Method for obtaining glyphosate-resistant trait without fitness cost

Abstract

Provided is a method for obtaining a glyphosate-resistant trait without a fitness cost. The method comprises the following steps: producing at least two copies of an endogenous epsps gene by means of in planta chromosomal segment duplication, wherein a point mutation conferring resistance to glyphosate occurs in at least one copy of the epsps gene, and at least one copy remains wild-type. Compared with a wild-type plant, the plant obtained by the method exhibits no significant loss of agronomic traits, demonstrates significantly improved resistance to glyphosate-based herbicides, and offers great application value.

Description

A method for obtaining glyphosate resistance without adaptation cost Technical Field

[0001] This invention relates to the fields of genetic engineering and bioinformatics, and more specifically, to a method for obtaining glyphosate resistance without aptemia cost. Background Technology

[0002] Rice (Oryza sativa) is one of the world's three most important food crops, providing a basic food source for more than half of the world's population. Weed control is a key challenge in rice cultivation, especially for upland rice production. Without effective weed control measures, rice yields can typically decrease by 5%-15%, and in severe cases, by as much as 15%-30%. To combat weed problems, farmers have to use herbicides extensively. Glyphosate is currently the most widely used herbicide in the world, and the development of resistant crop varieties has become a key focus for seed companies and research institutions both domestically and internationally.

[0003] Glyphosate is a systemic, broad-spectrum organophosphorus herbicide applied foliar. It is characterized by its broad spectrum, high efficiency, low toxicity, and easy degradation, and has rapidly gained dominance in the global herbicide market. Glyphosate is the only herbicide that targets 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in plant chloroplasts. EPSPS is widely distributed in plants and some fungi and bacteria, and is the sixth enzyme in the shikimate metabolic pathway. The shikimate pathway is most active at the plant's growing point and is extremely important to plants, contributing more than 35% of the plant's dry weight biomass. In the shikimate pathway, EPSPS is responsible for catalyzing the conversion of phosphoenolpyruvate (PEP) and phosphoshikimate (S3P) to 5-enolpyruvylshikimate-3-phosphate (EPSP). The mechanism of action of glyphosate is to bind with EPSPS in plants through competitive PEP and non-competitive S3P, forming a structurally stable EPSPS-S3P-glyphosate complex, thereby causing the loss of EPSPS activity, ultimately leading to inhibited plant growth or even death.

[0004] Currently, commercially available glyphosate-resistant crops are mainly produced by introducing the epsps gene from Agrobacterium CP4 strain. However, due to the presence of foreign genes in these crops, the large-scale commercialization of such transgenic crops has been significantly limited, given the increasing emphasis on biosafety considerations. Therefore, precisely editing the rice genome to cultivate new lines resistant to glyphosate not only helps overcome these obstacles but also has significant scientific research and practical application value. In 2016, Gao Caixia's research group used gene editing technology to simultaneously mutate threonine (T) at position 173 and proline (P) at position 177 of the EPSPS gene in rice to isoleucine (I) and serine (S), respectively, creating glyphosate-resistant EPSPS (TP-IS) rice. EPSPS (TP-IS) rice can only be stably inherited in a heterozygous form, accompanied by a significantly reduced seed setting rate, resulting in severe yield losses. Besides generating glyphosate-resistant plants through amino acid mutations at the EPSPS site, increasing the expression level of EPSPS is also an important means for plants in nature to produce glyphosate-resistant traits.

[0005] Given the numerous drawbacks of currently available non-Glyphosate-resistant rice, such as low resistance levels, inability to be stably inherited, and severe loss of agronomic traits, there is an urgent need in production for a non-Glyphosate-resistant rice variety with high resistance, stable inheritance, and no significant yield loss, along with its breeding method.

[0006] Invention Summary

[0007] To address the aforementioned problems in the prior art, the present invention provides a method for obtaining glyphosate resistance without adaptation cost, the method comprising the following steps:

[0008] At least two copies of the endogenous epsps gene are generated in the plant through chromosome fragment doubling, with at least one copy of the epsps gene generating a point mutation for glyphosate resistance and at least one copy retaining the wild type.

[0009] In one specific embodiment, the plant is a plant with a single copy of epsps.

[0010] In one specific embodiment, the method for generating at least two copies of the endogenous epsps gene by doubling chromosome fragments includes the following steps:

[0011] Simultaneous cleavage of the intergenic regions upstream and downstream of the epsps gene in the plant genome produces double-stranded DNA breaks, wherein the cleaved chromosomal fragments contain at least a complete epsps gene expression cassette or gene region. These DNA breaks are interconnected via non-homologous end joining (NHEJ) or homologous repair. Plants that produce doubled copies of the epsps gene and contain at least two copies of the endogenous epsps gene are identified and screened.

[0012] Simultaneous cleavage of the upstream and downstream intergenic regions of the epsps gene in the plant genome produces single-strand DNA breaks. The chromosome fragment between the two ends contains at least a complete epsps gene expression cassette or gene region. The 3' end of the DNA single-strand break is doubled through complementary amplification to identify and screen plants that produce doubled epsps gene and contain at least two copies of the endogenous epsps gene.

[0013] In one specific embodiment, the at least two copies of the endogenous epsps gene include at least two endogenous epsps gene expression cassettes or gene regions.

[0014] In one specific embodiment, the doubling of the chromosome segment also includes the further doubling of the epsps gene in combination with other endogenous genes.

[0015] In another specific embodiment, the other endogenous gene is the waxy gene.

[0016] In one specific embodiment, the expression cassette includes at least a promoter, a coding region, and a terminator; or the gene region includes at least a gene coding region.

[0017] In one specific embodiment, the glyphosate-resistant point mutation comprises one or more of the following mutation points:

[0018] In the rice EPSPS amino acid sequence corresponding to SEQ ID NO: 2, amino acid position 172 is mutated from glycine to alanine, amino acid position 173 is mutated from threonine to isoleucine, amino acid position 174 is mutated from alanine to valine, or amino acid position 177 is mutated from proline to serine.

[0019] In another specific embodiment, the glyphosate-resistant point mutation comprises:

[0020] In the rice EPSPS amino acid sequence corresponding to SEQ ID NO:2, amino acid 173 is mutated from threonine to isoleucine, amino acid 174 is mutated from alanine to valine, and amino acid 177 is mutated from proline to serine.

[0021] In the rice EPSPS amino acid sequence corresponding to SEQ ID NO: 2, amino acid position 172 is mutated from glycine to alanine, amino acid position 173 is mutated from threonine to isoleucine, and amino acid position 177 is mutated from proline to serine; or

[0022] In the rice EPSPS amino acid sequence corresponding to SEQ ID NO:2, amino acid position 173 is mutated from threonine to isoleucine and amino acid position 177 is mutated from proline to serine.

[0023] In one specific implementation, the point mutation or DNA break is achieved by delivering a gene-editing tool with targeting properties into plant cells to contact specific locations on the genomic DNA.

[0024] In one specific embodiment, the gene editing tool with targeting properties is selected from CRISPR / Cas, Meganuclease, Zinc finger nuclease, TALEN, CBE, ABE, or PE.

[0025] In another specific embodiment, the CRISPR / Cas tool enzyme is KingCas12 or Cas9.

[0026] In another specific embodiment, the amino acid sequence of KingCas12 is as shown in SEQ ID NO: 8 or the amino acid sequence of the Cas9 enzyme is as shown in SEQ ID NO: 18.

[0027] In one specific embodiment, the method for delivering a gene-editing tool with targeting properties into cells is selected from: 1) PEG-mediated cell transfection; 2) liposome-mediated cell transfection; 3) electroporation transformation; 4) microinjection; 5) gene gun bombardment; or 6) Agrobacterium-mediated transformation.

