Glutamine synthetase mutant and use thereof

Abstract

The present invention relates to the field of plant genetic engineering. Specifically, the present invention relates to a glutamine synthetase mutant that can confer herbicide resistance in a plant, the use thereof, and a method for producing a herbicide-resistant plant containing the glutamine synthetase (GS) mutant.

Description

Glutamine synthetase mutant and its application Technical Field

[0001] The present invention relates to the field of plant genetic engineering, in particular to a glutamine synthetase mutant capable of conferring herbicide resistance in plants, its use, and a method for producing herbicide-resistant plants comprising the glutamine synthetase (GS) mutant. Background Art

[0002] Weeds are a major stressor that severely impacts crop growth. They compete with crops for resources, limiting their growth and development. Weeds also harbor pathogens and pests, leading to frequent crop diseases and pests, which in turn directly impacts crop yield and quality. Therefore, effective weed management and control has become a critical issue in both scientific research and production.

[0003] Herbicides have become a widely used weed control method due to their broad spectrum, high efficiency and easy operation.

[0004] Glufosinate ammonium, also known as glufosinate, is a broad-spectrum, fast-acting, low-toxic, and environmentally compatible non-selective herbicide with broad application prospects. Glufosinate achieves its weed control effect by inhibiting the activity of glutamine synthetase (GS), a key enzyme in the plant's ammonium assimilation process, disrupting normal plant growth and ultimately causing plant death. However, since most common cultivated crops are not resistant to glufosinate, the use of glufosinate herbicides often results in accidental crop damage. Therefore, cultivating glufosinate-resistant crops is of great significance for ensuring my country's grain yield and quality and promoting the development of modern agriculture.

[0005] In this field, crop glutamine synthetase is modified from the perspective of directed evolution to obtain crops that are resistant to herbicides such as glufosinate ammonium that use glufosinate as an active ingredient.

[0006] Summary of the Invention

[0007] The present invention provides at least the following items:

[0008] Item 1. A glutamine synthetase (GS) or a functional fragment thereof, which has an amino acid mutation, such as an amino acid substitution, at position 309 relative to wild-type glutamine synthetase (GS), wherein the amino acid position is referenced to SEQ ID NO: 1.

[0009] Item 2. The glutamine synthetase or a functional fragment thereof of Item 1, wherein the wild-type glutamine synthetase comprises an amino acid sequence of one of SEQ ID NOs: 1, 5, 9, 13, 33-42, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity with an amino acid sequence of one of SEQ ID NOs: 1, 5, 9, 13, 33-42.

[0010] Item 3. The glutamine synthetase or a functional fragment thereof according to Item 1 or 2, wherein the alanine (A) at position 309 of the glutamine synthetase mutant or the functional fragment thereof is substituted relative to the wild-type glutamine synthetase, and the amino acid position is referenced to SEQ ID NO: 1.

[0011] Item 4. The glutamine synthetase or a functional fragment thereof of Item 3, wherein relative to the wild-type glutamine synthetase, the alanine (A) at position 309 of the glutamine synthetase mutant or the functional fragment thereof is replaced by aspartic acid (D), threonine (T) or valine (V), and the amino acid position is referred to SEQ ID NO: 1.

[0012] Item 5. The glutamine synthetase or a functional fragment thereof of Item 3, wherein relative to the wild-type glutamine synthetase, the glutamine synthetase mutant or the functional fragment thereof comprises at least an amino acid substitution A309D, A309T or A309V, and the amino acid position is referenced to SEQ ID NO: 1.

[0013] Item 6. The glutamine synthetase or a functional fragment thereof according to any one of items 1 to 5, wherein the glutamine synthetase mutant comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 2-4, 6-8, 10-12, and 14-16, or the glutamine synthetase mutant comprises the amino acid sequence shown in any one of SEQ ID NOs: 2-4, 6-8, 10-12, and 14-16.

[0014] Item 7. The glutamine synthetase or a functional fragment thereof according to any one of Items 1 to 6, wherein the glutamine synthetase mutant or the functional fragment thereof, when expressed in a eukaryotic organism such as yeast or a plant, can confer resistance to a herbicide, such as a glutamine synthetase-inhibiting herbicide on the eukaryotic organism such as yeast or a plant. Preferably, the glutamine synthetase-inhibiting herbicide is glufosinate-ammonium.

[0015] Item 8. A nucleic acid comprising a nucleotide sequence encoding the glutamine synthetase mutant or a functional fragment thereof according to any one of Items 1 to 7.

[0016] Item 9. The nucleic acid of Item 8, wherein the nucleotide sequence encoding the glutamine synthetase mutant or a functional fragment thereof is shown in any one of SEQ ID NOs: 18-20, 22-24, 26-28, 30-32.

[0017] Item 10. An expression cassette comprising a nucleotide sequence encoding any one of the glutamine synthetase mutants or a functional fragment thereof according to Items 1 to 7, operably linked to a regulatory sequence.

[0018] Item 11. The expression cassette of Item 10, wherein the nucleotide sequence encoding the glutamine synthetase mutant or a functional fragment thereof is shown in any one of SEQ ID NOs: 18-20, 22-24, 26-28, 30-32.

[0019] Item 12. An expression construct comprising the expression cassette of Item 10 or 11.

[0020] Item 13. Use of the glutamine synthetase mutant or a functional fragment thereof according to any one of Items 1 to 7, the nucleic acid according to Item 8 or 9, the expression cassette according to Item 10 or 11, or the expression construct according to Item 12 for producing herbicide-resistant plants.

[0021] Item 14. The use of Item 13, wherein the plant is selected from rice (Oryza sativa), wheat (Triticum aestivum), Brachypodium distachyon, corn (Zea mays), warty rice (Oryza meyeriana var. granulata), wild rice (Oryza barthii), African cultivated rice (Oryza glaberrima), Panicum hallii, millet (Panicum miliaceum), foxtail millet (Setaria italica), sorghum (Sorghum bicolor), false rice (Leersia perrieri), Urartu wheat (Triticum urartu), and barley (Hordeum vulgare).

[0022] Item 15. A herbicide-resistant plant comprising or expressing the glutamine synthetase mutant or a functional fragment thereof according to any one of Items 1 to 7.

[0023] Item 16. The herbicide-resistant plant of Item 15, wherein the plant is selected from the group consisting of rice (Oryza sativa), wheat (Triticum aestivum), Brachypodium distachyon, corn (Zea mays), warty rice (Oryza meyeriana var. granulata), wild rice (Oryza barthii), African cultivated rice (Oryza glaberrima), Panicum hallii, millet (Panicum miliaceum), foxtail millet (Setaria italica), sorghum (Sorghum bicolor), false rice (Leersia perrieri), Urartu wheat (Triticum urartu), and barley (Hordeum vulgare).

[0024] Item 17. A method for producing herbicide-resistant plants by transgenic means, comprising introducing the nucleic acid of item 8 or 9, the expression cassette of item 10 or 11 and / or the expression construct of item 12 into a plant, preferably, the introduction resulting in the plant comprising or expressing the glutamine synthetase mutant or a functional fragment thereof according to any one of items 1 to 7.

[0025] Item 18. A method for producing herbicide-resistant plants, the method comprising modifying (e.g., targeted modification) the endogenous glutamine synthetase coding sequence of the plant, thereby causing the endogenous glutamine synthetase (e.g., expressed endogenous glutamine synthetase) to undergo an amino acid mutation, such as an amino acid substitution, at position 309, the amino acid position being referenced to SEQ ID NO: 1.

[0026] Item 19. The method of Item 18, wherein the endogenous glutamine synthetase of the plant, for example, comprises the amino acid sequence of one of SEQ ID NOs: 1, 5, 9, 13, 33-42, or comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to the amino acid sequence of one of SEQ ID NOs: 1, 5, 9, 13, 33-42.

[0027] Item 20. The method of Item 18 or 19, wherein the modification results in the substitution of alanine (A) at position 309 of the endogenous glutamine synthetase (eg, expressed endogenous glutamine synthetase), the amino acid position being referenced to SEQ ID NO: 1.

[0028] Item 21. The method of Item 20, wherein the modification results in the endogenous glutamine synthetase (e.g., expressed endogenous glutamine synthetase) having alanine (A) at position 309 replaced by aspartic acid (D), threonine (T) or valine (V), and the amino acid position refers to SEQ ID NO: 1.

[0029] Item 22. The method of Item 20 or 21, wherein the modification causes the endogenous glutamine synthetase (eg, expressed endogenous glutamine synthetase) to comprise an amino acid substitution A309D, A309T, or A309V, wherein the amino acid position is referenced to SEQ ID NO: 1.

[0030] Item 23. The method of any one of items 18 to 22, wherein the modification results in the glutamine synthetase mutant of any one of items 1 to 7, such as results in the plant expressing the glutamine synthetase mutant of any one of items 1 to 7.

[0031] Item 24. The method of any one of Items 18-23, wherein the endogenous glutamine synthetase coding sequence of the plant is targeted for modification by homologous recombination or gene editing.

[0032] Item 25. The method of Item 24, wherein the gene editing comprises introducing a gene editing system targeting the endogenous glutamine synthetase coding region in the plant genome into the plant.

[0033] Item 26. The method of Item 25, wherein the gene editing system is a gene editing system based on CRISPR, ZFN or TALEN, preferably, the gene editing system is a gene editing system based on CRISPR.

[0034] Item 27. The method of Item 25 or 26, wherein the gene editing system is a base editing system or a prime editing system.

[0035] Item 28. A method for producing herbicide-resistant plants, comprising subjecting a population of the plants to physical or chemical mutagenesis, and screening the mutagenized plants for plants whose endogenous glutamine synthetase (e.g., expressed endogenous glutamine synthetase) comprises an amino acid mutation such as an amino acid substitution at at least position 309, wherein the amino acid position is referenced to SEQ ID NO: 1.

[0036] Item 29. The method of Item 28, wherein a plant is screened in which endogenous glutamine synthetase (eg, expressed endogenous glutamine synthetase) is substituted with alanine (A) at position 309, the amino acid position being referenced to SEQ ID NO: 1.

