Application of ZmNRL1 gene and its encoded protein in improving nitrogen use efficiency of plants

Abstract

The present invention discloses the application of the ZmNRL1 gene and its encoded protein in improving the nitrogen utilization efficiency of plants. The nucleotide sequence of the ZmNRL1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2. The present invention finds that the corn ZmNRL1 gene can effectively regulate the nitrogen utilization efficiency of plants. After mutating ZmNRL1, corn seedlings can exhibit a nitrogen starvation-sensitive phenotype, while overexpressing ZmNRL1 can improve the ability of corn seedlings and Arabidopsis plants to tolerate low nitrogen stress, while increasing plant biomass, nitrate reductase activity and nitrate nitrogen content. The present invention provides genetic resources for cultivating new low-nitrogen-tolerant corn germplasm, which is of great significance for increasing corn yield and improving nitrogen utilization efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to the application of the ZmNRL1 gene and its encoded protein in improving the nitrogen utilization efficiency of plants. Background Art

[0002] Nitrogen is a crucial nutrient for plant growth and a major factor limiting plant yield. In agricultural production, nitrogen fertilizer application plays a crucial role in ensuring stable crop yields worldwide. However, excessive nitrogen fertilizer application not only impairs crop absorption and utilization but also contributes to environmental problems such as soil acidification and water eutrophication.

[0003] Corn (Zea mays L.) is a widely cultivated crop used as food, feed, and industrial energy. In agricultural production, due to the limited availability of nitrogen in the soil, increasing corn yields relies heavily on the application of large amounts of nitrogen fertilizer. Therefore, to address the conflict between growing corn production and increasing environmental pollution, improving the nitrogen use efficiency of corn is crucial.

[0004] Plants absorb inorganic nitrogen from the soil primarily in the form of nitrate and ammonium. Maize, as a food crop, primarily absorbs and utilizes nitrate from the soil. With the continuous advancement of molecular biology research, significant progress has been made in understanding the physiological and molecular mechanisms of nitrogen uptake, transport, and utilization in plants. Several genes related to nitrogen uptake and utilization have been cloned in maize, including the nitrogen uptake and transport gene families NRT1 and NRT2, and the nitrogen assimilation-encoding gene GLN1. However, nitrogen uptake and utilization is a complex biological process involving the interaction of multiple nitrogen regulatory genes and signaling molecules. Although researchers have identified several genes associated with nitrogen use efficiency in maize, research on the molecular mechanisms regulating nitrogen use efficiency remains scarce, and efforts to genetically improve maize for efficient nitrogen fertilizer utilization are also limited. Therefore, it is crucial to identify new key loci affecting maize nitrogen use efficiency and to clarify the molecular mechanisms regulating this efficiency, in order to provide theoretical and technical support for molecular breeding to improve nitrogen use efficiency in maize. Summary of the Invention

[0005] The technical problem to be solved by the present invention is the above-mentioned defects and deficiencies in the prior art, and provides the application of the ZmNR L1 gene and its encoded protein in improving the nitrogen utilization efficiency of plants.

[0006] The first object of the present invention is to provide a protein having an amino acid sequence as shown in SEQ ID NO. 2 for use in improving plant nitrogen utilization efficiency and / or cultivating plants resistant to low nitrogen stress.

[0007] The second object of the present invention is to provide a use of a nucleic acid molecule encoding a protein having an amino acid sequence as shown in SEQ ID NO. 2 in improving plant nitrogen utilization efficiency and / or cultivating plants tolerant to low nitrogen stress.

[0008] The third object of the present invention is to provide a use of a vector containing the above-mentioned nucleic acid molecule in improving plant nitrogen utilization efficiency and / or cultivating plants resistant to low nitrogen stress.

[0009] The fourth object of the present invention is to provide the use of cells and / or recombinant bacteria containing the vector of the above-mentioned nucleic acid molecule in improving the nitrogen utilization efficiency of plants and / or cultivating plants resistant to low nitrogen stress.

