Wheat epsps mutant protein and use thereof in herbicide resistance

Abstract

The present invention relates to the fields of agriculture, plant biotechnology, and molecular biology. Specifically, the present invention relates to a wheat EPSPS mutant protein and use thereof in improving the tolerance of wheat to glyphosate. The mutant protein comprises an amino acid sequence having the following mutations compared to the EPSPS amino acid sequence of any genome of wild-type wheat: in the amino acid sequence set forth in SEQ ID NO: 2, the amino acid at position 168 is mutated from threonine to isoleucine, the amino acid at position 169 is mutated from alanine to valine, and the amino acid at position 172 is mutated from proline to serine. The mutant protein enables the production of stable glyphosate-resistant wheat, showing important significance for wheat breeding.

Description

Wheat EPSPS mutant protein and its application in herbicide resistance Technical Field

[0001] This invention relates to the fields of agriculture, plant biotechnology, and molecular biology. Specifically, this invention relates to a wheat EPSPS mutant protein and its application in improving wheat's tolerance to glyphosate. Background Technology

[0002] Wheat is a widely cultivated grass species worldwide, almost entirely used for food, with only about one-sixth used as animal feed. Previously, genetically modified glyphosate-resistant wheat had been developed, but it has not been commercially promoted due to consumer resistance to genetically modified foods. However, glyphosate-resistant wheat still has significant commercial value, making it crucial to explore methods for developing glyphosate-resistant wheat through non-GMO means.

[0003] Although previous studies have investigated the properties of EPSPS and the conserved nature of the glyphosate-binding pocket, the specific amino acids in the EPSPS enzyme that lead to resistance alterations vary between species. Wheat is an allohexaploid, and the EPSPS gene has three homologous copies in wheat: TaEPSPS-7A1, TaEPSPS-7D1, and TaEPSPS-4A1. Therefore, mutating these three homologous copies of the wheat EPSPS gene using non-transgenic methods to generate stable glyphosate-resistant wheat is of great significance for wheat breeding. Summary of the Invention

[0004] To address the aforementioned problems in the prior art, this invention provides a wheat EPSPS mutant protein and its application in improving wheat's tolerance to glyphosate.

[0005] A wheat EPSPS mutant protein comprising an amino acid sequence having the following mutations compared to the EPSPS amino acid sequence of any wild-type wheat genome: amino acid position 168 is mutated from threonine to isoleucine; amino acid position 169 is mutated from alanine to valine; and amino acid position 172 is mutated from proline to serine.

[0006] In one specific embodiment, the wheat EPSPS mutant protein comprises the following amino acid sequence:

[0007] In the amino acid sequence corresponding to wild-type wheat TaEPSPS-4A shown in SEQ ID NO:1, amino acid position 136 is mutated from threonine to isoleucine; amino acid position 137 is mutated from alanine to valine; and amino acid position 140 is mutated from proline to serine; or,

[0008] in the wild-type wheat TaEPSPS-7A amino acid sequence shown in SEQ ID NO: 2 is mutated from threonine to isoleucine at the 168th amino acid; and from alanine to valine at the 169th amino acid; and from proline to serine at the 172nd amino acid; or,

[0009] in the wild-type wheat TaEPSPS-7D amino acid sequence shown in SEQ ID NO: 3 is mutated from threonine to isoleucine at the 168th amino acid; and from alanine to valine at the 169th amino acid; and from proline to serine at the 172nd amino acid.

[0010] In another specific embodiment, the amino acid sequence of the wheat EPSPS mutein is as shown in SEQ ID NO: 6-8.

[0011] The present application also provides a combination of genes for improving the resistance of wheat EPSPS, wherein at least one wheat EPSPS gene comprises the mutein as claimed in any one of claims 1-3; preferably, it comprises the amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 8, the amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 7, the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 6, the amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 8, the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 8, the amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 7, or the amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

[0012] The present application also provides a genomic combination for improving EPSPS resistance of wheat and maintaining fertility, wherein one or two of the wheat TaEPSPS-4A, TaEPSPS-7A or TaEPSPS-7D genes comprises the mutant protein as claimed in any one of claims 1-3; preferably, it comprises the amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 8, the amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 7, the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 6, the amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 8, the amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 8, or the amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 7.

