A gene for regulating starch and protein content in corn kernels and application thereof

Abstract

The present application belongs to the field of biotechnology, and relates to application of EREB167 protein and a coding gene thereof in regulating starch and protein content of corn kernels. More specifically, the present application relates to use of activity or expression regulation of the EREB167 protein in regulating (for example, increasing) starch and protein content of plant kernels.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the use of the activity of EREB167 protein or the regulation of its expression in regulating (e.g., enhancing) the nutrients (e.g., starch and / or protein) of plant seeds. Background Technology

[0002] Gene editing technology is one of the most commonly used biotechnologies in the biological field, with applications spanning areas such as breeding, disease treatment, drug development, and molecular diagnostics. Currently, CRISPR / Cas systems are mainly classified into two categories based on the composition of their effector proteins: Class 2 systems composed of a single effector protein and Class 1 systems composed of multiple effector proteins. Each category is further divided into three types and multiple subtypes based on evolutionary and functional diversity. The well-known Cas9 system belongs to Type II of the Class 2 system. The CRISPR / Cas9 system requires the Cas9 protein and sgRNA composed of tracrRNA and crRNA to function and achieve cleavage of the target site. Due to its simple design and efficient cleavage, the CRISPR / Cas9 system has become the most widely used gene editing system.

[0003] Maize (Zea mays) is one of the world's most important crops, serving as a staple food, animal feed, and industrial raw material. The endosperm accounts for 90% of the entire dry seed, and its main storage substances are starch and protein. Gene editing technology is gradually emerging in maize genetic improvement breeding, allowing for the effective editing of target sequences to obtain genetically modified breeding materials with superior traits. For example, using CRISPR / Cas9 gene editing technology to edit LAC1 resulted in homozygous knockout lines exhibiting a "tight top, loose bottom" intelligent plant type phenotype, which can increase maize yield under high-density planting conditions (Tian et al. 2024); using CRISPR / Cas9 gene editing technology to edit the promoter region of the CLE7 gene created new maize materials with improved ear diameter, ear thickness, number of kernel rows, kernel depth, ear weight, and yield per ear (Liu et al. 2021); using CRISPR / Cas9 gene editing technology to precisely edit the key genes SBEI and SBEIIb in the maize starch synthesis pathway successfully cultivated new maize germplasm containing higher levels of amylose and resistant starch (Doe et al. 2024). Summary of the Invention

[0004] Through extensive research, the inventors of this application have discovered that plant plants with the endogenous EREB167 gene knocked out (e.g., corn plants) have higher nutritional content (e.g., higher starch content, higher protein content) in their grains compared to plants without the EREB167 gene knocked out, thus possessing better breeding value.

[0005] Therefore, in one aspect, this application provides the use of reagents for inhibiting EREB167 protein activity or expression in regulating (e.g., enhancing) the nutrients in plant seeds, wherein the reagents for inhibiting EREB167 protein activity or expression are reagents that inhibit or inactivate EREB167 protein activity and / or downregulate or prevent EREB167 protein expression.

[0006] In some implementations, the nutrient is carbohydrates.

[0007] In some implementations, the nutrient is protein.

[0008] In some implementations, the nutrient is fat.

[0009] In some implementations, the nutrient is a vitamin.

[0010] In some implementations, the nutrient is a mineral.

[0011] In some implementations, the nutrient is dietary fiber.

[0012] In some implementations, the nutrient is carotene.

[0013] In some embodiments, the carbohydrate is starch.

[0014] In some embodiments, the fat is selected from unsaturated fatty acids, linoleic acid, or any combination thereof.

[0015] In some embodiments, the vitamin is selected from vitamin B1, vitamin E, vitamin B2, vitamin B6, niacin, folic acid, vitamin K, or any combination thereof.

[0016] In some embodiments, the mineral is selected from potassium, magnesium, calcium, zinc, copper, manganese, iron, or any combination thereof.

[0017] In some embodiments, the plant is a monocotyledonous plant or a dicotyledonous plant.