[0010] A fifth object of the present invention is to provide a method for improving nitrogen utilization efficiency of plants.

[0011] A sixth object of the present invention is to provide a method for cultivating plants tolerant to low nitrogen stress.

[0012] In order to achieve the above object, the present invention is implemented through the following scheme:

[0013] The present invention mutates the maize ZmNRL1 gene and overexpresses the maize ZmNRL1 gene, obtaining ZmNRL1 mutant maize plants zmnrl1-e and zmnrl1-m, as well as ZmNRL1-overexpressing transgenic maize plants OE19 and OE111, respectively. The ZmNRL1 mutant maize plants and the ZmNRL1-overexpressing transgenic maize plants were cultured under varying nitrogen concentrations, and the biomass, nitrate nitrogen content, and nitrate reductase activity of different plant tissues were measured. The results showed that the ZmNRL1-overexpressing transgenic maize plants were significantly more tolerant to low nitrogen stress than wild-type maize B73 plants. Furthermore, the present invention also constructed ZmNRL1-overexpressing transgenic Arabidopsis plants and cultured them under varying nitrogen concentrations. The results showed that the ZmNRL1 gene also has relatively conserved regulatory functions in monocotyledonous model plants.

[0014] Therefore, the present invention claims the following:

[0015] Use of a protein having an amino acid sequence as shown in SEQ ID NO. 2 in improving plant nitrogen utilization efficiency and / or cultivating plants resistant to low nitrogen stress.

[0016] Use of a nucleic acid molecule encoding a protein with an amino acid sequence as shown in SEQ ID NO. 2 in improving plant nitrogen utilization efficiency and / or cultivating plants resistant to low nitrogen stress.

[0017] Preferably, the nucleic acid molecule is a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.1 or a nucleic acid molecule having a reverse complementary nucleotide sequence as shown in SEQ ID NO.1.

[0018] The use of a vector containing the nucleic acid molecule in improving plant nitrogen utilization efficiency and / or cultivating plants resistant to low nitrogen stress.

[0019] Use of cells and / or recombinant bacteria containing the vector of the nucleic acid molecule in improving plant nitrogen utilization efficiency and / or cultivating plants resistant to low nitrogen stress.

[0020] Preferably, the plant is maize and / or Arabidopsis thaliana.

[0021] Preferably, the improving plant nitrogen utilization efficiency is to increase plant biomass, nitrate nitrogen content and / or nitrate reductase activity.

[0022] A method for improving nitrogen utilization efficiency of plants, the method comprising overexpressing a protein having an amino acid sequence as shown in SEQ ID NO. 2 or a nucleic acid molecule encoding the protein having an amino acid sequence as shown in SEQ ID NO. 2.

[0023] Preferably, the plant is maize and / or Arabidopsis thaliana.

[0024] Preferably, the improving plant nitrogen utilization efficiency is to increase plant biomass, nitrate nitrogen content and / or nitrate reductase activity.

[0025] A method for cultivating plants resistant to low nitrogen stress comprises overexpressing a protein having an amino acid sequence as shown in SEQ ID NO. 2 or a nucleic acid molecule encoding the protein having an amino acid sequence as shown in SEQ ID NO. 2.

[0026] Preferably, the plant is maize and / or Arabidopsis thaliana.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention discloses the application of the ZmNRL1 gene in improving the nitrogen utilization efficiency of plants. The nucleotide sequence of the ZmNRL1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2. The present invention finds that the corn ZmNRL1 gene can effectively regulate the nitrogen utilization efficiency of plants. After mutating ZmNRL1, corn seedlings can exhibit a nitrogen starvation-sensitive phenotype, while overexpressing ZmNRL1 can improve the ability of corn seedlings and Arabidopsis plants to tolerate low nitrogen stress, while increasing plant biomass, nitrate reductase activity and nitrate nitrogen content. The present invention provides genetic resources for cultivating new low-nitrogen-tolerant corn germplasm, which is of great significance for increasing corn yield and improving nitrogen utilization efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1Detection and analysis of the mutation position of the ZmNRL1 gene and the expression level of the ZmNRL1 gene after mutation; A: Mutation position and sequencing peak diagram of the ZmNRL1 gene in the maize zmnrl1-e mutant, B: Schematic diagram of the insertion position of the Mu transposon and gene structure in the maize zmnrl1-m mutant, C: Gel electrophoresis diagram of the maize zmnrl1-m mutant, D: Analysis of the expression level of the ZmNRL1 gene in maize ZmNRL1 mutant plants, E: Analysis of the expression level of the ZmNRL1 gene in ZmNRL1 overexpressing transgenic maize plants.

