Method for increasing row number of corncobs

Abstract

The invention provides a method for increasing the row number of corn ears, increasing the width of the corn ears, increasing the weight of corn kernels or increasing the yield of corn. The method comprises the step of mutating CLE genes in the corn. After mutation of the CLE gene, the row number, the ear width, the grain weight and the yield of corn can be increased, and the CLE gene has an important application value in corn breeding.

Description

[0001] This application claims the priority of Chinese Patent Application CN202410364411.2 with an application date of March 28, 2024. This application incorporates the entire text of the above-mentioned Chinese patent application by reference. Technical Field

[0002] The present invention belongs to the fields of biotechnology and crop genetics and breeding, and relates to a method for increasing the number of kernel rows of maize, increasing the width of maize ears, increasing the weight of maize kernels, and increasing maize yield, especially a method for increasing the number of kernel rows of maize, increasing the width of maize ears, increasing the weight of maize kernels, and increasing maize yield by mutating the CLE gene. Background Art

[0003] Maize is the crop with the largest planting area worldwide, with an annual global planting area of over 2 billion mu and a total output of up to 1 billion tons. Its edible value, industrial value, and forage value have made it occupy an important position in China's food security guarantee system.

[0004] Ensuring and increasing maize yield is of great significance for national food security. Theoretically, increasing the number of kernel rows of maize (when the number of kernels per row does not decrease) is beneficial to increasing maize yield. In order to increase the number of kernel rows and yield of maize, we studied the CLE gene family in maize, hoping to obtain excellent maize germplasm resources. Summary of the Invention

[0005] The present invention provides a method for increasing the number of kernel rows of maize, increasing the width of maize ears, increasing the weight of maize kernels, or increasing maize yield.

[0006] On the one hand, the present invention provides a method for increasing the number of kernel rows of maize, increasing the width of maize ears, increasing the weight of maize kernels, or increasing maize yield, the method comprising the step of mutating the CLE gene in maize.

[0007] In one embodiment, the parental CLE gene is derived from maize.

[0008] In one embodiment, the CLE gene is selected from one or any combination of CLE18, CLE5, CLE26, CLE32, and CLE4A; preferably, the CLE18 gene.

[0009] In another preferred example, the amino acid sequence of the CLE18 gene is selected from the following group:

[0010] (i) a polypeptide having the amino acid sequence shown in SEQ ID NO.: 1;

[0011] (ii) A polypeptide derived from (i), which is formed by substitution, deletion or addition of one or several (such as 1-10) amino acid residues in the amino acid sequence shown in SEQ ID NO.: 1 and has the same or similar function.

[0012] Or (iii) a polypeptide having a homology of 50% or more (preferably more than 60%, more preferably more than 70%, more preferably more than 80%, still more preferably more than 90%, still more preferably more than 95%, most preferably more than 98%, such as 99% or 100%) with the amino acid sequence shown in SEQ ID NO.: 1 and having the same or similar function.

[0013] In another preferred embodiment, the nucleotide sequence of the CLE18 gene is selected from the following groups:

[0014] (a) A polynucleotide encoding the polypeptide shown in SEQ ID NO.: 1;

[0015] (b) A polynucleotide having the sequence shown in SEQ ID NO.: 2;

[0016] (c) A polynucleotide having a homology of ≥95% (preferably ≥98%, more preferably ≥99%) with the sequence shown in SEQ ID NO.: 2;

[0017] (d) A polynucleotide truncated or added with 1-60 (preferably 1-

[0018] 30, more preferably 1-10) nucleotides at the 5' end and / or 3' end of the polynucleotide shown in SEQ ID NO.: 2;

[0019] (e) A polynucleotide complementary to any one of the polynucleotides described in (a)-(d).

[0020] In one embodiment, the polynucleotide is selected from the following groups: genomic sequence, cDNA sequence, CDS sequence, RNA sequence, or a combination thereof.

[0021] In one embodiment, the parental CLE18 gene is derived from a naturally occurring maize line, and the nucleotide sequence of the parental CLE18 gene has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the sequence shown in SEQ ID No.2.

[0022] In one embodiment, the amino acid sequence of the parental CLE18 gene has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the sequence shown in SEQ ID No.1.

[0023] In another preferred example, the amino acid sequence of the CLE18 gene has at least 80% sequence identity compared to SEQ ID NO.:1.

[0024] In another preferred example, the nucleotide sequence of the CLE18 gene has at least 80% sequence identity compared to SEQ ID NO.:2.

[0025] In one embodiment, the nucleotide sequence of the parental CLE4A gene has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the sequence shown in SEQ ID No.4.

[0026] In one embodiment, the amino acid sequence of the parental CLE4A gene has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the sequence shown in SEQ ID No.3.

[0027] In one embodiment, the nucleotide sequence of the parental CLE5 gene has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the sequence shown in SEQ ID No.6.

[0028] In one embodiment, the amino acid sequence of the parental CLE5 gene has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the sequence shown in SEQ ID No.5.

[0029] In one embodiment, the nucleotide sequence of the parental CLE26 gene has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the sequence shown in SEQ ID No.8.

[0030] In one embodiment, the amino acid sequence of the parental CLE26 gene has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the sequence shown in SEQ ID No.7.

[0031] In one embodiment, the nucleotide sequence of the parental CLE32 gene has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID No. 10.

[0032] In one embodiment, the amino acid sequence of the parental CLE32 gene has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence shown in SEQ ID No. 9.

[0033] In one embodiment, the parental CLE18 gene is derived from a maize inbred line.

[0034] In one embodiment, the nucleotide sequence of the parental CLE18 gene is as shown in SEQ ID No. 2.

[0035] In one embodiment, the amino acid sequence of the parental CLE18 gene is as shown in SEQ ID No. 1.

[0036] In one embodiment, the NCBI accession number of the parental CLE18 gene is LOC111589273.

[0037] In another preferred example, the mutation is achieved by a method selected from the group consisting of: gene editing technology, gene mutation, gene knockout, gene disruption, RNA interference technology, or a combination thereof.

[0038] In another preferred example, the gene mutation is obtained by one or more of the following methods: natural variation, physical mutagenesis (such as ultraviolet mutagenesis, X-ray or Y-ray mutagenesis), chemical mutagenesis (such as nitrous acid, hydroxylamine, EMS, nitrosoguanidine, etc.), biological mutagenesis (such as virus- or bacteria-mediated mutagenesis), gene editing, or biosynthesis.

[0039] In another preferred example, the gene editing technology is selected from the group consisting of: CRISPR technology, TALEN technology, ZFN technology, or a combination thereof.

[0040] In another preferred example, the method comprises the steps of:

[0041] (i) providing a maize or maize cell; and

[0042] (ii) introducing into the maize or maize cell a gRNA targeting the CLE gene and the corresponding Cas protein; in a preferred embodiment, introducing into the maize or maize cell an expression vector containing the gRNA and the Cas protein.

