Method for increasing kernel row number of ear of corn

Abstract

Provided is a method for increasing the kernel row number of an ear of corn, increasing the width of the ear of corn, increasing corn kernel weight or improving corn yield. The method comprises the step of mutating the CLE gene in corn. The mutation of the CLE gene can lead to increases in the kernel row number of an ear of corn, ear width, kernel weight and yield thereof. Therefore, the method has important application value in corn breeding.

Description

A method for increasing the number of corn ear rows

[0001] This application claims priority to Chinese patent application CN202410364411.2, filed on March 28, 2024. This application incorporates the entire text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention belongs to the fields of biotechnology and crop genetic breeding, and relates to a method for increasing the number of corn cob rows, increasing the corn cob width, increasing the corn kernel weight, and improving corn yield, in particular, increasing the number of corn cob rows, increasing the corn cob width, increasing the corn kernel weight, and improving corn yield through a CLE gene mutation method. Background Art

[0003] Corn is the crop with the largest planting area in the world, with an annual global planting area of ​​more than 2 billion mu and a total output of 1 billion tons. Its edible value, industrial value and feed value make it occupy an important position in my country's food security system.

[0004] Ensuring and increasing corn yields is crucial for national food security. Theoretically, increasing the number of rows per corn ear (without reducing the number of kernels per row) is beneficial for increasing corn yield. To increase the number of rows per corn ear and yield, we are studying the CLE gene family in maize, hoping to obtain superior maize germplasm resources. Summary of the Invention

[0005] The invention provides a method for increasing the number of corn ear rows, increasing the corn ear width, increasing the corn kernel weight, or improving corn yield.

[0006] In one aspect, the present invention provides a method for increasing the number of corn ear rows, increasing the corn ear width, increasing the corn kernel weight, or improving corn yield, the method comprising the step of mutating the CLE gene in corn.

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

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

[0009] In another preferred embodiment, 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) having the same or similar function as described above, formed by substituting, deleting or adding one or several (e.g., 1-10) amino acid residues of the amino acid sequence shown in SEQ ID NO.: 1;

[0012] or (iii) a polypeptide having an amino acid sequence that is 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99% or 100%) identical to the amino acid sequence of SEQ ID NO.: 1 and having the same or similar functions.

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

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

[0015] (b) a polynucleotide whose sequence is shown in SEQ ID NO.: 2;

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

[0017] (d) a polynucleotide having 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide shown in SEQ ID NO.: 2;

[0018] (e) A polynucleotide complementary to the polynucleotide described in any one of (a) to (d).

[0019] In one embodiment, the polynucleotide is selected from the group consisting of a genomic sequence, a cDNA sequence, a CDS sequence, an RNA sequence, or a combination thereof.

[0020] In one embodiment, the parent CLE18 gene is derived from a naturally occurring corn line, and the nucleotide sequence of the parent CLE18 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. 2.

[0021] In one embodiment, the amino acid sequence of the parent CLE18 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. 1.

[0022] In another preferred embodiment, the amino acid sequence of the CLE18 gene has at least 80% sequence identity with SEQ ID NO.: 1.

[0023] In another preferred example, the nucleotide sequence of the CLE18 gene has at least 80% sequence identity with SEQ ID NO.: 2.

[0024] In one embodiment, the nucleotide sequence of the parent CLE4A 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. 4.

[0025] In one embodiment, the amino acid sequence of the parent CLE4A 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. 3.

[0026] In one embodiment, the nucleotide sequence of the parent CLE5 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. 6.

[0027] In one embodiment, the amino acid sequence of the parent CLE5 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. 5.

[0028] In one embodiment, the nucleotide sequence of the parent CLE26 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. 8.

[0029] In one embodiment, the amino acid sequence of the parent CLE26 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. 7.

[0030] In one embodiment, the nucleotide sequence of the parent 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.

[0031] In one embodiment, the amino acid sequence of the parent 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.

[0032] In one embodiment, the parent CLE18 gene is derived from a maize inbred line.

[0033] In one embodiment, the nucleotide sequence of the parent CLE18 gene is shown as SEQ ID No. 2.

[0034] In one embodiment, the amino acid sequence of the parent CLE18 gene is shown as SEQ ID No.1.

[0035] In one embodiment, the NCBI accession number of the parent CLE18 gene is LOC111589273.

[0036] In another preferred embodiment, 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.

[0037] In another preferred embodiment, 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 nitrite, hydroxylamine, EMS, nitrosoguanidine, etc.), biological mutagenesis (such as virus- or bacteria-mediated mutagenesis), gene editing or biosynthesis.

[0038] In another preferred embodiment, the gene editing technology is selected from the following group: CRISPR technology, TALEN technology, ZFN technology, or a combination thereof.