[0030] Figure 2 Schematic diagram of binary vectors, A: Schematic diagram of binary vector CPB:Ubipro-GFP, B: Schematic diagram of binary vector pGreenII 0229:35Spro-HA.

[0031] Figure 3 Schematic diagrams of the recombinant expression vectors CPB:Ubipro-ZmNRL1-GFP and pGreenII 0229:35Spro-ZmNRL1-HA, A: Schematic diagram of the recombinant expression vector CPB:Ubipro-ZmNRL1-GFP, B: Schematic diagram of the recombinant expression vector pGreenII0229:35Spro-ZmNRL1-HA.

[0032] Figure 4 Phenotypes of wild-type maize inbred line B73 and maize ZmNRL1 mutant plants in nutrient solutions containing different nitrogen concentrations; A: Phenotypes of plants treated with different nitrogen concentrations for 11 days, B: Leaf senescence phenotype, C: Statistical results of dry biomass weight, soluble protein content, nitrate nitrogen content in roots and leaves, and nitrate reductase activity of wild-type and maize ZmNRL1 mutant plants.

[0033] Figure 5 Figure 3. Phenotypes of wild-type maize inbred line B73 and ZmNRL1-overexpressing transgenic maize plants in nutrient solutions containing different nitrogen concentrations. A: Phenotypes of plants treated with different nitrogen concentrations for 11 days. B: Statistical results of root and aerial fresh weight, nitrate nitrogen content in roots and leaves, and nitrate reductase activity of wild-type and ZmNRL1-overexpressing transgenic maize plants.

[0034] Figure 6 Phenotypes of maize ZmNRL1 mutant plants and ZmNRL1 overexpressing transgenic maize plants under soil culture conditions; A: Phenotypic analysis and biomass dry weight statistics of maize ZmNRL1 mutant plants under different nitrogen concentrations; B: Phenotypic analysis and biomass dry weight statistics of ZmNRL1 overexpressing transgenic maize plants under different nitrogen concentrations.

[0035] Figure 7Figure 2 Growth phenotypes of ZmNRL1-overexpressing transgenic Arabidopsis plants; A: Phenotype and soluble protein content statistics of ZmNRL1-overexpressing transgenic Arabidopsis plants under different nitrogen concentrations; B: Phenotype, main inflorescence length, and dry biomass weight statistics of ZmNRL1-overexpressing transgenic Arabidopsis plants after 5 weeks of low nitrogen treatment; C: Phenotype, plant height, and seed weight per plant statistics of ZmNRL1-overexpressing transgenic Arabidopsis plants under normal growth conditions. DETAILED DESCRIPTION

[0036] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0038] The wild-type maize variety is the inbred line B73, and its genome sequence has been publicly released in the Maize Database (https: / / maizegdb.org).

[0039] Maize zmnrl1-e mutant plant seeds (stock ID: EMS-095209) were derived from the Maize EMS induced Mutant Database (MEMD). This mutant has a base mutation in the fourth exon of the ZmNRL1 gene from C to T, resulting in premature translation termination.

[0040] The seeds of the maize zmnrl1-m mutant plant (stock ID: 20346698) were derived from the maize Mutator transposon mutant library platform (ChinaMu). The mutant had a Mu transposon insertion at 1037 bp in the first exon of the ZmNRL1 gene.