[0043] In another preferred example, the mutations include insertion mutations, deletion mutations, frameshift mutations, and substitution mutations.

[0044] In a preferred embodiment, the method includes reducing or inhibiting the expression level and / or activity of the CLE gene.

[0045] In another preferred example, the reduction or inhibition means that, compared with the expression level E0 of the CLE gene in wild-type plants, the expression level E1 of the CLE gene in the plants is 0 - 80% of that of the wild type, preferably 0 - 60%, more preferably 0 - 40%, and even more preferably 0 - 30%.

[0046] In another preferred example, the reduction or inhibition of the expression and / or activity of the CLE gene is achieved by a method selected from the following group: gene mutation, gene knockout, gene disruption, RNA interference technology, gene editing technology, introduction of an inhibitor of a gene or protein, or a combination thereof.

[0047] In another preferred example, the reduction or inhibition of the expression level and / or activity of the CLE gene is achieved by mutating the CLE gene.

[0048] In another preferred example, the mutation results in a complete loss or partial loss of the function of the CLE gene.

[0049] In another preferred example, the mutation is that a part of the bases of the nucleotide sequence of the CLE18 gene is deleted relative to the sequence shown in SEQ ID NO.2.

[0050] In one embodiment, the nucleotide sequence of the mutated CLE18 gene is deleted corresponding to the 428 - 429th bases of the sequence shown in SEQ ID No.2 relative to the nucleotide sequence of the parental CLE18 gene.

[0051] In another preferred example, the maize is inbred maize.

[0052] In another preferred example, the increase in maize yield refers to an increase in plant height, ear height, leaf length, leaf width, number of kernel rows per ear, number of kernels per row, ear length, ear width, 100 - kernel weight, number of kernels per ear, number of kernels per mu, unit yield, or yield per mu of maize.

[0053] In one embodiment, the increase in the number of kernel rows per ear means that the maize containing the mutated CLE gene has at least a 3% increase, preferably a 5% increase, preferably an 8% increase, preferably a 10% increase, preferably a 15% increase, preferably a 20% increase, preferably a 30% increase, preferably a 40% increase, preferably a 50% increase, preferably a 60% increase, preferably an 80% increase, preferably a 100% increase in the number of kernel rows per ear compared to the parental maize (containing the wild-type CLE gene).

[0054] In one embodiment, the increase in ear width means that the ear width of maize containing the mutant CLE gene is at least 3%, preferably 5%, preferably 8%, preferably 10%, preferably 15%, preferably 20%, preferably 30%, preferably 40%, preferably 50%, preferably 60%, preferably 80%, preferably 100% greater than that of the parental maize (containing the wild-type CLE gene).

[0055] In one embodiment, the increase in grain weight means that the ear grain weight, single grain weight, 100-grain weight or 1000-grain weight of maize containing the mutant CLE gene is at least 5%, preferably 10%, preferably 12%, preferably 13%, preferably 15%, preferably 20%, preferably 25%, preferably 30%, preferably 40%, preferably 50%, preferably 100% greater than that of the parental maize (containing the wild-type CLE gene).

[0056] In one embodiment, the increase in yield means that the yield, per-plant yield or per-mu yield of maize containing the mutant CLE gene is at least 5%, preferably 10%, preferably 15%, preferably 20%, preferably 21%, preferably 25%, preferably 30%, preferably 40%, preferably 50%, preferably 60%, preferably 100% greater than that of the parental maize (containing the wild-type CLE gene).

[0057] On the other hand, the present invention provides a method for preparing maize cells, or maize seeds, or maize tissues, or maize parts, or maize with an increased number of ear rows, an increased ear width, an increased grain weight or an increased yield, comprising the steps of: mutating the CLE gene in maize cells, or maize seeds, or maize tissues, or maize parts, or maize, so as to obtain maize cells, or maize seeds, or maize tissues, or maize parts, or maize with an increased number of ear rows, an increased ear width, an increased grain weight or an increased yield.

[0058] In one embodiment, the CLE gene is selected from one or any combination of CLE18, CLE5, CLE26, CLE32 and CLE4A; preferably, the CLE18 gene.

[0059] In one embodiment, the mutating of the CLE gene in maize comprises the step of mutating the nucleotide sequence of the endogenous CLE gene in maize so as to introduce the mutant CLE gene.

[0060] In one embodiment, the method for introducing mutations includes natural variation, physical mutagenesis (such as ultraviolet mutagenesis, X-ray or Y-ray mutagenesis), chemical mutagenesis (such as nitrous acid, hydroxylamine, EMS, nitrosoguanidine, etc.), biological mutagenesis (such as virus- or bacteria-mediated mutagenesis), and gene editing.

[0061] In one embodiment, the mutated CLE gene is introduced into maize cells, maize seeds, maize tissues or maize parts by gene editing.

[0062] In one embodiment, the method includes the following steps:

[0063] (1) Introduce an expression vector containing a gene editing tool into maize cells, maize seeds, maize tissues or maize parts;

[0064] (2) Allow the gene editing tool to act on the nucleotide sequence of the endogenous CLE gene in maize and cause it to mutate;

[0065] (3) Screen for mutated maize cells, maize seeds, maize tissues, maize parts or maize;

[0066] (4) Isolate the gene editing tool.

[0067] In one embodiment, the gene editing tools include CRISPR, TALEN and ZFN.

[0068] In one embodiment, the gene editing tool is Cas9.

[0069] In a specific embodiment, the gene editing is carried out using a Cas enzyme in maize cells, maize seeds, maize tissues or maize parts.

[0070] In a specific embodiment, the Cas enzyme is linked with one or more NLS sequences. In one embodiment, the NLS sequence is linked to the N-terminus and / or C-terminus of the protein.

[0071] In one embodiment, introducing the mutated CLE gene includes the step of expressing the mutated CLE gene in maize cells, maize seeds, maize tissues, maize parts or maize.

[0072] In one embodiment, the gene editing method further includes the step of delivering the Cas enzyme into maize cells, maize seeds, maize tissues, maize parts or maize.

[0073] On the other hand, the present invention provides a maize plant with an increased number of kernel rows, an increased ear width, an increased grain weight or an increased yield, and the maize plant contains the above-mentioned mutated CLE gene.

[0074] On the other hand, the present invention provides a method for improving maize traits, and the method comprises the steps of:

[0075] (a) Mutating the CLE gene in maize cells, maize seeds, maize tissues or maize parts;

[0076] (b) Regenerating the maize cells, maize seeds, maize tissues or maize parts in step (a) into plants.

[0077] In one embodiment, the CLE gene is selected from one or any combination of CLE18, CLE5, CLE26, CLE32 and CLE4A; preferably, the CLE18 gene.

[0078] In another preferred example, in step (a), gene editing technology is used to transform the maize cells, maize seeds, maize tissues or maize parts, so that the CLE gene in the maize cells, maize seeds, maize tissues or maize parts is mutated.

