Application of ZmNRT2.7 gene or related biological material containing ZmNRT2.7 gene

Abstract

The invention belongs to the technical field of plant molecular biology, and particularly relates to application of a ZmNRT2.7 gene or a related biological material containing the ZmNRT2.7 gene. A zmnrt2.7 gene editing knockout mutant and a ZmNRT2.7 overexpression transgenic line are respectively obtained by taking a corn B73 inbred line as a transformation material, and the biological functions of the ZmNRT2.7 gene are clarified through methods such as phenotypic analysis, physiological and biochemical index determination and agronomic character investigation of the transgenic line, so that the ZmNRT2.7 gene is obtained. And an important new gene resource is provided for cultivating a new corn variety with high nitrogen utilization rate.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular biology, and in particular relates to the application of ZmNRT2.7 gene or biological materials containing ZmNRT2.7 gene. Background Art

[0002] Nitrogen is a large amount of mineral elements necessary for plant growth and development. In different soils, nitrogen can exist in the form of nitrates, ammonium salts, amino acids, proteins or other organic matter. Among them, nitrates are the main form of nitrogen absorbed and utilized by most terrestrial plants. Under natural conditions, the concentration of nitrates in the soil is low and easily lost, which cannot fully meet the needs of normal plant growth and development. In addition, the nitrogen utilization efficiency of major crops is low, and the yield is highly dependent on nitrogen fertilizer. In order to maintain high crop yields, excessive nitrogen fertilizers are usually required in agricultural production, which not only greatly increases the cost of planting, but also causes serious environmental pollution. Therefore, exploring important genes that regulate nitrogen utilization and cultivating new varieties with high nitrogen utilization rates are effective ways to achieve the goal of sustainable agricultural development of "less input, more output, and less pollution."

[0003] Plant roots can absorb nitrate nitrogen from the soil using nitrate transporters with different affinities and specificities, and distribute the nitrogen levels of their own sources and sinks according to the nitrogen concentration in the soil to meet the needs of growth and development. At present, some nitrate transporters have been identified in the model organism rice, such as OsNRT1.1B, OsNRT1.1A, OsNRT2.4, etc. The discovery and utilization of these key genes have significantly accelerated the process of rice nitrogen efficient breeding. In the model plant Arabidopsis, it has also been confirmed that AtNRT1.1, as a dual-affinity transporter of nitrate, can not only absorb nitrate from the soil, but also be responsible for transporting nitrate absorbed by the roots to the aboveground organs. In corn, the ZmNRT1.1B protein can affect nitrate transport and nitrogen signal transduction, thereby improving the nitrogen use efficiency of corn. These studies show that discovering new nitrate transporters in crops and analyzing their biological functions can provide important targets for genetic improvement of efficient nitrogen use in crops.

[0004] Nitrogen plays a key role in the formation of maize yield and quality. However, it is still unclear which other nitrate transporters in maize have the function of transporting nitrate nitrogen and regulating nitrogen utilization. Summary of the invention

[0005] In order to solve the defect of low nitrogen utilization rate of corn in the prior art, the present invention provides a new use of the ZmNRT2.7 gene, specifically relates to the use of the ZmNRT2.7 gene or biological materials containing the ZmNRT2.7 gene.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0007] The present invention provides the use of ZmNRT2.7 gene or biological materials containing ZmNRT2.7 gene in any of the following:

[0008] a. Application in improving nitrogen utilization efficiency of corn;

[0009] b. Application in cultivating corn with high nitrogen utilization efficiency;

[0010] c. Application in improving agronomic traits of corn;

[0011] The nucleotide sequence of the ZmNRT2.7 gene is shown in SEQ ID NO.1.

[0012] In some technical solutions of the present invention, the biomaterial includes any one of the following:

[0013] a. The protein encoded by the ZmNRT2.7 gene, the amino acid sequence of which is shown in SEQ ID NO.2;

[0014] b. a recombinant plasmid comprising the ZmNRT2.7 gene;

[0015] c. A recombinant microbial strain comprising the gene.

[0016] In some technical solutions of the present invention, the original vector of the recombinant plasmid is pCAMBIA1305.

[0017] In some technical solutions of the present invention, the starting strain of the recombinant microorganism is Escherichia coli.

[0018] In some embodiments of the present invention, the agronomic traits include: corn cob weight, corn cob length, corn cob diameter, and 100-kernel weight.

[0019] The present invention also provides a method for improving the nitrogen utilization rate of corn, comprising: overexpressing the ZmNRT2.7 gene in corn plants to obtain corn plants with high nitrogen utilization rate.

[0020] In some embodiments of the present invention, the steps include constructing a ZmNRT2.7 gene overexpression vector and transferring the ZmNRT2.7 gene overexpression vector into corn plants.

[0021] The present invention also provides a method for improving agronomic traits of corn, which enables the ZmNRT2.7 gene to be overexpressed in corn plants to obtain corn plants with excellent agronomic traits.

[0022] In some embodiments of the present invention, the steps include constructing a ZmNRT2.7 gene overexpression vector and transferring the ZmNRT2.7 gene overexpression vector into corn plants.

[0023] Compared with the prior art, the present invention has the following beneficial effects: the present invention uses corn B73-329 inbred line as transformation material, obtains zmnrt2.7 gene editing knockout mutant and ZmNRT2.7 overexpressing transgenic strain, respectively, and preliminarily clarifies the biological function of ZmNRT2.7 gene through phenotypic analysis, determination of physiological and biochemical indicators and investigation of agronomic traits of transgenic strain, providing important new gene resources for breeding new corn varieties with high nitrogen utilization efficiency.

[0024] The present invention creates a gene-edited mutant and an overexpression transgenic strain of the ZmNRT2.7 gene, proving that the ZmNRT2.7 gene plays an important role in the absorption, assimilation and transport of nitrate nitrogen, and confirming that the ZmNRT2.7 gene has important breeding value. So far, there has been no application of the ZmNRT2.7 gene in regulating nitrogen utilization in corn, and the present invention provides a theoretical basis for efficient nitrogen breeding in corn.

[0025] The present invention systematically analyzes the biological function of ZmNRT2.7, proves that ZmNRT2.7 plays an important role in the absorption, assimilation and transport of nitrate nitrogen, and also provides a theoretical basis for efficient nitrogen breeding of corn. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram for the construction of maize overexpression vector.