[0018] In some embodiments, the plant is a grass (Poaceae). For example, corn, wheat, rice, sorghum, and oats.

[0019] In some implementations, the plant is corn.

[0020] In some embodiments, the reagent used to inhibit EREB167 protein activity or expression contains an EREB167 protein activity inhibitor.

[0021] In some embodiments, the reagent for inhibiting EREB167 protein activity or expression comprises a reagent for knocking down the EREB167 gene or a fragment thereof.

[0022] In some embodiments, the reagent for inhibiting EREB167 protein activity or expression comprises a reagent for knocking out the EREB167 gene or a fragment thereof.

[0023] In some embodiments, the EREB167 gene has a sequence as shown in SEQ ID NO:1.

[0024] In some embodiments, the EREB167 protein has the sequence shown in SEQ ID NO:2.

[0025] In some embodiments, the fragment of the EREB167 gene has a sequence as shown in SEQ ID NO:6.

[0026] In some embodiments, the reagent for knocking down the EREB167 gene comprises: (i) an sgRNA targeting EREB167 mRNA, or (ii) a nucleic acid molecule encoding (i), or (iii) a vector comprising (i) or (ii). In some embodiments, the reagent for knocking down the EREB167 gene comprises a vector containing a nucleotide sequence encoding an sgRNA targeting EREB167 mRNA, and the vector contains a sequence homologous to the plant genome sequence in the nucleotide sequence encoding the sgRNA.

[0027] In some embodiments, the reagent for knocking out the EREB167 gene comprises a gene editing tool or its active element that targets and knocks out the EREB167 gene or a fragment thereof; for example, the reagent for knocking out the EREB167 gene comprises a CRISPR / Cas gene editing system (e.g., CRISPR / Cas9, CRISPR / Cas12a), a zinc finger protein-nuclease (ZFN) gene editing system, and / or a transcription activator effector nuclease (TALEN) gene editing system that targets the EREB167 gene, or the system comprises an element that targets and recognizes and / or binds to the EREB167 gene (e.g., sgRNA or crRNA targeting the EREB167 gene, zinc finger protein or zinc finger protein group targeting the EREB167 gene, TALE protein or TALE protein group targeting the EREB167 gene).

[0028] In some embodiments, the reagent for knocking out the EREB167 gene comprises: (i) a guide nucleic acid molecule (e.g., sgRNA or crRNA) that targets and recognizes and / or binds to the EREB167 gene or a fragment thereof; or (ii) a nucleic acid molecule encoding the guide nucleic acid molecule; or (iii) a vector containing the guide nucleic acid molecule or a nucleic acid molecule encoding the guide nucleic acid molecule or a guide protein.

[0029] In some embodiments, the reagent for knocking out the EREB167 gene comprises a first nucleic acid molecule encoding an sgRNA that targets and recognizes and / or binds to the EREB167 gene or a fragment thereof, and a second nucleic acid molecule encoding a cas effector protein corresponding to the sgRNA (e.g., cas9 corresponding to the sgRNA); and the first nucleic acid molecule and the second nucleic acid molecule are located in the same vector.

[0030] In some embodiments, the sgRNA has a sequence as shown in SEQ ID NO:9.

[0031] On the other hand, this application provides a kit containing reagents for knocking down or eliminating the EREB167 gene.

[0032] In some embodiments, the EREB167 gene has a sequence as shown in SEQ ID NO:1.

[0033] In some embodiments, the reagent for knocking down the EREB167 gene comprises: (i) an sgRNA targeting EREB167 mRNA, or (ii) a nucleic acid molecule encoding (i), or (iii) a vector comprising (i) or (ii). In some embodiments, the reagent for knocking down the EREB167 gene comprises a vector containing a nucleotide sequence encoding an sgRNA targeting EREB167 mRNA, and the vector contains a sequence homologous to the plant genome sequence flanking the nucleotide sequence encoding the sgRNA.