[0013] The present application also provides an isolated polynucleotide comprising a nucleic acid sequence encoding the mutant protein of wheat EPSPS or a complement thereof.

[0014] In one embodiment, the polynucleotide has a nucleotide sequence selected from the group consisting of:

[0015] (1) a nucleotide sequence encoding the amino acid sequence as shown in any one of SEQ ID NO: 6-8 or a complement thereof;

[0016] (2) a nucleotide sequence hybridizing to the sequence shown in (1) under stringent conditions; and / or

[0017] (3) a nucleotide sequence encoding the same amino acid sequence as shown in (1) due to the degeneracy of genetic code, or a complement thereof.

[0018] In another embodiment, the nucleotide sequence is optimized for expression in plant cells.

[0019] The present application also provides an expression vector comprising the polynucleotide and an expression regulatory element operably linked thereto.

[0020] The present application also provides a plant, seed, plant tissue, plant part or cell comprising the mutant protein of wheat EPSPS, the genomic combination, the polynucleotide or the expression vector.

[0021] In one embodiment, the plant is wheat.

[0022] In another embodiment, the mutation is included in each of the three EPSPS genes in the wheat.

[0023] The present application also provides a method for conferring glyphosate herbicide tolerance to a plant, seed, cell, or plant part, comprising expressing the wheat EPSPS mutant protein or the combination of genes in the plant, seed, cell, or plant part.

[0024] In one embodiment, the plant is wheat.

[0025] In another embodiment, the mutation is included in each of the three EPSPS genes in the wheat.

[0026] The present application also provides a method for producing a glyphosate herbicide tolerant transgenic plant, comprising transforming a plant cell or tissue with a polynucleotide encoding the wheat EPSPS mutant protein or the expression vector, and regenerating a glyphosate herbicide tolerant transgenic plant from the transformed plant cell or tissue.

[0027] The present application also provides a method for controlling weeds in a plant growing area, comprising contacting a plant growing area comprising a plant or seed with a glyphosate herbicide, the plant or seed comprising the wheat EPSPS mutant protein, the combination of genes, the polynucleotide, or the expression vector, and being tolerant to the glyphosate herbicide.

[0028] In one embodiment, the plant is wheat.

[0029] In another embodiment, the mutation is included in each of the three EPSPS genes in the wheat.

[0030] Some of the terms used in this specification are defined as follows.

[0031] The "wheat" as described herein refers to any plant of the species Triticum, as well as all plant varieties that can be mated therewith. In one embodiment, the wheat is Triticum aestivum.

[0032] The "comprising" as described herein means "including, but not limited to."

[0033] The "wild type" as described herein means a nucleic acid molecule or protein that can be found existing in nature.

[0034] The term "plant" as used herein refers to any differentiated multicellular organism capable of photosynthesis, particularly a monocotyledonous or dicotyledonous plant. In a particular embodiment, the plant is wheat.

[0035] The term "plant tissue" or "plant part" as used herein includes plant cells, protoplasts, plant tissue cultures, plant calli, plant clumps, and plant embryos, pollen, ovules, seeds, leaves, stems, flowers, shoots, seedlings, fruits, kernels, ears, roots, root tips, anthers, and the like.

[0036] The term "plant cell" as used herein is understood to be any cell from or found in a plant that is capable of forming, for example, undifferentiated tissue such as callus, differentiated tissue such as an embryo, a component part of a plant, a plant, or a seed.

[0037] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues, including polymers in which one or more of the amino acid residues are chemical analogs of naturally occurring amino acids. Proteins and polypeptides of the present application can be produced recombinantly or by chemical synthesis.

[0038] The terms "polynucleotide," "nucleic acid," "nucleic acid molecule," or "nucleic acid sequence" are used interchangeably herein to refer to an oligonucleotide, nucleotide, or polynucleotide and fragments or portions thereof, which can be single-stranded or double-stranded, and represent the sense or anti-sense strand. Nucleic acids include DNA, RNA or hybrids thereof, and can be of natural or synthetic origin. For example, a nucleic acid can comprise mRNA or cDNA. A nucleic acid can comprise a nucleic acid that has been amplified (e.g., using the polymerase chain reaction). The nucleotide designations "R" means a purine, e.g., guanine or adenine; "Y" means a pyrimidine, e.g., cytosine or thymine (uracil if RNA); "M" means adenine or cytosine; "K" means guanine or thymine; and "W" means adenine or thymine.