[0027] Figure 2 These are the Bar test results for the maize ZmNRT2.7 gene overexpression strain.

[0028] Figure 3 This is the structure diagram of the maize ZmNRT2.7 knockout gene.

[0029] Figure 4 This is a phylogenetic tree analysis of the maize NRT2s family.

[0030] Figure 5 This is a diagram for the functional domain analysis of corn NRT2s protein.

[0031] Figure 6 This is a diagram of the conserved motif analysis of corn NRT2s protein.

[0032] Figure 7 This is an analysis diagram of the cis-acting elements in the promoter region of the maize NRT2s gene family.

[0033] Figure 8This is the result of protein structure prediction for ZmNRT2.7; Figure 8 A in the figure indicates the predicted transmembrane structure of ZmNRT2.7 protein; Figure 8 B and C show the three-dimensional structure analysis of ZmNRT2.7 protein.

[0034] Fig. 9 This is the expression heat map of the ZmNRT2 subgene family.

[0035] Fig.10 This is a diagram showing the tissue expression pattern of ZmNRT2.7.

[0036] Fig.11 The in situ hybridization result of ZmNRT2.7 is shown; wherein, Fig.11 A indicates the cross section of leaves of B73 at the four-leaf stage (V4), and a blank probe was used as a negative control (NC); Fig.11 B represents the cross section of a leaf of B73 at the four-leaf stage using ZmNRT2.7 RNA as a probe; Fig.11 C indicates the cross section of the main root of B73 at the four-leaf stage using ZmNRT2.7 RNA as a probe; Fig.11 D indicates the cross section of the lateral root of B73 at the four-leaf stage using ZmNRT2.7 RNA as a probe; wherein VB indicates vascular bundle; EC indicates epidermal cell; and MC indicates mesophyll cell.

[0037] Fig.12 This is a diagram of the inducible expression pattern of ZmNRT2.7.

[0038] Fig.13 Subcellular localization of ZmNRT2.7 protein.

[0039] Fig.14 This is the map of the 1305-d35SGFP vector.

[0040] Fig.15 The RT-qPCR results of knockout and overexpression strains of ZmNRT2.7 gene; Fig.15 A indicates the relative expression levels of the two mutant strains and the wild-type ZmNRT2.7 gene; Fig.15 B shows the relative expression levels of the two overexpression strains and the wild-type ZmNRT2.7 gene.

[0041] Fig.16 This is a statistical diagram of the seedling phenotypes of zmnrt2.7 knockout mutants and overexpression strains under different nitrogen conditions; Fig.16 A in the figure represents the phenotypes of different plants under LN conditions; Fig.16 B in the figure represents the phenotypes of different plants under HN conditions; Fig.16C in the figure represents the statistical result of the fresh weight of the aboveground parts of different strains under different treatment conditions; Fig.16 D in the figure represents the statistical result of the fresh weight of underground parts of different strains under different treatment conditions; Fig.16 E in the figure represents the statistical result of the dry weight of the aboveground parts of different strains under different treatment conditions; Fig.16 F in the figure represents the statistical result of dry weight of underground parts of different strains under different treatment conditions.

[0042] Fig.17 Figure 2 is a graph of agronomic traits of zmnrt2.7 knockout mutants and overexpression lines; Fig.17 A and D in the figure represent ear phenotypes; Fig.17 B and E in the figure represent the grain length phenotype (10 grains); Fig.17 C and F in the figure represent the grain width phenotype diagram (10 grains).

[0043] Fig.18 The figure shows the measurement results of ear length, ear width, kernel length, kernel width and 100-kernel weight of zmnrt2.7 knockout mutant and overexpression strain corn; Fig.18 A represents the statistical result of ear weight of different strains;

[0044] Fig.18 B in the figure represents the statistical result of ear length of different strains; Fig.18 C in the figure represents the statistical result of ear width of different strains; Fig.18 D in the figure represents the statistical result of grain length of different strains; Fig.18 E in the figure represents the statistical result of grain width of different strains; Fig.18 F in the figure represents the statistical result of grain thickness of different strains; Fig.18 G in the figure represents the statistical result of 100-grain weight of different strains.

[0045] Fig.19 For plant pairs 15 Absorption measurement diagram of N; wherein, Fig.19 A in the figure indicates the aboveground parts of different strains. 15 N / 14 N ratio; Fig.19 The B in the figure indicates the difference between the aboveground and underground parts of different strains. 15 N / 14 N ratio.

[0046] Fig. 20 This is the result of the chlorate lethality test; Fig. 20 A in the figure indicates that WT and zmnrt2.7 mutants grow normally; Fig. 20 Panel B shows WT and zmnrt2.7 mutant treated with chlorate for 3 days; Fig. 20C in the figure indicates 7d of chlorate treatment of WT and zmnrt2.7 mutant; Fig. 20 D in the figure indicates that the WT and ZmNRT2.7 overexpressing lines grew normally for 8 days; Fig. 20 E in the figure indicates 3d chlorate treatment of WT and ZmNRT2.7 overexpressing strains; Fig. 20 F in the figure indicates 7d of chlorate treatment for WT and ZmNRT2.7 overexpressing strains; Fig. 20 G in the figure indicates lethality analysis of WT and zmnrt2.7 knockout mutant; Fig. 20 H in the figure indicates lethality analysis of WT and ZmNRT2.7 overexpressing strains. DETAILED DESCRIPTION

[0047] The present invention is described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0048] 1. Experimental Materials

[0049] 1. Vectors and strains

[0050] Escherichia coli cloning strain Trans-T1 was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0051] Escherichia coli cloned strain DH5α was purchased from Shanghai Weidi Biotechnology Co., Ltd.;

[0052] pEASY-Blunt Simple cloning vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0053] 2. Instruments and equipment

[0054] Table 1 Instruments and equipment

[0055] Instruments and Equipment Related information Agarose gel electrophoresis apparatus JUNY1JY600C Gel imaging analysis system GelDoc-It2Imager Real-time fluorescence quantitative PCR instrument RocheLightCycler480 Laser confocal microscopy Zeiss LSM800 Fluorescence microscopy LeciaDM5000B

[0056] 3. Enzymes and reagents

[0057] Prime STARMax DNA Polymerase was purchased from Bao Biotechnology Co., Ltd.;

[0058] Trizon was purchased from Bao Bioengineering Co., Ltd.;

[0059] Seamless Cloning and Assembly Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0060] The reverse transcription kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0061] DNA gel recovery kit was purchased from Axygen;

[0062] The plasmid extraction kit was purchased from Axygen;

[0063] The genome extraction kit was purchased from Axygen;

[0064] Various restriction endonucleases and their corresponding reaction buffers were purchased from NEB;

[0065] SYBR Green Realtime PCR MasterMix was purchased from Hoffmann-La Roche;

[0066] ChamQ Universal SYBR qPCR MasterMix was purchased from Novozymes Biotech Co., Ltd.

