EPSPS mutants and their application methods

Abstract

The present invention provides a plant EPSP synthase (EPSPS) mutant and its application in plants. The EPSPS mutant comprises a VA mutation at position 403 compared to wild-type EPSPS having the amino acid sequence shown in SEQ ID NO:1.

Description

[Technical field]

[0001] The field of the present invention relates to the field of molecular biology. More specifically, the present invention relates to amino acid mutations that confer glyphosate tolerance and their application in glyphosate tolerant plants. [Background technology]

[0002] 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), encoded by the 5-enolpyruvylshikimate-3-phosphate synthase gene, is the sixth enzyme in the shikimate pathway. This enzyme catalyzes the conversion of phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P) to phosphate and 5-enolpyruvylshikimate-3-phosphate (EPSP). The shikimate pathway is widely present in plants, bacteria, and fungi, but animals lack such a pathway. Glyphosate is one of the world's best-selling chemical herbicides. Its structure is a structural analogue of PEP. By competitively inhibiting the binding of EPSPS and PEP, it prevents the synthesis of downstream aromatic amino acids, resulting in an imbalance in downstream biosynthesis and metabolism, thereby effectively killing weeds. Glyphosate herbicides are nearly lethal to all plants. In addition to EPSPS in plants, some bacteria also have EPSPS that are highly sensitive to glyphosate. These EPSPS that are highly sensitive to glyphosate are classified as Class I, and the protein structure of EPSPS in some microorganisms is similar to that of EPSPS in plants, but the similarity to the amino acid sequence of the Class I EPSPS sequence is less than 50%, such as CP4EPSPS, G2-aroA, etc., and such EPSPS enzymes that are less sensitive to glyphosate are classified as Class II.

[0003] Glyphosate has become one of the most popular chemical herbicides due to its broad spectrum, high efficiency, low toxicity, and low cost. When used in agricultural fields, it can greatly save weeding costs and increase farmers' income. Glyphosate resistance is one of the most popular traits in plant varieties created by biotechnology. To date, about seven genes have been included in about 221 approved genetic modification events. Only two genes (mEPSPS and 2mEPSPS) are of plant origin, and the other five genes are of microbial origin. As the public pays more attention to the safety of introducing exogenous genes into genetically modified crops, the products of chemical mutagenesis and gene editing are gaining acceptance worldwide. Furthermore, gene editing technology has become a new direction for the development of glyphosate-resistant crops due to its high efficiency and site specificity. By mutating only a few plant genes, it is possible to obtain target plants with ideal traits and high commercial value.

[0004] The present inventors have discovered that some amino acid mutations in the original EPSPS enzyme of plants can confer glyphosate resistance to plants without affecting the function of the original enzyme. The present inventors have further developed the application of these protein mutants and their encoding genes in transgenic plants, gene-edited plants or other plant breeding. They can be used to breed plants, particularly crops such as agricultural plants, with glyphosate resistance. Summary of the Invention

[0005] The present disclosure relates to the amino acid mutation positions and mutation types of the mutated EPSPS. A method for producing a glyphosate-tolerant plant is further provided.

[0006] In one embodiment, the mutated EPSPS polypeptide comprises the mutation V403A, and the amino acid mutation position corresponds to the amino acid position shown in SEQ ID NO:1.

[0007] In one embodiment, the mutated EPSPS polypeptide comprises the mutations G172A and V403A, and the amino acid mutation positions correspond to the amino acid positions shown in SEQ ID NO:1.

[0008] In one embodiment, the mutated EPSPS polypeptide comprises the mutations P177S and V403A, and the amino acid mutation positions correspond to the amino acid positions shown in SEQ ID NO:1.

[0009] In one embodiment, the mutated EPSPS polypeptide comprises the mutations G172A, P177S and / or V403A, the amino acid mutation positions corresponding to the amino acid positions shown in SEQ ID NO:1.

[0010] In one embodiment, the mutated EPSPS polypeptide comprises the mutations T173I, P177S and / or V403A, the amino acid mutation positions corresponding to the amino acid positions shown in SEQ ID NO:1.

[0011] In one embodiment, the mutated EPSPS polypeptide comprises the mutations T173I, P177T and / or V403A, the amino acid mutation positions corresponding to the amino acid positions shown in SEQ ID NO:1.

[0012] In yet another embodiment, the mutated EPSPS polypeptide comprises a mutation V→A, the position corresponding to the position of the original EPSPS amino acid sequence aligned to position 403 of SEQ ID NO:1.

[0013] In yet another embodiment, the mutated EPSPS polypeptide contains the mutations G→A and V→A, which positions correspond to positions 172 and 403 of the original EPSPS amino acid sequence aligned to SEQ ID NO:1.

[0014] In yet another embodiment, the mutated EPSPS polypeptide contains the mutations P→S and V→A, which positions correspond to positions 177 and 403 of the original EPSPS amino acid sequence aligned to SEQ ID NO:1.

[0015] In yet another embodiment, the mutated EPSPS polypeptide contains mutations G→A, P→S and / or V→A, which positions correspond to positions in the original EPSPS amino acid sequence aligned to positions 172, 177 and / or 403 of SEQ ID NO:1.

[0016] In yet another embodiment, the mutated EPSPS polypeptide contains mutations T→I, P→S and / or V→A, which positions correspond to positions in the original EPSPS amino acid sequence aligned to positions 173, 177 and / or 403 of SEQ ID NO:1.

[0017] In yet another embodiment, the mutated EPSPS polypeptide contains mutations T→I, P→T and / or V→A, which positions correspond to positions of the original EPSPS amino acid sequence aligned to positions 173, 177 and / or 403 of SEQ ID NO:1.

[0018] In yet another embodiment, the mutated EPSPS polypeptide comprises the mutations G172A, P177S and / or V403A and any one of (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) Y252F, (17) Y252C, (18) Y252D, (19) Y252E, (20) Y252F, (21) Y252F, (22) Y252F, (23) Y252F, (24) Y252F, (25) Y252F, (26) Y252F, (27) Y252F, (28) Y252F, (29) Y252F, (30) Y252F, (31) Y252F, (32) Y252F, (33) Y252F, (34) Y252F, (35) Y252F, (36) Y252F, (37) Y252F, (38) Y252F, (39) Y252F, (40) Y252F, (41) Y252F, (42) Y252F, (43) Y252F, (44) Y252F, (45) Y252F, (46) Y252F, (47) Y252F, (48) and at least one amino acid mutation selected from among 16) L253M, (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I, and (31) R514S / K, wherein each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO:1.

[0019] In yet another embodiment, the mutated EPSPS polypeptide comprises the mutations G172A, P177S and / or V403A and any one of (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) Y252F, (17) Y252C, (18) Y252D, (19) Y252E, (20) Y252F, (21) Y252F, (22) Y252F, (23) Y252F, (24) Y252F, (25) Y252F, (26) Y252F, (27) Y252F, (28) Y252F, (29) Y252F, (30) Y252F, (31) Y252F, (32) Y252F, (33) Y252F, (34) Y252F, (35) Y252F, (36) Y252F, (37) Y252F, (38) Y252F, (39) Y252F, (40) Y252F, (41) Y252F, (42) Y252F, (43) Y252F, (44) Y252F, (45) Y252F, (46) Y252F, (47) Y252F, (48) and at least one or more analogous amino acid mutations selected from among 6) L253M, (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I, and (31) R514S / K, wherein each amino acid mutation position corresponds to the analogous amino acid position shown in SEQ ID NO:1.

[0020] In another embodiment, the mutated EPSPS polypeptide comprises the mutations A70V, G172A, P177S, V403A and / or R514S, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1.

[0021] In another embodiment, the mutated EPSPS polypeptide comprises the mutations A73T, G172A, P177S and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0022] In another embodiment, the mutated EPSPS polypeptide comprises the mutations A73T, E86G, G172A, P177S and / or V403A, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1.

[0023] In other embodiments, the mutated EPSPS polypeptide comprises the mutations A145G, G172A, P177S, V403A, T442S and / or D472E, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0024] In another embodiment, the mutated EPSPS polypeptide comprises the mutations G172A, P177S, V403A and / or F502V, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1.

[0025] In another embodiment, the mutated EPSPS polypeptide comprises the mutations G172A, P177S, Y252F and / or V403A, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1.

[0026] In another embodiment, the mutated EPSPS polypeptide comprises the mutations E137D, G172A, P177S, L253M and / or V403A, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1.

[0027] In another embodiment, the mutated EPSPS polypeptide comprises the mutations G172A, P177S, K399N, V403A and / or T466S, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0028] In other embodiments, the mutated EPSPS polypeptide comprises the mutations A73G, L134F, G172A, P177S, G265V, S374R and / or V403A, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1.

[0029] In other embodiments, the mutated EPSPS polypeptide comprises the mutations A73K, D114H, V136M, K161E, G172A, P177S, A260T and / or V403A, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1.

[0030] In other embodiments, the mutated EPSPS polypeptide comprises the mutations E163K, G172A, P177S, A216V, L253M, H389R and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0031] In another embodiment, the mutated EPSPS polypeptide comprises the mutations G172A, P177S, N246D and / or V403A, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1.

[0032] In other embodiments, the mutated EPSPS polypeptide comprises the mutations G172A, M175A, P177S, M258L, K356E, V403A and / or F512I, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0033] In another embodiment, the mutated EPSPS polypeptide comprises the mutations A73S, G172A, P177S, L214F and / or V403A, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1.

[0034] In another embodiment, the mutated EPSPS polypeptide comprises the mutations G172A, P177S, V208A, L264H, V403A and / or R514K, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1.

[0035] In another embodiment, the mutated EPSPS polypeptide comprises the mutations V113M, G172A, P177S and / or V403A, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1.

[0036] The present disclosure further provides a recombinant DNA construct comprising a polynucleotide for encoding a polypeptide described herein, and further provides a plant cell comprising a polynucleotide described herein or comprising a recombinant DNA construct of the polynucleotide. In some examples, the plant cell is a rice cell. In some embodiments, the plant is rice.

[0037] In one embodiment, the present specification provides a method for generating a glyphosate-tolerant plant, the method includes expressing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising V403A, each amino acid mutation position corresponds to the amino acid position shown in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0038] In one embodiment, the present disclosure provides a method for producing a glyphosate-tolerant plant, comprising expressing in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide comprising G172A and V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1, and producing a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0039] In one embodiment, the present disclosure provides a method for producing a glyphosate-tolerant plant, comprising expressing in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide comprising P177S and V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1, and producing a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0040] In some embodiments, the method includes expressing in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPS PS polypeptide comprising G172A, P177S and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0041] In some embodiments, the method includes expressing in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a plant EPSPS polypeptide comprising T173I, P177S and / or V403A, wherein each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0042] In some embodiments, the method includes expressing in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide comprising T173I, P177T and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0043] In some embodiments, the method comprises expressing in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide, the polypeptide comprising G172A, P177S, and / or V403A, and at least one of the following: (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) and at least one or more amino acid mutations selected from M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I, and (31) R514S / K, wherein each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO:1.

[0044] In one embodiment, the present specification provides a method for generating a glyphosate-tolerant plant, the method includes expressing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising amino acid mutations similar to V403A, each amino acid mutation position corresponds to the analogous amino acid position shown in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0045] In one embodiment, the present disclosure provides a method for generating a glyphosate-tolerant plant, comprising expressing in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide comprising an amino acid mutation similar to G172A and an amino acid mutation similar to V403A, each amino acid mutation position corresponding to the analogous amino acid position shown in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0046] In one embodiment, the present disclosure provides a method for producing a glyphosate-tolerant plant, comprising expressing in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide comprising an amino acid mutation similar to P177S and / or an amino acid mutation similar to V403A, each amino acid mutation position corresponding to the analogous amino acid position shown in SEQ ID NO:1, and producing a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0047] In some embodiments, the method includes expressing in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide comprising an amino acid mutation similar to G172A, an amino acid mutation similar to P177S, and / or an amino acid mutation similar to V403A, wherein each amino acid mutation position corresponds to the analogous amino acid position shown in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0048] In some embodiments, the method includes expressing in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide comprising an amino acid mutation similar to T173I, an amino acid mutation similar to P177S, and / or an amino acid mutation similar to V403A, wherein each amino acid mutation position corresponds to the analogous amino acid position shown in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0049] In some embodiments, the method includes expressing in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide comprising an amino acid mutation similar to T173I, an amino acid mutation similar to P177T, and / or an amino acid mutation similar to V403A, wherein each amino acid mutation position corresponds to the analogous amino acid position shown in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0050] In some embodiments, the method includes expressing in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide, the polypeptide comprising an amino acid mutation similar to G172A, an amino acid mutation similar to P177S, and / or an amino acid mutation similar to V403A, and an amino acid mutation similar to (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K and at least one or more amino acid mutations selected from SEQ ID NO:161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I, and (31) R514S / K, each amino acid mutation position being represented by the sequence of SEQ ID NO:161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I, and (31) R514S / K. corresponding to the analogous amino acid positions shown in ID NO:1, and generating a glyphosate-tolerant plant whose genome contains the recombinant DNA construct.

[0051] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising A70V, G172A, P177S, V403 and / or R514S, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0052] In another embodiment, the method comprises expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising A73T, G172A, P177S and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct. In another embodiment, the method comprises expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising A73T, E86G, G172A, P177S and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0053] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising A145G, G172A, P177S, V403A, T442S and / or D472E, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0054] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising G172A, P177S, V403A and / or F502V, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0055] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising G172A, P177S, Y252F and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0056] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising E137D, G172A, P177S, L253M and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0057] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising G172A, P177S, K399N, V403A and / or T466S, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0058] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising A73G, L134G, G172A, P177S, G265V, S374R and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0059] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising A73K, D114H, V136M, K161E, G172A, P177S, A260T and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0060] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising E163K, G172A, P177S, A216V, L253M, H389R and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0061] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising G172A, P177S, N247D and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0062] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising G172A, M175A, P177S, M258L, K356E, V403A and / or F512I, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0063] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising A73S, G172A, P177S, L214F and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0064] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising G172A, P177S, V208A, L264H, V403A and / or R514K, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0065] In another embodiment, the method includes expressing a recombinant DNA construct in a plant cell, the recombinant DNA construct comprising a polynucleotide encoding a plant EPSPS polypeptide, the plant EPSPS polypeptide comprising V113M, G172A, P177S and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.

[0066] In one aspect, the present specification provides a method of producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein comprising V403A, the amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0067] In another aspect, the specification provides a method of producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein comprising G172A and V403A, the amino acid mutation positions corresponding to the amino acid positions shown in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0068] In another aspect, the present specification provides a method for producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein comprising P177S and V403A, the amino acid mutation positions corresponding to the amino acid positions shown in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0069] In another aspect, the specification provides a method of producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein comprising G172A, P177S and / or V403A, the amino acid mutation positions corresponding to the amino acid positions shown in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0070] In another aspect, the specification provides a method of producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein comprising T173I, P177S and / or V403A, the amino acid mutation positions corresponding to the amino acid positions shown in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0071] In another aspect, the specification provides a method of producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein comprising T173I, P177T and / or V403A, the amino acid mutation positions corresponding to the amino acid positions shown in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0072] In yet another aspect, the present specification provides a method for producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein comprising at least one of the following amino acids: G172A, P177S, and / or V403A, and (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) M176A, (13) M177B, (14) M178B, (15) M179B, (16) M180B, (17) M181C, (18) M182C, (19) M183C, (20) M184C, (21) M185C, (22) M186C, (23) M187C, (24) M188C, (25) M189C, (26) M190C, (27) M191C, (28) M192C, (29) M193C, (30) M194C, (31) M195C, (32) M196C, (33) M197C, (34) M198C, (35) M199C, (36) M206C, (37) M205C, (38) M210C, (39) M221C, (40) M222C, (41) M230C, (42) M231C, and at least one amino acid selected from among (1) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I, and (31) R514S / K, wherein each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0073] In one aspect, the present specification provides a method of producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein containing an amino acid mutation similar to V403A, the amino acid mutation position corresponding to the analogous amino acid position shown in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0074] In another aspect, the specification provides a method of producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein containing an amino acid mutation similar to G172A and an amino acid mutation similar to V403A, the amino acid mutation positions corresponding to the analogous amino acid positions shown in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0075] In another aspect, the specification provides a method of producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein containing an amino acid mutation similar to P177S and an amino acid mutation similar to V403A, the amino acid mutation positions corresponding to the analogous amino acid positions shown in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0076] In another aspect, the specification provides a method of producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein containing an amino acid mutation similar to G172A, an amino acid mutation similar to P177S, and / or an amino acid mutation similar to V403A, wherein the amino acid mutation positions correspond to the analogous amino acid positions shown in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0077] In another aspect, the specification provides a method of producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein containing an amino acid mutation similar to T173I, an amino acid mutation similar to P177S, and / or an amino acid mutation similar to V403A, the amino acid mutation positions corresponding to the analogous amino acid positions shown in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0078] In another aspect, the specification provides a method of producing a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein containing an amino acid mutation similar to T173I, an amino acid mutation similar to P177T, and / or an amino acid mutation similar to V403A, the amino acid mutation positions corresponding to the analogous amino acid positions shown in SEQ ID NO:1, and growing a glyphosate-tolerant plant from the plant cell.