[0037] Item 30. The method of Item 29, wherein plants are screened in which the alanine (A) at position 309 of endogenous glutamine synthetase (e.g., expressed endogenous glutamine synthetase) is substituted with aspartic acid (D), threonine (T) or valine (V), wherein the amino acid position is referenced to SEQ ID NO: 1.

[0038] Item 31. The method of any one of items 28 to 30, wherein a plant is screened in which an endogenous glutamine synthetase (eg, an expressed endogenous glutamine synthetase) is mutated to the glutamine synthetase mutant of any one of items 1 to 7.

[0039] Item 32. A method for identifying herbicide-resistant plants, comprising detecting

[0040] i) the presence and / or amount of glutamine synthetase or a functional fragment thereof wherein amino acid position 309 is aspartic acid (D), threonine (T) or valine (V), wherein the amino acid position is referenced to SEQ ID NO: 1; or

[0041] ii) the presence and / or expression of a nucleotide sequence encoding a glutamine synthetase or a functional fragment thereof wherein the amino acid at position 309 is aspartic acid (D), threonine (T) or valine (V), the amino acid position being referenced to SEQ ID NO: 1,

[0042] The presence or increased amount of the glutamine synthetase or the presence and / or increased expression of the nucleotide sequence indicates the presence of resistance to glutamine synthetase-inhibiting herbicides and / or increased resistance to glutamine synthetase-inhibiting herbicides in the plant, in particular relative to a plant not comprising the glutamine synthetase or the encoding nucleotide sequence.

[0043] Item 33. The method of Item 32, wherein the glutamine synthetase comprises the amino acid sequence shown in any one of SEQ ID NOs: 2-4, 6-8, 10-12, and 14-16, or comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 2-4, 6-8, 10-12, and 14-16; or the encoding nucleotide sequence of the glutamine synthetase mutant or a functional fragment thereof is as shown in any one of SEQ ID NOs: 18-20, 22-24, 26-28, 30-32.

[0044] Item 34. A plant breeding method comprising crossing the herbicide-resistant plant of Item 15 or 16 or a first plant having herbicide resistance obtained by the method of any one of Items 17 to 32 with a second plant not having the herbicide resistance, thereby introducing the herbicide resistance into the second plant.

[0045] Item 35. The method of any one of Items 17 to 34, wherein the plant is selected from rice (Oryza sativa), wheat (Triticum aestivum), Brachypodium distachyon, corn (Zea mays), Oryza meyeriana var. granulata, wild rice (Oryza barthii), African cultivated rice (Oryza glaberrima), Panicum hallii, Panicum miliaceum, Setaria italica, sorghum (Sorghum bicolor), Leersia perrieri, Urartu wheat (Triticum urartu), and barley (Hordeum vulgare). BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1. In a competitive growth experiment using a yeast library of rice glutamine synthetase OsGS1 mutants under a glufosinate selection environment, sequencing and analysis revealed that the A309D, A309T, and A309V mutations at amino acid position 309, as well as the multiple mutant S29P / T104I / V172I / A309V, exhibited a stronger growth advantage than the wild-type OsGS1-WT (Ref). Specifically, the larger the value on the abscissa of the data point in the figure, the greater the growth advantage in the glufosinate selection environment, indicating greater resistance to glufosinate.

[0047] Competitive growth experiments were conducted in a rice glutamine synthetase (OsGS1) yeast mutants (A309D, A309T, and A309V) bearing amino acid mutations at position 309, compared to wild-type OsGS1-WT in YPD medium containing 10 g / L glufosinate ammonium (PPT). The abscissa represents time in hours, and the ordinate represents log2 (mutant / wild-type). The linear fit equation is shown in the figure; the greater the slope, the greater the growth advantage of the mutant relative to the wild-type under glufosinate ammonium culture conditions.

[0048] Figure 2. Competitive growth assay of yeast mutants (A309D, A309T, and A309V) harboring amino acid mutations at position 309 in rice glutamine synthetase (OsGS1) versus wild-type OsGS1-WT in a medium containing 0.05 g / L (NH₄)₂SO₄. The abscissa represents time in hours, and the ordinate represents log² (mutant / wild-type). The linear fit equation is shown in the figure. A slope less than 0 indicates a greater growth advantage for the wild-type relative to the mutant under low-ammonia conditions. Conversely, a slope greater than 0 indicates a greater growth advantage for the mutant over the wild-type under low-ammonia conditions.

[0049] Figure 3. Ratio of enzyme activity of rice glutamine synthetase OsGS1 A309D, A309T, A309V mutants to wild-type OsGS1.

[0050] Figure 4. Growth rates of three mutants and the wild type of three species in YPD medium containing 10 g / L glufosinate. The horizontal axis represents time in hours, and the vertical axis represents the number of generations.

[0051] FIG5 . Glufosinate-resistance phenotype of glutamine synthetase mutant transgenic plants. DETAILED DESCRIPTION

[0052] 1. Definition

[0053] In the present invention, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and routine procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described 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 (hereinafter referred to as "Sambrook"). At the same time, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0054] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0055] When the word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0056] "Exogenous" with respect to a sequence refers to a sequence that is from a foreign species, or, if from the same species, has been significantly altered from its native form in composition and / or locus through deliberate human intervention.

[0057] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid" are used interchangeably and are single-stranded or double-stranded polymers of RNA or DNA that optionally contain synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single-letter names as follows: "A" is adenosine or deoxyadenosine (RNA or DNA, respectively), "C" is cytidine or deoxycytidine, "G" is guanosine or deoxyguanosine, "U" is uridine, "T" is deoxythymidine, "R" is a purine (A or G), "Y" is a pyrimidine (C or T), "K" is G or T, "H" is A or C or T, "D" is A, T, or G, "I" is inosine, and "N" is any nucleotide.

[0058] Codon optimization refers to a process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with codons that are more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which is believed to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons that are most frequently used for peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp / codon / , and these tables can be adapted in various ways. See, Nakamura et al., 2002, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044 Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0059] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, 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 attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0060] Sequence "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. (See, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0061] In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Generally, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0062] As used herein, an "expression construct" refers to a vector, such as a recombinant vector, suitable for expressing a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to produce mRNA or functional RNA) and / or translation of RNA into a precursor or mature protein.

[0063] The "expression construct" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be RNA (such as mRNA) that can be translated, for example, RNA generated by in vitro transcription.

[0064] An "expression construct" of the present invention may comprise regulatory sequences and a nucleotide sequence of interest from different sources, or regulatory sequences and a nucleotide sequence of interest from the same source but arranged in a manner different from that normally found in nature.

[0065] "Regulatory sequence" and "regulatory element" are used interchangeably to refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0066] "Promoter" refers to a nucleic acid fragment that is capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the present invention, a promoter is a promoter that is capable of controlling the transcription of a gene in a cell, whether or not it is derived from the cell. A promoter can be a constitutive promoter, a tissue-specific promoter, a developmentally regulated promoter, or an inducible promoter.

[0067] "Constitutive promoter" refers to a promoter that will generally cause a gene to be expressed in most cell types under most circumstances. "Tissue-specific promoter" and "tissue-preferred promoter" are used interchangeably and refer to a promoter that is expressed primarily, but not necessarily exclusively, in one tissue or organ, and may also be expressed in one specific cell or cell type. "Developmentally regulated promoter" refers to a promoter whose activity is determined by developmental events. "Inducible promoter" selectively expresses an operably linked DNA sequence in response to endogenous or exogenous stimuli (environmental, hormonal, chemical signals, etc.).

[0068] Examples of promoters include, but are not limited to, polymerase (pol) I, pol II, or pol III promoters. When used in plants, the promoter can be cauliflower mosaic virus 35S promoter, maize Ubi-1 promoter, wheat U6 promoter, rice U3 promoter, maize U3 promoter, rice actin promoter.

[0069] As used herein, the term "operably linked" refers to the connection of a regulatory element (e.g., but not limited to, a promoter sequence, a transcription termination sequence, etc.) to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) 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" a nucleic acid molecule (e.g., a plasmid, a linear nucleic acid fragment, RNA, etc.) or a protein into an organism refers to transforming the cells of the organism with the nucleic acid or protein so that the nucleic acid or protein can function in the cell. "Transformation" as used in the present invention includes stable transformation and transient transformation. "Stable transformation" refers to the introduction of an exogenous nucleotide sequence into the genome, resulting in the stable inheritance of the exogenous gene. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof. "Transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell to perform a function without the stable inheritance of the exogenous gene. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.

[0071] As used herein, the term "plant" includes whole plants and any offspring, cells, tissues, or parts of plants. The term "plant part" includes any part of a plant, including, for example, but not limited to: seeds (including mature seeds, immature embryos without seed coats, and immature seeds); plant cuttings; plant cells; plant cell cultures; plant organs (e.g., pollen, embryos, flowers, fruits, buds, leaves, roots, stems, and related explants). Plant tissues or plant organs can be seed, callus, or any other plant cell colony organized into structural or functional units. Plant cells or tissue cultures can regenerate plants with the physiological and morphological characteristics of the plant from which the cells or tissues were derived, and can regenerate plants with substantially the same genotype as the plant. In contrast, some plant cells cannot regenerate to produce plants. The regenerable cell in plant cell or tissue culture can be an embryo, protoplast, meristematic cell, callus, pollen, leaf, anther, root, root tip, silk, flower, kernel, ear, cob, husk, or stem.

[0072] Plant "progeny" include any subsequent generation of the plant.

[0073] 2. Glutamine synthetase (GS) mutants

[0074] Glutamine synthetase (GS; EC 6.3.1.2) is a ubiquitous enzyme in many organisms, participating in nitrogen and carbon metabolism and serving as a key enzyme in nitrogen metabolism. In plants, GS is a crucial detoxifying enzyme, neutralizing the toxicity of ammonium released through nitrate reduction, amino acid degradation, and photorespiration.

[0075] The herbicide resistance described in various aspects of the present invention preferably refers to resistance to glutamine synthetase (GS)-inhibiting herbicides. Glutamine synthetase (GS)-inhibiting herbicides include, but are not limited to, glufosinate ammonium, bialaphos-sodium, or other herbicides with phosphinotricin as the primary active ingredient. For example, glufosinate ammonium can inhibit wild-type GS, leading to intracellular ammonia accumulation, inhibition of amino acid synthesis and photosynthesis, and chlorophyll destruction. This is primarily through rapid inhibition of RuBp carboxylase and photorespiration, causing plant death.

