Mutant miR396 and application thereof

Abstract

The invention provides mutated miR396 and application thereof, the mutated miR396 has base mutation relative to parent miR396, the parent miR396 is wild type miR396 derived from corn, the miR396 comprises miR396c and / or miR396d, and the mutated miR396 can enhance the disease resistance of the corn and increase the yield of the corn.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a mutant miR396 and an application thereof, and in particular to a mutant miR396 and an application thereof in enhancing corn disease resistance and improving corn yield. Background Art

[0002] Corn is an important food, feed and industrial raw material, and plays an important role in the world's grain production. The occurrence of corn diseases and insect pests has a great impact on corn production, including ear rot, stalk rot, bacterial wilt, corn leaf blight, southern rust, etc.

[0003] In order to obtain corn strains with excellent traits, we studied the relevant genes in corn. MicroRNA (miRNA) is a class of non-coding single-stranded small RNA molecules that bind to the mRNA of the target gene through base pairing, thereby regulating the target gene. During the growth of plants, plant miRNA plays a key role in regulating meristem characteristics, leaf polarity and flowering patterns. In the study of miRNA, we found that the mutation of corn miR396 can enhance corn disease resistance and increase corn yield. Summary of the invention

[0004] The purpose of the present invention is to provide a mutated miR396 and an application thereof.

[0005] On the one hand, the present invention provides a mutant miR396, wherein the mutant miR396 has a base mutation relative to the parent miR396, wherein the parent miR396 is a wild-type miR396 derived from corn, and the miR396 includes miR396c and / or miR396d.

[0006] In one embodiment, the base mutation includes base deletion, base insertion or base substitution.

[0007] In one embodiment, the mutated miR396 is selected from any one of the following (1)-(5):

[0008] (1) The nucleic acid sequence of the parent miR396c is as shown in SEQ ID No. 3, and the mutant miR396c has a base deletion relative to the sequence shown in SEQ ID No. 3;

[0009] (2) The nucleic acid sequence of the parent miR396d is as shown in SEQ ID No. 4, and the mutant miR396d has a base deletion relative to the sequence shown in SEQ ID No. 4;

[0010] (3) The nucleic acid sequence of the parent miR396d is as shown in SEQ ID No. 4, and the mutant miR396d has a base insertion relative to the sequence shown in SEQ ID No. 4;

[0011] (4) The nucleic acid sequence of the parent miR396c is as shown in SEQ ID No.3, and the mutant miR396c has a base deletion relative to the sequence shown in SEQ ID No.3; the nucleic acid sequence of the parent miR396d is as shown in SEQ ID No.4, and the mutant miR396d has a base insertion relative to the sequence shown in SEQ ID No.4;

[0012] (5) The nucleic acid sequence of the parent miR396c is as shown in SEQ ID No.3, and the mutant miR396c has a base deletion relative to the sequence shown in SEQ ID No.3; the nucleic acid sequence of the parent miR396d is as shown in SEQ ID No.4, and the mutant miR396d has a base deletion relative to the sequence shown in SEQ ID No.4.

[0013] In one embodiment, the mutated miR396c lacks bases 141-151 relative to the sequence shown in SEQ ID No. 3. Preferably, the nucleic acid sequence of the mutated miR396c is shown in SEQ ID No.5.

[0014] In one embodiment, the mutated miR396c lacks base 151 relative to the sequence shown in SEQ ID No. 3. Preferably, the nucleic acid sequence of the mutated miR396c is shown in SEQ ID No.8.

[0015] In one embodiment, the mutated miR396c lacks bases 146-156 relative to the sequence shown in SEQ ID No. 3. Preferably, the nucleic acid sequence of the mutated miR396c is shown in SEQ ID No.9.

[0016] In one embodiment, the mutated miR396d lacks bases 198-208 relative to the sequence shown in SEQ ID No. 4. Preferably, the nucleic acid sequence of the mutated miR396d is shown in SEQ ID No.6.

[0017] In one embodiment, the mutant miR396d has a base inserted after base 203 relative to the sequence shown in SEQ ID No. 4. Preferably, the nucleic acid sequence of the mutant miR396d is shown in SEQ ID No.7.

[0018] In one embodiment, the mutated miR396c lacks the 151st base relative to the sequence shown in SEQ ID No.3, and the mutated miR396d inserts a base after the 203rd base relative to the sequence shown in SEQ ID No.4. Preferably, the nucleic acid sequence of the mutated miR396c is shown in SEQ ID No.8, and the nucleic acid sequence of the mutated miR396d is shown in SEQ ID No.7.

[0019] In one embodiment, the mutated miR396c lacks bases 146-156 relative to the sequence shown in SEQ ID No. 3, and the mutated miR396d lacks bases 198-208 relative to the sequence shown in SEQ ID No. 4. Preferably, the nucleic acid sequence of the mutated miR396c is shown in SEQ ID No. 9, and the nucleic acid sequence of the mutated miR396d is shown in SEQ ID No. 6.

[0020] In one embodiment, the parent miR396 is wild-type miR396 derived from corn.

[0021] In one embodiment, the corn is the corn inbred line Chang 7-2.

[0022] In one embodiment, the parent miR396c is derived from corn and has 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%, or at least 99% sequence identity with the sequence shown in SEQ ID No.3.

[0023] In a specific embodiment, the sequence of the parent miR396c is shown as SEQ ID No.3.

[0024] In one embodiment, the parent miR396d is derived from corn and has 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%, or at least 99% sequence identity with the sequence shown in SEQ ID No.4.

[0025] In a specific embodiment, the sequence of the parent miR396d is shown as SEQ ID No.4.

[0026] On the other hand, the present invention provides the use of the above-mentioned mutant miR396, the use is to improve corn traits, or the use in preparing a reagent or kit for improving corn traits, wherein the improved trait is selected from one or more of the following (i)-(iv):

[0027] (i) Enhance disease resistance;

[0028] (ii) increase production;

[0029] (iii) enlarge the fruit cluster;

[0030] (iv) Increased grain weight.

