WO2024230371A1 - Herbicide-tolerant gene and method of using same - Google Patents

Abstract

Provided is a recombinant DNA molecule encoding an enzyme that degrades a synthetic hormone herbicide and/or an ACCase inhibitor herbicide. Further provided are a transgenic plant, transgenic plant part, seed, cell and plant part containing the recombinant DNA molecule, and a method of using the transgenic plant, transgenic plant part, seed, cell and plant part.

Description

Herbicide tolerance genes and methods of use thereof Technical Field

The present invention relates generally to the field of biotechnology. More specifically, the present invention relates to recombinant DNA molecules encoding enzymes that degrade synthetic hormones and/or ACCase inhibitor herbicides. The present invention also relates to transgenic plants, parts, seeds, cells and plant parts containing the recombinant DNA molecules, and methods of using the same.

Background Art

Crop production often utilizes transgenic traits produced using biotechnology methods. Heterologous genes (also referred to as transgenics) are introduced into plants to produce transgenic traits. The expression of transgenes in plants imparts desired traits to plants, such as herbicide tolerance. Examples of transgenic herbicide tolerance traits include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance. With the increase of weed species resistant to the most commonly used herbicides, new herbicide tolerance traits are needed in the field. Particularly interesting herbicides are synthetic hormone herbicides. Synthetic hormone herbicides provide control of a range of glyphosate-resistant weeds, thereby producing traits that confer these herbicide tolerances, particularly for use in crop systems combined with other herbicide tolerance traits.

The herbicide-eating Sphingobium herbicidovorans strain MH, isolated from a dichloroprop-degrading soil sample, was identified as being able to cleave the ether bonds of various phyenoxyalkanoic acid herbicides, thereby utilizing them as a sole carbon and energy source for its growth (HPE Kohler, Journal of Industrial Microbiology & Biotechnology (1999) 23:336-340). The catabolism of the herbicides is carried out by two different enantioselective α-ketoglutarate-dependent dioxygenases, RdpA (R-2,4-dichloropropionate dioxygenase) and SdpA (S-2,4-dichloropropionate dioxygenase). (A Westendorf, et al., Microbiological Research (2002) 157:317-322; Westendorf, et al., Acta Biotechnologica (2003) 23(1):3-17). RdpA has been expressed in Escherichia coli (GenBank accession AF516752 (DNA) and AAM90965 (protein)) and Delftia acidovorans (GenBank accession NG_036924 (DNA) and YP_009083283 (protein)) (T. A. Mueller, et al., Applied and Environmental Microbiology (2004) 70(10):6066-6075). 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). Improving the activity of the RdpA enzyme using protein engineering techniques to produce proteins for transgenic plants will allow higher herbicide application rates, thereby improving transgenic crop safety and weed control measures.

Brief description of the invention

The present invention provides a recombinant DNA molecule comprising a nucleic acid sequence encoding a polypeptide having 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% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133 or 137.

In a specific embodiment, the nucleic acid sequence is selected from the group consisting of: SEQ ID NO: 2, 3, 4, 6, 7, 8, 10, 11, 12, 14, 15, 16, 18, 19, 20, 22, 23, 24, 26, 27, 28, 30, 31, 32, 34, 35, 36, 38, 39, 40, 42, 43, 44, 46, 47, 48, 50, 51, 52, 54, 55, 56, 58, 59, 60, 62, 63, 64, 66, 67, 68, 70, 71, 72, 74, 75, 76, 78, 79, 80, 82, 83, 84, 86, 87, 88, 90, 91, 92, 94, 95, 96, 98, 99, 100, 102, 103, 104, 106, 107, 108, 110, 111, 112, 114, 115, 116, 118, 119, 120, 122, 123, 124, 126, 127 , 128, 130, 131, 132, 134, 135, 136, 138, 139, 140, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 or 167, and nucleic acid sequences that encode the same amino acid sequence as the shown sequence due to the degeneracy of the genetic code.

In a specific embodiment, the recombinant DNA molecule is operably linked to a heterologous promoter functional in plant cells.

In another specific embodiment, the recombinant DNA molecule is further operably linked to a DNA molecule encoding a chloroplast transit peptide.

The present invention also provides a DNA construct comprising a heterologous promoter functional in plant cells operably linked to the recombinant DNA molecule.

In a specific embodiment, it further comprises a DNA molecule encoding a chloroplast transit peptide operably linked to the recombinant DNA molecule.

In another specific embodiment, wherein said DNA construct is present in the genome of the transgenic plant.

The present invention provides a plant, seed, plant tissue, plant part or cell, which comprises the recombinant DNA molecule.

