A group of genes regulating maize flowering time and nitrogen use efficiency and their applications

Abstract

This invention discloses a set of genes regulating maize flowering time and nitrogen use efficiency, and their applications, belonging to the field of plant biotechnology breeding. The maize provided by this invention... ZmNNR1 and / or ZmNNR4 The gene, when overexpressed, has the function of promoting early flowering and increasing nitrogen use efficiency in plants. The nitrogen use efficiency improvement functions include enhanced nitrate nitrogen use efficiency, increased plant nitrogen content, increased chlorophyll content and / or increased maximum photochemical efficiency, and increased yield. The aforementioned gene resources and their application methods have broad application prospects in the field of maize breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology breeding, specifically involving the utilization of ZmNNR1 and ZmNNR4 Methods and applications for regulating maize flowering time and nitrogen use efficiency through gene overexpression or loss-of-function mutations. Background Technology

[0002] Early flowering of corn and improved nitrogen use efficiency are of great significance to agricultural production. Early flowering shortens the corn growing season, allowing farmers to harvest earlier and thus gain valuable time for planting the next crop. This changes the traditional crop rotation pattern, improves the annual utilization rate of land, enables multiple harvests per year, and increases the total crop yield per unit area. Simultaneously, early flowering extends the grain-filling period, allowing the kernels to accumulate sufficient nutrients, becoming fuller and more plump, directly increasing corn grain yield. Furthermore, early flowering means earlier kernel dehydration, resulting in lower moisture content at harvest, which facilitates mechanized corn harvesting, increases labor productivity, and reduces labor costs and harvesting losses.

[0003] Nitrogen plays a crucial role in many essential life processes of crops, ensuring their basic growth, development, and yield. Sufficient nitrogen fertilizer supply is a vital way to guarantee crop yield. From a crop perspective, improving the crop's nitrogen use efficiency can effectively reduce nitrogen fertilizer application and promote increased crop yield and efficiency. In terms of environmental protection, improving crop nitrogen use efficiency reduces the amount of nitrogen fertilizer used. Excessive nitrogen fertilizer not only increases economic costs but also enters the atmosphere and water bodies through gaseous emissions or rainwater runoff, causing environmental problems such as eutrophication and air pollution. When crop nitrogen use efficiency is improved, the amount of unused nitrogen remaining in the soil decreases, reducing the risk of soil and water pollution and contributing to the protection and sustainable development of the agricultural ecological environment.

[0004] Improving corn yield and quality is crucial for ensuring a stable food supply. Simultaneously advancing flowering and improving nitrogen use efficiency can effectively increase corn production, ensuring a sufficient supply in the market to meet the demands of daily consumption, feed, and industrial processing. Furthermore, higher nitrogen use efficiency promotes the increase of amino acid (protein) content in corn plants and kernels, improving the nutritional quality of corn and providing a higher-quality food and feed source for humans and animals, further solidifying the foundation of food security.

[0005] However, to date, no genes have been reported that can simultaneously promote early flowering, nitrogen use efficiency, and yield improvement in crops such as maize. Summary of the Invention

[0006] All references cited herein are incorporated herein by reference. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless otherwise stated, the techniques used or mentioned herein are standard techniques known to one of ordinary skill in the art. Materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0007] This application's embodiments, through a series of experimental studies, have discovered that [the following occurs in corn plants]... ZmNNR1 and / or ZmNNR4 When genes are overexpressed, they have unexpected technical effects. The overexpressed plants have phenotypes of early flowering and improved nitrogen use efficiency. The functions of improved nitrogen use efficiency include enhanced nitrate nitrogen use efficiency, increased plant nitrogen content, increased chlorophyll content and / or increased maximum photochemical efficiency, and increased yield. The aforementioned gene resources and functions are of great significance to maize breeding.

[0008] This application provides a method for producing maize plants with early flowering and / or increased nitrogen use efficiency phenotypes. The method includes the following steps:

[0009] (a) Enhancing the functional genes in maize plants ZmNNR1 and / or ZmNNR4 The expression level or protein content;

[0010] (b) Obtain at least one seed from the corn plant produced in step (a).

[0011] Optionally, the aforementioned gene that enhances the function of maize plants ZmNNR1 and / or ZmNNR4 Expression levels include the introduction of functional genes into maize plants. ZmNNR1 and / or ZmNNR4 Overexpression vectors, either utilizing natural variations or... ZmNNR1 and / or ZmNNR4 Promoters are used for gene editing, or for... ZmNNR1 and / or ZmNNR4 Modifications are made to the 5'-UTR or 3'-UTR of the gene, or... ZmNNR1 and / or ZmNNR4 Add enhancers to the promoter region of a gene to increase ZmNNR1 and / or ZmNNR4 The expression level or protein content.

[0012] Optionally, the increased nitrogen use efficiency includes enhanced nitrate nitrogen use efficiency, increased plant nitrogen content, increased chlorophyll content, increased maximum photochemical efficiency, and / or increased yield.

[0013] Alternatively, the term "early flowering" refers to the early flowering of female flowers or male spikes.

[0014] Optionally, the multinucleotide sequence of the ZmNNR1 gene is selected from one of the following groups of sequences:

[0015] (a) A polynucleotide sequence as shown in SEQ ID No: 1, 2, 3 or 4;

[0016] (b) The polynucleotide sequence that encodes an amino acid sequence as shown in SEQ ID No: 5, 6 or 7;

[0017] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under strict hybridization conditions, and the overexpression of the polynucleotide sequence exogenously in maize plants has the function of causing early flowering, increased nitrogen use efficiency, increased nitrogen content, increased chlorophyll content, increased maximum photochemical efficiency, enhanced nitrate nitrogen use efficiency and / or increased yield.

[0018] (d) A polynucleotide sequence that has at least 90%, 95%, or 98% similarity to any of the polynucleotide sequences shown in (a)-(c), wherein overexpression of the exogenous polynucleotide sequence in maize plants has the function of promoting early flowering, increasing nitrogen use efficiency, increasing nitrogen content, increasing chlorophyll content, increasing maximum photochemical efficiency, enhancing nitrate nitrogen use efficiency, and / or increasing yield; or

[0019] (e) A polynucleotide sequence complementary to any of the sequences described in (a)-(d);

[0020] (f) A sequence in which one or more nucleotides are substituted, deleted and / or added on any of the polynucleotide sequences described in (a)-(e).

[0021] Optionally, the aforementioned ZmNNR4 The gene's polynucleotide sequence is selected from one of the following groups of sequences:

[0022] (a) A polynucleotide sequence as shown in SEQ ID No: 8 or 9;

[0023] (b) Its encoding amino acid sequence is the polynucleotide sequence shown in SEQ ID No: 10;

[0024] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under strict hybridization conditions, and the overexpression of the polynucleotide sequence exogenously in maize plants has the function of causing early flowering, increased nitrogen use efficiency, increased nitrogen content, increased chlorophyll content, increased maximum photochemical efficiency, enhanced nitrate nitrogen use efficiency and / or increased yield.