[0031] In one embodiment, the enhanced disease resistance refers to enhanced disease resistance to bacterial wilt and / or stem rot.

[0032] In one embodiment, the enhanced disease resistance refers to enhanced resistance to Fusarium, Pythium or Anthracnose.

[0033] In one embodiment, the enhanced disease resistance means that the disease resistance of corn containing mutated miR396 is at least 10% enhanced, preferably 20% enhanced, preferably 30% enhanced, preferably 40% enhanced, preferably 50% enhanced, preferably 100% enhanced, preferably 200% enhanced, preferably 300% enhanced, preferably 400% enhanced, preferably 500% enhanced, preferably 500% enhanced, preferably 100% enhanced, preferably 200% enhanced, preferably 300% enhanced, preferably 400% enhanced, preferably 500% enhanced compared to the parent corn (containing wild-type miR396).

[0034] In one embodiment, the enhanced disease resistance means that the disease resistance level of corn containing mutated miR396 is increased by at least one level, preferably by 2 levels, preferably by 3 levels, and preferably by 4 levels compared to the parent corn (containing wild-type miR396).

[0035] In one embodiment, the increased yield means that the yield or yield per plant or yield per mu of corn containing mutated miR396 is at least 5% higher than that of the parent corn (containing wild-type miR396), preferably 10% higher, preferably 15% higher, preferably 20% higher, preferably 21% higher, preferably 25% higher, preferably 30% higher, preferably 40% higher, preferably 50% higher, preferably 60% higher, preferably 100% higher.

[0036] In one embodiment, the enlarged ears means that the ears of corn containing mutated miR396 are at least 3% more, preferably 5% more, preferably 6% more, preferably 10% more, preferably 15% more, preferably 20% more, preferably 23% more, preferably 25% more, preferably 30% more, preferably 40% more, preferably 50% more, preferably 100% more, than those of the parent corn (containing wild-type miR396).

[0037] In one embodiment, the increase in grain weight means that the single grain weight, 100-grain weight or 1,000-grain weight of corn containing mutated miR396 is at least 5% higher than that of the parent corn (containing wild-type miR396), preferably 10% higher, preferably 12% higher, preferably 13% higher, preferably 15% higher, preferably 20% higher, preferably 25% higher, preferably 30% higher, preferably 40% higher, preferably 50% higher, preferably 100% higher.

[0038] On the other hand, the present invention provides a method for preparing corn cells, or corn seeds, or corn tissues, or corn parts, or corn with improved traits, the method comprising the step of introducing the mutated miR396 into corn cells, or corn seeds, or corn tissues, or corn parts, or corn.

[0039] In one embodiment, introducing the mutant miR396 comprises the step of mutating endogenous miR396 of corn to thereby introduce the mutant miR396.

[0040] In one embodiment, the method of introducing mutations includes natural variation, physical mutagenesis (such as ultraviolet mutagenesis, X-ray or Y-ray mutagenesis), chemical mutagenesis (such as nitrite, hydroxylamine, EMS, nitrosoguanidine, etc.), biological mutagenesis (such as virus- or bacteria-mediated mutagenesis), and gene editing.

[0041] In one embodiment, the mutant miR396 is introduced into corn cells, corn seeds, corn tissues, corn parts, or corn by gene editing.

[0042] In one embodiment, the method comprises the following steps:

[0043] (1) introducing an expression vector containing a gene editing tool into a corn cell, corn seed, corn tissue, corn part, or corn;

[0044] (2) Using gene editing tools to act on endogenous miR396 in maize and cause it to mutate;

[0045] (3) screening for mutant maize cells, maize seeds, maize tissues, maize parts, or maize;

[0046] (4) Isolating the gene editing tool.

[0047] In one embodiment, the gene editing tools include CRISPR, TALEN and ZFN.

[0048] In one embodiment, the gene editing tool is a Cas enzyme.

[0049] In a specific embodiment, the gene editing is performed using a Cas enzyme in corn cells, or corn seeds, or corn tissues, or corn parts, or corn.

[0050] In one embodiment, the introducing of the mutated miR396 comprises the step of expressing the mutated miR396 in corn cells, or corn seeds, or corn tissues, or corn parts, or corn.

[0051] On the other hand, the present invention also provides a method for preparing corn with improved traits or improving corn traits, the method comprising the steps of: regenerating corn cells, corn seeds, corn tissues, or corn parts prepared by the above method into corn plants, thereby obtaining corn with improved traits.

[0052] On the other hand, the present invention also provides a method for gene editing of corn, the method comprising the steps of:

[0053] (a) using Cas enzymes and gRNA to perform gene editing in corn cells, corn seeds, corn tissues or corn parts to obtain gene-edited corn cells, corn seeds, corn tissues or corn parts;

[0054] (b) regenerating the gene-edited corn cell, corn seed, corn tissue or corn part in step (a) into a corn plant;

[0055] The gRNA targets miR396 in maize.

[0056] In one embodiment, the Cas enzyme is Cas9 or Cas12i or a Cas12i mutant protein (eg, Cas-SF01).

[0057] In one embodiment, the amino acid sequence of the Cas-SF01 is as shown in SEQ ID No.1.

[0058] In a specific embodiment, the Cas enzyme is connected to one or more NLS sequences. In one embodiment, the NLS sequence is connected to the N-terminus and / or C-terminus of the protein.

[0059] In one embodiment, the gRNA includes a first segment and a second segment; the first segment is also called a "skeleton region", "protein binding segment", "protein binding sequence", or "Direct Repeat sequence"; the second segment is also called a "targeting sequence for targeting nucleic acid" or a "targeting segment for targeting nucleic acid", or a "guide sequence for targeting a target sequence".