[0028] The present invention also provides a plant obtained according to any one of the methods and its application in breeding.

[0029] Invention Details

[0030] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. For example, the standard recombinant DNA and molecular cloning techniques used in this invention are well known to those skilled in the art and are described more fully in the following literature: Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989. Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0031] The term "fitness cost" used in this article, also known as "adaptation cost," refers to the price an organism may pay after acquiring a certain adaptive trait (such as antibiotic or herbicide resistance). This cost is usually manifested in the fact that, in an environment without selection pressure, the survival ability, reproductive capacity, or adaptability of an organism that has acquired an adaptive trait may decrease compared to an organism that has not acquired that trait. Adaptation costs can be divided into the following categories: (1) Ecological cost: After an organism acquires a certain adaptive trait, it may have a negative impact on its living environment, leading to a narrowing of its ecological niche and a decrease in its survival ability. (2) Physiological cost: After an organism acquires an adaptive trait, it may have a negative impact on its physiological functions, leading to a decrease in its physiological functions. (3) Evolutionary cost: After an organism acquires an adaptive trait, it may have a negative impact on its evolutionary potential, leading to a slowdown in its evolutionary rate. For example, after rice EPSPS produces the (TP-IS) mutation, the plant acquires glyphosate resistance, but the plant is stunted, the yield is reduced, and it is impossible to obtain a (TP-IS) homozygous line.

[0032] The term "genome" refers to the complete complement of genetic material (genes and non-coding sequences) present in every cell, virus, or organelle of an organism, and / or the complete set of chromosomes inherited as a unit (haploid) from a parent.

[0033] The term "gene editing" refers to strategies and techniques for targeted and specific modifications to any genetic information or genome of a living organism. Therefore, the term includes editing of gene-coding regions, but also editing of regions other than the gene-coding regions of the genome. It also includes editing or modifying the nucleus (if present) and other genetic information within the cell.

[0034] The term "chromosomal structural variation" refers to the phenomenon in eukaryotic organisms where the structure of chromosomes, the carriers of genetic material, changes, resulting in alterations in the traits of offspring. It is classified into several types, including chromosome segment duplication, deletion, inversion, and translocation.

[0035] The term "chromosome segment doubling" refers to the phenomenon of variation caused by the addition of the same segment to a chromosome. This can be an increase of 1, 2, 3, 4, 5, 10, 15, 20, 30 or more times.

[0036] The term "chromosomal segment deletion" refers to the phenomenon of a mutation caused by the loss of a segment of a chromosome.

[0037] The term "chromosomal segment inversion" refers to the phenomenon where a segment of a chromosome is rotated 180°, resulting in a variation.

[0038] The term "chromosome arm translocation" refers to the exchange of chromosome arms between non-homologous chromosomes, resulting in variation.

[0039] The term "chromosomal insertion translocation" refers to the phenomenon in which a segment of a chromosome is transferred to another non-homologous chromosome, thereby causing a variation. In this invention, "insertion translocation" and "knock-in" can be used interchangeably.

[0040] The term “CRISPR / Cas” can refer to a CRISPR-based nuclease or a nucleic acid sequence encoding it, including but not limited to: 1) Cas9, including SpCas9, ScCas9, SaCas9, xCas9, VRER-Cas9, EQR-Cas9, SpG-Cas9, SpRY-Cas9, SpCas9-NG, NG-Cas9, NGA-Cas9 (VQR), etc.; 2) Cas12, including LbCpf1, FnCpf1, AsCpf1, MAD7, KingCas12 (SEQ ID NO:8), etc.; or any variant or derivative of the aforementioned CRISPR-based nucleases. Preferably, the at least one of the CRISPR-based nucleases contains a mutation compared to the corresponding wild-type sequence, such that the obtained CRISPR-based nuclease recognizes a different PAM sequence.

[0041] The term "CRISPR" refers to a sequence-specific genetic manipulation technique that relies on clustered, regularly spaced short palindromic repeats, unlike RNA interference which regulates gene expression at the transcriptional level.

[0042] In this invention, "guide RNA" and "directing RNA" are used interchangeably. In this invention, the guide RNA is used to guide the Cas protein to a specific DNA sequence.

[0043] In some embodiments, guide RNA(one or more) and Cas9 or KingCas12 protein can be delivered to cells as a ribonucleoprotein (RNP) complex. The RNP consists of purified Cas9 or KingCas12 protein complexed with the guide RNA, and it is well known in the art that RNPs can be efficiently delivered to a variety of cell types, including but not limited to stem cells and immune cells (Addgene, Cambridge, MA; Mirus Bio LLC, Madison, WI).

[0044] In one specific implementation, the guide RNA is crRNA.

[0045] In one specific embodiment, the guide RNA consists of two parts: crRNA (CRISPR RNA) containing a sequence complementary to the target DNA; and transducer RNA (tracrRNA) that helps the crRNA bind to the Cas protein. The combination of these two components is referred to in this invention as guide RNA (gRNA).

[0046] In one specific implementation, crRNA and tracrRNA are designed into a single chimeric RNA, called sgRNA (single guide RNA).

[0047] In this article, the protospacer adjacent motif (PAM) refers to a short nucleotide sequence adjacent to the (targeted) target sequence (prespacer) recognized by the gRNA / Cas endonuclease system. If the target DNA sequence is not adjacent to a suitable PAM sequence, the Cas endonuclease may fail to recognize the target DNA sequence. The sequence and length of the PAM in this article can vary depending on the Cas protein or Cas protein complex used. The PAM sequence can be of any length, but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.

[0048] The term "gene" includes a segment of nucleic acid that expresses a functional molecule (such as, but not limited to, a specific protein), including regulatory sequences before (5' non-coding sequence) and after (3' non-coding sequence).

[0049] The DNA sequence that “encodes” a specific RNA is the DNA nucleic acid sequence that is transcribed into RNA. DNA polynucleotides can encode RNA (mRNA) that is translated into proteins, or DNA polynucleotides can encode RNA that is not translated into proteins (such as tRNA, rRNA, or RNA that targets DNA; also known as “non-coding” RNA or “ncRNA”).

[0050] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0051] For the terminology related to amino acid substitutions used in the specification, the first letter represents a naturally occurring amino acid at a specific position in a particular sequence, the following number represents the position relative to SEQ ID NO:2, and the second letter represents the different amino acid that replaces that natural amino acid. For example, T173I indicates that, relative to the amino acid sequence of SEQ ID NO:2, threonine at position 173 is replaced by isoleucine. For double or multiple mutations, the mutations are separated by " / ". For example, T173I / A174V indicates that, relative to the amino acid sequence of SEQ ID NO:2, threonine at position 173 is replaced by isoleucine, and alanine at position 174 is replaced by valine, and both mutations are present in the specific mutant OsEPSPS protein.

[0052] The terms "corresponding to" and "relative to" are used interchangeably. In this invention, the specific amino acid position (number) within the protein is determined using standard sequence alignment tools by comparing the amino acid sequence of the target protein with SEQ ID NO:2, etc. For example, the Smith-Waterman algorithm or the CLUSTALW2 algorithm can be used to align two sequences, where the sequence is considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80:726-730. In the ClustalW2 (1.82) algorithm, the default parameters are preferably used: protein gap opening penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNA GAPDIST = 4.

[0053] The AlignX program (part of the vectorNTI group) is preferably used with default parameters suitable for multiple alignments (gap opening penalty: 10; gap extension penalty: 0.05) to determine the position of specific amino acids in the protein of the present invention by comparing the amino acid sequence of the protein with SEQ ID NO:2.