[0079] In another preferred example, the gene editing technology is selected from the following group: CRISPR gene editing system, error-prone PCR, gene recombination, TALEN and ZFN.

[0080] In another preferred example, the trait improvement is to increase the number of kernel rows per ear of maize, increase the ear width of maize, increase the kernel weight of maize, or increase the yield of maize.

[0081] On the other hand, the present invention also provides a method for gene editing of maize, and the method comprises the steps of:

[0082] (a) Performing gene editing in maize cells, maize seeds, maize tissues or maize parts by using a Cas enzyme and a gRNA to obtain gene-edited maize cells, maize seeds, maize tissues or maize parts; the gRNA comprises a Scaffold sequence that binds to the Cas enzyme and a guiding sequence that hybridizes with a target sequence, and the gRNA targets the nucleotide sequence of the CLE gene in maize;

[0083] (b) Regenerating the gene-edited maize cells, maize seeds, maize tissues or maize parts in step (a) into maize plants.

[0084] In one embodiment, the gRNA comprises a first segment and a second segment; the first segment is also called the "backbone region", or the "Scaffold sequence"; the second segment is also called the "targeting sequence of the target nucleic acid" or the "targeting segment of the target nucleic acid", or the "guiding sequence", or the "spacer sequence".

[0085] The first segment, "backbone region", or "Scaffold sequence" of the gRNA can interact with the Cas enzyme of the present invention, enabling the formation of a complex between the Cas enzyme and the gRNA. The gRNA of the present invention guides the interacting Cas enzyme to a specific nucleotide sequence within the target nucleic acid through the action of the target sequence of the target nucleic acid.

[0086] The target sequence of the target nucleic acid of the present invention or the target segment of the target nucleic acid contains a nucleotide sequence complementary to the sequence in the target nucleic acid. In other words, the target sequence of the target nucleic acid of the present invention or the target segment of the target nucleic acid interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). Therefore, the target sequence of the target nucleic acid or the target segment of the target nucleic acid can be altered or modified to hybridize to any desired sequence within the target nucleic acid.

[0087] Preferably, the gRNA comprises a first segment and a second segment in the 5' to 3' direction.

[0088] In the present invention, the second segment can also be understood as a guiding sequence that hybridizes to the target sequence.

[0089] In a specific embodiment, the guiding sequence targeting the target sequence in the gRNA is as shown in any one of SEQ ID No. 11 - 15.

[0090] In a specific embodiment, the guiding sequence targeting the target sequence in the gRNA is as shown in SEQ ID No. 11.

[0091] In one embodiment, the nucleic acid sequence encoding the Cas enzyme and the nucleic acid encoding the guide RNA are artificially synthesized.

[0092] In one embodiment, the gene editing method of the present invention includes the step of delivering the Cas enzyme and the gRNA to maize cells, maize seeds, maize tissues, maize parts, or maize.

[0093] The above delivery can be carried out by any method known in the art. Such methods include, but are not limited to, transformation, transfection, electroporation, lipofection, microinjection, sonoporation, gene gun, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection, magnetofection, lipofection, puncture transfection, optical transfection, reagent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, vectors, viral vectors, artificial virions, etc.

[0094] In one embodiment, one or more AAV vectors, lentiviral vectors, nanoparticles, or a combination thereof are used to deliver one or more components of the Cas enzyme and the gRNA.

[0095] In one embodiment, the Cas enzyme and gRNA are delivered to maize cells, maize seeds, maize tissues, maize parts or maize by Agrobacterium-mediated transformation.

[0096] The above gRNA targets the nucleotide sequence of the CLE gene in maize cells and guides the Cas enzyme to the genomic locus site to modify, edit or cleave the target sequence, whereby the nucleotide sequence of the CLE gene is mutated.

[0097] In one embodiment, the gene-edited maize produces the mutated CLE gene.

[0098] In one embodiment, the ear row number, ear width, grain weight or yield of the gene-edited maize is increased.

[0099] On the other hand, the present invention provides a genetically engineered maize prepared by the above method.

[0100] On the other hand, the present invention provides a method for screening or identifying the ear row number, ear width, grain weight or yield of maize, the method comprising the step of detecting whether the CLE gene is mutated.

[0101] In another preferred example, the detection sites of the maize include maize callus, fruits, seeds, flowers, stems, leaves, ears, roots.

[0102] On the other hand, the present invention also provides a method for preparing maize with an increased ear row number, increased ear width, increased grain weight or increased yield, the method comprising the step of hybridizing the maize seeds or maize plants with increased ear row number, increased ear width, increased grain weight or increased yield with other maize to prepare maize with an increased ear row number, increased ear width, increased grain weight or increased yield.

[0103] On the other hand, the present invention provides a mutated CLE protein, and the nucleotide sequence encoding the mutated CLE protein has a base mutation relative to the nucleotide sequence of the parental CLE protein.

[0104] In one embodiment, the parental CLE protein is derived from maize.

[0105] In one embodiment, the CLE protein is selected from one or any combination of CLE18, CLE5, CLE26, CLE32 and CLE4A; preferably, CLE18 protein.

[0106] On the other hand, the present invention provides a gene editing reagent for increasing the number of rows of maize ears, increasing the width of maize ears, increasing the weight of maize kernels, or increasing maize yield, and the gene editing reagent can mutate the CLE gene in maize; the gene editing reagent includes a Cas enzyme and a gRNA, and the gRNA includes a guiding sequence targeting the nucleotide sequence of the CLE gene in maize.

[0107] In one embodiment, the CLE gene is selected from one or any combination of CLE18, CLE5, CLE26, CLE32, and CLE4A; preferably, the CLE18 gene.

[0108] In one embodiment, the guiding sequence contains the nucleotide sequence of a partial parental CLE gene, preferably at least 15 bp of the nucleotide sequence of the CLE gene, and more preferably at least 20 bp of the nucleotide sequence of the CLE gene.

[0109] In one embodiment, the gene editing reagent further includes a gene editing enzyme. The gene editing enzyme includes nucleases of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Transcription Activator-like (TAL) effector nucleases), and ZFN (Zinc finger nuclease) editing tools.

[0110] Preferably, the gene editing enzyme is a Cas protein, also known as a CRISPR enzyme or a Cas effector protein, and its types include but are not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, and FDK1 protein.

[0111] Preferably, the Cas protein is operably linked to a first regulatory element.

[0112] In one embodiment, the gene editing enzyme is a Cas9 protein, and the vector further includes a Scaffold sequence that can specifically bind to the Cas9 protein. After the Scaffold sequence is operably linked to the guiding sequence (or called the targeting sequence, spacer sequence), it constitutes a guiding sequence (gRNA). Preferably, the gRNA is operably linked to a second regulatory element.