[0060] The first segment, "skeleton region", "protein binding segment", "protein binding sequence", or "direct repeat sequence" of the gRNA can interact with the Cas enzyme of the present invention, so that the Ca enzyme and the gRNA form a complex. The gRNA of the present invention guides the interacting Cas enzyme protein to a specific nucleotide sequence in the target nucleic acid through the action of the targeting sequence of the targeting nucleic acid.

[0061] In one embodiment, the gRNA includes a direct repeat sequence that binds to the Cas enzyme and a guide sequence that hybridizes to the target sequence.

[0062] The targeting sequence of the targeting nucleic acid of the present invention or the targeting segment of the targeting nucleic acid comprises a nucleotide sequence complementary to the sequence in the target nucleic acid. In other words, the targeting sequence of the targeting nucleic acid of the present invention or the targeting segment of the targeting nucleic acid interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). Therefore, the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid can be changed, or can be modified to hybridize any desired sequence in the target nucleic acid.

[0063] Preferably, the gRNA comprises a first segment and a second segment from 5' to 3' direction.

[0064] In the present invention, the second segment can also be understood as a guide sequence that hybridizes with the target sequence.

[0065] In one embodiment, the guide sequence of the gRNA is shown as SEQ ID No.2.

[0066] In one embodiment, the direct repeat sequence of the gRNA is agagaaugugugcauagucacac.

[0067] In other embodiments, the direct repeat sequence of the gRNA may have a base deletion, substitution or addition on the basis of agagaaugugugcauagucacac, as long as it can ensure the binding ability with the Cas enzyme, for example, "agagaaugugugcauagucaacac" recorded in Chinese patent application (CN113337502A),

[0068] "agagaaugugugcauagucuacac", "agagaaugugugcauaguccacac" or "agagaaugugugcauagucgacac".

[0069] In one embodiment, the nucleic acid sequence encoding the Cas enzyme and the nucleic acid encoding the guide RNA are artificially synthesized.

[0070] In one embodiment, the gene editing method of the present invention includes the step of delivering a Cas enzyme and gRNA into corn cells, corn seeds, corn tissues, corn parts or corn.

[0071] The above-mentioned delivery can be delivered by any method known in the art. Such methods include, but are not limited to, transformation, transfection, electroporation, lipofection, microinjection, sonoporation, gene gun, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendritic transfection, heat shock transfection, nucleofection, magnetofection, lipofection, puncture transfection, optical transfection, agent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, vectors, viral vectors, artificial virions, etc.

[0072] In some embodiments, one or more components of the Cas enzyme and gRNA are delivered using one or more AAV vectors, lentiviral vectors, nanoparticles, or a combination thereof.

[0073] In one embodiment, the Cas enzyme and gRNA are delivered to corn cells, corn seeds, corn tissues, or corn parts or corn by Agrobacterium transformation.

[0074] The gRNA targets miR396 in corn cells and guides the Cas protein to the genomic locus to modify, edit or cut the target sequence, thereby causing a mutation in miR396.

[0075] On the other hand, the present invention also provides a method for preparing hybrid corn, which comprises the step of hybridizing the corn obtained by the above method with other corns to prepare hybrid corn.

[0076] Definition of terms

[0077] Unless defined otherwise, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0078] The terms "polynucleotide", "nucleotide sequence", "nucleic acid sequence", "nucleic acid molecule" and "nucleic acid" are used interchangeably and include DNA, RNA or hybrids thereof, which may be double-stranded or single-stranded.

[0079] The terms "protein", "polypeptide" and "peptide" are used interchangeably herein to refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of naturally occurring amino acid residues. The proteins and polypeptides of the present invention can be produced recombinantly or by chemical synthesis.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] The term "regulatory element", also known as "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, California (1990). In some cases, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and those sequences that direct the nucleotide sequence to be expressed 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, neuron, bone, skin, blood, a specific organ (e.g., liver, pancreas), or a specific cell type (e.g., lymphocyte). In some cases, regulatory elements can also direct expression in a timing-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).

[0084] As used herein, the term "promoter" has a meaning well known to those skilled in the art, and refers to a non-coding nucleotide sequence located upstream of a gene that can initiate expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of a 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 a gene product in the cell essentially only when the cell is a cell of the tissue type corresponding to the promoter.

[0085] The term "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for import into the nucleus by nuclear transport, i.e., a protein with an NLS is transported to the nucleus. Typically, an NLS comprises a positively charged Lys or Arg residue exposed on the surface of the protein. Exemplary nuclear localization sequences include, but are not limited to, NLSs from: SV40 large T antigen, EGL-13, c-Myc, and TUS proteins.

[0086] As used herein, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0087] wild type

[0088] As used herein, the term "wild type" has the meaning generally understood by those skilled in the art, which refers to the typical form of an organism, strain, gene, or the characteristics that distinguish it from mutant or variant forms when it exists in nature, which can be isolated from a source in nature and has not been intentionally modified by man.

[0089] Carrier

[0090] The term "vector" refers to a nucleic acid molecule that is capable of transporting another nucleic acid molecule to which it is attached. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, no free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and other various polynucleotides known in the art. The vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements it carries are expressed in the host cell. A vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc. as described herein (e.g., CRISPR transcripts, such as nucleic acid transcripts, proteins, or enzymes). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication initiation site.

[0091] One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.

[0092] Another type of vector is a viral vector, wherein a virally derived DNA or RNA sequence is present in a vector for packaging a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus). The viral vector also comprises a polynucleotide carried by a virus for transfection into a host cell. Some vectors (e.g., bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) can replicate autonomously in the host cell into which they are introduced.

[0093] Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, and are replicated together with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to as "expression vectors" herein.

[0094] Host cells

[0095] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, eukaryotic cells such as microbial cells, fungal cells, animal cells and plant cells.

[0096] Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression desired, and the like.