[0039] In another preferred embodiment, the method comprises the steps of:

[0040] (i) providing a corn or corn cell; and

[0041] (ii) using gRNA targeting the CLE gene and the corresponding Cas protein to introduce into the corn or corn cell; in a preferred embodiment, an expression vector containing the gRNA and Cas protein is introduced into the corn or corn cell.

[0042] In another preferred embodiment, the mutation includes insertion mutation, deletion mutation, frameshift mutation, and substitution mutation.

[0043] In a preferred embodiment, the method comprises reducing or inhibiting the expression level and / or activity of the CLE gene.

[0044] In another preferred embodiment, the reduction or inhibition refers to that compared with the expression level E0 of the CLE gene in the wild-type plant, the expression level E1 of the CLE gene in the plant is 0-80% of the wild-type, preferably 0-60%, more preferably 0-40%, and even more preferably 0-30%.

[0045] In another preferred embodiment, the reduction or inhibition of the expression and / or activity of the CLE gene is achieved by a method selected from the group consisting of gene mutation, gene knockout, gene interruption, RNA interference technology, gene editing technology, introduction of gene or protein inhibitors, or a combination thereof.

[0046] In another preferred embodiment, the reduction or inhibition of the expression level and / or activity of the CLE gene is achieved by mutating the CLE gene.

[0047] In another preferred embodiment, the mutation causes complete or partial loss of function of the CLE gene.

[0048] In another preferred embodiment, the mutation is that the nucleotide sequence of the CLE18 gene is missing some bases relative to the sequence shown in SEQ ID NO.2.

[0049] In one embodiment, the nucleotide sequence of the mutated CLE18 gene lacks bases 428-429 corresponding to the sequence shown in SEQ ID No. 2 relative to the nucleotide sequence of the parent CLE18 gene.

[0050] In another preferred embodiment, the corn is an inbred line of corn.

[0051] In another preferred example, the increasing corn yield refers to increasing corn plant height, ear height, leaf length, leaf width, number of ear rows, number of grains per row, ear length, ear width, 100-grain weight, number of grains per ear, number of grains per mu, unit yield or per mu yield.

[0052] In one embodiment, the increase in the number of ear rows means that the corn containing the mutated CLE gene has at least 3% more ear rows than the parent corn (containing the wild-type CLE gene), preferably 5% more, preferably 8% more, preferably 10% more, preferably 15% more, preferably 20% more, preferably 30% more, preferably 40% more, preferably 50% more, preferably 60% more, preferably 80% more, and preferably 100% more.

[0053] In one embodiment, the increased ear width means that the ear width of corn containing the mutated CLE gene is at least 3% increased, preferably 5% increased, preferably 8% increased, preferably 10% increased, preferably 15% increased, preferably 20% increased, preferably 30% increased, preferably 40% increased, preferably 50% increased, preferably 60% increased, preferably 80% increased, preferably 100% increased compared to the parent corn (containing the wild-type CLE gene).

[0054] In one embodiment, the increase in grain weight means that the kernel weight per ear, kernel weight per kernel, 100-kernel weight or 1000-kernel weight of corn containing a mutated CLE gene is at least 5% higher than that of the parent corn (containing a wild-type CLE gene), preferably 10% higher, preferably 12% higher, preferably 13% higher, preferably 15% higher, preferably 20% higher, preferably 25% higher, preferably 30% higher, preferably 40% higher, preferably 50% higher, and preferably 100% higher.

[0055] In one embodiment, the yield increase means that the yield or yield per plant or yield per mu of corn containing the mutated CLE gene is at least 5% higher than that of the parent corn (containing the wild-type CLE gene), preferably increased by 10%, preferably increased by 15%, preferably increased by 20%, preferably increased by 21%, preferably increased by 25%, preferably increased by 30%, preferably increased by 40%, preferably increased by 50%, preferably increased by 60%, preferably increased by 100%.

[0056] On the other hand, the present invention provides a method for preparing corn cells, or corn seeds, or corn tissues, or corn parts, or corn with increased number of ear rows, increased ear width, increased grain weight, or improved yield, comprising the steps of mutating the CLE gene in the corn cells, or corn seeds, or corn tissues, or corn parts, or corn, thereby obtaining corn cells, or corn seeds, or corn tissues, or corn parts, or corn with increased number of ear rows, increased ear width, increased grain weight, or improved yield.

[0057] In one embodiment, the CLE gene is selected from one or any several of CLE18, CLE5, CLE26, CLE32 and CLE4A; preferably, CLE18 gene.

[0058] In one embodiment, the mutating the CLE gene in corn comprises the step of mutating the nucleotide sequence of the endogenous CLE gene of corn to introduce the mutated CLE gene.

[0059] In one embodiment, the method of 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.

[0060] In one embodiment, the mutated CLE gene is introduced into corn cells, corn seeds, corn tissues or corn parts by gene editing.