[0054] Those skilled in the art will also understand that the structure of a protein can be altered without adversely affecting its activity and function. For example, one or more conserved amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional conformation of the protein molecule. Examples and implementations of conserved amino acid substitutions are familiar to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., a nonpolar amino acid residue can replace another nonpolar amino acid residue, a polar uncharged amino acid residue can replace another polar uncharged amino acid residue, a basic amino acid residue can replace another basic amino acid residue, and an acidic amino acid residue can replace another acidic amino acid residue. Conservative substitutions in which an amino acid is replaced by another amino acid belonging to the same group fall within the scope of this invention, provided that the substitution does not impair the biological activity of the protein.

[0055] Therefore, in addition to the mutations described above, the mutant proteins of the present invention may also contain one or more other mutations, such as conserved substitutions, in their amino acid sequences. Furthermore, the present invention also covers mutant proteins containing one or more other non-conserved substitutions, provided that such non-conserved substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention.

[0056] As is well known in the art, one or more amino acid residues can be deleted from the N and / or C-terminus of a protein while retaining its functional activity. Therefore, in another aspect, the present invention also relates to fragments of mutant proteins that have one or more amino acid residues deleted from their N and / or C-terminus while retaining their desired functional activity; these are also within the scope of the present invention and are referred to as bioactive fragments. In the present invention, a "bioactive fragment" refers to a portion of the mutant protein of the present invention that retains the biological activity of the mutant protein of the present invention. For example, a bioactive fragment of a mutant protein may be a portion of the protein in which one or more (e.g., 1-50, 1-25, 1-10, or 1-5, e.g., 1, 2, 3, 4, or 5) amino acid residues are deleted from the N and / or C-terminus, but which still retains the biological activity of the full-length protein.

[0057] The term "mutation" refers to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to the normal sequence, wild-type sequence, or reference sequence. In some embodiments, a mutation refers to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to the nucleotide or amino acid sequence of a non-herbicide-resistant EPSPS protein. In some embodiments, a mutation refers to one or more mutations at amino acid positions relative to a reference EPSPS amino acid sequence as shown in SEQ ID NO: 2 or at homologous positions in its different species homologs. In some embodiments, a mutation may include substitution, deletion, inversion, or insertion. In some embodiments, substitution, deletion, insertion, or inversion may include variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. In some embodiments, substitution, deletion, insertion, or inversion may include variations at amino acid positions 1, 2, 3, 4, 5, 6, 7, or 8.

[0058] The terms "polynucleotide" and "nucleic acid" are used interchangeably, including DNA, RNA, or their hybrids, which can be double-stranded or single-stranded.

[0059] The terms "nucleotide sequence" and "nucleic acid sequence" both refer to the sequence of bases in DNA or RNA.

[0060] As used in this invention, "expression cassette," "expression vector," and "expression construct" refer to vectors, such as recombinant vectors, suitable for expressing nucleotide sequences of interest in plants. "Expression" refers to the production of a functional product. For example, the expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to generate mRNA or functional RNA) and / or the translation of RNA into precursor or mature proteins.

[0061] The "expression construct" of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, a translatable RNA (such as mRNA).

[0062] The "expression construct" of the present invention may contain regulatory sequences and nucleotide sequences of interest from different sources, or regulatory sequences and nucleotide sequences of interest from the same source but arranged in a manner different from those normally found in nature.

[0063] The terms “recombinant expression vector” or “DNA construct” are used interchangeably herein and refer to a DNA molecule comprising a vector and at least one insert. Recombinant expression vectors are typically created for the purpose of expressing and / or propagating the insert or for constructing other recombinant nucleotide sequences. The insert may be operatively or inoperably linked to a promoter sequence and may be operatively or inoperably linked to a DNA regulatory sequence.

[0064] The terms "regulatory sequence" and "regulatory element" are used interchangeably, referring to nucleotide sequences located upstream (5' non-coding sequence), midway, or downstream (3' non-coding sequence) of a coding sequence that influence the transcription, RNA processing, stability, or translation of the relevant coding sequence. Plant expression regulatory elements are nucleotide sequences that can control the transcription, RNA processing, stability, or translation of the nucleotide sequence of interest in plants. Depending on their proximity to the sequence or gene they control, regulatory elements are also called cis- or trans-regulatory elements.

[0065] Regulatory sequences may include, but are not limited to, promoters, translational leader sequences, introns, and polyadenylation recognition sequences.

[0066] A "promoter" refers to a nucleic acid fragment that controls the transcription of another nucleic acid fragment. In some embodiments of the present invention, the promoter is a promoter capable of controlling gene transcription in plant cells, regardless of whether it originates from plant cells. The promoter can be a constitutive promoter, a tissue-specific promoter, a developmental regulatory promoter, or an inducible promoter.

[0067] "Constraint promoters" refer to promoters that generally cause gene expression in most cell types and under most conditions. "Tissue-specific promoters" and "tissue-preferred promoters" are used interchangeably and refer to promoters that are primarily, but not necessarily, expressed specifically in one tissue or organ, and may also be expressed in a specific cell type. "Developmental regulatory promoters" are promoters whose activity is determined by developmental events. "Inducible promoters" selectively express manipulated DNA sequences in response to endogenous or exogenous stimuli (environment, hormones, chemical signals, etc.).

[0068] The term "enhancer" refers to a DNA sequence that can stimulate promoter activity and can be an intrinsic element of the promoter or a heterologous element inserted to enhance the promoter level or tissue specificity.

[0069] As used herein, the term "operably linked" refers to the linking of a regulatory element (e.g., but not limited to, promoter sequences, transcription termination sequences, etc.) to a nucleic acid sequence (e.g., coding sequences or open reading frames) such that transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.

[0070] "Introducing" nucleic acid molecules (such as plasmids, linear nucleic acid fragments, RNA, etc.) or proteins into plants refers to transforming plant cells with the nucleic acids or proteins so that the nucleic acids or proteins can function in the plant cells. The term "transformation" as used in this invention includes both stable transformation and transient transformation.

[0071] "Stable transformation" refers to the introduction of a foreign nucleotide sequence into a plant genome, resulting in the stable inheritance of the foreign gene. Once stable transformation occurs, the foreign nucleic acid sequence is stably integrated into the genome of the plant and its subsequent generations.

[0072] "Transient transformation" refers to the introduction of nucleic acid molecules or proteins into plant cells to perform their functions without the foreign gene being stably inherited. In transient transformation, the foreign nucleic acid sequence does not integrate into the plant genome.

[0073] Altering the expression of endogenous genes in organisms involves both intensity and spatiotemporal characteristics. Intensity includes upregulation (knock-up), downregulation (knock-down), and zeroing (knockout); spatiotemporal specificity includes time (reproductive stage) specificity and space (tissue) specificity, as well as inducibility. Furthermore, altering protein targeting can be achieved, for example, changing a protein located in the cytoplasm to one located in chloroplasts or the nucleus.

[0074] The "knock-up" and "upregulation of gene expression level" mentioned in this invention refer to the increased expression level of the new gene relative to the corresponding endogenous wild-type gene of the organism, preferably an increase of at least 0.5 times, at least 1 time, at least 2 times, at least 3 times, at least 4 times or at least 5 times.