[0076] In the present invention, the inventors created and identified new GS mutants that are resistant to GS-inhibiting herbicides by screening in yeast.

[0077] In one aspect, the present invention provides a glutamine synthetase (GS) or a functional fragment thereof, which has an amino acid mutation at position 309 relative to wild-type glutamine synthetase (GS), wherein the amino acid position is referenced to SEQ ID NO: 1.

[0078] As used herein, "amino acid position referenced to SEQ ID NO: x" (SEQ ID NO: x is a specific sequence listed herein) means that the position number of the specific amino acid described is the position number of the amino acid corresponding to that amino acid in SEQ ID NO: x. The correspondence of amino acids in different sequences can be determined using sequence alignment methods known in the art. For example, amino acid correspondence can be determined using the EMBL-EBI online alignment tool (https: / / www.ebi.ac.uk / Tools / psa / ), where two sequences are aligned using the Needleman-Wunsch algorithm using default parameters. For example, if the alanine at position 310 from the N-terminus of polypeptide X is aligned with the amino acid at position 309 of SEQ ID NO: 1 in a sequence alignment, then the alanine in polypeptide X may also be described herein as "the alanine at position 309 of polypeptide X, said amino acid position referenced to SEQ ID NO: 1."

[0079] In some embodiments, the glutamine synthetase mutant or a functional fragment thereof, when expressed in a eukaryotic organism, such as yeast or a plant, can confer resistance to a herbicide, such as a glutamine synthetase-inhibiting herbicide on the eukaryotic organism, such as yeast or a plant. "Conferring resistance to a herbicide, such as a glutamine synthetase-inhibiting herbicide on the eukaryotic organism, such as yeast or a plant" means that the resistance of the eukaryotic organism, such as yeast or a plant, comprising or expressing the glutamine synthetase mutant or a functional fragment thereof, to a herbicide, such as a glutamine synthetase-inhibiting herbicide, is enhanced, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more, relative to a eukaryotic organism, such as yeast or a plant, that does not comprise or express the glutamine synthetase mutant or a functional fragment thereof, or that only comprises or expresses (an equivalent amount of) wild-type glutamine synthetase. Methods for determining resistance to glutamine synthetase-inhibiting herbicides are known in the art. Resistance can be readily determined by one skilled in the art for a particular eukaryotic organism, such as yeast or plant, and for a particular herbicide.

[0080] In addition, the resistance of the glutamine synthetase mutant or a functional fragment thereof of the present invention to a glutamine synthetase-inhibiting herbicide can be determined by detecting the growth of a eukaryotic organism / eukaryotic cell, such as a yeast cell, or a plant / plant cell, comprising the mutant or a functional fragment thereof in the presence of a glutamine synthetase-inhibiting herbicide, for example, by referring to the methods described in Example 2, 3, or 5. The better the growth of a eukaryotic organism / eukaryotic cell, such as a yeast cell, or a plant / plant cell, in the presence of a glutamine synthetase-inhibiting herbicide, the stronger the resistance.

[0081] The enzymatic activity characteristics of the glufosinate-resistant glutamine synthetase mutant or its functional fragment of the present invention can be determined in vitro. Specifically, the activity characteristics of the glufosinate-resistant glutamine synthetase mutant or its functional fragment can be determined in vitro. 2+ In the presence of glufosinate, the enzymatic activity of the mutant or its functional fragment in catalyzing the synthesis of glutamine from ammonium ions and glutamate is measured. For example, the method described in Example 2 can be used; a commercial glutamine synthetase activity assay kit can also be used for enzyme activity assay. The stronger the measured glutamine synthetase activity, the more likely the glutamine synthetase mutant or its functional fragment, while possessing glufosinate-resistant properties, has its own enzymatic activity and function unaffected or even enhanced.

[0082] In some embodiments, the wild-type glutamine synthetase comprises an amino acid sequence of one of SEQ ID NOs: 1, 5, 9, 13, 33-42. In some embodiments, the amino acid sequence of the wild-type glutamine synthetase is, for example, one of SEQ ID NOs: 1, 5, 9, 13, 33-42. In some embodiments, the wild-type glutamine synthetase comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity to the amino acid sequence of one of SEQ ID NOs: 1, 5, 9, 13, 33-42, and an alanine (A) at position 309 or not an aspartic acid (D), threonine (T), or valine (V) at position 309, the amino acid positions being referenced to SEQ ID NO: 1.

[0083] In some embodiments, the glutamine synthetase mutant is derived from a wild-type rice (Oryza sativa) glutamine synthetase. An exemplary wild-type rice glutamine synthetase comprises the amino acid sequence shown in SEQ ID NO: 1.

[0084] In some embodiments, the glutamine synthetase mutant is derived from a wild-type wheat (Triticum aestivum) glutamine synthetase. An exemplary wild-type wheat glutamine synthetase comprises the amino acid sequence shown in SEQ ID NO:5.

[0085] In some embodiments, the glutamine synthetase mutant is derived from a wild-type glutamine synthetase from Brachypodium distachyon. An exemplary wild-type glutamine synthetase from Brachypodium distachyon comprises the amino acid sequence shown in SEQ ID NO:9.

[0086] In some embodiments, the glutamine synthetase mutant is derived from a wild-type glutamine synthetase from maize (Zea mays). An exemplary wild-type glutamine synthetase from maize comprises the amino acid sequence shown in SEQ ID NO:13.

[0087] In some embodiments, the glutamine synthetase mutant is derived from wild-type glutamine synthetase from Oryza meyeriana var. granulate. An exemplary wild-type glutamine synthetase from Oryza meyeriana var. granulate comprises the amino acid sequence shown in SEQ ID NO:33.

[0088] In some embodiments, the glutamine synthetase mutant is derived from wild-type glutamine synthetase of Oryza barthii. An exemplary wild-type glutamine synthetase of Oryza barthii comprises the amino acid sequence shown in SEQ ID NO:34.

[0089] In some embodiments, the glutamine synthetase mutant is derived from a wild-type glutamine synthetase from Oryza glaberrima. An exemplary wild-type glutamine synthetase from Oryza glaberrima comprises the amino acid sequence shown in SEQ ID NO:35.

[0090] In some embodiments, the glutamine synthetase mutant is derived from a Panicum hallii wild-type glutamine synthetase. An exemplary Panicum hallii wild-type glutamine synthetase comprises the amino acid sequence shown in SEQ ID NO:36.

[0091] In some embodiments, the glutamine synthetase mutant is derived from a wild-type glutamine synthetase from Panicum miliaceum. An exemplary wild-type glutamine synthetase from Panicum miliaceum comprises the amino acid sequence set forth in SEQ ID NO:37.

[0092] In some embodiments, the glutamine synthetase mutant is derived from a wild-type glutamine synthetase from Staria italica (millet). An exemplary wild-type glutamine synthetase from millet comprises the amino acid sequence set forth in SEQ ID NO:38.

[0093] In some embodiments, the glutamine synthetase mutant is derived from a wild-type glutamine synthetase from Sorghum bicolor. An exemplary wild-type glutamine synthetase from Sorghum bicolor comprises the amino acid sequence shown in SEQ ID NO:39.

[0094] In some embodiments, the glutamine synthetase mutant is derived from a wild-type glutamine synthetase from Leersia perrieri. An exemplary wild-type glutamine synthetase from Leersia perrieri comprises the amino acid sequence shown in SEQ ID NO:40.

[0095] In some embodiments, the glutamine synthetase mutant is derived from a wild-type glutamine synthetase from Triticum urartu. An exemplary wild-type glutamine synthetase from Triticum urartu comprises the amino acid sequence shown in SEQ ID NO:41.

[0096] In some embodiments, the glutamine synthetase mutant is derived from a wild-type barley glutamine synthetase. An exemplary wild-type barley glutamine synthetase comprises the amino acid sequence shown in SEQ ID NO:42.

[0097] In some embodiments, the amino acid mutation is an amino acid substitution.

[0098] In some embodiments, relative to wild-type glutamine synthetase, the amino acid at position 309 of the glutamine synthetase mutant or a functional fragment thereof is substituted with aspartic acid (D), and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, relative to wild-type glutamine synthetase, the amino acid at position 309 of the glutamine synthetase mutant or a functional fragment thereof is substituted with threonine (T), and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, relative to wild-type glutamine synthetase, the amino acid at position 309 of the glutamine synthetase mutant or a functional fragment thereof is substituted with valine (V), and the amino acid position is referenced to SEQ ID NO: 1.

[0099] In some embodiments, relative to wild-type glutamine synthetase, the glutamine synthetase mutant or a functional fragment thereof is substituted with alanine (A) at position 309, wherein the amino acid position is referenced to SEQ ID NO: 1.

[0100] In some embodiments, relative to wild-type glutamine synthetase, the alanine (A) at position 309 of the glutamine synthetase mutant or a functional fragment thereof is substituted with aspartic acid (D), and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, relative to wild-type glutamine synthetase, the alanine (A) at position 309 of the glutamine synthetase mutant or a functional fragment thereof is substituted with threonine (T), and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, relative to wild-type glutamine synthetase, the alanine (A) at position 309 of the glutamine synthetase mutant or a functional fragment thereof is substituted with valine (V), and the amino acid position is referenced to SEQ ID NO: 1.

[0101] In some embodiments, relative to wild-type glutamine synthetase, the glutamine synthetase mutant or a functional fragment thereof comprises at least amino acid substitution A309D, A309T or A309V, wherein the amino acid position is referenced to SEQ ID NO: 1.

[0102] The expression of amino acid substitution is generally recognized in the art. For example, the amino acid substitution A309D means that the amino acid A at position 309 of the wild-type glutamine synthetase is replaced by D in the glutamine synthetase mutant, and the amino acid position is referenced in SEQ ID NO: 1.

[0103] As used herein, a "functional fragment" refers to a fragment that at least partially or fully retains the function of the full-length glutamine synthetase mutant from which it is derived.