[0025] (d) A polynucleotide sequence that has at least 90%, 95%, or 98% similarity to any of the polynucleotide sequences shown in (a)-(c), wherein overexpression of the exogenous polynucleotide sequence in maize plants has the function of promoting early flowering, increasing nitrogen use efficiency, increasing nitrogen content, increasing chlorophyll content, increasing maximum photochemical efficiency, enhancing nitrate nitrogen use efficiency, and / or increasing yield; or

[0026] (e) A polynucleotide sequence complementary to any of the sequences described in (a)-(d);

[0027] (f) A sequence in which one or more nucleotides are substituted, deleted and / or added on any of the polynucleotide sequences described in (a)-(e).

[0028] Optionally, the overexpression vector further includes a promoter that is operatively linked to the nucleotide sequence of the functional gene and enhances the expression level of the functional gene.

[0029] Optionally, the promoter is an overexpression or overexpression promoter such as cauliflower mosaic virus CaMV 35S, maize ubiquitin promoter, or rice Actin1 promoter. Optionally, the promoter can also be an inducible promoter or a tissue / organ-specific expression promoter.

[0030] Optionally, as those skilled in the art will know, the embodiments disclosed in this application... ZmNNR1 , ZmNNR4 After gene function is established, any technology that utilizes naturally occurring variations to enhance [gene function]... ZmNNR1 and / or ZmNNR4 Methods that increase the expression level of [specific ingredient] to promote early flowering and / or nitrogen efficiency in maize should also be covered within the scope of protection of this invention.

[0031] Optionally, embodiments of this application also provide a method for producing maize plants, wherein the maize plants have a late-flowering and / or reduced nitrogen use efficiency phenotype, the method comprising the steps of:

[0032] (a) Producing one or more maize plants, wherein the maize plants contain endogenous ZmNNR1 Genes and ZmNNR4 Each gene contains at least one loss-of-function mutation, or an endogenous mutation. ZmNNR1 Genes and ZmNNR4 Gene expression is suppressed;

[0033] (b) Obtain at least one seed from the corn plant produced in step (a).

[0034] Optionally, the reduction in nitrogen use efficiency includes a reduction in nitrogen content, a reduction in chlorophyll content, a reduction in maximum photochemical efficiency, and / or a reduction in yield.

[0035] Optionally, wherein theZmNNR1 The gene's polynucleotide sequence is selected from one of the following groups of sequences:

[0036] (a) A polynucleotide sequence as shown in SEQ ID No: 1, 2, 3 or 4;

[0037] (b) The polynucleotide sequence that encodes an amino acid sequence as shown in SEQ ID No: 5, 6 or 7;

[0038] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under strict hybridization conditions, and a functional loss mutation of the polynucleotide sequence in maize plants has the functions of late flowering, reduced nitrogen content, reduced chlorophyll content, reduced maximum photochemical efficiency, reduced nitrate nitrogen use efficiency and / or reduced yield.

[0039] (d) A polynucleotide sequence that has at least 90%, 95%, or 98% similarity to any of the polynucleotide sequences shown in (a)-(c), and a loss-of-function mutation of this polynucleotide sequence in the maize plant has the function of causing late flowering, reduced nitrogen content, reduced chlorophyll content, reduced maximum photochemical efficiency, reduced nitrate nitrogen use efficiency, and / or reduced yield; or

[0040] (e) A polynucleotide sequence complementary to any of the sequences described in (a)-(d).

[0041] Optionally, wherein the ZmNNR4 The gene's polynucleotide sequence is selected from one of the following groups of sequences:

[0042] (a) A polynucleotide sequence as shown in SEQ ID No: 8 or 9;

[0043] (b) Its encoding amino acid sequence is the polynucleotide sequence shown in SEQ ID No: 10;

[0044] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under strict hybridization conditions, and a loss-of-function mutation of the polynucleotide sequence endogenous in maize plants that causes late flowering, reduced nitrogen content, reduced chlorophyll content, reduced maximum photochemical efficiency, reduced nitrate nitrogen use efficiency and / or reduced yield.

[0045] (d) A polynucleotide sequence that has at least 90%, 95%, or 98% similarity to any of the polynucleotide sequences shown in (a)-(c), and a loss-of-function mutation of this polynucleotide sequence in the maize plant has the function of causing late flowering, reduced nitrogen content, reduced chlorophyll content, reduced maximum photochemical efficiency, reduced nitrate nitrogen use efficiency, and / or reduced yield; or

[0046] (e) A polynucleotide sequence complementary to any of the sequences described in (a)-(d).

[0047] Optionally, the embodiments provided in this application... ZmNNR1 or ZmNNR4 Genes, including homologous genes or the same gene from different varieties that have at least 80%, 85%, 90%, 95%, 98%, or 99% sequence similarity to their polynucleotide sequences, or genes disclosed in the embodiments of this invention. ZmNNR1 or ZmNNR4 The homologous gene or the same gene in different varieties has at least 90%, 95%, or 98% sequence similarity in its amino acid sequence. After an endogenous homozygous loss-of-function mutation, the homologous gene has the function of causing late flowering, reduced nitrogen use efficiency, reduced nitrogen content, reduced chlorophyll content, and / or reduced maximum photochemical efficiency, and reduced yield. After overexpression, it has the function of causing early flowering, increased nitrogen use efficiency, increased nitrogen content, increased chlorophyll content, and / or increased maximum photochemical efficiency, and increased yield. The homologous gene can be isolated from any plant.

[0048] Optionally, the method provided in this application can be applied to any substance containing ZmNNR1 or ZmNNR4 Plants with homologous genes. Preferably, the plants include monocotyledonous plants such as corn, millet, wheat, barley, rye, rice, and sorghum, and dicotyledonous plants such as cotton, corn, peanut, sunflower, sweet potato, potato, apple, and tobacco.

[0049] The percentage of sequence similarity described in this application can be obtained using well-known bioinformatics algorithms, including the Myers and Miller algorithm, the Needleman-Wunsch global alignment method, the Smith-Waterman local alignment method, the Pearson and Lipman similarity search method, and the Karlin and Altschul algorithm, which are well known to those skilled in the art.

[0050] Those skilled in the art should know that single nucleotide polymorphisms (SNPs) exist for the same gene among different varieties of the same plant, meaning that the nucleotide sequence of the same gene often differs by a few bases. However, there are many varieties of the same crop, and it is impossible for the inventors to list them all. The embodiments of this application only provide sequences of representative varieties of maize. Therefore, those skilled in the art should know that sequences from different varieties may differ from those disclosed in this invention. ZmNNR1 or ZmNNR4A nucleotide sequence in which a gene and its nucleotide sequence have SNPs, a method and application for obtaining phenotypes such as early flowering, increased nitrogen use efficiency, increased nitrogen content, increased chlorophyll content, and / or increased maximum photochemical efficiency, and increased yield by overexpressing it, or a method and application for obtaining traits such as late flowering, decreased nitrogen use efficiency, decreased nitrogen content, decreased chlorophyll content, and / or decreased maximum photochemical efficiency, and decreased yield by using its endogenous loss-of-function mutation are also within the scope of protection of the present invention.