[0061] In one embodiment, the method comprises the following steps:

[0062] (1) introducing an expression vector containing a gene editing tool into corn cells, corn seeds, corn tissues, or corn parts;

[0063] (2) Using gene editing tools to act on the nucleotide sequence of the endogenous CLE gene of maize and causing mutations;

[0064] (3) screening corn cells, corn seeds, corn tissues, corn parts or corn for mutations;

[0065] (4) Isolating the gene editing tool.

[0066] In one embodiment, the gene editing tools include CRISPR, TALEN and ZFN.

[0067] In one embodiment, the gene editing tool is Cas9.

[0068] In a specific embodiment, the gene editing is performed using a Cas enzyme in corn cells, corn seeds, corn tissues or corn parts.

[0069] In a specific embodiment, the Cas enzyme is connected to one or more NLS sequences. In one embodiment, the NLS sequence is connected to the N-terminus and / or C-terminus of the protein.

[0070] In one embodiment, the introducing the mutated CLE gene comprises the step of expressing the mutated CLE gene in corn cells, corn seeds, corn tissues, corn parts or corn.

[0071] In one embodiment, the gene editing method further includes the step of delivering a Cas enzyme into corn cells, corn seeds, corn tissues, corn parts or corn.

[0072] In another aspect, the present invention provides a corn plant with increased ear rows, increased ear width, increased grain weight or improved yield, wherein the corn plant contains the mutated CLE gene.

[0073] In another aspect, the present invention provides a method for improving corn traits, the method comprising the steps of:

[0074] (a) mutating a CLE gene in a corn cell, corn seed, corn tissue or corn part;

[0075] (b) regenerating the corn cell, corn seed, corn tissue or corn part in step (a) into a plant.

[0076] In one embodiment, the CLE gene is selected from one or any several of CLE18, CLE5, CLE26, CLE32 and CLE4A; preferably, CLE18 gene.

[0077] In another preferred embodiment, in step (a), the corn cell, corn seed, corn tissue or corn part is modified by gene editing technology, so that the CLE gene in the corn cell, corn seed, corn tissue or corn part is mutated.

[0078] In another preferred embodiment, the gene editing technology is selected from the following group: CRISPR gene editing system, error-prone PCR, gene recombination, TALEN and ZFN.

[0079] In another preferred embodiment, 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.

[0080] On the other hand, the present invention also provides a method for gene editing of corn, comprising the steps of:

[0081] (a) performing gene editing in a corn cell, corn seed, corn tissue, or corn part using a Cas enzyme and a guide RNA (gRNA) to obtain a gene-edited corn cell, corn seed, corn tissue, or corn part; the gRNA comprises a Scaffold sequence that binds to the Cas enzyme and a guide sequence that hybridizes with a target sequence, and the gRNA targets a nucleotide sequence of a CLE gene in corn;

[0082] (b) regenerating the gene-edited corn cell, corn seed, corn tissue or corn part of step (a) into a corn plant.

[0083] In one embodiment, the gRNA includes a first segment and a second segment; the first segment is also called a "skeleton region" or a "Scaffold sequence"; the second segment is also called a "targeting sequence for targeting nucleic acid" or a "targeting segment for targeting nucleic acid", or a "guide sequence", or a "spacer sequence".

[0084] The first segment, "skeleton region", or "scaffold sequence" of the gRNA can interact with the Cas enzyme of the present invention, thereby forming 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 targeting sequence of the target nucleic acid.

[0085] The targeting sequence of the targeting nucleic acid of the present invention or the targeting segment of the targeting nucleic acid comprises a nucleotide sequence that is complementary to a sequence in the target nucleic acid. In other words, the targeting sequence of the targeting nucleic acid of the present invention or the targeting segment of the targeting nucleic acid interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). Therefore, the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid can be changed or modified to hybridize to any desired sequence within the target nucleic acid.

[0086] Preferably, the gRNA comprises a first segment and a second segment from the 5' to the 3' direction.

[0087] In the present invention, the second segment can also be understood as a guide sequence that hybridizes with the target sequence.

[0088] In a specific embodiment, the guide sequence targeting the target sequence in the gRNA is shown in any one of SEQ ID No.11-15.

[0089] In a specific embodiment, the guide sequence targeting the target sequence in the gRNA is shown as SEQ ID No.11.

[0090] In one embodiment, the nucleic acid sequence encoding the Cas enzyme and the nucleic acid encoding the guide RNA are artificially synthesized.

[0091] In one embodiment, the gene editing method of the present invention includes the steps of delivering Cas enzymes and gRNA into corn cells, corn seeds, corn tissues, corn parts or corn.

[0092] The above-mentioned 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, lipofection, dendritic transfection, heat shock transfection, nucleofection, magnetofection, lipofection, puncture transfection, optical transfection, agent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, vectors, viral vectors, artificial virions, etc.

[0093] In one embodiment, one or more components of the Cas enzyme and gRNA are delivered using one or more AAV vectors, lentiviral vectors, nanoparticles, or a combination thereof.