[0079] In yet another aspect, the present specification provides a method for generating a glyphosate-tolerant plant, wherein an endogenous plant EPSPS gene is modified (in a plant cell) to encode a glyphosate-tolerant EPSPS protein, the protein having an amino acid mutation similar to G172A, an amino acid mutation similar to P177S, and / or an amino acid mutation similar to V403A, and / or an amino acid mutation similar to (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E1 and at least one similar amino acid mutation selected from SEQ ID NO:63K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I, and (31) R514S / K, and the amino acid mutation position is selected from SEQ ID NO:63K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I, and (31) R514S / K. corresponding to the analogous amino acid positions shown in ID NO:1, and from the plant cells glyphosate-tolerant plants are grown.

[0080] In another embodiment, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising A70V, G172A, P177S, V403 and / or R514S, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0081] In another embodiment, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising A73T, G172A, P177S and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0082] In another embodiment, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising A73T, E86G, G172A, P177S and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0083] In other embodiments, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising A145G, G172A, P177S, V403A, T442S and / or D472E, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0084] In another embodiment, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising G172A, P177S, V403A and / or F502V, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0085] In another embodiment, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising G172A, P177S, Y252F and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0086] In another embodiment, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising E137D, G172A, P177S, L253M and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0087] In another embodiment, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein containing G172A, P177S, K399N, V403A and / or T466S, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0088] In other embodiments, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising A73G, L134G, G172A, P177S, G265V, S374R and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0089] In other embodiments, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising A73K, D114H, V136M, K161E, G172A, P177S, A260T and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0090] In other embodiments, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising E163K, G172A, P177S, A216V, L253M, H389R and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0091] In another embodiment, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising G172A, P177S, N247D and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0092] In other embodiments, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein that includes G172A, M175A, P177S, M258L, K356E, V403A and / or F512I, where each amino acid mutation position corresponds to an amino acid position shown in SEQ ID NO:1.

[0093] In another embodiment, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising A73S, G172A, P177S, L214F and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0094] In other embodiments, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising G172A, P177S, V208A, L264H, V403A and / or R514K, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1.

[0095] In another embodiment, the modified endogenous plant EPSPS gene encodes a glyphosate-tolerant EPSPS protein comprising V113M, G172A, P177S and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO:1.

[0096] The endogenous plant EPSPS gene can be modified by any method known to one of skill in the art, including, but not limited to, CRISPR / Cas guide RNA-mediated systems, zinc finger nuclease-mediated systems, meganuclease-mediated systems, and / or oligonucleotide base-mediated systems.

[0097] The present specification provides a polynucleotide construct providing a guide RNA in a plant cell, wherein the guide RNA targets an endogenous EPSPS gene of the plant cell, and the polynucleotide construct further comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising V403A, wherein the amino acid mutation position corresponds to the amino acid shown in SEQ ID NO:1.

[0098] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising G172A and V403A, the amino acid positions corresponding to the amino acid mutation positions shown in SEQ ID NO:1.

[0099] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising P177S and V403A, the amino acid positions corresponding to the amino acid mutation positions shown in SEQ ID NO:1.

[0100] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising G172A, P177S and / or V403A, the amino acid positions corresponding to the amino acid mutation positions shown in SEQ ID NO:1.

[0101] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising T173I, P177S and / or V403A, the amino acid positions corresponding to the amino acid mutation positions shown in SEQ ID NO:1.

[0102] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising T173I, P177T and / or V403A, the amino acid positions corresponding to the amino acid mutation positions shown in SEQ ID NO:1.

[0103] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to provide one or more of the following modifications: G172A, P177S, and / or V403A, (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L25 and at least one or more amino acid mutations selected from among 3M, (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I and (31) R514S / K, wherein each amino acid position corresponds to an amino acid mutation position as set forth in SEQ ID NO:1.

[0104] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising an amino acid mutation similar to V403A, where the amino acid position corresponds to the analogous amino acid mutation position shown in SEQ ID NO:1.

[0105] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising an amino acid mutation similar to G172A and an amino acid mutation similar to V403A, the amino acid positions corresponding to the analogous amino acid mutation positions shown in SEQ ID NO:1.

[0106] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising an amino acid mutation similar to P177S and an amino acid mutation similar to V403A, the amino acid positions corresponding to the analogous amino acid mutation positions shown in SEQ ID NO:1.

[0107] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising an amino acid mutation similar to G172A, an amino acid mutation similar to P177S and / or an amino acid mutation similar to V403A, wherein each amino acid position corresponds to the analogous amino acid mutation position shown in SEQ ID NO:1.

[0108] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising an amino acid mutation analogous to T173I, an amino acid mutation analogous to P177S and / or an amino acid mutation analogous to V403A, wherein each amino acid position corresponds to the analogous amino acid mutation position shown in SEQ ID NO:1.

[0109] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising an amino acid mutation analogous to T173I, an amino acid mutation analogous to P177T and / or an amino acid mutation analogous to V403A, wherein each amino acid position corresponds to the analogous amino acid mutation position shown in SEQ ID NO:1.

[0110] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising A70V, G172A, P177S, V403A and / or R514S, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0111] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising A73T, G172A, P177S and / or V403A, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0112] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising A73T, E86G, G172A, P177S and / or V403A, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0113] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising A145G, G172A, P177S, V403A, T442S and / or D472E, each amino acid position corresponding to an amino acid mutation position shown in SEQ ID NO:1.

[0114] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising G172A, P177S, V403A and / or F502V, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0115] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising G172A, P177S, Y252F and / or V403A, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0116] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising E137D, G172A, P177S, L253M and / or V403A, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0117] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising G172A, P177S, K399N, V403A and / or T466S, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0118] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising A73G, L134G, G172A, P177S, G265V, S374R and / or V403A, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0119] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising A73K, D114H, V136M, K161E, G172A, P177S, A260T and / or V403A, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0120] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising E163K, G172A, P177S, A216V, L253M, H389R and / or V403A, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0121] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising G172A, P177S, N247D and / or V403A, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0122] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising G172A, M175A, P177S, M258L, K356E, V403A and / or F512I, each amino acid position corresponding to an amino acid mutation position shown in SEQ ID NO:1.

[0123] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising A73S, G172A, P177S, L214F and / or V403A, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0124] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising G172A, P177S, V208A, L264H, V403A and / or R514K, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0125] In some embodiments, the polynucleotide construct comprises one or more polynucleotide modification templates to generate a modified endogenous EPSPS gene encoding a plant EPSPS polypeptide comprising V113M, G172A, P177S and / or V403A, each amino acid position corresponding to the amino acid mutation position shown in SEQ ID NO:1.

[0126] Further provided herein is a glyphosate-tolerant plant expressing an endogenous EPSPS polypeptide having V403A, the amino acid positions corresponding to the amino acid positions set forth in SEQ ID NO:1.

[0127] Further provided herein is a glyphosate-tolerant plant expressing an endogenous EPSPS polypeptide having G172A and V403A, the amino acid positions corresponding to the amino acid positions set forth in SEQ ID NO:1.

[0128] Further provided herein is a glyphosate-tolerant plant expressing an endogenous EPSPS polypeptide having P177S and V403A, the amino acid positions corresponding to the amino acid positions shown in SEQ ID NO:1.

[0129] Further provided herein is a glyphosate-tolerant plant expressing an endogenous EPSPS polypeptide having G172A, P177S and / or V403A, the amino acid positions corresponding to the amino acid positions set forth in SEQ ID NO:1.

[0130] Further provided herein is a glyphosate-tolerant plant expressing an endogenous EPSPS polypeptide having T173I, P177S and / or V403A, the amino acid positions corresponding to the amino acid positions shown in SEQ ID NO:1.

[0131] Further provided herein is a glyphosate-tolerant plant expressing an endogenous EPSPS polypeptide having T173I, P177T and / or V403A, the amino acid positions corresponding to the amino acid positions shown in SEQ ID NO:1.

[0132] This specification relates to a combination of G172A, P177S and / or V403A, and (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) Y252F, (20) Y253M, (21) Y253M, (22) Y253M, (23) Y253M, (24) Y253M, (25) Y253M, (26) Y253M, (27) Y253M, (28) Y253M, (29) Y253M, (30) Y253M, (31) Y253M, (32) Y253M, (33) Y253M, (34) Y253M, (35) Y253M, (36) Y253M, (37) Y253M, (38) Y253M, (39) Y253M, (40) Y253M, (41) Y253M, (42) Y253M, (43) Y253M, (44) Y253M, (45) Y253M, (46) Y253M, (47) Y253M, (48) Y253M, (49) Y2 and (31) R514S / K, wherein each amino acid position corresponds to an amino acid position set forth in SEQ ID NO:1.

[0133] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having V403A, where the amino acid positions correspond to the amino acid positions set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant can express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO: 3.

[0134] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having G172A and V403A, where the amino acid positions correspond to the amino acid positions set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having the sequence set forth in SEQ ID NO:4.

[0135] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having P177S and V403A, where the amino acid positions correspond to the amino acid positions set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:5.

[0136] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having G172A, P177S and / or V403A, where the amino acid positions correspond to the amino acid positions set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:6.

[0137] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having T173I, P177S and / or V403A, where the amino acid positions correspond to the amino acid positions set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:37.

[0138] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having T173I, P177T and / or V403A, where the amino acid positions correspond to the amino acid positions set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:38.

[0139] This specification relates to a compound including G172A, P177S and / or V403A, and (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) L and (21) at least one or more amino acid mutations selected from among 264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I and (31) R514S / K, wherein each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO:1.

[0140] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having A70V, G172A, P177S, V403 and / or R514S, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO: 10.

[0141] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having A73T, G172A, P177S and / or V403A, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO: 11.

[0142] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having A73T, E86G, G172A, P177S and / or V403A, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO: 12.

[0143] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having A145G, G172A, P177S, V403A, T442S and / or D472E, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO: 13.

[0144] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having G172A, P177S, V403A and / or F502V, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO: 14.

[0145] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having G172A, P177S, Y252F and / or V403A, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO: 15.

[0146] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having E137D, G172A, P177S, L253M and / or V403A, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO: 16.

[0147] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having G172A, P177S, K399N, V403A and / or T466S, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO: 17.

[0148] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having A73G, L134G, G172A, P177S, G265V, S374R and / or V403A, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO: 18.

[0149] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having A73K, D114H, V136M, K161E, G172A, P177S, A260T and / or V403A, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:19.

[0150] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having E163K, G172A, P177S, A216V, L253M, H389R and / or V403A, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:20.

[0151] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having G172A, P177S, N247D and / or V403A, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:21.

[0152] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having G172A, M175A, P177S, M258L, K356E, V403A and / or F512I, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:22.

[0153] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having A73S, G172A, P177S, L214F and / or V403A, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:23.

[0154] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having G172A, P177S, V208A, L264H, V403A and / or R514K, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:24.

[0155] Further provided herein is a glyphosate-tolerant rice plant that expresses an endogenous EPSPS polypeptide having V113M, G172A, P177S and / or V403A, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO: 1. The glyphosate-tolerant rice plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:25.

[0156] Further provided herein is a glyphosate tolerant flax plant expressing an endogenous EPSPS polypeptide having V403A, the amino acid position corresponding to the amino acid position set forth in SEQ ID NO: 1. The glyphosate tolerant flax plant can express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO: 27.

[0157] Further provided herein is a glyphosate tolerant flax plant expressing an endogenous EPSPS polypeptide having T173I and P177T, where each amino acid mutation position corresponds to an amino acid position shown in SEQ ID NO: 1. The glyphosate tolerant flax plant can express a plant EPSPS polypeptide having a sequence shown in SEQ ID NO:28.

[0158] Further provided herein is a glyphosate tolerant flax plant expressing an endogenous EPSPS polypeptide having T173I, P177T and / or V403A, each amino acid mutation position corresponding to an amino acid position shown in SEQ ID NO: 1. The glyphosate tolerant flax plant can express a plant EPSPS polypeptide having a sequence shown in SEQ ID NO:29.

[0159] Further provided herein is a glyphosate tolerant flax plant expressing an endogenous EPSPS polypeptide having G172A, P177S and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO: 1. The glyphosate tolerant flax plant may express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:30.

[0160] This specification relates to a combination of G172A, P177S and / or V403A, and (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) L2 and at least one or more similar amino acid mutations selected from among 64H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I and (31) R514S / K, wherein each amino acid mutation position corresponds to the similar amino acid position shown in SEQ ID NO:1.

[0161] Further provided herein is a glyphosate tolerant wheat plant that expresses an endogenous EPSPS polypeptide having V403A, the amino acid position corresponding to the amino acid position set forth in SEQ ID NO: 1. The glyphosate tolerant wheat plant can express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:32.

[0162] This specification relates to a compound including G172A, P177S and / or V403A, and (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) L26 and at least one or more similar amino acid mutations selected from among the following: (1) glyphosate-tolerant wheat plants expressing an endogenous EPSPS polypeptide having at least one similar amino acid mutation selected from the following: (2) 4H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I and (31) R514S / K, wherein each amino acid mutation position corresponds to the similar amino acid position set forth in SEQ ID NO:1.

[0163] Further provided herein is a glyphosate tolerant soybean plant that expresses an endogenous EPSPS polypeptide having V403A, the amino acid position corresponding to the amino acid position set forth in SEQ ID NO: 1. The glyphosate tolerant soybean plant can express a plant EPSPS polypeptide having a sequence set forth in SEQ ID NO:34.

[0164] This specification relates to a combination of G172A, P177S and / or V403A, and (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) L2 and at least one or more analogous amino acid mutations selected from among 64H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I, and (31) R514S / K, wherein each amino acid mutation position corresponds to the analogous amino acid position set forth in SEQ ID NO: 1. Methods of producing glyphosate-tolerant soybean plants are further provided. The method includes introducing a recombinant oligonucleotide having a mutated EPSPS gene into a plant cell, wherein the mutated EPSPS gene expresses an EPSPS protein mutated at amino acid position V403 in the rice EPSPS protein (SEQ ID NO:1) or an analogous amino acid residue in an EPSPS homolog, where V403 is mutated to A, selecting plant cells having improved tolerance to glyphosate compared to corresponding wild-type plant cells, and regenerating herbicide-resistant or tolerant plants having the mutated EPSPS gene from the selected plant cells.

[0165] Further provided is a method for producing a glyphosate-tolerant plant, comprising: introducing a recombinant oligonucleotide having a mutated EPSPS gene into a plant cell, expressing an EPSPS protein mutated at amino acid positions G172 and V403 in the rice EPSPS protein (SEQ ID NO:1) or similar amino acid residues in an EPSPS homolog, where G172 is mutated to A and V403 is mutated to A, selecting a plant cell having improved tolerance to glyphosate compared to a corresponding wild-type plant cell, and regenerating a herbicide-resistant or herbicide-tolerant plant having the mutated EPSPS gene from the selected plant cell.

[0166] Further provided is a method for producing a glyphosate-tolerant plant, comprising: introducing a recombinant oligonucleotide carrying a mutated EPSPS gene into a plant cell, expressing an EPSPS protein mutated at amino acid positions P177 and V403 in the rice EPSPS protein (SEQ ID NO:1) or similar amino acid residues in an EPSPS homolog, where P177 is mutated to S and V403 is mutated to A; selecting a plant cell having improved tolerance to glyphosate compared to a corresponding wild-type plant cell; and regenerating a herbicide-resistant or herbicide-tolerant plant carrying the mutated EPSPS gene from the selected plant cell.

[0167] Further provided is a method for producing a glyphosate-tolerant plant, comprising: introducing a recombinant oligonucleotide carrying a mutated EPSPS gene into a plant cell, expressing an EPSPS protein mutated at amino acid positions G172, P177 and / or V403 in the rice EPSPS protein (SEQ ID NO:1) or analogous amino acid residues in an EPSPS homolog, where G172 is mutated to A, P177 is mutated to S and / or V403 is mutated to A; selecting a plant cell having improved tolerance to glyphosate compared to a corresponding wild-type plant cell; and regenerating a herbicide-resistant or herbicide-tolerant plant carrying the mutated EPSPS gene from the selected plant cell.

[0168] Further provided is a method for producing a glyphosate-tolerant plant, comprising: introducing a recombinant oligonucleotide carrying a mutated EPSPS gene into a plant cell, expressing an EPSPS protein mutated at amino acid positions T173, P177 and / or V403 in the rice EPSPS protein (SEQ ID NO:1) or analogous amino acid residues in an EPSPS homolog, where T173 is mutated to I, P177 is mutated to S and / or V403 is mutated to A; selecting a plant cell having improved tolerance to glyphosate compared to a corresponding wild-type plant cell; and regenerating a herbicide-resistant or herbicide-tolerant plant carrying the mutated EPSPS gene from the selected plant cell.

