Mutant della protein and use thereof

Abstract

A mutant DELLA protein and use thereof. The mutant DELLA protein lacks some amino acids compared to the parental DELLA protein. The mutant DELLA protein can improve corn yield and enhance the salt-alkali tolerance of corn, showing important application value in corn breeding.

Description

A mutant DELLA protein and its applications

[0001] This application claims priority to Chinese patent application CN202411600001.X, filed on November 11, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the fields of biotechnology and crop genetics and breeding, and relates to a mutant DELLA protein and its applications, especially the application of the mutant DELLA protein in increasing yield and enhancing salt and alkali tolerance. Background Technology

[0003] Corn is the most widely planted crop in the world, with an annual global planting area of ​​over 2 billion mu and a total output of 1 billion tons. Its edible, industrial, and feed value makes it an important part of my country's food security system.

[0004] Since the discovery that the plant hormone gibberellins (GAs) can function at various stages of plant growth and development, exploring and understanding their underlying molecular mechanisms has become a major research focus. The DELLA protein plays a crucial role in the gibberellin signaling pathway and has therefore been extensively studied. In maize, the DELLA protein is encoded by the D8 (Dwarf8) gene.

[0005] The purpose of this invention is to use CRISPR / Cas gene editing technology to edit the DELLA protein in maize to obtain maize varieties with superior traits. Summary of the Invention

[0006] This invention provides a mutant DELLA protein and its application in increasing yield and enhancing salt and alkali tolerance.

[0007] On one hand, the present invention provides an application of a mutant DELLA protein, wherein the mutant DELLA protein is missing a certain number of amino acids relative to the parental DELLA protein, and the application is for improving plant traits, wherein the improved plant traits are selected from any one or more of the following (a)-(d):

[0008] (a) Increase production;

[0009] (b) Increase spikelet length;

[0010] (c) Enhanced salt tolerance;

[0011] (d) Enhance alkali resistance.

[0012] In another preferred embodiment, the mutated DELLA protein is selected from any one of the following (1)-(2):

[0013] (1) Compared with the amino acid sequence of the parental DELLA protein, amino acids corresponding to positions 81-88 of the sequence shown in SEQ ID No. 1 are missing;

[0014] (2) Compared with the amino acid sequence of the parent DELLA protein, amino acids corresponding to positions 80-88 of the sequence shown in SEQ ID No.1 are missing.

[0015] In another preferred embodiment, the amino acid sequence of the parental 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 with SEQ ID No. 1.

[0016] In another preferred embodiment, the nucleotide sequence encoding the parental 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 with SEQ ID No. 2.

[0017] In another preferred embodiment, the parental DELLA protein is derived from monocotyledonous and / or dicotyledonous plants.

[0018] In another preferred embodiment, the parental DELLA protein is derived from corn.

[0019] In another preferred embodiment, the parental DELLA protein is derived from maize Zheng 58.

[0020] In another preferred embodiment, the amino acid sequence of the parental DELLA protein is shown in SEQ ID No. 1.

[0021] In another preferred embodiment, the nucleotide sequence encoding the parental DELLA protein is shown in SEQ ID No. 2.

[0022] In another preferred embodiment, the nucleotide sequence encoding the mutated DELLA protein is missing a portion of bases relative to the sequence shown in SEQ ID NO.2.

[0023] In another preferred embodiment, the nucleotide sequence encoding the mutated DELLA protein is missing bases 239-262 relative to the sequence shown in SEQ ID NO.2.

[0024] In another preferred embodiment, the nucleotide sequence encoding the mutated DELLA protein is missing bases 237-263 relative to the sequence shown in SEQ ID NO.2.

[0025] In another preferred embodiment, the nucleotide sequence of the mutated DELLA protein is shown in SEQ ID NO.4 or SEQ ID NO.6.

[0026] In another preferred embodiment, the amino acid sequence of the mutated DELLA protein is shown in SEQ ID NO.3 or SEQ ID NO.5.

[0027] In another preferred embodiment, the plant is a monocotyledonous plant and / or a dicotyledonous plant.