[0097] animal

[0098] For example, mammals, such as bovines, equines, ovines, porcines, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans. In certain embodiments, the subject (e.g., a human) suffers from a disorder (e.g., a disorder caused by a disease-related gene defect).

[0099] plant

[0100] 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.

[0101] 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.

[0102] 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.

[0103] Gene Editing

[0104] The term "gene editing" technology includes CRISPR technology, TALEN technology, and ZFN technology. CRISPR technology refers to clustered, regularly spaced short palindromic repeats (Clustered regularly interspaced short palindromicrepeats), which come from the immune system of microorganisms. Among them, gene editing tools include guideRNA, Cas proteins (such as Cas9, Cpf1, Cas12b, Cas12i, etc.). The gene editing tool referred to in TALEN technology is a restriction enzyme that can cut a specific DNA sequence, which includes a TAL effector DNA binding domain and a DNA cleavage domain. The gene editing tool referred to in ZFN technology is also a restriction enzyme that can cut a specific DNA sequence, which includes a zinc finger DNA binding domain and a DNA cleavage domain. It is well known to those skilled in the art that 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.

[0105] CRISPR system

[0106] As used herein, the terms "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system" or "CRISPR system" are used interchangeably and have the meaning generally understood by those skilled in the art, which generally includes transcription products or other elements related to the expression of CRISPR-associated ("Cas") genes, or transcription products or other elements capable of directing the activity of the Cas genes.

[0107] Cas proteins

[0108] Cas protein, Cas enzyme, or CRISPR-related protein refers to a nuclease suitable for the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system. Preferably, the Cas protein is a CRISPR enzyme, and its types include but are not limited to: Cas9 protein, Cas12 protein, Cas13 protein, Cas14 protein, Csm1 protein, FDK1 protein. The Cas protein may have different structures depending on its source, such as SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus aureus; it can also be classified according to structural features (such as domains), such as the Cas12 family including Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12i, etc. The Cas protein may have double-stranded or single-stranded or no cutting activity. The Cas protein of the present invention may be a wild type or a mutant thereof, and the mutation type of the mutant includes amino acid replacement, substitution or deletion, and the mutant may or may not change the enzymatic activity of the Cas protein. As known to those skilled in the art, a variety of Cas proteins with nucleic acid cleavage activity reported in the prior art, the known protein or its modified variant can achieve the function of the present invention, and are included in the scope of protection by reference herein.

[0109] It is well known in the art that one or more amino acid residues can be changed (replaced, deleted, truncated or inserted) from the N and / or C terminus of a protein while still retaining its functional activity. Therefore, proteins in which one or more amino acid residues are changed from the N and / or C terminus of a Cas protein while retaining its desired functional activity are also within the scope of the present invention. These changes may include changes introduced by modern molecular methods such as PCR, which includes PCR amplification of a protein coding sequence by means of including an amino acid coding sequence in an oligonucleotide used in PCR amplification to change or extend the protein coding sequence.

[0110] 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 the above-mentioned proteins can be prepared by mutations in the DNA. Other forms of mutagenesis and / or directed evolution can also be accomplished, for example, using known mutagenesis, recombination and / or shuffling methods, in combination with related screening methods, to perform single or multiple amino acid substitutions, deletions and / or insertions.

[0111] Those skilled in the art will appreciate that these minor amino acid changes in the Cas proteins of the present invention can occur (e.g., naturally occurring mutations) or be produced (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.

[0112] Those skilled in the art can identify the essential amino acids of the Cas mutant protein of the present invention according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of a bioinformatics system. The catalytic domain, active site or other functional domain of the protein can also be determined by physical analysis of the structure, such as by the following techniques: such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, combined with mutations of amino acids at putative key sites.

[0113] 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).

[0114] The biological functions of the Cas protein include, but are not limited to, the activity of binding to the guide RNA, the endonuclease activity, the activity of binding to and cutting a specific site of the target sequence under the guidance of the guide RNA, including but not limited to Cis cutting activity and Trans cutting activity.

[0115] Guide RNA (gRNA)

[0116] As used herein, the terms "guide RNA (gRNA)", "mature crRNA", "guide sequence" are used interchangeably and have meanings generally understood by those skilled in the art. In general, the guide RNA may comprise a direct repeat sequence and a guide sequence, or may consist essentially of or consist of a direct repeat sequence and a guide sequence.

[0117] In some cases, the guide sequence is any polynucleotide sequence that has sufficient complementarity with the target sequence to hybridize with the target sequence and guide the specific binding of the CRISPR / Cas complex to the target sequence. In one embodiment, when optimally aligned, the degree of complementarity between the guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining the optimal alignment is within the capabilities of ordinary technicians in this field. For example, there are publicly available and commercially available alignment algorithms and programs, such as, but not limited to, ClustalW, Smith-Waterman algorithm in matlab, Bowtie, Geneious, Biopython, and SeqMan.

[0118] Target sequence

[0119] "Target sequence" refers to a polynucleotide targeted by a guide sequence in a gRNA, such as a sequence having complementarity with the guide sequence, wherein hybridization between the target sequence and the guide sequence will promote the formation of a CRISPR / Cas complex (including Cas protein and gRNA). Complete complementarity is not required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR / Cas complex.

[0120] The target sequence can comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located inside or outside the cell. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. A sequence or template that can be used to recombine into a target locus comprising the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.

[0121] In the present invention, "target sequence" or "target polynucleotide" or "target nucleic acid" can be any endogenous or exogenous polynucleotide to a cell (e.g., a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). In some cases, the target sequence should be associated with a protospacer adjacent motif (PAM).

[0122] miR396

[0123] miR396 is a highly conserved endogenous non-coding single-stranded small RNA molecule with a length of 20-24 nucleotides. The plant miR396 family has very little sequence change in the long-term biological evolution and is highly conserved. miR396 binds to the mRNA of the target gene growth-regulating factors (GRFs) through base pairing, regulates its expression and affects the growth and development of various organs of the plant.