[0094] In one embodiment, the Cas enzyme and gRNA are delivered to corn cells, corn seeds, corn tissues, corn parts, or corn by Agrobacterium transformation.

[0095] The gRNA targets the nucleotide sequence of the CLE gene in corn cells and guides the Cas enzyme to the genomic locus to modify, edit or cut the target sequence, thereby mutating the nucleotide sequence of the CLE gene.

[0096] In one embodiment, the gene-edited corn produces the mutated CLE gene.

[0097] In one embodiment, the gene-edited corn has an increased number of ear rows, an increased ear width, an increased grain weight, or an improved yield.

[0098] In another aspect, the present invention provides a genetically engineered corn, which is prepared using the above method.

[0099] In another aspect, the present invention provides a method for screening or identifying corn ear row number, ear width, grain weight, or yield, the method comprising the step of detecting whether the CLE gene is mutated.

[0100] In another preferred embodiment, the detection parts of corn include corn callus, fruit, seeds, flowers, stems, leaves, ears, and roots.

[0101] On the other hand, the present invention also provides a method for preparing corn with increased number of ear rows, increased ear width, increased grain weight or improved yield, which method includes the steps of hybridizing the corn seeds or corn plants with increased number of ear rows, increased ear width, increased grain weight or improved yield with other corns to prepare corn with increased number of ear rows, increased ear width, increased grain weight or improved yield.

[0102] In another aspect, the present invention provides a mutant CLE protein, wherein the nucleotide sequence encoding the mutant CLE protein has a base mutation relative to the nucleotide sequence of the parent CLE protein.

[0103] In one embodiment, the parent CLE protein is derived from maize.

[0104] In one embodiment, the CLE protein is selected from one or any several of CLE18, CLE5, CLE26, CLE32 and CLE4A; preferably, CLE18 protein.

[0105] On the other hand, the present invention provides 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, wherein the gene editing reagent is capable of mutating the CLE gene in corn; the gene editing reagent includes a Cas enzyme and gRNA, and the gRNA includes a guide sequence targeting the nucleotide sequence of the CLE gene in corn.

[0106] In one embodiment, the CLE gene is selected from one or any several of CLE18, CLE5, CLE26, CLE32 and CLE4A; preferably, CLE18 gene.

[0107] In one embodiment, the guide sequence contains a portion of the nucleotide sequence of the parental CLE gene, preferably contains at least 15 bp of the nucleotide sequence of the CLE gene, and more preferably contains at least 20 bp of the nucleotide sequence of the CLE gene.

[0108] In one embodiment, the gene editing reagent further comprises a gene editing enzyme. The gene editing enzyme includes nucleases of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Tanscription Activator-like (TAL) effector nucleases), and ZFN (Zinc finger nuclease) editing tools.

[0109] Preferably, the gene editing enzyme is a Cas protein, also known as CRISPR enzyme or Cas effector protein, and its types include but are not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, FDK1 protein.

[0110] Preferably, the Cas protein is operably linked to a first regulatory element.

[0111] 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 connected to the guide sequence (or targeting sequence, spacer sequence), it constitutes a guide sequence (gRNA). Preferably, the gRNA is operably connected to the second regulatory element.

[0112] Such regulatory elements include promoters, terminator sequences, leader sequences, polyadenylation sequences, signal peptide coding regions, marker genes, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences).

[0113] On the other hand, the present invention provides a reagent or kit, which can be used to increase the number of corn ear rows, ear width, grain weight or improve corn yield, and the reagent or kit contains the above-mentioned gene editing reagent.

[0114] On the other hand, the present invention provides the use of the above-mentioned gene editing reagent in preparing corn with increased number of ear rows, increased ear width, increased grain weight or increased yield; or the use of the above-mentioned gene editing reagent in preparing a reagent or kit for preparing corn with increased number of ear rows, increased ear width, increased grain weight or increased yield.

[0115] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0116] The terms "polynucleotide," "nucleotide sequence," "nucleic acid sequence," "nucleic acid molecule," and "nucleic acid" are used interchangeably and include DNA, RNA, or hybrids thereof, which may be double-stranded or single-stranded.

[0117] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of naturally occurring amino acid residues. The proteins and polypeptides of the present invention can be produced recombinantly or by chemical synthesis. The term "mutant" or "mutant protein" refers to a protein that has one or more amino acid residue substitutions, insertions, deletions, and / or additions compared to the amino acid sequence of a parent protein.

[0118] The term "amino acid" refers to a carboxylic acid containing an amino group. Various proteins in living organisms are composed of 20 basic amino acids.

[0119] The term "encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in a biological process having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system.