[0169] Further provided is a method for producing a glyphosate-tolerant plant, comprising: introducing a recombinant oligonucleotide carrying a mutated EPSPS gene into a plant cell, expressing an EPSPS protein mutated at amino acid positions T173, P177 and / or V403 in the rice EPSPS protein (SEQ ID NO:1) or analogous amino acid residues in an EPSPS homolog, where T173 is mutated to I, P177 is mutated to T and / or V403 is mutated to A, selecting a plant cell having improved tolerance to glyphosate compared to a corresponding wild-type plant cell, and regenerating a herbicide-resistant or herbicide-tolerant plant carrying the mutated EPSPS gene from the selected plant cell. [Brief description of the drawings]

[0170] [Figure 1] 1 shows a multiple alignment between SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:31 and SEQ ID NO:33. [Diagram 2] 2 shows the growth of E. coli cells expressing rice EPSPS proteins with G172A, P177S and / or V403A mutations and combination mutants thereof in media with different glyphosate concentrations. "Glyphosate" in FIG. 2 is glyphosate. [Diagram 3] 3 shows the growth of E. coli cells expressing rice EPSPS proteins with T173I, P177S / T and / or V403A mutations and their combination mutants in media with different glyphosate concentrations. "Glyphosate" in FIG. 3 is glyphosate. [Figure 4] 4 is a diagram showing the growth status of Escherichia coli cells expressing flax, wheat or soybean EPSPS proteins with the V403A mutation in media with different glyphosate concentrations. "Glyphosate" in FIG. 4 is glyphosate. [Diagram 5]5 is a diagram showing a comparison of the growth conditions of E. coli cells expressing flax EPSPS proteins having G172A, P177S and V403A mutations and E. coli cells expressing CP4 EPSPS proteins in media with different glyphosate concentrations. "Glyphosate" in FIG. 5 is glyphosate. [Figure 6] 6 is a diagram showing the growth status of E. coli cells expressing flax EPSPS proteins with G172A, T173I, P177T / S and / or V403A mutations in media with different glyphosate concentrations. "Glyphosate" in FIG. 6 is glyphosate. [Figure 7] FIG. 1 shows a comparison of glyphosate resistance between E. coli cells expressing rice EPSPS protein and several mutants at a herbicide concentration of 125 ml glyphosate. [Figure 8] 8 is a diagram showing the construct pKED2 vector. In FIG. 8, "Operator" is an operator. [Figure 9] 9 is a diagram showing a pk510 vector including one embodiment of the present invention. In Fig. 9, "PolyA Signal" is a PolyA signal, and "kanamycin" is kanamycin. [Figure 10] Figure 10 shows the comparison between wild-type rice plants and transgenic rice plants and their glyphosate tolerance capabilities. In Figure 10, "wild-type rice" is wild-type rice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0171] The term "EPSPS homolog" or any variant refers to an EPSPS gene or EPSPS gene product found in another plant species, which performs the same or substantially the same biological function as the EPSPS gene disclosed herein, and the nucleic acid or polypeptide sequences (EPSPS gene products) described below are referred to as "identical" or at least 50% similar (also referred to as "percent identity" or "substantially identical"). Two polynucleotides or polypeptides are the same if the sequence of nucleotides or amino acid residues in the two polynucleotides or polypeptide sequences, respectively, are the same when aligned for maximum correspondence, as described below. In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "percent identity" refers to two or more sequences or subsequences that are the same, or have a specified percentage of amino acid residues or nucleotides that are identical, when compared and aligned for maximum correspondence over a comparison window, using one of the alignment algorithms described below, or by manual alignment and visual measurement. For polypeptides whose sequences differ in conservative substitutions, the sequence percent identity may be adjusted upwards to correct for the conservative nature of the substitution. Methods for this adjustment are well known to those skilled in the art. Usually, this involves increasing the percent sequence identity by scoring conservative substitutions as partial misfits rather than complete misfits. Thus, for example, where identical amino acids are assigned a score of 1 and non-conservative substitutions are assigned a score of zero, conservative substitutions are assigned a score between zero and 1. Scoring of conservative substitutions is performed, for example, according to the algorithm of Meyers & Miller, Computer Applic Biol. Sci. 4:11-17 (1988) eg, in the program PC / GENE (Intelligenetics, Mountain View, Calif., USA).

[0172] The phrases "substantially identical" and "percent identity" of two nucleic acids or polypeptides in this context refer to sequences or subsequences that have at least 50%, advantageously 60%, preferably 70%, more preferably 80% and most preferably 90%-95% nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window when aligned using one of the sequence comparison algorithms below or by manual alignment and visual measurement. The definition also refers to the complement of a test sequence having substantial sequence or subsequence complementarity when it has substantial identity with a reference sequence.

[0173] "Operably linked" refers to the juxtaposition of genetic elements, where the elements are in a relationship permitting them to function in their expected manner. For example, a 5' regulatory sequence is operably linked to a coding sequence if the function of the 5' regulatory sequence is to initiate transcription of the coding sequence.

[0174] As used herein, a "glyphosate-resistant" cell or plant refers to a cell or plant that can continue to survive or grow in the presence of a particular concentration of glyphosate that would normally kill or inhibit the growth of other cells or plants. For example, growth can include photosynthesis, increased rooting, increased height, increased mass, or new leaf development. In one embodiment, the glyphosate-resistant cell can grow and divide on a medium containing 50 mg / L or more of glyphosate. Preferably, the glyphosate-resistant cell can grow and divide on a medium containing 100 mg / L or more of glyphosate, for example 200 mg / L, 300 mg / L, or 400 mg / L. More preferably, the glyphosate-resistant cell can grow and divide on a medium containing 500 mg / L or more of glyphosate, for example 600 mg / L. For purposes of this invention, the term "glyphosate" includes any herbicidally effective form of N-phosphonomethylglycine, including any salt thereof, and other forms that result in the production of the glyphosate anion in plants.

[0175] Explanation of sequence listing SEQ ID NO:1 is the full-length amino acid sequence of the wild-type rice EPSPS protein (GenBank accession number XP_015643046.1), the N-terminus of which contains the chloroplast transit peptide. SEQ ID NO:1 is used herein as the reference EPSPS sequence, and all mutation positions are marked according to SEQ ID NO:1.

[0176] SEQ ID NO:2 is the amino acid sequence of the wild-type rice EPSPS protein. Compared to SEQ ID NO:1, the chloroplast transit peptide is deleted from SEQ ID NO:2.

[0177] SEQ ID NO:3 is a mutant EPSPS sequence from rice and contains the V403A mutation.

[0178] SEQ ID NO:4 is a mutant EPSPS sequence from rice and contains the G172A and V403A mutations.

[0179] SEQ ID NO:5 is a mutant EPSPS sequence from rice and contains the P177S and V403A mutations.

[0180] SEQ ID NO:6 is a mutant EPSPS sequence from rice and contains the G172A, P177S or V403A mutations.

[0181] SEQ ID NO:7 is a mutant EPSPS sequence from rice and contains a G172A mutation.

[0182] SEQ ID NO:8 is a mutant EPSPS sequence from rice and contains the P177S mutation.

[0183] SEQ ID NO:9 is a mutant EPSPS sequence from rice and contains the G172A and P177S mutations.

[0184] SEQ ID NO:10 is a mutant EPSPS sequence from rice and contains the A70V, G172A, P177S, V403A and R514S mutations.

[0185] SEQ ID NO:11 is a mutant EPSPS sequence from rice and contains the A73T, G172A, P177S and V403A mutations.

[0186] SEQ ID NO:12 is a mutant EPSPS sequence from rice and contains the A73T, E86G, G172A, P177S and V403A mutations.

[0187] SEQ ID NO:13 is a mutant EPSPS sequence from rice and contains the A145G, G172A, P177S, V403A, T442S and D472E mutations.

[0188] SEQ ID NO:14 is a mutant EPSPS sequence from rice and contains G172A, P177S, V403A and F502V mutations.

[0189] SEQ ID NO:15 is a mutant EPSPS sequence from rice and contains the G172A, P177S, Y252F and V403A mutations.

[0190] SEQ ID NO:16 is a mutant EPSPS sequence from rice and contains the E137D, G172A, P177S, L253M and V403A mutations.

[0191] SEQ ID NO:17 is a mutant EPSPS sequence from rice and contains G172A, P177S, K399N, V403A and T466S mutations.

[0192] SEQ ID NO:18 is a mutant EPSPS sequence from rice and contains A73G, L134F, G172A, P177S, G265V, S374R and V403A mutations.

[0193] SEQ ID NO:19 is a mutant EPSPS sequence from rice and contains the A73K, D114H, V136M, K161E, G172A, P177S, A260T and V403A mutations.

[0194] SEQ ID NO:20 is a mutant EPSPS sequence from rice and contains E163K, G172A, P177S, A216V, L253M, H389R and V403A mutations.

[0195] SEQ ID NO:21 is a mutant EPSPS sequence from rice and contains the G172A, P177S, N246D and V403A mutations.

[0196] SEQ ID NO:22 is a mutant EPSPS sequence from rice and contains G172A, M175A, P177S, M258L, K356E, V403A and F512I mutations.

[0197] SEQ ID NO:23 is a mutant EPSPS sequence from rice and contains the A73S, G172A, P177S, L214F and V403A mutations.

[0198] SEQ ID NO:24 is a mutant EPSPS sequence from rice and contains G172A, P177S, V208A, L264H, V403A and R514K mutations.

[0199] SEQ ID NO:25 is a mutant EPSPS sequence from rice and contains the V113M, G172A, P177S and V403A mutations.

[0200] SEQ ID NO:26 is the amino acid sequence of wild-type flax EPSPS2 translated from Lus10000788 (Sauer NJ, Narvaez-Vasquez, Javier, Mozoruk J, et al. Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants[J]. Plant Physiology, 2016:1917-1928.), where the chloroplast transit peptide has been removed from SEQ ID NO:26.

[0201] SEQ ID NO:27 is a mutant of the EPSPS sequence from flax and contains the V403A mutation.

[0202] SEQ ID NO:28 is a mutant EPSPS sequence from flax and contains T173I and P177T mutations.

[0203] SEQ ID NO:29 is a mutant EPSPS sequence from flax and contains T173I, P177T and V403A mutations.

[0204] SEQ ID NO:30 is a mutant EPSPS sequence from flax and contains the G172A, P177S and V403A mutations.

[0205] SEQ ID NO:31 is the amino acid sequence of the wild-type wheat EPSPS protein represented as GenBank entry ALK27163.1, with the chloroplast transit peptide removed from SEQ ID NO:31.

[0206] SEQ ID NO:32 is a mutant of the EPSPS sequence from wheat and contains a V403A mutation.

[0207] SEQ ID NO:33 is the amino acid sequence of the wild-type soybean EPSPS protein, represented as GenBank entry XP_003521857.1, with the chloroplast transit peptide removed from SEQ ID NO:33.

[0208] SEQ ID NO:34 is a mutant of the EPSPS sequence from soybean and contains a V403A mutation.

[0209] SEQ ID NO:35 is a mutant EPSPS sequence from rice and contains the T173I and P177S mutations.

[0210] SEQ ID NO:36 is a mutant EPSPS sequence from rice and contains T173I and P177T mutations.

[0211] SEQ ID NO:37 is a mutant EPSPS sequence from rice and contains the T173I, P177S and V403A mutations.

[0212] SEQ ID NO:38 is a mutant EPSPS sequence from rice and contains T173I, P177T and V403A mutations.

[0213] SEQ ID NO:39 is the amino acid sequence of wild-type EPSPS from CP4 (Agrobacterium).

[0214] 1. Configuration A. EPSPS Polypeptides Various methods and compositions using mutant polypeptides having EPSPS activity are provided. Such EPSPS polypeptides contain a V403A mutation, at amino acid positions corresponding to those shown in SEQ ID NO:1. Such EPSPS polypeptides contain G172A and V403A mutations, at amino acid positions corresponding to those shown in SEQ ID NO:1. Such EPSPS polypeptides contain P177S and V403A mutations, at amino acid positions corresponding to those shown in SEQ ID NO:1.

[0215] In some embodiments, the EPSPS polypeptide comprises G172A, P177S and / or V403A, each amino acid position corresponding to the amino acid position set forth in SEQ ID NO:1.

[0216] In some embodiments, the EPSPS polypeptide comprises T173I, P177S and / or V403A, each amino acid position corresponding to the amino acid position set forth in SEQ ID NO:1.

[0217] In some embodiments, the EPSPS polypeptide comprises T173I, P177T and / or V403A, each amino acid position corresponding to the amino acid position set forth in SEQ ID NO:1.

[0218] In other embodiments, the EPSPS polypeptide is selected from the group consisting of G172A, P177S, V403A, (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253 and at least one amino acid mutation selected from among M, (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I and (31) R514S / K, wherein each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO:1.

[0219] In another embodiment, the plant EPSPS polypeptide comprises an A70V, G172A, P177S, V403 and / or R514S mutation. In another embodiment, the plant EPSPS polypeptide comprises an A73T, G172A, P177S and / or V403A mutation. In another embodiment, the plant EPSPS polypeptide comprises an A73T, E86G, G172A, P177S and / or V403A mutation. In another embodiment, the plant EPSPS polypeptide comprises an A145G, G172A, P177S, V403A, T442S and / or D472E mutation. In another embodiment, the plant EPSPS polypeptide comprises a G172A, P177S, V403A and / or F502V mutation. In another embodiment, the plant EPSPS polypeptide comprises a G172A, P177S, Y252F and / or V403A mutation. In another embodiment, the plant EPSPS polypeptide comprises an E137D, G172A, P177S, L253M and / or V403A mutation. In another embodiment, the plant EPSPS polypeptide comprises a G172A, P177S, K399N, V403A and / or T466S mutation. In another embodiment, the plant EPSPS polypeptide comprises an A73G, L134F, G172A, P177S, G265V, S374R and / or V403A mutation. In another embodiment, the plant EPSPS polypeptide comprises an A73K, D114H, V136M, K161E, G172A, P177S, A260T and / or V403A mutation. In another embodiment, the plant EPSPS polypeptide comprises an E163K, G172A, P177S, A216V, L253M, H389R and / or V403A mutation. In another embodiment, the plant EPSPS polypeptide comprises a G172A, P177S, N246D and / or V403A mutation. In another embodiment, the plant EPSPS polypeptide comprises a G172A, M175A, P177S, M258L, K356E, V403A and / or F512I mutation. In another embodiment, the plant EPSPS polypeptide comprises an A73S, G172A, P177S, L214F and / or V403A mutation.In other embodiments, the plant EPSPS polypeptide comprises a G172A, P177S, V208A, L264H, V403A and / or R514K mutation, hi other embodiments, the plant EPSPS polypeptide comprises a V113M, G172A, P177S and / or V403A mutation.

[0220] The EPSPS polypeptide mutants disclosed herein may have improved catalytic ability in the presence of glyphosate compared to a wild-type EPSPS polypeptide.

[0221] As disclosed herein, the EPSPS mutant containing V403A can improve the glyphosate resistance of a plant while still maintaining the catalytic activity of its own biological enzyme. However, the position of such amino acid V403 in EPSPS may be different in other different plant species. A similar mutation V→A at a similar position can also improve the glyphosate resistance of other plants. The mutation position can be determined in two steps. The first step is to align the wild-type plant EPSPS with the rice EPSPS sequence (SEQ ID NO:1). The second step is to find the position corresponding to position 403 on the rice EPSPS sequence.

[0222] Previous studies have further disclosed that G172 and P177 are also two conserved positions on most EPSPS sequences. However, the position of the amino acid G172 or P177 on the EPSPS sequence may vary in some other species. Similar mutations G→A, P→S and / or V→A at similar positions can also improve the glyphosate resistance of some other plants. The mutation position can be determined in two steps. The first step is to align the wild-type plant EPSPS and rice EPSPS sequences. The second step is to find the positions corresponding to positions 172, 177 and / or 403 on the rice EPSPS sequence.

[0223] Previous studies have further disclosed that T173 and P177 are also two conserved positions on most EPSPS sequences. However, the position of the amino acid T173 or P177 on the EPSPS sequence may vary in some other species. Similar mutations T→I, P→S / T and / or V→A at similar positions can also improve the glyphosate resistance of some other plants. The mutation position can be determined in two steps. The first step is to align the wild-type plant EPSPS and rice EPSPS sequences. The second step is to find the positions corresponding to positions 173, 177 and / or 403 on the rice EPSPS sequence.

[0224] Various methods and compositions are provided that use mutant polypeptides having EPSPS activity. Such EPSPS polypeptides include an amino acid mutation analogous to V403A, where each amino acid position corresponds to the analogous amino acid position shown in SEQ ID NO:1. Such EPSPS polypeptides include an amino acid mutation analogous to G172A and an amino acid mutation analogous to V403A, where each amino acid position corresponds to the analogous amino acid position shown in SEQ ID NO:1. Such EPSPS polypeptides include an amino acid mutation analogous to P177S and an amino acid mutation analogous to V403A, where each amino acid position corresponds to the analogous amino acid position shown in SEQ ID NO:1.

[0225] In some embodiments, the EPSPS polypeptide comprises an amino acid mutation similar to G172A, an amino acid mutation similar to P177S, and an amino acid mutation similar to V403A, where each amino acid position corresponds to the analogous amino acid position shown in SEQ ID NO:1.

[0226] In some embodiments, the EPSPS polypeptide comprises an amino acid mutation similar to T173I, an amino acid mutation similar to P177S, and / or an amino acid mutation similar to V403A, where each amino acid position corresponds to the analogous amino acid position shown in SEQ ID NO:1.