[0028] In another preferred embodiment, the plant is selected from one or more plants from the group consisting of: legumes, cruciferous plants, grasses, solanaceous plants, cucurbitaceous plants, chenopodiaceae plants, polygonaceae plants, sesame plants, asteraceae plants, madder plants, roseaceae plants, sesame plants, convolvulaceae plants, dioscoreaceae plants, umbelliferous plants, lilyaceae plants, ginger family plants, and palm plants.

[0029] In another preferred embodiment, the plant is derived from one or more plants selected from the group consisting of: soybean, Arabidopsis thaliana, 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 combinations thereof.

[0030] In another preferred embodiment, the plant is selected from soybean, Arabidopsis thaliana, rice, tobacco, tomato, potato, corn, cotton, peanut, sorghum, cucumber, and coconut.

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

[0032] In another preferred embodiment, the plant is maize Zheng 58.

[0033] On the other hand, the present invention provides a method for improving plant traits, the method comprising the step of improving plant traits using the above-mentioned mutated DELLA protein, wherein the improved plant trait is selected from any one or more of the following (a)-(d):

[0034] (a) Increase production;

[0035] (b) Increase spikelet length;

[0036] (c) Enhanced salt tolerance;

[0037] (d) Enhance alkali resistance.

[0038] 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.

[0039] 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 gamma-ray mutagenesis), chemical mutagenesis (such as nitrite, hydroxylamine, EMS, nitrosoguanidine, etc.), biological mutagenesis (such as virus or bacterial-mediated mutagenesis), gene editing or biosynthesis.

[0040] In another preferred embodiment, the gene editing technology is selected from the group consisting of CRISPR technology, TALEN technology, ZFN technology, or combinations thereof.

[0041] In another preferred embodiment, the method includes the steps of:

[0042] (i) providing a plant or plant cell; and

[0043] (ii) The plant or plant cell is introduced with gRNA targeting a plant-endogenous DELLA protein encoding gene (e.g., the D8 gene in maize) and the corresponding Cas protein; in a preferred embodiment, an expression vector containing the gRNA and Cas protein is introduced into the plant or plant cell.

[0044] In another preferred embodiment, the Cas protein, also known as a CRISPR enzyme or Cas effector protein, includes, but is not limited to, Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, and FDK1 protein.

[0045] In one embodiment, the gene-editing enzyme is the Cas9 protein, and the vector further includes a scaffold sequence that specifically binds to the Cas9 protein. The scaffold sequence and the guide sequence are operatively linked to form a guide RNA (gRNA).

[0046] In other embodiments, the gene-editing enzyme is a Cas12 protein, such as Cas12a, Cas12b, or Cas12i, and the vector further includes a direct repeat sequence that specifically binds to the Cas12 protein. The direct repeat sequence and the guide sequence are operatively linked to form a guide RNA.

[0047] In another preferred embodiment, the introduction of the DELLA mutant protein includes the step of introducing the mutant protein by mutating a plant-endogenous DELLA protein-encoding gene (e.g., the D8 gene in maize).

[0048] On the other hand, the present invention provides a method for preparing plants with improved traits, the method comprising the step of introducing the above-mentioned mutated DELLA protein into plant cells, plant seeds, plant tissues, plant parts or plants, wherein the improved plant trait is selected from any one or more of the following (a)-(d):

[0049] (a) Increase production;

[0050] (b) Increase spikelet length;

[0051] (c) Enhance salt tolerance.

[0052] (d) Enhance alkali resistance.

[0053] In one embodiment, introducing the mutated DELLA protein includes the step of expressing the DELLA mutant protein in plant cells, plant seeds, plant tissues, plant parts, or plants, for example, by expressing the mutant protein through an expression vector, or by integrating the polynucleotide encoding the mutant protein into the plant genome for expression.

[0054] In another preferred embodiment, the method of introducing mutations includes natural variation, physical mutagenesis (such as ultraviolet mutagenesis, X-ray or gamma-ray mutagenesis), chemical mutagenesis (such as nitrite, hydroxylamine, EMS, nitrosoguanidine, etc.), biological mutagenesis (such as virus or bacterial-mediated mutagenesis), and gene editing.