[0039] The term "isolated" as used herein in reference to a nucleic acid means a nucleic acid that is separated from a substantial portion of the genomic sequence in which it naturally occurs and / or is substantially separated from other cellular components with which the nucleic acid is naturally associated. For example, any nucleic acid that has been produced by synthesis (e.g., by sequential base condensation) is considered isolated. Likewise, a recombinantly expressed nucleic acid, a cloned nucleic acid, a nucleic acid produced by a primer extension reaction (e.g., PCR), or a nucleic acid excised from a genome is also considered isolated.

[0040] "Transgene expression," "expressing a transgene," "protein expression," "polypeptide expression," "expressing a protein," and "expressing a polypeptide" as used herein means the production of a protein or polypeptide by the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into a polypeptide chain, which can ultimately fold into a protein. The DNA molecule encoding the protein or the DNA molecule encoding the polypeptide can be operably linked to a heterologous promoter in a DNA construct for expressing the protein or polypeptide in a cell transformed with the recombinant DNA molecule.

[0041] It is well understood by those skilled in the art that, due to the degeneracy of the genetic code, there are a number of different nucleic acid sequences that can encode the amino acid sequences disclosed herein. It is within the ability of one of ordinary skill in the art to generate other nucleic acid sequences that encode the same protein, and thus the present application encompasses nucleic acid sequences that encode the same amino acid sequence due to the degeneracy of the genetic code. For example, to achieve high expression of a heterologous gene in a target host organism, such as a plant, the gene can be optimized for codon preference of the host organism to better express the gene.

[0042] The regulatory sequences described in the present application include, but are not limited to, promoters, transit peptides, terminators, enhancers, leaders, introns, and other regulatory sequences operably linked to the wheat EPSPS mutein.

[0043] "Operably linked" as used herein refers to two DNA molecules being linked in a manner such that one DNA molecule can affect the function of the other DNA molecule. The operably linked DNA molecules can be part of a single contiguous molecule and can or can not be adjacent. For example, a promoter is operably linked to a DNA molecule encoding a protein or a DNA molecule encoding a polypeptide in a DNA construct, where the two DNA molecules are arranged such that the promoter can affect the expression of the transgene.

[0044] The coding gene of the wheat EPSPS mutein described in the present application can be introduced into a plant according to methods commonly used in the industry, and can be genetically manipulated by a suitable plant transformation expression vector.

[0045] Any suitable promoter can be used in the vector, including promoters commonly used in the art for plant transformation. For example, promoters commonly used in plant transformation include, but are not limited to, SP6 promoters, T7 promoters, T3 promoters, PM promoters, maize ubiquitin promoters, cauliflower mosaic virus (CaMV) 35S promoters, nopaline synthase (nos) promoters, figwort mosaic virus 35S promoters, sugarcane bacilliform virus promoters, maize streak virus promoters, light-inducible promoters, ribulose-1,5-bisphosphate carboxylase (ssRUBISCO small subunit) promoters, rice cytosolic triose phosphate isomerase (TPI) promoters, Arabidopsis adenine phosphoribosyltransferase (APRT) promoters, octopine synthase promoters, and BCB (blue-copper-binding protein) promoters.

[0046] Plant transformation vectors include polyadenylation signal sequences that cause 3'-end polyadenylation. For example, NOS 3'-end derivatives of the nopaline synthase gene of Agrobacterium, octopine synthase 3'-end derivatives of the octopine synthase gene of Agrobacterium, 3'-ends of the tomato or potato protease inhibitor I or II genes, CaMV PolyA signal sequences, 3'-ends of the rice alpha-amylase gene, and 3'-ends of the bean lectin gene.

[0047] Vectors also include coding genes for selectable markers or reporters. Examples of selectable markers include, but are not limited to, antibiotic (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, bleomycin, chloramphenicol, etc.) or herbicide (glyphosate, phosphinothricin, phosphinouricin, etc.) resistance genes.