[0067] 4. Culture medium preparation

[0068] Escherichia coli LB medium: each 100 mL dd H2O contains 1.0 g of tryptone, 0.5 g of yeast extract, 2.0 g of NaCl, and 1.5 g of agar (solid).

[0069] 5. Reagent configuration

[0070] 50×TAE solution (1 L): Tris 242 g, Na2EDTA·2H2O 37.2 g, dd H2O 800 mL, glacial acetic acid 57.1 mL.

[0071] WI solution formula (100 mL): mannitol 10.932 g, MES 0.085 g, KCl 0.149 g, adjust pH to 5.8 with KOH, sterilize at high temperature and high pressure for 20 min, and store at 4°C.

[0072] MMG solution formula (50 mL): MgCl2·6H2O 0.152 g, MES 0.05 g, mannitol 5.467 g, adjust pH to 5.8 with KOH, sterilize at high temperature and high pressure for 20 min, and store at 4°C.

[0073] 45% PEG4000 solution (5.5 mL): PEG4000 4.5 g, ddH2O 2 mL, 0.8 M mannitol 2.5 mL, 1 M CaCl2 1 mL.

[0074] Cellulase lysis solution (15 mL): CelluLase R10 dry powder 0.22 g, MeceroZyme R10 dry powder 0.075 g, mannitol dry powder 1.64 g, 0.30 M KCl mother solution 1 mL, 0.30 M MES mother solution 1 mL, add 10 mL of sterilized ddH2O, heat in a 55°C water bath for 10 min, cool to room temperature, add 0.15 M CaCl2 mother solution 1 mL, 1.50% BSA 1 mL, 75 mM β-mercaptoethanol mother solution 6 μL, and filter using a 0.45 μM filter membrane into a new 50 mL centrifuge tube.

[0075] W5 solution formula (500 mL): NaCl 4.5 g, CaCl2·2H2O 9.186 g, KCl 0.186 g, Glucose 0.45 g, MES 0.15 g.

[0076] TBST buffer (1 L): Add 201 mL of polysorbate to 1 L of TBS buffer.

[0077] Hoagland medium (1L): 0.75mM K2SO4, 0.65mM MgSO4·7H2O, 1×10 -3 mM H3BO3, 1×10 - 3 mM MnSO4·H2O, 1×10 -4 mM CuSO4·5H2O, 1×10 -3 mM ZnSO4·7H2O, 5×10 -6 mM(NH4)6Mo7O 24 4H2O, 2mM Ca(NO 3) 4H2O, 2mM Fe-EDTA, 0.25mM KH2PO4, 0.1mM KCl, adjust pH to 5.8-6.0.

[0078] Example 1: Construction of plant materials

[0079] 1. Obtaining plant materials:

[0080] (1) Construction of overexpression plants

[0081] 1) Cloning of ZmNRT2.7 gene

[0082] According to the fourth-generation cDNA database of maize B73, the most suitable and optimal transcript of ZmNRT2.7 (Zm00001d044504) was selected, and the CDS sequence of ZmNRT2.7 was obtained from the NCBI website. The sequence is shown in SEQ ID NO: 1, and the amino acid sequence encoded by it is shown in SEQ ID NO: 2. Using Primer Premier 5 software, several sets of upstream and downstream primers (ZmNRT2.7-F and ZmNRT2.7-R) of different lengths were designed. The sequences are shown in SEQ ID NO: 3-4 in Table 2. The specificity of the primers was checked by Primer-Blast on different websites, and the upstream and downstream primers with the best specificity were selected for cloning.

[0083] Table 2 Cloning primers

[0084] name sequence serial number ZmNRT2.7-F 5′-ATGGTGGCGATGGGGAAA-3′ SEQ ID NO:3 ZmNRT2.7-R 5′-TTTGAGGAGCGTGTAATCTCCAG-3′ SEQ ID NO:4

[0085] Select corn inbred line B73 of the same year, with full grains and relatively uniform size, and grow them in an incubator with 28℃ light for 16h and 22℃ dark for 8h, with a ratio of vermiculite to black soil of 1:1. When they grow to the three-leaf stage, cut the leaves to extract RNA, reverse transcribe into cDNA, and then use cDNA as a template for PCR. After the PCR reaction is completed, use agarose gel electrophoresis to detect whether the band is correct. If it is correct and there is a bright and clear single band, perform gel recovery (refer to the instructions of the DNA gel recovery kit for specific steps).

[0086] Take 4.5 μL of PCR gel to recover the product, add 0.5 μL of Blunt Simple, mix well, put in a 25℃ metal bath for 30 minutes, add 30 μL of E. coli Trans1-T1 competent cells, and after transformation, apply it on a LB solid plate with kanamycin resistance, put it in a 37℃ incubator and invert it for one day, pick the spots and shake the bacteria for sequencing. After the sequencing is correct, keep the bacteria and extract the plasmid (refer to the instructions of the plasmid extraction kit for specific steps), and ZmNRT2.7 is obtained.

[0087] 2) Taking pCAMBIA1305 vector as the vector required for the experiment, constructing d35S-ZmNRT2.7-GFP as an example, select the appropriate restriction site BamHI (GGATCC). Design the recombination primer sequence as shown in SEQ ID NO: 5-6 in Table 3:

[0088] Table 3 Recombination primers

[0089]

[0090] PCR amplification was performed, and the reaction conditions of Prime STAR Max DNA polymerase were as follows: 98℃ pre-denaturation for 10min; 98℃ denaturation for 10s, 64℃ annealing for 5s, 72℃ extension for 3min, 34 cycles; 72℃ total extension for 10min; 20℃ insulation. The pCAMBIA1305 vector was digested with a single enzyme, and the single enzyme digestion loading system was 16μL of vector plasmid, 2μL of BamHI, and 2μL of Buffer. Digestion was performed in a metal bath at 37℃ for 3 h. After the PCR was completed, agarose gel electrophoresis was performed together. The enzyme digestion should have an empty load as a control group to check whether the enzyme digestion was successful. If the band is correct, gel recovery was performed.

