Herbicide tolerance genes and methods of use thereof

Abstract

Provided in the present invention are polypeptides and recombinant DNA molecules suitable for conferring tolerance to pyridyloxy-acid herbicides, as well as herbicide-tolerant plants, seeds, cells, and plant parts containing said recombinant DNA molecules, and methods of use thereof.

Description

Technical Field

[0001] This invention generally relates to the field of biotechnology. More specifically, this invention relates to recombinant DNA molecules encoding enzymes that degrade herbicides. This invention also relates to transgenic plants, parts, seeds, cells, and plant parts containing recombinant DNA molecules, and methods of using them. Background Technology

[0002] Crop production often utilizes transgenic traits produced using biotechnological methods. A heterologous gene (also called a transgene) is introduced into a plant to produce a transgenic trait. Expression of the transgene in the plant confers the desired trait, such as herbicide tolerance. Examples of transgenic herbicide tolerance traits include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance. With the increasing number of weed species resistant to the most commonly used herbicides, new herbicide tolerance traits are needed in this field. Herbicides of particular interest are pyridyloxy acid herbicides. Pyridyloxy acid herbicides provide control over a range of glyphosate-resistant weeds, thus producing traits that confer tolerance to these herbicides, specifically for use in crop systems in combination with other herbicide tolerance traits.

[0003] The herbicide-eating Sphingobium herbicidovorans strain MH, isolated from soil samples degrading 2,4-dichloropropionic acid, was identified as capable of cleaving the ether bonds of various phyenoxyalkanoic acid herbicides, thereby utilizing them as its sole carbon and energy source for growth (HPE Kohler, Journal of Industrial Microbiology & Biotechnology (1999) 23:336-340). Herbicide catabolism occurs via two distinct enantioselective α-ketoglutarate-dependent dioxygenases, RdpA (R-2,4-dichloropropionic acid dioxygenase) and SdpA (S-2,4-dichloropropionic acid dioxygenase). (A Westendorf, et al., Microbiological Research (2002) 157:317-322; Westendorf, et al., Acta Biotechnologica (2003) 23(1):3-17). RdpA has been autophagous for herbicides by *Sphingobacterium* (GenBank records AF516752 (DNA) and AAM90965 (protein)) and *Delftia acidovorans* (GenBank records NG_036924 (DNA) and YP_009083283 (protein)) (TA Mueller et al., *Applied and Environmental Microbiology* (2004) 70(10): 6066-6075). The RdpA and SdpA genes have been used in plant transformation to confer herbicide tolerance to crops (TR Wright et al., *Proceedings of the National Academy of Sciences USA* (2010) 107(47): 20240-5). Using protein engineering techniques to improve the activity of the RdpA enzyme to produce proteins for transgenic plants will allow for higher herbicide application rates, thereby improving transgenic crop safety and weed control. Invention Summary

[0004] This invention provides a recombinant DNA molecule comprising a nucleic acid sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide, compared to the RdpA amino acid sequence shown in SEQ ID NO:1, has the following mutation: the amino acid at position 82 is changed from leucine to histidine. In one embodiment, the amino acid sequence of the polypeptide further comprises one or more mutations selected from the group consisting of: the amino acid at position 187 being changed from valine to leucine, methionine, or isoleucine; the amino acid at position 187 being changed from valine to leucine, and the amino acid at position 104 being changed from arginine to alanine, aspartic acid, or leucine; the amino acid at position 187 being changed from valine to leucine, and the amino acid at position 182 being changed from phenylalanine to tryptophan; the amino acid at position 187 being changed from valine to leucine, and the amino acid at position 103 being changed from glycine to leucine; the amino acid at position 187 being changed from valine to leucine, and the amino acid at position 182 being changed from phenylalanine to tryptophan; the amino acid at position 187 being changed from valine to leucine, and the amino acid at position 103 being changed from glycine to leucine; the amino acid at position 187 being changed from valine to leucine, and the amino acid at position 182 being changed from phenylalanine to tryptophan. The amino acid 104 is changed from arginine to glycine; the amino acid 187 is changed from valine to leucine, the amino acid 182 is changed from phenylalanine to tryptophan, and the amino acid 103 is changed from glycine to leucine; the amino acid 187 is changed from valine to leucine, the amino acid 182 is changed from phenylalanine to tryptophan, the amino acid 104 is changed from arginine to glycine, and the amino acid 112 is changed from threonine to serine; the amino acid 187 is changed from valine to leucine, the amino acid 182 is changed from phenylalanine to tryptophan, and the amino acid 104 is changed from arginine to glycine. The amino acid at position 80 is changed from valine to threonine; at position 187, valine is changed to leucine; at position 182, phenylalanine is changed to tryptophan; at position 104, arginine is changed to glycine; and at position 180, arginine is changed to tryptophan or methionine. The amino acid at position 187 is changed from valine to leucine; at position 182, phenylalanine is changed to tryptophan; at position 104, arginine is changed to glycine; and at position 108, aspartic acid is changed to cysteine. The amino acid at position 187 is changed from valine to leucine; and at position 182, phenylalanine is changed to tryptophan. The amino acid at position 104 is mutated from arginine to glycine, and the amino acid at position 109 is mutated from aspartic acid to glutamic acid; the amino acid at position 187 is mutated from valine to leucine, the amino acid at position 182 is mutated from phenylalanine to tryptophan, the amino acid at position 104 is mutated from arginine to glycine, and the amino acid at position 219 is mutated from glutamine to cysteine ​​or proline; the amino acid at position 187 is mutated from valine to leucine, the amino acid at position 182 is mutated from phenylalanine to tryptophan, the amino acid at position 103 is mutated from glycine to leucine, and the amino acid at position 180 is mutated from arginine to aspartic acid, glutamic acid, serine, leucine, tryptophan, or threonine;The amino acid at position 187 is mutated from valine to leucine, the amino acid at position 182 from phenylalanine to tryptophan, the amino acid at position 103 from glycine to leucine, and the amino acid at position 80 from valine to threonine; the amino acid at position 187 is mutated from valine to leucine, the amino acid at position 182 from phenylalanine to tryptophan, the amino acid at position 103 from glycine to leucine, and the amino acid at position 112 from threonine to alanine, serine, or methionine; the amino acid at position 187 is mutated from valine to leucine, the amino acid at position 182 from phenylalanine to tryptophan, the amino acid at position 103 from glycine to leucine, and the amino acid at position 247 from phenylalanine to tyros ... The amino acid at position 2 is mutated from phenylalanine to tryptophan, the amino acid at position 104 is mutated from arginine to glycine, and the amino acid at position 77 is mutated from valine to isoleucine; the amino acid at position 187 is mutated from valine to leucine, the amino acid at position 182 is mutated from phenylalanine to tryptophan, the amino acid at position 104 is mutated from arginine to glycine, the amino acid at position 112 is mutated from threonine to serine, and the amino acid at position 180 is mutated from arginine to lysine, methionine, tryptophan, or glutamine; and / or the amino acid at position 187 is mutated from valine to leucine, the amino acid at position 182 is mutated from phenylalanine to tryptophan, the amino acid at position 103 is mutated from glycine to leucine, the amino acid at position 104 is mutated from arginine to glycine, and the amino acid at position 105 is mutated from valine to tyrosine. In another embodiment, the amino acid sequence of the polypeptide further has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the RdpA amino acid sequence shown in SEQ ID NO:1.