[0055] In another preferred embodiment, the method includes the following steps:

[0056] (1) Introduce expression vectors containing gene editing tools into plant cells, plant seeds, plant tissues, and plant parts;

[0057] (2) The gene editing tool is applied to its endogenous DELLA protein-coding gene (e.g., the D8 gene in maize) and mutates it at the mutation site corresponding to SEQ ID No. 1.

[0058] (3) Screening for mutated plant cells, seeds, tissues, and parts;

[0059] (4) Isolate the gene editing tool.

[0060] In another preferred embodiment, the gene editing tools include CRISPR, TALEN, and ZFN.

[0061] In another preferred embodiment, the method includes the step of regenerating a plant from the plant cells, plant seeds, plant tissues, and plant parts.

[0062] On the other hand, the present invention provides a method for preparing plant cells, or plant seeds, or plant tissues, or plant parts with improved traits, the method comprising the step of introducing the aforementioned mutated DELLA protein into the plant cells, plant seeds, plant tissues, plant parts, or plant, wherein the improved plant trait is selected from any one or more of the following (a)-(d):

[0063] (a) Increase production;

[0064] (b) Increase spikelet length;

[0065] (c) Enhance salt tolerance.

[0066] (d) Enhance alkali resistance.

[0067] In another preferred embodiment, the method includes the step of regenerating a plant from the plant cells, plant seeds, plant tissues, and plant parts.

[0068] In another preferred embodiment, the method further includes the step of harvesting plant seeds from the plant that has been modified using the trait.

[0069] On the other hand, the present invention provides a trait-improved plant cell, or plant seed, or plant tissue, or plant part, or plant, wherein the plant cell, or plant seed, or plant tissue, or plant part, or plant is prepared by the above method.

[0070] On the other hand, the present invention provides a genetically engineered plant, which is prepared using the above-described method.

[0071] On the other hand, the present invention provides a method for preparing hybrid plants, the method comprising the step of hybridizing trait-improved plant seeds or trait-improved plants prepared by the above method with other plants to prepare hybrid plants.

[0072] In this invention, salt tolerance includes tolerance to NaCl, and alkali tolerance includes tolerance to sodium bicarbonate.

[0073] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described 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 described in detail here.

[0074] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0075] As used herein, the terms “polynucleotide,” “nucleotide sequence,” “nucleic acid sequence,” “nucleic acid molecule,” and “nucleic acid” are used interchangeably and include DNA, RNA, or their hybrids, which may be double-stranded or single-stranded.

[0076] The term "vector" refers to an element that allows the vector to integrate into the host cell's genome or to replicate autonomously within the cell independently of the genome. The vector may contain any element that guarantees self-replication. It typically carries a gene that is not part of the cell's central metabolism and is usually in the form of double-stranded DNA. The choice of vector generally depends on its compatibility with the host cell to which it 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, plasmid vectors may be used.

[0077] The "DELLA protein" is encoded by the D8 (Dwarf8) gene in maize. The Della protein in different maize lines can be obtained through genetic engineering techniques, such as genome sequencing and polymerase chain reaction (PCR).

[0078] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0079] Homology or identity can be calculated using known methods including, but not limited to, the following: Computational Molecular Biology (edited by Lesk, AM), Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (edited by Smith, DW), Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (edited by Griffin, AM and Griffin, HG), Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (edited by von Heinje, G.), Academic Press (1987); and Sequence Analysis Primer (edited by Gribskov, M. and Devereux, J.), Stockton. Stockton Press, New York (1991).