[0104] In some embodiments, the glutamine synthetase mutant or its functional fragment further comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) additional amino acid mutations, such as conservative amino acid substitutions, relative to wild-type glutamine synthetase. Furthermore, it will be appreciated by those skilled in the art that a small amount of amino acid insertions, deletions, or additions at the termini (C-terminus and / or N-terminus) of a protein generally does not significantly alter the function of the protein. For example, a tag, such as a histidine tag, may be added to the termini of the protein to facilitate protein purification and / or detection.

[0105] In some embodiments, relative to wild-type glutamine synthetase, the glutamine synthetase mutant or a functional fragment thereof further comprises one or more or all amino acid substitutions selected from S29P, T104I, and V172I.

[0106] In some embodiments, the glutamine synthetase mutant comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 2-4, 6-8, 10-12, and 14-16. In some embodiments, the glutamine synthetase mutant comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 2-4, 6-8, 10-12, and 14-16.

[0107] In another aspect, the present invention also provides a nucleic acid comprising a nucleotide sequence encoding a glutamine synthetase mutant or a functional fragment thereof of the present invention. In some embodiments, the nucleic acid is an isolated nucleic acid or a recombinant nucleic acid. In some embodiments, the nucleotide sequence encoding a glutamine synthetase mutant or a functional fragment thereof of the present invention is set forth in any one of SEQ ID NOs: 18-20, 22-24, 26-28, and 30-32.

[0108] In some embodiments, the nucleotide sequence encoding the glutamine synthetase mutant or a functional fragment thereof of the present invention can be codon-optimized for a eukaryotic organism of interest, such as yeast or plant.

[0109] In another aspect, the present invention also provides an expression cassette comprising a nucleotide sequence encoding a glutamine synthetase mutant or a functional fragment thereof operably linked to a regulatory sequence.

[0110] In another aspect, the present invention also provides an expression construct comprising the expression cassette of the present invention.

[0111] In another aspect, the present invention also provides use of the glutamine synthetase mutant of the present invention or a functional fragment thereof, the nucleic acid of the present invention, the expression cassette of the present invention or the expression construct of the present invention in producing herbicide-resistant plants.

[0112] In another aspect, the present invention also provides a herbicide-resistant plant comprising or expressing the glutamine synthetase mutant or a functional fragment thereof of the present invention.

[0113] The plants described in various aspects of the present invention can be monocots or dicots, preferably plants that are sensitive to glutamine synthetase inhibitors. Preferably, the plants are crop plants. Examples of suitable plants include, but are not limited to, rice (Oryza sativa), wheat (Triticum aestivum), Brachypodium distachyon, corn (Zea mays), Oryza meyeriana var. granulata, wild rice (Oryza barthii), African cultivated rice (Oryza glaberrima), Panicum hallii, millet (Panicum miliaceum), foxtail millet (Setaria italica), sorghum (Sorghum bicolor), pseudo-rice (Leersia perrieri), Urartu wheat (Triticum urartu), barley (Hordeum vulgare), etc. In some preferred embodiments, the plant is rice. In other preferred embodiments, the plant is wheat. In other preferred embodiments, the plant is corn. In other preferred embodiments, the plant is Brachypodium distachyon.

[0114] 3. Methods for producing herbicide-resistant plants through genetic modification

[0115] In another aspect, the present invention also provides a method for producing herbicide-resistant plants by transgenic means, comprising introducing into a plant a nucleic acid of the present invention, an expression cassette of the present invention, and / or an expression construct of the present invention. In some embodiments, introduction of the nucleic acid of the present invention, the expression cassette of the present invention, and / or the expression construct of the present invention results in the plant comprising or expressing a glutamine synthetase mutant of the present invention or a functional fragment thereof.

[0116] Nucleic acids of the invention, expression cassettes of the invention, and / or expression constructs of the invention can be introduced into the plant using various methods known in the art. Suitable introduction methods include, but are not limited to, biolistic methods, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube passage, and ovary injection.

[0117] In some embodiments, the nucleic acid of the present invention, the expression cassette of the present invention and / or the expression construct of the present invention are integrated into the genome of the plant. The isolated nucleic acid of the present invention, the expression cassette of the present invention and / or the expression construct of the present invention will confer resistance to herbicides that can inhibit glutamine synthetase activity on the plant.

[0118] In another aspect, the present invention also provides a herbicide-resistant plant comprising an expression cassette of the present invention, a nucleic acid of the present invention or an expression construct, or transformed with a nucleic acid of the present invention, an expression cassette of the present invention and / or an expression construct of the present invention. The present invention also encompasses progeny of the herbicide-resistant plant.

[0119] The plants described in various aspects of the present invention can be monocots or dicots, preferably plants that are sensitive to glutamine synthetase inhibitors. Preferably, the plants are crop plants. Examples of suitable plants include, but are not limited to, rice (Oryza sativa), wheat (Triticum aestivum), Brachypodium distachyon, corn (Zea mays), Oryza meyeriana var. granulata, wild rice (Oryza barthii), African cultivated rice (Oryza glaberrima), Panicum hallii, millet (Panicum miliaceum), foxtail millet (Setaria italica), sorghum (Sorghum bicolor), false rice (Leersia perrieri), Urartu wheat (Triticum urartu), barley (Hordeum vulgare), etc. In some preferred embodiments, the plant is rice. In other preferred embodiments, the plant is wheat. In other preferred embodiments, the plant is corn. In other preferred embodiments, the plant is Brachypodium distachyon.

[0120] 4. Method for Producing Herbicide-Resistant Plants by Targeted Modification of Endogenous Glutamine Synthetase

[0121] Based on the herbicide-resistant glutamine synthetase mutants and corresponding mutation sites identified in the present invention, endogenous glutamine synthetase in plants can be engineered and modified by targeted mutagenesis to produce herbicide-resistant plants.

[0122] Therefore, in one aspect, the present invention also provides a method for producing a herbicide-resistant plant, the method comprising modifying, for example, targeting the modification of an endogenous glutamine synthetase coding sequence of a plant, thereby causing the endogenous glutamine synthetase (e.g., expressed endogenous glutamine synthetase) to mutate an amino acid at position 309, the amino acid position being referenced to SEQ ID NO: 1.

[0123] The endogenous glutamine synthetase of the plant, for example, comprises an amino acid sequence of one of SEQ ID NOs: 1, 5, 9, 13, 33-42, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to an amino acid sequence of one of SEQ ID NOs: 1, 5, 9, 13, 33-42. In some embodiments, the endogenous glutamine synthetase has an alanine (A) at position 309 or is not an aspartic acid (D), a threonine (T), or a valine (V) at position 309, with reference to SEQ ID NO: 1.

[0124] In some embodiments, the amino acid mutation is an amino acid substitution.

[0125] In some embodiments, the modification results in a substitution of alanine (A) at position 309 of the endogenous glutamine synthetase (eg, expressed endogenous glutamine synthetase), wherein the amino acid position is referenced to SEQ ID NO: 1.

[0126] In some embodiments, the modification results in a substitution of an alanine (A) at position 309 of the endogenous glutamine synthetase (e.g., an expressed endogenous glutamine synthetase) with an aspartic acid (D), the amino acid position being referenced to SEQ ID NO: 1. In some embodiments, the modification results in a substitution of an alanine (A) at position 309 of the endogenous glutamine synthetase (e.g., an expressed endogenous glutamine synthetase) with a threonine (T), the amino acid position being referenced to SEQ ID NO: 1. In some embodiments, the modification results in a substitution of an alanine (A) at position 309 of the endogenous glutamine synthetase (e.g., an expressed endogenous glutamine synthetase) with a valine (V), the amino acid position being referenced to SEQ ID NO: 1.

[0127] In some embodiments, the modification results in the endogenous glutamine synthetase (eg, expressed endogenous glutamine synthetase) comprising an amino acid substitution A309D, A309T, or A309V, wherein the amino acid position is referenced to SEQ ID NO: 1.

[0128] In some embodiments, the modification results in the glutamine synthetase mutant described above in the present invention, for example, results in the plant expressing the glutamine synthetase mutant described above in the present invention.

[0129] Therefore, the present invention also provides a method for producing a herbicide-resistant plant, which method comprises modifying, for example, targeting the modification of an endogenous glutamine synthetase coding sequence of a plant, thereby resulting in the glutamine synthetase mutant described above in the present invention, for example, resulting in the plant expressing the glutamine synthetase mutant described above in the present invention.

[0130] "Herbicide-resistant plants" may refer to plants whose endogenous glutamine synthetase coding sequences have been targeted for modification and whose resistance to glutamine synthetase-inhibiting herbicides is enhanced, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more, relative to plants without such targeted modification. Methods for determining resistance to glutamine synthetase-inhibiting herbicides are known in the art. Resistance can be readily determined by those skilled in the art based on the specific plant and the specific herbicide.

[0131] In some embodiments, the endogenous glutamine synthetase coding sequence of the plant is targeted for modification by homologous recombination. Methods for modifying endogenous genes in plants by homologous recombination are well known to those skilled in the art.

[0132] In some embodiments, the endogenous glutamine synthetase coding sequence of the plant is targeted for modification by gene editing. In some embodiments, the endogenous glutamine synthetase coding sequence of the plant is targeted for modification by introducing a gene editing system targeted to the endogenous glutamine synthetase coding region in the plant genome into the plant.

[0133] In some embodiments, the introduction of the gene editing system results in an amino acid mutation at position 309 of the endogenous glutamine synthetase, wherein the amino acid position is referenced to SEQ ID NO:1.

[0134] In some embodiments, the introduction of the gene editing system results in the endogenous glutamine synthetase (e.g., expressed endogenous glutamine synthetase) being substituted with alanine (A) at position 309, and the amino acid position is referenced to SEQ ID NO:1.

[0135] In some embodiments, the introduction of the gene editing system results in the endogenous glutamine synthetase (e.g., the expressed endogenous glutamine synthetase) being substituted with aspartic acid (D) at alanine (A) at position 309, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the introduction of the gene editing system results in the endogenous glutamine synthetase (e.g., the expressed endogenous glutamine synthetase) being substituted with threonine (T) at alanine (A) at position 309, and the amino acid position is referenced to SEQ ID NO: 1. In some embodiments, the introduction of the gene editing system results in the endogenous glutamine synthetase (e.g., the expressed endogenous glutamine synthetase) being substituted with valine (V) at alanine (A) at position 309, and the amino acid position is referenced to SEQ ID NO: 1.