[0005] The present invention also provides a recombinant DNA molecule comprising a nucleic acid sequence encoding a polypeptide having at least about 92% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, and 142. In one embodiment, the recombinant DNA molecule comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: NO:3,4,5,7,8,9,11,12,13,15,16,17,19,20,21,23,24,25,27,28,29,31,32,33,35,36,37,39,40,41,43,44,45, 47, 48, 49, 51, 52, 53, 55, 56, 57, 59, 60, 61, 63, 64, 65, 67, 68, 69, 71, 72, 73, 75, 76, 77, 79, 80, 81, 83, 84, 85, 87, 88, 89, 91, 92, 93, 95, 96, 97, 99, 100, 101, 103, 104, 105, 107, 108, 109, 111, 112, 113, 115, 116, 117, 119, 120, 121, 123, 124, 125, 127, 128, 129, 131, 132, 133, 135, 136, 137, 139, 140, 141, and 143-181, and nucleic acid sequences that encode the same amino acid sequence as the illustrated sequence due to the degeneracy of the genetic code. In another embodiment, the recombinant DNA molecule encodes a polypeptide having oxygenase activity against at least one herbicide selected from the group consisting of pyridyloxy acid herbicides. In another embodiment, the recombinant DNA molecule is operatively linked to a heterologous promoter that is functional in plant cells. In another embodiment, the recombinant DNA molecule is operatively linked to a DNA molecule encoding a chloroplast transport peptide, the chloroplast transport peptide being used to localize the operatively linked polypeptide within the cell.

[0006] This invention provides a DNA construct comprising a functional heterologous promoter in a plant cell, the heterologous promoter being operatively linked to the recombinant DNA molecule of this invention. In one embodiment, the recombinant DNA molecule is operatively linked to a DNA molecule encoding a chloroplast transport peptide for localizing the operatively linked polypeptide within the cell. In another embodiment, expression of the polypeptide encoded by the recombinant DNA molecule in a transgenic plant confers herbicide tolerance. In yet another embodiment, the DNA construct is present in the genome of the transgenic plant.

[0007] This invention provides a transgenic plant, seed, cell, or plant part comprising the recombinant DNA molecule described herein. In one embodiment, the transgenic plant, seed, cell, or plant part comprises a transgenic trait of tolerance to at least one herbicide selected from the group consisting of pyridyloxy acid herbicides. In another embodiment, the transgenic plant, seed, cell, or plant part comprises the DNA construct of this invention. In yet another embodiment, the transgenic plant, seed, cell, or plant part comprises a polypeptide of this invention.

[0008] This invention provides a polypeptide whose amino acid sequence, compared to the RdpA amino acid sequence shown in SEQ ID NO:1, has the following mutation: the amino acid at position 82 is changed from leucine to histidine. In one embodiment, the amino acid sequence of the polypeptide further has one or more of the above-mentioned mutations. In another embodiment, the amino acid sequence of the polypeptide further has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the RdpA amino acid sequence shown in SEQ ID NO:1.

[0009] The present invention also provides a polypeptide having at least about 92% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, and 142. In one embodiment, the polypeptide has oxygenase activity against at least one herbicide selected from the group consisting of: pyridyloxy acid herbicides.

[0010] This invention provides a method for conferring herbicide tolerance to plants, seeds, cells, or plant parts, the method comprising expressing a polypeptide of the invention in the plant, seed, cell, or plant part. In one embodiment, the method for conferring herbicide tolerance is used with a transgenic plant, seed, cell, or plant part containing a transgenic trait comprising a recombinant DNA molecule of the invention. In one embodiment, the method for conferring herbicide tolerance is used with a herbicide selected from the group consisting of pyridyloxy acid herbicides.

[0011] This invention provides a plant transformation method comprising introducing a recombinant DNA molecule or DNA construct of the invention into plant cells or tissues and regenerating from therea a plant containing the recombinant DNA molecule or DNA construct and resistant to at least one herbicide selected from the group consisting of pyridyloxy acid herbicides. In one embodiment, the plant transformation method comprises hybridizing the regenerated plant with itself or with a second plant and collecting seeds from the hybridization.

[0012] The present invention provides a method for controlling weeds in a plant growth zone by contacting a plant growth zone containing the transgenic plant or seed of the present invention with at least one herbicide selected from the group consisting of: pyridyloxy acid herbicides, wherein the transgenic plant or seed is tolerant to the herbicide. Attached Figure Description

[0013] Figure 1 The image shows control Jinjing 818 plants and transgenic Jinjing 818 plants containing the protein-coding gene of SEQ ID NO:42 after 19 days of adding 0.5 μM and 1 μM compound B to the culture medium (the growth variation between plants expressing the same protein may be due to the design of the construct or the position of the transgene insertion, the same below).

[0014] Figure 2 The image shows control Jinjing 818 plants and transgenic Jinjing 818 plants containing the protein-coding gene of SEQ ID NO:42 after application of compound B at the corresponding number of days (DAT) at doses of 20 g, 40 g, 60 g, 80 g, 100 g, and 120 g / acre.

[0015] Figure 3 The image shows control wild-type and T2 generation transgenic Arabidopsis seeds containing the protein-coding gene of SEQ ID NO:46, after 11 days of screening with 0.15 μM compound A added to the culture medium.

[0016] Figure 4The image shows control Arabidopsis thaliana transgenic with the RdpA wild-type gene and Arabidopsis thaliana containing the protein-coding gene SEQ ID NO:42, after the plant leaves were sprayed with 40 g / mu of compound B for 12 days.

[0017] Figure 5 The image shows control Jinjing 818 plants and transgenic Jinjing 818 plants containing the protein-coding gene SEQ ID NO:138 / 86 after 17 days of adding 1 μM compound B to the culture medium.

[0018] Figure 6 The image shows control Jinjing 818 plants and transgenic Jinjing 818 plants containing the protein-coding gene of SEQ ID NO:102 after 22 days of adding 1 μM compound B to the culture medium.

[0019] Figure 7 The image shows control Jinjing 818 plants and transgenic Jinjing 818 plants containing the protein-coding gene of SEQ ID NO:82 after 17 days of adding 1 μM compound B to the culture medium.

[0020] Figure 8 The image shows control Jinjing 818 plants and transgenic Jinjing 818 plants containing the protein-coding gene of SEQ ID NO:106 after 22 days of adding 1 μM compound B to the culture medium.

[0021] Figure 9 The image shows control Jinjing 818 plants and transgenic Jinjing 818 plants containing the protein-coding gene of SEQ ID NO:126 after 26 days of adding 1 μM compound B to the culture medium.

[0022] Figure 10 The image shows control Jinjing 818 plants and transgenic Jinjing 818 plants containing the protein-coding gene of SEQ ID NO:142 after 26 days of adding 1 μM compound B to the culture medium.

[0023] Figure 11 The image shows control Jinjing 818 plants and transgenic Jinjing 818 plants containing the protein-coding gene of SEQ ID NO:38 after 22 days of adding 1 μM compound B to the culture medium.

[0024] Figure 12 The results show the test results of control Jinjing 818 plants and transgenic Jinjing 818 plants containing the SEQ ID NO:46 protein-coding gene after application of 0 g, 5 g, and 10 g / mu of the compound quizalofop-P-ethyl 20 DAT. (The boxed area in the figure represents wild-type control Jinjing 818 plants, and the rest are transgenic Jinjing 818 plants containing the SEQ ID NO:46 protein-coding gene.)