[0227] In some embodiments, the EPSPS polypeptide comprises an amino acid mutation similar to T173I, an amino acid mutation similar to P177T, and / or an amino acid mutation similar to V403A, where each amino acid position corresponds to the analogous amino acid position shown in SEQ ID NO:1.

[0228] In other embodiments, the EPSPS polypeptide has an amino acid mutation similar to G172A, an amino acid mutation similar to P177S, an amino acid mutation similar to V403A, or a combination of the following: (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V , (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I and (31) R514S / K, wherein each amino acid mutation position corresponds to the analogous amino acid position set forth in SEQ ID NO:1.

[0229] B. Plants and other host cells of interest Additionally, engineered host cells are provided that are transduced (transformed or transfected) with one or more EPSPS sequences or active variants or fragments thereof. EPSPS polypeptides can be expressed in any organism, including non-animal cells such as plants, yeast, fungi, bacteria, etc.

[0230] Further provided are plants, plant cells, plant parts, seeds and grains having the EPSPS sequences disclosed herein. In specific embodiments, the plants and / or plant parts already have at least one heterologous EPSPS polypeptide or active variant or fragment thereof stably linked thereto as disclosed herein. It should be noted that a plant or organism of interest may contain multiple EPSPS polynucleotides.

[0231] In specific embodiments, the heterologous plant EPSPS polynucleotide in the plant or plant part is operably linked to a heterologous regulatory element, such as, but not limited to, a constitutive, tissue-preferred or other promoter or constitutive enhancer for expression in the plant.

[0232] As used herein, the term "plant" includes plant cells, plant protoplasts, plant cell tissue cultures capable of regenerating plants, plant callus, plant colonies, and intact plant cells of a plant or plant parts, such as embryos, shoots, ovules, seeds, leaves, flowers, branches, kernels, seed pits, ears, stalks, husks, stems, roots, root tips, anthers, etc. Grain refers to mature seeds produced by commercial growers for purposes other than the cultivation or propagation of the seed. Progeny, variants and mutants of regenerated plants are also included within the scope of the present disclosure, provided that these parts contain the introduced polynucleotide.

[0233] The EPSPS sequences disclosed herein and active variants and fragments thereof can be used to transform any plant species, including, but not limited to, monocotyledonous and dicotyledonous plants. Examples of relevant plant species are maize (Zea mays), brassicas (e.g., B. napus, B. rapa, B. juncea), particularly those brassicas used as a source of seed oil, barley (Hordeum vulgare), oats (Avena sativa), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sweet sorghum (Sorghum dochna), foxtail millet (e.g., pearl millet (Pennisetum glaucum), millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), cannabis (Cannabis sativa L.), Kentucky bluegrass (Poa pratensis), tall fescue (Festuca arundinacea), bedweed (Agrostis stolonifera), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), flax (Linum usitatissimum), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), groundnut (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), tomato (Solanum lycopersicum), potato (Solanum tuberosum), apple tree (Malus pumila), Vitis vinifera (Vitis vinifera L.), cucumber (C. sativus), olive (Olea europaea), papaya (Caricapapaya), cashew nut (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), sugarcane (Saccharum spp.), vegetables, ornamentals, conifers, and turfgrasses (including cool season and hot season grasses).

[0234] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), potatoes (Solanum tuberosum), carrots (Daucus carota subsp. Sativus), onions (Allium cepa), and members of the melon genus, such as C. sativus, C. cantalupensis, and C. melo. Ornamental plants include azaleas (Rhododendron spp.), hydrangeas (Macrophylla hydrangea), hibiscus rosasanensis, roses (Rosa spp.), tulips (Tulipas pp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphorbia pulcherrima) and chrysanthemums.

[0235] Other related plants include species of the Poaceae family, such as goosegrass, Aegliops tauschilsubse.Tausch, foxtail millet, sorghum, wheat, ryegrass, wild rice subspecies, wheatgrass, brookweed, corn millet, Dichanthelium oligosanthes, barley, and millet. Other related plants include species of the Asteraceae family, such as Artemisia gracilis, Artemisia anguicida, and Asteraceae. Other related plants include species of the Palm family, such as the Guinea palm and the date palm. Other related plants include plants of the Orchidaceae family, such as Dendrobium nigricans, and Phalaenopsis orchid.

[0236] Other related plants include plants of the Rosaceae family, such as strawberry, Rosa chinensis. Other related plants include cereal plants, oilseed plants and legumes, which provide related seeds. Related seeds include cereal seeds, such as maize, wheat, barley, rice, sorghum and rye. Oilseed plants include cotton, soybean, safflower, sunflower, rapeseed, corn, alfalfa, palm, coconut, etc. Legumes include beans and peas, and legumes include guar beans, locust beans, fenugreek, soybeans, kidney beans, cowpeas, mung beans, lima beans, fava beans, lentils, chickpeas, etc.

[0237] Other host cells of interest may be eukaryotic cells, protoplasts, tissue culture cells, prokaryotic cells and / or bacterial cells, such as E. coli.

[0238] For example, in some embodiments, a glyphosate-tolerant rice plant is provided, the glyphosate-tolerant rice plant expressing an endogenous EPSPS polypeptide comprising V403A, where the amino acid positions correspond to those shown in SEQ ID NO:1. The glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising G172A, P177S and / or V403A, where each amino acid position corresponds to those shown in SEQ ID NO:1. The glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising T173I, P177S and / or V403A, where each amino acid position corresponds to those shown in SEQ ID NO:1. The glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising T173I, P177T and / or V403A, where each amino acid position corresponds to those shown in SEQ ID NO:1. Glyphosate-tolerant rice plants include those that are derived from G172A, P177S, and / or V403A, and are selected from the group consisting of (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) M259L, (19) M162F, (20) M175A, (21) M175A, (22) M208A, (23) M214F, (24) M216V, (25) M175A, (26) M258L, (27) M175A, (28) M175A, (29) M175A, (30) M175A, (31) M175A, (32) M175A, (33) M175A, (34) M175A, (35) M175A, (36) M175A, (37) M175A, (38) M175A, (39) M175A, (40) M175A, (41) M175A, (42) M175A, (43) M175A, (44) M175A, (45) M175A, (46) M175A, (47) M175 and at least one amino acid mutation selected from among 18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I and (31) R514S / K, wherein each amino acid mutation position corresponds to an amino acid position shown in SEQ ID NO:1.

[0239] In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising A70V, G172A, P177S, V403 and / or R514S mutations. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising A73T, G172A, P177S and / or V403A mutations. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising A73T, E86G, G172A, P177S and / or V403A mutations. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising A145G, G172A, P177S, V403A, T442S and / or D472E mutations. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, V403A and / or F502V mutation. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, Y252F and / or V403A mutation. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising an E137D, G172A, P177S, L253M and / or V403A mutation. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, K399N, V403A and / or T466S mutation. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising A73G, L134F, G172A, P177S, G265V, S374R and / or V403A mutations. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising A73K, D114H, V136M, K161E, G172A, P177S, A260T and / or V403A mutations. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising E163K, G172A, P177S, A216V, L253M, H389R and / or V403A mutations.In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, N246D and / or V403A mutation. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising a G172A, M175A, P177S, M258L, K356E, V403A and / or F512I mutation. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising an A73S, G172A, P177S, L214F and / or V403A mutation. In addition, the glyphosate-tolerant rice plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, V208A, L264H, V403A and / or R514K mutation. Additionally, glyphosate-tolerant rice plants can express endogenous EPSPS polypeptides that contain the V113M, G172A, P177S and / or V403A mutations.

[0240] For example, in some embodiments, a glyphosate-tolerant flax plant is provided, the glyphosate-tolerant flax plant expressing an endogenous EPSPS polypeptide comprising V403A, the amino acid positions corresponding to the amino acid positions set forth in SEQ ID NO:1, or the glyphosate-tolerant flax plant may express an endogenous EPSPS polypeptide comprising G172A, P177S and / or V403A, the amino acid positions corresponding to the amino acid positions set forth in SEQ ID NO:1. In addition, glyphosate-resistant flax plants include those that are resistant to G172A, P177S and / or V403A, and (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) M259L, (19) M260L, (20) M270L, (21) M271L, (22) M272L, (23) M275L, (24) M275L, (25) M275L, (26) M275L, (27) M275L, (28) M275L, (29) M280L, (30) M275L, (31) M275L, (32) M275L, (33) M275L, (34) M275L, (35) M275L, (36) M275L, (37) M275L, (38) M275L, (39) M275L, (40) M275L, (41) M275L, (42) M275L, (43) M275L, (44) M275L, (45) M275L, (46) M275L, (47) M275L, (48 and at least one amino acid mutation selected from among 18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I and (31) R514S / K, wherein each amino acid mutation position corresponds to an amino acid position shown in SEQ ID NO:1.

[0241] In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising A70V, G172A, P177S, V403 and / or R514S mutations. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising A73T, G172A, P177S and / or V403A mutations. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising A73T, E86G, G172A, P177S and / or V403A mutations. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising A145G, G172A, P177S, V403A, T442S and / or D472E mutations. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising G172A, P177S, V403A and / or F502V mutations. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising G172A, P177S, Y252F and / or V403A mutations. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising E137D, G172A, P177S, L253M and / or V403A mutations. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising G172A, P177S, K399N, V403A and / or T466S mutations. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising A73G, L134F, G172A, P177S, G265V, S374R and / or V403A mutations. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising A73K, D114H, V136M, K161E, G172A, P177S, A260T and / or V403A mutations. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising E163K, G172A, P177S, A216V, L253M, H389R and / or V403A mutations.In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising the G172A, P177S, N246D and / or V403A mutation. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising the G172A, M175A, P177S, M258L, K356E, V403A and / or F512I mutation. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising the A73S, G172A, P177S, L214F and / or V403A mutation. In addition, the glyphosate-tolerant flax plant can express an endogenous EPSPS polypeptide comprising the G172A, P177S, V208A, L264H, V403A and / or R514K mutation. Additionally, glyphosate-tolerant flax plants can express endogenous EPSPS polypeptides that contain the V113M, G172A, P177S and / or V403A mutations.

[0242] For example, in some embodiments, a glyphosate-tolerant wheat plant is provided that expresses an endogenous EPSPS polypeptide comprising V403A, where the amino acid position corresponds to the amino acid position set forth in SEQ ID NO:1, or can express an endogenous EPSPS polypeptide comprising G172A, P177S, and / or V403A, where the amino acid position corresponds to the amino acid position set forth in SEQ ID NO:1. In addition, glyphosate-tolerant wheat plants include those that are derived from G172A, P177S, and / or V403A, and (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) M259L, (19) M260L, (20) M270L, (21) M271L, (22) M272L, (23) M275L, (24) M275L, (25) M275L, (26) M275L, (27) M275L, (28) M275L, (29) M280L, (30) M281L, (31) M282L, (32) M283L, (33) M284L, (34) M285L, (35) M286L, (36) M287L, (37) M288L, (38) M289L, (39) M290L, (40) M291L, (41) M292L, (42) M293L, (43) M294L, (44) M295L, (45) M295L, (46) M296L, (47) M297L, (4 and at least one amino acid mutation selected from among 18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I and (31) R514S / K, wherein each amino acid mutation position corresponds to an amino acid position shown in SEQ ID NO:1.

[0243] In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising A70V, G172A, P177S, V403A and / or R514S mutations. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising A73T, G172A, P177S and / or V403A mutations. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising A73T, E86G, G172A, P177S and / or V403A mutations. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising A145G, G172A, P177S, V403A, T442S and / or D472E mutations. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, V403A and / or F502V mutation. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, Y252F and / or V403A mutation. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising an E137D, G172A, P177S, L253M and / or V403A mutation. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, K399N, V403A and / or T466S mutation. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising A73G, L134F, G172A, P177S, G265V, S374R and / or V403A mutations. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising A73K, D114H, V136M, K161E, G172A, P177S, A260T and / or V403A mutations. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising E163K, G172A, P177S, A216V, L253M, H389R and / or V403A mutations.In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, N246D and / or V403A mutation. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising a G172A, M175A, P177S, M258L, K356E, V403A and / or F512I mutation. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising an A73S, G172A, P177S, L214F and / or V403A mutation. In addition, the glyphosate-tolerant wheat plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, V208A, L264H, V403A and / or R514K mutation. Additionally, glyphosate-tolerant wheat plants can express endogenous EPSPS polypeptides that contain the V113M, G172A, P177S and / or V403A mutations.

[0244] For example, in some embodiments, glyphosate-tolerant soybean plants are provided that express an endogenous EPSPS polypeptide comprising V403A, where the amino acid positions correspond to the amino acid positions set forth in SEQ ID NO:1, or can express an endogenous EPSPS polypeptide comprising G172A, P177S and / or V403A, where the amino acid positions correspond to the amino acid positions set forth in SEQ ID NO:1. In addition, glyphosate-tolerant soybean plants include those derived from G172A, P177S and / or V403A, and (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) A216V, (15) Y252F, (16) L253M, (17) M258L, (18) M259L, (19) M261E, (20) M270L, (21) M271E, (22) M272F, (23) M273M, (24) M274L, (25) M275L, (26) M275L, (27) M275L, (28) M275L, (29) M275L, (30) M275L, (31) M275L, (32) M275L, (33) M275L, (34) M275L, (35) M275L, (36) M275L, (37) M275L, (38) M275L, (39) M275L, (40) M275L, (41) M275L, (42) M275L, (43) M275L, (44) M275L, (45) M275L, (46) M275L, (47) M275L, (4 and at least one amino acid mutation selected from among 18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I and (31) R514S / K, wherein each amino acid mutation position corresponds to an amino acid position shown in SEQ ID NO:1.

[0245] In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising A70V, G172A, P177S, V403A and / or R514S mutations. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising A73T, G172A, P177S and / or V403A mutations. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising A73T, E86G, G172A, P177S and / or V403A mutations. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising A145G, G172A, P177S, V403A, T442S and / or D472E mutations. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, V403A and / or F502V mutation. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, Y252F and / or V403A mutation. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising an E137D, G172A, P177S, L253M and / or V403A mutation. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, K399N, V403A and / or T466S mutation. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising A73G, L134F, G172A, P177S, G265V, S374R and / or V403A mutations. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising A73K, D114H, V136M, K161E, G172A, P177S, A260T and / or V403A mutations. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising E163K, G172A, P177S, A216V, L253M, H389R and / or V403A mutations.In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, N246D and / or V403A mutation. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising a G172A, M175A, P177S, M258L, K356E, V403A and / or F512I mutation. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising an A73S, G172A, P177S, L214F and / or V403A mutation. In addition, the glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide comprising a G172A, P177S, V208A, L264H, V403A and / or R514K mutation. Additionally, a glyphosate-tolerant soybean plant can express an endogenous EPSPS polypeptide that contains a V113M, G172A, P177S and / or V403A mutation.

[0246] C. Stacking other purpose features In one embodiment, the present invention relates to a transgenic plant having a polynucleotide construct comprising a promoter operably linked to the mutated EPSPS described above, wherein other herbicide tolerance proteins can be inserted into the transgenic plant to generate a multi-herbicide resistant plant.

[0247] As used herein, the term "stacked" includes the presence of multiple traits in the same plant or organism. A trait, as used herein, is a phenotype resulting from a particular sequence or group of sequences. In one embodiment, the molecular stack includes at least one additional polynucleotide that also confers tolerance to at least one sequence that confers glyphosate tolerance by the same and / or different mechanism, and / or at least one additional polynucleotide that confers tolerance to a second herbicide.

[0248] The glyphosate tolerance mechanism provided by the EPSPS sequences disclosed herein can be combined with other herbicide resistance modes to provide host cells, plants, plant explants, and plant cells that are tolerant to glyphosate and one or more other herbicides. For example, the mechanism by which EPSPS confers glyphosate tolerance can be combined with other glyphosate tolerance modes known in the art. In other embodiments, the plant or plant cell or plant part having the EPSPS sequence or its active variant or fragment can be stacked with one or more sequences that confer tolerance to, for example, ALS inhibitors, HPPD inhibitors, 2,4-D, other phenoxy auxin herbicides, aryloxyphenoxypropionic acid herbaceous plants, dicamba, glufosinate herbicides, protease-targeting herbicides, etc.

[0249] The plant or plant cell or plant part carrying the EPSPS sequence can also be combined with at least one other trait to produce a plant containing a desired combination of traits. For example, the plant or plant cell or plant part carrying the EPSPS sequence or an active variant or fragment thereof can be stacked with a polynucleotide encoding a polypeptide having insecticidal and / or pesticidal activity, or the plant or plant cell or plant part carrying the EPSPS sequence can be combined with a plant disease resistance gene.

[0250] These stacked combinations may be produced by any method, including but not limited to, growing plants by any conventional method or genetic transformation.

[0251] 2.How to use A. Methods for Generating Glyphosate-Tolerant Plants The terms "glyphosate tolerance" and "glyphosate resistance" may be used interchangeably herein.