[0091] After gel recovery, the gel recovery product was used for homologous recombination. The homologous recombination system was 5 μL of recombinase ( Seamless Cloning and Assembly Kit), 4 μL of gene gel to recover the product, 1 μL of carrier gel to recover the product. Then heat in a 50℃ water bath for 15 min. In the above 10μL system, add 30μL of E. coli Trans1-T1 competent cells, and after transformation, apply them on LB solid plates with kanamycin resistance, place them in a 37℃ incubator and invert for one day, pick spots and shake the bacteria for sequencing, and after sequencing is correct, keep the bacteria and extract the plasmid, which is the constructed d35S-ZmNRT2.7-GFP vector (such as Figure 1 shown).

[0092] 3) Genetic transformation

[0093] With B73 as the genetic background, Weimi Biotechnology (Jiangsu) Co., Ltd. carried out corn genetic transformation, and finally the company obtained the ZmNRT2.7 overexpression strain. After receiving the transgenic material seeds, the T0 generation seeds were planted, and positive seedlings were tested at the four-leaf to five-leaf stage. The ZmNRT2.7 gene corn overexpression plants were tested by Bar test strips (such as Figure 2 As shown in the figure), positive seedlings detected by genome PCR. Using Bar test strips, two bars indicate positive plants ZmNRT2.7 OE1 and ZmNRT2.7 OE2 .

[0094] (2) Construction of knockout plants

[0095] In order to analyze the biological function of the ZmNRT2.7 gene, the present invention uses the nucleotide sequence of the ZmNRT2.7 gene to design a specific sgRNA, and the targeting domain sequence of the sgRNA is shown in SEQ ID NO: 7-8 in Table 4 to construct a CRISPR / Cas9 vector, and entrusts Changzhou Weimi Biotechnology (Jiangsu) Co., Ltd. to genetically transform the maize B73 inbred line. Genetic transformation was carried out by a maize tissue culture method, and ZmNRT2.7 knockout plants, i.e., zmnrt2.7 mutants, were obtained after identification.

[0096] Table 4 Targeting domain sequences of sgRNA

[0097] name sequence serial number sgRNA1 5′-GAGAACTGCTGCTACGGCGTCGG-3′ SEQ ID NO:7 sgRNA2 5′-GGGCGACGACGCGGTGACCGCGG-3′ SEQ ID NO:8

[0098] After the company returns the seeds, they are planted in the soil. When they grow to the three-leaf stage, some leaves are cut to extract the genome. The genome is used as a template for PCR, verified by agarose gel electrophoresis, and the genome of the corn inbred line B73 is used as a control. After sequencing, the knockout status is determined. If no homozygous mutant is screened out, continue to plant and screen for generations until a homozygous mutant without Cas9 is screened out.

[0099] Finally, two strains with different knockout modes were obtained: ZmNRT2.7 KO1 and ZmNRT2.7 KO2 (like Figure 3 ZmNRT2.7 gene knockout maize plants were detected by genomic PCR.

[0100] 2. Extraction of Maize Genome

[0101] (1) Plant corn in the soil. When it grows to the three-leaf stage, cut 1 g of leaves with scissors, put them into liquid nitrogen and grind them in a pre-heated mortar. Transfer the powder to a 2 mL centrifuge tube. The height of the powder should reach 1 / 3 of the centrifuge tube. Do not add too much.

[0102] (2) The specific steps were carried out according to the instructions of the genome extraction kit of Axygen Company.

[0103] 3. Extraction of corn RNA

[0104] (1) Germinate corn in the soil and take samples when it reaches the three-leaf stage.

[0105] (2) Take samples as needed, wrap them in tin foil, clearly mark them, and quickly place them in liquid nitrogen to prevent RNA degradation.

[0106] (3) Heat the mortar in advance, turn on the 4°C centrifuge to precool, and use liquid nitrogen to precool the mortar and RNase free tube.

[0107] (4) Grind the sample thoroughly and quickly, and transfer the powder to a 2 mL RNase free tube. The powder should be 1 / 3 of the tube, but not too much. (Change a pair of plastic gloves for each sample to prevent cross contamination).

[0108] (5) Add 1 mL of Trizon solution, mix thoroughly using a vortex oscillator, and leave at room temperature in the dark for 10 min.

[0109] (6) Add 500 μL of nucleic acid extraction solution (chloroform:isoamyl alcohol = 24:1), mix thoroughly using a vortex oscillator, and place on ice for 10 min.

[0110] (7) Centrifuge at 12,000 rpm for 10 min at 4°C.

[0111] (8) Transfer 500 μL of the supernatant to a new 1.5 mL RNase free tube and add an equal volume of isopropanol. Mix thoroughly using a vortex oscillator and place on ice for 10 min.

[0112] (9) Centrifuge at 12,000 rpm for 10 min at 4°C.

[0113] (10) Discard the supernatant and add 1 mL of 75% ethanol prepared with DEPC water, rinse and mix well to suspend the precipitate.

[0114] (11) Centrifuge at 12,000 rpm for 5 min at 4°C and discard the supernatant.

[0115] (12) Repeat steps (10) and (11) once.

[0116] (13) Aspirate all the supernatant and empty the tube once.

[0117] (14) Add 50 μL of DEPC water to dissolve the RNA.

[0118] (15) Detect RNA concentration and OD value. The OD260 / OD280 value of good quality RNA is between 1.8 and 2.0. If it exceeds this value, the RNA is considered to be degraded and cannot be used. 1% agarose gel electrophoresis can also be performed. The brightness of the 28S rRNA band should be twice that of the 18S rRNA.

[0119] (16) After the RNA is dissolved, it is best to take 1-2 μL of RNA and perform reverse transcription (refer to the instructions of the reverse transcription kit for specific steps) into cDNA for storage.

[0120] (17) The remaining RNA sample should be immediately stored in a -80°C freezer.