[0120] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the 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 x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent 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)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0121] The term "regulatory element," also known as a "regulatory element," as used herein, is intended to include promoters, terminator sequences, leader sequences, polyadenylation sequences, signal peptide coding regions, marker genes, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences), which are described in detail in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CA (1990). In some cases, regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (e.g., lymphocytes). In some cases, regulatory elements can also direct expression in a temporally 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 WPRE; CMV enhancer; R-U5' fragment in the LTR of HTLV-I ((Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); SV40 enhancer; and intron sequences between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0122] As used herein, the term "promoter" has a meaning well known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of a gene that can initiate expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in 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, results in the production of the gene product in the 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, results in the production of the gene product in the cell substantially only when the cell is a cell of the tissue type corresponding to the promoter.

[0123] The term "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" proteins for import into the cell nucleus via nuclear transport. That is, proteins with an NLS are transported to the cell nucleus. Typically, an NLS comprises a positively charged Lys or Arg residue exposed on the protein surface. Exemplary NLSs include, but are not limited to, NLSs from the SV40 large T antigen, EGL-13, c-Myc, and TUS proteins.

[0124] As used herein, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows for 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).

[0125] wild type

[0126] As used herein, the term "wild type" has the meaning generally understood by those skilled in the art to refer to the typical form of an organism, strain, gene, or characteristic as it exists in nature, as distinguished from mutant or variant forms, which can be isolated from a source in nature and has not been intentionally modified by man.

[0127] carrier

[0128] The term "vector" refers to a nucleic acid molecule that is capable of transporting another nucleic acid molecule to which it is attached. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, or no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other various polynucleotides known in the art. A vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements it carries are expressed in the host cell. A vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including 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 a variety of 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.

[0129] One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, eg, by standard molecular cloning techniques.

[0130] Another type of vector is a viral vector, in which a virally derived DNA or RNA sequence is present in a vector for packaging a virus (e.g., a retrovirus, a replication-defective retrovirus, adenovirus, a replication-defective adenovirus, and adeno-associated virus). The viral vector also comprises a polynucleotide carried by a virus for transfection into a host cell. Some vectors (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) can replicate autonomously in the host cell into which they are introduced.

[0131] Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell and are thereby replicated along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0132] host cells

[0133] As used herein, the term "host cell" refers to cells that can be used to introduce 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.

[0134] Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression desired, and the like.

[0135] animal

[0136] For example, mammals, such as bovines, equines, ovines, porcines, canines, felines, lagomorphs, rodents (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-associated gene defect).

[0137] plant

[0138] The term "plant" is to be understood as meaning any differentiated multicellular organism capable of photosynthesis, including crop plants, in particular monocotyledonous or dicotyledonous plants, at any stage of maturity or development, vegetable crops, including artichokes, Brussels sprouts, rocket, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, romaine lettuce), bok choy, yellow taro, melons (e.g., cantaloupe, watermelon, Crenshaw melon, honeydew melon, cantaloupe), oilseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, bok choy), cardoon, carrot, napa, okra, onion, celery, parsley, chickpeas, parsnips, endive, peppers, potatoes, cucurbits (e.g., zucchini, cucumber, courgette, squash, pumpkin), radish, cabbage, Onions, rutabagas, eggplant (also known as eggplant), salsify, lettuce, shallots, endive, garlic, spinach, green onions, squash, greens, beets (sugar beets and fodder beets), sweet potatoes, Swiss chard, horseradish, tomatoes, turnips, and spices; fruits and / or vines such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, y), cranberries, currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pome fruits, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, meadowsweet, corn / maize (feed corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereals (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, and legumes (beans, lentils, peas, soybeans) , oil plants (rapeseed, mustard, poppy, olive, sunflower, coconut, castor oil plant, cocoa bean, peanut), Arabidopsis, fiber plants (cotton, flax, hemp, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plant; and / or bedding plants, such as flowering plants, cacti, succulents and / or ornamental plants, as well as trees such as forests (broadleaf trees and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other seedlings.

[0139] The term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant pieces, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, ears, roots, root tips, anthers, and the like.

[0140] The term "plant cell" is to be understood as any cell from or found in a plant, which is capable of forming, for example, undifferentiated tissue such as callus, differentiated tissue such as embryos, plant components, plants or seeds.

[0141] gene editing

[0142] The term "gene editing" technology includes CRISPR technology, TALEN technology, and ZFN technology. CRISPR technology refers to clustered, regularly interspaced short palindromic repeats, which come 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 a specific DNA sequence, 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 a specific DNA sequence, which includes a zinc finger DNA binding domain and a DNA cleavage domain. It is well known to those skilled in the art that by constructing the nucleotides encoding the gene editing tool and other regulatory elements into a suitable vector and then transforming the cell, the editing of the genome in the cell can be achieved. The types of editing include gene knockout, insertion, and base editing.

[0143] CRISPR system

[0144] 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 associated with the expression of CRISPR-associated ("Cas") genes, or transcripts or other elements capable of directing the activity of the Cas genes.

