Application of ZmNRL1 gene and encoding protein thereof in improving nitrogen utilization efficiency of plant

Abstract

The invention discloses a ZmNRL1 gene and an application of a protein encoded by the ZmNRL1 gene in improving the nitrogen utilization efficiency of plants, the nucleotide sequence of the ZmNRL1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the ZmNRL1 gene is shown as SEQ ID NO.2. It is found that the corn ZmNRL1 gene can effectively regulate and control the nitrogen utilization efficiency of plants, corn seedlings can present a nitrogen starvation sensitive phenotype after ZmNRL1 mutation, the low nitrogen stress resistance of the corn seedlings and arabidopsis plants can be improved after ZmNRL1 overexpression, and meanwhile the plant biomass, the nitrate reductase activity and the nitrate nitrogen content are increased. The invention provides a gene resource for cultivating new low-nitrogen-resistant corn germplasm, and has important significance for increasing the yield of corn and improving the 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 ZmNRL1 gene and its encoded protein in improving the nitrogen utilization efficiency of plants. Background Art

[0002] Nitrogen is an important nutrient required for plant growth and is also the main limiting factor in determining plant yield. In agricultural production, the application of nitrogen fertilizer plays a very important role in ensuring stable crop yields around the world. However, excessive application of nitrogen fertilizer not only affects the absorption and utilization of nitrogen fertilizer by crops, but also causes environmental problems such as soil acidification and water eutrophication.

[0003] Corn (Zea mays L.) is a global food, feed and industrial energy crop with a wide planting area. In agricultural production, due to the very limited available nitrogen sources in the soil, the increase in corn yield depends largely on the large input of nitrogen fertilizer. Therefore, in order to avoid the contradiction between the growing demand for corn production and the intensification of environmental pollution, it is very important to improve the nitrogen use efficiency of corn.

[0004] The inorganic nitrogen sources absorbed by plants in the soil mainly include nitrate nitrogen and ammonium nitrogen. As a food crop, corn mainly absorbs and utilizes nitrate nitrogen in the soil. With the continuous deepening of molecular biology research, important progress has been made in the study of the physiological and molecular mechanisms of plant nitrogen absorption, transport and utilization. Several nitrogen absorption and utilization-related genes have also been cloned in corn, such as the nitrogen absorption and transport gene family NRT1 and NRT2, and the nitrogen assimilation-related coding gene GLN1. However, nitrogen absorption and utilization is an intricate biological process involving the interaction of multiple nitrogen regulatory genes and signal molecules. Although researchers have identified multiple genes related to corn nitrogen use efficiency, there is still a lack of research reports on the molecular mechanism of nitrogen use efficiency regulation, and researchers have also limited genetic improvement of corn for efficient use of nitrogen fertilizer. Therefore, it is particularly important to discover new key gene loci that affect corn nitrogen use efficiency, explore their molecular mechanisms for regulating corn nitrogen efficient utilization, and provide theoretical and technical support for molecular breeding to improve corn nitrogen use efficiency. 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 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 nucleic acid molecule encoding a protein with 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.

[0008] The third object of the present invention is to provide a vector containing the above-mentioned nucleic acid molecule for use 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 the nitrogen utilization efficiency of plants.

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

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

[0013] The present invention obtains corn ZmNRL1 mutant plants zmnrl1-e and zmnrl1-m, and ZmNRL1 overexpressing transgenic corn plants OE19 and OE111 by mutating the corn ZmNRL1 gene and overexpressing the corn ZmNRL1 gene. The corn ZmNRL1 mutant plants and ZmNRL1 overexpressing transgenic corn plants are cultured under different nitrogen concentration conditions, and the biomass of different tissues, the nitrate nitrogen content and the nitrate reductase activity in the plants are detected. The results show that the ZmNRL1 overexpressing transgenic corn plants are significantly more resistant to low nitrogen stress than the wild-type corn B73 plants. In addition, the present invention also constructs ZmNRL1 overexpressing transgenic Arabidopsis plants, and cultured them under different nitrogen concentration conditions. The results show that the ZmNRL1 gene also has a relatively conservative regulatory function in monocotyledonous model plants.