[0041] The wild type Arabidopsis thaliana Col-0 is the Columbia ecotype.

[0042] Example 1 Identification of mutation sites in ZmNRL1 mutant plants

[0043] 1. Experimental Methods

[0044] Maize zmnrl1-e mutant plant seeds (stock ID: EMS-095209) were obtained from the Maize EMS induced Mutant Database (MEMD). This mutant had a base mutation in the fourth exon of the ZmNRL1 gene (Zm00001d019173, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2), from C to T, resulting in premature translation termination.

[0045] Maize zmnrl1-m mutant plant seeds (stock ID: 20346698) were obtained from the maize Mutator transposon mutant library platform (ChinaMu). The mutant had a Mu transposon insertion at the 1037 bp of the first exon of the ZmNRL1 gene.

[0046] The conventional maize inbred line B73 was used as the wild-type control (WT). Maize seeds were sown, and genomic DNA was extracted from the maize plants after they grew up. PCR amplification was then performed. The polymerase used in the reaction system was Taq DNA polymerase (Vazyme). Specifically, the following was used: 4 μL DNA template, 25 μL 2× Taq Master Mix, 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), and water was added to 50 μL.

[0047] The reaction conditions were: 94°C for 3 min, 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min / Kb, 32 cycles, and extension at 72°C for 5 min;

[0048] PCR primers are shown in Table 1. The maize zmnrl1-e mutant plants were amplified using primers EMS-F (SEQ ID NO. 3) and EMS-R (SEQ ID NO. 4). The PCR products were purified and sent to the company for sequencing. The sequencing results were compared with the wild-type B73 plant reference genome sequence to analyze the mutation.

[0049] Identification of maize zmnrl1-m mutant plants was performed by PCR amplification using gene-specific primers and insert-specific primers. Gene-specific primers P1F (SEQ ID NO. 5) and P2R (SEQ ID NO. 6) were used to amplify the ZmNRL1 gene, and gene-specific primer P1F (SEQ ID NO. 5) and insert-specific primer Mu67 (SEQ ID NO. 7) were used to amplify the insert.

[0050] Table 1 PCR primers

[0051]

[0052]

[0053] 2. Experimental Results

[0054] The results are as follows Figure 1 As shown in A to C in Figure 2. Sequencing results showed that the maize zmnrl1-e mutant plant had a base mutation in the fourth exon of the ZmNRL1 gene, from C to T ( Figure 1 A).

[0055] The PCR amplification results of maize zmnrl1-m mutant plants showed that primers P1F (SEQ ID NO.5) and P2R (SEQ ID NO.6) could not amplify a band, while primers P1F (SEQ ID NO.5) and Mu67 (SEQ ID NO.7) could amplify a band, indicating that the Mu transposon insertion exists in maize zmnrl1-m mutant plants ( Figure 1 B and C in ).

[0056] Example 2 Construction of ZmNRL1 overexpressing plants

[0057] 1. Experimental Methods

[0058] 1. Construction of ZmNRL1 overexpression vector

[0059] (1) Root tissues of maize inbred line B73 seedlings at the V2 stage were collected, and total RNA was extracted using a plant RNA extraction kit (Plant RNA Kit, Beijing Tianenze Reagent), and reverse transcribed to obtain cDNA;

[0060] (2) Overexpression vector primers were designed using the ZmNRL1 gene (its nucleotide sequence is shown in SEQ ID NO.1) as the target sequence. Primers ZmNRL1-F1 (SEQ ID NO.8) and ZmNRL1-R1 (SEQ ID NO.9) were used to construct the maize ZmNRL1 overexpression vector, and primers ZmNRL1-F2 (SEQ ID NO.10) and ZmNRL1-R2 (SEQ ID NO.11) were used to construct the Arabidopsis overexpression vector. The primer sequences are shown in Table 2.