[0034] In some embodiments, the reagent for knocking out the EREB167 gene comprises a gene editing tool or its active element that targets and knocks out the EREB167 gene or a fragment thereof; for example, the reagent for knocking out the EREB167 gene comprises a CRISPR / Cas gene editing system (e.g., CRISPR / Cas9, CRISPR / Cas12a), a zinc finger protein-nuclease (ZFN) gene editing system, and / or a transcription activator effector nuclease (TALEN) gene editing system that targets the EREB167 gene, or the system comprises an element that targets and recognizes and / or binds to the EREB167 gene (e.g., sgRNA or crRNA targeting the EREB167 gene, zinc finger protein or zinc finger protein group targeting the EREB167 gene, TALE protein or TALE protein group targeting the EREB167 gene).

[0035] In some embodiments, the reagent for knocking out the EREB167 gene comprises: (i) a guide nucleic acid molecule (e.g., sgRNA or crRNA) that targets and recognizes and / or binds to the EREB167 gene or a fragment thereof; or (ii) a nucleic acid molecule encoding the guide nucleic acid molecule; or (iii) a vector containing the guide nucleic acid molecule or a nucleic acid molecule encoding the guide nucleic acid molecule or a guide protein.

[0036] In some embodiments, the reagent for knocking out the EREB167 gene comprises a first nucleic acid molecule encoding an sgRNA that targets and recognizes and / or binds to the EREB167 gene or a fragment thereof, and a second nucleic acid molecule encoding a cas effector protein corresponding to the sgRNA (e.g., cas9 corresponding to the sgRNA); and the first nucleic acid molecule and the second nucleic acid molecule are located in the same vector.

[0037] In some embodiments, the sgRNA has a sequence as shown in SEQ ID NO:9.

[0038] On the other hand, this application provides a plant cell in which the endogenous EREB167 gene is knocked down or eliminated.

[0039] In some embodiments, the plant cells contain reagents for knocking down or eliminating the EREB167 gene. In some embodiments, the reagents are as defined above.

[0040] In some embodiments, the endogenous EREB167 gene in the plant cells is knocked down.

[0041] In some embodiments, the plant cells contain sgRNA that targets EREB167 mRNA, or the coding sequence of the sgRNA.

[0042] In some embodiments, the genome of the plant cell contains a DNA sequence encoding an sgRNA that targets EREB167 mRNA.

[0043] In some embodiments, the endogenous EREB167 gene in the plant cells is knocked out.

[0044] In some embodiments, the plant cells contain one or more endogenous EREB167 genes, and all of the one or more endogenous EREB167 genes are knocked out.

[0045] In some embodiments, the plant cells are derived from monocotyledonous or dicotyledonous plants.

[0046] In some embodiments, the plant cells are derived from gramineous plants.

[0047] In some embodiments, the plant cells are derived from corn.

[0048] In some implementations, the plant cells have the ability to develop into a complete plant.

[0049] In some implementations, the plant cells do not have the ability to develop into a complete plant.

[0050] On the other hand, this application provides a method for preparing plant cells as described above, comprising:

[0051] (1) Provide plant cells to be treated;

[0052] (2) Introduce the reagent used to knock down or eliminate the EREB167 gene into the plant cells to be treated;

[0053] This allows us to obtain cells in which the endogenous EREB167 gene is knocked down or eliminated.

[0054] In some embodiments, the reagents used to knock down or eliminate the EREB167 gene are as defined above.

[0055] In some embodiments, the plant cells to be treated are cells derived from monocotyledonous or dicotyledonous plants. In some embodiments, the plant cells to be treated are cells derived from grasses (Poaceae). In some embodiments, the plant cells to be treated are cells derived from maize.

[0056] In some embodiments, the method involves introducing the reagent for knocking down or eliminating the EREB167 gene into the cells to be treated via Agrobacterium-mediated transformation. In some embodiments, the cells to be treated are plant callus cells.

[0057] Without being limited by theory, the method of introducing the reagent for knocking down or knocking out the EREB167 gene into the cells to be treated can be any conventional method in the art, such as using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, Agrobacterium-mediated transformation and other conventional biological methods.