In a specific embodiment, wherein the plant, seed, plant tissue, plant part or cell comprises tolerance to at least one herbicide selected from the group consisting of: synthetic hormone herbicides, ACCase inhibitor herbicides.

The present invention also provides a plant, seed, plant tissue, plant part or cell, which comprises the DNA construct.

The present invention also provides a plant, seed, plant tissue, plant part or cell, which comprises the polypeptide encoded by the recombinant DNA molecule.

The present invention also provides a polypeptide having at least 98% or at least 99% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133 or 137.

In a specific embodiment, the polypeptide has oxygenase activity against at least one herbicide selected from the group consisting of synthetic hormone herbicides and ACCase inhibitor herbicides.

The present invention also provides a method for conferring herbicide tolerance to a plant, seed, cell or plant part, the method comprising expressing the polypeptide in the plant, seed, cell or plant part.

In a specific embodiment, the plant, seed, cell or plant part comprises a DNA construct comprising a heterologous promoter functional in plant cells operably linked to a recombinant DNA molecule encoding the polypeptide.

In another specific embodiment, wherein said plant, seed, cell or plant part comprises tolerance to at least one herbicide selected from the group consisting of: synthetic hormone herbicides, ACCase inhibitor herbicides.

The present invention also provides a method for producing herbicide-tolerant transgenic plants, which comprises transforming plant cells or tissues with the recombinant DNA molecule or the DNA construct, and regenerating herbicide-tolerant transgenic plants from the transformed plant cells or tissues.

In a specific embodiment, the herbicide-tolerant transgenic plant comprises tolerance to at least one herbicide selected from the group consisting of synthetic hormone herbicides and ACCase inhibitor herbicides.

The present invention also provides a method for controlling weeds in a plant growth area, the method comprising contacting a plant growth area including plants or seeds with at least one herbicide selected from the group consisting of synthetic hormone herbicides and ACCase inhibitor herbicides, wherein the plants or seeds contain the recombinant DNA molecule and are tolerant to the at least one herbicide.

Compared with the wild-type RdpA protein, the engineered protein of the present invention not only retains the original resistance to synthetic hormone herbicides such as phenoxycarboxylic acids and/or ACCase inhibitor herbicides, but also increases the resistance to pyridyloxy acid herbicides, thereby broadening the resistance spectrum to herbicides.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions and methods are provided to better define the present invention and guide those skilled in the art to implement the present invention. Unless otherwise specified, terms should be understood according to conventional usage by those skilled in the relevant art.

Engineered proteins and recombinant DNA molecules

The present 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 usually found in nature and produced by human intervention. "Engineered proteins" are proteins whose polypeptide sequences are 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 are provided herein as SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133 and 137.

The engineered proteins provided by the present invention are enzymes with oxygenase activity. As used herein, the term "oxygenase activity" means the ability to oxidize a substrate by transferring oxygen from molecular oxygen to a substrate, a byproduct, or an intermediate. The oxygenase activity of the engineered proteins provided by the present invention can inactivate one or more of the synthetic hormone herbicides and/or the ACCase inhibitor herbicides.

As used herein, "wild-type" means naturally occurring. As used herein, a "wild-type DNA molecule," "wild-type polypeptide," or "wild-type protein" is a naturally occurring DNA molecule, polypeptide, or protein, i.e., a DNA molecule, polypeptide, or protein that pre-exists in nature. The wild-type version of a polypeptide, protein, or DNA molecule can be useful for comparison with an engineered protein or gene. The wild-type version of a protein or DNA molecule can be useful as a control in an experiment.

As used herein, "control" means an experimental control designed for comparison purposes. For example, a control plant in a transgenic plant analysis is a plant of the same type as the experimental plant (i.e., the plant to be tested) but without the transgenic insert, recombinant DNA molecule, or DNA construct of the experimental plant. An example of a control plant suitable for comparison with a transgenic corn plant is non-transgenic LH244 corn (U.S. Pat. No. 6,252,148) and an example of a control plant suitable for comparison with a transgenic soybean plant is non-transgenic A3555 soybean (U.S. Pat. No. 7,700,846).