[0025] Figure 13The image shows a comparison of root length between T1 generation transgenic Jinjing 818 seeds containing different protein-coding genes and wild-type Jinjing 818 seeds after soaking for 20 days with 0.3 μM compound B.

[0026] Figure 14 The image shows a comparison of the germination rates of T2 generation transgenic Arabidopsis seeds containing the SEQ ID NO:42 protein-coding gene with wild-type Arabidopsis (first row) after 20 days of screening with 0.15 μM compound A.

[0027] Figure 15 The image shows a comparison of the germination rates of T2 generation transgenic Arabidopsis seeds containing the SEQ ID NO:78 protein-coding gene with wild-type Arabidopsis (first row) after 19 days of screening with 0.15 μM compound A.

[0028] Figure 16 The image shows a comparison of the germination rates of T2 generation transgenic Arabidopsis seeds containing the SEQ ID NO:82 protein-coding gene with wild-type Arabidopsis (first row) after 24 days of screening with 0.15 μM compound A.

[0029] Figure 17 The image shows a comparison of the germination rates of T2 generation transgenic Arabidopsis seeds containing the SEQ ID NO:86 protein-coding gene with wild-type Arabidopsis (first row) after 21 days of screening with the addition of 0.15 μM compound A.

[0030] Figure 18 The image shows a comparison of the germination rates of T2 generation transgenic Arabidopsis seeds containing the SEQ ID NO:98 protein-coding gene with wild-type Arabidopsis (first row) after 19 days of screening with 0.15 μM compound A.

[0031] Figure 19 The image shows a comparison of the germination rates of T2 generation transgenic Arabidopsis seeds containing the SEQ ID NO:102 protein-coding gene with wild-type Arabidopsis (first row) after 19 days of screening with 0.15 μM compound A.

[0032] Figure 20 The image shows a comparison of the germination rates of T2 generation transgenic Arabidopsis seeds containing the SEQ ID NO:126 protein-coding gene with wild-type Arabidopsis (first row) after 20 days of screening with 0.15 μM compound A.

[0033] Figure 21 The image shows a comparison of the germination rates of T2 generation transgenic Arabidopsis seeds containing the SEQ ID NO:138 protein-coding gene with wild-type Arabidopsis (first row) after 19 days of screening with 0.15 μM compound A.

[0034] Figure 22The image shows a comparison of the germination rates of T2 generation transgenic Arabidopsis seeds containing the SEQ ID NO:142 protein-coding gene with wild-type Arabidopsis (first row) after 24 days of screening with 0.15 μM compound A.

[0035] Figure 23 The image shows a comparison of resistance in T0 generation transgenic soybean containing the protein-coding gene of SEQ ID NO:46 with wild-type soybean (compound C10g).

[0036] Figure 24 The image shows a comparison of resistance in T1 generation transgenic soybean containing the protein-coding gene of SEQ ID NO:46 with wild-type soybean (compound C10g).

[0037] Figure 25 The image shows a comparison of resistance in T1 generation transgenic soybean containing the protein-coding gene of SEQ ID NO:42 with wild-type soybean (compound C10g).

[0038] Figure 26 The image shows a comparison of resistance between T1 generation transgenic soybean containing the protein-coding gene of SEQ ID NO:42 and wild-type soybean (compound C 20g, 40g, 80g).

[0039] Figure 27 The results of spraying 150g, 250g, 400g, 600g, and 800g of 30% glyphosate compound C(25+5)ME onto T0 generation transgenic maize seedlings are shown.

[0040]

[0041]

[0042]

[0043]

[0044] Invention Details

[0046] The following definitions and methods are provided to better define the invention and to guide those skilled in the art in carrying it out. Unless otherwise stated, the terminology should be understood according to its conventional usage by those skilled in the art.

[0047] Engineered proteins and recombinant DNA molecules

[0048] This invention provides novel engineered proteins and recombinant DNA molecules encoding them. As used herein, the term "engineered" refers to non-natural DNA, proteins, or organisms that are not normally found in nature and are produced through human intervention. An "engineered protein" is a protein whose polypeptide sequence is conceived and created in the laboratory using one or more protein engineering techniques, such as protein design using site-directed mutagenesis and directed evolution using random mutagenesis and DNA shuffling. For example, an engineered protein may have one or more deletions, insertions, or substitutions relative to the coding sequence of a wild-type protein, and each deletion, insertion, or substitution may consist of one or more amino acids. Examples of engineered proteins provided herein are SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138 and 142.

[0049] The engineered protein provided by this invention is an enzyme with oxygenase activity. As used herein, the term "oxygenase activity" refers to the ability to oxidize a substrate by transferring oxygen from molecular oxygen to the substrate, byproduct, or intermediate. The oxygenase activity of the engineered protein provided by this invention can inactivate one or more pyridyloxy acid herbicides.

[0050] As used herein, "wildtype" means naturally occurring. As used herein, "wildtype DNA molecule," "wildtype polypeptide," or "wildtype protein" refers to a naturally occurring DNA molecule, polypeptide, or protein, that is, a DNA molecule, polypeptide, or protein that is pre-existing in nature. The wild-type form of a polypeptide, protein, or DNA molecule can be used for comparison with engineered proteins or genes. The wild-type form of a protein or DNA molecule can be used as a control in experiments.

[0051] As used herein, “control” means an experimental control designed for comparative purposes. For example, a control plant in transgenic plant analysis is a plant of the same type as the experimental plant (i.e., the plant being tested) but without the transgenic insert, recombinant DNA molecule, or DNA construct of the experimental plant. Examples of control plants suitable for comparison with transgenic maize plants are non-transgenic LH244 maize (US Patent No. 6,252,148) and non-transgenic A3555 soybean (US Patent No. 7,700,846).

[0052] As used herein, the term "recombinant" refers to non-natural DNA, polypeptides, or proteins that are the result of genetic engineering and therefore not typically found in nature and are produced through human intervention. A "recombinant DNA molecule" is a DNA molecule containing a DNA sequence that is not naturally occurring and is therefore the result of human intervention, such as a DNA molecule encoding an engineered protein. Another example is a DNA molecule consisting of a combination of at least two heterologous DNA molecules (such as a DNA molecule encoding a protein and a heterologous promoter operatively linked). Examples of recombinant DNA molecules are DNA molecules containing at least one sequence selected from: SEQ ID NO:3,4,5,7,8,9,11,12,13,15,16,17,19,20,21,23,24,25,27,28,29,31,32,33,35,36,37,39,40,41,4 3, 44, 45, 47, 48, 49, 51, 52, 53, 55, 56, 57, 59, 60, 61, 63, 64, 65, 67, 68, 69, 71, 72, 73, 75, 76, 77, 79, 80, 81 83, 84, 85, 87, 88, 89, 91, 92, 93, 95, 96, 97, 99, 100, 101, 103, 104, 105, 107, 108, 109, 111, 112, 113, 115, 116, 117, 119, 120, 121, 123, 124, 125, 127, 128, 129, 131, 132, 133, 135, 136, 137, 139, 140, 141, and 143-181. A “recombinant polypeptide” or “recombinant protein” is a polypeptide or protein that contains an amino acid sequence that is not naturally occurring and is therefore the result of human intervention, such as engineered proteins.