[0051] Optionally, the loss-of-function mutation described in the present application is obtained by mutation, and the mutation includes substitution, deletion, and / or addition of one or more nucleotides in the nucleotide sequence of the gene.

[0052] Optionally, the loss-of-function mutation includes, but is not limited to, being obtained by methods such as physical mutagenesis, chemical mutagenesis, and gene editing. Physical mutagenesis includes, but is not limited to, radiation mutagenesis, space breeding, etc.; the method of chemical mutagenesis includes mutagenesis caused by treating with mutagens such as EMS; the method of gene editing includes, but is not limited to, methods such as ZFN, TALEN, and / or CRISPR / Cas.

[0053] Those skilled in the art know that the main principle of the CRISPR / Cas gene editing system or gene editing method is to find the position to be gene-edited in the host genome through a nucleic acid fragment called guide RNA (gRNA), that is, the target DNA sequence, and then cut the DNA through the Cas protein. In the present application, the Cas protein includes, but is not limited to, proteins such as Cas9, Cas12, Cas12a, Cas12j, Cas12e, Cas13, and / or Cas14.

[0054] Optionally, when the gene editing system used is CRISPR / Cas9, the gene mutant sequence obtained by the CRISPR / Cas9 method, the target sequence used in the CRISPR / Cas9 technology is selected from one of the sequences in the following group:

[0055] (a) A fragment conforming to the sequence arrangement rule of 5'-Nx-NGG-3' in the nucleotide sequences shown in SEQ ID No: 1, 2, 3, 4, 8, or 9, where N represents any one of A, G, C, and T, 14 < X < 30, and X is an integer, and Nx represents X consecutive nucleotides; or

[0056] (b) A nucleotide sequence complementary to the polynucleotide sequence described in (a).

[0057] Optionally, the maize plant has ZmNNR1 and / or ZmNNR4 a gene mutant, whereZmnnr1 The mutation characteristics are: ZmNNR1 A large deletion occurs between bases 648 and 3187 of the genomic DNA; or in the transcript. ZmNNR1 A large deletion occurs between bases 487 and 1089 of the CDS of _T1; or in the transcript. ZmNNR1 A large deletion occurs between bases 487 and 816 of the CDS of _T2; or ZmNNR1 A 4bp deletion between bases 644 and 647 of the genomic DNA; or a deletion in the transcript. ZmNNR1 A 4bp deletion occurs between bases 483 and 486 of the CDS of _T1; or in the transcript. ZmNNR1 A 4bp deletion occurs between bases 483 and 486 of the CDS of _T2. Zmnnr4 The mutation characteristics are: ZmNNR4 A large deletion occurs in the genomic DNA between bases 570 and 3046; or ZmNNR4 The CDS contains a large deletion between bases 459 and 1289; or ZmNNR4 A 1bp deletion occurs between bases 569 and 571 in the genomic DNA; or ZmNNR4 A 1bp deletion occurs between bases 458 and 460 of the CDS.

[0058] Optionally, the loss-of-function mutant or overexpression plant described in the embodiments of this application can also be obtained by hybridizing with maize plants that have the loss-of-function mutant or overexpression.

[0059] Optionally, the method described in the embodiments of this application, wherein the reduction or suppression ZmNNR1 and / or ZmNNR4 Normal expression or protein function of homologous genes can be achieved through RNA interference (RNAi) and / or mutation, or by altering the promoter of functional genes using natural variation, molecular biology methods, or gene editing to obtain a phenotype with reduced expression levels or protein content. Those skilled in the art will recognize that RNAi technology is a conventional technique in the field, which involves the specific binding of 21-23 bp short double-stranded RNA (siRNA: small interfering RNA) or long double-stranded RNA (dsRNA: double-stranded RNA) to the homologous region of the mRNA expressing the target gene, causing mRNA degradation and thus inhibiting gene expression.

[0060] Alternatively, in this application, endogenous maize can be inhibited using RNAi. ZmNNR1 Genes and / or ZmNNR4Gene expression is affected, thereby influencing the activity of the aforementioned genes. Inhibiting gene expression results in maize plants exhibiting phenotypes such as late flowering, reduced nitrogen content, reduced chlorophyll content, and / or reduced maximum photochemical efficiency.

[0061] Optionally, this application also provides ZmNNR1 and / or ZmNNR4 Application of genes in producing maize plants with early flowering, increased nitrogen content, increased chlorophyll content, and / or increased maximum photochemical efficiency.

[0062] Optionally, this application also provides a method for obtaining this application by means of any of the foregoing. ZmNNR1 and / or ZmNNR4 The application of gene loss mutants in maize breeding, preferably, includes, but is not limited to, the application in regulating traits such as late flowering, reduced nitrogen use efficiency, reduced nitrogen content, reduced chlorophyll content and / or reduced maximum photochemical efficiency, and reduced yield.

[0063] Optionally, embodiments of this application also provide feed, coarse flour, protein, or oil products made from corn, wherein the feed, coarse flour, protein, or oil product contains... ZmNNR1 Genes and / or ZmNNR4 Increased gene expression; or the feed, roughage, protein, or oil product contains... ZmNNR1 Genes and / or ZmNNR4 Loss-of-function mutants of genes, the ZmNNR1 Genes and / or ZmNNR4 Each gene contains at least one loss-of-function mutation, or an endogenous mutation. ZmNNR1 Genes and / or ZmNNR4 Gene expression is suppressed. The loss-of-function mutation includes substitution, deletion, and / or addition of one or more nucleotides in the nucleotide sequence of the aforementioned gene. Embodiments of this application also provide a corn-based feed, meal, protein, or oil product containing… ZmNNR1 Genes and / or ZmNNR4 Gene overexpression constructs.

[0064] Optionally, embodiments of this application also provide a gene mutant, wherein the gene mutant is... ZmNNR1 or ZmNNR4 A loss-of-function mutant, characterized by endogenous mutations in the maize plant. ZmNNR1 and / or ZmNNR4 After gene mutation, it has the function of causing late flowering, reduced nitrogen use efficiency, reduced nitrogen content, reduced chlorophyll content and / or reduced maximum photochemical efficiency, and reduced yield. ZmNNR1 The gene's polynucleotide sequence is selected from one of the following groups of sequences:

[0065] (a) A polynucleotide sequence as shown in SEQ ID No: 1, 2, 3 or 4;

[0066] (b) The polynucleotide sequence that encodes an amino acid sequence as shown in SEQ ID No: 5, 6 or 7;

[0067] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under strict hybridization conditions, wherein a loss-of-function mutation of the polynucleotide sequence endogenous in maize plants has the function of causing late flowering, reduced nitrogen content, reduced chlorophyll content, reduced maximum photochemical efficiency, reduced nitrate nitrogen use efficiency and / or reduced yield.