[0121] 4. Basic information of NRT2 family

[0122] The NRT2 protein sequence of maize was obtained from the Phytozome (https: / / phytozome-next.jgi.doe.gov / ) database. The ExPASy (https: / / www.expasy.org / ) online software was used to analyze the structural physicochemical properties of the NRT2 family members, including the number of amino acids, molecular weight, isoelectric point (pI), number of introns, etc. The CELLO (http: / / cello.life.nctu.edu.tw / ) online website was used to analyze the subcellular localization of the NRT2 family.

[0123] 5. Phylogenetic tree analysis of the NRT2 family

[0124] In order to understand the NRTs gene family in maize, the amino acid sequences of the NRTs family members of maize, rice, sorghum and Arabidopsis were used for multiple sequence alignment and phylogenetic tree construction. First, the protein sequences of the NRT2 family in maize, Arabidopsis, rice and sorghum were obtained. Then, the protein sequences of the NRT2 family in maize, Arabidopsis, rice and sorghum were multiple aligned using DNAMAN software. All NRT2 family genes were integrated together according to similarity. Their full-length protein sequences were imported into MAGE7.0 software to start building an evolutionary tree. The result of the evolutionary tree was copied and saved as nwk format, and the nwk format tree text was uploaded using the iTOL (https: / / itol.embl.de / ) online tool, and the evolutionary tree was beautified.

[0125] 6. Analysis of cis-acting elements in the promoter of maize NRT2 gene family

[0126] The 2000 bp sequence upstream of the start codon of maize NRT2 and the maize NRT2 gene family IDs were extracted and submitted to the PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) online database to predict the cis-acting elements in the promoter region. After obtaining the results, the cis-acting elements were counted.

[0127] 7. Analysis of functional domains of maize NRT2 protein

[0128] The maize NRT2 protein sequence was analyzed using the NCBI database online software to obtain the protein domain information of each member of the maize family, and the ChiPlot (https: / / www.chiplot.online / ) online tool was used to draw a domain distribution map to visualize the results.

[0129] 8. Conserved Motif Analysis of Maize NRT2 Protein

[0130] The maize NRT2 protein sequence was uploaded using the MEME (http: / / meme-suite.org / tools / meme) online tool to analyze the motifs with high similarity in the family. The maximum number of identifications of conservative sequences was set to 10, and the other parameters were set as default. After all parameters were set, the conserved motifs in the maize NRT2 protein sequence were searched. TBtools software was used to draw relevant motif visualization images and analyze their conservation.

[0131] 9. Protein structure prediction of ZmNRT2.7 gene

[0132] The transmembrane helices of maize NRT2 protein were predicted using DeepTMHMM (https: / / dtu.biolib.com / DeepTMHMM). The structure of ZmNRT2.7 protein was predicted on the AlphaFold website (https: / / www.alphafold.ebi.ac.uk / ). The 3D protein structure was predicted by AlphaFold based on the protein sequence of the B73-RefGen_v4 gene model.

[0133] 10. Analysis of Inducible Expression Pattern

[0134] B73 corn seeds were planted in small pots with a ratio of vermiculite to black soil of 1:1 and grown in a white incubator under the conditions of 28°C for 16h of light and 22°C for 8h of darkness. After germination for 4 days, the vermiculite and black soil on the roots of the corn seedlings were cleaned and then transferred to a hydroponic box for 3 days of hydroponics in tap water. The corn endosperm was removed and the corn seedlings were transferred to nitrogen-deficient MS culture solution for 1 day. After that, the corn seedlings were transferred to MS culture solution containing 2.5mM KNO3 for 6 days, and the culture solution was changed every 2 days. After growing for 6 days, transfer to nitrogen-deficient MS culture medium, and sample three plants at 0h, 0.5h, 1h, 2h, 4h, and 8h, respectively. Then divide the corn seedlings into two parts, one part is transferred to 10mM KCl MS culture medium, and the other part is transferred to 10mM KNO3 MS culture medium. Sample three plants at 0.5h, 1h, 2h, 4h, and 8h of treatment, respectively. Each sample is wrapped in tin foil, quickly frozen with liquid nitrogen, and stored in a -80℃ refrigerator.

[0135] Take the sample out of the -80℃ refrigerator, heat the mortar in advance, turn on the 4℃ centrifuge for precooling, use liquid nitrogen to precool the mortar and RNase free tubes, and extract RNA. Reverse transcribe the extracted RNA. Dilute the cDNA obtained by reverse transcription to 200ng / μL~300ng / μL and store it in a -20℃ refrigerator for use. Take out the diluted cDNA and perform RT-qPCR. Add samples according to the RT-qPCR reaction system, and carry out the whole process on ice and in the dark. Set the reaction program according to the reaction program of Novozymes' qPCR enzyme. Use the Ct method to process the qPCR data, summarize the processed data, and use GraphPad Prism 9.0.0 software to draw the graph.

[0136] RT-qPCR reaction system: ChamQ Universal SYBR qPCR Master Mix 10 μL, Forward Primer 0.8 μL, Reverse Primer 0.8 μL, RNase free water 6.8 μL, Template Plasmid 1.6 μL.

[0137] 11. Subcellular localization

[0138] The localization of ZmNRT2.7 protein in cells was further observed by using corn protoplast transient transformation experiments. Corn seeds were cultured according to the steps (1) in the analysis of induced expression patterns to obtain corn seedlings. After the seedlings had just sprouted out of the soil, they were transferred to a dark environment and grown for about 10 days. Then, the yellowed seedling leaves were cut, the leaf tips, bases and veins were removed, and the tenderer parts, about 1 g, were torn off, cut into small leaves and placed in a round culture dish. 15 μL of cellulase lysis solution was added, and the culture dish was gently shaken to evenly disperse the leaves in the cellulase lysis solution. The culture dish was wrapped in tin foil and enzymatically hydrolyzed at 40 r / min for 6 hours. After the enzymatic hydrolysis was completed, the culture dish was gently shaken to promote the release of protoplasts. Then, according to the method described in the article by Lei Haiying, Bai Fenglin, Feng Yu, et al., "Preparation of corn leaf protoplasts and establishment of transient transformation system", 2018, 35(02): 11-14., protoplasts were induced at room temperature for 24-36 hours. After the induction, the localization of ZmNRT2.7 protein in the cells was observed using a laser confocal microscope and photos were taken.