[0252] I. Introduction A variety of methods can be used to introduce a sequence of interest into a host cell, plant, or plant part. The methods of the present disclosure do not depend on a particular method of introducing a sequence into an organism or plant or plant part, but only on the polynucleotide or polypeptide entering at least one cell of the organism or plant. Methods for introducing a polynucleotide or polypeptide into various organisms, including plants, are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0253] In specific embodiments, the EPSPS sequence can be provided to a plant using a number of transient transformation methods, including but not limited to directly introducing the EPSPS protein or active variants and fragments thereof into the plant, including, for example, microinjection or particle bombardment.

[0254] In other embodiments, the EPSPS polynucleotides disclosed herein or active variants thereof may be introduced into a plant by contacting the plant with a virus or viral nucleic acid. Typically, these methods involve incorporating the nucleotide constructs of the invention into a DNA or RNA molecule.

[0255] Methods are known in the art for targeted insertion of a polynucleotide into a specific location in a plant genome. In one embodiment, a site-specific recombination system is used to achieve insertion of the polynucleotide into the desired genomic location.

[0256] The transformed cells may be grown into plants according to conventional methods. These plants may then be grown or pollinated with the same transformant strain or a different strain, and the resulting progeny may be identified as having constitutive expression of the desired phenotypic trait. They may be grown for two or more generations to ensure stable maintenance and inheritance of the expression of the desired phenotypic trait, and then the seeds may be harvested to ensure realization of the expression of the desired phenotypic trait. In this manner, the present invention provides transformed seeds (also referred to as transgenic seeds), the genome of which stably incorporates the polynucleotides disclosed herein, for example as part of an expression cassette.

[0257] Transformed plant cells derived from plant transformation techniques (including those techniques discussed above) may be cultured to regenerate whole plants, carrying the glyphosate or glyphosate analog transformation genotype.

[0258] One skilled in the art will recognize that after an expression cassette containing an EPSPS gene has been incorporated into a transgenic plant and confirmed to be operative, it can be introduced into other plants by sexual crossing. Depending on the species being crossed, any one of a variety of standard breeding techniques can be used.

[0259] In some embodiments, the method comprises introducing in a manner that allows expression in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide comprising V403A, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct. In some embodiments, the method comprises introducing in a manner that allows expression in a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide comprising G172A, P177S and / or V403A, where each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising T173I, P177S and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising T173I, P177T and / or V403A, each amino acid mutation position corresponding to an amino acid position set forth in SEQ ID NO:1, and generating a glyphosate-tolerant plant whose genome comprises the recombinant DNA construct.In some embodiments, the method comprises introducing into a regenerable plant cell in a manner that permits expression of a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, the polynucleotide encoding a plant EPSPS polypeptide, the polypeptide comprising G172A, P177S, and / or V403A, and at least one of the following sequences: (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E16 and at least one amino acid mutation selected from SEQ ID NO:3, (10) M175A, (11) V208A, (12) L214F, (13) A216V, (14) Y252F, (15) L253M, (16) M258L, (17) A260T, (18) L264H, (19) G265V, (20) N316D, (21) K356E, (22) S374R, (23) H389R, (24) K399N, (25) T442S, (26) T466S, (27) D472E, (28) F502V, (29) F512I, and (30) R514S / K, each amino acid mutation position being represented by the sequence of SEQ ID NO:3. corresponding to the amino acid positions set forth in ID NO:1, and generating a glyphosate-tolerant plant whose genome contains the recombinant DNA construct.

[0260] In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising A70V, G172A, P177S, V403 and / or R514S mutations. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising A73T, G172A, P177S and / or V403A mutations. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising A73T, E86G, G172A, P177S and / or V403A mutations. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising an A145G, G172A, P177S, V403A, T442S and / or D472E mutation. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising a G172A, P177S, V403A and / or F502V mutation.In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising a G172A, P177S, Y252F and / or V403A mutation. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising an E137D, G172A, P177S, L253M and / or V403A mutation. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising a G172A, P177S, K399N, V403A and / or T466S mutation. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising A73G, L134F, G172A, P177S, G265V, S374R and / or V403A mutations. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising A73K, D114H, V136M, K161E, G172A, P177S, A260T and / or V403A mutations.In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising an E163K, G172A, P177S, A216V, L253M, H389R and / or V403A mutation. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising an G172A, P177S, N246D and / or V403A mutation. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising a G172A, M175A, P177S, M258L, K356E, V403A and / or F512I mutation. In some embodiments, the method comprises introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising a A73S, G172A, P177S, L214F and / or V403A mutation. In some embodiments, the method includes introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising a G172A, P177S, V208A, L264H, V403A and / or R514K mutation.In some embodiments, the method includes introducing a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence in a manner that allows expression in a regenerable plant cell, the polynucleotide encoding a plant EPSPS polypeptide comprising a V113M, G172A, P177S and / or V403A mutation.

[0261] II. Qualification Generally, methods for modifying or altering host genomic DNA are known and available to those skilled in the art. For example, existing or endogenous EPSPS sequences in a host plant are modified or altered in a site-specific manner using one or more site-specific engineering systems. This includes modifying the host's DNA sequence or existing recombinant sequences, including regulatory elements, coding sequences and non-coding sequences. These methods are also used to target nucleic acids to pre-designed target recognition sequences in the genome. For example, the genetically modified cells or plants described herein are generated using "custom" or engineered endonucleases, such as meganucleases, generated by modifying the plant genome. Another site-directed engineering is to recognize the restriction properties of a binding restriction enzyme using a zinc finger domain. Transcription activator-like (TAL) effector DNA-modifying enzymes (TALEs or TALENs) are also used to engineer plant genomes.

[0262] Site-specific modification of plant genomes may be performed using the CRISPR system (clustered regularly interspaced short palindromic repeats). For example, researchers can use the CRISPR system to generate DNA double-strand breaks (DSBs) at specific sites in the plant genome and generate the disclosed mutations using the error-prone non-homologous end joining (NHEJ) mechanism. Specific mutations may be obtained by precise base replacement under the guidance of a foreign DNA donor (donor). The base editing tools include, but are not limited to, cytosine base editors (CBEs) and / or adenine base editors (ABEs). Alternatively, a primer editing system may be used, where a PBS (primer binding site) sequence in the pegRNA generates these mutations by introducing the mutations into the genome using a reverse transcription template (RT template) containing the target editing sequence.

[0263] An endogenous plant EPSPS gene in a plant cell can be modified to encode a glyphosate-tolerant EPSPS protein, the protein comprising V403A, where each amino acid mutation position corresponds to an amino acid position shown in SEQ ID NO: 1. An endogenous plant EPSPS gene in a plant cell can be modified to encode a glyphosate-tolerant EPSPS protein, the protein comprising G172A, P177S and / or V403A, where each amino acid mutation position corresponds to an amino acid position shown in SEQ ID NO: 1. An endogenous plant EPSPS gene in a plant cell can be modified to encode a glyphosate-tolerant EPSPS protein, the protein comprising T173I, P177S and / or V403A, where each amino acid mutation position corresponds to an amino acid position shown in SEQ ID NO: 1. An endogenous plant EPSPS gene in a plant cell can be modified to encode a glyphosate-tolerant EPSPS protein, which contains T173I, P177T and / or V403A, each amino acid mutation position corresponding to the amino acid position shown in SEQ ID NO:1. An endogenous plant EPSPS gene in a plant cell can be modified to encode a glyphosate-tolerant EPSPS protein, the protein comprising at least one of the following amino acids: G192A, P177S and / or V403A, (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) V208A, (13) L214F, (14) L220F, (15) L214F, (16) L220G, (17) L220H, (18) L220I, (19) L220I, (20) L220I, (21) L220I, (22) L220I, (23) L220I, (24) L220I, (25) L220I, (26) L220I, (27) L220I, (28) L220I, (29) L220I, (30) L220I, (31) L220I, (32) L220I, (33) L220I, (34) L220I, (35) L220I, (36) L220I, (37) L220I, (38) L220I, (39) L220I, (40) L220I, (41) L220I, (42) L220I, (43) L220I, (44) L220I, (45) L220I and at least one amino acid mutation selected from among A216V, (15) Y252F, (16) L253M, (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I and (31) R514S / K, wherein each amino acid mutation position corresponds to an amino acid position set forth in SEQ ID NO:1.

[0264] An endogenous plant EPSPS gene in a plant cell can be modified to encode a glyphosate-tolerant EPSPS protein, which contains an amino acid mutation similar to V403A, where the amino acid mutation positions correspond to the analogous amino acid positions shown in SEQ ID NO: 1. An endogenous plant EPSPS gene in a plant cell can be modified to encode a glyphosate-tolerant EPSPS protein, which contains an amino acid mutation similar to G172A, an amino acid mutation similar to P177S, and / or an amino acid mutation similar to V403A, where each amino acid mutation position corresponds to the analogous amino acid position listed in SEQ ID NO: 1. An endogenous plant EPSPS gene in a plant cell can be modified to encode a glyphosate-tolerant EPSPS protein, the protein comprising an amino acid mutation similar to G172A, an amino acid mutation similar to P177S, and / or an amino acid mutation similar to V403A, and / or an amino acid mutation similar to (1) A70V, (2) A73T / V / S / K / G, (3) V113M, (4) D114H, (5) L134F, (6) V136M, (7) E137D, (8) A145G, (9) K161E, (10) E163K, (11) M175A, (12) (17) M258L, (18) A260T, (19) L264H, (20) G265V, (21) N316D, (22) K356E, (23) S374R, (24) H389R, (25) K399N, (26) T442S, (27) T466S, (28) D472E, (29) F502V, (30) F512I and (31) R514S / K, wherein each amino acid mutation position corresponds to the analogous amino acid position shown in SEQ ID NO:1.

[0265] The endogenous plant EPSPS gene in the plant cell can be modified to encode a glyphosate-tolerant EPSPS protein, the protein comprising A70V, G172A, P177S, V403 and / or R514S mutations.The endogenous plant EPSPS gene in the plant cell can be modified to encode a glyphosate-tolerant EPSPS protein, the protein comprising A73T, G172A, P177S and / or V403A mutations.The endogenous plant EPSPS gene in the plant cell can be modified to encode a glyphosate-tolerant EPSPS protein, the protein comprising A73T, E86G, G172A, P177S and / or V403A mutations. The endogenous plant EPSPS gene in the plant cell can be modified to code for a glyphosate-tolerant EPSPS protein, and the protein comprises A145G, G172A, P177S, V403A, T442S and / or D472E mutation.The endogenous plant EPSPS gene in the plant cell can be modified to code for a glyphosate-tolerant EPSPS protein, and the protein comprises G172A, P177S, V403A and / or F502V mutation.The endogenous plant EPSPS gene in the plant cell can be modified to code for a glyphosate-tolerant EPSPS protein, and the protein comprises G172A, P177S, Y252F and / or V403A mutation. The endogenous plant EPSPS gene in the plant cell can be modified to code for a glyphosate-tolerant EPSPS protein, and the protein comprises E137D, G172A, P177S, L253M and / or V403A mutations.The endogenous plant EPSPS gene in the plant cell can be modified to code for a glyphosate-tolerant EPSPS protein, and the protein comprises G172A, P177S, K399N, V403A and / or T466S mutations.The endogenous plant EPSPS gene in the plant cell can be modified to code for a glyphosate-tolerant EPSPS protein, and the protein comprises A73G, L134F, G172A, P177S, G265V, S374R and / or V403A mutations.The endogenous plant EPSPS gene in the plant cell can be modified to code for a glyphosate-tolerant EPSPS protein, and the protein comprises A73K, D114H, V136M, K161E, G172A, P177S, A260T and / or V403A mutation.The endogenous plant EPSPS gene in the plant cell can be modified to code for a glyphosate-tolerant EPSPS protein, and the protein comprises E163K, G172A, P177S, A216V, L253M, H389R and / or V403A mutation.The endogenous plant EPSPS gene in the plant cell can be modified to code for a glyphosate-tolerant EPSPS protein, and the protein comprises G172A, P177S, N246D and / or V403A mutation. The endogenous plant EPSPS gene in the plant cell can be modified to code for a glyphosate-tolerant EPSPS protein, the protein comprising G172A, M175A, P177S, M258L, K356E, V403A and / or F512I mutation.The endogenous plant EPSPS gene in the plant cell can be modified to code for a glyphosate-tolerant EPSPS protein, the protein comprising A73S, G172A, P177S, L214F and / or V403A mutation.The endogenous plant EPSPS gene in the plant cell can be modified to code for a glyphosate-tolerant EPSPS protein, the protein comprising G172A, P177S, V208A, L264H, V403A and / or R514K mutation. An endogenous plant EPSPS gene in a plant cell can be modified to encode a glyphosate-tolerant EPSPS protein, which contains the V113M, G172A, P177S and / or V403A mutations.

[0266] The endogenous plant EPSPS gene can be modified by a CRISPR / Cas guide RNA-mediated system, a zinc finger nuclease-mediated system, a meganuclease-mediated system, an oligonucleotide base-mediated system, TALEN, or any gene modification system known to one of skill in the art. For purposes of this specification, the endogenous plant EPSPS gene also includes the coding DNA and genomic DNA within and surrounding the coding DNA, such as, for example, promoter, intron, and terminator sequences.

[0267] Working Example In the following examples, it should be understood that these examples are merely illustrative and illustrative of the embodiments of the present invention. Based on the above discussion and these examples, those skilled in the art can make various changes and modifications to the present invention to suit various applications and conditions. Such modifications are also intended to fall within the scope of the accompanying examples.

[0268] Example 1: Alignment of rice EPSPS sequences with other plant EPSPS sequences Based on the rice (Oryza sativa) full-length EPSPS (SEQ ID NO:1), the rice EPSPS (SEQ ID NO:2), flax EPSPS (SEQ ID NO:26), wheat EPSPS (SEQ ID NO:31) and soybean EPSPS (SEQ ID NO:33) were aligned. The alignment results are shown in Figure 1, which shows the described mutation positions and the corresponding positions in each plant species. Based on SEQ ID NO:1, all disclosed mutation positions are marked.

[0269] Example 2: Glyphosate resistance test at mutation site 403 of each rice EPSPS mutant The inventors discovered that V403 on the rice EPSPS sequence plays a role in sensitivity to glyphosate. The inventors discovered whether mutation of the site could reduce the sensitivity of rice EPSPS to glyphosate by performing saturation mutagenesis at position 403 of the native rice EPSPS sequence. The amino acid V at position 403 of the rice EPSPS polypeptide was mutated to other 19 amino acids using NNK (N stands for adenine, thymine, guanine and cytosine, and K stands for thymine and thymine nucleotide) primer as the degenerate codon for the position to be mutated. The construct pKED 2 (SEQ ID NO:2) (FIG. 8) expressing OsWt was used as the template, V403 F61 was used as the forward primer, and V403 R61 was used as the reverse primer to amplify the mutated PCR product. The V403 F61 primer sequence was AATGCCTGATNNKGCCATGACCC. The V403 R61 primer sequence was TTGTTCATGTTGACATCAACAG.

[0270] The experimental steps and results were as follows.

[0271] a) PCR system: According to the mixed PCR system of primer and template corresponding to the target vector in the table above, forward primer was 1.25μl, reverse primer was 1.25μl, template plasmid was 2μl, Q5 PCR master mix was 12.5μl, and ddH2O was 18μl.

[0272] b) The PCR reaction conditions were as follows: [Table 1]

[0273] c) 5 μl of the PCR product was taken and added to the agarose gel electrophoresis buffer. The PCR product was about 4.5 kb in size, indicating that the amplification was successful.

[0274] d) The template in the PCR product was removed using KLD enzyme mix from NEB, and then the product was ligated. The ligation system was 5 μl KLD buffer, 1 μl KLD mix, 1 μl PCR product, and 3 μl ddH2O. After ligation for 5 min at room temperature, the PCR product was transformed into E. coli.

[0275] e) The ligation product was transformed into EPSPS-deficient competent E. coli Dh5α, and an EPSPS-deficient E. coli strain was constructed by the known Red homologous recombination method. (Guangxing Bai, Zhiwei Sun, Ying Huang, et al. Knockout of E. coli ClpP gene using Red recombination system [J]. Chinese Journal of Biochemistry and Molecular Biology (1): 45-48.) The E. coli transformation method and the detection method are both conventional and well-known methods in the field. After resuscitation, the cells were washed twice with M9 medium, and then the washed cells were spread on M9 medium containing 0.2 mM glyphosate and cultured for 48 hours. Each grown single colony was sequenced (sequencing and its analysis were performed by Tsingke Biological Technology Co., Ltd.), and it was found that only the V403A mutant was resistant to 0.2 mM glyphosate.

[0276] Example 3: Comparison of glyphosate resistance in rice EPSPS mutants.

[0277] Example 2 showed that the mutation V403A on rice EPSPS could affect the sensitivity of rice EPSPS to glyphosate. The inventor designed this experiment to test whether any mutation at other positions could also improve the sensitivity of mutant rice EPSPS to glyphosate.