[0124] In one embodiment, the sequence of corn miR396c is shown as SEQ ID No.3; the sequence of corn miR396d is shown as SEQ ID No.4.

[0125] The sequence involved in the present invention is as follows:

[0126] SEQ ID No. Sequence Description 1 Amino acid sequence of Cas-SF01 2 Guide sequence of gRNA 3 Nucleotide sequence of wild-type miR396c 4 Nucleotide sequence of wild-type miR396d 5 Nucleic acid sequence of mutated miR396c 1 6 Nucleic acid sequence of mutated miR396d 1 7 Nucleic acid sequence of mutated miR396d2 8 Nucleic acid sequence of mutated miR396c 2 9 Nucleic acid sequence of mutated miR396c 3

[0127] The main advantages of the present invention are:

[0128] The present invention uses gene editing technology to directionally edit corn miR396, and finds that after miR396c and / or miR396d mutate, the disease resistance of corn is enhanced and the yield is increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] Figure 1 .Comparison of ears between edited plants and wild-type plants, where the control is a wild-type corn plant (Chang 7-2) and KO1-KO5 are edited plants.

[0130] Figure 2 .Comparison of 100-grain weight between edited plants and wild-type plants, where the control is wild-type corn plant (Chang 7-2) and KO1-KO5 are edited plants.

[0131] Figure 3 .Comparison of single plant yield between edited plants and wild-type plants, where the control is a wild-type corn plant (Chang 7-2) and KO1-KO5 are edited plants.

[0132] Figure 4 .Comparison of bacterial wilt incidence between edited plants and wild-type plants, where the control is a wild-type corn plant (Chang 7-2) and KO1-KO5 are edited plants. Implementation

[0134] 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.

[0135] Example 1. Obtaining Gene-Edited Corn

[0136] 1. Target design and vector construction

[0137] In this example, Cas-SF01 and gRNA targeting miR396c and miR396d are used to edit the above two genes in corn, and the specific operation method can be carried out in a conventional manner in the art. Among them, Cas-SF01 is a Cas protein BC26312 with an amino acid mutation in CN116004573B, and its amino acid sequence is shown in SEQ ID No.1.

[0138] The nucleic acid sequences of corn miR396c and miR396d are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively. The gRNA was designed based on the sequences, and the guide sequence of the designed gRNA is as follows:

[0139]

[0140]

[0141] The direct repeat sequence of the gRNA is agagaaugugugcauagucacac (5' to 3'). The gRNA includes the direct repeat sequence and the guide sequence in sequence from 5' to 3'.

[0142] After the above gRNA targets the precursor sequences of miRNA396c and miRNA396d, the mature sequences (UCCACAGGCUUUCUUGAACUGU) identical to those of miRNA396c and miRNA396d can be mutated, thereby achieving the purpose of inactivating miR396c and miR396d.

[0143] Amino acid sequence of Cas-SF01 (SEQ ID No. 1):

[0144] MKKVEVRRPYQSLLLPNHRKFKYLDETWNAYKSVKSLLHRFLVCAYGAVPFNKFVEVVE

[0145] KVDNDQLVLAFAVRLFRLVPVESTSFAKVDKANLAKSLANHLPVGTAIPANVQSYFDSNF

[0146] DPKKYMWIDCAWEADRLAREMGLSASQFSEYATTMLWEDWLPLNKDDVNGWGSVSGL

[0147] FGEGKKEDRQQKVKMLNNLLNGIKKNPPKDYTQYLKILLNAFDAKSHKEAVKNYKGRS

[0148] TGRTASYLSEKSGEITELMLEQLMSNIQRDIGRKQKEISLPKKDVVKKYLESESGVPYDQN

[0149] LWSQAYRNAASSIKKTTDRNFNSTLEKFKNEVELRGLLSEGDDVEILRSKFFSSEFHKTPD

[0150] KFVIKPEHIGFNRKYNVVAELYKLKAEATDFESAFATFVKDEFEEKGIKHPIKNILEYIWNNE

[0151] VPVEKWGRVARFNQREEKLLRIKANPTVECNQGMTFGNSAMVGEVLRSNYVSKKGALV

[0152] SGEHGGRLIGQNNMIWLEMRLLNKKGKWETHHVPTHNMKFFEEVHAYNPSLADSVNVRN

[0153] RLYRSEDYTQLPSSITDGLKGNPKAKLLKRQHCALNNMTANVLNPKLSFTINKKNDDYTV

[0154] IIVHSVEVSKPRREVLVGDYLVGMDQNQTASNTYAVMQVVKPKSTDAIPFRNMWVRFVE

[0155] SGSIESRTLNSRGEYVDQLNHDGVDLFEIGDTEWVDSARKFFNKLGVKHKDGTLVDLSTA

[0156] PRKAYAFNNFYFKTMLNHLRSNEVDLTLLRNEILRVANGRFSPMRLGSLSWTTLKALGSF

[0157] KSLVLSYFDRLGAKEMVDKEAKDKSLFDLLVAINNKRSNKREERTSRIASSLMTVAQKYK

[0158] VDNAVVHVVVEGNLSSTDRSASKAHNRNTMDWCSRAVVKKLEDMCNLYGFNIKGVPAF

[0159] YTSHQDPLVHRADYDDPKPALRCRYSSYSRADFSKWGQNALAAVVRWASNKKSNTCYK

[0160] VGAVEFLKQHGLFADKKLTVEQFLSKVKDEEILIPRRGGRVFLTTHRLLAESTFVYLNGVKYHSCNADEVAAVNICLNDWVIPCKKKMKEESSASG;

[0161] Nucleic acid sequence of miR396c (SEQ ID No.3):