[0136] In some embodiments, introduction of the gene editing system results in the endogenous glutamine synthetase (e.g., expressed endogenous glutamine synthetase) comprising an amino acid substitution A309D, A309T, or A309V, wherein the amino acid position is referenced to SEQ ID NO: 1.

[0137] In some embodiments, introduction of the gene editing system results in the glutamine synthetase mutant described above in the present invention, for example, results in the plant expressing the glutamine synthetase mutant described above in the present invention.

[0138] The gene editing system that can be used in the present invention can be various gene editing systems known in the art, as long as it can perform targeted genome editing in plants. The gene editing system can be a gene editing system based on CRISPR, ZFN or TALEN. Preferably, the gene editing system is a gene editing system based on CRISPR.

[0139] In some preferred embodiments, the gene editing system is a base editing system. The base editing system useful in the present invention can be various base editing systems known in the art, as long as it can perform targeted genomic base editing in plants. For example, the base editing system includes but is not limited to those described in WO 2018 / 056623, WO 2019 / 120283, and WO 2019 / 120310.

[0140] In some embodiments, the base editing system comprises a base editing fusion protein or an expression construct comprising a nucleotide sequence encoding the same, and at least one guide RNA or an expression construct comprising a nucleotide sequence encoding the same, for example, the system comprises at least one of the following i) to v):

[0141] i) a base editing fusion protein, and at least one guide RNA;

[0142] ii) an expression construct comprising a nucleotide sequence encoding a base editor fusion protein, and at least one guide RNA;

[0143] iii) a base editing fusion protein, and an expression construct comprising a nucleotide sequence encoding at least one guide RNA;

[0144] iv) an expression construct comprising a nucleotide sequence encoding a base editor fusion protein, and an expression construct comprising a nucleotide sequence encoding at least one guide RNA;

[0145] v) an expression construct comprising a nucleotide sequence encoding a base editor fusion protein and a nucleotide sequence encoding at least one guide RNA;

[0146] The base editing fusion protein comprises a CRISPR effector protein and a deaminase domain, and the at least one guide RNA is capable of targeting the base editing fusion protein to an endogenous glutamine synthetase coding sequence in the plant genome.

[0147] In the embodiments herein, "base editing fusion protein" and "base editor" are used interchangeably to refer to a protein that can mediate the replacement of one or more nucleotides in a target sequence in the genome in a sequence-specific manner.

[0148] As used herein, the term "CRISPR effector protein" generally refers to a nuclease (CRISPR nuclease) present in a naturally occurring CRISPR system or a functional variant thereof. The term encompasses any effector protein based on the CRISPR system that is capable of achieving sequence-specific targeting within a cell.

[0149] As used herein, a "functional variant" with respect to a CRISPR nuclease means that it at least retains the sequence-specific targeting ability mediated by the guide RNA. Preferably, the functional variant is a variant in which the nuclease is inactivated, i.e., it lacks double-stranded nucleic acid cleavage activity. However, CRISPR nucleases lacking double-stranded nucleic acid cleavage activity also encompass nickases, which form a nick in a double-stranded nucleic acid molecule but do not completely cut the double-stranded nucleic acid. In some preferred embodiments of the present invention, the CRISPR effector protein of the present invention has nickase activity. In some embodiments, the functional variant recognizes a different PAM (protospacer adjacent motif) sequence relative to the wild-type nuclease.

[0150] "CRISPR effector protein" can be derived from a Cas9 nuclease, including a Cas9 nuclease or a functional variant thereof. The Cas9 nuclease can be a Cas9 nuclease from a different species, such as spCas9 from Streptococcus pyogenes (S.pyogenes) or SaCas9 derived from Staphylococcus aureus (S.aureus). "Cas9 nuclease" and "Cas9" are used interchangeably herein and refer to an RNA-guided nuclease comprising a Cas9 protein or a fragment thereof (e.g., a protein comprising an active DNA cleavage domain of Cas9 and / or a gRNA binding domain of Cas9). Cas9 is a component of the CRISPR / Cas (clustered regularly interspaced short palindromic repeats and related systems) genome editing system that can target and cut a DNA target sequence to form a DNA double-strand break (DSB) under the guidance of a guide RNA.

[0151] The "CRISPR effector protein" can also be derived from a Cpf1 nuclease, including a Cpf1 nuclease or a functional variant thereof. The Cpf1 nuclease can be a Cpf1 nuclease from a different species, such as Francisella novicida U112, Acidaminococcus sp. BV3L6, and Lachnospiraceae bacterium ND2006.

[0152] Useful "CRISPR effector proteins" can also be derived from Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Csn2, Cas4, C2c1, C2c3 or C2c2 nucleases, for example, including these nucleases or functional variants thereof.

[0153] In some embodiments, the CRISPR effector protein is a nuclease-inactivated Cas9. The DNA cleavage domain of the Cas9 nuclease is known to comprise two subdomains: the HNH nuclease subdomain and the RuvC subdomain. The HNH subdomain cleaves the strand complementary to the guide RNA, while the RuvC subdomain cleaves the non-complementary strand. Mutations in these subdomains can inactivate the nuclease activity of Cas9, resulting in a "nuclease-inactivated Cas9." This nuclease-inactivated Cas9 still retains the ability to bind DNA under the guidance of the guide RNA.

[0154] The nuclease-inactivated Cas9 of the present invention can be derived from Cas9 of different species, for example, derived from Streptococcus pyogenes (S. pyogenes) Cas9 (SpCas9), or derived from Staphylococcus aureus (S. aureus) Cas9 (SaCas9). Simultaneously mutating the HNH nuclease subdomain and the RuvC subdomain of Cas9 (for example, comprising mutations D10A and H840A) inactivates the nuclease of Cas9, resulting in a nuclease-dead Cas9 (dCas9). Mutating and inactivating one of the subdomains can impart nickase activity to Cas9, i.e., obtaining a Cas9 nickase (nCas9), for example, nCas9 having only the D10A mutation.

[0155] When Cas9 nuclease is used for gene editing, it is usually required that the target sequence has a 5'-NGG-3' PAM (protospacer adjacent motif) sequence at the 3' end. However, this PAM sequence occurs very rarely in certain species such as rice, greatly limiting gene editing in these species such as rice. For this reason, the present invention preferably uses CRISPR effector proteins that recognize different PAM sequences, such as functional variants of Cas9 nucleases with different PAM sequences.

[0156] In some preferred embodiments, the CRISPR effector protein is a Cas9 variant that recognizes the PAM sequence 5'-NG-3'. In some preferred embodiments, the CRISPR effector protein is a nuclease-inactivated Cas9 variant that recognizes the PAM sequence 5'-NG-3'.

[0157] The deaminase domain described herein can be a cytosine deamination domain or an adenine deamination domain.

[0158] As used herein, "cytosine deamination domain" refers to a domain that can accept single-stranded DNA as a substrate and catalyze the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively.

[0159] Examples of cytosine deaminases that can be used in the present invention include, but are not limited to, APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, CDA1, human APOBEC3A deaminase, or functional variants thereof. In some embodiments, the cytosine deaminase is human APOBEC3A deaminase or a functional variant thereof.

[0160] As used herein, "adenine deamination domain" refers to a domain that can accept single-stranded DNA as a substrate and catalyze the conversion of adenosine or deoxyadenosine (A) to inosine (I). In some embodiments, the adenine deaminase is a variant of Escherichia coli tRNA adenine deaminase TadA (ecTadA).

[0161] As used herein, “guide RNA” and “gRNA” are used interchangeably and refer to an RNA molecule that can form a complex with a CRISPR effector protein and can target the complex to a target sequence due to a certain homology with the target sequence. The guide RNA targets the target sequence by base pairing with the complementary strands of the target sequence. For example, the gRNA used by the Cas9 nuclease or its functional variants is generally composed of crRNA and tracrRNA molecules that are partially complementary to form a complex, wherein the crRNA comprises a guide sequence (also known as a seed sequence) that has sufficient homology to the target sequence so as to hybridize with the complementary strand of the target sequence and guide the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to the target sequence sequence. However, it is known in the art that single guide RNA (sgRNA) can be designed, which contains the features of crRNA and tracrRNA at the same time. The gRNA used by the Cpf1 nuclease or its functional variants is generally composed of only mature crRNA molecules, which may also be referred to as sgRNA. It is within the capabilities of those skilled in the art to design a suitable gRNA based on the CRISPR nuclease used and the target sequence to be edited.

[0162] In some embodiments, the at least one gRNA of the present invention comprises a target sequence in the endogenous glutamine synthetase coding region, and the amino acid sequence encoded by the target sequence comprises amino acids at positions 365 and 309 of the endogenous glutamine synthetase, and the amino acid positions refer to SEQ ID NO: 1.

[0163] In some embodiments, the gene editing system is a so-called prime editing system. The system includes a fusion of a Cas nuclease (e.g., Cas9-H840A) with target chain nicking activity and a reverse transcriptase (e.g., M-MLV reverse transcriptase), and a pegRNA (prime editing gRNA) with a 3' end having a repair template (RT template) and a binding region (PBS) for a free single strand. The system combines the free single strand produced by the Cas nickase (e.g., Cas9-H840A) with the PBS and transcribes a single-stranded DNA sequence according to a given RT template. After cell repair, any change in the DNA sequence located downstream of the PAM sequence -3 position can be achieved in the genome. For example, the prime editing system can be described in Anzalone, A. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature https: / / doi.org / 10.1038 / s41586-019-1711-4 (2019).

[0164] In the methods of the present invention, the gene editing system can be introduced into plants using various methods well known to those skilled in the art. Methods for introducing the gene editing system of the present invention into plants include, but are not limited to, biolistic methods, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube access, and ovary injection.

[0165] In the method of the present invention, target sequence modification can be achieved simply by introducing or generating the gene editing system into plant cells, and the modification is stably inherited, without the need for stably transforming the gene editing system into plants. This avoids potential off-target effects of a stable gene editing system and prevents integration of exogenous nucleotide sequences into the plant genome, thereby enhancing biosafety.

[0166] In some preferred embodiments, the introduction is performed in the absence of selection pressure, thereby avoiding integration of the exogenous nucleotide sequence into the plant genome.