[0053] The term "transgenic" refers to a DNA molecule artificially incorporated into the genome of an organism as a result of human intervention (such as through plant transformation). As used herein, the term "transgenic" means containing a transgenic gene; for example, a "transgenic plant" refers to a plant whose genome contains a transgenic gene, and a "transgenic trait" refers to a characteristic or phenotype transmitted or conferred by the presence of a transgenic gene incorporated into the plant's genome. As a result of such genomic alteration, the transgenic plant is a plant that is significantly different from the associated wild-type plant, and the transgenic trait is a trait not naturally found in wild-type plants. The transgenic plant of the present invention comprises the recombinant DNA molecule and engineered protein provided by the present invention.

[0054] As used herein, the term "heterologous" refers to a relationship between two or more substances that originate from different sources and are therefore not typically related in nature. For example, a recombinant DNA molecule encoding a protein is heterologous relative to a promoter that is operatively linked, if such a combination is not typically found in nature. Furthermore, a particular recombinant DNA molecule can be heterologous relative to the cell or organism in which it is inserted when it is not naturally present in that particular cell or organism.

[0055] As used herein, the terms “DNA molecule encoding a protein” or “DNA molecule encoding a polypeptide” refer to a DNA molecule that contains a nucleotide sequence encoding a protein or polypeptide. “Sequence encoding a protein” or “sequence encoding a polypeptide” means the DNA sequence encoding a protein or polypeptide. “Sequence” means the sequential arrangement of nucleotides or amino acids. The boundaries of a protein-coding sequence or a polypeptide-coding sequence are typically determined by a 5'-terminal translation start codon and a 3'-terminal translation stop codon. A molecule encoding a protein or a polypeptide may contain a DNA sequence encoding a protein or polypeptide sequence. As used herein, “transgenic expression,” “expressing a transgenic,” “protein expression,” “polypeptide expression,” “expressing a protein,” and “expressing a polypeptide” refer to the production of a protein or polypeptide through the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into a polypeptide chain (which may eventually fold into a protein). A DNA molecule encoding a protein or a DNA molecule encoding a polypeptide may be operatively linked to a heterologous promoter in a DNA construct for the expression of a protein or polypeptide in cells transformed with a recombinant DNA molecule. As used herein, “operably linked” refers to two DNA molecules linked in such a way that one DNA molecule can influence the function of the other DNA molecule. Operablely linked DNA molecules can be part of a single, continuous molecule and may or may not be adjacent. For example, a promoter is operably linked to a DNA molecule encoding a protein or a DNA molecule encoding a polypeptide in a DNA construct, wherein the two DNA molecules are arranged such that the promoter can influence the expression of the transgene.

[0056] As used herein, a “DNA construct” is a recombinant DNA molecule containing two or more heterologous DNA sequences. DNA constructs are suitable for transgenic expression and can be contained in vectors and plasmids. DNA constructs can be used in vectors for transformation purposes (i.e., the introduction of heterologous DNA into host cells) to produce transgenic plants and cells, and therefore can also be contained in plasmid DNA or genomic DNA of transgenic plants, seeds, cells, or plant parts. As used herein, “vector” means any recombinant DNA molecule that can be used for plant transformation purposes. Recombinant DNA molecules, as shown in the sequence listing, can be inserted into vectors, for example, as part of a construct having a recombinant DNA molecule operatively linked to a promoter that functions in the plant to drive the expression of an engineered protein encoded by the recombinant DNA molecule. Methods for constructing DNA constructs and vectors are well known in the art. Components of a DNA construct or a vector containing a DNA construct generally include, but are not limited to, one or more of the following: a suitable promoter for expressing operatively linked DNA, an operatively linked non-human DNA molecule encoding a protein, and a 3' untranslated region (3'-UTR). Promoters suitable for practicing the present invention include promoters that function in plants to express operatively linked polynucleotides. Such promoters are diverse and well-known in the art, and include inducible, viral, synthetic, constitutive, time-regulated, space-regulated, and / or spatiotemporally regulated promoters. Additional optional components include, but are not limited to, one or more of the following elements: 5'-UTR, enhancer, leader sequence, cis-acting element, intron, chloroplast transport peptide (CTP), and one or more selectively labeled transgenes.

[0057] The DNA constructs of the present invention may comprise CTP molecules operatively linked to DNA molecules encoding proteins provided by the present invention. CTPs suitable for practicing the present invention include those for promoting the intracellular localization of engineered protein molecules. By promoting intracellular protein localization, CTPs can increase the accumulation of engineered proteins, protect them from proteolytic degradation, enhance herbicide tolerance levels, and thereby reduce damage levels following herbicide application. CTP molecules used in the present invention are known in the art, including but not limited to Arabidopsis EPSPS CTP (Klee et al., 1987), petunia EPSPS CTP (della-Cioppa et al., 1986), maize cab-m7 signal sequence (Becker et al., 1992; PCT WO 97 / 41228), and pea glutathione reductase signal sequence (Creissen et al., 1991; PCT WO 97 / 41228).

[0058] The recombinant DNA molecules of the present invention can be synthesized and modified, either wholly or partially, by methods known in the art, particularly when it is desirable to provide sequences suitable for DNA manipulation (such as restriction enzyme recognition sites or recombinant gene cloning sites), plant-preferred sequences (such as plant codon usage or Kozak concordant sequences), or sequences suitable for DNA construct design (such as spacer regions or adapter sequences). The present invention includes recombinant DNA molecules and engineered proteins that have at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, and about 99% sequence identity with any of the recombinant DNA molecules or engineered protein sequences provided herein, for example, with recombinant DNA molecules containing sequences selected from the group consisting of: SEQ ID NO:3,4,5,7,8,9,11,12,13,15,16,17,19,20,21,23,24,25,27,28,29,31,32,33,35,36,37,39,40,41,4 3, 44, 45, 47, 48, 49, 51, 52, 53, 55, 56, 57, 59, 60, 61, 63, 64, 65, 67, 68, 69, 71, 72, 73, 75, 76, 77, 79, 80, 81 83, 84, 85, 87, 88, 89, 91, 92, 93, 95, 96, 97, 99, 100, 101, 103, 104, 105, 107, 108, 109, 111, 112, 113, 115, 116, 117, 119, 120, 121, 123, 124, 125, 127, 128, 129, 131, 132, 133, 135, 136, 137, 139, 140, 141 and 143-181. As used herein, the terms "percentage sequence identity" or "% sequence identity" refer to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of the reference ("query") sequence (or its complement) compared to the test ("subject") sequence (or its complement) when two sequences are optimally aligned (with a total of less than 20% of appropriate nucleotide or amino acid insertions, deletions, or vacancies within the comparison window). Optimal sequence alignment for the comparison window is well known to those skilled in the art and can be implemented using tools such as Smith and Waterman's local homology algorithm, Needleman and Wunsch's homology alignment algorithm, and Pearson and Lipman's similarity search method, and is implemented through computerized implementations of these algorithms, such as using default parameters. Wisconsin GAP, BESTFIT, FASTA, and TFASTA are available for comparison with parts of (Accelrys Inc., San Diego, CA), MEGAlign (DNAStar, Inc., 1228 S. Park St., Madison, Wis. 53715), and MUSCLE (version 3.6) (RCEdgar, Nucleic Acids Research (2004) 32(5): 1792-1797). The “identity score” of the aligned fragments of the test and reference sequences is calculated by dividing the number of identical components shared by the two aligned sequences by the total number of components in the reference sequence fragment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The sequence identity percentage is expressed as the identity score multiplied by 100. Comparisons of one or more sequences can be made against the full-length sequence or a portion thereof, or against a longer sequence.