[0075] As used herein, the term "plant" includes the whole plant and any offspring, plant cells, tissues, or parts. The term "plant part" includes any part of a plant, including, for example, but not limited to: seeds (including mature seeds, immature embryos without a seed coat, and immature seeds); plant cuttings; plant cells; plant cell cultures; plant organs (e.g., pollen, embryo, flower, fruit, bud, leaf, root, stem, and related explants). Plant tissues or plant organs can be seeds, callus, or any other population of plant cells organized into structural or functional units. Some plant cells or tissue cultures are capable of regenerating plants with the physiological and morphological characteristics of the plant from which the cell or tissue originated, and are capable of regenerating plants with substantially the same genotype as that plant. In contrast, some plant cells are incapable of regenerating plants. Regenerative cells in plant cells or tissue cultures can be embryos, protoplasts, meristems, callus, pollen, leaves, anthers, roots, root tips, filaments, flowers, kernels, spikes, rachis, shells, or stems.

[0076] Plant parts include harvestable parts and parts that can be used to propagate offspring. Plant parts that can be used for propagation include, for example, but not limited to: seeds; fruits; cuttings; seedlings; tubers; and rootstocks. Harvestable parts of a plant can be any useful part of the plant, including, for example, but not limited to: flowers; pollen; seedlings; tubers; leaves; stems; fruits; seeds; and roots.

[0077] Plant cells are the structural and physiological units of a plant. As used herein, plant cells include protoplasts and protoplasts with a partial cell wall. Plant cells can be in the form of isolated single cells or aggregates of cells (e.g., loose callus and cultured cells) and can be part of higher-level tissue units (e.g., plant tissues, plant organs, and whole plants). Thus, a plant cell can be a protoplast, a gamete-producing cell, or a cell or collection of cells capable of regenerating into a whole plant. Therefore, in the embodiments described herein, a seed comprising multiple plant cells and capable of regenerating into a whole plant is considered a “plant part”.

[0078] As used herein, the term "protoplast" refers to a plant cell whose cell wall has been completely or partially removed, exposing its lipid bilayer membrane. Typically, a protoplast is an isolated plant cell without a cell wall, which has the potential to regenerate cell cultures or whole plants.

[0079] Plant "offspring" includes any subsequent generations of a plant.

[0080] In this invention, "plant" should be understood as any differentiated multicellular organism capable of photosynthesis, particularly monocotyledonous or dicotyledonous plants, such as: (1) food crops: *Oryza* spp., for example, rice (*Oryza sativa*), broadleaf rice (*Oryza latifolia*), paddy rice (*Oryza sativa*), and gleaming rice (*Oryza glaberrima*); *Triticum* spp., for example, common wheat (*Triticum aestivum*), and durum wheat (*T. turgidum ssp. durum*); *Hordeum* spp., for example, barley (*Hordeum vulgare*), Arizona barley (*Hordeum arizonicum*); rye (*Secale cereale*); *Avena* spp., for example, oats (*Avena sativa*), wild oats (*Avena fatua*), and byzantina oats (*Avena byzantina*). fatuavar. sativa, hybrid oats (Avena hybrida); barnyard grass (Echinochloa spp.), for example, pearl millet (Pennisetum glaucum), sorghum (Sorghum bicolor, Sorghum vulgare)), black wheat, maize or corn, millet, rice, foxtail millet, sorghum, millet, buckwheat (Fagopyrum spp.), millet (Panicum miliaceum), millet (Setaria italica), marsh grass (Zizania palustris), Ethiopian thrush (Eragrostis tef), millet (Panicum miliaceum), dragon claw millet (Eleusine coracana); (2) legumes: soybean (Glycine spp.), for example, soybean (Glycine max), soybean (Soja hispida, Soja The genera *Vicia*, *Vigna*, *Pisum*, *Field Bean*, *Lupinus*, *Vicia*, *Tamarindus indica*, *Lens culinaris*, and *Lathyrus* are all related to the pea family.), lentils (Lablab), broad beans, mung beans, red beans, chickpeas; (3) oil crops: peanuts (Arachis hypogaea), peanuts (Arachis spp.), sesame (Sesamum spp.), sunflowers (Helianthus spp.) (e.g. sunflowers (Helianthus annuus)), oil palms (Elaeis) (e.g. oil palms (Elaeis guineensis), American oil palms (Elaeis oleifera)), soybeans (soybeans), rapeseed (Brassica napus), brassica, sesame, mustard greens (Brassica juncea), rapeseed rape (oilseed rape), camellia, oil palm, olive, castor bean, European rapeseed (Brassica napus L.), canola (canola); (4) fiber crops: sisal (Agave sisalana), cotton (cotton, sea island cotton (Gossypium barbadense), upland cotton (Gossypium hirsutum), kenaf, sisal, abaca, flax (Linum usitatissimum), jute, ramie, hemp (Cannabis sativa), fire hemp; (5) Fruit crops: Ziziphus spp., Cucumis spp., Passiflora edulis, Vitis spp., Vaccinium spp., Pyrus communis, Prunus spp., Psidium spp., Punica granatum, Malus spp., Citrullus lanatus, Citrus spp., Ficus carica, Fortunella spp., Fragaria spp., Crataegus spp., Diospyros spp.), red fruit (Eugenia unifora), loquat (Eriobotrya japonica), longan (Dimocarpus longan), papaya (Carica papaya), coconut (Cocos spp.), star fruit (Averrhoa carambola), monkey fruit (Actinidia spp.), almond (Prunus amygdalus), banana (Musa spp.).(Banana), Avocado (Persea spp.), Avocado (Persea americana)), Guava (Psidium guajava), Mammea americana, Mango (Mangifera indica), Olive (Olea europaea)), Papaya (Carica papaya), Coconut (Cocos nucifera), Malpighia emarginata, Sapodilla (Manilkara zapota), Pineapple (Ananas comosus), Anchovy (Annona spp.), Citrus (Citrus spp.), Jackfruit (Artocarpus spp.), Litchi (Litchi chinensis), Ribes (Ribes spp.), Rubus (Rubus) (6) Root and tuber crops: cassava (Manihot spp.), sweet potato (Ipomoea batatas), taro (Colocasia esculenta), pickled mustard greens, onion, water chestnut, sedge, yam; (7) Vegetable crops: spinach (Spinacia spp.), common bean (Phaseolus spp.), lettuce (Lactuca sativa), bitter melon (Momordica spp.), parsley (Petroselinum crispum), pepper (Capsicum spp.), solanum (Solanum spp.) (e.g., potato (Solanum tuberosum), red eggplant (Solanum integrifolium) or tomato (Solanum lycopersicum)), tomato (Lycopersicon) (e.g., tomato (Lycopersicon esculentum), tomato (Lycopersicon lycopersicum), pear-shaped tomato (Lycopersicon pyriforme)), and hard-skinned bean (Macrotyloma spp.).), headless cabbage, angular loofah, lentil, okra, onion, potato, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, carrot, cauliflower, celery, collard greens, zucchini, winter melon (Benincasa hispida), asparagus officinalis, celery (Apium graveolens), amaranth (Amaranthus spp.), allium (Allium spp.), okra (Abelmoschus spp.), endive (Cichorium endivia), squash (Cucurbita spp.), coriander (Coriandrum) (8) Flowering crops: Tropaeolum minus, Tropaeolum majus, Canna indica, Opuntia spp., Tagetes genus (e.g., European rapeseed, Brassica napus, Brassica rapa ssp., Canola, Oilseed rape, Turnip rape, Mustard, Black mustard, Rapeseed rape), Brussels sprouts, Solanaceae plants (eggplant), sweet pepper, cucumber, loofah, Chinese cabbage, rapeseed, cabbage, gourd, leek, lotus, lotus root, lettuce; (8) Flowering crops: Tropaeolum minus, Tropaeolum majus, Canna indica, Opuntia spp., Tagetes genus spp.), orchid, spider lily, clivia, amaryllis, rose, rose, jasmine, tulip, cherry blossom, morning glory, marigold, lotus, daisy, carnation, petunia, lily, plum blossom, daffodil, winter jasmine, primrose, daphne, camellia, white magnolia, purple magnolia, viburnum, clivia, crabapple, peony, peony, lilac, azalea, Western azalea, Michelia figo, Bauhinia, Kerria japonica, weigela, forsythia, jasmine, broom, cyclamen, phalaenopsis, dendrobium, hyacinth, iris, calla lily, marigold, lotus, begonia, fuchsia, begonia maculata, geranium, pothos; (9) medicinal crops: safflower (Carthamus tinctorius), mint (Mentha spp.).), Rheum rhabarbarum, Crocus sativus, Lycium barbarum, Polygonatum odoratum, Polygonatum sibiricum, Anemarrhena asphodeloides, Ophiopogon japonicus, Fritillaria cirrhosa, Curcuma longa, Amomum villosum, Polygonum multiflorum, Rheum palmatum, Glycyrrhiza uralensis, Astragalus membranaceus, Panax ginseng, Panax notoginseng, Acanthopanax senticosus, Angelica sinensis, Ligusticum chuanxiong, Bupleurum chinense, Datura stramonium, Datura stramonium, Mentha haplocalyx, Leonurus japonicus, Pogostemon cablin, Scutellaria baicalensis, Prunella vulgaris, Pyrethrum salicaria, Ginkgo biloba, Cinchona japonica, Rubia cordifolia, Alfalfa, Pepper, Isatis indigotica, Atractylodes macrocephala; (10) Raw material crops: Rubber, Ricinus communis, Tung oil tree, Mulberry, Hops, Birch, Alder, Lacquer tree; (11) Forage crops: Agropyron spp., Trifolium spp., Miscanthus sinensis, Pennisetum sp., Phalaris (12) Sugar crops: sugarcane (species of the genus Saccharum), beet (species of the genus Beta vulgaris); (13) Beverage crops: large-leaved tea (Camellia sinensis), tea (Camellia sinensis), tea tree (tea), coffee (species of the genus Coffea), cocoa (species of the genus Coffea), alfalfa, timothy grass, alfalfa, sweet clover, milkvetch, tamarisk, sesbania, duckweed, water hyacinth, purple locust, lupin, clover, alfalfa, water hyacinth, water peanut, ryegrass; (14) Sugar crops: sugarcane (species of the genus Saccharum), beet (Beta vulgaris); (15) Beverage crops: large-leaved tea (Camellia sinensis), tea (Camellia sinensis), tea tree (tea), coffee (species of the genus Coffea), cocoa tree (Theobroma) (14) Turfgrass: Ammophila arenaria, Poa spp. (Poa pratensis) (bluegrass), Agrostis spp. (Agrostis palustris) (Agrostis palustris) (Agrostis spp.) ...Bermuda grass, bermudagrass, Stenotaphrum secundatum (Stenotaphrum secundatum) (St. Augustine grass), Paspalum spp. (Paspalum), Eremochloa ophiuroides (Centipede grass), Axonopus spp. (Carpet grass), Bouteloua dactyloides (Buffalo grass), Bouteloua var. spp. (Granmar grass), Digitaria sanguiinalis, Cyperus rotundus, Kylling abrevifolia, Cyperus amuricus, Erigeron canadensis, Hydrocotyle sibthorpioides, Kummerowia striata, Euphorbia humifusa), Viola arvensis, white sedge, heterospike, turf; (15) Tree crops: Pinus spp., Salix sp., Acer spp., Hibiscus spp., Eucalyptus sp., Ginkgo biloba, Bambusa sp., Populus spp., Prosopis spp., Quercus spp., Phoenix spp., Fagus spp., Ceiba pentandra, Cinnamomum spp., Corchorus sp., Phragmites australis), Physalis spp., Desmodium spp., Poplar, Ivy, Populus, Coral Tree, Ginkgo, Oak, Ailanthus altissima, Schima superba, Holly, Sycamore, Privet, Buxus macrocarpa, Larch, Black thorn, Masson pine, Simao pine, Yunnan pine, South Asian pine, Pinus tabuliformis, Red pine, Black walnut, Lemon, Sycamore, Syzygium spp., Davidia involucrata, Bombax ceiba, Javanese kapok, Bauhinia purpurea, Bauhinia purpurea, Rain tree, Albizia julibrissin, Agrimonia pilosa, Erythrina crista-galli, Magnolia grandiflora, Cycas revoluta, Lagerstroemia indica, Conifers, Trees, Shrubs; (16) Nut crops: Brazil chestnut (Bertholletia excelsea), Castanea spp., Corylus spp.), Carya spp., Juglans spp., Pistacia vera, Anacardium occidentale, Macadamia integrifolia, Pecan, Macadamia nut, Pistachio, Almond and other nut-producing plants; (17) Others: Arabidopsis thaliana, Brachypodium album, Tribulus terrestris, Large foxtail grass, Goosegrass, Cadaba farinosa, Algae, Carex elata, Ornamental plants, Carissa macrocarpa, Cynara spp., Daucus carota, Dioscorea spp., Erianthus sp., Festuca arundinacea, Hemerocallis fulva, Lotus spp., Luzula *Sylvatica*, *Medicago sativa*, *Melilotus* spp., *Morus nigra*, *Nicotiana* spp., *Olea* spp., *Ornithopus* spp., *Pastinaca sativa*, *Sambucus* spp., *Sinapi* ssp., *Syzygium* spp., *Tripsacum dactyloides*, *Triticosecale rimpaui*, *Viola odorata*, etc.