[0145] Cas proteins

[0146] Cas protein, or CRISPR-related protein refers to a nuclease suitable for 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 depending on its source, such as SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus; it may also be classified according to structural features (such as domains), such as the Cas12 family including Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12i, etc. The Cas protein may have double-stranded or single-stranded or no cutting activity. The Cas protein of the present invention may be a wild type or a mutant thereof, and the mutation type of the mutant includes amino acid replacement, substitution or deletion, and the mutant may or may not change the enzymatic 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. The known protein or its modified variant can achieve the function of the present invention, and is herein incorporated by reference into the scope of protection.

[0147] Those skilled in the art will appreciate that protein structure can be altered without adversely affecting its activity and functionality. For example, one or more conservative amino acid substitutions can be introduced into a protein's amino acid sequence without adversely affecting the activity and / or three-dimensional structure of the protein molecule. Examples and implementations of conservative amino acid substitutions will be apparent to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the substituted residue, i.e., a non-polar amino acid residue can be substituted for another non-polar amino acid residue, a polar uncharged amino acid residue can be substituted for another polar uncharged amino acid residue, a basic amino acid residue can be substituted for another basic amino acid residue, and an acidic amino acid residue can be substituted for another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions, where one amino acid is replaced with another amino acid belonging to the same group, fall within the scope of the present invention, as long as the substitution does not inactivate the biological activity of the protein. Therefore, the proteins of the present invention may contain one or more conservative substitutions in their amino acid sequences, preferably generated by substitutions according to Table 1. Furthermore, the present invention also encompasses proteins containing one or more other non-conservative substitutions, as long as such non-conservative substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention.

[0148] Conservative amino acid replacement can be carried out 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 by an amino acid residue with a similar side chain.Amino acid replacement can be carried out in the non-conserved region of the above-mentioned Cas mutant protein.In general, such replacement is not carried out for conserved amino acid residues, or is not carried out for amino acid residues located within a conserved motif, where such residues are required for protein activity.However, it will be appreciated by those skilled in the art that functional variants can have less conservative or non-conservative changes in conserved regions.

[0149] Table 1

[0150] It is well known in the art that one or more amino acid residues can be changed (replaced, deleted, truncated or inserted) from the N and / or C terminus of a protein while still retaining its functional activity. Therefore, proteins in which one or more amino acid residues are changed 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 changes may include changes introduced by modern molecular methods such as PCR, which includes PCR amplification of a protein coding sequence by means of including an amino acid coding sequence among the oligonucleotides used in the PCR amplification.

[0151] It will 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-described proteins can be prepared by mutations in the DNA. Other forms of mutagenesis and / or directed evolution can also be accomplished, for example, using known mutagenesis, recombination, and / or shuffling methods, in combination with relevant screening methods, to perform single or multiple amino acid substitutions, deletions, and / or insertions.

[0152] It will be appreciated by those skilled in the art 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 domains of the protein, the properties of the polypeptide may be changed, but the polypeptide may retain its activity. If the mutations present are not close to the catalytic domain, active site, or other functional domains, a smaller effect can be expected.

[0153] Those skilled in the art can identify the essential amino acids of the Cas mutant protein of the present invention according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of bioinformatics systems. The catalytic domain, active site or other functional domain of the protein can also be determined by physical analysis of the structure, such as by the following techniques: such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, combined with mutations of amino acids at putative key sites.

[0154] In the present invention, amino acid residues can be represented by single letters or three letters, 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).

[0155] The term "AxxB" means that the amino acid A at position xx is changed to amino acid B. Unless otherwise specified, the amino acid A at position xx from the N-terminus is changed to amino acid B.

[0156] Those skilled in the art can use commonly used software in the art, such as Clustal Omega, to compare and align the amino acid sequence of any parent CLE18 protein with SEQ ID No. 1 for sequence identity, thereby obtaining the amino acid sites in the parent CLE18 protein corresponding to the amino acid sites defined based on SEQ ID No. 1 described in this application.

[0157] Guide RNA (gRNA)

[0158] As used herein, the terms "guide RNA (gRNA)", "mature crRNA", and "guide sequence" are used interchangeably and have meanings generally understood by those skilled in the art. Generally speaking, a guide RNA may comprise a Scaffold sequence and a spacer sequence (or referred to as a targeting sequence, a guide sequence), or may consist essentially of or consist of a Scaffold and a spacer sequence.

[0159] 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 guide the specific binding of the CRISPR / Cas complex to the target sequence. In one embodiment, when optimally aligned, the degree of complementarity between a 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 optimal alignment is within the capabilities 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, Smith-Waterman in matlab, Bowtie, Geneious, Biopython, and SeqMan.

[0160] Target sequence

[0161] "Target sequence" refers to a polynucleotide targeted by a guide sequence in a gRNA, such as a sequence having complementarity with the guide sequence, wherein hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR / Cas complex (including Cas protein and gRNA). Complete complementarity is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR / Cas complex.