[0058] In some embodiments, the method involves introducing the reagent for knocking down or eliminating the EREB167 gene into the cells to be treated via Agrobacterium-mediated transformation. In some embodiments, the cells to be treated are plant callus cells.

[0059] On the other hand, this application provides a plant tissue containing, or composed of, the cells described above.

[0060] In some embodiments, the plant tissue is plant callus (e.g., embryogenic callus).

[0061] In some implementations, the plant tissue has the ability to develop into a complete plant.

[0062] In some implementations, the plant tissue does not have the ability to develop into a complete plant.

[0063] On the other hand, this application provides a plant in which the endogenous EREB167 gene is knocked down or eliminated.

[0064] In some embodiments, the plant is an endogenous EREB167 gene knockout (or knockdown) homozygous mutant plant or a heterozygous mutant plant.

[0065] In some embodiments, the endogenous EREB167 gene knockout homozygous mutant plant refers to a plant in which all EREB167 alleles have been knocked out. In some embodiments, the endogenous EREB167 gene knockdown homozygous mutant plant refers to a plant in which all EREB167 alleles have been knocked down.

[0066] Those skilled in the art will readily understand that the knockout or knockdown homozygous mutant plant does not mean that all the EREB167 alleles contained in the plant maintain the same nucleotide sequence; it only requires that all the EREB167 alleles contained therein lose their corresponding function or have their function suppressed. For example, the EREB167 alleles of the knockout homozygous mutant plant are all knocked out, and the endogenous EREB167 alleles contained therein contain the same or different mutations that lead to the loss of EREB167 gene function (e.g., insertion, deletion, substitution, rearrangement, etc.).

[0067] On the other hand, this application provides a method for preparing the plant as described above, comprising:

[0068] (1) Provide plant cells (e.g., protoplasts) or plant callus (e.g., embryogenic callus) to be treated;

[0069] (2) Introduce the reagent used to knock down or eliminate the EREB167 gene into the plant cells to be treated or into the cells of the plant callus.

[0070] (3) Culture plant cells or plant callus that have been treated in step (2) to obtain plant plants in which the endogenous EREB167 gene is knocked down or removed.

[0071] In some embodiments, the reagents used to knock down or eliminate the EREB167 gene are as defined above.

[0072] In some embodiments, the method has one or more features selected from the following:

[0073] (a) After step (2) and before step (3), the method further includes screening for positively transformed plant cells or plant callus;

[0074] (b) After step (3), the method further includes screening for homozygous mutants with EREB167 gene knockdown or knockout.

[0075] In some embodiments, the plant cells or plant callus are derived from monocotyledonous or dicotyledonous plants.

[0076] In some embodiments, the plant cells or plant callus are derived from grasses.

[0077] In some embodiments, the plant cells or plant callus are derived from maize.

[0078] On the other hand, this application provides a method for obtaining a plant, the method comprising culturing plant cells as described above into a plant.

[0079] On the other hand, this application provides a transgenic plant, a portion thereof, or its seeds obtained by the method described above.

[0080] On the other hand, this application provides an article of manufacture made from the plant or its parts or its seeds as described above.

[0081] In some embodiments, the article is a food product and is made from the edible parts of the plant as described above.

[0082] Terminology Definition

[0083] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virological, biochemical, and immunological laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0084] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0085] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0086] As used herein, the term "EREB167 protein" includes a polypeptide product encoded by the gene numbered "Zm00001d032095" in the maize genome (LH244), as well as orthologs, homologs, and variants of said polypeptide (e.g., orthologs, homologs, and variants of said polypeptide derived from other maize materials other than LH244 or other plants other than maize). In some embodiments, said EREB167 protein includes a polypeptide product encoded by the gene shown on chromosome 1 of the maize genome (LH244) at 212,364,330-212,367,222 bp (5'-3') (e.g., a gene having the sequence shown in SEQ ID NO:2), as well as orthologs, homologs, and variants of said polypeptide (e.g., orthologs, homologs, and variants of said polypeptide derived from other maize materials other than LH244 or other plants other than maize).