[0068] (d) A polynucleotide sequence that has at least 90%, 95%, or 98% similarity to any of the polynucleotide sequences shown in (a)-(c), wherein a loss-of-function mutation of this polynucleotide sequence in the maize plant has the function of causing late flowering, reduced nitrogen content, reduced chlorophyll content, reduced maximum photochemical efficiency, reduced nitrate nitrogen use efficiency, and / or reduced yield; or

[0069] (e) A polynucleotide sequence complementary to any of the sequences described in (a)-(d);

[0070] The ZmNNR4 The gene's polynucleotide sequence is selected from one of the following groups of sequences:

[0071] (a) A polynucleotide sequence as shown in SEQ ID No: 8 or 9;

[0072] (b) Its encoding amino acid sequence is the polynucleotide sequence shown in SEQ ID No: 10;

[0073] (c) A polynucleotide sequence that can hybridize with the polynucleotide sequence described in (a) or (b) under strict hybridization conditions, and a loss-of-function mutation of the polynucleotide sequence endogenous in maize plants that causes late flowering, reduced nitrogen content, reduced chlorophyll content, reduced maximum photochemical efficiency, reduced nitrate nitrogen use efficiency and / or reduced yield.

[0074] (d) A polynucleotide sequence that has at least 90%, 95%, or 98% similarity to any of the polynucleotide sequences shown in (a)-(c), and a loss-of-function mutation of this polynucleotide sequence in the maize plant has the function of causing late flowering, reduced nitrogen content, reduced chlorophyll content, reduced maximum photochemical efficiency, reduced nitrate nitrogen use efficiency, and / or reduced yield; or

[0075] (e) A polynucleotide sequence complementary to any of the sequences described in (a)-(d).

[0076] Optionally, the methods described in the embodiments of this application for transferring nucleotide sequences, vectors, constructs, or expression cassettes into plants, introducing them into plants, or transforming plants all refer to transferring the target nucleotide sequence, construct, vector, or expression cassette into recipient cells or recipient plants through conventional transgenic methods or methods of hybridization with target transgenic plants. Any transgenic method known to those skilled in the art can be used to transform recombinant expression vectors into plant cells to produce transgenic plants or mutants of the embodiments of this application. Transformation methods may include direct or indirect transformation methods. Specifically, the transformation methods include, but are not limited to, polyethylene glycol-induced DNA uptake, liposome-mediated transformation, gene gun introduction, electroporation, microinjection, and Agrobacterium-mediated plant transformation methods.

[0077] Compared with the prior art, this application has the following beneficial effects:

[0078] (1) This application provides a method for producing maize plants and its application, by improving the quality of maize plants. ZmNNR1 and / or ZmNNR4 By adjusting gene expression levels, plants exhibiting superior agronomic traits such as early flowering, increased nitrogen use efficiency, increased nitrogen content, increased chlorophyll content, increased maximum photochemical efficiency, and / or increased yield can be obtained. The aforementioned genes, methods, and applications provide new germplasm resources and breeding strategies for maize breeding, which are of great significance to global food security and sustainable agricultural development.

[0079] (2) It was clarified that corn ZmNNR1 Genes and ZmNNR4 Gene overexpression and mutation functions provide new genetic resources for crop species;

[0080] (3) By overexpressing or repressing two genes, breeding materials with application value can be obtained, providing new ideas for crop breeding and the study of gene action mechanism networks.

[0081] Term definitions involved in the present invention

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods, apparatus, and materials are now described.

[0083] In the context of this application, the terms "polynucleotide" or "nucleotide" mean deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers in single-stranded or double-stranded form. Unless specifically limited, the term covers nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0084] In this application, the term "homologous gene" refers to two or more gene sequences with a sequence similarity of 80%, including orthologous genes (also known as vertical homologous genes, positive homologous genes, or directed evolutionary homologous genes), transverse homologous genes (also known as paralogous genes, paralogous homologous genes, or parallel evolutionary homologous genes), and / or heterologous genes.

[0085] The term "sequence similarity" refers to the degree of similarity between two sequences. It is a quantitative concept used to compare the similarity between different sequences, thereby discovering and analyzing the association between the two sequences. Sequence similarity can be used to compare gene sequences, protein sequences, DNA sequences, etc.

[0086] The term "strict hybridization conditions" as used in this application refers to conditions of low ionic strength and high temperature known in the art. Typically, under strict conditions, the detectability of a probe hybridizing with its target sequence is significantly higher than that with other sequences (e.g., at least twice the background level). Strict hybridization conditions are sequence-dependent and will vary under different environmental conditions; longer sequences hybridize specifically at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the strictness of hybridization or washing conditions. Detailed guidance on nucleic acid hybridization can be found in relevant literature (Tijssen, ...). Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, (Overview of principles of hybridization and the strategy of nucleic acid assays. 1993). More specifically, the stringent conditions are typically chosen to be below the melting point (T0) of the specific sequence at a specified ionic strength pH. m Approximately 5-10℃. m The temperature at which 50% of the probe complementary to the target sequence hybridizes to the target sequence under equilibrium conditions (at specified ionic strength, pH, and nucleic acid concentration) (because the target sequence is present in excess, therefore at T...). m(Under equilibrium conditions, 50% of the probe is occupied). Strict conditions may include: a salt concentration of less than about 1.0 M sodium ions at pH 7.0 to 8.3, typically about 0.01 to 1.0 M sodium ions (or other salts), and a temperature of at least about 30°C for short probes (including, but not limited to, 10 to 50 nucleotides) and at least about 60°C for long probes (including, but not limited to, greater than 50 nucleotides). Strict conditions can also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, the positive signal may be at least twice the background hybridization, and, where appropriate, 10 times the background hybridization. Exemplary strict hybridization conditions may be: 50% formamide, 5×SSC and 1% SDS, incubated at 42°C; or 5×SSC, 1% SDS, incubated at 65°C, washed in 0.2×SSC and washed in 0.1% SDS at 65°C. The washing can be performed for 5, 15, 30, 60, 120 minutes or longer.

[0087] The term "recombinant expression vector" refers to one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, along with vectors containing helper plasmids, are commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: the cis-acting sequence required for T-DNA transfer, a selection marker engineered for expression in plant cells, and the heterologous DNA sequence to be transcribed.

[0088] The term "hybridization" is used in a broad sense to refer to the process by which gametes from different populations or genotypes combine to produce hybrids. Depending on the parental relationship, it includes close hybridization and distant hybridization.

[0089] The term "mutation" as used in this application refers to a "loss-of-function mutation" or "loss-of-function mutation," which is a mutation in the coding sequence of a gene that causes a reduction or complete loss of function in the gene product (usually a protein). Loss-of-function mutations can be caused, for example, by truncation of the gene product (due to frameshift or nonsense mutations), and the phenotype associated with an allele having a loss-of-function mutation can be recessive or dominant.