[0167] In some embodiments, the introduction comprises transforming the gene editing system of the present invention into isolated plant cells or tissues, and then regenerating the transformed plant cells or tissues into whole plants. Preferably, the regeneration is performed in the absence of selective pressure, that is, no selective agent targeting the selection gene carried on the expression vector is used during the tissue culture process. The absence of a selective agent can improve the regeneration efficiency of the plant and obtain herbicide-resistant plants that do not contain exogenous nucleotide sequences.

[0168] In other embodiments, the gene editing system of the present invention can be transformed into specific parts of intact plants, such as leaves, stem tips, pollen tubes, young ears, or hypocotyls. This is particularly suitable for the transformation of plants that are difficult to regenerate through tissue culture.

[0169] In some embodiments of the present invention, in vitro expressed proteins and / or in vitro transcribed RNA molecules are directly transformed into the plant. The proteins and / or RNA molecules can achieve gene editing in plant cells and are subsequently degraded by the cells, thereby avoiding the integration of exogenous nucleotide sequences into the plant genome.

[0170] In another aspect, the present invention also provides a herbicide-resistant plant produced by the method of the present invention for targeted modification of endogenous glutamine synthetase in a plant. The present invention also encompasses progeny of the herbicide-resistant plant.

[0171] In some embodiments of the invention, the herbicide-resistant plants are non-transgenic.

[0172] The plants described in various aspects of the present invention can be monocots or dicots, preferably plants that are sensitive to glutamine synthetase inhibitors. Preferably, the plants are crop plants. Examples of suitable plants include, but are not limited to, rice (Oryza sativa), wheat (Triticum aestivum), Brachypodium distachyon, corn (Zea mays), Oryza meyeriana var. granulata, wild rice (Oryza barthii), African cultivated rice (Oryza glaberrima), Panicum hallii, millet (Panicum miliaceum), foxtail millet (Setaria italica), sorghum (Sorghum bicolor), false rice (Leersia perrieri), Urartu wheat (Triticum urartu), barley (Hordeum vulgare), etc. In some preferred embodiments, the plant is rice. In other preferred embodiments, the plant is wheat. In other preferred embodiments, the plant is corn. In other preferred embodiments, the plant is Brachypodium distachyon.

[0173] V. Methods for producing herbicide-resistant plants through physical or chemical mutagenesis

[0174] In another aspect, the present invention also provides a method for producing herbicide-resistant plants, comprising subjecting a population of said plants to physical or chemical mutagenesis, and screening for plants whose endogenous glutamine synthetase (e.g., expressed endogenous glutamine synthetase) comprises an amino acid mutation at at least position 309, wherein the amino acid position refers to SEQ ID NO: 1.

[0175] In some embodiments, plants are screened for endogenous glutamine synthetase (eg, expressed endogenous glutamine synthetase) substituted with alanine (A) at position 309, the amino acid position being referenced to SEQ ID NO: 1.

[0176] In some embodiments, plants are screened for an endogenous glutamine synthetase (e.g., an expressed endogenous glutamine synthetase) in which the alanine (A) at position 309 is substituted with aspartic acid (D), as referenced to the amino acid position in SEQ ID NO: 1. In some embodiments, plants are screened for an endogenous glutamine synthetase (e.g., an expressed endogenous glutamine synthetase) in which the alanine (A) at position 309 is substituted with threonine (T), as referenced to the amino acid position in SEQ ID NO: 1. In some embodiments, plants are screened for an endogenous glutamine synthetase (e.g., an expressed endogenous glutamine synthetase) in which the alanine (A) at position 309 is substituted with valine (V), as referenced to the amino acid position in SEQ ID NO: 1.

[0177] In some embodiments, plants are screened for endogenous glutamine synthetase (eg, expressed endogenous glutamine synthetase) comprising the amino acid substitution A309D, A309T, or A309V, with reference to the amino acid position in SEQ ID NO: 1.

[0178] In some embodiments, plants are screened for plants whose endogenous glutamine synthetase (eg, expressed endogenous glutamine synthetase) is mutated to a glutamine synthetase mutant as described above in the present invention.

[0179] Therefore, the present invention also provides a method for producing herbicide-resistant plants, comprising subjecting a population of said plants to physical or chemical mutagenesis, and screening for plants comprising or expressing the glutamine synthetase mutant of the present invention as described above.

[0180] "Herbicide-resistant plants" may refer to plants whose endogenous glutamine synthetase has been mutagenized and whose resistance to glutamine synthetase-inhibiting herbicides is increased, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more, relative to plants that do not contain the mutation in the endogenous glutamine synthetase. Methods for determining resistance to glutamine synthetase-inhibiting herbicides are known in the art. Resistance can be readily determined by those skilled in the art based on the specific plant and the specific herbicide.

[0181] In some embodiments, the physical mutagenesis can be achieved by irradiating the plant population (e.g., irradiating plant seeds) with radioactive irradiation. In some implementation methods, the chemical mutagenesis can be achieved by treating the plant population (e.g., treating plant seeds) with ethyl methanesulfonate (EMS).

[0182] In some embodiments, the screening can be achieved by sequencing the coding sequence of the endogenous glutamine synthetase.

[0183] In another aspect, the present invention also provides a herbicide-resistant plant produced by the physical or chemical mutagenesis method of the present invention. The present invention also encompasses the progeny of the herbicide-resistant plant.

[0184] The plants described in various aspects of the present invention can be monocots or dicots, preferably plants that are sensitive to glutamine synthetase inhibitors. Preferably, the plants are crop plants. Examples of suitable plants include, but are not limited to, rice (Oryza sativa), wheat (Triticum aestivum), Brachypodium distachyon, corn (Zea mays), Oryza meyeriana var. granulata, wild rice (Oryza barthii), African cultivated rice (Oryza glaberrima), Panicum hallii, millet (Panicum miliaceum), foxtail millet (Setaria italica), sorghum (Sorghum bicolor), pseudo-rice (Leersia perrieri), Urartu wheat (Triticum urartu), barley (Hordeum vulgare), etc. In some preferred embodiments, the plant is rice. In other preferred embodiments, the plant is wheat. In other preferred embodiments, the plant is corn. In other preferred embodiments, the plant is Brachypodium distachyon.

[0185] 6. Plant Breeding Methods

[0186] In another aspect, the present invention provides a plant breeding method, comprising crossing a first plant having herbicide resistance obtained by the above-mentioned method of the present invention with a second plant not having the herbicide resistance, thereby introducing the herbicide resistance into the second plant.

[0187] The plants described in various aspects of the present invention can be monocots or dicots, preferably plants that are sensitive to glutamine synthetase inhibitors. Preferably, the plants are crop plants. Examples of suitable plants include, but are not limited to, rice (Oryza sativa), wheat (Triticum aestivum), Brachypodium distachyon, corn (Zea mays), Oryza meyeriana var. granulata, wild rice (Oryza barthii), African cultivated rice (Oryza glaberrima), Panicum hallii, millet (Panicum miliaceum), foxtail millet (Setaria italica), sorghum (Sorghum bicolor), pseudo-rice (Leersia perrieri), Urartu wheat (Triticum urartu), barley (Hordeum vulgare), etc. In some preferred embodiments, the plant is rice. In other preferred embodiments, the plant is wheat. In other preferred embodiments, the plant is corn. In other preferred embodiments, the plant is Brachypodium distachyon.

[0188] VII. Methods for Identifying Herbicide-Resistant Plants

[0189] In another aspect, the present invention provides a method for identifying herbicide-resistant plants, comprising detecting

[0190] i) the presence and / or amount of glutamine synthetase or a functional fragment thereof wherein amino acid position 309 is aspartic acid (D), threonine (T) or valine (V), wherein the amino acid position is referenced to SEQ ID NO: 1; or

[0191] ii) the presence and / or expression of a nucleotide sequence encoding a glutamine synthetase or a functional fragment thereof wherein the amino acid at position 309 is aspartic acid (D), threonine (T) or valine (V), wherein the amino acid position is referenced to SEQ ID NO: 1.

[0192] In some embodiments, the glutamine synthetase comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity to any one of SEQ ID NOs: 2-4, 6-8, 10-12, and 14-16. In some embodiments, the glutamine synthetase comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 2-4, 6-8, 10-12, and 14-16.

[0193] In some embodiments, the nucleotide sequence encoding glutamine synthetase or a functional fragment thereof is shown in any one of SEQ ID NOs: 18-20, 22-24, 26-28, 30-32.

[0194] In some embodiments, the presence or increased amount of the glutamine synthetase or the presence and / or increased expression of the nucleotide sequence indicates the presence of resistance to glutamine synthetase-inhibiting herbicides and / or increased resistance to glutamine synthetase-inhibiting herbicides in the plant, especially relative to a plant not comprising the glutamine synthetase or encoding nucleotide sequence.

[0195] Example

[0196] Example 1. Screening of glufosinate-resistant mutants of rice glutamine synthetase OsGS1

[0197] The OsGS1 yeast mutant library was removed from a -80°C freezer and revived overnight in SC medium. The cells were then transferred to SD-Leu-Ura medium (a liquid medium deficient in leucine and uracil) and continued to resuscitate until the yeast cells reached a healthy logarithmic growth phase. At this time, the yeast mutant library was sampled and collected as the time 0 sample. Simultaneously, ~0.128 x 10^7 yeast cells were transferred to SD-Leu-Ura liquid medium containing 0.5 g / L glufosinate for resistance screening, with two replicates performed simultaneously. Every 8 hours of culture, cells were harvested as the time 0 sample. Simultaneously, 0.128 x 10^7 yeast cells were transferred to fresh screening medium and continued screening. After 68 hours of screening, a total of 18 samples were collected at nine time points, with two replicates at each time point.

[0198] DNA from these 18 yeast samples was extracted and the barcode corresponding to the time point and the UMI corresponding to the different mutants were added through 2 cycles of PCR. Then, 24 cycles of PCR were performed with Gibson ligation of homology arms and the DNA was cloned into the pUC19 vector via Gibson ligation. Escherichia coli DH5α was transformed, and 20,000-30,000 clones were collected from the plates. The plasmids were extracted, restriction enzyme digestion was performed, and the products were sequenced by third-generation sequencing.