[0048] Methods for transforming vectors include Agrobacterium-mediated transformation, electroporation, microprojectile bombardment, polyethylene glycol-mediated uptake, etc. Plant transformation recipients in the present invention include plant cells (including suspension culture cells), protoplasts, calli, hypocotyls, seeds, cotyledons, shoots, and mature plants.

[0049] The scope of transgenic plants includes not only the plants obtained in the generation in which the genes were introduced, but also the clones and progeny (T1, T2, or subsequent generations) thereof. The scope of the present invention also includes all mutants and variants of the above transgenic plants that exhibit the characteristics of the primary transgenic plants through hybridization and fusion. The scope of the present invention also includes parts of plants, such as seeds, flowers, stems, fruits, leaves, roots, tubers, and corms, which are derived from plants that have been previously genetically modified by the methods described in the present invention, or the progeny thereof, and which are composed of at least a portion of the genetically modified cells.

[0050] The term "transgenic" plant refers to a plant that comprises a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated into the genome such that the polynucleotide is transmitted to successive generations. The heterologous polynucleotide can be integrated into the genome individually or as part of a recombination expression cassette. "Transgenic" is used herein to refer to any cell, cell line, callus, tissue, plant part or plant that contains a heterologous nucleic acid that was not in an organism at the time of the first generation. This term includes transgenic organisms, cells or plants that are initially so changed, as well as those produced by the breeding or asexual propagation of the initial transgenic organism or cell. The term "transgenic" as used herein is not intended to encompass changes to the genome (chromosomal or extrachromosomal) by conventional plant breeding methods (e.g., cross breeding) or by naturally occurring events (e.g., self-fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation). The transgenic plant can be at any stage of development.

[0051] The genes and proteins described in the present application include not only the specific example sequences, but also portions and fragments (including those with terminal deletions) of the specific examples that retain the herbicide tolerance activity characteristics of the proteins of the specific examples, variants, mutants, substitutions (proteins with alternative amino acids), chimeras and fusion proteins. The "variant" or "variation" refers to a nucleotide sequence that encodes the same protein or an equivalent protein with herbicide tolerance activity. The "equivalent protein" refers to a protein that has the same or substantially the same herbicide tolerance biological activity as the proteins of the claims.

[0052] The "herbicide" as described in the present application refers to an active ingredient that is capable of killing or controlling or being detrimental to the growth of plants.

[0053] "Herbicide-tolerant," "herbicide-resistant," "herbicide tolerance," or "herbicide resistance" as described herein means the ability of a plant, seed, plant tissue, plant part, or cell to withstand the toxic effects of one or more herbicides. Herbicide tolerance of a plant, seed, plant tissue, plant part, or cell can be measured by comparing the plant, seed, plant tissue, plant part, or cell to a suitable control. For example, herbicide tolerance can be measured by applying an herbicide to a plant comprising a nucleotide encoding a protein capable of conferring herbicide tolerance (test plant) and a plant not comprising a nucleotide encoding a protein capable of conferring herbicide tolerance (control plant), and then comparing the plant damage of both plants, wherein herbicide tolerance of the test plant is indicated by a reduced rate of damage compared to the rate of damage of the control plant. A herbicide-tolerant plant, seed, plant tissue, plant part, or cell displays a reduced response to the toxic effects of a herbicide when compared to a control plant, seed, plant tissue, plant part, or cell. As used herein, a "herbicide tolerance trait" is a transgenic trait that confers improved herbicide tolerance to a plant compared to a wild-type plant or control plant.

[0054] The term "controlling weeds" will be understood to mean killing and / or retarding or inhibiting the normal growth of weeds. In the broadest sense, weeds are understood to mean all plants which are known to grow where they are not wanted, for example at (crop) plant cultivation sites. Weeds according to the present application include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus and Taraxacum.Monocotyledonous weeds include, but are not limited to, weeds of the genera Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and Apera. Additionally, weeds of the present application can include, for example, crop plants growing in an undesirable location. For example, if corn plants are not desired in a field of wheat plants, a volunteer corn plant present in a field that primarily comprises wheat plants can be considered a weed.