[0087] In this paper, EREB167 protein belongs to the transcriptional repressor class, which are proteins that can inhibit gene transcription. They prevent gene transcription through multiple mechanisms. EREB167 protein can bind directly to DNA, thereby preventing transcription from occurring.

[0088] Those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions, such as EREB167 proteins from different species) can be naturally generated or artificially introduced into the amino acid sequence of the EREB167 protein without affecting its biological function. Therefore, in this invention, the term "EREB167 protein" should include all EREB167 proteins corresponding to such sequences, including, for example, the EREB167 protein as shown in SEQ ID NO: 2 and its natural or artificial variants. In some embodiments, the EREB167 protein has: (a) the amino acid sequence as shown in SEQ ID NO: 2; (b) an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 2; or (c) a sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conserved substitutions), additions, or deletions compared to the amino acid sequence shown in SEQ ID NO: 2.

[0089] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit is occupied (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0090] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0091] As used herein, the term "EREB167 gene" refers to the DNA segment necessary to produce the EREB167 protein. For example, a gene having the sequence shown in SEQ ID NO:1, or its orthologs, homologs, and variants (e.g., DNA segments encoding the EREB167 protein in the genomes of other species). For example, a gene having the sequence shown in SEQ ID NO:3, or its orthologs, homologs, and variants (e.g., DNA segments encoding the EREB167 protein in the genomes of other species).

[0092] As used herein, the term "cereal crops" has the meaning commonly understood by those skilled in the art, and generally includes all grasses cultivated for grain purposes, including but not limited to rice, wheat, barley, maize, sorghum, millet, etc.

[0093] As used herein, the term “gene knockdown” has the meaning commonly understood by those skilled in the art, and generally refers to the reduction of the expression level of a target gene in a cell or organism by means of methods such as RNA interference (RNAi), thereby inhibiting its function.

[0094] As used herein, the term “gene knockout” has the meaning commonly understood by those skilled in the art, and typically refers to the complete deletion or inactivation of the DNA sequence of a target gene from a cell or organism through means such as genome editing technology, thereby blocking its transcription and translation processes and rendering the target gene completely nonfunctional.

[0095] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: binary Agrobacterium vectors and vectors that can be used for plant microbombardment, such as pCAMBIA-1300-221, pGreen0029, pCAMBIA3301, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN, or other derived expression vectors; plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage, and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site.

[0096] As used herein, when the terms “increased” or “enhanced” are used to describe nutrients, they mean an increase of at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, or more compared to a control. It is known in the art that an increase of 5% in the content of nutrients (e.g., starch, protein) in plant (e.g., maize) kernels is considered a significant breakthrough. Therefore, when the terms “increased” or “enhanced” are used to describe the content of nutrients (e.g., starch, protein) in plant kernels, they mean an increase of at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% compared to the nutrient content in control plant kernels.

[0097] As used herein, when describing a polypeptide or protein, the term “fragment” or “part” may refer to a polypeptide or protein that is shorter than a reference polypeptide or protein and that contains, is substantially composed of, and / or is composed of the same amino acid sequence of consecutive amino acids as, is substantially composed of, and is identical to the corresponding part of the reference polypeptide or protein (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).

[0098] As used herein, the term "parts of a plant" includes, but is not limited to, reproductive tissues (e.g., petals, sepals, stamens, pistils, receptacle, anthers, pollen, flowers, fruits, buds, ovules, seeds, and embryos); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stems, buds, branches, bark, apical meristems, axillary buds, cotyledons, hypocotyls, and leaves); and vascular tissues (e.g., phloem and xylem).

[0099] As used herein, the term "plant cell" refers to a structural and physiological unit of a plant, which typically includes a cell wall but also includes a protoplast. The plant cells of the present invention may be in the form of isolated single cells, or may be cultured cells, or may be part of a higher tissue unit, such as plant tissue (including callus) or plant organ.