[0061] Table 2 Primer sequences

[0062]

[0063] (3) Perform PCR reaction. Amplify the target fragment according to the above primers. The polymerase used in the PCR reaction is HS DNA polymerase (Takara). The reaction system is 50 μL. The reaction system is prepared according to the instructions, specifically: 2 μL cDNA template, 25 μL PrimerSTAR GC buffer, 4 μL dNTP, 2 μL upstream primer (10 μM), 2 μL downstream primer (10 μM), Add 0.2 μL of HS DNA polymerase and add water to 50 μL.

[0064] The PCR reaction procedure was as follows: initial denaturation at 98°C for 2 minutes; 35 cycles of 98°C for 10 seconds, 60°C for 10 seconds, and 72°C for 1 minute / kb; and extension at 72°C for 5 minutes. After the reaction, the PCR product was recovered and purified. The nucleotide sequence of the amplified target fragment is shown in SEQ ID NO. 1.

[0065] (4) CPB:Ubipro-GFP (this vector is the same vector as CPB-Ubi-EGFP in DOI:10.1093 / plcell / koac296 and the modified pCAMBIA vector in Chinese patent CN111763682A) and pGreenII0229:35Spro-HA (this vector is the same vector as OE-6HA in DOI:10.1016 / j.molp.2020.02.011) are binary vectors used to construct overexpression vectors in maize and Arabidopsis plants, respectively. The vector schematics are shown in Figure 4. Figure 2 As shown in A and B.

[0066] The CPB:Ubipro-GFP binary vector was digested into a linearized vector using BamHI. The purified fragment amplified with primers ZmNRL1-F1 (SEQ ID NO. 8) and ZmNRL1-R1 (SEQ ID NO. 9) was inserted into the center of the BamHI restriction site of the CPB:Ubipro-GFP vector using homologous recombination.

[0067] Homologous recombination was performed using T5 exonuclease ligation. The ligation system was as follows: 4 μL 5× TEDA Cloning Mix, 30 ng linearized CPB:Ubipro-GFP binary vector, 90 ng target fragment, and ddH2O was added to 20 μL. Recombination was carried out at 37°C for 40 min to obtain the recombinant expression vector CPB:Ubipro-ZmNRL1-GFP (the vector is the target fragment with the nucleotide sequence shown in SEQ ID NO.1 inserted into the BamHI restriction site of the CPB:Ubipro-GFP binary vector). The vector schematic diagram is shown in FIG. Figure 3 As shown in A.

[0068] The pGreenII 0229:35Spro-HA binary vector was digested into a linearized vector using XhoI and SpeI enzymes. The purified fragments amplified with primers ZmNRL1-F2 (SEQ ID NO. 10) and ZmNRL1-R2 (SEQ ID NO. 11) were inserted between the XhoI and SpeI restriction sites of the pGreenII 0229:35Spro-HA vector using the T4 ligation method.

[0069] The ligation system is as follows: 1 μL of 10× Ligase buffer; 0.4 μL of T4 DNA Ligase; 30 ng of the linearized pGreenII 0229:35Spro-HA vector fragment and 100 ng of the target fragment, diluted to 10 μL. Ligate overnight at 16°C to obtain the recombinant expression vector pGreenII 0229:35Spro-ZmNRL1-HA (this vector is the target fragment with the nucleotide sequence shown in SEQ ID NO. 1 inserted between the XhoI and SpeI restriction sites of the pGreenII 0229:35Spro-HA binary vector). The vector schematic is shown below. Figure 3 As shown in B.

[0070] After transformation and colony PCR analysis of the above recombinant expression vector, positive clones were obtained for sequencing detection and positive plasmids were extracted.

[0071] 2. Obtaining and identifying ZmNRL1-overexpressing transgenic maize lines

[0072] The constructed recombinant expression vector CPB:Ubipro-ZmNRL1-GFP was introduced into the maize inbred line B73 using Agrobacterium tumefaciens GV3101-mediated genetic transformation (DOI: 10.1038 / nprot.2007.241). T0-generation transgenic maize seeds were obtained after the plants grew to maturity. These T0-generation transgenic maize seeds were sown and screened for resistance by spraying with a 0.1% v / v solution of glufosinate ammonium (Basta). Plants that tested positive for the Bar resistance gene showed no leaf wilt. PCR was also performed using primers designed to detect the Bar resistance gene (primer sequences are shown in Table 3). Once positive plants were obtained, individual plants were harvested and seeded until the T2 generation, resulting in two homozygous overexpressing transgenic maize lines, OE19 and OE111.