[0162] The target sequence can comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located inside or outside the cell. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. A sequence or template that can be used to recombine into a target locus comprising the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.

[0163] In the present invention, a "target sequence" or "target polynucleotide" or "target nucleic acid" can be any polynucleotide that is endogenous or exogenous to a cell (e.g., a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide that is 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 associated with a protospacer adjacent motif (PAM).

[0164] CLE gene

[0165] The CLAVATA3 / EMBRYOSURROUNDINGREGION (CLE) family of genes encodes small secreted proteins that play a variety of biological roles in plant growth and development and in response to abiotic stresses. Literature has shown that some genes in the CLE family act as feedback signals from stem cells to tissue centers, inhibiting WUS expression and limiting stem cell populations.

[0166] The maize CLE family contains nearly 50 related genes, and the functions of most of the related genes and their effects on plant growth and development are still unclear.

[0167] In the present application, the NCBI accession number of the corn CLE18 gene is LOC111589273, the amino acid sequence is shown in SEQ ID No. 1, and the nucleotide sequence is shown in SEQ ID No. 2; the NCBI accession number of the corn CLE4A gene is LOC103644198, the amino acid sequence is shown in SEQ ID No. 3, and the nucleotide sequence is shown in SEQ ID No. 4; the NCBI accession number of the corn CLE5 gene is LOC100216662, the amino acid sequence is shown in SEQ ID No. 5, and the nucleotide sequence is shown in SEQ ID No. 6; the NCBI accession number of the corn CLE26 gene is LOC100277806, the amino acid sequence is shown in SEQ ID No. 7, and the nucleotide sequence is shown in SEQ ID No. 8; the NCBI accession number of the corn CLE32 gene is LOC100277301, the amino acid sequence is shown in SEQ ID No. 9, and the nucleotide sequence is shown in SEQ ID No. 10.

[0168] The main advantages of the present invention are:

[0169] The present invention has found through research that, compared with wild-type corn plants, corn plants with CLE18 gene mutations have increased ear rows, increased ear width, increased grain weight, and improved yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0170] Figure 1 is the phylogenetic tree of the CLE gene.

[0171] Figure 2 is a comparison of the number of ear rows between different mutant lines and the parental corn inbred line.

[0172] FIG3 is a comparison of the number of ear rows between the mutant line 3840AR108-16 and the parental corn inbred line.

[0173] FIG4 is a comparison of ear lengths of different mutant lines and the parental corn inbred line.

[0174] FIG5 is a comparison of ear width between different mutant lines and the parental corn inbred line.

[0175] FIG6 is a comparison of 100-grain weight of different mutant strains and the parental corn inbred line.

[0176] FIG7 is a comparison of plant heights of different mutant lines and the parental corn inbred line.

[0177] FIG8 is a comparison of kernel weight per ear between the mutant line 3840AR108-16 and the parental corn inbred line.

[0178] Sequence Listing DETAILED DESCRIPTION

[0179] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0180] Example 1. Target design and vector construction

[0181] The sequences of CLE gene family members were downloaded from the gene database, and the protein sequences were compared and analyzed to construct a phylogenetic tree. The phylogenetic tree is shown in Figure 1.

[0182] 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 Shown in No.10.

[0183] Amino acid sequence of CLE18:

[0184] Nucleotide sequence of CLE18:

[0185] Amino acid sequence of CLE4A:

[0186] Nucleotide sequence of CLE4A:

[0187] Amino acid sequence of CLE5:

[0188] Nucleotide sequence of CLE5:

[0189] Amino acid sequence of CLE26:

[0190] Nucleotide sequence of CLE26:

[0191] Amino acid sequence of CLE32:

[0192] Nucleotide sequence of CLE32:

[0193] A multi-gene knockout vector was constructed using Cas9 protein and gRNA targeting a maize CLE gene in the phylogenetic tree. The specific operation method can be carried out according to conventional methods in the art. Vector construction can be referenced in 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).

[0194] Specifically, in this embodiment, the guide sequence of gRNA (sequence that hybridizes with the target sequence) is as follows:

[0195] The vector was introduced into a maize inbred line via Agrobacterium infection. In this embodiment, the parent maize inbred line F003 was used as the recipient. PCR was used to identify gene-edited positive material. Two to three generations of self-pollination were then performed to increase the mutant population and obtain homozygous edited seedlings without exogenous gene insertion.

[0196] After identification and field screening, the above vectors were used to transform pure and mutant strains 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57. The specific editing types are:

[0197] The line 3840AR108-16 is a CLE18 single mutant plant, and the nucleotide sequence of its CLE18 gene is deleted at bases 428-429 relative to the sequence shown in SEQ ID No. 2.