[0100] Beneficial effects of the invention

[0101] The endogenous EREB167 gene knockout plant plants (e.g., maize plants) provided in this application have kernels with higher nutritional content (e.g., higher starch content, higher protein content) compared to plants with the EREB167 gene not knocked out. Therefore, endogenous EREB167 gene knockout plant plants (e.g., maize plants) have significant potential for breeding yield-increasing plant varieties.

[0102] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0103] Figure 1 Analysis of EREB167 expression in maize.

[0104] Figure 2 Subcellular localization results for EREB167.

[0105] Figure 3 This study analyzed the transcriptional activity of EREB167.

[0106] Figure 4 The results show the sequence alignment of wild-type and CRISPR / Cas9 gene knockout mutants. The red bases are the inserted bases.

[0107] Figure 5 Phenotypic identification of maize kernels in the T3 generation of ereb167-C1 and ereb167-C2 mutants.

[0108] Figure 6 The size of starch grains in the seeds of ereb167-C1 and ereb167-C2 mutants was observed.

[0109] Figure 7 The determination and analysis of 100-grain weight, starch content and protein content of ereb167-C1 and ereb167-C2 mutant seeds.

[0110] Figure 8 RNA-seq data analysis of wild-type and ereb167-C1 mutant endosperm 8 days after pollination.

[0111] Sequence information

[0112] The descriptions of the sequences involved in this application are provided in the table below.

[0113] Table 1: Sequence Information

[0114]

[0115]

[0116] Detailed Implementation

[0117] The present invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it). Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products. Those skilled in the art will understand that the embodiments are described by way of example and are not intended to limit the scope of protection claimed by the present invention.

[0118] Example 1. Analysis of the EREB167 gene and its expression in maize.

[0119] The coding sequence (CDS) of the EREB167 gene in maize variety LH244 (details described in the literature "Zhuoyang Li, Diyi Fu, Xi Wang, Rong Zeng, Xuan Zhang, Jinge Tian, ​​Shuisong Zhang, Xiaohong Yang, Feng Tian, ​​Jinsheng Lai, Yiting Shi, Shuhua Yang, The transcription factor bZIP68 negatively regulates cold tolerance in maize, The Plant Cell, Volume 34, Issue 8, August 2022, Pages 2833-2851") is shown in SEQ ID NO:1, the encoded amino acid sequence is shown in SEQ ID NO:2, and the genomic sequence encoding the EREB167 protein is shown in SEQ ID NO:3. First, transcriptome data from different maize tissues were collected (Chen et al. 2014). Analysis of gene expression in different tissues revealed that EREB167 is an endosperm-specific gene. Figure 1 (A) By collecting materials from different maize tissues and performing RNA extraction, reverse transcription, and RT-qPCR experiments (RT-qPCR primers are as follows: qEREB167-F: SEQ ID NO:4; qEREB167-R: SEQ ID NO:5), it was further confirmed that EREB167 is an endosperm-specific expression gene. Figure 1 (B in the middle).

[0120] EREB167 subcellular localization analysis

[0121] EREB167 is an AP2 / ERF family transcription factor. To further confirm its transcription factor function, its subcellular localization was first analyzed. A vector expressing GFP and EREB167 via a 35S promoter fusion was constructed and transformed into maize protoplasts for localization observation. AHL-22RFP was used as a control protein for nuclear localization. The results showed that EREB167 was localized in the nucleus of maize protoplasts. Figure 2 (A in the text). Simultaneously, a vector fused with the 35S promoter expressing GFP and EREB167 was transformed into Agrobacterium GV3101 and injected into tobacco leaves. The expression site of EREB167 in the tobacco leaves was observed using a fluorescence microscope. The results showed that EREB167 was specifically expressed in the cell nucleus. Figure 2 (B in the middle).

