNA has a sequence as set forth in SEQ ID NO:

9.

10. A NLP4 gene edited soybean plant, characterized in that, The soybean plant has a silenced expression of NLP4, and the soybean has improved symbiotic nitrogen fixation efficiency; wherein the improved symbiotic nitrogen fixation efficiency comprises: improved 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 response; Preferably, the NLP4 gene-edited soybean plant is prepared by the method of claim 9. Preferably, the nitrogen element is in the form of nitrate, and the high level 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; 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.

Images