[0090] The term "RNA interference" (RNAi) is a gene blocking technique that uses a double-stranded RNA (dsRNA) molecule to block or silence the expression of a specific gene at the mRNA level; it is also known as sequence-specific post-transcriptional gene silencing (PTGS).

[0091] "Overexpression," also known as "overexpression," refers to the process of operatively linking the full-length sequence of a target gene with a constitutive promoter, an inducible promoter, or a tissue-specific promoter, and then transferring it into a plant through transformation technology, thereby causing the gene product to accumulate in large quantities in the plant. Attached Figure Description

[0092] Figure 1 yes ZmNNR1 and ZmNNR4 A schematic diagram of a gene-edited mutant.

[0093] Figure 2 yes ZmNNR1 , ZmNNR4 Identification of overexpression events.

[0094] Figure 3 It is wild type, ZmNNR1 and ZmNNR4 Field photos of gene-edited mutants and overexpressed materials during their flowering period.

[0095] Figure 4 It is wild type, ZmNNR1 and ZmNNR4 Statistical analysis of the pollen shedding and silking stages of gene-edited mutant materials.

[0096] Figure 5 It is wild type, ZmNNR1 and ZmNNR4 Phenotypic statistics of the pollen shedding and silking stages of the expressed material.

[0097] Figure 6 It is wild type, ZmNNR1 and ZmNNR4 Statistics on nitrogen content in leaves of gene-edited mutant materials.

[0098] Figure 7 It is wild type, ZmNNR1 and ZmNNR4 The nitrogen content of leaves exceeding the expression material was statistically analyzed.

[0099] Figure 8 It is wild type, ZmNNR1 and ZmNNR4 Statistics on chlorophyll content in leaves of gene-edited mutant materials.

[0100] Figure 9 It is wild type, ZmNNR1 and ZmNNR4 Statistics on leaf chlorophyll content exceeding that of the expressed material.

[0101] Figure 10 It is wild type, ZmNNR1 and ZmNNR4 Statistical analysis of the maximum photochemical efficiency of leaves from gene-edited mutant materials.

[0102] Figure 11 It is wild type, ZmNNR1 and ZmNNR4 Statistical analysis of the maximum photochemical efficiency of leaves exceeding that of the expressed material.

[0103] Figure 12 It is wild type, ZmNNR1 and ZmNNR4 Statistics on single-ear yield of gene-edited mutants and overexpression materials under low nitrogen conditions.

[0104] Figure 13 It is wild type, ZmNNR1 and ZmNNR4 Statistics on single-ear yield of gene-edited mutants and overexpression materials under normal nitrogen application conditions.

[0105] Figure 14 It is wild type, ZmNNR1 and ZmNNR4 The yield performance of gene-edited mutants and overexpression materials under different nitrogen application conditions.

[0106] Figure 15 It is wild type, ZmNNR1 and ZmNNR4 Gene-edited mutants and overexpression materials under chlorate treatment conditions.

[0107] Figure 16 In natural groups ZmNNR4 Gene expression levels are negatively correlated with flowering period and positively correlated with leaf nitrogen content.

[0108] Figure 17 The correlation analysis shows ZmNNR4 There are natural variations near the gene that can regulate its expression. Detailed Implementation

[0109] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0110] The inbred lines and maize varieties used in the following examples can be obtained from the "China Crop Germplasm Information Network" and the corresponding seeds can be obtained by applying for them.

[0111] Example 1: ZmNNR1 and ZmNNR4 Creation of mutants and overexpression materials

[0112] ZmNNR1The genomic DNA sequence is shown in SEQ ID NO:1, and the CDS sequences of the three transcripts are shown in SEQ ID NO:2, 3, and 4, respectively. The protein amino acid sequences corresponding to the three transcripts are shown in SEQ ID NO:5, 6, and 7, respectively. ZmNNR4 The genomic DNA sequence is shown in SEQ ID NO:8, its corresponding CDS sequence is shown in SEQ ID NO:9, and the protein amino acid sequence is shown in SEQ ID NO:10. This study aims to investigate the role of [the protein in maize]. ZmNNR1 and ZmNNR4 To determine the biological function of sgRNA, we designed a series of steps, including using SnapGene Viewer software, assessing multiple copies of sequences in the B73 v5 genome, and predicting the secondary structure of sgRNA. ZmNNR1 and ZmNNR4 The optimal gene editing target sequences were determined, with two target sites designed for each gene. The sequences are as follows:

[0113] ZmNNR1-T1 :5'-AGGTGACGGGCCTCGTGAGGCGG-3' (SEQ ID NO:11)

[0114] ZmNNR1-T2 :5'-CCTCCACCTCGACGGACCAGAAG-3' (SEQ ID NO:12)

[0115] ZmNNR4-T1 :5'-AGGTGACGGGCCTCGTGAGGCGG-3' (SEQ ID NO:13)

[0116] ZmNNR4-T2 :5'-CCTCCACCTCGACGGACCAGAAG-3' (SEQ ID NO:14)

[0117] Using the aforementioned target sequence and the pCPB-ZmUbi:hSpCas9 vector backbone, a simultaneous targeting... ZmNNR1 and ZmNNR4 The CRISPR / Cas9 gene editing vector was obtained and genetically transformed into the maize inbred line ZC01. Zmnnr1 Single mutant, Zmnnr4 Single mutant and Zmnnr1 Zmnnr4 Two independent lines of each double mutant ( Figure 1 They were named as follows: Zmnnr1-1 , Zmnnr1-2 , Zmnnr4-1 , Zmnnr4-2 , ​ , ​Amino acid sequence analysis showed that these mutants... ​ or ​ Premature termination or frameshift mutations occurred in the protein, among which ZmNNR1 Two dominant transcripts of the gene have been mutated.

[0118] in Zmnnr1 The mutation characteristics are:

[0119] ZmNNR1 A large deletion occurs between bases 648 and 3187 of the genomic DNA, or

[0120] Transcript ZmNNR1 A large deletion occurs between bases 487 and 1089 of the CDS (SEQ ID NO:2) of _T1, or

[0121] Transcript ZmNNR1 A large deletion occurs between bases 487 and 816 of the CDS (SEQ ID NO:3) of _T2, or

[0122] ZmNNR1 A 4bp deletion between bases 644 and 647 of the genomic DNA, or

[0123] Transcript ZmNNR1 A 4bp deletion occurs between bases 483 and 486 of the CDS in _T1, or

[0124] Transcript ZmNNR1 A 4bp deletion occurs between bases 483 and 486 of the CDS of _T2.

[0125] Zmnnr4 The mutation characteristics are:

[0126] ZmNNR4 A large deletion occurs in the genomic DNA between bases 570 and 3046; or

[0127] ZmNNR4 The CDS contains a large deletion between bases 459 and 1289; or

[0128] ZmNNR4 A 1bp deletion occurs between bases 569 and 571 in the genomic DNA; or

[0129] ZmNNR4 A 1bp deletion occurs between bases 458 and 460 of the CDS.