[0278] The detailed experimental steps and test results are as follows:

[0279] [Table 2]

[0280] The specific experimental steps were as follows:

[0281] i. Acquisition and conservation of the required rice EPSPS mutants a) PCR system: According to the mixed PCR system of primer and template corresponding to the target vector in the table above, forward primer was 1.25μl, reverse primer was 1.25μl, template plasmid was 2μl, Q5 PCR master mix was 12.5μl, and ddH2O was 18μl. [Table 3]

[0282] b) The PCR reaction conditions were as follows: The annealing temperature was set based on the annealing temperature corresponding to the target vector in the table above.

[0283] *Annealing temperatures were set according to the table above.

[0284] c) 5 μl of the resulting PCR product was taken and added to the application buffer for agarose gel electrophoresis. A band of approximately 4.5 kb in size was considered successful.

[0285] d) The template was removed and the PCR product was ligated using NEB's KLD enzyme mix. The ligation system was 5 μl KLD buffer, 1 μl KLD mix, 1 μl PCR product and 3 μl ddH2O, ligated for 5 minutes at room temperature, and then transformed into E. coli.

[0286] e) The ligation product was transformed into EPSPS-deficient Dh5α competent E. coli. After resuscitation, the cells were washed twice with M9 medium, and then the washed cells were spread on M9 medium and cultured for 48 hours. The grown single colonies were sequenced (Tsingke Biological Technology Co., Ltd.), and the correct mutants were selected and stored for use.

[0287] The following mutants were selected for testing:

[0288] OsWt represents wild type rice EPSPS (SEQ ID NO:2); OsVA represents mutant rice EPSPS with V403A mutation (SEQ ID NO:3); OsGA represents mutant rice EPSPS with G172A mutation (SEQ ID NO:7); OsPS represents mutant rice EPSPS with P177S mutation (SEQ ID NO:8); OsGAPS represents mutant rice EPSPS with G172A and P177S mutations (SEQ ID NO:9); OsGAVA represents mutant rice EPSPS with G172A and V403A mutations (SEQ ID NO:4); OsPSVA represents mutant rice EPSPS with P177S and V403A mutations (SEQ ID NO:5). OsGPV represents a mutant rice EPSPS with mutations G172A, P177S and V403A (SEQ ID NO:6). OsTIPS represents a mutant rice EPSPS with mutations T173I and P177S (SEQ ID NO:35). OsTIPT represents a mutant rice EPSPS with mutations T173I and P177T (SEQ ID NO:36). OsTIPSVA represents a mutant rice EPSPS with mutations T173I, P177S and V403A (SEQ ID NO:37). OsTIPTVA represents a mutant rice EPSPS with mutations T173I, P177T and V403A (SEQ ID NO:38). cp4 represents a wild type EPSPS from Agrobacterium (SEQ ID NO:39).

[0289] ii: Comparison of glyphosate resistance in rice EPSPS mutants Each product obtained in the above steps was transformed into E. coli lacking EPSPS. The glyphosate resistance of the rice EPSPS mutant was verified by detecting the growth of the transformed E. coli on M9 medium with different glyphosate concentrations (0 mM, 0.5 mM, 2 mM, 4 mM, 5 mM, 25 mM, 75 mM, 100 mM), where 1 mM glyphosate was equivalent to 169 mg / mL glyphosate acid. The results are shown in Figure 2.

[0290] Except for the EPSPS-deficient E. coli including OsGAPS, which could not grow on the medium containing 0 mM glyphosate, all the strains expressing other genes could grow normally. This result indicated that all the other rice EPSPS mutants except for the OsGAPS mutant could be expressed in E. coli and compensated for the function of the original EPSPS in E. coli.

[0291] OsGA, OsPS and OsVA EPSPS mutants could grow on 0.5 mM glyphosate medium, but OsWt could not, indicating that G172A, P177S or V403A single mutation could improve the glyphosate resistance of rice EPSPS mutants, where V403A was a newly discovered mutation that could improve the tolerance of EPSPS to glyphosate.

[0292] Figure 2 shows that OsGAVA can grow on 75 mM glyphosate medium or any medium containing less than 75 mM glyphosate, and OsPSVA can grow on 4 mM glyphosate medium or any medium containing less than 4 mM glyphosate. The combination of the V403A mutation with the G172A or P177S mutation could improve the glyphosate resistance of the rice EPSPS mutant.

[0293] Figure 2 further showed that OsGPV could grow on 100 mM glyphosate medium and the glyphosate resistance was comparable to that of EPSPS derived from CP4. Further mutation of V403A could improve the glyphosate resistance of OsGAVA or OsGAPS.

[0294] 3 further showed that OsTIPTVA could grow on 100 mM glyphosate medium and OsTIPTVA could grow on 75 mM glyphosate medium. Further mutation of V403A could improve the glyphosate resistance of OsTIPS or OsTIPT.

[0295] Example 4: Glyphosate resistance of flax EPSPS mutants i: Obtaining flax EPSPS mutants The flax (Linum usitatissimum) EPSPS mutant sequence (SEQ ID NO:27) was synthesized and cloned into pKED2 vector to obtain vector LuVA. After introducing the A403V mutation by PCR, vector LuWt was constructed.

[0296] The specific experimental steps were as follows: [Table 4]

[0297] a) PCR system: According to the mixed PCR system of primer and template corresponding to the target vector in the table above, forward primer was 1.25μl, reverse primer was 1.25μl, template plasmid was 2μl, Q5 PCR master mix was 12.5μl, and ddH2O was 18μl. [Table 5]

[0298] b) The PCR reaction conditions were as follows: The annealing temperature corresponding to the target vector in the table above was set as the annealing temperature.

[0299] c) 5 μl of the resulting PCR product was taken and added to the application buffer for agarose gel electrophoresis. A band with a size of approximately 4.5 kb was considered successful.

[0300] d) The template was removed using NEB's KLD enzyme mix, followed by ligation of the PCR product. The ligation system was 5 μl KLD buffer, 1 μl KLD mix, 1 μl PCR product and 3 μl ddH2O. After ligation for 5 minutes at room temperature, it was transformed into E. coli.

[0301] e) The ligation product was transformed into EPSPS-deficient Dh5α competent E. coli. After resuscitation, the cells were washed twice with M9 medium, and then the washed cells were spread on M9 medium and cultured for 48 h. The grown single colonies (Tsingke Biological Technology Co., Ltd.) were sequenced, and the correct mutants were selected and stored for use.

[0302] ii: Resistance testing of flax EPSPS mutants and comparison with wild type The resistance results were shown in Figure 4. LuWt (SEQ ID NO:26) represented the wild type flax EPSPS gene, and LuVA (SEQ ID NO:27) represented the V403A single point mutant of flax EPSPS.

[0303] In the presence of 1 mM and 2 mM glyphosate, the growth status of LuVA was significantly better than that of LuWt, indicating that the V403A mutation could improve the glyphosate resistance of flax EPSPS.

[0304] Example 5: Glyphosate resistance of soybean EPSPS mutants i. Obtaining the desired soybean EPSPS mutant Based on the wild-type sequence information of soybean (Glycine max) EPSPS (SEQ ID NO: 36), the gene was synthesized and inserted into vector pKED2 to obtain vector GmWt. The V403 site of GmWt was mutated to A by PCR to obtain vector GmVA, which expresses soybean EPSPS mutant protein containing V403A mutation. The specific experimental steps were as follows: [Table 6]

[0305] a) PCR system: According to the mixed PCR system of primer and template corresponding to the target vector in the table above, forward primer was 1.25μl, reverse primer was 1.25μl, template plasmid was 2μl, Q5 PCR master mix was 12.5μl, and ddH2O was 18μl. [Table 7]

[0306] b) The PCR reaction conditions were as follows: The annealing temperature corresponding to the target vector in the above table was set as the annealing temperature.

[0307] c) 5 μl of the PCR product was removed and added to the application buffer for agarose gel electrophoresis. A band of approximately 4.5 kb in size was considered successful.

[0308] d) The template was removed and the PCR product was ligated using NEB's KLD enzyme mix. The ligation system was 5 μl KLD buffer, 1 μl KLD mix, 1 μl PCR product and 3 μl ddH2O. After ligation for 5 minutes at room temperature, it was transformed into E. coli.

[0309] e) The ligation product was transformed into EPSPS-deficient Dh5α competent E. coli. After resuscitation, the cells were washed twice with M9 medium, and then the washed cells were spread on M9 medium and cultured for 48 h. The grown single colonies (Tsingke Biological Technology Co., Ltd.) were sequenced, and the correct mutants were selected and stored for use.

[0310] ii: Resistance test of soybean EPSPS mutants and comparison with wild type The resistance results are shown in Figure 4. GmWt (SEQ ID NO:33) represents the wild-type soybean EPSPS gene, and GmVA (SEQ ID NO:34) represents the V403A single point mutant of soybean EPSPS.

[0311] Under the pressure of 0.5 mM glyphosate, the growth condition of GmVA was significantly better than that of GmWt, indicating that the V403A mutation could improve the glyphosate resistance of soybean EPSPS.

[0312] Example 6: Glyphosate resistance of wheat EPSPS mutants i: Experimental steps to obtain wheat EPSPS mutants Based on the V403A mutation sequence information (SEQ ID NO: 32) of the EPSPS gene on chromosome 7 of the wheat A genome, the gene was synthesized and inserted into vector pKED2 to obtain a wheat (Triticum vulgare) EPSPS mutation expression vector TaVA containing the V403A mutation. The A403 site of TaVA was mutated to V by PCR to obtain the TaWt vector expressing the EPSPS wild-type gene on chromosome 7 of the wheat A genome.

[0313] The specific experimental steps were as follows: [Table 8]

[0314] a) PCR system: According to the mixed PCR system of primer and template corresponding to the target vector in the table above, the forward primer was 1.25μl, the reverse primer was 1.2μl, the template plasmid was 2μl, the Q5-PCR master mix was 12.5μl, and the ddH2O was 18μl. [Table 9]

[0315] b) The PCR reaction conditions were as follows: The annealing temperature corresponding to the target vector in the above table was set as the annealing temperature.

[0316] c) 5 μl of the resulting PCR product was taken and added to the application buffer for agarose gel electrophoresis. A band with a size of approximately 4.5 kb was considered successful.

[0317] d) The template was removed and the PCR product was ligated using NEB's KLD enzyme mix. The ligation system was 5 μl KLD buffer, 1 μl KLD mix, 1 μl PCR product and 3 μl ddH2O. After ligation for 5 minutes at room temperature, it was transformed into E. coli.

[0318] e) The ligation product was transformed into EPSPS-deficient Dh5α competent E. coli. After resuscitation, the cells were washed twice with M9 medium, and then the washed cells were spread on M9 medium and cultured for 48 h. The grown single colonies (Tsingke Biological Technology Co., Ltd.) were sequenced, and the correct mutants were selected and stored for use.

[0319] ii: Resistance measurement of wheat EPSPS mutants and their comparison with the wild type The resistance results are shown in Figure 4. TaWt (SEQ ID NO:31) represents the wild-type EPSPS gene on chromosome 7 of the wheat A genome, and TaVA (SEQ ID NO:32) represents the V403A single point mutant of wheat EPSPS.

[0320] Under the pressure of 0.2 mM glyphosate, the growth condition of TaVA was significantly better than that of TaWt, indicating that the V403A mutation could improve the glyphosate resistance of wheat EPSPS.

[0321] Example 7: Resistance identification of flax EPSPS mutants LuGPV, LuTIPT, and LuTIPTVA The G172 and P177 positions of LuVA were simultaneously mutated to A and S by PCR to obtain a three-point mutant LuGPV.

[0322] The T173 and P177 positions of LuWT were simultaneously mutated to I and T by PCR to obtain the triple point mutant LuTIPT.

[0323] The T173 and P177 positions of LuVA were simultaneously mutated to I and T by PCR, resulting in a triple point mutant LuTIPTVA.

[0324] The specific experimental steps were as follows: [Table 10]

[0325] a) PCR system: According to the mixed PCR system of primer and template corresponding to the target vector in the table above, forward primer was 1.25μl, reverse primer was 1.25μl, template plasmid was 2μl, Q5 PCR master mix was 12.5μl, and ddH2O was 18μl. [Table 11]

[0326] b) The PCR reaction conditions were as follows: The annealing temperature corresponding to the target vector in the table above was set as the annealing temperature.

[0327] c) 5 μl of the resulting PCR product was taken and added to the application buffer for agarose gel electrophoresis. A band with a size of approximately 4.5 kb was considered successful.

[0328] d) The template was removed and the PCR product was ligated using NEB's KLD enzyme mix. The ligation system was 5 μl KLD buffer, 1 μl KLD mix, 1 μl PCR product and 3 μl ddH2O, and the ligation was carried out at room temperature for 1 min, followed by transformation into E. coli.

[0329] e) The ligation product was transformed into EPSPS-deficient Dh5α competent E. coli. After resuscitation, the cells were washed twice with M9 medium, and then the washed cells were spread on M9 medium and cultured for 48 h. The grown single colonies (Tsingke Biological Technology Co., Ltd.) were sequenced, and the correct mutants were selected and stored for use.

[0330] ii: Resistance testing of flax EPSPS mutants and comparison with the wild type The resistance results were shown in Figure 5. LuWt (SEQ ID NO:26) represented flax EPSPS2 gene, and LuGPV (SEQ ID NO:30) represented flax G172A+P177S+V403A triple point mutant. The resistance results were shown in Figure 6. LuTIPT (SEQ ID NO:28) was flax T173I+P177S double point mutant, and LuTIPTVA (SEQ ID NO:29) was flax T173I+P177T+V403A triple point mutant.

[0331] The results showed that flax GPV mutants had relatively strong glyphosate resistance. Based on the colony saturation rate at 100 mM glyphosate pressure, the resistance of LuGPV was higher than that of CP4, the resistance of LuTIPTVA was higher than that of LuTIPT, and V403A could also significantly improve the resistance level of mutants in flax EPSPS gene.

[0332] Example 7: Resistance testing of other rice mutants To further improve the glyphosate resistance of rice and breed highly resistant rice EPSPS mutants that can meet commercial production requirements, we performed random mutations using OsGPV as a template and high-concentration glyphosate screening to identify EPSPS mutants that can enhance glyphosate resistance.

[0333] i. Obtaining rice mutants 1) Error-prone PCR Error-prone PCR was performed using OsGPV (SEQ ID NO:6) as a template (GeneMorph II Random Mutation Kit, #200550, Agilent Technologies).

[0334] The reaction conditions were as follows: [Table 12]

[0335] The primer sequences were as follows:

[0336] EP OsF:tgtgaattcatggcggcgaaggcggaggatc EP OsR:gatcctgcaggtcagttcctgacgaaagtgc

[0337] The PCR reaction steps were as follows. [Table 13]

[0338] 2) Recovery and purification of the resulting PCR products 3) Digestion of the harvested product and vector backbone a) The recovered product was digested with EcoRI and SbfI, and then ligated into pKED2 vector after the same double digestion with T4 DNA ligase to obtain the ligation product, thereby obtaining a vector carrying the rice EPSPS gene mutant.

[0339] b) The above vector was transformed into E. coli DH5α with EPSPS gene knocked out. The transformation steps were as follows. 50 μl of the above ligation product was added to E. coli DH5α competent cells with EPSPS gene knocked out, mixed evenly, incubated on ice for 30 minutes, then heat-shocked at 42°C for 90 seconds, then removed and immediately placed on ice for 2 minutes ice bath. After the ice bath, 500 μl of LB liquid medium was added and revived at 37°C, 180r / min for 45 minutes. The transformed E. coli was a mutant library expressing rice EPSPS mutant genes obtained using OsGPV as a template.

[0340] 4) Resistance screening The transformation product obtained above was centrifuged at 3000 rpm for 5 minutes, the supernatant was discarded, and then resuspended in 1 ml of M9 liquid medium, resuspended by adding 200 μl of M9 liquid medium, and inoculated onto an M9 plate containing 75 mM glyphosate. Incubated at 37 degrees for 24 hours.

[0341] 5) Single colonies that grew on the above screening plates were selected and stored for further resistance identification and sequencing.

[0342] ii. Resistance test of rice mutants. In this example, the wild-type rice EPSPS gene OsWT obtained in this study, the most widely used commercial glyphosate resistance gene CP4EPSPS, the rice mutant OsTIPS with the same mutation type as the commercial glyphosate-resistant maize GA21, and the rice mutant OsGPV with a highly glyphosate-resistant rice EPSPS mutant gene were transformed as controls, and the newly obtained rice EPSPS mutant was transformed as an experimental group, and the glyphosate resistance of the control and mutants was compared. The monoclonal bacteria of the control group and the experimental group were inoculated into media containing 0, 100 mM and 125 mM glyphosate, respectively, and the growth status of the colonies of each group was observed.