[0199] UMIC-seq sequencing and analysis results showed that the single mutants A309D, A309V, and A309T, as well as the multiple mutant S29P / T104I / V172I / A309V, all exhibited significant resistance to glufosinate, as shown in Figure 1. Therefore, primers containing base mutations were designed, and the corresponding mutations were introduced by PCR. The PCR products were then transformed into yeast to obtain yeast mutants carrying different OsGS1 mutations, resulting in a total of three mutants (A309D, A309V, and A309T).

[0200] Table 1. Summary of mutation sites and types of glufosinate-resistant mutants of OsGS1

[0201] Table 2. Primer information used to construct mutants

[0202] Example 2. Characterization of OsGS1 mutants

[0203] 2.1 Competitive growth experiment under glufosinate conditions

[0204] To verify the glufosinate resistance of the above mutants, competitive growth experiments were conducted with yeast expressing the wild-type OsGS1 sequence and GFP. The competitive growth experiments were conducted in YPD liquid medium containing 10 g / L glufosinate. Three biological replicates and two technical replicates were performed for each yeast mutant. After recovery to the logarithmic phase, 0.128*10^7 cells of each yeast mutant and wild-type were added to YPD liquid medium containing 10 g / L glufosinate to initiate competitive growth, with this time being designated as time 0. Flow cytometry was used for quantitative analysis, and the ratio of fluorescent to non-fluorescent cells was calculated to obtain the initial ratio of mutant to wild-type. Samples were then collected every hour and quantitatively analyzed as described above. The competitive experiments lasted for 5-7 hours, with data collected at 5-7 time points. The difference in growth rate between the mutant and wild-type yeast under glufosinate conditions was determined, reflecting the mutant's sensitivity to glufosinate.

[0205] The results are shown in Figure 1. The yeast mutants A309D, A309T, and A309V with mutations at amino acid position 309 of rice glutamine synthetase OsGS1 all showed a strong growth advantage over the wild type in YPD medium containing 10 g / L glufosinate, and their resistance to glufosinate was greatly improved compared to the wild type.

[0206] 2.2. Verification of ammonium assimilation efficiency - competitive growth experiment under low ammonium conditions

[0207] To verify the ammonium assimilation efficiency of the above mutants, competitive growth experiments were conducted with yeast expressing the wild-type OsGS1 sequence and GFP in YNB liquid medium containing 0.5 g / L (NH₄)₂SO₄. Three biological replicates and two technical replicates were performed for each mutant. After recovery to logarithmic phase, 0.128*10⁷ cells of each mutant and wild-type yeast were added to YNB liquid medium containing 0.5 g / L ammonium sulfate to initiate competitive growth, with this time being designated as time 0. Flow cytometric analysis was performed, and the ratio of fluorescent to nonfluorescent cells was calculated to determine the initial ratio of mutant to wild-type cells. Samples were then collected every 8-12 hours and quantitatively analyzed as described above. The competition experiment lasted approximately 60 hours, with data collected at 6-7 time points. The differences in growth rates between the mutant and wild-type yeast under low ammonium conditions were determined, reflecting the ammonium assimilation efficiency of the mutants.

[0208] The results are shown in Figure 2. The yeast mutants A309D, A309T, and A309V with mutations at amino acid position 309 of rice glutamine synthetase OsGS1 had slightly or no significant decrease in growth rate compared with the wild type in YNB medium containing 0.05 g / L (NH4)2SO4, which to some extent reflects that the ammonium assimilation efficiency of the mutants was not significantly affected.

[0209] 2.3. Glutamine synthetase activity detection of mutants - ferric chloride colorimetric method

[0210] Reagent preparation:

[0211] Extraction Buffer:

[0212] The pH was adjusted to 8.0.

[0213] Reaction buffer:

[0214] Before the assay, add hydroxylamine hydrochloride (H₃NO₄HCl, molecular weight 69.49) and ATP (molecular weight 507.18); for a 15 mM hydroxylamine hydrochloride concentration, add 0.1042 g per 100 ml; for a 15 mM ATP concentration, add 0.7608 g per 100 ml. For the control reaction buffer, do not add ATP. Adjust the pH to 6.8 with 1 M Tris.

[0215] Stop Buffer:

[0216] Because the addition of TCA dilutes the stop buffer concentration, the stop buffer stock concentration should be 10 / 9X:

[0217] 10X TCA (molecular weight 163.3797): dilute 1.6338 g TCA to 5 ml ddH2O and store at -20°C in the dark. Add to the stop buffer proportionally when needed to a final concentration of 200 mM.

[0218] Enzyme activity assay:

[0219] 1: Enzyme extraction

[0220] Grind the sample to be tested into powder with liquid nitrogen, take 200 mg into a centrifuge tube, add 900 μl extraction buffer, vortex and oscillate for 1 minute, place on ice for 10 minutes, centrifuge at >13500 rpm and 4°C for 3-5 minutes, and take the supernatant into a new centrifuge tube. If there are impurities in the supernatant, repeat the centrifugation and take the supernatant again.

[0221] 2: Reaction

[0222] Experimental group: 250 μl enzyme solution was added to 500 μl reaction buffer (containing ATP), mixed well,

[0223] Control group: 250 μl enzyme solution was added to 500 μl reaction buffer (without ATP), mixed well,

[0224] 37℃, reaction for 30min,

[0225] After the reaction is completed, add 250 μl of the prepared stop buffer and mix well.

[0226] The mixture was centrifuged at 4000 rpm for 5 min, the supernatant was transferred to an ELISA plate (250 μl per well, 3 replicates), and the absorbance was measured at 498 nm.

[0227] 3: Protein concentration detection

[0228] The protein concentration was determined using the Braford method.

[0229] Prepare BSA solutions at varying concentrations (0.0625 mg / ml to 1 mg / ml), sample 3 μl of each solution, and as a blank control, sample 3 μl of extraction buffer. Add 300 μl of Coomassie Brilliant Blue G-250. Measure each sample in duplicate, mix thoroughly, and transfer 250 μl to an ELISA plate. Measure absorbance at 595 nm. Draw a standard curve using absorbance as the y-axis and BSA concentration as the x-axis.

[0230] Take 3 μl of enzyme solution, add 300 μl of Coomassie Brilliant Blue G-250, detect the absorbance at 595 nm, and calculate the protein concentration.

[0231] 4: Calculation

[0232] The absorbance of the reaction solution was measured at 498 nm. The absorbance can be used to indirectly reflect the activity of GS:

[0233] GS activity (A mg -1 -h -1 )=A / (PVT)

[0234] A is the absorbance measured at 498 nm, P is the measured protein concentration, V is the volume of sample added to the reaction system, and T is the reaction time.

[0235] The results are shown in Figure 3. The enzyme activities of the A309D and A309T mutants of rice glutamine synthetase OsGS1, as measured by the ferric chloride colorimetric method, were higher than those of the wild type. The A309D mutant had an activity increased by 2.5 times, and the A309T mutant had an activity increased by 2 times. The A309V mutant had an activity close to that of the wild type.

[0236] Example 3: Glufosinate resistance experiments on GS1 mutants of corn, wheat, and Brachypodium distichum

[0237] Mutants of homologous glutamine synthetases from maize (Zea mays), wheat (Triticum aestivum), and Brachypodium distachyon were constructed, with the mutations at A309D, A309V, and A309T, respectively. Primers containing these base mutations were designed and introduced via PCR. Yeast mutants carrying these mutations were then transformed with the PCR products.

[0238] Two replicates of each mutant were selected for growth competition experiments in YPD medium supplemented with 10 g / L glufosinate. OD values ​​were measured at 660 nm every 1-2 hours for 5-6 hours, and the growth of yeast cells compared to the zero time point was calculated.

[0239] The results are shown in Figure 4. The yeast mutants A309D, A309T, and A309V with amino acid mutations at position 309 of glutamine synthetase in corn, wheat, and Brachypodium distichum all showed a strong growth advantage over the wild type in YPD medium containing 10 g / L glufosinate, and their resistance to glufosinate was significantly improved compared to the wild type.

[0240] Example 4: Glufosinate-resistance experiment of transgenic plants

[0241] The experimental steps are as follows:

[0242] The 35S promoter sequence and the target gene CDS sequence were connected by fusion PCR to obtain 35S-OsGS1-WT and 35S-OsGS1-A309D;

[0243] Vector enzyme digestion: Extract the plasmid of the empty vector XF675 to be constructed, perform double enzyme digestion, and run gel recovery;

[0244] Vector construction: The above fragments were connected to the vector by homologous recombination, transformed into competent E. coli, and plated on LB medium resistant to the vector;

[0245] Pick a single colony, culture it and perform PCR identification, sequence the PCR product of the correct size and compare the sequencing results;

[0246] Transformation of Agrobacterium: Culture and extract plasmids from the correctly sequenced single colonies, and then transform them into Agrobacterium;

[0247] Infection: After transformation, Agrobacterium is plated on LB plates containing hygromycin resistance. Single colonies are picked for bacterial identification, cultured, and used to infect Arabidopsis (inflorescence dip method).

[0248] Screening positive seedlings: After infection, obtain T0 seeds from Arabidopsis thaliana. Sow the T0 seeds on MS medium containing antibiotics, screen out T1 seedlings containing hygromycin-resistant transgenes, and transplant them into plug trays.

[0249] Identification: PCR identification was performed using primers on the vector and fragment to obtain T1 positive seedlings. Seedlings about 2-3 weeks old were selected for glufosinate spraying experiments.

[0250] The spraying conditions were: 150 mg / L glufosinate solution, 10 sprays per pot, approximately a total volume of 1.5 mL, and an active ingredient of ~0.225 mg;

[0251] The phenotypic changes of the plants were then tracked and recorded.

[0252] The results are shown in Figure 5. On the second day after spraying with glufosinate ammonium, all wild-type plants (35S-OsGS1-WT) and some smaller mutant plants (35S-OsGS1-A309D) began to show symptoms of yellowing leaves; on the 13th day after spraying, all wild-type seedlings had died, while most of the leaves of the mutant seedlings remained green and did not turn yellow, and they were still alive even on the 16th day after spraying.

[0253] It can be seen that Arabidopsis plants transformed with the rice glutamine synthetase A309D mutant are more resistant to glufosinate than Arabidopsis plants transformed with the rice glutamine synthetase wild type.

