Mutated DELLA protein and application thereof

Abstract

The invention provides a mutated DELLA protein and application thereof. The mutated DELLA protein lacks part of amino acid relative to parent DELLA protein. The DELLA mutant protein can improve the corn yield and enhance the saline-alkaline resistance of corn, and has important application value in corn breeding.

Description

Technical Field

[0001] The invention belongs to the fields of biotechnology and crop genetic breeding, and relates to a mutant DELLA protein and an application thereof, in particular to the application of the mutant DELLA protein in increasing yield and enhancing salt-alkali tolerance. Background Art

[0002] 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.

[0003] Since the plant hormone gibberellins (GAs) were found to play a role in various stages of plant growth and development, exploring and understanding their underlying molecular mechanisms has become the main research direction of researchers. DELLA proteins are a key link in the gibberellin signal transduction pathway and have therefore been studied in depth. In corn, DELLA proteins are encoded by the D8 (Dwarf8) gene.

[0004] The purpose of the present invention is to edit corn DELLA protein using CRISPR / Cas gene editing technology to obtain corn strains with excellent traits. Summary of the invention

[0005] The invention provides a mutant DELLA protein and application of the mutant protein in improving yield and enhancing salt-alkali resistance.

[0006] In one aspect, the present invention provides an application of a mutant DELLA protein, wherein the mutant DELLA protein lacks some amino acids relative to the parent DELLA protein, and the application is to improve plant traits, wherein the improved plant traits are selected from any one or more of the following (a)-(d):

[0007] (a) Increase production;

[0008] (b) increase ear length;

[0009] (c) Enhance salt tolerance;

[0010] (d) Enhanced alkali resistance.

[0011] In another preferred embodiment, the mutated DELLA protein is selected from any one of the following (1)-(2):

[0012] (1) Compared with the amino acid sequence of the parent DELLA protein, amino acids 81 to 88 corresponding to the sequence shown in SEQ ID No. 1 are missing;

[0013] (2) Compared with the amino acid sequence of the parent DELLA protein, the amino acids 80-88 corresponding to the sequence shown in SEQ ID No. 1 are missing.

[0014] In another preferred example, the amino acid sequence of the parent DELLA protein 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.

[0015] In another preferred example, the nucleotide sequence encoding the parent DELLA protein 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.

[0016] In another preferred embodiment, the parent DELLA protein is derived from monocotyledonous plants and / or dicotyledonous plants.

[0017] In another preferred example, the parent DELLA protein is derived from corn.

[0018] In another preferred embodiment, the parent DELLA protein is derived from Zea mays Zheng 58.

[0019] In another preferred example, the amino acid sequence of the parent DELLA protein is shown in SEQ ID No.1.

[0020] In another preferred example, the nucleotide sequence encoding the parent DELLA protein is shown as SEQ ID No.2.

[0021] In another preferred embodiment, the nucleotide sequence encoding the mutant DELLA protein lacks some bases relative to the sequence shown in SEQ ID NO.2.

[0022] In another preferred embodiment, the nucleotide sequence encoding the mutant DELLA protein lacks bases 239 to 262 relative to the sequence shown in SEQ ID NO.2.

[0023] In another preferred embodiment, the nucleotide sequence encoding the mutant DELLA protein lacks bases 237 to 263 relative to the sequence shown in SEQ ID NO.2.

[0024] In another preferred example, the nucleotide sequence of the mutated DELLA protein is shown as SEQ ID NO.4 or SEQ ID NO.6.

[0025] In another preferred example, the amino acid sequence of the mutated DELLA protein is shown as SEQ ID NO.3 or SEQ ID NO.5.

[0026] In another preferred embodiment, the plant is a monocotyledon and / or a dicotyledon.

[0027] 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, Dioscoreaceae, Umbelliferae, Liliaceae, Zingiberaceae, and Palmaceae.

[0028] In another preferred embodiment, the plant is derived from one or more plants selected from the following group: 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.

[0029] In another preferred embodiment, the plant is selected from soybean, Arabidopsis, rice, tobacco, tomato, potato, corn, cotton, peanut, sorghum, cucumber and coconut.

[0030] In another preferred example, the plant is corn.

[0031] In another preferred embodiment, the plant is corn Zheng 58.