[0343] Sample order (left to right): 1st row: 1-8,1-13,1-19,1-24,1-27,1-37,1-38 2nd row: 1-42, 1-68, 1-88, 1-93, 1-98, 1-125, 1-139 3rd row: 1-193,1-150,1-217,1-246,1-250,1-265,1-386 4th line: 1-411,1-547,1-552,1-599,1-614,1-631,1-130 5th row: 2-5, 2-43, 2-45, 2-46, 2-47, 2-63, 2-121 6th row: 2-114, 2-133, 3-8, 3-25, 3-27, 3-30, 3-32 Row 7:3-33,OsTIPS,3-36,CP4,Os Wt,Os GPV

[0344] In the above results, except for 3-32 and OsTIPS, which did not grow under the pressure of 100 mM glyphosate, all other experimental and control samples could tolerate / resist glyphosate, but the colony growth status was not consistent. Based on the colony growth status under 125 mM glyphosate pressure, 16 mutants (1-68 (SEQ ID NO:10), 1-98 (SEQ ID NO:11), 1-139 (SEQ ID NO:12), 1-246 (SEQ ID NO:13), 1-386 (SEQ ID NO:14), 1-411 (SEQ ID NO:15), 1-547 (SEQ ID NO:16), 1-552 (SEQ ID NO:17), 1-599 (SEQ ID NO:18), (SEQ ID NO:19), 2-5 (SEQ ID NO:20), 2-46 (SEQ ID NO:21), 2-47 (SEQ ID NO:22), (SEQ ID NO:23), 3-33 (SEQ ID NO:24), 3-36 (SEQ ID NO:25), 3-40 (SEQ ID NO:26), 3-42 (SEQ ID NO:27), 3-46 (SEQ ID NO:28), 3-48 (SEQ ID NO:29), 3-50 (SEQ ID NO:30), 3-51 (SEQ ID NO:31), 3-52 (SEQ ID NO:32), 3-53 (SEQ ID NO:33), 3-54 (SEQ ID NO:34), 3-55 (SEQ ID NO:35), 3-56 (SEQ ID NO:36), 3-57 (SEQ ID NO:37), 3-59 (SEQ ID NO:38), 3-60 (SEQ ID NO:39), 3-61 (SEQ ID NO:40), 3-62 (SEQ ID NO:41), 3-63 (SEQ ID NO:42), 3-64 (SEQ ID NO:43), 3-65 (SEQ ID NO:44), The colony growth status of NO:25) was significantly better than OsGPV, and the resistance of some mutants was significantly higher than CP4, indicating improved resistance. The colony growth status of the other 16 mutants showed lower resistance than OsGPV. In Figure 7, there was no significant difference in the resistance of the other 12 mutants to OsGPV.

[0345] Example 8: Resistance of transgenic rice to glyphosate herbicide

[0346] The rice seeds used in this example were the publicly available RICE KENG XIANG DAO08-169.

[0347] 1. Obtaining an expression vector.

[0348] Based on conventional methods, the nucleic acid sequences encoding OsGPV mutants (including G172A, P177S and V403A) (SEQ ID NO:6) were inserted into a plant expression vector to obtain the pk510 vector expressing OsGPV genes (see FIG. 9).

[0349] 2. Genetic modification experiments in rice callus.

[0350] 1) The recombinant vector pk510 was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

[0351] 2) One to five days before the transformation experiment, the recombinant Agrobacterium was inoculated onto YEB solid medium containing 50 mg / L kanamycin and 15 mg / L rifampicin. The medium was sealed in a culture dish with 3M tape, and the culture dish was inverted and cultured at 28°C in the dark for one to five days.

[0352] 3) The Z-shaped tails of the bacteria were scraped off with an inoculation ring and lightly suspended in 100 μm NB liquid medium containing 2 mg / L 2,4-dichlorophenoxyacetic acid to a bacterial liquid concentration of about OD600=0.1, and then the prepared Agrobacterium infection solution was stored.

[0353] 4) After removing the rice seed coat, the rice seeds were placed in an Erlenmeyer flask and washed three times with sterile water. 75% ethanol was added to soak the seeds, gently shaken for 1 minute, and the ethanol was discarded. Then, 2.5% aqueous sodium hydrochloride solution was added, and the solution was shaken at 150-170 rpm for 25 minutes. Then, sterile water was added, gently shaken 5-8 times, and then the water was poured off. The seeds were inoculated into a callus induction medium and cultured at 30°C in the dark for 4-6 weeks to obtain rice callus.

[0354] 5) The rice callus obtained in step 4 was immersed in the Agrobacterium infection solution for 10 minutes, then placed on a co-culture medium covered with two sheets of sterile filter paper, and cultured at 22°C in the dark for 3 days.

[0355] 6) The rice callus obtained in step 5 was placed on resuscitation medium and cultured at 30°C for 4-7 days.

[0356] 7) The rice callus obtained in step 6 was placed on hygromycin selection medium and cultured at 30°C for 2 weeks.

[0357] 8) The rice callus obtained in step 7 was placed on hygromycin selection medium and cultured at 30°C for 2 weeks.

[0358] 9) The vigorous growing calli were transferred to differentiation medium components and cultured at 30°C for 20-30 days. Plants were generated from the yellow and green calli.

[0359] 10) Healthy green regenerated seedlings greater than 3 cm in height were selected, transferred to rooting medium, and cultured for 7-14 days to obtain rice T0 seedlings.

[0360] The medium components were as follows:

[0361] Co-medium components: NB solid medium containing 2 mg / L 2,4-dichlorophenoxyacetic acid Resuscitation medium components: NB solid medium containing 200mg / L timentin Hygromycin selective medium components: NB solid medium containing 30-50mg / L hygromycin Differentiation medium components: NB solid medium containing 0.5 g / L glutamine, 0.5 g / L proline, 2 mg / L kanamycin, and 0.2 mg / L α-naphthaleneacetic acid. Rooting medium components: MS solid medium containing 0.5 g / L glutamine, 0.5 g / L proline, and 0.2 mg / L α-naphthaleneacetic acid.

[0362] The transgenic rice T0 seedlings were cultured in an artificial climate chamber until harvest, and T1 generation rice seeds were obtained for glyphosate resistance testing.

[0363] 2. Identification of glyphosate resistance in T1 transgenic rice

[0364] The transgenic rice T0 seedlings were cultured in an artificial climate chamber until harvest, and T1 generation rice seeds were obtained for glyphosate resistance testing.

[0365] 1) We selected T1 generation seeds that were positive for genetic recombination under ultraviolet light. The plant expression vector was constructed by expressing OsGPV and the DsRed gene, which can express red fluorescent protein, in series, so that the T1 generation seeds could be differentiated by exposing them to ultraviolet light. The seeds under red light were the seeds that contained both the DsRed and OsGPV vectors.

[0366] 2) Germination promotion: 20 seeds each from non-transgenic control seeds and T1 transgenic seeds containing OsGPV gene were collected and soaked in purified water for 4 hours to allow the seeds to fully absorb water. The seeds were then transferred to a culture dish with two layers of filter paper at the bottom and a small amount of purified water was added to keep them moist. The amount of water should not overfill the seeds.

[0367] 3) After culturing for 2 days according to the above method, the seeds were transferred to a culture dish (diameter 9 cm) with two layers of filter paper at the bottom, and 5 ml of 100 ppm glyphosate aqueous solution was added (Round up, diluted 10,000 times by adding 41% isopropylamine salt solution) and cultivated for 10 days.

[0368] 4) Ten rice seedlings were randomly selected and photographed, and the photographs are shown in Figure 10.

[0369] Figure 10 shows that the growth of the control wild-type rice in the 100 ppm glyphosate solution was significantly inhibited, and the root length was also significantly inhibited. Subsequent observations showed that the wild-type rice plants were all dead and had rotten roots. The transgenic rice plants containing the OsGPV mutant were not significantly affected by the glyphosate herbicide and continued to grow, indicating that the rice line containing the OsGPV mutant is tolerant / resistant to glyphosate.

[0370] FIG. 1 shows a multiple alignment between SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:31 and SEQ ID NO:33.

[0371] FIG. 2 shows the growth of E. coli cells expressing rice EPSPS protein with G172A, P177S and / or V403A mutations and combination mutations thereof in media with different glyphosate concentrations.

[0372] FIG. 3 shows the growth of E. coli cells expressing rice EPSPS proteins with T173I, P177S / T and / or V403A mutations and combination mutations thereof in media with different glyphosate concentrations.

[0373] FIG. 4 shows the growth status of E. coli cells expressing flax or wheat or soybean EPSPS proteins with the V403A mutation in media with different glyphosate concentrations.

[0374] FIG. 5 shows a comparison of the growth conditions of E. coli cells expressing flax EPSPS proteins having the G172A, P177S and V403A mutations and E. coli cells expressing CP4 EPSPS protein in media with different glyphosate concentrations.

[0375] FIG. 6 shows the growth status of E. coli cells expressing flax EPSPS proteins with G172A, T173I, P177T / S and / or V403A mutations in media with different glyphosate concentrations.

[0376] FIG. 7 shows a comparison of glyphosate resistance between E. coli cells expressing rice EPSPS protein and several mutants at a herbicide concentration of 125 ml glyphosate.

[0377] FIG. 8 shows the construct pKED2 vector.

[0378] FIG. 9 shows a pk510 vector comprising one embodiment of the present invention.

[0379] FIG. 10 shows a comparison between wild type and transgenic rice plants and their glyphosate tolerance capabilities.

[0380] SEQ ID NO:1 is the full-length amino acid sequence of the wild-type rice EPSPS protein (GenBank accession number XP_015643046.1), the N-terminus of which contains the chloroplast transit peptide. SEQ ID NO:1 is used herein as the reference EPSPS sequence, and all mutation positions are marked according to SEQ ID NO:1.

[0381] maatmasnaaaaaavsldqavaasaafssrkqIrlpaaarggmrvrvrargrreavvvasassssvaapaakaeeivlqpireisgavqlpgskslsnrilllsalsegttv v dnllnsedvhymlealkalglsveadkvakravv v gcggkfpvekdakeevq l flgnagtamrpltaavtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcflgtecppvrvkgigglpggkvklsgsisqylsallmaaplalgdveieiidklisipyvemt l rlmerfgvkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdvamtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyci i tppeklnitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0382] SEQ ID NO:2 is the amino acid sequence of the wild-type rice EPSPS protein. Compared to SEQ ID NO:1, the chloroplast transit peptide has been deleted from SEQ ID NO:2.

[0383] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttvvdnlInsedvhymlealkalglsveadkvakravvvgcggkfpvekdakeevqlflgnagtamrplta avtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcflgtecppvrvkgigglpggkvklsgissqylsallmaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdva mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppeklnitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0384] SEQ ID NO:3 is a mutant EPSPS sequence from rice and contains the V403A mutation.

[0385] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttvvdnlInsedvhymlealkalglsveadkvakravyvgcggkfpvekdakeevqlflgngatamrplta avtaaggnatyvldgvprmrerpigdlyvglkqlgadvdcflgtecppvrvkgigglpggkvklsgsissqylsallmaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppeklnitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0386] SEQ ID NO:4 is a mutant EPSPS sequence from rice and contains the G172A and V403A mutations.

[0387] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnllinsedvhymlealkalglsveadkvakrawwvgcggkfpvekdakeevqlflgnaatamrplt aavtaaggnatyvldgvprmrerpigdlvvglkqlgadvdeflgtecppvrvkgigglpggkvklsgissqylsallmaaplalgdveieiidklisipyvemtirlmerf gvkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpda amtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppeklnitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0388] SEQ ID NO:5 is a mutant EPSPS sequence from rice and contains the P177S and V403A mutations.

[0389] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnllnsedvhymlealkalglsveadkvakrawwvgcggkfpvekdakeevqlflgngatamrslta avtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcfigtecppvrvkgigglpggkvklsgissqylsallmaaplalgdveieiidklisipyvemtIrlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0390] SEQ ID NO:6 is a mutant EPSPS sequence from rice and contains the G172A, P177S or V403A mutations.

[0391] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttvvdnlInsedvhymlealkalglsveadkvakravyvgcggkfpvekdakeevqlflgnaatamrslta avtaaggnatyvldgvprmrerpigdlwvglkqlgadvdcfilgtecppvrvkgigglpggkvklsgsissqylsallmaaplalgdveieiidklisipyvemtirlmerf gvkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpda amtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekinitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0392] SEQ ID NO:7 is a mutant EPSPS sequence from rice and contains a G172A mutation.

[0393] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnllnsedvhymlealkalglsveadkvakrawwvgcggkfpvekdakeevqlflgnaatamrplta avtaaggnatyvldgvprmrerpigdlyvglkqlgadvdcfilgtecppvrvkgigglpggkvklsgsissqylsallmaaplalgdveieiidklisipyvemtirlmerf gvkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdv amtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppeklnitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0394] SEQ ID NO:8 is a mutant EPSPS sequence from rice and contains the P177S mutation.

[0395] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnllnsedvhymlealkalglsveadkvakrawwvgcggkfpvekdakeevqlflgnagtamrslta avtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcfigtecppvrvkgigglpggkvklsgissqylsallmaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdva mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0396] SEQ ID NO:9 is a mutant EPSPS sequence from rice and contains the G172A and P177S mutations.

[0397] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnllinsedvhymlealkalglsveadkvakrawwvgcggkfpvekdakeevqlflgnaatamrslt aavtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcflgtecppvrvkgigglpggkvklsgissqylsallmaaplalgdveieiidklisipyvemtirlmerf gvkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdv amtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppeklnitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0398] SEQ ID NO:10 is a mutant EPSPS sequence from rice and contains the A70V, G172A, P177S, V403A and R514S mutations.

[0399] mvakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnllnsedvhymlealkalglsveadkvakravwvvgcggkfpvekdakeevqlflgnaatamrslt aavtaaggnatyvldgvprmrerpigdlwvglkqlgadvdcfilgtecppvrvkgigglpggkvklsgissqylsallmaaplalgdveieiidklisipyvemtirlmerf gvkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpda amtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvsn*

[0400] SEQ ID NO:11 is a mutant EPSPS sequence from rice and contains the A73T, G172A, P177S and V403A mutations.

[0401] maakteeivlqpireisgavqlpgskskslsnrillsalsegttuwvdnlInsedvhymlealkalglsveadkvakrawwvgcggkfpvekdakeevqlflgnaatamrslta avtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcflgtecppvrvkgigglpggkvklsgsissqylsallmaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0402] SEQ ID NO:12 is a mutant EPSPS sequence from rice and contains the A73T, E86G, G172A, P177S and V403A mutations.

[0403] maakteeivlqpireisgavqlpgskslsnrillsalsegttwdnlInsedvhymlealkalglsveadkvakrawwvgcggkfpvgkdakeevqlflgnaatamrsltaa vtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcflgtecppvrvkgigglpggkvklsgissqylsalImaaplalgdveieidklisipyvemtirlmerfgv kaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0404] SEQ ID NO:13 is a mutant EPSPS sequence from rice and contains the A145G, G172A, P177S, V403A, T442S and D472E mutations.

[0405] maakaeeivlqpireisgavqlpgskskslsnrilllsalsegttyvdnllinsedvhymlealkalglsveadkvakrgwyvgcggkfpvekdakeevqlflgnaatamrslt aavtaaggnatyvldgvprmrerpigdlvglkqlgadvdcflgtecppvrvkgigglpggkvklsgissqylsallmaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairtelsklgasveegpdyciitppekInitaidtyedhrmamafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0406] SEQ ID NO:14 is a mutant EPSPS sequence from rice and contains G172A, P177S, V403A and F502V mutations.

[0407] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttvvdnlInsedvhymlealkalglsveadkvakravvvgcggkfpvekdakeevqlfignaatamrslta avtaaggnatyvldgvprmrerpigdlyvglkqlgadvdcflgtecppvrvkgigglpggkvklsgsissqylsallmaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktvpnyfdvlstfvrn*

[0408] SEQ ID NO:15 is a mutant EPSPS sequence from rice and contains the G172A, P177S, Y252F and V403A mutations.

[0409] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnllinsedvhymlealkalglsveadkvakrawwvgcggkfpvekdakeevqlflgnaatamrslt aavtaaggnatyvldgvprmrerpigdlwvglkqlgadvdcfilgtecppvrvkgigglpggkvklsgissqflsallmaaplalgdveieidklisipyvemtirlmerf gvkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpda amtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0410] SEQ ID NO:16 is a mutant EPSPS sequence from rice and contains the E137D, G172A, P177S, L253M and V403A mutations.

[0411] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnllnsedvhymlealkalglsvdadkvakravvgcggkfpvekdakeevqlflgnaatamrslta avtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcflgtecppvrvkgigglpggkvklsgissqymsallmaaplalgdveieiidklisipyvemtirlmerfg yvkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpda amtlavvalfadgptairdvaswrvketermvairteltklgasveegpdycitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0412] SEQ ID NO:17 is a mutant EPSPS sequence from rice and contains the G172A, P177S, K399N, V403A and T466S mutations.

[0413] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnllnsedvhymlealkalglsveadkvakrawwvgcggkfpvekdakeevqlflgnaatamrslt aavtaaggnatyvldgvprmrerpigdlwvglkqlgadvdcflgtecppvrvkgigglpggkvklsgissqylsallmaaplalgdveieiidklisipyvemtirlmerf gvkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnnmpda amtlavvalfadgptairdvaswrvketermvairteltkigasveegpdycitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0414] SEQ ID NO:18 is a mutant EPSPS sequence from rice and contains A73G, L134G, G172A, P177S, G265V, S374R and V403A mutations.

[0415] maakgeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnlInsedvhymlealkalgfsveadkvakravwvgcggkfpvekdakeevqlflgnaatamrslt aavtaaggnatyvldgvprmrerpigdlvglkqlgadvdcflgtecppvrvkgigglpggkvklsgissqylsallmaaplalvdveieidklisipyvemtIrlmerfgv kaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtrvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppeklnitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0416] SEQ ID NO:19 is a mutant EPSPS sequence from rice and contains the A73K, D114H, V136M, K161E, G172A, P177S, A260T and V403A mutations.