[0055] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined in this specification otherwise, the terms "a", "an" and "the" as used herein are inclusive of both singular and plural forms. The terms "comprises", "comprising", "includes", "including" and "having" as used herein, specifically set out the presence of stated features, integers, components and / or elements, but do not preclude the presence or addition of one or more other features, integers, components, elements or groups thereof. The term "and / or", as used herein, includes any and all combinations of one or more of the associated listed items.

[0056] The present application has been described in detail by way of several embodiments, and indeed many modifications and variations of the application can be apparent to those skilled in the art having the benefit of this disclosure. Accordingly, the scope of the present application is not intended to be limited to the described embodiments, but is instead defined by the following claims. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 Growth of TaEPSPS-7A mutant and wild-type transgenic Arabidopsis thaliana material after 21 days of foliage treatment with glyphosate.

[0058] Figure 2 Growth status of gene edited wheat 169V, 168I+172S, M1-M3 after 28 days of treatment with different concentrations of glyphosate herbicide (leftmost in each group of photos is wild type control after spraying, the rest are current test samples).

[0059] Figure 3 Growth status of gene edited wheat M4-M7 materials after 28 days of treatment with different concentrations of glyphosate herbicide (leftmost in each group of photos is wild type control without spraying, second left is wild type control after spraying (this control does not exist for the group without spraying), the rest are current test samples).

[0060] Sequence Description DETAILED DESCRIPTION

[0061] The application will be further described in conjunction with the preferred embodiments thereof, given only by way of illustration of the present application, and not intended to limit the scope of the present application. The experimental methods in the following examples are all conventional methods, unless otherwise specified. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0062] Example 1 Cloning of Wheat 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Gene

[0063] 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the synthesis of aromatic amino acid pathway in plants, bacteria and fungi. Common wheat (Triticum aestivum) is a typical allohexaploid (AABBDD) plant, so it has three homologous genes of EPSPS, namely TaEPSPS-7A (NCBI number LOC100192183), TaEPSPS-4A (NCBI number LOC123088173) and TaEPSPS-7D (NCBI number LOC123165688). Using the cDNA of common wheat as a template, Kod DNA polymerase was used for PCR amplification, and the amplified fragments showed about 1.5Kb in agarose gel electrophoresis, and the gene sequences of TaEPSPS-7A, TaEPSPS-4A and TaEPSPS-7D were obtained, respectively.

[0064] The TaEPSPS-7A gene amplified was used as a template to synthesize primers TaEPSPS-7A 169V-F / TaEPSPS-7A 169V-R, TaEPSPS-7A 168I+172S-F / TaEPSPS-7A 168I+172S-R and TaEPSPS-7A 168I+169V+172S-F / TaEPSPS-7A 168I+169V+172S-R, respectively, and then PCR amplification was performed using Kod DNA polymerase. After detection by agarose gel electrophoresis, the correct band with a size of about 1.5 kb was recovered. The gene sequences of TaEPSPS-7A 169V, TaEPSPS-7A 168I+172S and TaEPSPS-7A 168I+169V+172S were obtained, respectively.

[0065] Table 1 Primers for preparing wheat EPSPS mutants

[0066] Example Two, Tolerance of Overexpression of Different Mutants of Wheat EPSPS-7A to Glyphosate in Arabidopsis

[0067] In order to quickly verify the tolerance of different mutants of wheat EPSPS-7A to glyphosate in plants, vectors overexpressing wild-type and mutant wheat EPSPS-7A genes were constructed, respectively.

[0068] 1. Construction of overexpression vector of Arabidopsis

[0069] The EPSPS fragment recovered after XbaI and SacI digestion and the plasmid pHSE401V were used to construct the overexpression vector using the HB-infusion™ Seamless Cloning Kit of Hanheng Biotechnology (Shanghai) Co., Ltd. After transformation into competent E. coli DH5a, positive clones were obtained, which were then verified by sequencing and restriction enzyme digestion before being transformed into Agrobacterium for use.