[0032] In another aspect, the present invention provides a method for improving plant traits, the method comprising the step of using the above-mentioned mutant DELLA protein to improve plant traits, wherein the improved plant traits are selected from any one or more of the following (a)-(d):

[0033] (a) Increase production;

[0034] (b) increase ear length;

[0035] (c) Enhance salt tolerance;

[0036] (d) Enhanced alkali resistance.

[0037] In another preferred embodiment, the mutated DELLA protein is obtained by a method selected from the group consisting of gene mutation, gene knockout, gene editing technology, or a combination thereof.

[0038] 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.

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

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

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

[0042] (ii) using a gRNA targeting a plant endogenous DELLA protein encoding gene (e.g., the D8 gene in corn) and a 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.

[0043] In another preferred embodiment, the Cas protein, also known as CRISPR enzyme or Cas effector protein, includes but is not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, FDK1 protein.

[0044] In one embodiment, the gene editing enzyme is a Cas9 protein, and the vector further includes a Scaffold sequence that can specifically bind to the Cas9 protein. After the Scaffold sequence is operably linked to the guide sequence, it constitutes a guide sequence (gRNA).

[0045] In other embodiments, the gene editing enzyme is a Cas12 protein, for example, Cas12a, Cas12b, Cas12i, and the vector further includes a unidirectional repeat sequence (DirectRepeat) that specifically binds to the Cas12 protein. After the unidirectional repeat sequence is operably connected to the guide sequence, a guide guide sequence (gRNA) is formed.

[0046] In another preferred embodiment, the introduction of the DELLA mutant protein comprises the step of mutating the plant endogenous DELLA protein encoding gene (eg, the D8 gene in corn) to introduce the mutant protein.

[0047] In another aspect, the present invention provides a method for preparing a plant with improved traits, the method comprising the step of introducing the above-mentioned mutant DELLA protein into a plant cell, a plant seed, a plant tissue, a plant part or a plant, wherein the improved plant trait is selected from any one or more of the following (a)-(d):

[0048] (a) Increase production;

[0049] (b) increase ear length;

[0050] (c) Enhance salt tolerance.

[0051] (d) Enhanced alkali resistance.

[0052] In one embodiment, introducing the mutant DELLA protein includes the step of expressing the DELLA mutant protein in plant cells, plant seeds, plant tissues, plant parts or plants, for example, expressing the mutant protein through an expression vector, or integrating the polynucleotide encoding the mutant protein into the plant genome for expression.

[0053] In another preferred embodiment, in the described method, the method of introducing mutations includes 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), and gene editing.

[0054] In another preferred embodiment, the method comprises the following steps:

[0055] (1) Introducing expression vectors containing gene editing tools into plant cells, plant seeds, plant tissues, or plant parts;

[0056] (2) using a gene editing tool to act on an endogenous DELLA protein encoding gene (e.g., the D8 gene in corn) and causing a mutation at the mutation site corresponding to SEQ ID No. 1;

[0057] (3) Screening for mutant plant cells, plant seeds, plant tissues, and plant parts;

[0058] (4) Isolating the gene editing tool.

[0059] In another preferred embodiment, the gene editing tools include CRISPR, TALEN and ZFN.

[0060] In another preferred embodiment, the method comprises the step of regenerating the plant cells, plant seeds, plant tissues, and plant parts into plants.

[0061] In another aspect, the present invention provides a method for preparing a plant cell, plant seed, plant tissue, plant part, or plant with improved traits, the method comprising the step of introducing the above-mentioned mutant DELLA protein into the plant cell, plant seed, plant tissue, plant part, or plant, wherein the improved plant trait is selected from any one or more of the following (a)-(d):

[0062] (a) Increase production;

[0063] (b) increase ear length;

[0064] (c) Enhance salt tolerance.

[0065] (d) Enhanced alkali resistance.

[0066] In another preferred embodiment, the method comprises the step of regenerating the plant cells, plant seeds, plant tissues, and plant parts into plants.

[0067] In another preferred embodiment, the method further comprises the step of harvesting plant seeds from the plants improved with the trait.

[0068] On the other hand, the present invention provides a plant cell, or plant seed, or plant tissue, or plant part, or plant with improved traits, wherein the plant cell, or plant seed, or plant tissue, or plant part, or plant is prepared by the above method.