[0130] To further determine ZmNNR1 and ZmNNR4 To understand the biological functions of [the gene / organism], the plant overexpression vector backbone CPB-UBI-EGFP was used to construct [various gene / organisms].ZmNNR1 and ZmNNR4 Overexpression vectors, in which ZmNNR4 Overexpression vectors were constructed using its unique transcript. ZmNNR1 Overexpression vectors were constructed using the dominant transcript T2 (SEQ ID NO:3). The constructed overexpression vector was then genetically transformed into the maize inbred line ZC01 to obtain... ZmNNR1 and ZmNNR4 Gene overexpression materials. Two independent lines were obtained from each material. Figure 2 Two overexpression materials were planted at the Langfang experimental base in the summer of 2024. Leaf samples were taken at the V10 stage, flash-frozen in liquid nitrogen, and RNA was extracted using the TRIzol method. Specific primers were then used (…). ZmNNR1 Expression level identification: ZmNNR1-1F : 5'-GAGGACCACACGGAGATG-3' (SEQ ID NO:15), ZmNNR1- 1R : 5'-CCAACGCTGTACTTCCAC-3' (SEQ ID NO: 16); ZmNNR4 Expression level identification: ZmNNR4-1F : 5'-CCGCATGGTCAAGTGGCT-3' (SEQ ID NO:17), ZmNNR4-1R qRT-PCR quantitative analysis was performed on 5'-TTGTACCACCACGCCTCAG-3' (SEQ ID NO:18) to identify... ZmNNR1 and ZmNNR4 Two overexpression lines were identified; they were named respectively. ZmNNR1-OE-1 , ZmNNR1-OE-2 , ZmNNR4-OE-1 , ZmNNR4-OE-2 ;in ZmNNR1-OE-1 and ZmNNR1-OE-2 The expression levels were 38.233 and 810.65 times higher than those of the wild type, respectively. ZmNNR4-OE-1 and ZmNNR4-OE-2 The overexpression was 181.17 and 167.84 times higher than that of the wild type, respectively. Figure 2 ).

[0131] Example 2: ZmNNR1 and ZmNNR4 Regulating the flowering period of corn

[0132] In the winter of 2024, at the Ledong Experimental Station in Hainan, wild-type... Zmnnr1 Single mutant, Zmnnr4 Single mutant, Zmnnr1Zmnnr4 Double mutants and ZmNNR1 , ZmNNR4Overexpression materials were planted, and flowering time data of wild-type and above genetic materials were statistically analyzed. The results showed that, compared with the wild-type, Zmnnr1 Single mutant, Zmnnr4 Single mutant, Zmnnr1Zmnnr4 The flowering period of the double mutant is significantly later. Figure 3 and Figure 4 The late-flowering days during the pollen shedding period were 6-8, 6-8, and 10-11 days, respectively, and the late-flowering days during the silking period were 5-7, 7-8, and 10-11 days, respectively; among which... Zmnnr1Zmnnr4 The flowering period of the double mutant is longer than Zmnnr1 Single mutant and Zmnnr4 Single mutants all came later, indicating ZmNNR1 and ZmNNR4 Genes play an important role in regulating the flowering period of maize, and there is some functional redundancy between the two.

[0133] Further ZmNNR1 Phenotypic analysis of the overexpression materials revealed that, compared to the negative control materials, ZmNNR1 The overexpression material showed significantly earlier flowering, with the number of days between pollen shedding and flowering being 3-5 days earlier, and the number of days between silking and flowering being 4-6 days earlier. Figure 3 and Figure 5 Similarly, for ZmNNR4 Phenotypic analysis of the overexpression materials revealed that, compared to the negative control materials, ZmNNR4 The overexpression materials also showed significantly earlier flowering, with the number of days earlier flowering during the pollen shedding stage being 1-2 days and the number of days earlier flowering during the silking stage being 3-4 days. Figure 3 , Figure 5 ).illustrate ZmNNR1 and ZmNNR4 Both genes promote the flowering period of maize, and increasing the expression level of either one can promote early flowering of maize.

[0134] Example 3 ZmNNR1 and ZmNNR4 Promote high nitrogen efficiency in corn

[0135] In the winter of 2024, wild-type and... Zmnnr1 Single mutant, Zmnnr4 Single mutant, Zmnnr1Zmnnr4 Double mutants and ZmNNR1 , ZmNNR4 Overexpression materials were planted, and the silking date was strictly recorded at flowering time. Leaf nitrogen content was measured using an N-Pen N110 handheld nitrogen meter at the time of female ear silking. To ensure accuracy, measurements were taken at the base, middle, and tip of the ear leaves, and averages were calculated. At least six plants of each material were measured. Statistical analysis of the obtained data revealed that… Zmnnr1 Single mutant, Zmnnr4Single mutant, Zmnnr1Zmnnr4 The leaf nitrogen content of the double mutants was significantly lower than that of the wild type, with the percentages lower being 6.0%–12.2%, 6.0%–12.6%, and 13.0%–13.8%, respectively. Figure 6 );in Zmnnr1Zmnnr4 Leaf nitrogen content ratio of double mutant Zmnnr1 Single mutant and Zmnnr4 Single mutants all had lower levels; leaf nitrogen content is an important indicator of nitrogen assimilation and utilization efficiency in maize plants. The changes in leaf nitrogen content in the above mutants indicate that... [[ID=8Q]]ZmNNR1 and ZmNNR4 Genes play an important role in regulating nitrogen use efficiency in maize, and there is some functional redundancy between the two.

[0136] Further ZmNNR1 Phenotypic analysis of nitrogen content in leaves during the silking stage using overexpression materials revealed that, compared to the negative control materials, ZmNNR1 The nitrogen content in the leaves of the overexpression material was significantly increased, with an increase of 21.6%–27.2%. Figure 7 Similarly, for ZmNNR4 Phenotypic analysis of the overexpression materials revealed that, compared to the negative control materials, ZmNNR4 The nitrogen content in the leaves of the overexpression material was also significantly increased, with an increase of 15.6%–48.3%. Figure 7 ).illustrate ZmNNR1 and ZmNNR4 Both genes play an important role in promoting nitrogen use efficiency in maize, and increasing the expression level of either one can significantly improve nitrogen use efficiency in maize.