[0139] 12. In situ hybridization

[0140] In order to study the tissue expression of ZmNRT2.7, a ZmNRT2.7 specific probe was designed as shown in Table 5. Corn was planted in soil and grown to the V4 stage. The leaves (V4), main roots (V4) and lateral roots (V4) of corn B73 under normal growth conditions were fixed in FAA fixative containing 50% ethanol, 5% acetic acid and 37% formaldehyde and sent to Sewell Biotech for in situ hybridization detection.

[0141] Table 5 ZmNRT2.7 specific probes

[0142]

[0143] 13. Chlorate lethality experiment

[0144] B73, ZmNRT2.7 KO1 、ZmNRT2.7 KO2 、ZmNRT2.7 OE1 and ZmNRT2.7 OE2 The seeds of five strains were planted in trays filled with vermiculite and placed in a white incubator for seed germination. The culture conditions were: light 16h (30℃) / dark 8h (28℃), and humidity was about 60%. After 3-4 days, the plants with good growth and uniform size were selected, the vermiculite at the roots was washed off, the endosperm was removed, and the plants were moved to a hydroponic box. Three pots were moved to ensure three replicates. The seedlings were placed in Hoagland's culture solution containing 4mM KNO3 for 8 days, then treated with 4mM chlorate for 7 days, and then placed in Hoagland's culture solution containing 4mM KNO3 for 2 days to recover, and then the mortality of each strain was counted.

[0145] 14. Identification of knockout and overexpression strain phenotypes

[0146] (1) Different maize strains were cultured in soil under nitrogen-sufficient (nitrogen content of 4 mM) and nitrogen-deficient (nitrogen content of 0.04 mM) conditions, and their aboveground lengths, aboveground and underground fresh weights, and dry weights were measured.

[0147] (2) After different maize strains were cultured to the four-leaf stage under normal conditions, they were replaced with MS nutrient solution containing 10 mM potassium nitrate 15N and cultured hydroponically under normal conditions for 24 h. They were then fixed with 0.1 mM CaSO4 solution for 1 min and dried in an oven at 80°C until the plant mass was constant. The roots and leaves of 2-3 plants were ground into powder of about 0.5 g, and three biological replicates were taken from each group and sent to Qingdao Kechuang Quality Inspection Co., Ltd. for testing. 15 N absorption;

[0148] (3) After the corn ears grow to maturity and are harvested, the agronomic traits such as ear length, ear width, ear weight, 100-kernel weight, kernel length, kernel width, and kernel thickness are counted.

[0149] 15. Results Analysis

[0150] (1) Basic information of corn NRT2s family

[0151] The whole genome file and GFF annotation file of maize NRT2 family genes were downloaded from NCBI database, combined with transcriptome sequencing data of various maize tissues, and finally 7 ZmNRT2 genes (named ZmNRT2.4A, ZmNRT2.4B, ZmNRT2.4C, ZmNRT2.5A, ZmNRT2.7, ZmNRT2.7A and ZmNRT2.7C) were obtained through domain verification. The members and physicochemical properties of maize NRT2s family are shown in Table 6. The number of amino acids of NRT2s is mostly concentrated between 485–542; the molecular weight is between 49.8–59.6 kDa; the theoretical isoelectric point is basically between 8.2–9.17; the protein subcellular localization prediction software CELLO v2.5 Server was used for further analysis to predict the possible protein localization of each different candidate NRTs in maize. The results showed that NRT2s protein is basically located in the cell membrane.

[0152] Table 6 Maize NRT2s gene family

[0153]

[0154] (2) Phylogenetic tree analysis of maize NRT2 family

[0155] like Figure 4 As shown in the figure, the evolutionary tree was divided into three branches by the neighbor-joining method. The ZmNRT2.7 protein in maize belongs to the same branch as the OsNRT2.4 protein. Studies have shown that OsNRT2.4 is a dual-affinity nitrate transporter that can transport nitrate under low and high nitrate concentrations. ZmNRT2.7 is closely related to OsNRT2.4, and it is speculated that ZmNRT2.7 may also have the function of nitrate transport.

[0156] (3) Analysis of the functional domains of maize NRT2 protein

[0157] Domain analysis of ZmNRT2 proteins showed that all ZmNRT2s contained MFS superfamily domains, except for ZmNRT2.7A and ZmNRT2.7C, which contained NARK domains (e.g. Figure 5As shown in Figure 1 ). MFS is one of the largest known membrane transporter superfamilies and is widely present in the biological world. As a membrane transporter, the main function of MFS is to help supplement the multi-membrane transport of substances. NARK in plants has the function of transporting nitrite, and most ZmNRT2 proteins have NARK domains. It is speculated that the presence of these conserved domains allows ZmNRT2 proteins to transport nitrate.

[0158] (4) Conserved sequence analysis of maize NRT2 protein

[0159] The amino acid sequence of the maize NRT2 gene was analyzed for conserved motifs (e.g. Figure 6 The results showed that the ZmNRT2 gene contained 10 conserved motifs, which were named motif1-motif10; among them, motif1-motif5 and motif8 were both 50 amino acids (as shown in Figure 6 As shown in the figure). In the ZmNRT2s protein sequence, it can be clearly seen that motif10 is missing in some sequences, and the arrangement positions of other motifs in the ZmNRT2 protein family are consistent. Therefore, it is shown that motif1–motif10 is a relatively conservative motif, and it is speculated that the protein motif motif1–motif10 obtained by analysis is likely to be closely related to certain specific functions.

[0160] (5) Analysis of cis-acting elements in the promoter region of the maize NRT2 gene family

[0161] The cis-acting elements in the maize promoter region (2000 bp before ATG) were analyzed. Figure 7 As shown in the figure, the cis-acting elements contained are mainly divided into three categories, namely, elements of abiotic stress and biotic stress, plant hormone response elements, and elements related to plant growth and development. The maize NRT2 gene mainly contains light response elements; ABRE elements are present in the promoter regions of all ZmNRT2s genes, among which ZmNRT2.7 is rich in ABRE elements; LTR elements and TC-rich repeats are present in the promoter regions of ZmNRT2.4A, ZmNRT2.5A and ZmNRT2.7C; MYB elements are present in the promoters of all genes. It is speculated that the presence of these stress resistance elements enables plants to respond to various adversities more quickly.