[0069] In another aspect, the present invention provides a genetically engineered plant, wherein the plant is prepared by the above method.

[0070] In another aspect, the present invention provides a method for preparing hybrid plants, comprising the step of hybridizing plant seeds with improved traits or plants with improved traits prepared by the above method with other plants to prepare hybrid plants.

[0071] In the present invention, salt resistance includes resistance to NaCl, and alkali resistance includes resistance to sodium bicarbonate.

[0072] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0073] Unless defined otherwise, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0074] 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.

[0075] The term "vector" is a vector that contains elements that allow the vector to be integrated into the host cell genome or to replicate autonomously in the cell independently of the genome. The vector may contain any element that ensures 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.

[0076] "DELLA protein" is encoded by the D8 (Dwarf8) gene in corn; Della proteins from different corn strains can be obtained through genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc.

[0077] As used herein, the term "identity" is used to refer to the matching of sequences 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 a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by 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.

[0078] "Homology" or "identity" can be calculated by known methods including, but not limited to, Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton, New Jersey (1993). Stockton Press, New York (1991).

[0079] The specific amino acid positions (numbers) of the Della proteins of the present invention are determined by aligning the target amino acid sequences using standard sequence alignment tools, such as using the Smith-Waterman algorithm or using the CLUSTALW2 algorithm to align two sequences, 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. The default parameters are preferably 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 with default parameters suitable for multiple alignment (gap opening penalty: 10og gap extension penalty 0.05). By comparing the amino acid sequences of the Della proteins of different maize inbred lines or varieties, the specific positions corresponding to the amino acids of the Della proteins of different parents and SEQ ID No.1 are determined; through sequence alignment methods known in the art, those skilled in the art can obtain the amino acid correspondence between the Della protein sequences of different varieties of maize and SEQ ID No.1.

[0080] The term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant pieces, and plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, ears, roots, root tips, anthers, etc.

[0081] 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.

[0082] 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, 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 the nucleotides encoding the gene editing tool and other regulatory elements are constructed in a suitable vector, and then the cell is transformed to achieve the editing of the genome in the cell. The types of editing include gene knockout, insertion, and base editing.

[0083] It is clear to those skilled in the art that the structure of a protein can be changed without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional structure of the protein molecule. Examples and embodiments of conservative amino acid substitutions are clear to those skilled in the art. Specifically, the amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, that is, 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. As long as the substitution does not result in the inactivation of the biological activity of the protein, a conservative substitution in which an amino acid is replaced by other amino acids belonging to the same group falls within the scope of the present invention. Therefore, the protein of the present invention may contain one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably generated by substitution according to Table 1. In addition, the present invention also covers proteins that also contain one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired function and biological activity of the protein of the present invention. Conservative amino acid substitutions can be made at one or more predicted non-essential amino acid residues. "Non-essential" amino acid residues are amino acid residues that can be changed (deleted, substituted or replaced) without changing the biological activity, while "essential" amino acid residues are required for biological activity. "Conservative amino acid substitutions" are substitutions in which amino acid residues are replaced by amino acid residues with similar side chains. Amino acid substitutions can be made in the non-conserved regions of Della protein. In general, such substitutions are not made to conserved amino acid residues, or to amino acid residues located within conserved motifs, where such residues are required for protein activity. However, it will be appreciated by those skilled in the art that functional variants may have fewer conservative or non-conservative changes in conserved regions.

[0084] It is well known in the art that one or more amino acid residues can be altered (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 altered from the N and / or C terminus of a Della protein while retaining its desired functional activity are also within the scope of the present invention. These alterations may include alterations introduced by modern molecular methods such as PCR, which includes PCR amplification of a protein coding sequence by means of including the amino acid coding sequence in the oligonucleotides used in the PCR amplification to alter or extend the protein coding sequence.

[0085] It will be appreciated that proteins can be altered in a variety of 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 Della proteins can be prepared by mutations in 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 related screening methods, to perform single or multiple amino acid substitutions, deletions and / or insertions.