[0137] Example 4: ZmNNR1 and ZmNNR4 Regulating maize chlorophyll content and photosynthesis

[0138] In the winter of 2024, at the Ledong experimental station in Hainan, similar studies were conducted on wild-type and... ZmNNR1 and ZmNNR4 Strictly recording the silking period of different genetic materials and observing leaf color revealed that... Zmnnrl Single mutant, Zmnnr4 Single mutant, ZmnnrlZmnnr4 The leaves of the double mutant are significantly yellower than those of the wild type, among which ZmnnrlZmnnr4 The double mutant exhibited more severe yellowing; further measurements of chlorophyll content and maximum photochemical efficiency were conducted using a SPAD-502 chlorophyll meter and a FluorPen FP110 handheld chlorophyll fluorescence meter, respectively. To ensure accuracy, measurements were taken at the base, middle, and tip of the panicle leaves, and averages were calculated. At least six plants of each material were measured. Statistical analysis of the obtained data revealed that... Zmnnr1 Single mutant,Zmnnr4 Single mutant, Zmnnr1Zmnnr4 The chlorophyll content and maximum photochemical efficiency of the leaves of the double mutants were significantly lower than those of the wild type. The percentages with lower chlorophyll content were 10.5%–18.3%, 12.1%–19.2%, and 15.2%–22.0%, respectively, and the percentages with lower maximum photochemical efficiency were 5.3%–6.8%, 6.7%–7.3%, and 13.3%–14.2%, respectively. Figure 8 and Figure 10 );in Zmnnr1Zmnnr4 The leaf chlorophyll content of the double mutant is higher than that of the double mutant. Zmnnr1 Single mutant and Zmnnr4 Single mutants all showed lower values; leaf chlorophyll content and maximum photochemical efficiency are important indicators of photosynthetic efficiency in maize plants. The changes in these photosynthetic parameters in the leaves of the above mutants indicate that... ZmNNR1 and ZmNNR4 Genes play an important role in regulating maize photosynthetic efficiency, and there is some functional redundancy between the two.

[0139] Further ZmNNR1 Phenotypic analysis of chlorophyll content and maximum photochemical efficiency in leaves during the silking stage of the overexpression materials revealed that, compared with the negative control materials, ZmNNR1 The chlorophyll content and maximum photochemical efficiency of the leaves of the overexpression material were significantly increased, with chlorophyll content increasing by 14.7%–17.4% and maximum photochemical efficiency increasing by 6.7%–9.1%. Figure 9 and Figure 11 Similarly, for ZmNNR4 Phenotypic analysis of the overexpression materials revealed that, compared to the negative control materials, ZmNNR4 The chlorophyll content and maximum photochemical efficiency of the leaves of the overexpression material were also significantly increased, with chlorophyll content increasing by 10.8%–11.0% and maximum photochemical efficiency increasing by 7.9%–9.5%. Figure 9 and Figure 11 ).illustrate ZmNNR1 and ZmNNR4 Both genes play a crucial role in promoting maize photosynthetic efficiency; increasing the expression level of either gene can significantly enhance photosynthetic efficiency. It is worth noting that nitrogen in plants has a significant impact on many processes of photosynthesis, and in this example... ZmNNR1 and ZmNNR4 The gene's effect on maize photosynthetic efficiency is most likely due to the improved nitrogen use efficiency of the plant.

[0140] Example 5. ZmNNR1 and ZmNNR4 Regulating corn yield

[0141] In the winter of 2024, wild-type and... Zmnnr1 Single mutant, Zmnnr4 Single mutant, Zmnnr1Zmnnr4 Double mutants and ZmNNR1 , ZmNNR4 Overexpression materials were planted in low-nitrogen fields and under normal nitrogen fertilization conditions, and open pollination was performed. Harvesting was conducted 45 days after pollination, and yield data were collected under both low-nitrogen and normal nitrogen fertilization conditions. At least six plants were measured for each material. Analysis revealed that... Zmnnr1 Single mutant, Zmnnr4 Single mutant, Zmnnr1Zmnnr4 The single-ear yield of the double mutant was significantly lower than that of the wild type under both low-nitrogen and normal nitrogen fertilization conditions. The percentage reductions under low-nitrogen conditions were 4.3%–5.0%, 5.2%–5.5%, and 8.6%–9.0%, respectively. Figure 12 Under normal nitrogen fertilizer application conditions, the percentages of lower concentrations were 1.9%–4.0%, 2.2%–3.8%, and 5.8%–5.3%, respectively. Figure 13 ); regardless of whether the nitrogen fertilizer is applied under low nitrogen or normal nitrogen fertilizer conditions, Zmnnr1Zmnnr4 The yield per ear of the double mutant Zmnnr1 Single mutant and Zmnnr4 Single mutants all showed lower yields. Yield performance under normal and low-nitrogen conditions is the most effective indicator of plant nitrogen use efficiency, and the above results indicate that… ZmNNR1 , ZmNNR4 They play an important role in regulating plant nitrogen use efficiency, and there is a certain degree of functional redundancy between them.

[0142] Further studies were conducted on crops planted in low-nitrogen fields and under normal nitrogen fertilization conditions. ZmNNR1 Analysis of single-ear yield data from overexpression materials revealed that, compared to wild-type control materials, ZmNNR1 Overexpression materials showed a significant increase in single-ear yield in low-nitrogen fields, with an increase of 3.4%–3.5%. Figure 12 and Figure 14 Under normal nitrogen fertilizer application conditions, ZmNNR1 The overexpression materials also showed a significant increase in yield compared to the control, with a 1.6%–1.7% increase in yield per ear. Figure 13 and Figure 14 Similarly, for crops planted in low-nitrogen fields and under normal nitrogen fertilization conditions... ZmNNR4 Analysis of single-ear yield data from overexpression materials revealed that, compared to wild-type control materials, ZmNNR4 Overexpression materials significantly increased single-ear yield in low-nitrogen fields, with an increase of 4.1%–4.6%. Figure 12 and Figure 14 Under normal nitrogen fertilizer application conditions, ZmNNR4The overexpression materials also showed a significant increase in yield compared to the control, with a 1.4%–1.6% increase in yield per ear. Figure 13 and Figure 14 Under both normal and low-nitrogen conditions, yields increased compared to the control, indicating that... ZmNNR1 and ZmNNR4 Increased gene expression levels can significantly increase yield. The results also show that under low nitrogen conditions, two genes (…) ZmNNR1 and ZmNNR4 The yield increase (compared to their respective wild-type controls) of overexpression materials was greater than that under normal nitrogen conditions; yield performance under normal and low nitrogen conditions is the most effective indicator of plant nitrogen use efficiency, and the above results indicate that... ZmNNR1 , ZmNNR4 Increased expression levels of these substances can improve nitrogen use efficiency in plants.

[0143] Example 6. ZmNNR1 and ZmNNR4 Regulating nitrogen absorption and utilization efficiency in maize

[0144] To further determine ZmNNR1 and ZmNNR4 Regarding its role in nitrogen use efficiency in maize, we used chlorate to assess the effects on wild-type maize, ZmNNR1 , ZmNNR4 The gene-edited and overexpression materials were treated with chlorate (ClO3). - and nitrate NO3 - The molecular properties of nitrate and chlorate are quite similar, and they have similar absorption and transport pathways in organisms. Therefore, the absorption of chlorate by plants can be used to determine their ability to absorb and utilize nitrate. Normally, chlorate, once absorbed into a plant, is toxic. Therefore, the efficiency of nitrate absorption and utilization can be determined by treating plants with chlorate and assessing the degree of chlorate toxicity after treatment. The greater the chlorate toxicity after treatment, the higher the efficiency of nitrate absorption and utilization; conversely, the less chlorate toxicity, the lower the efficiency.