[0122] EREB167 transcriptional activity analysis

[0123] To determine whether EREB167 possesses transcriptional activation activity, we first conducted an experiment in a yeast system. The full-length coding sequence of EREB167 (SEQ ID NO:1) was cloned into the expression vector pBD-GAL4 and fused with the GAL4 DNA-binding domain. pBD-EREB167, along with positive (pGAL4) and negative control plasmids (pBD-GAL4), were transformed into yeast strain YRG-2. Compared to the positive control, transfected yeast cells containing pBD-EREB167 and the negative control grew vigorously on SD medium lacking Trp, but failed to grow on SD medium without Trp and His, indicating that EREB167 lacks transcriptional activation activity. Figure 3 (A in the original text). Subsequently, we evaluated the transcriptional repressive activity of EREB167 in maize protoplasts using the GUS / LUC system. The results showed that the GUS / LUC value was significantly reduced when co-expressed with EREB167 ( ). Figure 3 (B in the text). These results indicate that EREB167 has the function of a transcriptional repressor.

[0124] Example 2. Construction of CRISPR / Cas9 gene editing vector and genetic transformation of maize

[0125] Based on the sequence in Example 1, and utilizing the characteristic of CRISPR / Cas9 to recognize the 5'-NGG PAM site, the target sequence selected in the first exon of EREB167 is (SEQ ID NO: 6). An expression vector fusing Cas9 and sgRNA (SEQ ID NO: 9) was constructed, following the method described in (Xing et al. 2014). The constructed recombinant vector was transformed into Agrobacterium EHA105, and the Agrobacterium was then transformed into wild-type maize LH244 to obtain T0 generation transgenic maize carrying the EREB167 gene.

[0126] Example 3. Identification of EREB167 gene knockout materials

[0127] Based on the location of the target site, primer sequences were designed approximately 260 bp upstream and 430 bp downstream. The primer sequences are as follows:

[0128] EREB167-jc-F: SEQ ID NO:7; EREB167-jc-R: SEQ ID NO:8.

[0129] DNA was extracted from maize leaves of the T0 generation transgenic EREB167 gene using the CTAB method. This DNA was then used as a template for PCR amplification using primers EREB167-jc-F and EREB167-jc-R, yielding a 680 bp PCR product. First-generation sequencing analysis of the PCR product was performed to obtain the base sequence of the transgenic plant.

[0130] Compared to the wild-type sequence, the transgenic plant ereb167-C1 has an inserted nucleotide T between positions 99 and 100, and the transgenic plant ereb167-C2 has an inserted nucleotide A between positions 101 and 102. Figure 4 A and Figure 4 (B in the middle).

[0131] Example 4. Identification of the kernel phenotype of EREB167 gene knockout material maize

[0132] To further confirm the role of endosperm-specific expression of the transcription factor EREB167 in maize kernel development, we conducted phenotypic observations on T3 generation seeds of the EREB167 knockout mutants ereb167-C1 and ereb167-C2. The results showed that loss of function of the EREB167 gene resulted in larger maize kernels, with both kernel length and width exceeding those of the wild type. Figure 5 A and Figure 5 (B in the middle).

[0133] Example 5. Observation of starch grain size in EREB167 gene knockout materials

[0134] We observed whether there were differences in the morphology of starch grains and proteosomes in endosperm-specific expression loss-of-function mutant materials of transcription factor EREB167. Mature wild-type and mutant seeds were examined by scanning electron microscopy. The results showed that the starch grains of the ereb167-C1 and ereb167-C2 mutants were significantly larger than those of the wild type. Figure 6 (A) Seeds of wild-type and mutant strains 14 days after pollination were observed by transmission electron microscopy. The results showed that the starch granules of the ereb167-C1 and ereb167-C2 mutants were significantly larger (A). Figure 6 (B in the middle).

[0135] Example 6. Determination and analysis of 100-grain weight, starch content, and protein content of EREB167 gene knockout material

[0136] To further confirm the effect of EREB167 on maize kernel development, we measured the 100-kernel weight of mature kernels from the ereb167-C1 and ereb167-C2 mutants. The results showed that the 100-kernel weight of the EREB167 loss-of-function mutant was significantly increased. Figure 7 (A) Starch content was determined in mature grains of the ereb167-C1 and ereb167-C2 mutants. The results showed that the starch content of the EREB167 loss-of-function mutant was significantly increased. Figure 7 (B in the text). Protein content was determined in mature seeds of ereb167-C1 and ereb167-C2 mutants. The results showed that the protein content of the EREB167 loss-of-function mutant was significantly increased. Figure 7 (C in the middle).