[0073] 3. Detection of ZmNRL1 gene expression

[0074] Leaf tissues of homozygous overexpressing transgenic maize lines OE19 and OE111 germinated in soil were collected to extract total RNA and obtain cDNA after reverse transcription. The expression levels of the target gene ZmNRL1 in the homozygous overexpressing transgenic maize lines OE19 and OE111 were identified by qRT-PCR (see Table 3 for qRT-PCR primer sequences). The maize inbred line B73 was used as the wild-type control (WT).

[0075] In addition, leaf tissues of the maize zmnrl1-e mutant plants and zmnrl1-m mutant plants obtained in Example 1 were taken, total RNA was extracted and reverse transcribed to obtain cDNA, and the expression level of the target gene ZmNRL1 in the maize zmnrl1-e mutant plants and zmnrl1-m mutant plants was identified by qRT-PCR (see Table 3 for qRT-PCR primer sequences), and the maize inbred line B73 was used as a wild-type control (WT).

[0076] Table 3 Primer sequences

[0077]

[0078] 2. Experimental Results

[0079] The expression levels of ZmNRL1 in maize inbred line B73 (WT), maize zmnrl1-e mutant plants and zmnrl1-m mutant plants were detected as shown in Figure 2. Figure 1 As shown in D. The results showed that the expression level of the target gene ZmNRL1 was significantly reduced in the mutant plants zmnrl1-e and zmnrl1-m.

[0080] The expression levels of ZmNRL1 in maize inbred line B73 (WT) and homozygous overexpression transgenic maize lines OE19 and OE111 were detected as follows: Figure 1 As shown in Figure E. The results showed that the target gene ZmNRL1 was highly expressed in the homozygous overexpression transgenic maize lines OE19 and OE111, but was less expressed in the maize inbred line B73, indicating that the ZmNRL1 overexpression transgenic maize line was successfully obtained.

[0081] Example 3 Phenotypes of ZmNRL1 mutant maize plants and ZmNRL1 overexpressing transgenic maize plants in nutrient solutions containing different nitrogen concentrations

[0082] 1. Experimental Methods

[0083] Seeds of the maize mutant plants zmnrl1-e and zmnrl1-m obtained in Example 1, the ZmNRL1 overexpressing transgenic maize lines OE19 and OE111 obtained in Example 2, and the wild-type maize inbred line B73 were soaked and germinated to the three-leaf, one-heart stage. Excess nutrient solution was washed off with dH2O, and after removing the endosperm, the seeds were cultured in dH2O for 1 day. They were then transplanted to modified Hoagland nutrient solution containing 0 mM, 0.15 mM, and 15 mM KNO3 and cultured for 11 days (the formula of the normal Hoagland nutrient solution is shown in Table 4. The nitrate nutrient solutions of different concentrations were prepared based on the normal Hoagland nutrient solution, with KNO3 additionally added or reduced to the required concentration, and the reduced portion was replaced by KCl or CaCl2 to ensure ion concentration balance). The phenotypes of the different plants in response to nitrogen stress were observed.

