Method for improving insect resistance and disease resistance of plant

Abstract

A method for improving plant traits. The method comprises the step of mutating a BX13 gene and a BX14 gene. Upon mutation of the BX13 gene and the BX14 gene, for a plant, the insect resistance is improved, the disease resistance is improved, the fruit cluster is increased, the grain is increased, the hundred-grain weight is increased, and the yield is improved, achieving important application value in plant breeding.

Description

A method for improving plant insect resistance and disease resistance

[0001] This application claims priority to Chinese patent application CN202311094713.4, filed on August 29, 2023. 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 improving the insect resistance, disease resistance and yield of corn, in particular, improving the insect resistance and disease resistance of corn, enlarging corn ears and grains, increasing the 100-grain weight of corn, and improving corn yield by mutating the BX13 gene and the BX14 gene. Background Art

[0003] Corn is an important food, feed, and industrial raw material, playing a crucial role in global grain production. Corn production is significantly impacted by the occurrence of corn pests and diseases. Currently, major corn pests include the corn armyworm, corn borer, fall armyworm, and cotton bollworm. Corn diseases include ear rot, stalk rot, corn leaf spot, and southern rust.

[0004] In order to improve the insect resistance and disease resistance of corn, we study the relevant genes in corn in order to expand the insect resistance and pathogen resistance spectrum of corn and obtain corn strains with excellent traits.

[0005] Summary of the Invention

[0006] The object of the present invention is to provide a method for improving plant traits.

[0007] A first aspect of the present invention provides a method for improving plant traits, comprising the step of mutating the BX13 gene and / or the BX14 gene.

[0008] In another preferred embodiment, the amino acid sequence encoded by the BX13 gene is selected from the following group:

[0009] (i) a polypeptide having the amino acid sequence shown in SEQ ID NO.: 1;

[0010] (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;

[0011] 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.

[0012] In another preferred embodiment, the amino acid sequence encoded by the BX14 gene is selected from the following group:

[0013] (i) a polypeptide having the amino acid sequence shown in SEQ ID NO.: 2;

[0014] (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.: 2;

[0015] 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.: 2 and having the same or similar functions.

[0016] In another preferred embodiment, the nucleotide sequence of the BX13 gene is selected from the following group:

[0017] (a) a polynucleotide encoding the polypeptide shown in SEQ ID NO.: 1;

[0018] (b) a polynucleotide whose sequence is shown in SEQ ID NO.: 3;

[0019] (c) a polynucleotide having a nucleotide sequence homology ≥95% (preferably ≥98%, more preferably ≥99%) to the sequence shown in SEQ ID NO.: 3;

[0020] (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 represented by SEQ ID NO.: 3;

[0021] (e) A polynucleotide complementary to the polynucleotide described in any one of (a) to (d).

[0022] In another preferred embodiment, the nucleotide sequence of the BX14 gene is selected from the following group:

[0023] (a) a polynucleotide encoding the polypeptide shown in SEQ ID NO.: 2;

[0024] (b) a polynucleotide whose sequence is shown in SEQ ID NO.: 4;

[0025] (c) a polynucleotide having a nucleotide sequence homology of ≥95% (preferably ≥98%, more preferably ≥99%) to the sequence shown in SEQ ID NO.: 4;

[0026] (d) a polynucleotide having 1-60 (preferably 1-30, more preferably 1-10) nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide shown in SEQ ID NO.: 4;

[0027] (e) A polynucleotide complementary to the polynucleotide described in any one of (a) to (d).

[0028] In another preferred embodiment, the amino acid sequence encoded by the BX13 gene has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity compared to SEQ ID No. 1. In another preferred embodiment, the amino acid sequence encoded by the BX13 gene is as shown in SEQ ID No. 1. In another preferred embodiment, the accession number of the BX13 gene is Zm00001d007718.

[0029] In another preferred embodiment, the amino acid sequence encoded by the BX14 gene has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity compared to SEQ ID No. 2. In another preferred embodiment, the amino acid sequence encoded by the BX14 gene is as shown in SEQ ID No. 2. In another preferred embodiment, the accession number of the BX14 gene is Zm00001d004921.

[0030] In another preferred embodiment, the improved plant traits are selected from one or more of the following (i)-(vi):

[0031] (i) Improve plant resistance to insects;

[0032] (ii) improving plant disease resistance;

[0033] (iii) enlarge the fruit cluster;

[0034] (iv) enlarge the grain size;

[0035] (v) Increase 100-grain weight;

[0036] (vi) Increase plant yield.

[0037] In another preferred embodiment, the improving the insect resistance of plants refers to increasing the resistance of plants to chewing pests, piercing-sucking pests, or boring pests.

[0038] In another preferred embodiment, the improving the insect resistance of plants refers to increasing the resistance of plants to chewing pests.

[0039] In another preferred embodiment, the chewing pests are armyworms, fall armyworms, corn borers, sawflies, locusts, yellow stem borers, corn borers, cutworms, cotton bollworms, or cabbage worms.

[0040] In another preferred embodiment, the chewing pests are armyworms, fall armyworms, or corn borers.