[0059] Engineered proteins can be produced by altering (i.e., modifying) wild-type proteins to generate novel proteins with a novel combination of useful protein characteristics, such as altered Vmax, Km, substrate specificity, substrate selectivity, and protein stability. Modifications can be made at specific amino acid positions in the protein, and can involve replacing amino acids found in nature (i.e., in wild-type proteins) at those positions with different amino acids. The amino acid sequence of the wild-type protein RdpA suitable for protein engineering is shown in SEQ ID NO:1. Engineered proteins are designed to have at least about 92% sequence identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, and 142, and contain at least one of these amino acid mutations. Therefore, the engineered proteins provided by this invention offer novel proteins with one or more altered protein characteristics relative to wild-type proteins found in nature. In one embodiment of the invention, the engineered protein has altered protein characteristics, such as improved or reduced activity or improved protein stability against one or more herbicides, compared to similar wild-type proteins or any combination of such characteristics. In one embodiment, the present invention provides an engineered protein and a recombinant DNA molecule encoding the same, having at least about 80% sequence identity, about 85% sequence identity, about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, and about 99% sequence identity with an engineered protein sequence selected from the group consisting of: SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, and 142. Amino acid mutations can be performed as a single amino acid substitution in a protein or in combination with one or more other mutations, such as substitutions, deletions, or additions of one or more other amino acids. Mutations can be performed as described herein or by any other method known to those skilled in the art.

[0060] Genetically modified plants

[0061] One aspect of the present invention includes transgenic plant cells, transgenic plant tissues, transgenic plants, and transgenic seeds comprising the recombinant DNA molecules and engineered proteins provided by the present invention. These cells, tissues, plants, and seeds comprising the recombinant DNA molecules and engineered proteins exhibit tolerance to one or more pyridyloxy acid herbicides.

[0062] Suitable methods for transforming host plant cells for use in this invention include virtually any method that introduces DNA into cells (e.g., in which a recombinant DNA construct is stably integrated into the plant chromosome) and are known in the art. Exemplary and widely used methods for introducing recombinant DNA constructs into plants include the *Agrobacterium* transformation system, which is well known to those skilled in the art. Transgenic plants can be regenerated from transformed plant cells via plant cell culture. Regarding transgenic homozygous plants (i.e., two allele copies of the transgene), R1 seeds can be produced by self-pollinating (self-crossing) a transgenic plant containing a single transgenic allele with itself (e.g., an R0 plant). One-quarter of the resulting R1 seeds will be homozygous for the transgene. The conjugability of plants grown from germinating R1 seeds is typically tested using SNP assays, DNA sequencing, or thermoamplification assays that allow the distinction between heterozygotes and homozygotes; this is called a conjugability assay.

[0063] The plants, seeds, plant parts, plant tissues, and cells provided by this invention exhibit tolerance to one or more pyridyloxy acid herbicides. Pyridyloxy acid herbicides are synthetic plant growth hormones similar to the plant growth hormone indoleacetic acid (IAA). Broadleaf plants are sensitive to these herbicides, which induce rapid, uncontrolled growth and ultimately kill the plants.

[0064] Examples of pyridyloxy acid herbicides include, but are not limited to, compounds as shown in Formula I and their salts and ester derivatives.

[0065]

[0066] Wherein, A and B independently represent halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, and C3-C6 cycloalkyl, respectively;

[0067] C represents hydrogen, halogen, C1-C6 alkyl, and halogenated C1-C6 alkyl;

[0068] Q represents C1-C6 alkyl, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogen, cyano, amino, nitro, formyl, C1-C6 alkoxy, C1-C6 alkylthio, C1-C6 alkoxycarbonyl, hydroxy C1-C6 alkyl, C1-C6 alkoxy C1-C2 alkyl, cyano C1-C2 alkyl, C1-C6 alkylamino C1-C2 alkyl, benzyl, naphthyl, furanyl, thiophene, thiazolyl, pyridinyl, pyrimidinyl, and unsubstituted or substituted C1-C6 alkyl groups. Phenyl groups that are unsubstituted or substituted by at least one of C1-C6 alkyl, halo-C1-C6 alkyl, halogen, and C1-C6 alkoxy groups;

[0069] Y represents amino, C1-C6 alkylamino, C1-C6 alkylcarbonylamino, phenylcarbonylamino, benzylamino, unsubstituted or halogenated C1-C6 alkyl-substituted furanylmethyleneamino;

[0070] The salt is a metal salt or an ammonium salt (NH4). + Primary amine salt RNH2, secondary amine salt (R)2NH, tertiary amine salt (R)3N, quaternary amine salt (R)4N + Morpholine salts, piperidine salts, pyridine salts, aminopropyl morpholine salts, Jeffamine D-230 salts, salts of 2,4,6-tris(dimethylaminomethyl)phenol and sodium hydroxide, C1-C14 alkyl sulfonium salts, C1-C14 alkyl oxide sulfonium salts, C1-C14 alkyl phosphonium salts, C1-C14 alkanol phosphonium salts;

[0071] Wherein, R independently represents an unsubstituted C1-C14 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C12 cycloalkyl or phenyl, and the C1-C14 alkyl is optionally substituted by one or more of the following groups: halogen, hydroxyl, C1-C6 alkoxy, C1-C6 alkylthio, hydroxyC1-C6 alkoxy, amino, C1-C6 alkylamino, aminoC1-C6 alkylamino, phenyl;

[0072] The ester is Where X represents O or S;

[0073] M represents C1-C18 alkyl, halo-C1-C8 alkyl, C3-C6 cycloalkyl, C2-C6 alkenyl, halo-C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, C1-C6 alkylsulfonyl, cyano-C1-C2 alkyl, nitro-C1-C2 alkyl, C1-C6 alkoxy-C1-C2 alkyl, C1-C6 alkoxycarbonyl-C1-C2 alkyl, C2-C6 alkenoxycarbonyl-C1-C2 alkyl, -(C1-C2 alkyl)-Z. Tetrahydrofuranyl, pyridyl, naphthyl, furanyl, thiophenyl and unsubstituted or C1-C6 alkyl-substituted Phenyls that are not substituted or are substituted by C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkylamino, halogen or C1-C6 alkoxy;

[0074] Z represents Tetrahydrofuranyl, pyridyl Thiophene, furanyl, naphthyl, and phenyl groups that are unsubstituted or substituted with at least one of C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkyl, cyano, and halogen;

[0075] R3 independently represents C1-C6 alkyl groups;

[0076] R4, R5, and R6 independently represent hydrogen, C1-C6 alkyl, and C1-C6 alkoxycarbonyl, respectively;

[0077] R' represents hydrogen, C1-C6 alkyl, or halogenated C1-C6 alkyl.

[0078] In one embodiment, both general formula compounds I and I-1 are in the R configuration (the carbon atom at * is the chiral center). In another embodiment, in general formula compound I, A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, and Q represents methyl, and it is in the R configuration (the carbon atom at * is the chiral center) (i.e., compound A); in general formula compound I-1, A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, Q represents methyl, X represents O, and M represents methyl, and it is in the R configuration (the carbon atom at * is the chiral center) (i.e., compound B); or in general formula compound I-1, A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, Q represents methyl, X represents O, and M represents tetrahydrofuran-2-ylmethyl It is in the R configuration (i.e., compound C) (* indicates that the carbon atom is the chiral center).