[0070] 2. Transformation of Arabidopsis and screening of transgenic lines

[0071] The transformation of Arabidopsis was performed using the flower dipping method. After the seeds matured, they were harvested and dried in an oven at 30°C for about a week to obtain T1 seeds. After disinfection, the T1 seeds were sown on MS screening solid medium (containing 30 mg / L Hyg) to screen positive plants. The positive seedlings of overexpressed wheat TaEPSPS-7A 169V, TaEPSPS-7A 168I+172S, TaEPSPS-7A 168I+169V+172S and wild-type (WT) Arabidopsis were transplanted into flowerpots filled with nutrient soil and placed in a phytotron for cultivation. After 2 months, T2 seeds were harvested.

[0072] 3. Transgenic Arabidopsis thaliana glyphosate resistance test

[0073] The obtained T2 generation of Arabidopsis thaliana seeds overexpressing wheat EPSPS mutants and wild type were sown in nutrient soil, and glyphosate resistance test was carried out when they grew to 5 leaf stage. The above-mentioned three overexpression transgenic materials and wild type Arabidopsis seedlings were sprayed with different concentrations of glyphosate, and were treated according to 0 mg / L, 10 mg / L, 50 mg / L, 100 mg / L four concentration gradients, wherein the critical lethal concentration of wild type seedlings was about 10 mg / L, and the growth of seedlings was observed after herbicide treatment for 21 days (Figure 1).

[0074] Compared with wild type Arabidopsis thaliana, transgenic Arabidopsis thaliana overexpressing wheat EPSPS has certain tolerance / resistance to glyphosate. Among them, under the concentration of 100 mg / L glyphosate, transgenic Arabidopsis thaliana overexpressing TaEPSPS-7A 168I+172S and TaEPSPS-7A 169V has already died, while transgenic Arabidopsis thaliana overexpressing TaEPSPS-7A 168I+169V+172S can still grow normally, indicating that the resistance of TaEPSPS-7A 168I+169V+172S mutant to glyphosate is higher than that of TaEPSPS-7A 168I+172S and TaEPSPS-7A 169V mutants.

[0075] Example Three, PE-mediated wheat EPSPS mutant editing

[0076] It is found by comparison that the amino acid sequences of the three EPSPS genes of wheat are very conservative. Therefore, it is speculated that the biological effects of mutations of wheat TaEPSPS-7A corresponding sites are consistent in the other two genes.

[0077] 1. Construction of gene editing vector

[0078] After designing the pegRNA expression frame of different wheat EPSPS mutants for synthesis, the editing vector was constructed for Agrobacterium-mediated genetic transformation of wheat.

[0079] 2. Agrobacterium-mediated wheat immature embryo transformation and screening

[0080] The editing vector is introduced into the wheat recipient by Agrobacterium-mediated wheat young embryo transformation method. The callus after recovery culture is moved to the screening medium for the first round of screening (containing 5 mg / L glufosinate) (2 weeks); the newly grown callus after the first round of screening is moved to the screening medium (containing 10 mg / L glufosinate) for the second round of screening (3 weeks); after the screening is completed, the callus with good growth state is picked for differentiation. After 10 days, 1 cm or so seedlings can be obtained; the differentiated seedlings are moved to the rooting medium for rooting culture; after the rooting is completed, the seedlings are moved to the flowerpot containing soil and placed in the greenhouse for culture; the edited seedlings or events of transgenic wheat are obtained.

[0081] Example Four, Glyphosate Resistance Test of T0 Generation of Gene Edited Wheat

[0082] After the wheat seedlings are moved into the greenhouse for culture, the genomic DNA of the transgenic wheat is extracted, and the DNA is used as a template for PCR amplification detection, and the T0 generation of wheat strains edited at different sites of three wheat EPSPS genes is successfully obtained. In order to test the resistance of the edited wheat material to glyphosate, the T0 generation of gene edited wheat seedlings are treated with glyphosate at concentrations of 0 g a.i / mu, 20 g a.i / mu, 40 g a.i / mu and 80 g a.i / mu, respectively, and the growth conditions of the gene edited wheat 169V, 168I+172S, M1-M7 (Table 2) after 28 days of glyphosate herbicide treatment are observed, and whether phytotoxicity occurs.