[0041] In another preferred embodiment, the chewing pests are armyworms.

[0042] In another preferred embodiment, the improving the insect resistance of plants refers to increasing the resistance of plants to armyworms.

[0043] In another preferred embodiment, the improving plant disease resistance refers to increasing the plant's resistance to stem rot.

[0044] In another preferred embodiment, the improving plant disease resistance refers to increasing the plant's resistance to stem rot-related pathogens.

[0045] In another preferred embodiment, the improving plant disease resistance refers to increasing the plant's resistance to Fusarium graminearum, Fusarium graminearum, Penicillium, Aspergillus, Cladosporium, or Monosporus.

[0046] In another preferred embodiment, the improving plant disease resistance refers to increasing the plant's resistance to Fusarium graminearum.

[0047] In another preferred embodiment, the mutation 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.

[0048] 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.

[0049] In another preferred embodiment, the gene editing technology is selected from the group consisting of CRISPR technology, TALEN technology, ZFN technology, or a combination thereof. In another preferred embodiment, the method comprises administering an inhibitor of the BX13 gene and / or the BX14 gene.

[0050] In another preferred embodiment, the inhibitor is selected from the following group: antisense nucleic acid, antibody, small molecule compound, CRISPR reagent, small molecule ligand, or a combination thereof.

[0051] In another preferred embodiment, the method comprises the steps of:

[0052] (i) providing a plant or plant cell; and

[0053] (ii) using gRNA targeting the BX13 gene and / or the BX14 gene and the corresponding Cas protein to introduce into the plant or plant cell; in a preferred embodiment, an expression vector containing the gRNA and Cas protein is introduced into the plant or plant cell.

[0054] In another preferred embodiment, the Cas protein is Cas9.

[0055] In another preferred embodiment, the method comprises the steps of:

[0056] (i) providing a plant or plant cell; and

[0057] (ii) introducing an inhibitor of the BX13 gene and / or the BX14 gene into the plant or plant cell, thereby obtaining a modified plant or plant cell.

[0058] In another preferred embodiment, the mutation includes insertion mutation, deletion mutation, frameshift mutation, and substitution mutation.

[0059] In a preferred embodiment, the method comprises reducing or inhibiting the expression level and / or activity of the BX13 gene and / or the BX14 gene.

[0060] In another preferred example, the reduction or inhibition refers to that the expression level E1 of the BX13 gene and / or BX14 gene in the plant is 0-80% of the wild type, preferably 0-60%, more preferably 0-40%, and more preferably 0-30% compared with the expression level E0 of the BX13 gene and / or BX14 gene in the wild type plant.

[0061] In another preferred embodiment, the reduction or inhibition of the expression and / or activity of the BX13 gene and / or BX14 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.

[0062] In another preferred embodiment, the mutation causes complete loss of function or partial loss of function of the BX13 gene; preferably, the mutation causes complete loss of function of the BX13 gene.

[0063] In another preferred embodiment, the mutation causes complete loss of function or partial loss of function of the BX14 gene; preferably, the mutation causes complete loss of function of the BX14 gene.

[0064] In another preferred embodiment, the mutated nucleotide sequence of the BX13 gene is missing some bases relative to the sequence shown in SEQ ID NO. 3. In another preferred embodiment, the mutated nucleotide sequence of the BX13 gene is missing 242 bases after base 144 relative to the sequence shown in SEQ ID NO. 3.

[0065] In another preferred embodiment, the nucleotide sequence of the BX14 gene mutated is a deletion of some bases relative to the sequence shown in SEQ ID NO. 4. In another preferred embodiment, the nucleotide sequence of the BX14 gene mutated is a deletion of two bases after base 572 relative to the sequence shown in SEQ ID NO. 4. In another preferred embodiment, the nucleotide sequence of the BX14 gene mutated is a deletion of 197 bases after base 376 relative to the sequence shown in SEQ ID NO. 4.

[0066] In another preferred embodiment, the nucleotide sequence of the BX13 gene mutated has one or more bases inserted relative to the sequence shown in SEQ ID NO. 3. In another preferred embodiment, the nucleotide sequence of the BX13 gene mutated has one base inserted after base 145 relative to the sequence shown in SEQ ID NO. 3.

[0067] In another preferred embodiment, the mutation is a nucleotide sequence of the BX14 gene in which one or more bases are inserted relative to the sequence shown in SEQ ID NO.4.

[0068] In another preferred embodiment, the nucleotide sequence of the mutated BX13 gene is shown in either SEQ ID NO.5 or SEQ ID NO.7.

[0069] In another preferred embodiment, the nucleotide sequence of the mutated BX14 gene is shown in either SEQ ID NO.6 or SEQ ID NO.8.

[0070] In another preferred embodiment, the plants include monocotyledonous plants and dicotyledonous plants.

[0071] In another preferred embodiment, the plant is selected from one or more plants of the following group: Leguminosae, Cruciferae, Poaceae, Solanaceae, Cucurbitaceae, Chenopodiaceae, Polygonaceae, Pedulaceae, Asteraceae, Malvaceae, Rosaceae, Pedulaceae, Convolvulaceae, Dioscoreaaceae, Apiaceae, Liliaceae, Zingiberaceae, and Palmaceae.