[0079] Herbicides can be applied to plant growth areas containing the plants and seeds provided by this invention as a method of weed control. The plants and seeds provided by this invention contain herbicide-tolerant traits and are therefore resistant to the application of one or more pyridyloxy acid herbicides. When the herbicide is applied, the plant growth area may or may not include weed plants.

[0080] Herbicide application may be sequentially mixed with one, two, or a combination of several pyridyloxy acid herbicides or any other compatible herbicides. Multiple applications of one herbicide or a combination of two or more herbicides, or individually, may be used during the growing season in areas containing the transgenic plants of the present invention for the control of broad-spectrum dicotyledonous weeds, monocotyledonous weeds, or both, for example, two applications (e.g., pre-planting and post-emergence or pre-emergence and post-emergence) or three applications (e.g., pre-planting, pre-emergence and post-emergence or pre-emergence and two post-emergence applications).

[0081] As used herein, “tolerance” or “herbicide tolerance” means the ability of a plant, seed, plant tissue, plant part, or cell to resist the toxic effects of one or more herbicides. Herbicide tolerance in a plant, seed, plant tissue, plant part, or cell can be measured by comparing the plant, seed, plant tissue, plant part, or cell to a suitable control. For example, herbicide tolerance can be measured by applying a herbicide to a plant containing a recombinant DNA molecule encoding a protein that confers herbicide tolerance (test plant) and a plant not containing a recombinant DNA molecule encoding a protein that confers herbicide tolerance (control plant), and then comparing the plant damage to the two plants, wherein the herbicide tolerance of the test plant is indicated by a reduced damage rate compared to the damage rate of the control plant. Herbicide-tolerant plants, seeds, plant tissues, plant parts, or cells show a reduced response to the toxic effects of herbicides when compared to control plants, seeds, plant tissues, plant parts, or cells. As used herein, a “herbicide tolerance trait” is a transgenic trait that confers improved herbicide tolerance in a plant compared to a wild-type plant or a control plant.

[0082] The transgenic plants, offspring, seeds, plant cells, and plant parts of the present invention may also contain one or more additional transgenic traits. Additional transgenic traits can be introduced by hybridizing a plant containing a transgenic trait comprising a recombinant DNA molecule provided by the present invention with another plant containing an additional transgenic trait. As used herein, “hybridization” means breeding two separate plants to produce offspring plants. Thus, two transgenic plants can be hybridized to produce offspring containing the transgenic trait. As used herein, “offspring” means any generation of the parent plant, and the transgenic offspring contain a DNA construct provided by the present invention and inherited from at least one parent plant. Alternatively, additional transgenic traits can be introduced by co-transforming a DNA construct of the additional transgenic trait with a DNA construct comprising a recombinant DNA molecule provided by the present invention (e.g., wherein all DNA constructs are portions of the same vector used for plant transformation) or by inserting the additional trait into a transgenic plant containing a DNA construct provided by the present invention, or vice versa (e.g., by using any method of plant transformation of transgenic plants or plant cells). Such additional transgenic traits include, but are not limited to, increased insect resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, and herbicide tolerance, wherein the trait is measured relative to a wild-type plant or a control plant. Such additional transgenic traits are known to those skilled in the art; for example, the USDA Animal and Plant Health Inspection Service (APHIS) provides a list of such traits, which can be found on their website www.aphis.usda.gov.

[0083] Transgenic plants and progeny containing the transgenic traits provided by this invention can be used with any breeding method generally known in the art. In plant lines containing two or more transgenic traits, the transgenic traits can be independently isolated, linked, or combined in plant lines containing three or more transgenic traits. Backcrossing with parental plants and crosscrossing with non-transgenic plants, as well as asexual reproduction, are also considered. Descriptions of breeding methods commonly used for different traits and crops are well known to those skilled in the art. To confirm the presence of transgenes in a particular plant or seed, various assays can be performed. Such assays include, for example, molecular biological assays such as DNA blotting and RNA blotting, PCR and DNA sequencing; biochemical assays such as, for example, detection of the presence of protein products by immunological methods (ELISA and Western blotting) or by enzyme function; plant part assays such as leaf or root assays; and also by analyzing the phenotype of the whole plant.

[0084] As a result of backcrossing transformation, transgenic traits are introduced into plant genotypes. Plant genotypes that have undergone transgenic introgression can be called backcrossed genotypes, lines, inbred plants, or hybrids. Similarly, plant genotypes lacking the desired transgenic trait can be called untransformed genotypes, lines, inbred plants, or hybrids.

[0085] As used in this article, the term "comprising" means "including but not limited to". Detailed Implementation

[0086] The following embodiments are included to demonstrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that work well in the practice of the invention and can therefore be considered to constitute preferred modes of practice of the invention. However, based on this disclosure, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed and still yielding similar or analogous results without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant reagents can be substituted for those described herein to obtain the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0087] Example 1. Initial protein engineering and enzyme analysis

[0088] By modeling and molecular docking of RdpA homologous proteins, and combining sequence alignment results, candidate sites are mutated using methods known to those skilled in the art, such as alanine scanning mutation, homology scanning mutation, Pro / Gly scanning mutation, region exchange or mutation, and combinations of these techniques (see M Lehmann and M Wyss, Current Opinion in Biotechnology (2001) 12(4):371-375; B Van den Burg and VGH Eijsink, Current Opinion in Biotechnology (2002) 13(4):333-337; and Weiss et al., Proc Natl Acad Sci USA (2000) 97(16):8950–8954).

[0089] High-throughput protein expression was achieved by cloning the genes encoding each engineered protein into bacterial expression vectors with C-terminal histidine tags (His-tags). The vectors were transformed into *Escherichia coli* (E. coli), and the expression of the engineered proteins was induced. *E. coli* cultures were cultured overnight in centrifuge tubes with substrate and IPTG added. The cultures were centrifuged the next day to precipitate the bacteria. Alternatively, *E. coli* cultures were cultured overnight in centrifuge tubes, and the substrate was added the next day, followed by centrifugation to precipitate the bacteria. The supernatant of the reaction solution was transferred to 96-well plates. The absorbance of 4-aminoantipyrine and potassium ferricyanide at 510 nm was measured using an endpoint colorimetric method to detect phenolic products. The oxygenase activity (i.e., enzyme activity) of the engineered proteins was measured by detecting substrate loss and product production using high-performance liquid chromatography. Protein activities were compared by calculating the conversion rate. The results are shown in Table 1.

[0090] Conversion rate = (Initial substrate peak area - Substrate peak area after reaction) / Initial substrate peak area * 100%

[0091] Table 1. Transformation rates of each mutant

[0092]

[0093]

[0094] Note: Reaction condition 1: Incubate bacteria overnight, add substrate compound A and IPTG and react overnight; Reaction condition 2: Incubate bacteria overnight, add 8 times the dose of substrate compound A from reaction condition 1 on the second day and react for 3 hours.

[0095] Based on the results from the high-throughput liquid chromatography system, representative engineered proteins were selected for protein purification. Further protein characterization, including Km, Vmax, and Kcat, was performed using eight engineered proteins from Table 2. Protein purification was performed using conventional Ni column affinity chromatography. Protein extract purity was assessed by SDS-PAGE analysis. Enzyme activity was measured after protein concentration was determined by the BCA method, with purified wild-type enzymes as controls. Enzyme kinetics were determined using compound A at concentrations of 0, 5, 10, 20, 50, 200, 500, or 1000 μM. Table 2 shows the measured Km, Vmax, Kcat, and Kcat / Km for the eight proteins using compound A as a substrate. These enzyme kinetic parameters for the eight engineered proteins indicate that protein engineering can significantly improve the enzyme activity, specifically Km and Kcat.