[0080] The specific amino acid positions (numbers) of the Della protein described in this invention are determined by comparing the target amino acid sequences using standard sequence alignment tools, such as the Smith-Waterman algorithm or the CLUSTALW2 algorithm. The sequence is 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. In the ClustalW2 (1.82) algorithm, the default parameters are preferably used: protein nick opening penalty = 10.0; protein nick extension penalty = 0.2; protein matrix = Gonnet; protein / DNA terminal gap = -1; protein / DNA GAPDIST = 4. The AlignX program (part of the vectorNTI group) is preferably used with default parameters suitable for multiple alignments (gap opening penalty: 10og gap extension penalty 0.05). By comparing the amino acid sequences of the Della protein from different maize inbred lines or varieties, the specific positions of the amino acids in the Della protein of different parents corresponding to SEQ ID No. 1 are determined. Using sequence alignment methods known in the art, those skilled in the art can determine the amino acid correspondence between the Della protein sequences of different maize varieties and SEQ ID No. 1.

[0081] The term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant masses, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0082] The term “plant cell” should be understood as any cell that is derived from or found in a plant and is capable of forming, for example: undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, or seeds.

[0083] The term "gene editing" technology includes CRISPR, TALEN, and ZFN technologies. CRISPR technology refers to clustered, regularly interspaced short palindromic repeats derived from the microbial immune system. Gene editing tools include guide RNA and Cas proteins (such as Cas9, Cpf1, and Cas12b). The gene editing tools referred to in TALEN technology are restriction enzymes capable of cleaving specific DNA sequences, comprising a TAL effector DNA-binding domain and a DNA-cleaving domain. The gene editing tools referred to in ZFN technology are also restriction enzymes capable of cleaving specific DNA sequences, comprising a zinc finger DNA-binding domain and a DNA-cleaving domain. Those skilled in the art know that by constructing nucleotides and other regulatory elements encoding gene editing tools into suitable vectors and then transforming them into cells, intracellular genome editing can be achieved, including gene knockout, insertion, and base editing.

[0084] Those skilled in the art will understand that the structure of a protein can be altered without adversely affecting its activity and function. For example, one or more conserved 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 implementations of conserved amino acid substitutions are familiar to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., replacing another nonpolar amino acid residue with a nonpolar amino acid residue, replacing another polar uncharged amino acid residue with a polar uncharged amino acid residue, replacing another basic amino acid residue with a basic amino acid residue, and replacing another acidic amino acid residue with an acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions, where an amino acid is replaced by another amino acid belonging to the same group, fall within the scope of this invention, provided that the substitution does not lead to the inactivation of the protein's biological activity. Therefore, the proteins of this invention can contain one or more conserved substitutions in their amino acid sequence, preferably generated by substitutions according to Table 1. Furthermore, this invention also covers proteins that also contain one or more other nonconservative substitutions, provided that such nonconservative substitutions do not significantly affect the desired function and biological activity of the proteins of this invention. Conserved amino acid substitutions can occur at one or more predicted non-essential amino acid residues. “Non-essential” amino acid residues are those that can be altered (deleted, substituted, or replaced) without changing biological activity, while “essential” amino acid residues are required for biological activity. A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Amino acid substitutions can occur in non-conserved regions of the Della protein. Generally, such substitutions are not performed on conserved amino acid residues, or on amino acid residues located within conserved motifs, where such residues are required for protein activity. However, those skilled in the art will understand that functional variants may have fewer conserved or non-conserved alterations in conserved regions.

[0085] As is well known in the art, one or more amino acid residues can be altered (replaced, deleted, truncated, or inserted) from the N and / or C ends of a protein while retaining its functional activity. Therefore, proteins in which one or more amino acid residues have been altered from the N and / or C ends while retaining their desired functional activity are also within the scope of this invention. These alterations can include those introduced by modern molecular methods such as PCR, which includes PCR amplification that alters or lengthens the protein-coding sequence by means of oligonucleotides containing amino acid-coding sequences used in the PCR amplification.

[0086] It should be recognized that proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions, and methods for such operations are generally known in the art. For example, amino acid sequence variants of the Della protein can be prepared by mutating DNA. This can also be accomplished through other forms of mutagenesis and / or directed evolution, for example, using known mutagenesis, recombination, and / or shuffling methods, combined with relevant screening methods, to perform single or multiple amino acid substitutions, deletions, and / or insertions.