[0072] In another preferred embodiment, the plant is derived from one or more plants selected from the group consisting of soybean, Arabidopsis, rice, tobacco, tomato, potato, corn, cotton, alfalfa, sorghum, barley, wheat, millet, sweet potato, quinoa, lettuce, rapeseed, cabbage, spinach, beet, peanut, watermelon, cabbage, strawberry, cucumber, coconut, or a combination thereof.

[0073] In another preferred embodiment, the plant is selected from soybean, Arabidopsis, rice, tobacco, tomato, potato, corn, cotton, peanut, sorghum, cucumber, and coconut.

[0074] In another preferred embodiment, the plant is corn.

[0075] In another preferred embodiment, the corn is Wannuo 2000 female parent W67.

[0076] A second aspect of the present invention provides a composition for improving plant traits, comprising:

[0077] (a) an inhibitor of the BX13 gene and / or the BX14 gene; and, optionally,

[0078] (b) an agriculturally acceptable carrier.

[0079] In another preferred example, the amino acid sequence encoded by the BX13 gene has at least 70% sequence identity with SEQ ID NO.: 1; the amino acid sequence encoded by the BX14 gene has at least 70% sequence identity with SEQ ID NO.: 2.

[0080] In a preferred embodiment, the inhibitor can reduce or inhibit the expression level and / or activity of the BX13 gene and / or the BX14 gene.

[0081] In another preferred embodiment, the composition comprises an agricultural composition.

[0082] In another preferred embodiment, the inhibitor includes an agricultural inhibitor.

[0083] In another preferred embodiment, the dosage form of the composition is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.

[0084] In another preferred embodiment, the inhibitor is selected from the group consisting of gene editing reagents, antisense nucleic acids, antibodies, small molecule compounds, Crispr reagents, small molecule ligands, or a combination thereof.

[0085] In one embodiment, the gene editing reagent includes a Cas enzyme and a gRNA capable of targeting a target gene.

[0086] In another preferred embodiment, the antisense nucleic acid is selected from the group consisting of antisense RNA, antisense DNA, interfering RNA, ribozyme, or a combination thereof.

[0087] In another preferred embodiment, the interfering RNA is selected from the group consisting of siRNA, shRNA, RNAi, miRNA, dsRNA, hpRNA, ihpRNA, or a combination thereof.

[0088] In another preferred embodiment, the composition further comprises other substances capable of improving plant traits.

[0089] In another preferred embodiment, the composition further comprises other substances that improve the insect resistance and / or disease resistance of the plant.

[0090] The third aspect of the present invention provides a use of the composition according to the second aspect of the present invention for improving plant traits.

[0091] In another preferred embodiment, the composition is used as a reagent or kit for preparing plants with improved traits.

[0092] A fourth aspect of the present invention provides a method for preparing a plant cell, plant seed, plant tissue, plant part, or plant with improved traits, comprising the steps of:

[0093] The expression and / or activity of the BX13 gene and / or the BX14 gene in plant cells, plant seeds, plant tissues, plant parts, or plants is reduced or inhibited, thereby obtaining plant cells, plant seeds, plant tissues, plant parts, or plants with improved traits.

[0094] In another preferred example, the amino acid sequence encoded by the BX13 gene has at least 70% sequence identity with SEQ ID NO.: 1; the amino acid sequence encoded by the BX14 gene has at least 70% sequence identity with SEQ ID NO.: 2.

[0095] A fifth aspect of the present invention provides a method for improving plant traits, comprising the steps of:

[0096] The plant cells, plant seeds, plant tissues, or plant parts with improved traits prepared by the method of the fourth aspect of the present invention are regenerated into plants, thereby obtaining plants with improved traits.

[0097] In another preferred embodiment, the method further comprises the step of harvesting plant seeds from the plant improved with the trait.

[0098] The sixth aspect of the present invention provides a plant cell, or plant seed, or plant tissue, or plant part, or plant with improved traits, characterized in that the plant cell, or plant seed, or plant tissue, or plant part, or plant is prepared by the method described in the fourth or fifth aspect of the present invention.

[0099] A seventh aspect of the present invention provides a method for improving plant traits, the method comprising the steps of:

[0100] (a) providing a plant cell, plant tissue, or plant part, and introducing an inhibitor of the BX13 gene and / or the BX14 gene into the plant cell, plant tissue, or plant part; or reducing or inhibiting the expression and / or activity of the BX13 gene and / or the BX14 gene in the plant cell, plant tissue, or plant part;

[0101] (b) regenerating the plant cells, plant tissues, or plant parts of step (a) into plants.

[0102] In another preferred example, the amino acid sequence encoded by the BX13 gene has at least 70% sequence identity with SEQ ID NO.: 1; the amino acid sequence encoded by the BX14 gene has at least 70% sequence identity with SEQ ID NO.: 2.

[0103] In another preferred embodiment, in step (a), the plant cells, plant tissues, and plant parts are modified using gene editing technology, thereby reducing the expression or activity of the BX13 gene and / or BX14 gene in the plant cells, plant tissues, and plant parts.