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

[0015] The use of 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.

[0016] Application 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 is used to improve the nitrogen utilization efficiency of plants and / or cultivate plants resistant to low nitrogen stress.

[0019] The use of cells and / or recombinant bacteria containing the vector of the nucleic acid molecule in improving the nitrogen utilization efficiency of plants 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 plant nitrogen utilization efficiency, the method comprising overexpressing a protein with an amino acid sequence as shown in SEQ ID NO.2 or a nucleic acid molecule encoding a protein with 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, the method comprising overexpressing a protein with an amino acid sequence as shown in SEQ ID NO.2 or a nucleic acid molecule encoding a protein with 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 ZmNRL1 gene in improving the nitrogen utilization efficiency of plants. The nucleotide sequence of 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 ZmNRL1 is mutated, the corn seedlings can show a nitrogen starvation sensitive phenotype, and after overexpressing ZmNRL1, the ability of corn seedlings and Arabidopsis plants to withstand low nitrogen stress can be improved, while increasing plant biomass, nitrate reductase activity and nitrate nitrogen content. The present invention provides gene resources for cultivating new low-nitrogen resistant corn germplasm, which is of great significance to 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 of the ZmNRL1 gene in the maize zmnrl1-e mutant and sequencing peak diagram detection, B: Mu transposon insertion position and gene structure diagram 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 The 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 weight of biomass, 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 The 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 fresh weight of roots and aerial parts, nitrate nitrogen content in roots and leaves, and nitrate reductase activity of wild-type and ZmNRL1-overexpressing transgenic maize plants.

[0034] Figure 6 The 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 shows the growth phenotype of ZmNRL1 overexpressing transgenic Arabidopsis plants; A: phenotype and soluble protein content of ZmNRL1 overexpressing transgenic Arabidopsis plants under different nitrogen concentrations, B: phenotype, main inflorescence length and biomass dry weight of ZmNRL1 overexpressing transgenic Arabidopsis plants after 5 weeks of low nitrogen treatment, C: phenotype, plant height and single seed weight 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, but the examples do not limit the present invention in any form. 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 are commercially available.

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

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

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

[0041] The Arabidopsis wild type 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). The mutant had a base mutation in the fourth exon of the ZmNRL1 gene (Zm00001d019173, whose nucleotide sequence 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), from C to T, resulting in premature termination of translation.

[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 corn inbred line B73 was used as the wild type control (WT), and corn seeds were sown. After the plants grew up, the genomic DNA of the corn plants was extracted, and then PCR amplification was performed. The polymerase used in the reaction system was Taq DNA polymerase (Vazyme Company), specifically: DNA template 4μL, 2×Taq Master Mix 25μL, upstream primer (10μM) 2μL, downstream primer (10μM) 2μL, and water was added to 50μL;

[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, where the maize zmnrl1-e mutant plants were amplified using primers EMS-F (SEQ ID NO.3) and EMS-R (SEQ ID NO.4), and the PCR products were purified and sent to the company for sequencing, and 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, wherein 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 1. The 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 the maize zmnrl1-m mutant plants showed that primers P1F (SEQ ID NO.5) and P2R (SEQ ID NO.6) could not amplify the band, while primers P1F (SEQ ID NO.5) and Mu67 (SEQ ID NO.7) could amplify the band, indicating that the Mu transposon insertion was present in the 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 (whose nucleotide sequence is shown in SEQ ID NO.1) as the target sequence, wherein 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 using 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: cDNA template 2 μL, PrimerSTAR GC buffer 25 μL, dNTP 4 μL, upstream primer (10 μM) 2 μL, downstream primer (10 μM) 2 μL, HS DNA polymerase 0.2μL, add water to 50μL.