[0198] The line 3263AR65-1 is a CLE32 single mutant plant, and the nucleotide sequence of its CLE32 gene has an insertion of a base A after the 377th base of the sequence shown in SEQ ID No. 10.

[0199] The line 3263AR67-15 is a CLE32 single mutant plant, and the nucleotide sequence of its CLE32 gene is deleted at base 379 relative to the sequence shown in SEQ ID No. 10.

[0200] The line 3094AR52-15 is a CLE26 single mutant plant, and the nucleotide sequence of its CLE26 gene has a base C inserted after the 356th base of the sequence shown in SEQ ID No. 8.

[0201] The line 3262AR114-59 is a CLE5 single mutant plant, and the nucleotide sequence of its CLE5 gene is deleted at bases 381 to 418 relative to the sequence shown in SEQ ID No. 6.

[0202] The line 3262AR19-57 is a CLE4A single mutant plant, and the nucleotide sequence of its CLE4A gene is deleted at base 1121 relative to the sequence shown in SEQ ID No. 4.

[0203] The editing types of mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 are summarized as follows:

[0204] The traits of the parental maize inbred lines (F003 in Figures 2-7 and WT in Figure 8) and the mutant lines were observed and statistically analyzed, including plant height, ear length, ear width, number of ear rows, kernel weight per ear, and 100-kernel weight.

[0205] Figure 2 shows a comparison of the number of ear rows of mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 with their parental maize inbred lines. As can be seen from Figure 2, the number of ear rows of mutant lines 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 showed no significant difference from their parental maize inbred lines; however, the number of ear rows of mutant line 3840AR108-16 was significantly increased compared to the parental maize inbred line (in this example, the number of ear rows refers to the number of rows in the thickest part of the middle of the maize ear).

[0206] FIG3 is a comparison of the number of ear rows of the mutant line 3840AR108-16 (cle18 in FIG3 ) and the parental corn inbred line; FIG3 shows that the number of ear rows of the mutant line 3840AR108-16 is significantly increased compared with the parental corn inbred line.

[0207] Figure 4 shows a comparison of ear lengths between the mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 and their parental maize inbred lines. As can be seen from Figure 4, there are no significant differences in ear lengths between the mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 and their parental maize inbred lines.

[0208] Figure 5 shows a comparison of ear widths of mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 with their parental maize inbred lines. As can be seen from Figure 5, the ear widths of mutant lines 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 were not significantly different from those of their parental maize inbred lines; however, the ear width of mutant line 3840AR108-16 was significantly greater than 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).

[0209] Figure 6 shows a comparison of 100-kernel weight between the mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 and their parental maize inbred lines. As shown in Figure 6, the 100-kernel weight of the mutant lines 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 showed no significant difference from that of the parental maize inbred lines; however, the 100-kernel weight of the mutant line 3840AR108-16 was significantly higher than that of the parental maize inbred line.

[0210] Figure 7 shows a comparison of plant height between the mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 and their parental maize inbred lines. As can be seen from Figure 7, there is no significant difference in plant height between the mutant lines 3840AR108-16, 3263AR65-1, 3263AR67-15, 3094AR52-15, 3262AR114-59, and 3262AR19-57 and their parental maize inbred lines.

[0211] FIG8 is a comparison of the grain weight per ear of the mutant line 3840AR108-16 and the parental corn inbred line (WT in FIG8 ); as can be seen from FIG8 , the grain weight per ear of the mutant line 3840AR108-16 is significantly increased compared with the parental corn inbred line.

[0212] Therefore, the cle18 mutant line 3840AR108-16 had increased ear row number, ear width, kernel weight per ear, 100-kernel weight, and yield.

[0213] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for increasing the number of corn ear rows, increasing the width of corn ears, increasing the weight of corn kernels, or improving corn yield, characterized in that: The method includes 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: Including steps: mutating a 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 a corn cell, corn seed, corn tissue or corn part; (b) regenerating the corn cells, corn tissues, or corn parts from 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 a corn cell, corn seed, corn tissue, or corn part using a Cas enzyme and a guide RNA (gRNA) to obtain a gene-edited corn cell, corn seed, corn tissue, or corn part; wherein the gRNA comprises a guide sequence that targets a nucleotide sequence of a CLE18 gene in corn; (b) regenerating the gene-edited corn cell, corn seed, corn tissue or corn part of 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 ear rows, increasing corn ear width, increasing corn kernel weight, or improving corn yield, characterized in that: The gene editing reagent is capable of mutating the CLE18 gene in corn; the gene editing reagent includes a Cas enzyme and a gRNA, and the gRNA includes a guide sequence that targets the 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 according to claim 8 in preparing corn with increased number of ear rows, increased ear width, increased grain weight or improved yield; or use of the gene editing reagent according to claim 8 in preparing a reagent or kit for producing corn with increased number of 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 the method according to any one of claims 1 to 7 with other corns to produce corn with increased ear rows, increased ear width, increased grain weight or increased yield.

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