[0104] 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.

[0105] In another preferred embodiment, the improved trait is improved insect resistance and / or disease resistance.

[0106] The eighth aspect of the present invention provides a genetically engineered plant, which is prepared using the method described in the seventh aspect of the present invention.

[0107] A ninth aspect of the present invention provides a method for screening or identifying insect resistance and / or disease resistance of a plant, the method comprising the step of detecting the expression level of the BX13 gene and / or the BX14 gene.

[0108] In another preferred example, the amino acid sequence encoded by the BX13 gene has at least 70% sequence identity with SEQ ID NO.: 1; the amino acid sequence encoded by the BX14 gene has at least 70% sequence identity with SEQ ID NO.: 2.

[0109] In another preferred embodiment, the detection parts of the detection plant include plant callus, fruit, seeds, flowers, stems, leaves, ears, and roots.

[0110] In another preferred embodiment, the plant is selected from corn.

[0111] A tenth aspect of the present invention provides a method for preparing plant seeds with improved traits, the method comprising the step of preparing plant seeds with improved traits using the plants prepared by the above method.

[0112] The eleventh aspect of the present invention further provides a plant seed with improved traits, wherein the plant seed with improved resistance traits is prepared using the above method for preparing plant seeds with improved traits.

[0113] The twelfth aspect of the present invention also provides a method for preparing hybrid plants, which includes the steps of hybridizing plant seeds with improved traits prepared by the above method or plants with improved traits with other plants to prepare hybrid plants.

[0114] The thirteenth aspect of the present invention further provides a method for inhibiting the growth of pests or killing pests, the method comprising the steps of:

[0115] (a) preparing plant seeds, plant tissues, plant parts, or plants with improved traits using the above method;

[0116] (b) using the plant seeds, plant tissues, plant parts, or plants obtained in step (a) to feed pests.

[0117] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. DETAILED DESCRIPTION

[0118] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0119] As used herein, the "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.

[0120] The terms "homology" or "identity" are used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in the two 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. Between two sequences. Typically, comparison is made when the two sequences are aligned for maximum identity. Alignment methods are conventional methods known to those skilled in the art, such as the BLAST algorithm.

[0121] The term "genetic engineering" refers to the technology of artificially modifying and utilizing the nucleotides that control the genetic information of organisms to obtain new genetic traits, new varieties, or new products. This includes all genetic modification technologies disclosed in the art, such as gene mutagenesis, transgenic technology, and gene editing. Genetic mutagenesis methods include, but are not limited to, physical mutagenesis (such as ultraviolet mutagenesis), chemical mutagenesis (such as acridine dyes), and biological mutagenesis (such as viral and bacteriophage mutagenesis).

[0122] Specific amino acid positions (numbers) within the proteins of the present invention are determined by aligning the amino acid sequence of the target protein with any of SEQ ID Nos. 1-2 using standard sequence alignment tools, such as the Smith-Waterman algorithm or the CLUSTALW2 algorithm, wherein the sequences are considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80: 726-730. Preferably, the default parameters are used in the ClustalW2 (1.82) algorithm: protein gap open penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNAGAPDIST = 4. Preferably, the AlignX program (part of the vectorNTI group) is used to determine the position of specific amino acids in the protein of the present invention by comparing the amino acid sequence of the protein with any of the sequences of SEQ ID No. 1-2 using default parameters suitable for multiple alignment (gap opening penalty: 10; gap extension penalty 0.05).

[0123] 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.

[0124] 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.

[0125] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues, including polymers in which one or more amino acid residues is a chemical analog of a naturally occurring amino acid residue. The proteins and polypeptides of the present invention can be produced recombinantly or by chemical synthesis.

[0126] 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).

[0127] 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).

[0128] 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 essentially 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 essentially only when the cell is a cell of the tissue type corresponding to the promoter.

[0129] 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.

[0130] 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).

[0131] The term "vector" refers to a vector that contains elements that allow the vector to be integrated into the host cell genome or to replicate autonomously within the cell independently of the genome. The vector may contain any elements that ensure self-replication. It usually carries genes that are not part of the central metabolism of the cell and is usually in the form of double-stranded DNA. The choice of vector usually depends on the compatibility of the vector with the host cell into which the vector is to be introduced. If a vector is used, the choice of vector depends on methods well known to those skilled in the art for transforming host cells. For example, a plasmid vector can be used.

[0132] Vectors suitable for use in the present invention include commercially available plasmids such as, but not limited to, pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, WI, USA), pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174, pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8, pCM7, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0133] The nucleic acid sequence, nucleic acid construct or expression vector of the present invention can be introduced into the host cell by a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun or Ti-plasmid-mediated gene delivery, as well as calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofection or electroporation.

[0134] 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.

[0135] 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.

[0136] 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, ry), 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 (fodder corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereals (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, beans), oil plants (rapeseed, mustard, poppy, olive, sunflower, coconut, castor oil plant, cocoa bean, peanut), Arabidopsis, fiber plants (cotton, flax, 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 forest (broadleaf trees and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other seedlings.

[0137] 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, Cas12j, 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.