[0113] The above-mentioned regulatory elements include promoters, terminator sequences, leader sequences, polyadenylation sequences, signal peptide coding regions, marker genes, enhancers, internal ribosome entry sites (IRESs), and other expression control elements (such as transcription termination signals, such as polyadenylation signals and poly-U sequences).

[0114] On the other hand, the present invention provides a reagent or kit that can be used to increase the number of rows of corn ears, ear width, grain weight, or increase the yield of corn, and the reagent or kit contains the above-mentioned gene editing reagent.

[0115] On the other hand, the present invention provides the use of the above-mentioned gene editing reagent in the preparation of corn with an increased number of rows of ears, an increased ear width, an increased grain weight, or an increased yield; or the use in the preparation of a reagent or kit for corn with an increased number of rows of ears, an increased ear width, an increased grain weight, or an increased yield.

[0116] Unless otherwise defined, the 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 belongs.

[0117] The terms "polynucleotide", "nucleotide sequence", "nucleic acid sequence", "nucleic acid molecule" and "nucleic acid" can be used interchangeably and include DNA, RNA or their hybrids, and can be double-stranded or single-stranded.

[0118] The terms "protein", "polypeptide" and "peptide" can be used interchangeably in the present invention and refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues. The proteins and polypeptides of the present invention can be produced recombinantly or by chemical synthesis. The term "mutant protein" or "mutant-type protein" refers to such a protein that has one or more amino acid residue substitutions, insertions, deletions, and / or additions compared to the amino acid sequence of the parental protein.

[0119] The term "amino acid" refers to a carboxylic acid containing an amino group. All kinds of proteins in organisms are composed of 20 basic amino acids.

[0120] The term "encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, as a template for synthesizing other polymers and macromolecules in a biological process with a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence and its resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to this gene produce a protein in a cell or other biological system, this gene encodes this protein.

[0121] As used herein, the term "identity" is used to refer to the sequence match between two polypeptides or between two nucleic acids. When a position in each of two sequences being compared is occupied by the same base or amino acid monomer subunit (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), then the molecules are identical at that position. The "percent 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 6 of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (3 of a total of 6 positions match). Typically, comparison is made when the two sequences are aligned to yield maximum identity. Such alignment can be accomplished by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.). Also, the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), incorporated into the ALIGN program (version 2.0), can be used with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, the algorithm of Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)), incorporated into the GAP program in the GCG software package (available at www.gcg.com), can be used with a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences.

[0122] The term "regulatory element" is also referred to as "regulatory element", as used herein, and is intended to include promoters, terminator sequences, leader sequences, polyadenylation sequences, signal peptide coding regions, marker genes, enhancers, internal ribosome entry sites (IRESs), and other expression control elements (e.g., transcriptional termination signals such as polyadenylation signals and poly-U sequences), the detailed description of which can be found in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). In some cases, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and those sequences that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can predominantly direct expression in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes). In some cases, regulatory elements can also direct expression in a time-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell type-specific. In some cases, the term "regulatory element" encompasses enhancer elements such as the WPRE; the CMV enhancer; the R-U5' fragment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0123] As used herein, the term "promoter" has the meaning well known to those skilled in the art, which refers to a non-coding nucleotide sequence located upstream of a gene that can initiate the expression of the downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, causes the production of the gene product in a cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, causes the production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, causes the production of the gene product in a cell substantially only when the cell is of the tissue type corresponding to the promoter.

[0124] The term "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for import into the nucleus by nuclear transport, i.e., a protein with an NLS is transported to the nucleus. Typically, an NLS contains positively charged Lys or Arg residues that are exposed on the surface of the protein. Exemplary nuclear localization sequences include, but are not limited to, the NLS from the following: SV40 large T antigen, EGL-13, c-Myc, and the TUS protein.

[0125] As used herein, the term "operably linked" is intended to mean that a nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that permits the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0126] Wild type

[0127] As used herein, the term "wild-type" has the meaning generally understood by those skilled in the art, which refers to the typical form of a biological organism, strain, or gene or the characteristics that distinguish it from mutant or variant forms when it exists in nature, and which can be isolated from natural sources and has not been deliberately modified artificially.

[0128] Vector

[0129] The term "vector" refers to a nucleic acid molecule that is capable of transporting another nucleic acid molecule to which it is linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, no free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and other diverse polynucleotides known in the art. A vector can be introduced into a host cell by transformation, transduction, or transfection, enabling the genetic material elements it carries to be expressed in the host cell. A vector can be introduced into a host cell and thereby produce transcripts, proteins, or peptides, including those from proteins, fusion proteins, isolated nucleic acid molecules, etc. as described herein (e.g., CRISPR transcripts such as nucleic acid transcripts, proteins, or enzymes). A vector can contain various elements for controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector can also contain an origin of replication.

[0130] One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques.

[0131] Another type of vector is a viral vector, in which virus-derived DNA or RNA sequences are present in a vector used for packaging a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus). A viral vector also contains polynucleotides carried by the virus for transfection into a host cell. Certain vectors (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in the host cells into which they are introduced.

[0132] Other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the host cell after being introduced into the host cell and are thereby replicated together with the host genome. Moreover, certain vectors are capable of directing the expression of genes operably linked to them. Such vectors are herein referred to as "expression vectors".

[0133] Host cell

[0134] As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, including but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, and eukaryotic cells such as microbial cells, fungal cells, animal cells, and plant cells.

[0135] Those skilled in the art will understand that the design of an expression vector can depend on factors such as the choice of host cell to be transformed and the desired level of expression.

[0136] Animal

[0137] For example, mammals such as bovines, equines, ovines, suines, canines, felines, lagomorphs (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans. In certain embodiments, the subject (e.g., a human) has a disorder (e.g., a disorder caused by a disease-related gene defect).

[0138] Plant

[0139] The term "plant" shall be understood to mean any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops including artichoke, kohlrabi, arugula, leek, asparagus, lettuce (e.g., iceberg lettuce, leaf lettuce, romaine lettuce), bok choy, taro, melons (e.g., cantaloupe, watermelon, crenshaw, honeydew, casaba), brassica crops (e.g., brussels sprouts, cabbage, cauliflower, broccoli, kale, collards, Chinese cabbage, pak choi), cardoon, carrot, napa, okra, onion, celery, parsley, chickpea, parsnip, chicory, pepper, potato, gourds (e.g., zucchini, cucumber, pattypan, squash, pumpkin), radish, dry bulb onion, rutabaga, eggplant (also known as aubergine), salsify, endive, scallions, escarole, garlic, spinach, green onion, squash, greens, beets (sugar beet and fodder beet), sweet potato, lamb's lettuce, wasabi, tomato, turnip, and spices; fruit and / or vine crops such as apple, apricot, cherry, nectarine, peach, pear, plum, prune, cherry, quince, almond, chestnut, hazelnut, pecan, pistachio, walnut, citrus, blueberry, boysenberry, cranberry, currant, loganberry, raspberry, strawberry, blackberry, grape, avocado, banana, kiwi, persimmon, pomegranate, pineapple, tropical fruit, pome, melon, mango, papaya, and lychee; field crops such as clover, alfalfa, evening primrose, camelina, corn / maize (fodder corn, sweet corn, popcorn), hops, jojoba, peanut, rice, safflower, small grain cereals (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), oil plants (rape, mustard, poppy, olive, sunflower, coconut, castor oil plant, cocoa bean, groundnut), Arabidopsis, fiber plants (cotton, flax, hemp, jute), lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plants; and / or flower bed plants such as flowering plants, cacti, succulents and / or ornamental plants, and trees such as forest (broadleaf and evergreen trees such as conifers), fruit trees, ornamental trees, and nut-bearing trees, and shrubs and other nursery stock.