[0137] Example 7. Transcriptomic analysis of ereb167-C1 mutant material

[0138] Transcription factors are primarily involved in the regulation of gene expression. To further identify the regulatory role of EREB167 in gene expression during endosperm development, we collected endosperm materials from wild-type and ereb167-C1 mutants 8 days after pollination for RNA extraction, library construction, and next-generation sequencing analysis. The results showed that compared to the wild-type, the ereb167-C1 mutant upregulated 1521 genes and downregulated 317 genes, indirectly indicating that EREB167 mainly plays a negative regulatory role in gene expression during endosperm development. Figure 8 (A) Among the upregulated genes are those involved in the transmission of cell development-related genes MRP-1 and MN1, and nutrient transport-related genes NRT1.1, NRT1.2, SUGCAR1, SUT2, SUT4, and HAK5, etc. Figure 8(B in the text). To further confirm the increased expression levels of these nutrient transport-related genes in the mutants, we collected endosperm materials from wild-type and ereb167-C1 mutants 8 days after pollination for RNA extraction, reverse transcription, and RT-qPCR validation. The results showed that 9 out of the 10 selected genes were expressed at increased levels in the ereb167-C1 mutant, further confirming the reliability of the RNA-seq data. Figure 8 (C in the middle).

[0139] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. The use of reagents for inhibiting EREB167 protein activity or its expression in improving the nutritional content of maize kernels, wherein, The reagent that inhibits the activity or expression of EREB167 protein is a reagent that inactivates the activity of EREB167 protein and / or a reagent that prevents the expression of EREB167 protein. The nutrients are starch and / or protein; the sequence of the EREB167 protein is shown in SEQ ID NO:

2.

2. The use as described in claim 1, wherein, The reagent used to inhibit the activity or expression of EREB167 protein is a reagent used to knock out the EREB167 gene or a fragment thereof.

3. The use as described in claim 2, wherein, The sequence of the EREB167 gene is shown in SEQ ID NO:

1.

4. The use as described in claim 2, wherein, The sequence of the EREB167 gene fragment is shown in SEQ ID NO:

6.

5. The use as described in claim 2, wherein, The reagents used to knock out the EREB167 gene comprise: (i) an sgRNA that targets EREB167 mRNA, or (ii) a nucleic acid molecule encoding (i), or (iii) a vector comprising (i) or (ii).

6. The use as described in claim 2, wherein, The reagent used to knock out the EREB167 gene contains a gene editing tool or its active element that targets and knocks out the EREB167 gene or a fragment thereof.

7. The use as described in claim 2, wherein, The reagent for knocking out the EREB167 gene comprises: (i) a guide nucleic acid molecule that targets and / or binds to the EREB167 gene or a fragment thereof, or (ii) a nucleic acid molecule encoding the guide nucleic acid molecule, or (iii) a vector containing the guide nucleic acid molecule or a nucleic acid molecule encoding the guide nucleic acid molecule or a guide protein.

8. The use as described in claim 6, wherein, The reagent used to knock out the EREB167 gene contains a CRISPR / Cas gene editing system that targets the EREB167 gene.

9. The use as described in claim 6, wherein, The reagent for knocking out the EREB167 gene comprises a first nucleic acid molecule encoding an sgRNA that targets and recognizes and / or binds to the EREB167 gene or a fragment thereof, and a second nucleic acid molecule encoding a cas effector protein corresponding to the sgRNA; and the first nucleic acid molecule and the second nucleic acid molecule are located in the same vector.

10. The use as described in claim 7, wherein, The guide nucleic acid molecule is sgRNA or crRNA.

11. The use as described in claim 7, wherein, The guide nucleic acid molecule is sgRNA, and its sequence is shown in SEQ ID NO: 9.

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