[0145] We are working on two different editing types Zmnnr1Zmnnr4 The double mutant materials were treated with chlorate. The treatment method involved cultivating maize seedlings on germination paper until they reached the one-leaf-one-heart stage, then treating them with 1 mol / L sodium chlorate for 10 days. The growth inhibition was then observed by photographing the seedlings. Phenotypic analysis after chlorate treatment revealed that, compared to the wild type, the double mutant materials exhibited greater tolerance to chlorate toxicity. Figure 15 Therefore, it can be determined that... Zmnnr1Zmnnr4 The nitrogen uptake and utilization efficiency of the double mutant is reduced.

[0146] Further comparisons with wild-type controls,ZmNNR1 and ZmNNR4 Overexpression materials were subjected to the same chlorate treatment. Phenotypic analysis revealed that, compared with the wild type, after treatment, ZmNNR1 and ZmNNR4 All overexpression materials showed reduced tolerance to chlorate toxicity. Figure 15 Therefore, it can be determined that... ZmNNR1 and ZmNNR4 Increased expression levels can effectively improve nitrogen uptake and utilization efficiency. Combined with previously observed chlorophyll content, maximum photochemical efficiency, and maize yield performance under different nitrogen treatments, it can be determined that... ZmNNR1 and ZmNNR4 It positively regulates the efficiency of nitrogen absorption and utilization in maize.

[0147] Example 7. In natural populations ZmNNR4 The expression level of [a specific substance] is negatively correlated with maize flowering period and positively correlated with nitrogen content.

[0148] We previously collected 137 maize inbred lines from different breeding periods in China, including early (CN1960 & 70s), mid-term (CN1980 & 90s), and current (CN2000 & 10s) periods. Seedlings and ear leaves of these 137 inbred lines were sampled and analyzed using RNA-seq. Data on pollen shedding and silking stages, as well as the phenotype of nitrogen content in ear leaves at flowering stage, were also collected. Correlation analysis revealed that in natural populations… ZmNNR4 Gene expression levels showed a significant negative correlation with the flowering period of maize, while exhibiting a significant positive correlation with the nitrogen content of maize leaves. Figure 16 These results are consistent with previously found... ZmNNR1 and ZmNNR4 The results showed that increased expression levels led to earlier flowering and improved nitrogen uptake and utilization efficiency in maize.

[0149] Further utilization ZmNNR4 Genome-wide association study (GWAS) analysis of gene expression data and genotype and population structure data of 137 maize inbred lines revealed that, ZmNNR4 Significant GWAS (Genome-Wide Association Studies) loci were found near the gene. Figure 17 This suggests that there are controllable natural variations in nature. ZmNNR4 Changes in gene expression levels can, in turn, regulate early flowering and nitrogen use efficiency in maize.

Claims

1. A method for producing corn plants, characterized in that, The corn plants exhibit phenotypes of early flowering, increased yield, and / or increased nitrogen use efficiency, and the method includes the following steps: (a) Enhancing the functional genes in maize plants ZmNNR1 or ZmNNR4 The amount of expression; (b) Obtain at least one seed from the corn plant produced in step (a); The ones mentioned therein ZmNNR1 The gene's polynucleotide sequence is selected from one of the following groups of sequences: (a) A polynucleotide sequence as shown in SEQ ID No: 1, 2, 3 or 4; or (b) The polynucleotide sequence that encodes an amino acid sequence as shown in SEQ ID No: 5, 6 or 7; The ones mentioned therein ZmNNR4 The gene's polynucleotide sequence is selected from one of the following groups of sequences: (a) A polynucleotide sequence as shown in SEQ ID No: 8 or 9; or (b) Its encoded amino acid sequence is the polynucleotide sequence shown in SEQ ID No:

10.

2. The method according to claim 1, wherein the method for enhancing functional genes in maize plants... ZmNNR1 or ZmNNR4 Expression levels include the introduction of functional genes into maize plants. ZmNNR1 or ZmNNR4 Overexpression vectors, either utilizing natural variations or... ZmNNR1 or ZmNNR4 Promoters are used for gene editing, or for... ZmNNR1 or ZmNNR4 Modifications are made to the 5'-UTR or 3'-UTR of the gene, or... ZmNNR1 or ZmNNR4 Add enhancers to the promoter region of a gene to increase ZmNNR1 or ZmNNR4 The expression level or protein content.

3. The method according to claim 1 or 2, wherein the increased nitrogen use efficiency includes enhanced nitrate nitrogen use efficiency, increased plant nitrogen content, increased chlorophyll content, and / or increased maximum photochemical efficiency.

4. The method according to claim 1 or 2, wherein early flowering refers to the early flowering of female flowers or male spikes.

5. The method according to claim 2, wherein the overexpression vector further comprises a promoter operably linked to the nucleotide sequence of the functional gene and increasing the expression level of the functional gene.

6. The method according to claim 5, wherein the promoter is cauliflower mosaic virus CaMV 35S, maize ubiquitin promoter Ubiquitin, or rice Actin1 promoter.

7. A method for producing corn plants, characterized in that, The corn plant exhibits a late-flowering phenotype, and the method includes the following steps: (a) Producing one or more maize plants, wherein the maize plants contain endogenous genes ZmNNR1 and / or ZmNNR4 Contains at least one loss-of-function mutation, or endogenous ZmNNR1 Genes and / or ZmNNR4 Gene expression is suppressed; (b) Obtain at least one seed from the corn plant produced in step (a); The ones mentioned therein ZmNNR1 The gene's polynucleotide sequence is selected from one of the following groups of sequences: (a) A polynucleotide sequence as shown in SEQ ID No: 1, 2, 3 or 4; or (b) The polynucleotide sequence that encodes an amino acid sequence as shown in SEQ ID No: 5, 6 or 7; The ones mentioned therein ZmNNR4 The gene's polynucleotide sequence is selected from one of the following groups of sequences: (a) A polynucleotide sequence as shown in SEQ ID No: 8 or 9; or (b) Its encoded amino acid sequence is the polynucleotide sequence shown in SEQ ID No:

10.

8. The method of claim 7, wherein the loss-of-function mutation includes ZmNNR1 and / or ZmNNR4 The substitution, deletion, and / or addition of one or more nucleotides in the polynucleotide sequence of a gene; wherein the gene expression is suppressed by altering the gene promoter through RNA interference, utilizing natural variation, molecular biology methods, or gene editing.

9. The application of the method according to any one of claims 1-6 in producing maize plants with early flowering, increased nitrogen use efficiency, increased nitrogen content, increased chlorophyll content, increased maximum photochemical efficiency, and / or increased yield.

10. The application of the method according to any one of claims 7-8 in the production of late-flowering maize plants.

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