[0140] The term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant clumps, and plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, ears, roots, root tips, anthers, etc.

[0141] The term "plant cell" should be understood to mean any cell derived from or found in a plant that is capable of forming, for example: undifferentiated tissues such as callus, differentiated tissues such as embryos, parts of a plant, a plant or a seed.

[0142] Gene editing

[0143] The term "gene editing" technology includes CRISPR technology, TALEN technology, and ZFN technology. CRISPR technology refers to Clustered regularly interspaced short palindromic repeats, which are from the immune system of microorganisms. Among them, gene editing tools include guideRNA, Cas proteins (such as Cas9, Cpf1, Cas12b, Cas12i, etc.). The gene editing tool referred to in TALEN technology is a restriction enzyme that can cut specific DNA sequences, which includes a TAL effector DNA-binding domain and a DNA cleavage domain. The gene editing tool referred to in ZFN technology is also a restriction enzyme that can cut specific DNA sequences, which includes a zinc finger DNA-binding domain and a DNA cleavage domain. As is well known to those skilled in the art, by constructing nucleotides encoding gene editing tools and other regulatory elements into a suitable vector and then transforming the cells, editing of the intracellular genome can be achieved, and the types of editing include gene knockout, insertion, and base editing.

[0144] CRISPR system

[0145] As used herein, the terms "clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system" or "CRISPR system" are used interchangeably and have the meaning commonly understood by those skilled in the art, which generally includes transcripts or other elements related to the expression of CRISPR-associated ("Cas") genes, or transcripts or other elements capable of guiding the activity of the Cas genes.

[0146] Cas protein

[0147] Cas proteins, or CRISPR-associated proteins, refer to nucleases applicable to the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system. Preferably, the Cas protein is a CRISPR enzyme, and its types include but are not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, FDK1 protein. The Cas protein may have different structures according to its source, such as SpCas9 derived from Streptococcus pyogenes and SaCas9 derived from Staphylococcus aureus; it can also be subclassified according to structural characteristics (such as domains), for example, the Cas12 family includes Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12i, etc. The Cas protein may have double-stranded or single-stranded or no cleavage activity. The Cas protein of the present invention may be wild-type or its mutant, and the mutation types of the mutant include amino acid substitution, replacement or deletion, and the mutant may or may not change the cleavage activity of the Cas protein. As known to those skilled in the art, a variety of Cas proteins with nucleic acid cleavage activity have been reported in the prior art, and the known protein or its modified variant can achieve the function of the present invention, and it is incorporated into the protection scope by reference herein.

[0148] Those skilled in the art are aware that the structure of a protein can be altered without adversely affecting its activity and functionality. For example, one or more conservative amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional structure of the protein molecule. Those skilled in the art are aware of examples and embodiments of conservative amino acid substitutions. Specifically, an amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, i.e., a nonpolar amino acid residue is replaced with another nonpolar amino acid residue, a polar uncharged amino acid residue is replaced with another polar uncharged amino acid residue, a basic amino acid residue is replaced with another basic amino acid residue, and an acidic amino acid residue is replaced with another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. As long as the substitution does not result in the inactivation of the biological activity of the protein, conservative substitutions in which one amino acid is replaced with another amino acid belonging to the same group fall within the scope of the present invention. Thus, the proteins of the present invention can contain one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably made according to Table 1. In addition, the present invention also encompasses proteins that further contain one or more other non-conservative substitutions, as long as such non-conservative substitutions do not significantly affect the desired functions and biological activities of the proteins of the present invention.

[0149] Conservative amino acid replacements can be made at one or more predicted non-essential amino acid residues. A "non-essential" amino acid residue is an amino acid residue that can be changed (deleted, substituted, or replaced) without changing biological activity, while an "essential" amino acid residue is required for biological activity. A "conservative amino acid replacement" is a replacement in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Amino acid replacements can be made in the non-conserved regions of the above-described Cas mutant proteins. Generally, such replacements are not made to conserved amino acid residues or to amino acid residues located within conserved motifs, where such residues are required for protein activity. However, those skilled in the art will understand that functional variants can have fewer conservative or non-conservative changes in the conserved regions.

[0150] Table 1

[0151] Initial residue Representative substitution Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0152] As is well known in the art, one or more amino acid residues can be altered (substituted, deleted, truncated or inserted) from the N- and / or C-terminus of a protein while still retaining its functional activity. Thus, proteins in which one or more amino acid residues have been altered from the N- and / or C-terminus of a Cas protein while retaining its desired functional activity are also within the scope of the present invention. These alterations can include those introduced by modern molecular methods such as PCR, which includes PCR amplification of a protein coding sequence that is altered or extended by virtue of inclusion of an amino acid coding sequence among the oligonucleotides used in the PCR amplification.

[0153] It should be appreciated that proteins can be altered in various ways, including amino acid substitutions, deletions, truncations and insertions, and methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the above proteins can be prepared by mutagenesis of the DNA. It can also be accomplished by other forms of mutagenesis and / or by directed evolution, for example, using known mutagenesis, recombination and / or shuffling methods, in combination with relevant screening methods, to effect single or multiple amino acid substitutions, deletions and / or insertions.

[0154] Those skilled in the art will appreciate that these minor amino acid changes in the Cas proteins of the present invention can occur (e.g., naturally occurring mutations) or be generated (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site or other functional domain of the protein, the properties of the polypeptide may be altered, but the polypeptide may retain its activity. If the mutations present are not close to the catalytic domain, active site or other functional domain, a lesser effect can be expected.

[0155] Those skilled in the art can identify the essential amino acids of the Cas mutant proteins of the present invention according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis by bioinformatics. The catalytic domain, active site or other functional domain of a protein can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in combination with mutations of putative key site amino acids.

[0156] In the present invention, amino acid residues can be represented by single-letter codes or three-letter codes. For example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R).

[0157] The term "AxxB" means that the amino acid A at the xx-th position is changed to amino acid B. Unless otherwise specified, it is the amino acid A at the xx-th position starting from the N-terminus that is changed to amino acid B.