[0138] As used herein, the term "gene editing enzyme" refers to nucleases suitable for editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), TALEN (Transcription Activator-like (TAL) effector nuclease technology), and ZFN (Zinc finger nuclease technology). Preferably, the gene editing enzyme is a CRISPR enzyme, also known as a Cas protein, and its types include but are not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, and FDK1 protein. The Cas protein refers to a family of proteins, which may have different structures depending on their sources, such as SpCas9 derived from Streptococcus pyogenes, SaCas9 derived 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 mutant 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 reported in the prior art, the known protein or its modified variants can achieve the function of the present invention, and are herein incorporated by reference into the scope of protection.

[0139] As used herein, the terms "guide RNA," "mature crRNA," and "guide sequence" are used interchangeably and have meanings commonly understood by those skilled in the art.

[0140] BX gene

[0141] The protein encoded by the BX13 gene is a 2-oxoglutarate-dependent dioxygenase. The amino acid sequence of the BX13 gene in maize is shown in SEQ ID NO.:1, and the nucleotide sequence is shown in SEQ ID NO.:3. The protein encoded by the BX14 gene is a methyltransferase. The amino acid sequence of the BX14 gene in maize is shown in SEQ ID NO.:2, and the nucleotide sequence is shown in SEQ ID NO.:4.

[0142] It should be understood that although the genes provided in the examples of the present invention are derived from corn, gene sequences derived from other similar plants and having a certain homology (e.g., having a sequence identity of 70% or more, such as 70%, 75%, 80%, 85%, 90%, 95%, or even 98%, 99%, or 100%) with the sequences of the present invention (preferably, the sequences are shown in any one of SEQ ID NOs.: 1-4) are also included within the scope of the present invention, as long as those skilled in the art can easily isolate the sequence from other plants based on the information provided in this application after reading this application. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0143] The "same or similar functions" mainly refer to the activity of improving plant insect resistance, improving plant disease resistance, increasing ear size, increasing grain size, increasing 100-grain weight or increasing plant yield through the loss of protein activity.

[0144] Agricultural inhibitors

[0145] The active substances of the present invention (such as inhibitors of BX13 genes and / or BX14 genes) can be prepared into agricultural preparations by conventional methods, such as solutions, emulsions, suspensions, powders, foams, pastes, granules, aerosols, natural and synthetic materials impregnated with active substances, microcapsules in polymers, and coatings for seeds.

[0146] These preparations can be produced by known methods, for example, by mixing the active substance with an extender, i.e., a liquid, liquefied gas, or solid diluent or carrier, and optionally a surfactant, i.e., an emulsifier and / or dispersant and / or foam former. When, for example, water is used as the extender, an organic solvent can also be used as an auxiliary agent.

[0147] When a liquid solvent is used as a diluent or carrier, it is basically suitable, such as: aromatic hydrocarbons, for example xylene, toluene or alkylnaphthalene; chlorinated aromatic or chlorinated aliphatic hydrocarbons, for example chlorobenzene, vinyl chloride or dichloromethane; aliphatic hydrocarbons, for example cyclohexane or paraffin, for example mineral oil fractions; alcohols, for example ethanol or ethylene glycol and their ethers and lipids; ketones, for example acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone; or less commonly used polar solvents, for example dimethylformamide and dimethyl sulfoxide, and water.

[0148] As for the diluent or carrier of liquefied gas, it refers to the liquid that will become gas at normal temperature and pressure, such as aerosol propellants, such as halogenated hydrocarbons and butane, propane, nitrogen and carbon dioxide.

[0149] Solid carriers can be ground natural minerals such as kaolin, clay, talc, quartz, atavistic clay, montmorillonite, or diatomaceous earth, and ground synthetic minerals such as highly dispersed silicic acid, alumina, and silicates. Solid carriers for particles are crushed and graded natural zircons such as calcite, marble, pumice, sepiolite, and dolomite, as well as particles synthesized from inorganic and organic coarse powders, and particles of organic materials such as sawdust, coconut shells, corn cobs, and tobacco stems.

[0150] Nonionic and anionic emulsifiers can be used as emulsifiers and / or foam formers. Examples include polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, such as alkylaryl polyethylene glycol ethers, alkyl sulfonates, alkyl sulfates, aryl sulfonates, and albumin hydrolysates. Dispersants include, for example, lignin sulfite waste liquor and methylcellulose.

[0151] Binders such as carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or emulsions, such as gum arabic, polyvinyl alcohol and polyvinyl acetate, may be used in the formulations.

[0152] Colorants such as inorganic dyes, such as iron oxide, cobalt oxide and Prussian blue; organic dyes, such as azo dyes or metal phthalocyanine dyes; and trace nutrients such as salts of iron, manganese, boron, copper, cobalt, aluminum and zinc can be used.

[0153] In the present invention, the "agricultural preparation" is generally an agricultural plant growth regulator, which contains inhibitors of the BX13 gene and / or the BX14 gene as active ingredients for improving plant traits (e.g., improving plant insect resistance or disease resistance, increasing cobs, increasing grains, increasing 100-grain weight, and increasing plant yield); and an agriculturally acceptable carrier.