[0084] Table 4 Formula of normal Hoagland nutrient solution

[0085] Components concentration <![CDATA[KNO3]]> 2mM <![CDATA[CaCl2]]> 4mM <![CDATA[Ca(NO3)2·4H2O]]> 1mM <![CDATA[MgSO4·7H2O]]> 2mM <![CDATA[KH2PO4]]> 1mM KCl 3mM <![CDATA[EDTA-FeNa2]]> 0.02mM <![CDATA[MnSO4·H2O]]> 1.5 μM <![CDATA[ZnSO4·H2O]]> 1.5 μM <![CDATA[CuSO4·5H2O]]> 0.5μM <![CDATA[NaB4O7·10H2O]]> 0.5μM <![CDATA[(NH4)6MO7O 24 ·4H2O]]> 0.15μM

[0086] 2. Experimental Results

[0087] like Figure 4 As shown in A to C, in 15mM NO3 - The growth of maize mutant plants zmnrl1-e and zmnrl1-m under treatment showed no significant difference compared with the wild-type maize inbred line B73, but under nitrogen deficiency (0 mM NO3 - ) and low nitrogen (0.15mMNO3 - ) conditions, the maize mutant plants zmnrl1-e and zmnrl1-m showed a phenotype that was sensitive to nitrogen starvation, characterized by accelerated leaf senescence, and significantly lower biomass, nitrate nitrogen content, and nitrate reductase activity than those of the wild-type maize inbred line B73.

[0088] In addition, from Figure 5 As shown in Figures A to B, under nitrogen starvation conditions, the two ZmNRL1 overexpressing transgenic maize lines OE19 and OE111 were more tolerant to low nitrogen stress than the wild type, with their leaves remaining green longer, and their root and aboveground biomass, nitrate nitrogen content, and nitrate reductase activity significantly higher than those of the wild-type maize inbred line B73. These results indicate that overexpression of the ZmNRL1 gene can promote the assimilation of nitrate nitrogen in plants under low nitrogen conditions, thereby improving nitrogen utilization efficiency ( Figure 5 ).

[0089] Example 4 Phenotypes of ZmNRL1 mutant maize plants and ZmNRL1 overexpressing transgenic maize plants under soil culture conditions

[0090] 1. Experimental Methods

[0091] Seeds of the maize mutant plants zmnrl1-e and zmnrl1-m obtained in Example 1, the ZmNRL1 overexpressing transgenic maize lines OE19 and OE111 obtained in Example 2, and the wild-type maize inbred line B73 were sown in low-nutrient soil (containing no macronutrients and trace nutrients, brand: Jiffy seedling peat soil 300L filled with 0-20mm fertilizer-free 810, item number: 1137467-300), cultured to the V2 stage, and then fertilized with 0.15mM and 15mM NO3, respectively. - Different strains were irrigated with modified Hoagland's nutrient solution (the formula for normal Hoagland's nutrient solution is shown in Table 4 in Example 3. Nitrate nutrient solutions of varying concentrations were prepared by adding or reducing KNO3 to the desired concentration based on normal Hoagland's nutrient solution. The reduced amount was replaced with KCl or CaCl2 to ensure ion balance). Irrigation was performed every three days for 21–24 days. Phenotypes were recorded and biomass of each strain was counted.

[0092] 2. Experimental Results

[0093] The results are as follows Figure 6 As shown in A to B in Figure 3, under low nitrogen conditions, the growth conditions of ZmNRL1 overexpressing transgenic maize lines OE19 and OE111 were better than those of the wild-type maize inbred line B73, and the aboveground biomass was significantly higher than that of the wild-type maize inbred line B73.

[0094] Example 6 Construction of ZmNRL1-overexpressing transgenic Arabidopsis

[0095] The recombinant expression vector pGreenII 0229:35Spro-ZmNRL1-HA constructed in Example 2 was electroporated into Agrobacterium GV3101 and then transformed into wild-type Arabidopsis Col-0 plants using the Arabidopsis floral dip method (DOI: 10.1046 / j.1365-313x.1998.00343.x). The seeds were plated onto MS medium containing 50 mg / L Basta (see Table 5 for the MS medium recipe) and positive seedlings were selected for the ZmNRL1 gene. Individual seeds were harvested and screened for Basta resistance to obtain two independently expressed homozygous transgenic Arabidopsis lines, AtOE1 and AtOE2.