[0158] Those skilled in the art can use commonly used software in the art, such as Clustal Omega, to compare the amino acid sequence of any parental CLE18 protein with SEQ ID No.1 for sequence identity and alignment, and thus obtain the amino acid sites in the parental CLE18 protein corresponding to the amino acid sites defined based on SEQ ID No.1 in the present application.

[0159] Guide RNA (guideRNA, gRNA)

[0160] As used herein, the terms "guide RNA (gRNA)", "mature crRNA", and "guide sequence" are used interchangeably and have the meanings commonly understood by those skilled in the art. Generally, a guide RNA can contain a Scaffold sequence and a spacer sequence (or referred to as a targeting sequence, guiding sequence), or consist essentially of or consist of a Scaffold and a spacer sequence.

[0161] In some cases, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target sequence to hybridize with the target sequence and direct the specific binding of the CRISPR / Cas complex to the target sequence. In one embodiment, when optimally aligned, the degree of complementarity between the guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining the optimal alignment is within the ability of one of ordinary skill in the art. For example, there are publicly available and commercially available alignment algorithms and programs, such as but not limited to ClustalW, the Smith-Waterman algorithm in matlab, Bowtie, Geneious, Biopython, and SeqMan.

[0162] Target sequence

[0163] A "target sequence" refers to a polynucleotide targeted by the guide sequence in the gRNA, e.g., a sequence complementary to the guide sequence, wherein hybridization between the target sequence and the guide sequence will promote the formation of a CRISPR / Cas complex (including the Cas protein and the gRNA). Perfect complementarity is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR / Cas complex.

[0164] The target sequence can comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is intracellular or extracellular. In some cases, the target sequence is in the nucleus or cytoplasm of a cell. In some cases, the target sequence can be within an organelle of a eukaryotic cell, such as a mitochondrion or a chloroplast. A sequence or template that can be used for recombination into a target locus containing the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In one embodiment, the editing template is exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.

[0165] In the present invention, a "target sequence" or "target polynucleotide" or "target nucleic acid" can be any endogenous or exogenous polynucleotide to a cell (e.g., a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). In some cases, the target sequence should be related to a protospacer adjacent motif (PAM).

[0166] CLE gene

[0167] Genes of the CLAVATA3 / EMBRYOSURROUNDINGREGION (CLE) family encode small secreted proteins and play multiple biological roles in plant growth and development and responses to abiotic stresses. It has been documented in the literature that some genes of the CLE family inhibit the expression of WUS by acting as feedback signals from stem cells to the organizing center, restricting the number of stem cells.

[0168] The maize CLE family contains nearly 50 related genes, and the functions of most related genes and their roles in plant growth and development are still unclear.

[0169] In this application, the NCBI accession number of the maize CLE18 gene is LOC111589273, and its amino acid sequence is shown in SEQ ID No.1, and its nucleotide sequence is shown in SEQ ID No.2; the NCBI accession number of the maize CLE4A gene is LOC103644198, and its amino acid sequence is shown in SEQ ID No.3, and its nucleotide sequence is shown in SEQ ID No.4; the NCBI accession number of the maize CLE5 gene is LOC100216662, and its amino acid sequence is shown in SEQ ID No.5, and its nucleotide sequence is shown in SEQ ID No.6; the NCBI accession number of the maize CLE26 gene is LOC100277806, and its amino acid sequence is shown in SEQ ID No.7, and its nucleotide sequence is shown in SEQ ID No.8; the NCBI accession number of the maize CLE32 gene is LOC100277301, and its amino acid sequence is shown in SEQ ID No.9, and its nucleotide sequence is shown in SEQ ID No.10.

[0170] Main advantages of the present invention:

[0171] Through research, the present invention found that compared with wild-type maize plants, maize plants with CLE18 gene mutations have an increased number of ear rows, increased ear width, increased grain weight, and increased yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0172] Figure 1 It is a phylogenetic tree of CLE genes.

[0173] Figure 2 It is a comparison of the number of ear rows of different mutant lines and the parental maize inbred line.

[0174] Figure 3 It is a comparison of the number of ear rows of the mutant line 3840AR108-16 and the parental maize inbred line.

[0175] Figure 4 It is a comparison of the ear length of different mutant lines and the parental maize inbred line.

[0176] Figure 5Comparison of ear width between different mutant lines and parental maize inbred lines.

[0177] Figure 6 Comparison of 100-kernel weight between different mutant lines and parental maize inbred lines.

[0178] Figure 7 Comparison of plant height between different mutant lines and parental maize inbred lines.

[0179] Figure 8 Comparison of grain weight per ear between mutant line 3840AR108-16 and parental maize inbred lines.

[0180] Sequence Listing

[0181] SEQ ID No. Description 1 Amino acid sequence of CLE18 2 Nucleotide sequence of CLE18 3 Amino acid sequence of CLE4A 4 Nucleotide sequence of CLE4A 5 Amino acid sequence of CLE5 6 Nucleotide sequence of CLE5 7 Amino acid sequence of CLE26 8 Nucleotide sequence of CLE26 9 Amino acid sequence of CLE32 10 Nucleotide sequence of CLE32 11 Guide sequence of gRNA1 12 Guide sequence of gRNA2 13 Guide sequence of gRNA3 14 Guide sequence of gRNA4 15 Guide sequence of gRNA5 Detailed Embodiments

[0182] The present invention will be further described below in conjunction with embodiments. The following description is only for the preferred embodiments of the present invention, and does not limit the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make equivalent changes to equivalent embodiments. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0183] Example 1: Target Design and Vector Construction

[0184] Download the sequences of CLE gene family members from the gene database, compare and analyze the protein sequences, and construct a phylogenetic tree. The phylogenetic tree is as Figure 1 shown.

[0185] The NCBI accession number of the maize CLE18 gene is LOC111589273, the amino acid sequence is shown in SEQ ID No.1, and the genomic nucleotide sequence (genomic DNA) is shown in SEQ ID No.2; the NCBI accession number of the maize CLE4A gene is LOC103644198, the amino acid sequence is shown in SEQ ID No.3, and the genomic nucleotide sequence (genomic DNA) is shown in SEQ ID No.4; the NCBI accession number of the maize CLE5 gene is LOC100216662, the amino acid sequence is shown in SEQ ID No.5, and the genomic nucleotide sequence (genomic DNA) is shown in SEQ ID No.6; the NCBI accession number of the maize CLE26 gene is LOC100277806, the amino acid sequence is shown in SEQ ID No.7, and the genomic nucleotide sequence (genomic DNA) is shown in SEQ ID No.8; the NCBI accession number of the maize CLE32 gene is LOC100277301, the amino acid sequence is shown in SEQ ID No.9, and the genomic nucleotide sequence (genomic DNA) is shown in SEQ ID No.10.