[0086] Those skilled in the art will appreciate that these minor amino acid changes in the Della protein of the present invention can occur (e.g., naturally occurring mutations) or be generated (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domain of the protein, the properties of the polypeptide may be changed, but the polypeptide may retain its activity. If the mutations present are not close to the catalytic domain, active site, or other functional domain, lesser effects may be expected.

[0087] Those skilled in the art can identify the essential amino acids of the Della protein 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 domains 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.

[0088] Table 1

[0089]

[0090]

[0091] The terms "protein", "polypeptide" and "peptide" are used interchangeably in the present invention and refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues. The proteins and polypeptides of the present invention can be produced recombinantly or by chemical synthesis. The term "mutant protein" or "mutant protein" refers to a protein that has one or more amino acid residues substituted, inserted, deleted and / or added compared to the amino acid sequence of the parent protein. As used herein, the terms "DELLA mutant protein", "mutated DELLA polypeptide", "mutant DELLA polypeptide", "mutant DELLA protein", "mutant protein", "mutant polypeptide" and the like are used interchangeably.

[0092] The term "encoding" 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 biological properties it produces. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system.

[0093] 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.

[0094] The term "plant" is to be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichokes, Brussels sprouts, rocket, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, romaine lettuce), bok choy, yellow taro, melons (e.g., melon, watermelon, Crenshaw melon, honeydew melon, cantaloupe), rapeseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, pak choi), cardoon, carrot, napa, okra, onion, celery, parsley, chickpeas, parsnips, endive, peppers, potatoes, cucurbits (e.g., zucchini, cucumber, courgette, squash, pumpkin), radish, bulb onions, rutabagas, eggplant (also called 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, erry), cranberries, currants, loganberries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pomegranates, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, silver grass, 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, soybeans), oil plants (rapeseed, mustard, olives, sunflowers, coconuts, castor oil plants, cocoa beans, peanuts), Arabidopsis, fiber plants (cotton, flax, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plants; and / or bedding plants, such as flowering plants, cacti, succulents and / or ornamental plants, and 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.

[0095] The main advantages of the present invention are:

[0096] The present invention has found through research that the yield of corn plants with DELLA protein mutations is increased and the salt-alkali tolerance is enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 , ear length of different edited plants ( Figure 1A and B), ear row number ( Figure 1 C) and single plant yield ( Figure 1 D) comparison.

[0098] Figure 2 , edit the salt and alkali tolerance of the plant. DETAILED DESCRIPTION

[0099] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0100] Example 1. Obtaining Gene-Edited Corn

[0101] The applicant edited the gene D8 (Dwarf8) encoding the corn DELLA protein and transformed Zheng 58 to obtain a variety of edited plants, including the edited plant Q15 and the edited plant N17. The CDS sequence of the DELLA protein of the edited plant Q15 lacked (24bp) bases from position 239 to position 262, resulting in the amino acid sequence of the DELLA protein lacking amino acids from position 81 to position 88. The CDS sequence of the DELLA protein of the edited plant N17 lacked (27bp) bases from position 237 to position 263, resulting in the amino acid sequence of the DELLA protein lacking amino acids from position 80 to position 88 (refer to CN113788889B).

[0102] The amino acid sequence of the DELLA protein of the wild-type corn Zheng 58 is shown in SEQ ID No.1, and the CDS sequence is shown in SEQID No.2; the amino acid sequence of the DELLA protein of the edited plant Q15 is shown in SEQ ID No.3, and the CDS sequence is shown in SEQID No.4; the amino acid sequence of the DELLA protein of the edited plant N17 is shown in SEQ ID No.5, and the CDS sequence is shown in SEQID No.6.

[0103] Amino acid sequence of the DELLA protein of wild-type corn Zheng 58 (SEQ ID No. 1):