[0162] tcaaccacctccctcctctctctccacaccacagttccagtccccctccctctccgtgggcggcgtagaattgtggtcggcgcgtgtccctcgc

[0163] cggaggaacggggtgtgggacgtgtggagaaaggtgcgcgggcatgctttccacaggctttcttgaactgtgaactcgtgggtggtggtggt

[0164] atgtcggatggaatgcttggctcctcccagctagcaattctcttcccccatgcaccggccgtgaattgagctgcggagcttccccgcagcctcg

[0165] ctctcggccggctctacctgttgatggttcaagaaagcccatggaaaccatgcagcctcccctcttgtgccagcatcgccgagaattcagccggttgaggggaatttaggtcctcccgctc;

[0166] Nucleic acid sequence of miR396d (SEQ ID No.4):

[0167] cggttgggtggttatacttgccgcaactagcagctatttatatgccccagcctctgtgggccgtttgcactcactccaaagcaaagcaaatctctct

[0168] ctctctctccatctctccacacacagccccttcgctgccgaggtccctcgccggacggggggtgaaggacgaaggggaagaaaggtgcgc

[0169] gggcatgctttccacaggctttcttgaactgtgaactcgtgggcgtggtggtggtggtgctggtgctgggtgtgtgggacggaatgattggctc

[0170] ctcccagtcccagtcccagtcccaccccaccaattctcttccccccgaccggccgtgaattgaattgaatcgaatcgcatcgctgagcttccccc

[0171] cggctcgctcacgcgacctgttgatggttcaagaaagcccatggaaaccatgcagcctccctcctgtgcagctgtgctctggtggcggagaattccgccggttccgggtgagggaattcagcagaccagccacccccgatgcggattcgtgtgtggaggaggtgagttg.

[0172] Annealing primers are designed according to the target. After primer annealing, the gene editing backbone vector is homologously connected by T5 nuclease, and the gene editing vector contains Cas-SF01 protein and the above-mentioned gRNA.

[0173] The above ligation products were transformed into competent E. coli Trans-T1, spread on Kan plates for culture, and 8 colonies were selected for liquid culture for 2 hours. PCR bacterial liquid detection was performed, and 2 correct monoclonal clones were selected and sent for bacterial liquid testing.

[0174] Select the single clone with correct sequencing for expansion, bacterial preservation and plasmid extraction.

[0175] 2. Genetic transformation

[0176] The maize inbred line Chang 7-2 was selected and gene editing was performed using the vector in step 1.

[0177] Transformation of Agrobacterium: The vector in step 1 was transformed into the Agrobacterium strain EHA105 by heat shock method, and a single clone was selected for liquid culture and PCR identification and then stored in a -80°C refrigerator for future use.

[0178] Activation of bacteria: Take out the bacteria from the refrigerator and streak culture on YEP solid culture medium.

[0179] Prepare Agrobacterium infection medium: scrape fresh bacteria from the reactivated bacterial plate and resuspend them in the infection medium.

[0180] Taking young embryos of corn: Take corn ears about 10 days after pollination, remove the bracts and filaments, pick out young embryos, and place them in an infection medium (without Agrobacterium) containing AS.

[0181] Infection: Wash the young embryos to be transformed with infection solution 3 times until the infection solution is clear, pour out the infection solution and add 1 ml of bacterial solution, gently invert 10 times, and let it stand for 5-10 minutes. Take a clean culture dish and put 3 sterilized filter papers. After infection, invert it several times and quickly pour the bacterial solution on the filter paper. Hold the culture dish and change the direction to evenly distribute the bacterial solution carrying the young embryos on the filter paper.

[0182] Co-culture: When the bacterial liquid can no longer be seen on the top layer of filter paper, use tweezers to pick up the upper layer of filter paper, and stick the side stained with immature embryos on the co-culture medium. Use tweezers to drive out the bubbles between the filter paper and the medium, then use tweezers to quickly peel off a corner of the filter paper. Use an embryo peeling knife to transfer the immature embryos remaining on the filter paper to the medium, with the shield side of the immature embryo facing up, and culture in the dark at 22°C for 3 days.

[0183] Recovery culture: After 3 days of co-culture, young embryos are transferred to recovery medium.

[0184] Differentiation: The young embryos transformed with the vector are directly differentiated on differentiation medium.

[0185] Rooting: Transfer the differentiated seedlings to the rooting medium, 3 to 4 seedlings per bottle, and culture them at 25 to 28°C until they grow into complete plants. After 7 days of rooting culture, small white roots will grow and can be sampled for testing.

[0186] 3. Screening of positive seedlings and editing types of edited plants

[0187] For the regenerated seedlings, a small amount of leaves were taken to extract genomic DNA using the CTAB method.

[0188] For plants transformed with the vector, primers (CasF: CTGCTGAGGATCAAGGCCAA and Cas R: CCCACATGTTCCTGAACGGA) are used to detect if the target product can be amplified, and the regenerated seedlings are transgenic positive seedlings.

[0189] For transgenic positive seedlings, the corresponding fragment of the gene was amplified with primers. The amplified product was sequenced by the Sanger method to confirm the editing form. If the sequencing result showed double peaks, the PCR product was connected to the T vector, and 5 clones were selected for sequencing to confirm the editing form. The sequencing primers are as follows:

[0190] Sequencing genes Primer Sequence(5'-3') miR396c P1F gtgggcggcgtagaattgtg miR396c P1R gcgggaggacctaaattccc miR396d P2F gttacttgccgcaactagca miR396d P2R gctggtctgctgaattccct

[0191] After 2-3 generations of self-pollination, homozygous edited seedlings without exogenous gene insertion were obtained.

[0192] The miRNA396c and miRNA396d of the inbred line Chang 7-2 were edited, and the edited plants KO1-KO5 were obtained.