[0186] Amino acid sequence of CLE18:

[0187] MRRRRWAPLAAACLVALAALLAAVHGGGGGGGARGAAAPMPATRRPEASARVAAF DAARCKRPRNRAGAACARLPAAGAGDDDDDKRVVPTGSNPLHNR (SEQ ID No.1);

[0188] Nucleotide sequence of CLE18:

[0189] ctcacttcacttcacccttcttctgtctgcttcgacgcctcgacgccacacttggcagcagctcgcactcgacagcagccgctgctgctactctgcgacgcggtctgcgccattgtgacagctagcgctgcactggtctcgccctcgccctcgcctccgcctccgcccaccgcgcgcccacgcagcctacacgtacacgcccacgtcctcctacacccctgctccctcccgccatgaggaggcgacggtgggcgccgctcgccgccgcgtgcctcgtcgcgctcgcggcgctcctggccgccgtccacggcggcggcggtggcggcgctcggggcgccgccgcgccaatgcccgcgacccgccgcccggaggcgagcgcgcgcgtcgccgccttcgacgccgcgcggtgcaagaggcCgcggaacagagctggcgccgcctgcgcgatgctgcccgccgccgctgcggccggcggcggcggcgacgacgacaagcgggtcgtgccgacaggctccaaccccctgcacaaccgatgacaagatcttgcacgattgcagaggcaaggacgtggaggatctccgtcgcacttgctctgcgtgcgtcgtgctcgtgcgtcattccatggcggattggttggcggcgcgaggggaagtgtagcggaggagaactccatggccactgatctagctagcctccagctagggagtagtaggattatagtagcgtatataaccagttactactaactaactagtggttcttgttccatggccgctctggtatcggcgttcgtcagagccgacgaactgttgtaagccacggtttggaacgccggaatttcgtgggatttccaaggaaattgcatgtttctcccgtgaaattctcgcgttccaaacaagacctcaagactgggaaaagatgctggtttcctcttatcttctctggt(SEQ ID No.2);

[0190] Amino acid sequence of CLE4A:

[0191] MIPSRTAREGCPMALILSTTEEPASQDDRRDEHDVGSAHAEAWKLPNPKRPAIATCWERRLANRRCVALHTCIAWRKRNRQEVHRGVVGLAGMLGVTRGRWPALSSSKHCYLRRF(SEQ ID No.3);

[0192] Nucleotide sequence of CLE4A:

[0193]

[0194] Amino acid sequence of CLE5:

[0195] MAGRLHACLLICTVLSSAAIMASCTRPAAGTVGGAAPVAATTPATSSAGSGVRPPAPP PPATLATTAGTNKAPPAVQQQQSPLDSKRKVPNGPDPIHNRRARWGEAPSKRV(SEQ ID No.5);

[0196] Nucleotide sequence of CLE5:

[0197]

[0198] Amino acid sequence of CLE26:

[0199] MSAGRWLRLALLLSLVPLALRAASLLLGVPALASPSSSSRQSWTTDPHERAAAAAVS VWPARDRDRPYRHARHRRRAGSGSAASSSDDGARRRLRQAAAGDWFEDDKRLAPTGSN PLHNLR (SEQ ID No.7);

[0200] Nucleotide sequence of CLE26:

[0201]

[0202] Amino acid sequence of CLE32:

[0203] MGARPDGAPGAVECCGRRLVRLLAFLFLVCACLVMAAMVATTDGGASLAGPSSNSA ASSATKTGGSPAWRSGGTAADAFRSSERRIPKGPDPIHNRRAGKTTTAPRRRD(SEQ ID No.9);

[0204] Nucleotide sequence of CLE32:

[0205] agtgcctctgcttcatcgtgcgagcgagggcatttggattcggattcgaggcggtgcactgaagtgcctagctagtgcagggagaagcagcaaagctctctctctctctctcccttatatgggagcaaggccggacggggcgccgggggctgtggagtgctgcggccgcaggctggtacgactgctagccttcctcttcctggtctgcgcttgcctcgtgatggccgccatggtggcgacgacggacggcggagcgagcttagcagggccgtcgtcgaacagcgctgcgtcgtccgccacgaagacgggcggctcgccggcgtggcgttcaggcgggacggccgcagacgcgttccggagcagcgagaggaggatccccaaggggcccgacccgattcacaacaggcgtgccgggaagacgacgaccgcgccgcgccggagagactagctagccggaggccggccgggggcggcggcggcggcggcaacagaagctaccactgccggtgctcatcgtcctgcacctgaagcagcagcagcagctgatctgatcgacatacagttcgtgttcggcagctagcccctagccatgctcggagaggtttaattaattacgcagcggactccttcgatgggggtggatttttggtgatcattctgggggtatgtataaacctgcaacctacccttgcaaggattagtttttcttcttctgtttttcttctttcaagtttcaccgaggtggagtggaggttgaccgacattctgttcttcccgcatattcttgtgagattttgattccaatcagtgtctatcaattcaatt(SEQ ID No.10).

[0206] Construct a multi-gene knockout vector using Cas9 protein and gRNA targeting the CLE genes in the maize phylogenetic tree. The specific operation method can be carried out in a conventional manner in this field. The vector construction can refer to the reference ("High-efficiency CRISPR / Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize", Weiwei Qi et al., 《BMC Biotechnology》, 2016).

[0207] Specifically, in this embodiment, the guiding sequence of the gRNA (the sequence hybridizing with the target sequence) is as follows:

[0208] gRNA Sequence(5’-3’) Target gene PAM(5’-3’) gRNA1 aagaggccgcggaacagagc(SEQ ID No.11) CLE18 TGG gRNA2 caaggcucaugaucccuucc(SEQ ID No.12) CLE4A AGG gRNA3 ugccugcuugcucaucugca(SEQ ID No.13) CLE5 CGG gRNA4 gcugcugucgcucgucccgc(SEQ ID No.14) CLE26 TGG gRNA5 cagcgagaggaggaucccca(SEQ ID No.15) CLE32 AGG

[0209] Introduce the above vector into the maize inbred line by the method of Agrobacterium infection. In this embodiment, the parental maize inbred line receptor is designated as F003, and the gene-edited positive materials are identified by PCR. Then, through 2-3 generations of self-crossing and propagation, the mutant type population is increased to obtain homozygous edited seedlings without foreign gene insertion.

[0210] After identification and field screening, the homozygous mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 are obtained by transforming with the above vector. The specific editing types are as follows:

[0211] The line 3840AR108-16 is a single mutant plant of CLE18, and the nucleotide sequence of its CLE18 gene has a deletion of the 428-429th bases relative to the sequence shown in SEQ ID No. 2.

[0212] The line 3263AR65-1 is a single mutant plant of CLE32, and one base A is inserted after the 377th base of the nucleotide sequence of its CLE32 gene relative to the sequence shown in SEQ ID No. 10.

[0213] The line 3263AR67-15 is a single mutant plant of CLE32, and the nucleotide sequence of its CLE32 gene has a deletion of the 379th base relative to the sequence shown in SEQ ID No. 10.