[0064] PCR reaction program: pre-denaturation at 98°C for 2 min; 98°C for 10 s, 60°C for 10 s, 72°C for 1 min / Kb, 35 cycles; extension at 72°C for 5 min. After the reaction, the PCR product was recovered and purified, and the nucleotide sequence of the amplified target fragment was shown in SEQ ID NO.1.

[0065] (4) CPB:Ubipro-GFP (this vector is the same 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 as OE-6HA in DOI:10.1016 / j.molp.2020.02.011) are binary vectors used to construct maize and Arabidopsis overexpression vectors, respectively. The vector schematic diagrams are shown in Figure 2. Figure 2 As shown in A and B.

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

[0067] Homologous recombination was carried out by ligation using T5 exonuclease. The ligation system was: 4 μL 5×TEDA Cloning Mix, 30 ng linearized CPB:Ubipro-GFP binary vector, 90 ng target fragment, ddH2O was added to 20 μL, and the mixture was recombined at 37°C for 40 min to obtain the recombinant expression vector CPB:Ubipro-ZmNRL1-GFP (the vector was the target fragment with the nucleotide sequence as 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 the figure Figure 3 As shown in A.

[0068] The pGreenII 0229:35Spro-HA binary vector was cut into a linearized vector using XhoI and SpeI enzymes, and the purified fragments amplified by 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: 1 μL of 10×Ligase buffer; 0.4 μL of T4 DNA Ligase; 30 ng of pGreenII 0229:35Spro-HA linearized vector fragment, 100 ng of target fragment, and water supplemented to 10 μL. Ligate overnight at 16°C to obtain the recombinant expression vector pGreenII 0229:35Spro-ZmNRL1-HA (the vector is the target fragment with the nucleotide sequence as 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 diagram is shown in 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 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 based on the method of genetic transformation mediated by Agrobacterium GV3101 (DOI: 10.1038 / nprot.2007.241), and the T0 generation transgenic maize seeds were obtained after the plants grew up. The T0 generation transgenic maize seeds were sown and resistance screening was performed by spraying 0.1% glufosinate Basta solution (v / v). If the plant was positive, the leaves would not wither. At the same time, primers for detecting the Bar resistance gene were designed for PCR detection (primer sequences are shown in Table 3). After obtaining positive plants, individual plants were harvested until the T2 generation, thereby obtaining two homozygous overexpression transgenic maize lines OE19 and OE111.

[0073] 3. Detection of ZmNRL1 gene expression

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

[0076] Table 3 Primer sequences

[0077]

[0078] 2. Experimental Results

[0079] The expression results of ZmNRL1 in maize inbred line B73 (WT), maize zmnrl1-e mutant plants and zmnrl1-m mutant plants are 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 maize zmnrl1-e mutant plants and zmnrl1-m mutant plants.