[0081] In one specific embodiment, the "plant with a single copy of EPSPS" is selected from rice, corn, millet, sorghum, tomato, chili pepper, oil-free camphor (Amborella trichopoda), asparagus (Asparagus officinalis), flaxseed (Camelina sativa), quinoa (Chenopodium quinoa), watermelon (Citrullus lanatus), kumquat (Citrus clementina), medium-grain coffee (Coffea canephora), jute (Corchorus capsularis), European hazel (Corylus avellana), lemon eucalyptus (Corymbia citriodora), cucumber (Cucumis sativus), carrot (Daucus carota), Dioscorea rotundata, eucalyptus (Eucalyptus grandis), salt mustard (Eutrema salsugineum), walnut (Juglans regia), wild pea (Lathyrus sativus), alfalfa (Medicago truncatula), and small-fruited wild banana (Musa). Acuminata, short-tongued wild rice (Oryza barthii), glossy rice (Oryza glaberrima), wild rice with spreading awns (Oryza glumipatula), southern wild rice (Oryza meridionalis), spotted rice (Oryza punctata), common wild rice (Oryza rufipogon), common bean (Phaseolus vulgaris), pistachio (Pistacia vera), pea (Pisum sativum), hairy poplar (Populus trichocarpa), European sweet cherry (Prunus avium), almond (Prunus dulcis), peach (Prunus persica), rose (Rosa chinensis), sweetgrass (Saccharum spontaneum), southern selaginella (Selaginella moellendorffii), red clover (Trifolium pratense), timopheevii wheat (Triticum timopheevii), broad bean (Vicia faba), mung bean (Vigna) radiata, cowpea (Vigna unguiculata), etc.

[0082] The terms "herbicide tolerance" and "herbicide resistance" are used interchangeably, both referring to tolerance to and resistance to herbicides. "Improved herbicide tolerance" and "improved herbicide resistance" refer to increased tolerance or resistance to the herbicide compared to plants containing wild-type genes.

[0083] The term "wild type" refers to nucleic acid molecules or proteins that can be found in nature.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are described hereafter.