[0083] As shown in FIG. 2, compared with the wild type wheat, the 169V, 168I+172S, M1-M3 mutants of the gene edited wheat EPSPS all exhibit certain tolerance / resistance to glyphosate. Among them, under the concentration of 40 g a.i / mu of glyphosate, the edited materials of TaEPSPS-7A 168I+172S and TaEPSPS-7A 169V appear serious phytotoxicity, the growth of the materials is obviously inhibited, the leaves appear dry, and part of the plants appear death, while the edited materials of M1-M3 can still grow normally, indicating that the resistance of TaEPSPS-4A T136I+A137V+P140S, TaEPSPS-7A 168I+169V+172S and TaEPSPS-7D 168I+169V+172S mutants to glyphosate is higher than that of TaEPSPS-7A 168I+172S and TaEPSPS-7A 169V mutants.

[0084] Table 2 Wheat EPSPS Mutant Types

[0085] Example Five, Comparison of Prowess and Glyphosate Resistance of Wheat EPSPS Combined Mutants

[0086] In order to further verify the heredity and glyphosate resistance of the different EPSPS combination mutant wheat materials, the harvested seeds of different EPSPS combination edited types M4-M7 (Table 2) were sowed for generation, and the M4-M7 wheat materials were tested for glyphosate resistance and fertility during growth, and the fertility and glyphosate resistance of different EPSPS combination mutant wheat materials were observed and compared. Taking the wild type wheat material as a control, after the seedling height reached the three-leaf-one-heart stage, herbicide resistance test was carried out by spraying glyphosate, and the drug concentration was set as four gradients of 0 g a.i / mu, 30 g a.i / mu, 60 g a.i / mu and 120 g a.i / mu.

[0087] The investigation results after 28 days of drug application showed that the wild type control wheat seedlings were dry and dead at a dose of 30 g a.i / mu, but the edited seedlings of M4-M7 could grow normally at a dose of 60 g a.i / mu, and began to appear plant dwarfing at a dose of 120 g a.i / mu, but could recover growth later, as shown in Figure 3. The results showed that different combinations of editing wheat EPSPS gene could significantly improve the resistance of wheat to glyphosate.

[0088] The fertility of the mutant edited plants shown in Table 2 was observed, and the M7 edited seedlings of homozygous edited plants had poor fertility and could not obtain seeds; the remaining mutant edited plants could obtain homozygous plants and normally set seeds, and the fertility of the offspring was normal.

[0089] While the application satisfies through a number of different forms of implementation, as described in detail with respect to the preferred embodiments of the application, it should be understood that the disclosure is to be considered as an exemplification of the principles of the application and is not intended to limit the application to the specific embodiments illustrated and described. Those skilled in the art will be able to make many modifications and variations without departing from the spirit of the application. The scope of the application is to be judged from the claims and their equivalents. The abstract and title are not to be construed as limiting the scope of the application, as their purpose is to enable proper officials and the general public to quickly ascertain the general nature of the application.

Claims

1. A wheat EPSPS mutein comprising an amino acid sequence having the following mutations compared to the EPSPS amino acid sequence of any one of the wild type wheat genomes: the amino acid at position 168 is mutated from threonine to isoleucine; and the amino acid at position 169 is mutated from alanine to valine; and the amino acid at position 172 is mutated from proline to serine in the amino acid sequence set forth in SEQ ID NO: 2; and comprising the amino acid sequence of: in the wild type wheat TaEPSPS-4A amino acid sequence set forth in SEQ ID NO: 1, the amino acid at position 136 is mutated from threonine to isoleucine; and the amino acid at position 137 is mutated from alanine to valine; and the amino acid at position 140 is mutated from proline to serine; or, in the wild type wheat TaEPSPS-7A amino acid sequence set forth in SEQ ID NO: 2, the amino acid at position 168 is mutated from threonine to isoleucine; and the amino acid at position 169 is mutated from alanine to valine; and the amino acid at position 172 is mutated from proline to serine; or, in the wild type wheat TaEPSPS-7D amino acid sequence set forth in SEQ ID NO: 3, the amino acid at position 168 is mutated from threonine to isoleucine; and the amino acid at position 169 is mutated from alanine to valine; and the amino acid at position 172 is mutated from proline to serine.