[0104] MKREYQDAGGSGGDMGSSKDKMMAAAAGAGEQEEEDVDELLAALGYKVRSSDMADV

[0105] AQKLEQLEMAMGMGGVGGAGATADDGFVSHLATDTVHYNPSDLSSWVESMLSELNAP

[0106] PAPLPPATPAPRLASTSSTVTSGAAAGAGYFDLPPAVDSSSSTYALKPIPSPVAAPSADPST

[0107] DSAREPKRMRTGGGSTSSSSSSSSSMDGGRTRSSVVEAAPPATQASAAANGPAVPVVVV

[0108] DTQEAGIRLVHALLACAEAVQQENFSAAEALVKQIPMLASSQGGAMRKVAAYFGEALA

[0109] RRVYRFRPPPDSSLLDAAFADLLHAHFYESCPYLKFAHFTANQAILEAFAGCRRVHVVDF

[0110] GIKQGMQWPALLQALALRPGGPPSFRLTGVGPPQPDETDALQQVGWKLAQFAHTIRVDF

[0111] QYRGLVAATLADLEPFMLQPEGDDTDDEPEVIAVNSVFELHRLLAQPGALEKVLGTVRA

[0112] VRPRIVTVVEQEANHNSGTFLDRFTESLHYYSTMFDSLEGAGAGSGQSTDASPAAAGGT

[0113] DQVMSEVYLGRQICNVVACEGAERTERHETLGQWRSRLGGSGFAPVHLGSNAYKQASTLLALFAGGDGYRVEEKDGCLTLGWHTRPLIATSAWRVAAAAAP*;

[0114] CDS sequence of the DELLA protein of wild - type maize Zheng 58 (SEQ ID No.2):

[0115]

[0116] Edit the amino acid sequence of the DELLA protein of plant Q15 (SEQ ID No. 3): MKREYQDAGGSGGDMGSSKDKMMAAAAGAGEQEEEDVDELLAALGYKVRSSDMADVAQKLEQLEMAMGMGGVGGAGATAATDTVHYNPSDLSSWVESMLSELNAPPAPLPPATPAPRLASTSSTVTSGAAAGAGYFDLPPAVDSSSSTYALKPIPSPVAAPSADPSTDSAREPKRMRTGGGSTSSSSSSSSSMDGGRTRSSVVEAAPPATQASAAANGPAVPVVVVDTQEAGIRLVHALLACAEAVQQENFSAAEALVKQIPMLASSQGGAMRKVAAYFGEALARRVYRFRPPPDSSLLDAAFADLLHAHFYESCPYLKFAHFTANQAILEAFAGCRRVHVVDFGIKQGMQWPALLQALALRPGGPPSFRLTGVGPPQPDETDALQQVGWKLAQFAHTIRVDFQYRGLVAATLADLEPFMLQPEGDDTDDEPEVIAVNSVFELHRLLAQPGALEKVLGTVRAVRPRIVTVVEQEANHNSGTFLDRFTESLHYYSTMFDSLEGAGAGSGQSTDASPAAAGGTDQVMSEVYLGRQICNVVACEGAERTERHETLGQWRSRLGGSGFAPVHLGSNAYKQASTLLALFAGGDGYRVEEKDGCLTLGWHTRPLIATSAWRVAAAAAP*;

[0117]

[0118] Edit the amino acid sequence of the DELLA protein of plant N17 (SEQ ID No. 5): MKREYQDAGGSGGDMGSSKDKMMAAAAGAGEQEEEDVDELLAALGYKVRSSDMADVAQKLEQLEMAMGMGGVGGAGATATDTVHYNPSDLSSWVESMLSELNAPPAPLPPATPAPRLASTSSTVTSGAAAGAGYFDLPPAVDSSSSTYALKPIPSPVAAPSADPSTDSAREPKRMRTGGGSTSSSSSSSSSMDGGRTRSSVVEAAPPATQASAAANGPAVPVVVVDTQEAGIRLVHALLACAEAVQQENFSAAEALVKQIPMLASSQGGAMRKVAAYFGEALARRVYRFRPPPDSSLLDAAFADLLHAHFYESCPYLKFAHFTANQAILEAFAGCRRVHVVDFGIKQGMQWPALLQALALRPGGPPSFRLTGVGPPQPDETDALQQVGWKLAQFAHTIRVDFQYRGLVAATLADLEPFMLQPEGDDTDDEPEVIAVNSVFELHRLLAQPGALEKVLGTVRAVRPRIVTVVEQEANHNSGTFLDRFTESLHYYSTMFDSLEGAGAGSGQSTDASPAAAGGTDQVMSEVYLGRQICNVVACEGAERTERHETLGQWRSRLGGSGFAPVHLGSNAYKQASTLLALFAGGDGYRVEEKDGCLTLGWHTRPLIATSAWRVAAAAAP*;