[0096] Table 2. Determination results of engineered proteins

[0097]

[0098] Note: N / D indicates that the enzyme activity is too low to determine its enzyme kinetic parameters.

[0099] Example 2. Expression of engineered proteins in rice

[0100] Plant transformation vectors were constructed, each containing a recombinant DNA molecule encoding an engineered protein (SEQ ID NO: 42 / 38 / 82 / 86 / 102 / 106 / 126 / 138 / 142) optimized for monocotyledonous expression. These vectors were used to transform rice (Jinjing 818) callus using *Agrobacterium tumefaciens* and standard methods known in the art. Different concentrations of the hormone herbicide compound B were added to the rice seedlings during the rooting stage to test hormone resistance. The test results for control Jinjing 818 plants and transgenic Jinjing 818 plants containing the SEQ ID NO: 42 protein-coding gene after 19 days of adding 0.5 μM and 1 μM compound B to the culture medium are shown below. Figure 1 As shown. The test results of control Jinjing 818 plants and transgenic Jinjing 818 plants containing the protein-coding gene SEQ ID NO:38 / 82 / 86 / 102 / 106 / 126 / 138 / 142 after adding 1 μM compound B to the culture medium are shown in the figure. Figure 5-11 As shown. Compared to wild-type plants, the Jinjing 818 plant containing the protein-coding gene of SEQ ID NO:42 / 38 / 82 / 86 / 102 / 106 / 126 / 138 / 142 showed superior herbicide resistance, indicating that plants expressing engineered proteins showed tolerance to herbicide compound B when screened in at least 1 μM compound B medium.

[0101] The resulting regenerated T0 generation transgenic plantlets were then grown in a greenhouse and sprayed with compound B at approximately the two-leaf-one-heart stage. The degree of plant resistance was recorded and evaluated after spraying. The test results for control Jinjing 818 plants and transgenic Jinjing 818 plants containing the SEQ ID NO:42 protein-coding gene were compared after application of 20g, 40g, 60g, 80g, 100g, and 120g / mu (1 mu = 1 / 15 hectare) of compound B for the corresponding number of days (DAT). Figure 2 As shown, compared with wild-type plants, the Jinjing 818 plant containing the protein-coding gene of SEQ ID NO:42 showed superior herbicide resistance, indicating that the plant expressing the engineered protein showed tolerance to the herbicide compound B when sprayed with at least 120 g / acre of compound B on its leaves.

[0102] In summary, in both the T0 generation culture medium dosing test and the greenhouse spraying test, the vector containing the engineered protein (SEQ ID NO: 42 / 38 / 82 / 86 / 102 / 106 / 126 / 138 / 142) recombinant DNA molecule showed significant tolerance compared to the wild type.

[0103] The obtained regenerated T0 generation transgenic plantlets were grown in a greenhouse and sprayed with the compound quizalofop-P-ethyl at the approximately two-leaf-one-heart stage. The degree of plant resistance was recorded and evaluated after spraying. The test results for control Jinjing 818 plants and transgenic Jinjing 818 plants containing the protein-coding gene of SEQ ID NO:46 after application of 0 g, 5 g, and 10 g / mu of quizalofop-P-ethyl 20 DAT are as follows: Figure 12 As shown, compared with wild-type plants, the Jinjing 818 plant containing the protein-coding gene of SEQ ID NO:46 showed better resistance to herbicides. This indicates that the plant expressing the engineered protein showed tolerance to the compound quizalofop-p-ethyl when sprayed with at least 10 g / acre of the compound quizalofop-p-ethyl as a foliar spray.

[0104] The transformed T0 transgenic plants were grown in a greenhouse. Seeds of T1 generation rice plants generated from all constructs were collected. The resistance levels of each construct were compared by soaking the T1 generation seeds in water with 0.3 μM compound B. Root lengths of each construct were also measured for comparison. Figure 13 As shown, after soaking seeds with 0.3 μM compound B for 20 days, the T1 generation transgenic Jinjing 818 seeds containing different protein-coding genes all showed better herbicide resistance than wild-type Jinjing 818, exhibiting longer roots. This indicates that plants expressing engineered proteins showed tolerance to the herbicide compound B during hydroponic seed soaking screening with 0.3 μM compound B.

[0105] Example 3. Expression of engineered proteins in Arabidopsis thaliana

[0106] Engineered proteins were selected for Arabidopsis transformation and plant analysis. The DNA constructs were used to transform Arabidopsis using *Agrobacterium tumefaciens* and standard methods known in the art.

[0107] Transformed T0 transgenic plantlets were grown in a greenhouse. After approximately 60 days of growth, seeds of T1 generation Arabidopsis thaliana plants produced from all constructs were harvested. Transgenic T1 generation Arabidopsis thaliana plants were screened by adding HYG to the T1 generation culture medium. T1 plants were then self-pollinated to produce T2 generation Arabidopsis thaliana seeds. Compound A was added to the culture medium to screen for T2 generation Arabidopsis thaliana seeds from all constructs that exhibited a unique event indicating successful T1 generation screening, such as… Figure 3 , 14As shown in Figure 22, after screening with 0.15 μM compound A for the corresponding number of days, T2 generation transgenic Arabidopsis seeds containing the protein-coding gene of SEQ ID NO:46 / 42 / 78 / 82 / 86 / 98 / 102 / 126 / 138 / 142 showed superior herbicide resistance compared to wild-type Arabidopsis, exhibiting longer roots and larger leaves. This indicates that plants expressing engineered proteins showed tolerance to herbicide compound A during screening with 0.15 μM compound A medium.

[0108] In addition, T2 generation Arabidopsis thaliana plants containing unique events selected through T1 generation screening were tested by spraying compound B on some constructs. The test results for control Arabidopsis thaliana transgenic with the RdpA wild-type gene and Arabidopsis thaliana plants containing the protein-coding gene of SEQ ID NO:42 were as follows: Figure 4 As shown, compared to wild-type plants, Arabidopsis plants containing the protein-coding gene of SEQ ID NO:42 exhibited superior herbicide resistance, indicating that plants expressing engineered proteins showed tolerance to herbicide compound B when foliar sprayed at 40 g / acre.

[0109] Example 4. Expression of engineered proteins in soybeans

[0110] Engineered proteins were selected for soybean transformation and plant analysis. The DNA constructs were used to transform soybeans using *Agrobacterium tumefaciens* and standard methods known in the art.

[0111] The transformed T0 transgenic plantlets were grown in a greenhouse. 10g of compound C was sprayed onto the T0 transgenic plantlets for testing. (For example...) Figure 23 As shown, transgenic soybeans containing the protein-coding gene of SEQ ID NO:46 exhibited significant resistance compared to wild-type soybeans. Seeds of T1 generation soybean plants generated from all construct transformations were collected, and 10g of compound C was sprayed onto the T1 seedlings for testing. Figure 24 As shown, transgenic soybeans containing the protein-coding gene of SEQ ID NO:46 still exhibit significant resistance compared to wild-type soybeans. Spraying some T1 generation transgenic plants with 10g, 20g, 40g, and 80g of compound C, as shown... Figure 25-26 Transgenic soybeans containing the protein-coding gene SEQ ID NO:42 exhibit superior resistance to compound C compared to wild-type soybeans at a minimum of 40g.