[0417] maakkeeivlqpireisgavqlpgskslsnrilllsalsegttyvhnlInsedvhymlealkalglsmeadkvakravwvgcggkfpvekdaeeevqlflgnaatamrslta avtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcflgtecppvrvkgigglpggkvklsgsissqylsallmatplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0418] SEQ ID NO:20 is a mutant EPSPS sequence from rice and contains E163K, G172A, P177S, A216V, L253M, H389R and V403A mutations.

[0419] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttvvdnlInsedvhymlealkalglsveadkvakravvvgcggkfpvekdakekvqlflgnaatamrslta avtaaggnatyvldgvprmrerpigdlvvglkqlgvdvdcflgtecppvrvkgigglpggkvklsgissqymsallmaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkrlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0420] SEQ ID NO:21 is a mutant EPSPS sequence from rice and contains the G172A, P177S, N246D and V403A mutations.

[0421] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttvvdnlnsedvhymlealkalglsveadkvakravyvgcggkfpvekdakeevqlflgnaatamrslta avtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcflgtecppvrvkgigglpggkvklsgsissqylsallmaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgdayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppeklnitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0422] SEQ ID NO:22 is a mutant EPSPS sequence from rice and contains G172A, M175A, P177S, M258L, K356E, V403A and F512I mutations.

[0423] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnllnsedvhymlealkalglsveadkvakrawwvgcggkfpvekdakeevqlflgnaataarslta avtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcflgtecppvrvkgigglpggkvklsgsissqylsalllaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvefaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstivrn*

[0424] SEQ ID NO:23 is a mutant EPSPS sequence from rice and contains the A73S, G172A, P177S, L214F and V403A mutations.

[0425] maakseeivlqpireisgavqlpgskslsnrillsalsegttvvdnlInsedvhymlealkalglsveadkvakravwvgcggkfpvekdakeevqlflgnaatamrslta avtaaggnatyvldgvprmrerpigdlwvglkqfgadvdcfigtecppvrvkgigglpggkvklsgissqylsallmaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0426] SEQ ID NO:24 is a mutant EPSPS sequence from rice and contains G172A, P177S, V208A, L264H, V403A and R514K mutations.

[0427] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnllinsedvhymlealkalglsveadkvakrawwvgcggkfpvekdakeevqlflgnaatamrslt aavtaaggnatyvldgvprmrerpigdlavglkqlgadvdcfigtecppvrvkgigglpggkvklsgissqylsallmaaplahgdveieiidklisipyvemtIrlmerf gvkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpda amtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvkn*

[0428] SEQ ID NO:25 is a mutant EPSPS sequence from rice and contains the V113M, G172A, P177S and V403A mutations.

[0429] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttvmdnlInsedvhymlealkalglsveadkvakravyvgcggkfpvekdakeevqlflgnaatamrslta avtaaggnatyvldgvprmrerpigdluvglkqlgadvdcflgtecppvrvkgigglpggkvklsgsissqylsallmaaplalgdveieiidklisipyvemtIrlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdykfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppeklnitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0430] SEQ ID NO:26 is the amino acid sequence of wild-type flax EPSPS2 and was translated from Lus10000788 (Sauer NJ, Narvaez-Vasquez, Javier, Mozoruk J, et al. Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants[J]. Plant Physiology, 2016:1917- 1928.), with the chloroplast transit peptide removed from SEQ ID NO:26.

[0431] mtvpeeivlqpikdisgivtlpgskslsnrillaalsegktyvdnlInsddvhymlgalktlgInvehsseqkraivegrggvfpvgklgkndielflgnagtamrpltaa vtaaggnssyildgvprmrerpigdlvvglkqlgadvscsstscppvhvnakgglpggkvklsgissqyltallmaaplalgdveieivdklisvpyvdmtlklmerfgv avehsgswdrffvkggqkykspgnayvegdassasyflagaaitggtitvegcgtssIqgdvkfaevlekmgakvtwtetsvtvtgpprdasgkkhlravdvnmnkmpdva mtlavvalyadgptairdvaswrvketermiavctelrklgatveegpdyciitppeklsiaeidtyddhrmafslaacadvpvtirdpgctkktfpdyfevlerytkh*

[0432] SEQ ID NO:27 is a mutant of the EPSPS sequence from flax and contains the V403A mutation.

[0433] mtvpeeivlqpikdisgivtlpgskslsnrillaalsegktyvdnlInsddvhymlgalktlgInvehsseqkraivegrggvfpvgklgkndielflgnagtamrpltaa vtaaggnssyildgvprmrerpigdlvvglkqlgadvscsstscppvhvnakgglpggkvklsgissqyltallmaaplalgdveieivdklisvpyvdmtlklmerfgv avehsgswdrffvkggqkykspgnayvegdassasyflagaaitggtitvegcgtssIqgdvkfaevlekmgakvtwtetsvtvtgpprdasgkkhlravdvnmnkmpdaa mtlavvalyadgptairdvaswrvketermiavctelrklgatveegpdyciitppeklsiaeidtyddhrmafslaacadvpvtirdpgctkktfpdyfevlerytkh*

[0434] SEQ ID NO:28 is a mutant EPSPS sequence from flax and contains T173I and P177T mutations.

[0435] mtvpeeivlqpikdisgivtlpgskslsnrilllaalsegktvvdnlInsddvhymlgalktlgInvehsseqkraivegrggvfpvgklgkndielflgnagiamrtlta avtaaggnssyildgvprmrerpigdlvvglkqlgadvscsstscppvhvnakgglpggkvklsgsissqyltallmaaplalgdveieivdklisvpyvdmtiklmerfgv avehsgswdrffvkggqkykspgnayvegdassasyflagaaitggtitvegcgtssIqgdvkfaevlekmgakvtwtetsvtvtgpprdasgkkhlravdvnmnkmpdva mtlavvalyadgptairdvaswrvketermiavctelrklgatveegpdyciitppeklsiaeidtyddhrmafslaacadvpvtirdpgctkktfpdyfevlerytkh*

[0436] SEQ ID NO:29 is a mutant EPSPS sequence from flax and contains T173I, P177T and V403A mutations.

[0437] mtvpeeivlqpikdisgivtlpgskslsnrillaalsegktvvdnllnsddvhymlgalktlgInvehsseqkraivegrggvfpvgklgkndielflgnagiamrtltaa vtaaggnssyildgvprmrerpigdlvvglkqlgadvscsstscppvhvnakgglpggkvklsgsisqyltallmaaplalgdveieivdklisvpyvdmtikImerfgv avehsgswdrffvkggqkykspgnayvegdassasyflagaaitggtitvegcgtsslqgdvkfaevlekmgakvtwtetsvtvtgpprdasgkkhlravdvnmnkmpdaa mtlavvalyadgptairdvaswrvketermiavctelrklgatveegpdyciitppeklsiaeidtyddhrmafslaacadvpvtirdpgctkktfpdyfevlerytkh*

[0438] SEQ ID NO:30 is a mutant EPSPS sequence from flax and contains the G172A, P177S and V403A mutations.

[0439] mtvpeeivlqpikdisgivtlpgskslsnrillaalsegktvvdnlInsddvhymlgalktlgInvehsseqkraivegrggvfpvgklgkndielflgnaatamrsltaa vtaaggnssyildgvprmrerpigdlvvglkqlgadvscsstscppvhvnakgglpggkvklsgissqyltallmaaplalgdveieivdklisvpyvdmtiklmerfgv avehsgswdrffvkggqkykspgnayvegdassasyflagaaitggtitvegcgtssIqgdvkfaevlekmgakvtwtetsvtvtgpprdasgkkhiravdvnmnkmpdaa mtlavvalyadgptairdvaswrvketermiavctelrklgatveegpdycitppeklsiaeidtyddhrmafslaacadvpvtirdpgctkktfpdyfevlerytkh*

[0440] SEQ ID NO:31 is the amino acid sequence of the wild-type wheat EPSPS protein, represented as GenBank entry ALK27163.1, with the chloroplast transit peptide removed from SEQ ID NO:31.

[0441] magaeevwlqpireisgavqlpgskslsnrillsalsegttuvdnllnsedvhymlealealglsveadkvakrawwgcggrfpvekdakeevklfignagtamrpltaav vaaggnatyvldgvprmrerpigdlvvglqqlgadvdcfigtncppvringkgglpggkvklsgissqylssllmaaplaledveieiidklisvpyvemtlklmehfgv taehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvegcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprqpfgrkhlkavdvnmnkmpdva mtlavvalfadgptairdvaswrvketermvairteltklgatveegpdycitppekinitaidtyddhrmafslaacaevpvtirdpgctrktfpnyfdvlstfvkn*

[0442] SEQ ID NO:32 is a mutant of the EPSPS sequence from wheat and contains a V403A mutation.

[0443] magaeevvlqpireisgavqlpgskslsnrilllsalsegttvvdnlInsedvhymlealealglsveadkvakravvgcggrfpvekdakeevklflgnagtamrpltaa vvaaggnatyvldgvprmrerpigdlvvglqqlgadvdcfigtncppvringkgglpggkvklsgissqylssllmaaplaledveieiidklisvpyvemtlkimehfg vtaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvegcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprqpfgrkhlkavdvnmnkmpda amtlavvalfadgptairdvaswrvketermvairteltklgatveegpdycitppekinitaidtyddhrmafslaacaevpvtirdpgctrktfpnyfdvlstfvkn*

[0444] SEQ ID NO:33 is the amino acid sequence of the wild-type soybean EPSPS protein, represented as GenBank entry XP_003521857.1, with the chloroplast transit peptide removed from SEQ ID NO:33.

[0445] maaaekpstapeivlepikdisgtitlpgskslsnrilllaalsegttvvdnllysedihymlgalrtlgIrveddqttkqaivegcgglfptikeskdeinlflgnagtamr pltaavvaaggnasyvldgvprmrerpigdlvaglkqlgadvdcflgtncppvrvngkgglpggkvklsgsissqyltallmaaplalgdveieivdklisvpyvemtlklme rfgvsvehsgnwdkflvhggqkykspgnafvegdassasyflagaavtggtitvngcgtnslqgdvkfaevlekmgakvtwsensvtvtgppqdssgqkvlqgidvnmnkmpd vamtlavvalfangqtairdvaswrvketermiaictelrklgatveegpdycvitppekInvtaidtyddhrmafslaacgdvpvtikdpgctrktfpdyfevlerftrh* SEQ ID NO:34 is a mutant of the EPSPS sequence from soybean and contains a V403A mutation.

[0446] maaaekpstapeivlepikdisgtitlpgskslsnrillaalsegttyvdnllysedihymlgalrtlgIrveddqttkqaivegcgglfptikeskdeinlflgnagtamrp ltaavvaaggnasyvldgvprmrerpigdlvaglkqlgadvdcfigtncppvrvngkgglpggkvklsgissqyltallmaaplalgdveieivdklisvpyvemtilklme rfgvsvehsgnwdkfivhggqkykspgnafvegdassasyflagaavtggtitvngcgtnslqgdvkfaevlekmgakvtwsensvtvtgppqdssgqkvlqgidvnmnkmpd aamtlavvalfangqtairdvaswrvketermiaictelrklgatveegpdycvitppekInvtaidtyddhrmafslaacgdvpvtikdpgctrktfpdyfevlerftrh*

[0447] SEQ ID NO:35 is a mutant EPSPS sequence from rice and contains the T173I and P177S mutations.

[0448] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttvvdnlnsedvhymlealkalglsveadkvakravyvgcggkfpvekdakeevqlflgnagiamrslta avtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcflgtecppvrvkgigglpggkvklsgissqylsallmaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdv amtlavvalfadgptairdvaswrvketermvairteltklgasveegpdycitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0449] SEQ ID NO:36 is a mutant EPSPS sequence from rice and contains T173I and P177T mutations.

[0450] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnllinsedvhymlealkalglsveadkvakrawwvgcggkfpvekdakeevqlflgnagiamrtlt aavtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcfigtecppvrvkgigglpggkvklsgissqylsallmaaplalgdveieiidklisipyvemtirlmerf gvkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdv amtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppeklnitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0451] SEQ ID NO:37 is a mutant EPSPS sequence from rice and contains the T173I, P177S and V403A mutations.

[0452] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttvvdnlInsedvhymlealkalglsveadkvakravwvgcggkfpvekdakeevqlflgnagiamrslta avtaaggnatyvldgvprmrerpigdlvvglkqlgadvdcflgtecppvrvkgigglpggkvklsgsissqylsallmaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0453] SEQ ID NO:38 is a mutant EPSPS sequence from rice and contains T173I, P177T and V403A mutations.

[0454] maakaeeivlqpireisgavqlpgskslsnrilllsalsegttyvdnlInsedvhymlealkalglsveadkvakravvvgcggkfpvekdakeevqlflgnagiamrtlt aavtaaggnatyvldgvprmrerpigdlvglkqlgadvdcfigtecppvrvkgigglpggkvklsgissqylsallmaaplalgdveieiidklisipyvemtirlmerfg vkaehsdswdrfyikggqkykspgnayvegdassasyflagaaitggtvtvqgcgttslqgdvkfaevlemmgakvtwtdtsvtvtgpprepygkkhlkavdvnmnkmpdaa mtlavvalfadgptairdvaswrvketermvairteltklgasveegpdyciitppekInitaidtyddhrmafslaacadvpvtirdpgctrktfpnyfdvlstfvrn*

[0455] SEQ ID NO:39 is the amino acid sequence of wild-type EPSPS from CP4 (Agrobacterium).

[0456] mdpgmshgassrpatarkssglsgtvripgdksishrsfmfgglasgetritgllegedintgkamqamgarirkegdtwiidgvgnggllapeapldfgnaatgcrltmglvgvydfdstfigdasltkrpmgrvInpIremgvqvksedgdrlpvtlrgpktptpityrvpmasaqvksavllagIntpgittviepimtrdhtekmlqgfganltvetdadgvrtirlegrgkitgqvidvpgdpsstafplvaallvpgsdvtilnvImnptrtgliltlqemgadievinprlaggedvadIrvrsstlkgvtvpedrapsmideypilavaaafaegatvmngleeIrvkesdrsavanglkIngvdcdegetslvvrgrpdgkglgnasgaavathldhriamsflvmglvsenpvtvddatmiatsfpefmdlmaglgakielsdtkaa

Claims

1. A polynucleotide encoding a plant EPSP synthase (EPSPS) polypeptide, said plant EPSPS polypeptide comprising a mutation: The mutation in the plant EPSPS polypeptide is V403A, and the position of the mutated amino acid corresponds to the position of the amino acid in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are G172A and V403A, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutation in the plant EPSPS polypeptide is P177S and V403A, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are G172A, P177S and V403A, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are T173I, P177S and V403A, and the amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are T173I, P177T and V403A, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are A70V, G172A, P177S, V403A and R514S, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are A73T, G172A, P177S and V403A, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are A73T, E86G, G172A, P177S and V403A, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are A145G, G172A, P177S, V403A, T442S and D472E, and the positions of the mutated amino acids correspond to the positions of the amino acids in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are G172A, P177S, V403A and F502V, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are G172A, P177S, Y252F and V403A, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are E137D, G172A, P177S, L253M and V403A, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are G172A, P177S, K399N, V403A and T466S, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are A73G, L134G, G172A, P177S, G265V, S374R and V403A, and the positions of the mutated amino acids correspond to the positions of the amino acids in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are A73K, D114H, V136M, K161E, G172A, P177S, A260T and V403A, and the positions of the mutated amino acids correspond to the positions of the amino acids in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are E163K, G172A, P177S, A216V, L253M, H389R and V403A, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are G172A, P177S, N247D and V403A, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are G172A, M175A, P177S, M258L, K356E, V403A and F512I, and the positions of the mutated amino acids correspond to the positions of the amino acids in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are A73S, G172A, P177S, L214F and V403A, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, the mutations in the plant EPSPS polypeptide are G172A, P177S, V208A, L264H, V403A and R514K, and the mutated amino acid positions correspond to the amino acid positions in SEQ ID NO: 1; Or, said mutations in said plant EPSPS polypeptide are V113M, G172A, P177S and V403A, and said mutated amino acid positions are positions corresponding to the amino acid positions in SEQ ID NO:

1.

2. The polynucleotide of claim 1, wherein the sequence of the plant EPSPS polypeptide is set forth in any one of SEQ ID NOs. 3-6, 10-25, 27, 29, 30, 32, 34, 37, 38.

3. The polynucleotide of claim 2, wherein the sequence of the plant EPSPS polypeptide is as set forth in SEQ ID NO.

4. A plant comprising in its genome the polynucleotide according to any one of claims 1 to 3, The plant is corn, rice, wheat, barley, oats, sorghum, foxtail millet, flax, hemp, Kentucky bluegrass, tall fescue, bush grass, brassica, sugar beet, soybean, pea, alfalfa, tobacco, cotton, sunflower, tomato, potato, apple tree, Vitis vinifera L., citrus tree, cucumber, or petunia.

5. The plant according to claim 4, which is rice.

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