[0162] (6) Protein transmembrane structure of ZmNRT2.7 gene

[0163] The protein structure of ZmNRT2.7 was predicted, such as Figure 8As shown in Figures AC, it was found that the ZmNRT2.7 protein has a total of 10 transmembrane domains. The three-dimensional structural analysis of the ZmNRT2.7 protein shows that it can form a pore, anchored on the membrane, open or close the channel in the presence or absence of nitrate, and transport nitrate.

[0164] (7) Expression analysis of ZmNRT2 family genes

[0165] In order to study the response of ZmNRT2 family genes to nitrate, we selected wild-type and nitrate-treated leaves at the three-leaf stage under normal nitrogen conditions, extracted RNA, and conducted RT-qPCR experiments after reverse transcription. Three corn plants were selected from each group as three biological replicates to ensure the reliability of the results. Fig. 9 As shown, the results showed that among the NRT2 family genes, the expression of ZmNRT2.7 was significantly upregulated after nitrate treatment, indicating that the expression of ZmNRT2.7 gene was significantly induced by nitrate at the transcriptional level.

[0166] (8) Analysis of tissue expression pattern of ZmNRT2.7 gene

[0167] In order to explore the expression pattern of ZmNRT2.7 gene in different tissues of maize, a series of tissues were obtained at the vegetative stage (V7) and reproductive stage (R1) of maize growth, including roots (V4), leaves (V4), bracts (V17), cobs (V17), filaments (V17), pollen (R1) and embryos 30 days after pollination (DAP) of B73 inbred line, RNA was extracted, and the relative expression level of ZmNRT2.7 was detected by RT-qPCR experiment (e.g. Fig.10 The relative expression levels of ZmNRT2.7 gene in different tissues were significantly different. ZmNRT2.7 was expressed at higher levels in leaves (V4) and embryos 30 days after pollination (DAP) of maize, and was expressed in bracts (V17), filaments (V17), and pollen (R1), while it was relatively low in roots (V4) and cobs (V17).

[0168] (9) In situ hybridization of ZmNRT2.7 gene

[0169] In addition, RNA in situ hybridization was used to further detect the expression sites of ZmNRT2.7 transcripts in the vegetative organs of maize B73 inbred lines at different developmental stages (e.g. Fig.11(shown in AD in the figure), it was found that ZmNRT2.7 was expressed in all vegetative organs. It was expressed in the vascular bundles, epidermal cells and mesophyll cells of leaves (V4). At the same time, ZmNRT2.7 transcripts were detected in the vascular bundles of the main roots and lateral roots at the four-leaf stage, and the epidermal cells of the lateral roots also had a higher expression level. Therefore, ZmNRT2.7 is highly expressed in the vascular tissues, mesophyll cells and epidermis of different tissues, suggesting that ZmNRT2.7 may be involved in the transport and metabolism of nutrients.

[0170] (10) Inducible expression pattern of ZmNRT2.7 gene

[0171] RT-qPCR was used to detect the expression of ZmNRT2.7 under nitrogen-sufficient and nitrogen-deficient conditions. Fig.12 As shown in the figure, the relative expression of ZmNRT2.7 continued to rise under continuous nitrogen deficiency. After 8 hours of nitrogen deficiency, the relative expression of ZmNRT2.7 was significantly upregulated by the recovery treatment of different nitrogen sources (10mM KNO3 or 10mM NH4Cl). After 0.5 hours of nitrate treatment, the relative expression was the highest, which was 4 times that under normal nitrogen conditions. However, the expression of ammonium salt was significantly downregulated after 8 hours of nitrate treatment, indicating that ZmNRT2.7 was induced and regulated by nitrate at the transcriptional level.

[0172] (11) Subcellular localization of ZmNRT2.7 protein

[0173] Results Fig.13 , with 1305 (d35S-GFP) empty vector (such as Fig.14 The results showed that the expression of GFP green fluorescent protein in the cells was stable, while the ZmNRT2.7 protein was mainly located on the cell membrane.

[0174] (13) Expression levels of ZmNRT2.7 mutants and overexpression strains

[0175] The expression levels in zmnrt2.7 mutants and overexpression lines were detected by RT-qPCR. Fig.15 As shown in A and B in the figure, the expression level of ZmNRT2.7 was downregulated in the zmnrt2.7 mutant, proving that the CRISPR-Cas9 technology used did not have off-target effects, while in the overexpression strain, the mRNA expression level of ZmNRT2.7 was significantly upregulated, indicating that the knockout mutants and overexpression materials obtained were reliable and authentic.

[0176] (14) Statistical analysis of seedling phenotypes of zmnrt2.7 mutants and overexpression strains

[0177] Different corn plants were treated with nutrient solutions containing 0.04 mM potassium nitrate (LN) and 4 mM potassium nitrate (HN) to simulate low-nitrogen and high-nitrogen environments, respectively. The phenotypes of corn at the seedling stage were observed, and the biomass of the aboveground and underground parts were counted.

[0178] As the nitrogen level in the environment increases, the biomass of the plants also increases. Fig.16 As shown in AF, in the LN treatment group, compared with the wild type, the aboveground fresh weight of the overexpression strain increased significantly by 39%, and the underground fresh weight increased significantly by 26%; compared with the wild type, the aboveground fresh weight of the zmnrt2.7 mutant decreased by 4.7%, and the underground fresh weight decreased by 7%. In the HN treatment group, compared with the wild type, the aboveground fresh weight of the overexpression strain increased significantly by 39%, and the underground fresh weight increased significantly by 31%.

[0179] In the HN and LN treatment groups, the aboveground and underground biomass of the zmnrt2.7 mutant decreased compared with the WT, but the difference was not significant. The biomass of the overexpression strain, both aboveground and underground, was significantly higher than that of the wild type (Student's t-test, P<0.01). Therefore, under nitrogen-sufficient and nitrogen-deficient conditions, the functional loss of the ZmNRT2.7 gene did not have much effect on the accumulation of aboveground and underground biomass in the maize seedling stage, and the biomass of the overexpression strain increased significantly. Overexpression of ZmNRT2.7 can increase the biomass of maize.

[0180] (15) Agronomic traits of zmnrt2.7 mutants and overexpression lines

[0181] The agronomic traits of B73, zmnrt2.7 mutants and overexpressed strains were observed and recorded. Fig.17 AF and Fig.18 As shown in AG, after seeds matured, compared with wild-type B73, the ear weight and ear length of zmnrt2.7 mutants were reduced, while the ear weight and ear length of overexpressing ears were increased compared with wild-type. Compared with the wild-type, the ear length of zmnrt2.7 mutants decreased significantly by about 7%, and the ear weight decreased significantly by about 11%.