[0112] Example 5. Expression of engineered proteins in maize

[0113] Engineered proteins were selected for maize transformation and plant analysis. The DNA constructs were used to transform maize using *Agrobacterium tumefaciens* and standard methods known in the art.

[0114] The transformed T0 transgenic plantlets were grown in a greenhouse. The T0 transgenic plantlets were sprayed with 150g, 250g, 400g, 600g, and 800g of 30% glyphosate compound C(25+5)ME for testing. Figure 27 As shown, wild-type plants died at all concentration treatments, while transgenic small plants showed no obvious phytotoxicity at concentrations below 600g (data below 600g are represented in the figure). Figure 27 (As shown on the far left); at 600g, the stem base swells slightly, overall plant growth is significantly inhibited, and the plant is in normal condition; at 800g, the stem base swells significantly, overall plant growth is further inhibited, and the leaves become lighter and duller. Therefore, it can be concluded that transgenic maize containing the protein-coding gene of SEQ ID NO:46 exhibits superior resistance to glyphosate at 600g with 30% glyphosate compound C(25+5)ME compared to wild-type maize.

[0115] Furthermore, numerous tests have revealed that introducing the recombinant DNA molecule described in this invention into model plants such as Arabidopsis thaliana and Brachypodium distichum resulted in increased resistance to pyridinyl oxyacid herbicides at corresponding levels. Therefore, it can be concluded that transgenic introduction into other plants, such as grain crops, legumes, oil crops, fiber crops, fruit crops, root and tuber crops, vegetable crops, flowering crops, medicinal crops, raw material crops, forage crops, sugar crops, beverage crops, turfgrass, trees, and nut crops, will also produce corresponding resistance traits, demonstrating significant industrial value.

Claims

1. A recombinant DNA molecule comprising a nucleic acid sequence encoding a polypeptide, said polypeptide having, compared to the RdpA amino acid sequence shown in SEQ ID NO: 1, a mutation in which leucine is mutated to histidine at amino acid position 82, wherein said amino acid sequence of the polypeptide is selected from the group consisting of: SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, and 142.

2. The recombinant DNA molecule of claim 1, wherein the nucleic acid sequence is selected from the group consisting of: SEQ ID NO: 3, 4, 5, 7, 8, 9, 11, 12, 13, 15, 16, 17, 19, 20, 21, 23, 24, 25, 27, 28, 29, 31, 32, 33, 35, 36, 37, 39, 40, 41, 43, 44, 45, 47, 48, 49, 51, 52, 53, 55, 56, 57, 59, 60, 61, 63, 64, 65, 67, 68, 69, 71, 72, 73, 75, 76, 77, 79, 80, 81, 83, 84, 85, 87, 88, 8 9, 91, 92, 93, 95, 96, 97, 99, 100, 101, 103, 104, 105, 107, 108, 109, 111, 112, 113, 115, 116, 117, 119, 120, 121, 123, 124, 125, 127, 128, 129, 131, 132, 133, 135, 136, 137, 139, 140, 141 and 143-181, and nucleic acid sequences that encode the same amino acid sequence as the sequence shown due to the degeneracy of the genetic code.

3. The recombinant DNA molecule of claim 1, wherein the recombinant DNA molecule is operatively linked to a heterologous promoter that is functional in plant cells.

4. The recombinant DNA molecule of claim 3, wherein the recombinant DNA molecule is further operatively ligated to a DNA molecule encoding a chloroplast transport peptide.

5. A DNA construct comprising a heterologous promoter operatively linked to a recombinant DNA molecule as claimed in any one of claims 1-4, which is functional in plant cells, and the recombinant DNA molecule as claimed in any one of claims 1-4.

6. The DNA construct of claim 5, further comprising a DNA molecule encoding a chloroplast transport peptide operably linked to the recombinant DNA molecule.

7. The DNA construct of claim 5, wherein the DNA construct is present in the genome of the transgenic plant.

8. A polypeptide having, compared with the amino acid sequence of RdpA as shown in SEQ ID NO: 1, a mutation in which leucine is mutated to histidine at amino acid position 82, wherein the amino acid sequence of the polypeptide is selected from the group consisting of: SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138 and 142.

9. A method for conferring herbicide tolerance on a plant, seed, cell, or plant part, the method comprising expressing the polypeptide of claim 8 in the plant, seed, cell, or plant part, wherein the herbicide is selected from: quizalofop-P-ethyl, compound A, compound B, and / or compound C; wherein, Compound A is a compound of the general formula I Compound A is a compound in which substituent A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, and Q represents methyl, and the carbon atom at * is a chiral center with an R configuration; compound B is a compound of the general formula I-1. Compound I-1 is a compound in which substituent A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, Q represents methyl, X represents O, M represents methyl, and the carbon atom at * is a chiral center with an R configuration; Compound C is a compound of general formula I-1 in which substituent A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, Q represents methyl, X represents O, M represents tetrahydrofuran-2-ylmethyl, and the carbon atom at * is a chiral center with an R configuration.

10. The method of claim 9, wherein the plant, seed, cell, or plant part comprises a DNA construct comprising a heterologous promoter operatively linked to a recombinant DNA molecule functional in a plant cell and the recombinant DNA molecule comprising a nucleic acid sequence encoding the polypeptide of claim 8.

11. A method for producing herbicide-tolerant transgenic plants, the method comprising transforming plant cells or tissues with a recombinant DNA molecule as described in any one of claims 1-4 or a DNA construct as described in any one of claims 5-7, and regenerating herbicide-tolerant transgenic plants from said transformed plant cells or tissues, said herbicide being selected from: quizalofop-P-ethyl, compound A, compound B, and / or compound C; wherein, Compound A is a compound of the general formula I Compound A is a compound in which substituent A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, and Q represents methyl, and the carbon atom at * is a chiral center with an R configuration; compound B is a compound of the general formula I-1. Compound I-1 is a compound in which substituent A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, Q represents methyl, X represents O, M represents methyl, and the carbon atom at * is a chiral center with an R configuration; Compound C is a compound of general formula I-1 in which substituent A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, Q represents methyl, X represents O, M represents tetrahydrofuran-2-ylmethyl, and the carbon atom at * is a chiral center with an R configuration.

12. A method for controlling weeds in a plant growth area, the method comprising contacting a plant growth area comprising plants or seeds with at least one herbicide selected from the group consisting of pyridyloxy acid herbicides, said plants or seeds comprising a recombinant DNA molecule as described in any one of claims 1-4 and resistant to said at least one herbicide, said herbicide being selected from: quizalofop-P-ethyl, compound A, compound B and / or compound C; wherein, Compound A is a compound of the general formula I Compound A is a compound in which substituent A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, and Q represents methyl, and the carbon atom at * is a chiral center with an R configuration; compound B is a compound of the general formula I-1. Compound I-1 is a compound in which substituent A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, Q represents methyl, X represents O, M represents methyl, and the carbon atom at * is a chiral center with an R configuration; Compound C is a compound of general formula I-1 in which substituent A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, Q represents methyl, X represents O, M represents tetrahydrofuran-2-ylmethyl, and the carbon atom at * is a chiral center with an R configuration.

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