[0154] As used herein, the term "agriculturally acceptable carrier" is an agrochemically acceptable solvent, suspending agent, or excipient for delivering the active substance of the present invention to plants. The carrier can be liquid or solid. Suitable agriculturally acceptable carriers for use in the present invention are selected from the group consisting of water, buffer, DMSO, surfactants such as Tween-20, or combinations thereof. Any agriculturally acceptable carrier known to those skilled in the art can be used in the present invention.

[0155] The agriculturally acceptable inhibitors of the present invention may comprise agricultural compositions.

[0156] The agricultural formulation of the present invention can be used in combination with other substances that improve plant resistance to insects or diseases, increase ear size, increase seed size, increase 100-kernel weight, or increase plant yield. The other substances that improve plant resistance to insects or diseases, increase ear size, increase seed size, increase 100-kernel weight, or increase plant yield can be plant growth regulators known to those skilled in the art.

[0157] The agricultural formulation of the present invention can be in a variety of forms, as long as the active ingredients can effectively reach the plant body. From the standpoint of ease of preparation and application, the preferred agricultural formulation is a spray or solution formulation.

[0158] The agricultural formulations of the present invention typically contain 0.0001-99 wt%, preferably 0.1-90 wt%, of the active ingredient of the present invention, based on the total weight of the agricultural formulation. The concentration of the active ingredient of the present invention in commercial formulations or dosage forms can vary over a wide range. The concentration of the active ingredient of the present invention in commercial formulations or dosage forms can range from 0.0000001-100% (g / v), preferably between 0.0001 and 50% (g / v).

[0159] The main advantages of the present invention are:

[0160] The present invention has found through research that inhibiting the expression or activity of BX13 and BX14 genes can improve plant insect resistance and disease resistance, increase the size of ears and grains, increase 100-grain weight, and improve yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0161] Figure 1. Schematic diagram of gene editing vectors.

[0162] Figure 2. Growth curves of armyworms, where WT is armyworms fed on wild-type corn plants, L1 is armyworms fed on 74-3618 corn plants, L2 is armyworms fed on 75-3672 corn plants, and L3 is armyworms fed on 77-3740 corn plants.

[0163] Figure 3. The state of corn after armyworms gnawed on wild-type corn plants and gene-edited corn plants. WT is the wild-type corn plant, and bx13 / 14 is the corn plant in which both BX13 and BX14 were edited.

[0164] Figure 4. Diseased corn stalk interior 12 days after inoculation with Fusarium graminearum. The corn stalk sections shown in the figure are from top to bottom: the section above the inoculated site, the inoculated section, and the section below the inoculated site.

[0165] Figure 5. Ear and grain phenotypes of the wild-type Wannuo 2000 female parent W67 and edited plants 74-3618, 75-3672, and 77-3740.

[0166] Figure 6. Statistical bar chart of 100-grain weight of wild-type Wannuo 2000 female parent W67 and edited plants 74-3618 and 77-3740.

[0167] DETAILED DESCRIPTION

[0168] 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.

[0169] Example 1: Obtaining Gene-Edited Corn

[0170] In this example, Cas9 and sgRNA targeting the BX13 and BX14 genes were used to edit the above two genes in maize. The specific operation method can be carried out according to conventional methods in the art. The accession number of the BX13 gene is Zm00001d007718, the amino acid sequence is shown in SEQ ID No. 1, and the CDS sequence is shown in SEQ ID No. 3; the accession number of the BX14 gene is Zm00001d004921, the amino acid sequence is shown in SEQ ID No. 2, and the CDS sequence is shown in SEQ ID No. 4.

[0171] In this embodiment, a schematic diagram of the constructed gene editing vector is shown in Figure 1; wherein, ZmU6 promoter is U6 promoter, Gly-tRNA is glycine tRNA, and UBI promoter is UBI promoter; vector construction can also refer to the reference literature ("High-efficiency CRISPR / Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize", Weiwei Qi et al., "BMC Biotechnology", 2016).

[0172] Amino acid sequence encoded by the BX13 gene:

[0173] Amino acid sequence encoded by the BX14 gene:

[0174] CDS sequence of the BX13 gene:

[0175] CDS sequence of the BX14 gene:

[0176] In this embodiment, sgRNAs targeting the BX13 and BX14 genes were designed using target Design (http: / / skl.scau.edu.cn / targetdesign / ). The sgRNA sequences are shown in Table 1.

[0177] Table 1 sgRNA sequence information

[0178] The constructed vector was transferred into Agrobacterium and used to infect maize embryos. The embryos were cultured, screened, differentiated, and rooted to grow into complete plants. The primers in Table 2 were then used to confirm the editing form of the gene-edited maize (the maize variety used in this experiment was the wild type of Wannuo 2000 parent W67).