[0193] The editing type of the edited plant KO1 is: single mutation of miRNA396c, miRNA396c lacks 11 bases at positions 141-151, the nucleic acid sequence of miRNA396c is shown in SEQ ID No.5, and miRNA396d is wild type;

[0194] The editing type of the edited plant KO2 is: miRNA396d single mutation, miRNA396d lacks 11 bases at positions 198-208, the nucleic acid sequence of miRNA396d is shown in SEQ ID No.6, and miRNA396c is wild type;

[0195] The editing type of the edited plant KO3 is: single mutation of miRNA396d, G is inserted after the 203rd base of miRNA396d, the nucleic acid sequence of miRNA396d is shown in SEQ ID No.7, and miRNA396c is wild type;

[0196] The editing type of the edited plant KO4 is: double mutation of miRNA396c and miRNA396d, miRNA396c lacks the 151st base, and the nucleic acid sequence of miRNA396c is shown in SEQ ID No.8; and, the 203rd base of miRNA396d is inserted with G, and the nucleic acid sequence of miRNA396d is shown in SEQ ID No.7;

[0197] The editing type of the edited plant KO5 is: double mutation of miRNA396c and miRNA396d, miRNA396c lacks 11 bases at positions 146-156, and the nucleic acid sequence of miRNA396c is shown in SEQ ID No.9; and miRNA396d lacks 11 bases at positions 198-208, and the nucleic acid sequence of miRNA396d is shown in SEQ ID No.6.

[0198] Nucleic acid sequence of miR396c of KO1 (SEQ ID No.5):

[0199] tcaaccacctccctcctctctctccacaccacagttccagtccccctccctctccgtgggcggcgtagaattgtggtcggcgcgtgtccctcgc

[0200] cggaggaacggggtgtgggacgtgtggagaaaggtgcgcgggcatgctttcttgaactgtgaactcgtgggtggtggtggtatgtcggatg

[0201] gaatgcttggctcctcccagctagcaattctcttcccccatgcaccggccgtgaattgagctgcggagcttccccgcagcctcgctctcggcc

[0202] ggctctacctgttgatggttcaagaaagcccatggaaaccatgcagcctcccctcttgtgccagcatcgccgagaattcagccggttgaggggaatttaggtcctcccgctc;

[0203] Nucleic acid sequence of miRNA396d of KO2 (SEQ ID No.6):

[0204] cggttgggtggttacttgccgcaactagcagctatttatatgccccagcctctgtgggccgtttgcactcactccaaagcaaagcaaatctctct

[0205] ctctctctccatctctccacacacagccccttcgctgccgaggtccctcgccggacggggggtgaaggacgaaggggaagaaaggtgcgc

[0206] gggcatgctttcttgaactgtgaactcgtgggcgtggtggtggtggtgctggtgctgggtgtgtgggacggaatgattggctcctcccagtccc

[0207] agtcccagtcccacccaccaattctcttccccccgaccggccgtgaattgaattgaatcgaatcgcatcgctgagcttccccccggctcgctca

[0208] cgcgacctgttgatggttcaagaaagcccatggaaaccatgcagcctccctcctgtgcagctgtgctctggtggcggagaattccgccggttccgggtgagggaattcagcagaccagccaccccgatgcggattcgtgtgtggaggaggtgagttg;

[0209] Nucleic acid sequence of miRNA396d of KO3 (SEQ ID No.7):

[0210] cggttgggtggttacttgccgcaactagcagctatttatatgccccagcctctgtgggccgtttgcactcactccaaagcaaagcaaatctctct

[0211] ctctctctccatctctccacacacagccccttcgctgccgaggtccctcgccggacggggggtgaaggacgaaggggaagaaaggtgcgc

[0212] gggcatgctttccacagggctttcttgaactgtgaactcgtgggcgtggtggtggtggtgctggtgctgggtgtgtgggacggaatgattggct

[0213] cctcccagtcccagtcccagtcccacccaccaattctcttccccccgaccggccgtgaattgaattgaatcgaatcgcatcgctgagcttcccc

[0214] ccggctcgctcacgcgacctgttgatggttcaagaaagcccatggaaaccatgcagcctccctcctgtgcagctgtgctctggtggcggagaattccgccggttccgggtgagggaattcagcagaccagccaccccgatgcggattcgtgtgtggaggaggtgagttg;

[0215] Nucleotide sequence of miR396c of KO4 (SEQ ID No.8):

[0216] Tcaaccacctccctcctctctctccacaccacagttccagtccccctccctctccgtgggcggcgtagaattgtggtcggcgcgtgtccctcgc

[0217] cggaggaacggggtgtgggacgtgtggagaaaggtgcgcgggcatgctttccacagctttcttgaactgtgaactcgtgggtggtggtggta

[0218] tgtcggatggaatgcttggctcctcccagctagcaattctcttcccccatgcaccggccgtgaattgagctgcggagcttccccgcagcctcgc

[0219] tctcggccggctctacctgttgatggttcaagaaagcccatggaaaccatgcagcctcccctcttgtgccagcatcgccgagaattcagccggttgaggggaatttaggtcctcccgctc;

[0220] Nucleotide sequence of miR396c of KO5 (SEQ ID No.9):

[0221] tcaaccacctccctcctctctctccacaccacagttccagtccccctccctctccgtgggcggcgtagaattgtggtcggcgcgtgtccctcgc

[0222] cggaggaacggggtgtgggacgtgtggagaaaggtgcgcgggcatgctttcttgaactgtgaactcgtgggtggtggtggtatgtcggatg

[0223] gaatgcttggctcctcccagctagcaattctcttcccccatgcaccggccgtgaattgagctgcggagcttccccgcagcctcgctctcggcc

[0224] ggctctacctgttgatggttcaagaaagcccatggaaaccatgcagcctcccctcttgtgccagcatcgccgagaattcagccggttgaggggaatttaggtcctcccgctc.