Claims

1. A glutamine synthetase (GS) or a functional fragment thereof, which has an amino acid mutation such as an amino acid substitution at position 309 relative to a wild-type glutamine synthetase (GS), wherein the amino acid position refers to SEQ ID NO:

1.

2. The glutamine synthetase or a functional fragment thereof of claim 1, wherein the wild-type glutamine synthetase comprises an amino acid sequence of one of SEQ ID NOs: 1, 5, 9, 13, 33-42, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity with an amino acid sequence of one of SEQ ID NOs: 1, 5, 9, 13, 33-42.

3. The glutamine synthetase or a functional fragment thereof according to claim 1 or 2, wherein relative to the wild-type glutamine synthetase, the glutamine synthetase mutant or the functional fragment thereof is substituted with alanine (A) at position 309, and the amino acid position is referenced to SEQ ID NO:

1.

4. The glutamine synthetase or a functional fragment thereof according to claim 3, wherein relative to the wild-type glutamine synthetase, the alanine (A) at position 309 of the glutamine synthetase mutant or the functional fragment thereof is substituted by aspartic acid (D), threonine (T) or valine (V), and the amino acid position is referenced to SEQ ID NO:

1.

5. The glutamine synthetase or a functional fragment thereof according to claim 3, wherein relative to the wild-type glutamine synthetase, the glutamine synthetase mutant or the functional fragment thereof comprises at least an amino acid substitution A309D, A309T or A309V, wherein the amino acid position refers to SEQ ID NO:

1.

6. The glutamine synthetase or a functional fragment thereof according to any one of claims 1 to 5, wherein the glutamine synthetase mutant comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with any one of SEQ ID NOs: 2-4, 6-8, 10-12, and 14-16, or the glutamine synthetase mutant comprises the amino acid sequence shown in any one of SEQ ID NOs: 2-4, 6-8, 10-12, and 14-16.

7. The glutamine synthetase or a functional fragment thereof according to any one of claims 1 to 6, wherein the glutamine synthetase mutant or the functional fragment thereof, when expressed in a eukaryotic organism such as yeast or a plant, can confer resistance to a herbicide, such as a glutamine synthetase-inhibiting herbicide, on the eukaryotic organism such as yeast or a plant. Preferably, the glutamine synthetase-inhibiting herbicide is glufosinate-ammonium.

8. A nucleic acid comprising a nucleotide sequence encoding the glutamine synthetase mutant or a functional fragment thereof according to any one of claims 1 to 7.

9. The nucleic acid of claim 8, wherein the nucleotide sequence encoding the glutamine synthetase mutant or a functional fragment thereof is shown in any one of SEQ ID NOs: 18-20, 22-24, 26-28, 30-32.

10. An expression cassette comprising a nucleotide sequence encoding any one of the glutamine synthetase mutants according to claims 1 to 7 or a functional fragment thereof operably linked to a regulatory sequence.

11. The expression cassette of claim 10, wherein the nucleotide sequence encoding the glutamine synthetase mutant or a functional fragment thereof is shown in any one of SEQ ID NOs: 18-20, 22-24, 26-28, 30-32.

12. An expression construct comprising the expression cassette of claim 10 or 11.

13. Use of the glutamine synthetase mutant or a functional fragment thereof according to any one of claims 1 to 7, the nucleic acid according to claim 8 or 9, the expression cassette according to claim 10 or 11 or the expression construct according to claim 12 for producing herbicide-resistant plants.

14. The method of claim 13, wherein the plant is selected from the group consisting of rice (Oryza sativa), wheat (Triticum aestivum), Brachypodium distachyon, corn (Zea mays), Oryza meyeriana var. granulata, wild rice (Oryza barthii), African cultivated rice (Oryza glaberrima), Panicum hallii, Panicum miliaceum, Setaria italica, sorghum (Sorghum bicolor), Leersia perrieri, Urartu wheat (Triticum urartu), and barley (Hordeum vulgare).

15. A herbicide-resistant plant comprising or expressing the glutamine synthetase mutant or a functional fragment thereof according to any one of claims 1 to 7.

16. The herbicide-resistant plant of claim 15, wherein the plant is selected from the group consisting of rice (Oryza sativa), wheat (Triticum aestivum), Brachypodium distachyon, corn (Zea mays), Oryza meyeriana var. granulata, wild rice (Oryza barthii), African cultivated rice (Oryza glaberrima), Panicum hallii, Panicum miliaceum, millet (Setaria italica), sorghum (Sorghum bicolor), pseudo-rice (Leersia perrieri), Urartu wheat (Triticum urartu), barley (Hordeum vulgare).

17. A method for producing herbicide-resistant plants by transgenic means, comprising introducing into a plant a nucleic acid according to claim 8 or 9, an expression cassette according to claim 10 or 11 and / or an expression construct according to claim 12, preferably, said introduction causing said plant to comprise or express a glutamine synthetase mutant or a functional fragment thereof according to any one of claims 1 to 7.

18. A method for producing a herbicide-resistant plant, the method comprising modifying, for example, targeting the modification of an endogenous glutamine synthetase coding sequence of a plant, thereby causing an amino acid mutation, such as an amino acid substitution, at position 309 of the endogenous glutamine synthetase (e.g., expressed endogenous glutamine synthetase), the amino acid position being referenced to SEQ ID NO:

1.

19. The method of claim 18, wherein the endogenous glutamine synthetase of the plant comprises, for example, an amino acid sequence of one of SEQ ID NOs: 1, 5, 9, 13, 33-42 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity to an amino acid sequence of one of SEQ ID NOs: 1, 5, 9, 13, 33-42.

20. The method of claim 18 or 19, wherein the modification results in a substitution of alanine (A) at position 309 of the endogenous glutamine synthetase (eg, expressed endogenous glutamine synthetase), the amino acid position being referenced to SEQ ID NO:

1.

21. The method of claim 20, wherein the modification results in the endogenous glutamine synthetase (e.g., expressed endogenous glutamine synthetase) having alanine (A) substituted with aspartic acid (D), threonine (T) or valine (V) at position 309, the amino acid position being referenced to SEQ ID NO:

1.

22. The method of claim 20 or 21, wherein the modification results in the endogenous glutamine synthetase (eg, expressed endogenous glutamine synthetase) comprising at least the amino acid substitution A309D, A309T or A309V, the amino acid position being referenced to SEQ ID NO:

1.

23. The method of any one of claims 18-22, wherein the modification results in the glutamine synthetase mutant of any one of claims 1-7, such as results in the plant expressing the glutamine synthetase mutant of any one of claims 1-7.

24. The method of any one of claims 18 to 23, wherein the endogenous glutamine synthetase coding sequence of the plant is targeted for modification by homologous recombination or gene editing.

25. The method of claim 24, wherein the gene editing comprises introducing into the plant a gene editing system targeted to an endogenous glutamine synthetase coding region in the plant genome.

26. The method of claim 25, wherein the gene editing system is a gene editing system based on CRISPR, ZFN or TALEN, preferably, the gene editing system is a gene editing system based on CRISPR.

27. The method of claim 25 or 26, wherein the gene editing system is a base editing system or a prime editing system.

28. A method for producing herbicide-resistant plants, comprising subjecting a population of said plants to physical or chemical mutagenesis, and selecting from the mutagenized plants plants whose endogenous glutamine synthetase (e.g., expressed endogenous glutamine synthetase) comprises an amino acid mutation, such as an amino acid substitution, at least at position 309, wherein the amino acid position refers to SEQ ID NO:

1.

29. The method of claim 28, wherein plants are screened for endogenous glutamine synthetase (eg, expressed endogenous glutamine synthetase) having alanine (A) substituted at position 309, the amino acid position being referenced to SEQ ID NO:

1.

30. The method of claim 29, wherein the plant is screened for an endogenous glutamine synthetase (e.g., an expressed endogenous glutamine synthetase) in which the alanine (A) at position 309 is substituted with aspartic acid (D), threonine (T) or valine (V), the amino acid position being referenced to SEQ ID NO:

1.

31. The method of any one of claims 28-30, wherein a plant is screened for one in which an endogenous glutamine synthetase (eg, an expressed endogenous glutamine synthetase) is mutated to a glutamine synthetase mutant according to any one of claims 1-7.

32. A method for identifying herbicide-resistant plants, comprising detecting i) the presence and / or amount of glutamine synthetase or a functional fragment thereof, wherein the amino acid at position 309 is aspartic acid (D), threonine (T) or valine (V), wherein the amino acid position is referenced to SEQ ID NO: 1; or ii) the presence and / or expression of a nucleotide sequence encoding a glutamine synthetase or a functional fragment thereof wherein the amino acid at position 309 is aspartic acid (D), threonine (T) or valine (V), wherein the amino acid position is referenced to SEQ ID NO: 1, The presence or increased amount of said glutamine synthetase or the presence and / or increased expression of said nucleotide sequence indicates the presence of resistance to glutamine synthetase-inhibiting herbicides and / or increased resistance to glutamine synthetase-inhibiting herbicides in said plant, especially relative to a plant not comprising said glutamine synthetase or encoding nucleotide sequence.

33. The method of claim 32, wherein the glutamine synthetase comprises an amino acid sequence as shown in any one of SEQ ID NOs: 2-4, 6-8, 10-12, and 14-16, or comprises an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NOs: 2-4, 6-8, 10-12, and 14-16; or the encoding nucleotide sequence of the glutamine synthetase mutant or a functional fragment thereof is shown in any one of SEQ ID NOs: 18-20, 22-24, 26-28, 30-32.

34. A method of plant breeding, comprising crossing the herbicide-resistant plant of claim 15 or 16 or a first plant having herbicide resistance obtained by the method of any one of claims 17 to 33 with a second plant not having said herbicide resistance, thereby introducing said herbicide resistance into the second plant.

35. The method of any one of claims 17-34, wherein the plant is selected from the group consisting of Oryza sativa, Triticum aestivum, Brachypodium distachyon, Zea mays, Oryza meyeriana var. granulata, Oryza barthii, Oryza glaberrima, Panicum hallii, Panicum miliaceum, Setaria italica, Sorghum bicolor, Leersia perrieri, Triticum urartu, and Hordeum vulgare.

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