[0085] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials relating to the publications referenced. Any reference to a publication is made prior to the filing date and should not be construed as an admission that the invention does not predate such publication. Furthermore, the publication date provided may differ from the actual publication date, which may require independent verification.

[0086] Unless specifically stated or implied, as used herein, the terms “a,” “an,” and “described” mean “at least one.” All patents, patent applications, and publications mentioned or cited herein are incorporated herein by reference in their entirety as if they were individually cited separately. Attached Figure Description

[0087] Figure 1. Schematic diagram of OsEPSPS chromosome fragment doubling editing;

[0088] Figure 2. Schematic diagram of OsEPSPS-OsWaxy chromosome fragment doubling editing;

[0089] Figure 3. Schematic diagram of OsEPSPS-IVS amino acid substitution editing;

[0090] Figure 4. Map of the OsEPSPS chromosome fragment doubling editing vector p1;

[0091] Figure 5. Map of the OsEPSPS-OsWaxy chromosome fragment doubling editing vector p2;

[0092] Figure 6. Map of the OsEPSPS-IVS amino acid substitution editing vector p4 mediated by homologous recombination;

[0093] Figure 7. Growth of T0 generation p1 and p2 edited seedlings after 14 days of treatment with 900g ae / ha glyphosate; the left side shows the growth of p1, and the right side shows the growth of p2; in the same group of experiments, the left side is the wild-type control, and the right side is the gene-edited rice;

[0094] Figure 8. Growth of T0 generation p4 edited seedlings after 14 days of treatment with 900g ae / ha glyphosate; from left to right: wild type without pesticide, wild type with glyphosate treatment, and p4 type with glyphosate treatment;

[0095] Figure 9. OsEPSPS expression levels in T1 generation p1 and p2 homozygous edited seedlings; the left side represents p1 homozygous edited seedlings, and the right side represents p2 homozygous edited seedlings; in the same group of test results, WT represents wild-type Suijing 18 control, and L1-L10 represent 10 homozygous edited single plants;

[0096] Figure 10. Growth status and agronomic traits of T1 generation p1 homozygous edited seedlings at the yellow maturity stage; from left to right, growth status, plant height, number of effective panicles and yield per plant are shown. In the same group of experiments, the left side is the wild type control and the right side is p1.

[0097] Figure 11. Growth status and agronomic traits of T1 generation p2 homozygous edited seedlings at the yellow maturity stage; from left to right, growth status, plant height, number of effective panicles and yield per plant are shown. In the same group of experiments, the left side is the wild type control and the right side is p2.

[0098] Figure 12. Growth status and agronomic traits of T1 generation p4 heterozygous edited seedlings at the yellow maturity stage; from left to right, growth status, plant height, number of effective panicles and yield per plant are shown. In the same group of experiments, the left side is the wild type control and the right side is p4.

[0099] Figure 13. Schematic diagram of the polymeric editing of OsEPSPS-OsWaxy chromosome fragment doubling and OsEPSPS-IVS amino acid substitution;

[0100] Figure 14. Growth of T0 generation p4@p2 homozygous edited seedlings after 14 days of treatment with 900g ae / ha glyphosate; from left to right: wild type without glyphosate, wild type with glyphosate treatment, and p4@p2 glyphosate treatment;

[0101] Figure 15. Growth status and agronomic traits of T1 generation p4@p2 homopolymer seedlings at the yellow maturity stage; from left to right, growth status, plant height, number of effective panicles and yield per plant are shown. In the same group of experiments, the left side is the wild-type control and the right side is p4@p2.

[0102] Figure 16. Schematic diagram of AE-mediated OsEPSPS-OsWaxy chromosome fragment doubling editing;

[0103] Figure 17. Map of the AE-mediated OsEPSPS-OsWaxy chromosome fragment doubling vector p5.

[0104] Sequence Description Detailed Implementation

[0105] The present invention will be further illustrated below with reference to examples. The following description is by way of example, but the scope of protection of the present invention should not be limited thereto.

[0106] Example 1: Gene Function Analysis

[0107] The Osepsps gene and adjacent gene sequences were analyzed using the NCBI database. The Osepsps gene (Gene ID: LOC4340026, SEQ ID NO: 1) is located at 1973395-1977064 bp on chromosome 6 of rice. The gene sequence, from start to stop codon, is 3267 bp in length, containing 8 exons and 7 introns, encoding the protein OsEPSPS (XP_015643046.1, SEQ ID NO: 2), which contains 515 amino acids. Functional analysis of neighboring genes revealed that the Oswaxy gene (Gene ID: LOC4340018, SEQ ID NO: 3) is located approximately 50 kb upstream of the 5' end of the Osepsps gene. Oswaxy controls the synthesis of amylose in rice, and the activity of Oswaxy is positively correlated with the content of amylose and resistant starch in rice grains (Wang A, Jing Y, Cheng Q, et al. Loss of function of SSIIIa and SSIIIb coordinately confers high RS content in cooked rice. Proc Natl Acad Sci USA. 2023; 120(19):e2220622120).

[0108] Example 2: Obtaining and testing rice with high expression of endogenous epsps gene or point mutation.

[0109] As shown in Figures 1-2, Osepsps gene knock-up expression was achieved by doubling chromosome segments, resulting in OsEPSPS chromosome segment doubling and OsEPSPS-OsWaxy chromosome segment doubling Osepsps gene knock-up rice. As shown in Figure 3, amino acid substitutions of T173I / A174V / P177S were introduced at the OsEPSPS site through homologous recombination-mediated gene knock-in editing, resulting in OsEPSPS-IVS amino acid substitution edited rice.

[0110] 1. Construction of gene editing vectors and genetic transformation in rice:

[0111] First, appropriate target sites were selected using the CRISPOR online tool (http: / / crispor.tefor.net / crispor.py). The target site downstream of the Osepsps gene terminator was named OsEPSPS-crRNA1: 5'-TTTCtctgactctctcctatggccgcg-3' (SEQ ID NO: 4), the target site upstream of the Osepsps gene promoter was named OsEPSPS-crRNA2: 5'-TTTCccgtatgaccggccgtccccacc-3' (SEQ ID NO: 5), the target site upstream of the Oswaxy gene promoter was named OsWaxy-crRNA1: 5'-CTTAattatccggccccggccgattct-3' (SEQ ID NO: 6), and the target site near the T173 / A174 / P177 sites of the OsEPSPS gene was named OsEPSPS-IVS-crRNA1: 5'-CTTGgggaacgctggaactgcaatgcg-3' (SEQ ID NO: 7).

[0112] The vector was constructed according to the method described in "Zhang Q, Zhang Y, Lu MH, et al. A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR / Cas Delivery in Maize. Plant Physiol. 2019; 181(4):1441-1448." Primers were designed for the above targets to amplify the crRNA fragments corresponding to the targets, and crRNA expression cassettes were constructed. KingCas12 was used as the Cas enzyme, and its amino acid sequence is shown in SEQ ID NO: 8. Among them, the homologous recombination-mediated OsEPSPS-IVS amino acid substitution editing rice used the sequence from 1000bp upstream to 1000bp downstream of T173I / A174V / P177S as the homologous substitution donor template. The template introduced the substitution of amino acids of T173I / A174V / P177S (p4-donor (SEQ ID NO: 9)). In the intermediate vector that introduced the target, the HDR homologous substitution donor sequence was linked. The final vector-related information is shown in Table 1, and the vector map is shown in Figures 4-6.