[0087] Those skilled in the art will understand 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 domains of the protein, the properties of the polypeptide may be altered, but the polypeptide may retain its activity. If the mutations are not located near the catalytic domain, active site, or other functional domains, a smaller impact can be expected.

[0088] Those skilled in the art can identify the essential amino acids of the Della protein using methods known in the art, such as localized mutagenesis, protein evolution, or bioinformatics analysis. The protein's catalytic domains, active sites, or other functional domains can also be determined through physical structural analysis, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations in presumed key site amino acids.

[0089] Table 1

[0090] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably in this invention to refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of native amino acid residues. The proteins and polypeptides of this invention can be generated recombinantly or chemically synthesized. The term “mutant protein” or “mutant protein” refers to a protein that, compared to the amino acid sequence of a parent protein, has one or more substitutions, insertions, deletions, and / or additions of amino acid residues. As used herein, the terms “DELLA mutant protein,” “mutant DELLA polypeptide,” “mutant DELLA polypeptide,” “mutant DELLA protein,” “mutant protein,” “mutant polypeptide,” etc., are used interchangeably.

[0091] The term "encoding" refers to the inherent characteristics of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, which serves as a template for the synthesis of other polymers and macromolecules in biological processes that have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the biological characteristics they produce. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes that protein.

[0092] 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.

[0093] The term "plant" should be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, particularly monocotyledonous or dicotyledonous plants, vegetable crops including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e.g., head lettuce, leaf lettuce, longleaf lettuce), bok choy, taro, cucurbits (e.g., melons, watermelons, crenshaw, cantaloupes, Roman melons), rapeseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, headless cabbage, Chinese cabbage, bok choy), artichokes, carrots, napa cabbage, okra, onions, celery, parsley, chickpeas, parsnip, chicory, peppers, potatoes, gourds (e.g., zucchini, cucumbers, baby zucchini, squash, pumpkin), radishes, dried artichokes, etc. Onions, turnips, purple eggplant (also known as eggplant), ginseng, lettuce, scallions, chicory, garlic, spinach, green onions, squash, leafy greens, beets (sugar beets and fodder beets), sweet potatoes, romaine lettuce, wasabi, tomatoes, turnips, and spices; fruits and / or vine crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus fruits, blueberries, boysenberry. y), cranberries, currants, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pears, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, miscanthus, corn / maize (feed corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereals (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans). Oil-bearing plants (rapeseed, mustard, poppy, olive, sunflower, coconut, castor oil plants, cocoa beans, peanuts), Arabidopsis, fiber plants (cotton, flax, hemp, jute), Lauraceae (cinnamon, camphor), or a plant such as coffee, sugarcane, 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.

[0094] The main advantages of this invention are:

[0095] The present invention has found that maize plants with DELLA protein mutations have increased yield and enhanced salt and alkali tolerance. Attached Figure Description

[0096] Figure 1. Comparison of spike length (Figure 1A and B), number of spike rows (Figure 1C), and yield per plant (Figure 1D) of different edited plants.

[0097] Figure 2. Editing the salt and alkali tolerance of the plant. Detailed Implementation

[0098] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0099] Example 1: Obtaining Gene-Edited Maize

[0100] The applicant edited the D8 (Dwarf8) gene encoding the maize DELLA protein and transformed it into Zheng 58, obtaining several edited plants, including edited plant Q15 and edited plant N17. The CDS sequence of the DELLA protein in edited plant Q15 was deleted (24 bp) from bases 239 to 262, resulting in a deletion of amino acids 81 to 88 in the DELLA protein's amino acid sequence. The CDS sequence of the DELLA protein in edited plant N17 was deleted (27 bp) from bases 237 to 263, resulting in a deletion of amino acids 80 to 88 in the DELLA protein's amino acid sequence (see CN113788889B).

[0101] The amino acid sequence of the DELLA protein from wild-type maize Zheng 58 is shown in SEQ ID No. 1, and the CDS sequence is shown in SEQ ID No. 2; the amino acid sequence of the DELLA protein from edited plant Q15 is shown in SEQ ID No. 3, and the CDS sequence is shown in SEQ ID No. 4; the amino acid sequence of the DELLA protein from edited plant N17 is shown in SEQ ID No. 5, and the CDS sequence is shown in SEQ ID No. 6.