[0179] Table 2 Primer sequence information used to detect editing types

[0180] The maize plants 74-3618, 75-3672, and 77-3740 in which both BX13 and BX14 genes were edited were obtained by the above method. The editing types are shown in Tables 3 to 6, respectively:

[0181] The editing result of the edited plant 74-3618 was a 242-nucleotide deletion after +144nt in the CDS sequence of the BX13 gene (SEQ ID No. 3), resulting in a frameshift mutation in the subsequent coding region, which would lead to premature termination of protein translation. The two bases AC were deleted after +572nt in the CDS sequence of the BX14 gene (SEQ ID No. 4), resulting in a frameshift mutation thereafter, which would lead to premature termination of protein translation. The CDS sequence of the BX13 gene of the edited plant 74-3618 is shown in SEQ ID No. 5, and the CDS sequence of the BX14 gene is shown in SEQ ID No. 6.

[0182] The editing result of the edited plant 75-3672 was a 242-nt deletion after +144nt in the CDS sequence of the BX13 gene (SEQ ID No. 3), resulting in a frameshift mutation in the subsequent coding region, which would lead to premature termination of protein translation. The CDS sequence of the BX13 gene of the edited plant 75-3672 is shown in SEQ ID No. 5, and the CDS sequence of the BX14 gene is shown in SEQ ID No. 8.

[0183] The editing result of the edited plant 77-3740 is the addition of a T base after +145nt in the CDS sequence of the BX13 gene (SEQ ID No. 3), resulting in a frameshift mutation in the subsequent coding region, which will lead to premature termination of protein translation. The two bases AC are deleted after +572nt in the CDS sequence of the BX14 gene (SEQ ID No. 4), resulting in a frameshift mutation thereafter, which will lead to premature termination of protein translation. The CDS sequence of the BX13 gene of the edited plant 77-3740 is shown in SEQ ID No. 7, and the CDS sequence of the BX14 gene is shown in SEQ ID No. 6.

[0184] Table 3 Gene sequence alignment of BX13 gene at sgRNA1 in different edited plants

[0185] Table 4 Gene sequence alignment of BX13 gene at sgRNA2 in different edited plants

[0186] Table 5 Gene sequence alignment of BX14 gene at sgRNA1 in different edited plants

[0187] Table 6 Gene sequence alignment of BX14 gene at sgRNA2 in different edited plants

[0188] Note: The underlined part is the PAM sequence.

[0189] Edit the CDS sequence of the BX13 gene of plants 74-3618 and 75-3672:

[0190] Edit the CDS sequence of the BX14 gene of plants 74-3618 and 77-3740:

[0191] Edit the CDS sequence of the BX13 gene of plant 77-3740:

[0192] Edit the CDS sequence of the BX14 gene of plant 75-3672:

[0193] Example 2: Gene-edited corn for insect resistance

[0194] Feeding experiment: Wild-type maize plants (Wild-type Wannuo 2000 parent W67) and maize plants with both BX13 and BX14 edits (i.e., edited plants 74-3618, 75-3672, and 77-3740) were grown and tested for resistance to Mythimna separata walker larvae (a chewing pest). Well-grown and consistent larvae were selected, their initial weights recorded on a scale, and then individually placed in air- and light-permeable incubation boxes. Larvae in different boxes were fed leaves from different plant strains. Young, tender leaves from the same part of the plant were selected for feeding. The weight of each insect was recorded daily, and throughout the experiment, edible leaves were always available in each box to ensure the larvae's normal growth.

[0195] The results of the feeding experiment are shown in Figure 2. Compared with the wild-type corn plants (WT in Figure 2), the weight gain and growth rate of the armyworms fed with leaves of the edited plants 74-3618 (L1 in Figure 2), 75-3672 (L2 in Figure 2) and 77-3740 (L3 in Figure 2) were slower; that is, the corn plants in which BX13 / BX14 were edited (edited plants 74-3618, 75-3672 and 77-3740) had increased resistance to armyworms and improved insect resistance.

[0196] After the armyworms were inoculated on corn plants growing in the field and fed on them, the condition of the corn plants was observed. The results are shown in Figure 3. Compared with the wild-type corn plant W67 (WT in Figure 3), the leaves of the corn plant 74-3618 (bx13 / 14 in Figure 3) in which both BX13 / BX14 were edited were more intact and significantly less affected by insect pests. This also proves that the insect resistance of the corn plants in which both BX13 / BX14 were edited was improved.

[0197] Therefore, the mutation and inactivation of BX13 and BX14 genes can improve the insect resistance of plants, especially the resistance of plants to armyworms.

[0198] Example 3: Gene-edited corn for stalk rot resistance

[0199] Resistance testing was performed using Fusarium graminearum on wild-type maize plants (wild-type Wannuo 2000 female parent W67) and maize plants with both BX13 and BX14 edited (i.e., edited plants 74-3618 and 77-3740). The main steps are as follows:

[0200] (1) Culture of pathogenic bacteria spores:

[0201] The Fusarium graminearum cells grown on PDA medium were inoculated into the culture medium and cultured under dark conditions at 28°C and 200 rpm for about 72 h. After the culture was completed, the spore solution was filtered with double gauze and enriched or diluted to the working concentration. The number of spores was about 10 7 / mL.