[0113] Table 1. Gene Editing Vector Information

[0114] Rice callus was transformed using Agrobacterium-mediated genetic transformation, as shown in Figures 1-2. Specific primers were designed for molecular identification of the transformed materials. After two rounds of screening, the expected positive bands were detected in the transformed samples, and the positive bands were subjected to Sanger first-generation sequencing. The editing interface of p1 and p2 transformed positive callus was identified as seamless doubling editing, while p4 transformed positive callus underwent substitution editing at three sites: T173I, A174V, and P177S (simultaneously preserving heterozygous and homozygous edited materials). The sequencing results are shown in SEQ ID NO: 10-12, respectively.

[0115] PCR-positive callus was transferred to a new selection medium for a third round of screening and expansion culture. After the callus diameter was greater than 5 mm, the expanded callus was subjected to a second round of molecular identification using the first round of identification primers. Yellowish-white callus tissue with good growth condition that was positive in the second round of identification was selected and transferred to differentiation medium for differentiation. Seedlings of about 1 cm were obtained after 3-4 weeks. The differentiated seedlings were transferred to rooting medium for rooting culture. After the rooted seedlings were hardened off, they were transferred to flower pots filled with soil and placed in a greenhouse for cultivation.

[0116] 2. Molecular detection and glyphosate resistance testing of T0 generation edited seedlings:

[0117] One positive callus from an edit event differentiated into several T0 generation seedlings after two rounds of molecular identification. A third round of molecular identification was then performed on the T0 generation seedlings using primers from the first round of identification. Positive seedlings were then transferred to a greenhouse for cultivation. Two weeks later, glyphosate resistance testing was conducted.

[0118] The representative results of different doses of glyphosate treatment after 14 days are shown in Figures 7-8. Wild-type seedlings died completely after treatment with 450 g ae / ha, and both p1 and p2 edited seedlings died completely after treatment with 900 g ae / ha. No obvious phytotoxicity was observed in p4 edited seedlings after treatment with 900 g ae / ha.

[0119] 3. Identification and agronomic trait evaluation of T1 generation edited seedlings:

[0120] T0 generation seedlings with positive editing events were transplanted into a greenhouse for cultivation. Except for the p4 homozygous edited T0 lines, whose fertility was affected and could not produce seeds, all other positive T0 lines with editing types produced seeds normally. Therefore, only heterozygous edited seedlings of the p4 edited material were retained for further research. T1 generation non-transgenic single plants were sown in Qingdao during the peak season and tested. These non-transgenic single plants were further identified using a first-round primer assay, yielding several positive single plants with expected homozygous editing of chromosome segments and heterozygous editing of OsEPSPS-IVS amino acid substitution. Notably, in the T1 population progeny of the p4 heterozygous edited T0 single plants, only two genotypes were identified: OsEPSPS-IVS amino acid substitution heterozygous editing and wild-type, with a ratio of approximately 1:1, further indicating that the OsEPSPS-IVS homozygous genotype cannot be stably inherited.

[0121] Ten T1 generation T1 plants with positive OsEPSPS editing were randomly selected for OsEPSPS gene expression level detection, with wild-type Suijing 18 as a control. The rice ubi5 gene (SEQ ID NO: 13) was used as an internal reference gene, and primers were designed using synthesized cDNA as a template for quantitative real-time PCR. Figure 9 shows that, compared with the wild type, the OsEPSPS expression level in p1 and p2 edited seedlings was upregulated by approximately 2-10 times.

[0122] The identified positive seedlings were transplanted into the field, with Suijing 18 planted nearby as a control. Routine management was implemented throughout the growing season. At the seed ripening stage, 10 seedlings were randomly selected to record plant height and number of effective panicles. Yield of these 10 seedlings was measured after harvest. The results are shown in Figures 10-12. Compared to the wild type, homozygous edited seedlings (p1 and p2) and heterozygous edited seedlings (p4) showed no significant loss of agronomic traits, including plant height, number of effective panicles, and yield per plant.

[0123] In summary, although the expression levels of OsEPSPS were significantly increased in OsEPSPS-knock-up edited materials compared to wild-type rice without significant loss of agronomic traits, the resistance improvement in p1 and p2 edited materials was not significant; although the glyphosate resistance level of the p4 OsEPSPS-IVS amino acid substitution heterozygous edited material was significantly improved, stable and heritable homozygous edited materials could not be obtained.

[0124] Example 3: Obtaining and testing rice with knock-up and point mutation aggregation.

[0125] By combining Osepsps knock-up editing with OsEPSPS-IVS amino acid substitution editing, a hybrid edited rice with OsEPSPS-OsWaxy chromosome fragment doubling and OsEPSPS-IVS amino acid substitution editing was obtained.

[0126] 1. Aggregate editing of rice acquisition

[0127] After inducing callus using homozygous edited seeds from the T2 generation of p2, the p4 vector was transformed using Agrobacterium-mediated rice genetic transformation, and named p4@p2. Callus-specific primers with a diameter greater than 3 mm were used for molecular identification. After two rounds of screening, the expected positive band was detected in the transformed samples, and the positive band was subjected to Sanger sequencing. As shown in Figure 13, p4@p2 is a material with homozygous amino acid substitutions at three sites (T173I, A174V, and P177S) at the OsEPSPS site, while no amino acid substitution occurred at the other site. The sequencing results are shown in SEQ ID NO: 14-15.

[0128] Positive callus was transferred to a new screening medium for a third round of screening and expansion culture. After the callus diameter was greater than 5 mm, the expanded p4@p2 callus was subjected to a second round of molecular identification using the first round of identification primers. Yellowish-white callus tissue with good growth status expected to be edited in the second round of identification was selected and transferred to differentiation medium for differentiation. Seedlings of about 1 cm in length could be obtained after 3-4 weeks. The differentiated seedlings were transferred to rooting medium for rooting culture. After the rooted seedlings were hardened off, they were transferred to flower pots filled with soil and placed in a greenhouse for cultivation.

[0129] 2. Molecular detection and glyphosate resistance testing of T0 generation edited seedlings:

[0130] One positive callus from an edit event differentiated into several T0 generation seedlings after two rounds of molecular identification. A third round of molecular identification was then performed on the T0 generation seedlings using the same primers from the second round. Positive seedlings were then transferred to a greenhouse for cultivation. Two weeks later, glyphosate resistance was tested.

[0131] Figure 14 shows the results after 14 days of treatment with different doses of glyphosate. The p4@p2 edited seedlings grew completely normally under the treatment of 900g ae / ha.

[0132] 3. Identification and agronomic trait evaluation of T1 generation edited seedlings:

[0133] After transplanting T0 generation seedlings that were positive for editing events to a greenhouse, all positive T0 lines were able to produce seeds normally, with no significant impact on the fertility of the T0 generation plants. T1 generation non-transgenic single plants were sown in Qingdao during the regular season and tested. These non-transgenic single plants were further identified using the aforementioned molecular identification primers, yielding several homozygous positive single plants with expected OsEPSPS-OsWaxy chromosome fragment doubling and OsEPSPS-IVS amino acid substitution polymerization editing.

[0134] The identified positive seedlings were transplanted into the field, with Suijing 18 planted next to them as a control. Routine management was implemented throughout the growing season. At the seed ripening stage, 10 individual plants were randomly selected to record plant height and number of effective panicles. The yield of these 10 individual plants was measured after seed harvest. The results are shown in Figure 15. Compared with the wild type, the p4@p2 edited seedlings did not show significant loss of agronomic traits, including plant height, number of effective panicles, and yield per plant, all of which were not significantly different from the wild type.