[0080] The expression results of ZmNRL1 in maize inbred line B73 (WT) and homozygous overexpression transgenic maize lines OE19 and OE111 are shown in Figure 2. Figure 1 As shown in 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 grow to the three-leaf and one-heart stage, and the excess nutrient solution was washed away with dH2O. After removing the endosperm, they were cultured in dH2O for 1 day, and then transplanted to the improved Hoagland nutrient solution containing 0mM, 0.15mM and 15mM KNO3 and cultured for 11 days (the formula of the normal Hoagland nutrient solution is shown in Table 4. The preparation of nitrate nutrient solutions of different concentrations is based on the normal Hoagland nutrient solution, and KNO3 is additionally increased or decreased to the required concentration. The reduced part is replaced by KCl or CaCl2 to ensure the balance of ion concentration), and the phenotypes of different plants in response to nitrogen stress are 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]]> 1.5μM <![CDATA[ZnSO4·H2O]]> 1.5μM <![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 status of the maize mutant plants zmnrl1-e and zmnrl1-m 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, as evidenced 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 resistant to low nitrogen stress than the wild type, mainly showing that the leaves remained green for a long time, the biomass of the roots and aboveground parts, the nitrate nitrogen content, and the nitrate reductase activity were significantly higher than those of the wild-type maize inbred line B73. The results showed that under low nitrogen conditions, the overexpression of the ZmNRL1 gene can promote the assimilation of nitrate nitrogen by plants, thereby making nitrogen utilization more efficient ( 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 with 0-20mm fertilizer-free 810, item number: 1137467-300), and after culturing to the V2 stage, the seeds were irrigated with 0.15mM and 15mM NO3, respectively. - Different strains were irrigated with modified Hoagland nutrient solution (the formula of normal Hoagland nutrient solution is shown in Table 4 of Example 3. The nitrate nutrient solutions of different concentrations were prepared based on the normal Hoagland nutrient solution, with KNO3 added or reduced to the required concentration, and the reduced part was replaced by KCl or CaCl2 to ensure the balance of ion concentration). The different strains were irrigated every 3 days, and the treatment lasted for 21 to 24 days. The phenotypes were recorded and the 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 the figure, under low nitrogen conditions, the growth conditions of ZmNRL1 overexpressing transgenic corn lines OE19 and OE111 were better than those of the wild-type corn inbred line B73, and the aboveground biomass was significantly higher than that of the wild-type corn 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 transformed into Agrobacterium GV3101 by electroporation, and then transformed into Arabidopsis wild-type Col-0 plants based on the Arabidopsis floral dip method (DOI: 10.1046 / j.1365-313x.1998.00343.x). After receiving the seeds, they were plated in MS medium containing 50 mg / L Basta (MS medium formula is shown in Table 5), and Arabidopsis positive seedlings with ZmNRL1 gene were screened. Single plants were harvested, and after Basta resistance screening, two independently expressed homozygous overexpression transgenic Arabidopsis strains AtOE1 and AtOE2 were obtained.

[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 in MS medium containing different nitrogen concentrations

[0099] 1. Experimental Methods

[0100] Seeds of wild-type Arabidopsis thaliana Col-0 and homozygous overexpression transgenic Arabidopsis thaliana strains 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 sole nitrogen source, containing 10 mM NO3 - The formula of MS solid medium is shown in Table 6, containing 0.1 mM NO3 - The MS solid medium is prepared by reducing KNO3 to 0.1 mM on the basis of this medium, and the reduced part is replaced by KCl or CaCl2 to ensure the balance of ion concentration, and the phenotype of each strain is observed.

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

[0102]

[0103]

[0104] 2. Experimental Results

[0105] The results are as follows Figure 7 As shown in 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 with wild-type Arabidopsis Col-0 ( Figure 7 B). Figure 7 The results in C showed that the homozygous transgenic Arabidopsis lines AtOE1 and AtOE2 grew better above ground and had increased grain yield per plant under normal growth conditions in the soil. These data further indicate that the ZmNRL1 gene has a relatively conservative regulatory function in both monocotyledonous and dicotyledonous model plants, which provides an important theoretical basis and gene resource for this gene 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. 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.

2. 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 resistant to low nitrogen stress.

3. The use according to claim 2, characterized in that: The nucleic acid molecule is a nucleic acid molecule whose nucleotide sequence is shown in SEQ ID NO.1 or a nucleic acid molecule whose reverse complementary nucleotide sequence is shown in 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.

5. Use of cells and / or recombinant bacteria containing the 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.

6. The use according to any one of claims 1 to 5, characterized in that: The plant is maize and / or Arabidopsis thaliana.

7. A method for improving nitrogen utilization efficiency of plants, characterized in that: The method is to overexpress the protein with an amino acid sequence as shown in SEQ ID NO.2 or a nucleic acid molecule encoding the protein with an amino acid sequence as shown in SEQ ID NO.

2.

8. The method according to claim 7, characterized in that The plant is maize and / or Arabidopsis thaliana.

9. A method for cultivating plants resistant to low nitrogen stress, characterized in that: The method is to overexpress the protein with an amino acid sequence as shown in SEQ ID NO.2 or a nucleic acid molecule encoding the protein with an amino acid sequence as shown in SEQ ID NO.

2.

10. The method according to claim 9, characterized in that The plant is maize and / or Arabidopsis thaliana.

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