[0202] (2) Vaccination identification:

[0203] Before the corn plants bloom, a 3 cm long and 0.5 cm deep wound is made with a scalpel in the middle of the fourth stem node near the ground. 2 mL of spore liquid is drawn with a syringe and inserted downward at a 45-degree angle at the wound for injection (the negative control group is a wild-type corn plant that was not injected with spore liquid but with the same volume of water). After inoculation, the field is watered once, and normal fertilizer and water management is carried out thereafter. 12 days after inoculation with the pathogen, the interior of the stems of wild-type corn plants and corn plants that have undergone BX13 / BX14 editing are photographed to identify the degree of disease susceptibility.

[0204] The results are shown in Figure 4. The stems of the negative control group (the HO-injected control in Figure 4) showed no obvious bacterial plaque and were not infected. The stems of the wild-type maize plants inoculated with the pathogen (W67-WT in Figure 4) were clearly infected, with the largest bacterial plaque area. Compared with the stems of wild-type maize plants, the stems of maize plants with both BX13 and BX14 edited (74-3618 and 77-3740 in Figure 4) were less infected, mainly manifested by lighter bacterial plaque color and smaller area. This indicates that both BX13 and BX14 editing can improve maize resistance to stalk rot; the inactivation of BX13 and BX14 genes can improve plant disease resistance, especially to stalk rot.

[0205] Example 4: Investigation of agronomic traits of gene-edited corn

[0206] Wild-type maize plants (wild-type Wannuo 2000 female parent W67) and BX13 / BX14 edited plants (i.e., edited plants 74-3618, 75-3672, and 77-3740) were sown in the field and normal water and fertilizer management was implemented. The seeds were harvested, dried, and threshed 45 days after artificial pollination, and the phenotypic analysis of ears, grains, and 100-grain weight was performed.

[0207] The results of ears and kernels are shown in Figure 5. The ears and kernels of wild-type corn plants (W67-WT in Figure 5) are smaller (due to the W67 inbred line); compared with wild-type corn plants, the ears and kernels of BX13 / BX14 edited plants 74-3618, 75-3672 and 77-3740 are significantly larger.

[0208] The results of 100-grain weight are shown in FIG6 . Compared with the wild-type maize plant (W67-WT in FIG6 ), the 100-grain weight of the BX13 / BX14 edited plants 74-3618 and 77-3740 was significantly increased.

[0209] This shows that the mutation and inactivation of BX13 and BX14 genes can increase the size of the ear, the size of the grain and the weight of 100 grains, thereby improving yield.

[0210] 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 improving plant traits, characterized in that: The method comprises the steps of mutating the BX13 gene and / or the BX14 gene; The amino acid sequence encoded by the BX13 gene has at least 70% sequence identity compared with SEQ ID NO.:1; the amino acid sequence encoded by the BX14 gene has at least 70% sequence identity compared with SEQ ID NO.:

2.

2. The method according to claim 1, characterized in that The improved plant traits are selected from one or more of the following (i)-(vi): (i) Improve plant resistance to insects; (ii) improving plant disease resistance; (iii) enlarge the fruit cluster; (iv) enlarge the grain size; (v) increase 100-grain weight; (vi) Increase plant yield.

3. The method according to claim 2, characterized in that The improved insect resistance of plants refers to the increased resistance of plants to chewing pests; the improved disease resistance of plants refers to the increased resistance of plants to stem rot.

4. The method according to any one of claims 1 to 3, characterized in that: The plant is a monocotyledonous plant; Preferably, the plant is corn.

5. A method for preparing a plant cell, plant seed, plant tissue, plant part, or plant with improved traits, characterized in that: Includes steps: Reducing or inhibiting the expression level and / or activity of the BX13 gene and / or the BX14 gene in plant cells, plant seeds, plant tissues, plant parts, or plants; The amino acid sequence encoded by the BX13 gene has at least 70% sequence identity compared with SEQ ID NO.:1; the amino acid sequence encoded by the BX14 gene has at least 70% sequence identity compared with SEQ ID NO.:

2.

6. The method according to claim 5, characterized in that The reduction or inhibition of the expression and / or activity of the BX13 gene and / or BX14 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.

7. A method for preparing a plant with improved traits, characterized in that: Includes steps: Regenerating the plant cell, plant seed, plant tissue, or plant part prepared by the method of any one of claims 5 to 6 into a plant body, thereby obtaining the plant with improved traits; Preferably, the method further comprises the step of harvesting plant seeds from the plant improved with the trait.

8. A method for preparing a hybrid plant, the method comprising the step of hybridizing a plant with improved traits prepared by the method of claim 7 with other plants to prepare a hybrid plant.

9. Use of the composition, characterized in that The composition is used for improving plant traits; or a reagent or kit for preparing a plant with improved traits; The composition comprises: (a) an inhibitor of the BX13 gene and / or the BX14 gene; and, optionally, (b) an agriculturally acceptable carrier; The amino acid sequence encoded by the BX13 gene has at least 70% sequence identity compared with SEQ ID NO.:1; the amino acid sequence encoded by the BX14 gene has at least 70% sequence identity compared with SEQ ID NO.:

2.

10. The use according to claim 9, characterized in that The inhibitor is selected from the following group: gene editing reagents, antisense nucleic acids, antibodies, small molecule compounds, Crispr reagents, small molecule ligands, or a combination thereof.

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