[0102] The amino acid sequence of the DELLA protein from wild-type maize Zheng 58 (SEQ ID No. 1):

[0103] The CDS sequence (SEQ ID No. 2) of the DELLA protein in wild-type maize Zheng 58:

[0104] Edit the amino acid sequence of the DELLA protein in plant Q15 (SEQ ID No. 3):

[0105] Edit the CDS sequence (SEQ ID No. 4) of the DELLA protein in plant Q15:

[0106] Edit the amino acid sequence of the DELLA protein in plant N17 (SEQ ID No. 5):

[0107] Edit the CDS sequence (SEQ ID No. 6) of the DELLA protein in plant N17:

[0108] Example 2: Agronomic traits of gene-edited maize

[0109] Compared with the wild-type Zheng 58, edited plants Q15 and N17 showed significantly increased ear length and yield per plant, with a slight increase in the number of ear rows, but no statistical difference, as shown in Figures 1A-D. In Figure 1, Zheng 58 refers to the wild-type Zheng 58 control group, KO1 refers to edited plant Q15, and KO2 refers to edited plant N17. This indicates that the deletion of amino acids 80-88 or 81-88 in the DELLA protein amino acid sequence can increase maize yield.

[0110] In addition, the salt and alkali tolerance of wild-type Zheng 58 and edited plant Q15 were tested. Specifically, uniformly sized maize seeds were selected and divided into three groups. These groups were planted in soil soaked with water, 100 mM NaCl solution, and 100 mM NaHCO3 solution, respectively. After 14 days of growth, their condition was observed, and their salt and alkali phenotypes were photographed and recorded. The results are shown in Figure 2. In Figure 2, Zheng 58 refers to the wild-type Zheng 58 control group, and KO refers to the edited plant Q15. It can be seen that there was no significant difference in the growth status of wild-type Zheng 58 and edited plant Q15 after planting in soil soaked with aqueous solution; however, after planting in soil soaked with 100mM NaCl solution and 100mM NaHCO3 solution, wild-type Zheng 58 plants were relatively short and had poor salt and alkali tolerance, while edited plant Q15 had better growth status and stronger salt and alkali tolerance; this indicates that the deletion of amino acids 81-88 in the amino acid sequence of DELLA protein can enhance the salt and alkali tolerance of maize.

[0111] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of a mutated DELLA protein, characterized in that, The mutant DELLA protein is deficient in part of the amino acids relative to the parent DELLA protein, the application is to improve plant traits, the improved plant traits are selected from any one or several of the following (a)-(d): (a) increase yield; (b) increase ear length; (c) enhance salt tolerance; (d) enhance alkali tolerance.

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

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

1.

4. Use according to claim 1, characterized in that, The plant is a dicotyledon and / or monocotyledon.

5. A method for improving a plant trait, comprising, The method comprises the step of using the mutant DELLA protein of any one of claims 1-3 to improve the traits of the plant, and the improved plant traits are selected from any one or several of the following (a)-(d): (a) increase yield; (b) increase ear length; (c) enhance salt tolerance; (d) enhance alkali tolerance.

6. The method of claim 5, wherein, The mutant 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 of claim 5, wherein, The introduction of the DELLA mutant protein includes the step of mutating the plant endogenous DELLA protein coding 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 1-3 into a plant cell, plant seed, plant tissue, plant part, or plant, and the improved plant traits are selected from any one or several of the following (a)-(d): (a) increase yield; (b) increase ear length; (c) enhance salt tolerance. (d) enhance alkali tolerance.

9. The method of claim 8, wherein, The method comprises the step of regenerating the plant cell, plant seed, plant tissue, plant part into a plant.

10. A method of making a hybrid plant, characterized in that, The method comprises the step of crossing the plant seed with improved traits or the plant with improved traits prepared by any one of the methods of claims 5-9 with other plants to prepare hybrid plants.

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