2. The wheat EPSPS mutein according to claim 1, characterized in that, 3. The wheat EPSPS mutein according to claim 1 or 2, having an amino acid sequence as set forth in SEQ ID NO: 6-8.

4. A combination of genes for increasing the resistance of wheat EPSPS, wherein at least one wheat EPSPS gene comprises a mutein according to any one of claims 1-3; preferably it comprises an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 8, as set forth in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 7, as set forth in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 6, as set forth in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 8, as set forth in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 8, as set forth in SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 7 or as set forth in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO:

8. ​ ​ ​ ​ 5. A genetic combination for conferring EPSPS resistance to wheat while maintaining fertility, wherein one or two of the wheat TaEPSPS-4A, TaEPSPS-7A or TaEPSPS-7D genes comprise a mutein according to any one of claims 1 to 3; preferably it comprises an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 8, as set forth in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 7, as set forth in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 6, as set forth in SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 8, as set forth in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 8 or as set forth in SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO:

7.

6. An isolated polynucleotide comprising a nucleic acid sequence encoding a wheat EPSPS mutein according to any one of claims 1 to 3 or the complement thereof.

7. The polynucleotide of claim 6, wherein having a nucleotide sequence selected from the group consisting of: (1) a nucleotide sequence encoding an amino acid sequence as set forth in any one of SEQ ID NO: 6 to 8 or the complement thereof; (2) a nucleotide sequence that hybridizes to the sequence set forth in (1) under stringent conditions; and / or (3) a nucleotide sequence that, due to the degeneracy of the genetic code, encodes the same amino acid sequence as the sequence set forth in (1), or the complement thereof.

8. The polynucleotide of claim 6 or 7, wherein the nucleotide sequence is optimized for expression in a plant cell.

9. An expression vector comprising the polynucleotide of any one of claims 6 to 8 operably linked to expression control elements.

10. A plant, seed, plant tissue, plant part or cell comprising a wheat EPSPS mutein according to any one of claims 1 to 3, a genetic combination according to any one of claims 4 to 5, a polynucleotide according to any one of claims 6 to 8 or an expression vector according to claim 9.

11. A method for conferring glyphosate herbicide tolerance to a plant, seed, cell or plant part, the method comprising expressing a wheat EPSPS mutein according to any one of claims 1 to 3 or a genetic combination according to any one of claims 4 to 5 in the plant, seed, cell or plant part.

12. A method for producing a glyphosate herbicide-tolerant transgenic plant, the method comprising transforming a plant cell or tissue with a polynucleotide encoding a wheat EPSPS mutein according to any one of claims 1 to 3 or an expression vector according to claim 9, and regenerating a glyphosate herbicide-tolerant transgenic plant from the transformed plant cell or tissue.

13. A method for controlling weeds in a plant growing area, said method comprising contacting a plant growing area comprising a plant or seed with a glyphosate herbicide, said plant or seed comprising a wheat EPSPS mutein according to any one of claims 1 to 3, a genetic combination according to any one of claims 4 to 5, a polynucleotide according to any one of claims 6 to 8 or an expression vector according to claim 9 and being tolerant to said glyphosate herbicide.

14. The plant, seed, plant tissue, plant part, or cell of claim 10, or the method of any one of claims 11-13, wherein, Said plant is wheat and preferably it comprises a genetic combination according to any one of claims 4 to 5.

14. A method for producing a wheat plant that is tolerant to a glyphosate herbicide, said method comprising introducing into a wheat plant a nucleic acid molecule encoding a wheat EPSPS mutein according to any one of claims 1 to 3, a genetic combination according to any one of claims 4 to 5, a polynucleotide according to any one of claims 6 to 8 or an expression vector according to claim 9.

15. A wheat plant that is tolerant to a glyphosate herbicide, said plant comprising a nucleic acid molecule encoding a wheat EPSPS mutein according to any one of claims 1 to 3, a genetic combination according to any one of claims 4 to 5, a polynucleotide according to any one of claims 6 to 8 or an expression vector according to claim 9.

16. A wheat plant that is tolerant to a glyphosate herbicide, said plant comprising a nucleic acid molecule encoding

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