[0096] Table 5 MS medium formula

[0097] Reagents Dosage MS powder 4.43g / L MES 0.5g / L Sucrose 10g / L Agar 7g / L

[0098] Example 7 Phenotypes of ZmNRL1-overexpressing transgenic Arabidopsis thaliana grown in MS medium containing different nitrogen concentrations

[0099] 1. Experimental Methods

[0100] Seeds of wild-type Arabidopsis thaliana Col-0 and homozygous overexpressing transgenic Arabidopsis thaliana lines AtOE1 and AtOE2 obtained in Example 6 were sown on MS medium and grown for 5 days. - and 10 mM NO3 - The cells were cultured horizontally in MS solid medium for 8 days (KNO3 was used as the only nitrogen source, containing 10 mM NO3 - The formula of MS solid medium is shown in Table 6, containing 0.1mM NO3 - The MS solid medium is based on this medium and KNO3 is reduced to 0.1mM. The reduced part is replaced by KCl or CaCl2 to ensure the balance of ion concentration. The phenotype of each strain is observed.

[0101] Table 6 contains 10mM NO3 - MS solid culture medium recipe

[0102]

[0103]

[0104] 2. Experimental Results

[0105] The results are as follows Figure 7 As shown in Figures A to C, compared with wild-type Arabidopsis Col-0, homozygous overexpressing transgenic Arabidopsis lines AtOE1 and AtOE2 showed a phenotype insensitive to nitrogen starvation, and the protein content in leaves was significantly higher than that in wild-type Arabidopsis Col-0 ( Figure 7 After 5 weeks of low nitrogen treatment, the homozygous transgenic Arabidopsis lines AtOE1 and AtOE2 showed increased plant height and aboveground biomass compared to wild-type Arabidopsis Col-0 ( Figure 7 B) in. Figure 7 Results in C showed that homozygous transgenic Arabidopsis lines AtOE1 and AtOE2 exhibited improved aboveground growth and increased grain yield per plant under normal soil growth conditions. These data further demonstrate that the ZmNRL1 gene has relatively conserved regulatory functions in both monocotyledonous and dicotyledonous model plants, providing important theoretical basis and genetic resources for its use in breeding.

[0106] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of a protein having an amino acid sequence as shown in SEQ ID NO. 2 for improving plant nitrogen utilization efficiency and / or cultivating plants tolerant to low nitrogen stress, characterized in that: The plant is maize and / or Arabidopsis thaliana.

2. Use of a nucleic acid molecule encoding a protein having an amino acid sequence as shown in SEQ ID NO. 2 for improving plant nitrogen utilization efficiency and / or cultivating plants tolerant to low nitrogen stress, characterized in that: The plant is maize and / or Arabidopsis thaliana.

3. The use according to claim 2, characterized in that The nucleic acid molecule is a nucleic acid molecule whose nucleotide sequence is shown as SEQ ID NO.

1.

4. Use of a vector containing the nucleic acid molecule according to claim 2 or 3 in improving plant nitrogen utilization efficiency and / or cultivating plants resistant to low nitrogen stress, characterized in that: The plant is maize and / or Arabidopsis thaliana.

5. Use of a cell and / or recombinant bacterium containing a vector of the nucleic acid molecule according to claim 2 or 3 in improving plant nitrogen utilization efficiency and / or cultivating plants resistant to low nitrogen stress, characterized in that: The plant is maize and / or Arabidopsis thaliana.

6. A method for improving nitrogen utilization efficiency of plants, characterized in that: The method comprises overexpressing a protein having an amino acid sequence as shown in SEQ ID NO. 2 or a nucleic acid molecule encoding a protein having an amino acid sequence as shown in SEQ ID NO. 2; The plant is maize and / or Arabidopsis thaliana.

7. A method for cultivating plants tolerant to low nitrogen stress, characterized in that: The method comprises overexpressing a protein having an amino acid sequence as shown in SEQ ID NO. 2 or a nucleic acid molecule encoding a protein having an amino acid sequence as shown in SEQ ID NO. 2; The plant is maize and / or Arabidopsis thaliana.

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