[0225] The editing types of edited plants KO1-KO5 are summarized as follows:

[0226]

[0227] Example 2: Agronomic traits of gene-edited corn

[0228] The agronomic traits of wild-type maize plants (Chang 7-2) and edited plants KO1-KO5 were counted.

[0229] Compared with wild-type maize plants, the ears of edited materials KO1-KO5 were larger ( Figure 1 ), 100-grain weight increased significantly ( Figure 2 ), the yield per plant increased significantly ( Figure 3 ).

[0230] Natural occurrence of bacterial wilt and stem rot in the field:

[0231] Corn bacterial wilt is caused by several Fusarium or Pythium fungi, either alone or in combination. The disease begins to show symptoms during the grain filling stage, with the peak of symptoms from the milky stage to the waxy stage. The statistics of natural field disease incidence during the waxy stage showed that compared with wild-type corn plants, the resistance to bacterial wilt of the edited plants KO1-KO5 was significantly improved, and the resistance of the double mutant plants of miRNA396c and miRNA396d (KO4 and KO5) to bacterial wilt was more significantly improved than that of the single mutant plants of miRNA396c (KO1) and the single mutant plants of miRNA396d (KO2 and KO3) ( Figure 4 ).

[0232] Corn stalk rot is a type of disease caused by multiple pathogens alone or in combination, which causes root and stem base rot. It is mainly caused by infection with Pythium, Colletotrichum, and Fusarium. The natural occurrence of the disease in the field was counted at the waxy and trumpet stages. The stalk rot infection of the plant was determined by whether there were irregular water-soaked lesions in the middle of the stem and leaf sheath, whether the plant was wilting, and whether the leaves were dying from top to bottom; and whether there were irregular brown spots extending longitudinally at the internodes of the stem base, and whether the interior was hollow. The results showed that compared with wild-type corn plants, the stalk rot resistance of the edited plants KO1-KO5 was significantly improved, and the resistance of the miRNA396c and miRNA396d double mutant plants (KO4 and KO5) to stalk rot was more significantly improved than that of the miRNA396c single mutant plants (KO1) and miRNA396d single mutant plants (KO2 and KO3).

[0233] The resistance of wild-type corn plants (Chang 7-2) and edited plants KO1-KO5 to bacterial wilt and stem rot is shown in the following table:

[0234] Edit Plant Wild-type control KO1 KO2 KO3 KO4 KO5 Bacterial wilt resistance feel Zhongkang Zhongkang Zhongkang High resistance High resistance Stem rot resistance feel Zhongkang Zhongkang Zhongkang anti- anti-

[0235] Disease resistance level: High resistance refers to an incidence rate of 0.0-5.0%; resistant refers to an incidence rate of 5.1-10.0%; moderate resistance refers to an incidence rate of 10.1-30.0%; susceptible refers to 30.1-40.0%; highly susceptible refers to above 40.1%.

[0236] This suggests that corn miR396c and / or miR396d mutations can increase corn yield and enhance corn disease resistance.

[0237] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A mutant miR396, wherein the mutant miR396 has a base mutation relative to the parent miR396, the parent miR396 is a wild-type miR396 derived from corn, and the miR396 includes miR396c and / or miR396d.

2. The mutated miR396 according to claim 1, characterized in that The mutated miR396 is selected from any one of the following (1)-(5): (1) The mutated miR396c has a base deletion relative to the sequence shown in SEQ ID No. 3; (2) the mutated miR396d has a base deletion relative to the sequence shown in SEQ ID No. 4; (3) the mutated miR396d has a base insertion relative to the sequence shown in SEQ ID No. 4; (4) the mutated miR396c has a base deletion relative to the sequence shown in SEQ ID No. 3, and the mutated miR396d has a base insertion relative to the sequence shown in SEQ ID No. 4; (5) The mutated miR396c has a base deletion relative to the sequence shown in SEQ ID No.3, and the mutated miR396d has a base deletion relative to the sequence shown in SEQ ID No.

4.

3. The use of the mutated miR396 according to any one of claims 1-2, characterized in that: The use is to improve corn traits, or to prepare a reagent or kit for improving corn traits, wherein the improved traits are selected from one or more of the following (i)-(iv): (i) Enhance disease resistance; (ii) increase production; (iii) enlarge the fruit cluster; (iv) Increased grain weight.

4. A method for preparing a corn cell, corn seed, corn tissue, corn part, or corn with improved traits, characterized in that: The method comprises the step of introducing the mutated miR396 according to any one of claims 1 to 2 into corn cells, corn seeds, corn tissues, corn parts, or corn.

5. The method according to claim 4, characterized in that The introduction of the mutated miR396 according to any one of claims 1 to 2 comprises the step of mutating the endogenous miR396 of corn to thereby introduce the mutated miR396.

6. The method according to claim 4, characterized in that The mutant miR396 is introduced into corn cells, corn seeds, corn tissues, corn parts, or corn by gene editing.

7. A method for preparing corn with improved traits or improving corn traits, characterized in that: The method comprises the steps of: The corn cells, corn seeds, corn tissues, or corn parts prepared by the method of any one of claims 4 to 6 are regenerated into corn plants to obtain corn with improved traits.

8. A method for gene editing of corn, characterized in that: The method comprises the steps of: (a) using Cas enzymes and gRNA to perform gene editing in corn cells, corn seeds, corn tissues or corn parts to obtain gene-edited corn cells, corn seeds, corn tissues or corn parts; (b) regenerating the gene-edited corn cell, corn seed, corn tissue or corn part in step (a) into a corn plant; The gRNA targets miR396 in maize.

9. The method according to claim 8, characterized in that The guide sequence in the gRNA that hybridizes with the target sequence is shown as SEQ ID No.

2.

10. A method for preparing hybrid corn, the method comprising the step of hybridizing corn prepared by the method according to any one of claims 4 to 9 with other corn to prepare hybrid corn.

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