[0214] The line 3094AR52-15 is a single mutant plant of CLE26, and one base C is inserted after the 356th base of the nucleotide sequence of its CLE26 gene relative to the sequence shown in SEQ ID No. 8.

[0215] The strain 3262AR114-59 is a CLE5 single mutant plant, and the nucleotide sequence of its CLE5 gene has a deletion of bases at positions 381-418 relative to the sequence shown in SEQ ID No. 6.

[0216] The strain 3262AR19-57 is a CLE4A single mutant plant, and the nucleotide sequence of its CLE4A gene has a deletion of the 1121st base relative to the sequence shown in SEQ ID No. 4.

[0217] The editing types of the mutant strains 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 are summarized as follows:

[0218]

[0219]

[0220] Observe and count the trait performances of the parental maize inbred lines ( Figure 2 - Figure 7 F003 in Figure 8 WT in

[0221] Figure 2 ), and the above mutant strains, including plant height, ear length, ear width, number of rows of kernels on the ear, ear grain weight, and 100-kernel weight. Figure 2 It can be seen from

[0222] Figure 3 that there is no significant difference in the number of rows of kernels on the ear between the mutant strains 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 and the parental maize inbred lines; the number of rows of kernels on the ear of the mutant strain 3840AR108-16 is significantly increased compared with that of the parental maize inbred line (in this example, the number of rows of kernels on the ear refers to the number of rows at the thickest part in the middle of the maize ear).

[0222] Figure 3 is the comparison of the number of rows of kernels on the ear between the mutant strain 3840AR108-16 ( Figure 3 cle18 in Figure 3 and the parental maize inbred line; it can be seen from

[0223] Figure 4 Figure 3 that the number of rows of kernels on the ear of the mutant strain 3840AR108-16 is significantly increased compared with that of the parental maize inbred line.

[0223] Figure 4Comparison of ear length between mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, 3262AR19-57 and their parental maize inbred lines. From Figure 4 It can be seen that there is no significant difference in ear length between mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, 3262AR19-57 and their parental maize inbred lines.

[0224] Figure 5 Comparison of ear width between mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59 and 3262AR19-57 and their parental maize inbred lines. From Figure 5 It can be seen that there is no significant difference in ear width between mutant lines 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, 3262AR19-57 and their parental maize inbred lines; the ear width of mutant line 3840AR108-16 is significantly increased compared with that of its parental maize inbred line (in this example, ear width refers to the outer width of the thickest part of the maize ear).

[0225] Figure 6 Comparison of 100-kernel weight between mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59 and 3262AR19-57 and their parental maize inbred lines. From Figure 6 It can be seen that there is no significant difference in 100-kernel weight between mutant lines 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, 3262AR19-57 and their parental maize inbred lines; the 100-kernel weight of mutant line 3840AR108-16 is significantly increased compared with that of its parental maize inbred line.

[0226] Figure 7 Comparison of plant height between mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59 and 3262AR19-57 and their parental maize inbred lines. From Figure 7 It can be seen that there is no significant difference in plant height between mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59 and 3262AR19-57 and their parental maize inbred lines.

[0227] Figure 8 Comparison of the grain weight per ear between the mutant line 3840AR108-16 and the parental maize inbred line ( Figure 8 WT in Figure 8 It can be seen that the grain weight per ear of the mutant line 3840AR108-16 is significantly increased compared with that of the parental maize inbred line.

[0228] Therefore, the ear row number, ear width, grain weight per ear, 100-grain weight and yield of the cle18 mutant line 3840AR108-16 are increased.

[0229] All documents mentioned in the present invention are cited herein by reference as if each individual document was specifically and individually cited herein. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A method for increasing the number of corn cob rows, increasing the width of corn cobs, increasing the weight of corn kernels, or improving corn yield, characterized in that: The method comprises the steps of mutating the CLE18 gene in corn; The amino acid sequence of the CLE18 gene has at least 80% sequence identity with SEQ ID NO.:

1.

2. The method according to claim 1, characterized in that The mutation is achieved by a method selected from the group consisting of gene editing technology, gene mutation, gene knockout, gene interruption, RNA interference technology, or a combination thereof.

3. A method for preparing corn cells, corn seeds, corn tissues, corn parts, or corn with increased ear rows, increased ear width, increased grain weight, or improved yield, characterized in that: Includes steps: Mutating the CLE18 gene in a corn cell, a corn seed, a corn tissue, a corn part, or a corn; The amino acid sequence of the CLE18 gene has at least 80% sequence identity with SEQ ID NO.:

1.

4. A method for improving corn traits, characterized in that: The method comprises the steps of: (a) mutating the CLE18 gene in corn cells, corn seeds, corn tissues or corn parts; (b) regenerating the corn cells, corn tissues, or corn parts of step (a) into corn plants; The amino acid sequence of the CLE18 gene has at least 80% sequence identity with SEQ ID NO.:

1.

5. The method according to claim 4, characterized in that The trait improvement is to increase the number of corn ear rows, increase the corn ear width, increase the corn kernel weight, or increase the corn yield.

6. A method for gene editing of corn, characterized in that: The method comprises the steps of: (a) performing gene editing in corn cells, corn seeds, corn tissues or corn parts using a Cas enzyme and a gRNA to obtain gene-edited corn cells, corn seeds, corn tissues or corn parts; the gRNA comprises a guide sequence targeting a nucleotide sequence of a CLE18 gene in corn; (b) regenerating the gene-edited corn cell, corn seed, corn tissue or corn part in step (a) into a corn plant; The amino acid sequence of the CLE18 gene has at least 80% sequence identity with SEQ ID NO.:

1.

7. The method according to claim 6, characterized in that The guide sequence in the gRNA that hybridizes with the target sequence is shown as SEQ ID No.

11.

8. A gene editing reagent for increasing the number of corn cob rows, increasing the width of corn cobs, increasing the weight of corn kernels, or improving corn yield, characterized in that: The gene editing reagent is capable of mutating the CLE18 gene in corn; the gene editing reagent comprises a Cas enzyme and a gRNA, and the gRNA comprises a guide sequence targeting a nucleotide sequence of the CLE18 gene in corn; The amino acid sequence of the CLE18 gene has at least 80% sequence identity with SEQ ID NO.:

1.

9. Use of the gene editing reagent of claim 8 in preparing corn with increased ear rows, increased ear width, increased grain weight or improved yield; or use of the gene editing reagent in preparing a reagent or kit for producing corn with increased ear rows, increased ear width, increased grain weight or improved yield.

10. A method for preparing corn with increased ear rows, increased ear width, increased grain weight or improved yield, characterized in that: The method comprises the steps of hybridizing corn seeds or corn plants obtained by any one of the methods described in claims 1 to 7 with other corns to produce corns with increased ear rows, increased ear width, increased grain weight or increased yield.

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