[0135] In summary, the aforementioned editing events are stably inherited. The homozygous materials of OsEPSPS-OsWaxy chromosome fragment doubling and OsEPSPS-IVS amino acid substitution polymerization editing are fully tolerant to 900 g ae / ha glyphosate treatment without significant loss of agronomic traits, making them a glyphosate-resistant rice variety without any adaptation cost.

[0136] Meanwhile, glyphosate resistance tests and agronomic trait assessments were also conducted on OsEPSPS chromosome fragment doubling and OsEPSPS-IVS amino acid substitution polymer editing homozygous materials (p4@p1). The results showed that resistance was significantly improved without any adaptation cost.

[0137] In addition, through extensive experimental verification, after knocking up the epsps gene, all copies of epsps (including 2, 3, 4, 5, 6, etc.) were subjected to point mutations. However, the results showed that materials with all copies of the epsps gene mutated could not produce seeds normally, which had a great impact on fertility. Only when at least one copy of the epsps gene was mutated to produce a glyphosate-resistant point mutation and at least one copy remained wild-type could glyphosate-resistant edited materials without the cost of adaptation be produced.

[0138] Example 4: Creating a genotype with doubled editing of the OsEPSPS-OsWaxy chromosome fragment using the AE editing tool

[0139] As shown in Figure 16, “referring to the method of Zhang et al. Amplification editing enables efficient and precise duplication of DNA from short sequence to megabase and chromosomal scale. Cell, 2024; 187, 1-17.”, the genotype of doubled OsEPSPS-OsWaxy chromosome segment is expected to be obtained in rice protoplasts using the AE (Amplification editing) tool.

[0140] Using the CRISPOR online tool (http: / / crispor.tefor.net / crispor.py), suitable targets were selected. The downstream target of the Osepsps gene terminator was named OsEPSPS-AE-sgRNA1: 5'-agtaatttgagaagcatatgTGG-3' (SEQ ID NO: 16), and the upstream target of the Oswaxy gene promoter was named OsWaxy-AE-sgRNA1: 5'-gattcttaattatccggcccCGG-3' (SEQ ID NO: 17). Primers were designed to amplify the corresponding sgRNA fragments for these targets, and sgRNA expression cassettes were constructed. The amino acid sequence of the Cas9 enzyme used is shown in SEQ ID NO: 18. The final vector was named p5, and its map is shown in Figure 17.

[0141] The p5 vector was transformed into rice protoplasts via PEG-mediated transformation, and the doubling fragment interface was identified using specific primers. As shown in Figure 16, a seamless doubling editing event of the OsEPSPS-OsWaxy chromosome fragment mediated by Agrobacterium was successfully obtained in rice protoplasts, and the sequencing results are shown in SEQ ID NO: 19. The Agrobacterium-mediated rice genetic transformation yielded the expected OsEPSPS-OsWaxy chromosome fragment doubling edited material, whose glyphosate resistance level and agronomical performance were consistent with those of the p2 transformed material.

[0142] Furthermore, extensive experiments have shown that in other EPSPS crops (especially single-copy EPSPS crops such as rice, corn, millet, sorghum, tomato, and pepper), by using the above method to double the expression of the epsps gene on a chromosome segment, and then introducing single-point mutations corresponding to OsEPSPS (SEQ ID NO: 2) such as G172A, T173I, A174V, and P177S, or any combination thereof, the glyphosate resistance level was correspondingly increased without significant loss of agronomic traits. Therefore, applying the method of this invention to other plants can also obtain glyphosate resistance without adaptation costs, demonstrating good industrial value.

[0143] All publications and patent applications mentioned in the specification are incorporated herein by reference as if each publication or patent application were individually and specifically incorporated herein by reference.

[0144] Although the invention has been described in considerable detail by way of example and embodiments for clarity, it will be apparent that certain changes and modifications may be made within the scope of the appended claims, and all such changes and modifications are within the scope of the invention.

Claims

1. A method for obtaining glyphosate resistance without adaptation cost, the method comprising the following steps: At least two copies of the endogenous epsps gene are generated in the plant through chromosome segment doubling, with at least one copy of the epsps gene generating a glyphosate-resistant point mutation and at least one copy maintaining the wild type. Preferably, the plant is a plant with a single copy of epsps.

2. The method according to claim 1, characterized in that, The method for generating at least two copies of the endogenous epsps gene by doubling chromosome segments includes the following steps: Simultaneous cleavage of the intergenic regions upstream and downstream of the epsps gene in the plant genome produces double-stranded DNA breaks, wherein the cleaved chromosomal fragments contain at least a complete epsps gene expression cassette or gene region. These DNA breaks are interconnected via non-homologous end joining (NHEJ) or homologous repair. Plants that produce doubled copies of the epsps gene and contain at least two copies of the endogenous epsps gene are identified and screened. Simultaneous cleavage of the upstream and downstream intergenic regions of the epsps gene in the plant genome produces single-strand DNA breaks. The chromosome fragment between the two ends contains at least a complete epsps gene expression cassette or gene region. The 3' end of the DNA single-strand break is doubled through complementary amplification to identify and screen plants that produce doubled epsps gene and contain at least two copies of the endogenous epsps gene.

3. The method according to claim 2, characterized in that, The at least two copies of the endogenous epsps gene include at least two endogenous epsps gene expression cassettes or gene regions.

4. The method according to claim 2 or 3, characterized in that, The doubling of the chromosome segment also includes the further doubling of the epsps gene in combination with other endogenous genes; preferably, the other endogenous genes are the waxy gene.

5. The method according to any one of claims 2-4, characterized in that, The expression cassette includes at least a promoter, a coding region, and a terminator; or the gene region includes at least a gene coding region.

6. The method according to any one of claims 1-5, characterized in that, The glyphosate-resistant point mutation includes one or more of the following mutation points: In the rice EPSPS amino acid sequence corresponding to SEQ ID NO: 2, amino acid 172 is mutated from glycine to alanine, amino acid 173 is mutated from threonine to isoleucine, amino acid 174 is mutated from alanine to valine, or amino acid 177 is mutated from proline to serine. Preferably, the point mutation for glyphosate resistance comprises: In the rice EPSPS amino acid sequence corresponding to SEQ ID NO:2, amino acid 173 is mutated from threonine to isoleucine, amino acid 174 is mutated from alanine to valine, and amino acid 177 is mutated from proline to serine. In the rice EPSPS amino acid sequence corresponding to SEQ ID NO: 2, amino acid position 172 is mutated from glycine to alanine, amino acid position 173 is mutated from threonine to isoleucine, and amino acid position 177 is mutated from proline to serine; or In the rice EPSPS amino acid sequence corresponding to SEQ ID NO:2, amino acid position 173 is mutated from threonine to isoleucine and amino acid position 177 is mutated from proline to serine.

7. The method according to any one of claims 1-6, characterized in that, The point mutation or DNA break is achieved by delivering a gene-editing tool with targeting properties into plant cells to contact specific locations on the genomic DNA; preferably, the gene-editing tool with targeting properties is selected from CRISPR / Cas, Meganuclease, Zinc finger nuclease, TALEN, CBE, ABE, or PE; more preferably, the CRISPR / Cas tool enzyme is KingCas12 or Cas9; even more preferably, the amino acid sequence of KingCas12 is as shown in SEQ ID NO: 8 or the amino acid sequence of the Cas9 enzyme is as shown in SEQ ID NO:

18.

8. The method according to claim 7, characterized in that, Methods for delivering targeted gene-editing tools into cells include: 1) PEG-mediated cell transfection; 2) liposome-mediated cell transfection; and 3) electroporation. 4) Microinjection; 5) Gene gun bombardment; or 6) Agrobacterium-mediated transformation.

9. Plants obtained by the method according to any one of claims 1-8 and their application in breeding.

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