[0182] The grain length, width, thickness and 100-grain weight of each strain were statistically analyzed. Fig.18 As shown in DG, compared with WT, the phenotypes of zmnrt2.7 mutant and overexpressed kernels were found to have significant effects on 100-grain weight. The 100-grain weight of zmnrt2.7 mutant was downregulated compared with wild type, and ZmNRT2.7 KO1 It was not very significant, but the 100-grain weight of the overexpressing strains increased.

[0183] It can be seen that compared with the wild type, some indicators such as ear weight, grain volume, and grain weight of the mutant are not very significant, resulting in no significant decrease in the yield of the mutant; at the same time, some indicators such as ear weight, grain volume, and grain weight of the overexpressed ones are significantly increased, indicating that overexpression of ZmNRT2.7 can increase corn yield.

[0184] (16) Determination of physiological parameters of zmnrt2.7 mutants and overexpression strains

[0185] use 15 The nitrogen isotope tracer method was used to measure the nitrogen absorption of zmnrt2.7 mutant and overexpression strains at the seedling stage. 15 N / 14 The N ratio of the overexpression strain was not significantly different from that of the wild type, but 15 N / 14 The N ratio was significantly higher than that of the wild type ( Fig.19 In addition, the aboveground / underground parts of the zmnrt2.7 mutant 15 N / 14 The N ratio was not significantly different from that of the wild type (e.g. Fig.19 ), while the overexpression lines 15 N / 14 The N ratio was significantly increased compared with the wild type. At the same time, the results of in situ hybridization showed that the ZmNRT2.7 gene was expressed in large quantities in the vascular bundles of the leaves (such as Fig.11 The vascular bundle is responsible for transporting nitrate, which means that the up-regulated expression of ZmNRT2.7 in the overexpression line may affect the transport process of nitrate absorbed by the roots to the aboveground parts, thereby increasing the accumulation of nitrogen in the aboveground parts.

[0186] (17) Chlorate lethality of zmnrt2.7 knockout mutants and overexpression strains

[0187] Chlorate is a structural analog of nitrate. Plants can use the nitrate system to transport and assimilate chlorate, and when chlorate is reduced to chlorite, it will be toxic to plants. In order to study the role of ZmNRT2.7 in regulating nitrogen absorption and transport, a chlorate sensitivity experiment was conducted. Fig. 20 As shown in Figures A-H, it was found that the mortality rate of the zmnrt2.7 knockout mutant was not much different from that of the wild type, but the mortality rate of the overexpression strain increased significantly. Since plants absorb chlorate and nitrate in the same way, the sensitivity to chlorate can be expressed as the plant's ability to absorb and transport nitrate, so ZmNRT2.7 can positively regulate the nitrate absorption process. 15N isotope tracing revealed that the aboveground part of the ZmNRT2.7 overexpressing strain 15 The nitrogen content increased significantly, which further proved that ZmNRT2.7 has the function of transporting nitrate and showed that ZmNRT2.7 positively regulates the uptake of nitrate.

[0188] The present invention studies the nitrate transporter ZmNRT2.7 in corn. Through the spatiotemporal expression pattern, it was found that ZmNRT2.7 was mainly expressed in leaves and embryos through the tissue expression pattern. Through in situ hybridization, it was found that ZmNRT2.7 was mainly highly expressed in epidermal cells, vascular cells and mesophyll cells of different tissues. This suggests that ZmNRT2.7 may be involved in the transport and metabolism of substances. After nitrogen deficiency of corn seedlings for 8 hours, nitrogen supplementation was performed, and the expression level of ZmNRT2.7 was detected. It was found that after adding nitrate for 0.5 hours, the expression level of ZmNRT2.7 was significantly upregulated, indicating that ZmNRT2.7 was induced by nitrate at the transcriptional level. In addition, subcellular localization analysis of ZmNRT2.7 was performed, and it was found that ZmNRT2.7 was located in the cell membrane, and the membrane-localized ZmNRT2.7 may play the function of transporting nitrate.

[0189] The present invention also constructed knockout plants and overexpression plants of the zmnrt2.7 mutant, and treated the knockout plants, overexpression plants and wild-type plants with different concentrations of nitrogen. The results showed that under normal water and fertilizer management, the biomass of the overexpression strain increased compared with the wild-type B73, indicating that overexpression of ZmNRT2.7 can increase corn yield.

[0190] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0191] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of ZmNRT2.7 gene or biological materials containing ZmNRT2.7 gene in any of the following: a. Application in improving nitrogen utilization efficiency of corn; b. Application in cultivating corn with high nitrogen utilization efficiency; c. Application in improving agronomic traits of corn; The nucleotide sequence of the ZmNRT2.7 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The biological material includes any one of the following: a. The protein encoded by the ZmNRT2.7 gene, the amino acid sequence of which is shown in SEQ ID NO.2; b. a recombinant plasmid comprising the ZmNRT2.7 gene; c. A recombinant microbial strain comprising the gene.

3. The use as claimed in claim 2, characterized in that The original vector of the recombinant plasmid is pCAMBIA1305.

4. The use according to claim 2, characterized in that The starting strain of the recombinant microorganism is Escherichia coli.

5. The use according to claim 1, characterized in that The agronomic traits include: corn cob weight, corn cob length, corn cob diameter, and 100-grain weight.

6. A method for improving nitrogen utilization efficiency of corn, characterized in that: include: The ZmNRT2.7 gene described in claim 1 is overexpressed in corn plants to obtain corn plants with high nitrogen utilization efficiency.

7. The method according to claim 6, characterized in that The method comprises constructing a ZmNRT2.7 gene overexpression vector and transferring the ZmNRT2.7 gene overexpression vector into corn plants.

8. A method for improving agronomic traits of corn, characterized in that: The ZmNRT2.7 gene described in claim 1 is overexpressed in corn plants to obtain corn plants with excellent agronomic traits.

9. The method according to claim 8, characterized in that The method comprises constructing a ZmNRT2.7 gene overexpression vector and transferring the ZmNRT2.7 gene overexpression vector into corn plants.

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