[0119]

[0120] Example 2: Agronomic traits of gene-edited corn

[0121] Compared with the wild type Zheng 58, the ear length and yield per plant of the edited plants Q15 and N17 were significantly increased, and the number of ear rows increased slightly, but there was no statistical difference. Figure 1 As shown in AD. Figure 1 In the figure, Zheng 58 refers to the wild-type Zheng 58 control group, KO1 refers to the edited plant Q15, and KO2 refers to the edited plant N17. This indicates that the amino acid sequence of DELLA protein lacking amino acids 80 to 88 or 81 to 88 can increase corn yield.

[0122] In addition, the salt and alkali tolerance of wild-type Zheng 58 and edited plant Q15 were tested. The specific method was as follows: corn seeds of the same size were selected and divided into a third group. They were planted in soil soaked in water, 100mM NaCl solution, and 100mM NaHCO3 solution, respectively. After 14 days of growth, their status was observed and their salt-alkali phenotypes were photographed and recorded. Figure 2 As shown, in Figure 2 In the figure, Zheng 58 refers to the wild-type Zheng 58 control group, and KO refers to the edited plant Q15. It can be seen that after planting in soil soaked with water solution, there is no significant difference in the growth status of wild-type Zheng 58 and edited plant Q15; however, after planting in soil soaked with 100mM NaCl solution and 100mM NaHCO3 solution, wild-type Zheng 58 plants are relatively short and have poor salt and alkali tolerance, while edited plant Q15 has better growth status and stronger salt and alkali tolerance; this indicates that the amino acid sequence of DELLA protein lacks amino acids 81-88, which can enhance the salt and alkali tolerance of corn.

[0123] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An application of a mutant DELLA protein, characterized in that: The mutant DELLA protein lacks some amino acids relative to the parent DELLA protein, and the application is to improve plant traits, wherein the improved plant traits are selected from any one or more of the following (a)-(d): (a) Increase production; (b) increase ear length; (c) Enhance salt tolerance; (d) Enhanced alkali resistance.

2. The use according to claim 1, characterized in that: The mutated DELLA protein is selected from any one of the following (1)-(2): (1) Compared with the amino acid sequence of the parent DELLA protein, amino acids 81 to 88 corresponding to the sequence shown in SEQ ID No. 1 are missing; (2) Compared with the amino acid sequence of the parent DELLA protein, the amino acids 80-88 corresponding to the sequence shown in SEQ ID No. 1 are missing.

3. The use according to claim 1, characterized in that: The amino acid sequence of the parent DELLA protein has at least 70% sequence identity with SEQ ID NO.:

1.

4. The use according to claim 1, characterized in that: The plants are dicotyledonous plants and / or monocotyledonous plants.

5. A method for improving plant traits, characterized in that: The method comprises the step of using the mutant DELLA protein in any one of claims 1 to 3 to improve plant traits, wherein the improved plant traits are selected from any one or more of the following (a) to (d): (a) Increase production; (b) increase ear length; (c) Enhance salt tolerance; (d) Enhanced alkali resistance.

6. The method according to claim 5, characterized in that The mutated DELLA protein is obtained by a method selected from the group consisting of gene mutation, gene knockout, gene editing technology, or a combination thereof.

7. The method according to claim 5, characterized in that The introduction of the DELLA mutant protein includes the step of mutating the plant endogenous DELLA protein encoding gene to introduce the mutant protein.

8. A method for preparing a plant with improved traits, the method comprising the step of introducing the mutant DELLA protein of any one of claims 1 to 3 into a plant cell, plant seed, plant tissue, plant part or plant, wherein the improved plant trait is selected from any one or more of the following (a) to (d): (a) Increase production; (b) increase ear length; (c) Enhance salt tolerance. (d) Enhanced alkali resistance.

9. The method according to claim 8, characterized in that The method comprises the step of regenerating the plant cell, plant seed, plant tissue or plant part into a plant.

10. A method for preparing a hybrid plant, characterized in that: The method comprises the step of hybridizing plant seeds or plants with improved traits prepared by the method according to any one of claims 5 to 9 with other plants to prepare hybrid plants.

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