As used herein, the term "recombinant" refers to a non-natural DNA, polypeptide or protein that is the result of genetic engineering and therefore is not normally found in nature and is produced by human intervention. A "recombinant DNA molecule" is a DNA molecule that contains a DNA sequence that does not occur naturally and is therefore the result of human intervention, such as a DNA molecule encoding an engineered protein. Another example is a DNA molecule composed of a combination of at least two DNA molecules that are heterologous to each other (e.g., a DNA molecule encoding a protein and an operably linked heterologous promoter). An example of a recombinant DNA molecule is a DNA molecule comprising at least one sequence selected from the group consisting of: SEQ ID NO:2,3,4,6,7,8,10,11,12,14,15,16,18,19,20,22,23,24,26,27,28,30,31,32,34,35,36,38,39,40,42,43,44,46,47,48,50,51,52,54,55,56, 58, 59, 60, 62, 63, 64, 66, 67, 68, 70, 71, 72, 74, 75, 76, 78, 79, 80, 82, 83, 84, 86, 87, 88, 90, 91, 92, 94, 95, 96, 98, 99, 100, 102, 103, 104, 106, 107, 108, 110, 111, 112, 114, 115, 116, 118, 119, 120, 122, 123, 124, 126, 127, 128, 130, 131, 132, 134, 135, 136, 138, 139, 140, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 and 167.

A "recombinant polypeptide" or "recombinant protein" is a polypeptide or protein that comprises an amino acid sequence that does not occur in nature and, thus, is the result of human intervention, eg, an engineered protein.

The term "transgenic" refers to a DNA molecule artificially incorporated into the genome of an organism as a result of human intervention (such as by a plant transformation method). As used herein, the term "transgenic" means comprising a transgene, for example, a "transgenic plant" refers to a plant comprising a transgene in its genome, and a "transgenic trait" refers to a characteristic or phenotype transmitted or conferred by the presence of a transgene incorporated into the plant genome. As a result of this 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 the wild-type plant. The transgenic plant of the present invention comprises the recombinant DNA molecules and engineered proteins provided by the present invention.

As used herein, the term "heterologous" refers to the relationship between two or more substances that are derived from different sources and are therefore not generally related in nature. For example, a recombinant DNA molecule encoding a protein is heterologous with respect to an operably linked promoter if such a combination does not normally occur in nature. In addition, a particular recombinant DNA molecule may be heterologous with respect to a cell or organism into which it is inserted when it does not naturally occur in the particular cell or organism.

As used herein, the term "DNA molecule encoding a protein" or "DNA molecule encoding a polypeptide" refers to a DNA molecule comprising a nucleotide sequence encoding a protein or polypeptide. "Sequence encoding a protein" or "sequence encoding a polypeptide" means a DNA sequence encoding a protein or polypeptide. "Sequence" means the sequential arrangement of nucleotides or amino acids. The boundaries of a sequence encoding a protein or a sequence encoding a polypeptide are usually determined by a translation start codon at the 5'-end and a translation stop codon at the 3'-end. A molecule encoding a protein or a molecule encoding a polypeptide may comprise 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" mean that a protein or polypeptide is produced by a process of transcribing a DNA molecule into a messenger RNA (mRNA) and translating mRNA into a polypeptide chain (which can ultimately fold into a protein). A DNA molecule encoding a protein or a DNA molecule encoding a polypeptide can be operably linked to a heterologous promoter in a DNA construct for expression of a protein or polypeptide in a cell transformed with a recombinant DNA molecule. As used herein, "operably linked" refers to two DNA molecules that are linked in a manner such that one DNA molecule can affect the function of the other DNA molecule. Operably 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 protein-encoding DNA molecule or a polypeptide-encoding DNA molecule in a DNA construct, wherein the two DNA molecules are arranged such that the promoter can affect the expression of a transgene.

As used herein, "DNA construct" is a recombinant DNA molecule comprising two or more heterologous DNA sequences. DNA constructs are suitable for transgenic expression and can be included in vectors and plasmids. DNA constructs can be used in vectors for the purpose of transformation (i.e., introducing heterologous DNA into host cells) to produce transgenic plants and cells, and therefore can also be included 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 such as those shown in the sequence table can be inserted into a vector as part of a construct, and the construct has a recombinant DNA molecule that is operably connected to a promoter, and the promoter works in plants to drive the expression of engineered proteins encoded by the recombinant DNA molecule. Methods for constructing DNA constructs and vectors are well known in the art. The components of a DNA construct or a vector comprising a DNA construct generally include, but are not limited to, one or more of the following: a suitable promoter for expressing operably connected DNA, an operably connected non-human DNA molecule encoding a protein, and a 3' untranslated region (3'-UTR). Promoters suitable for practicing the present invention include promoters that work in plants to express operably connected polynucleotides. Such promoters are varied and well known in the art and include inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated and/or spatiotemporally regulated. 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 transit peptide (CTP) and one or more selectable marker transgenes.

The DNA construct of the present invention may include a CTP molecule operably linked to a protein-encoding DNA molecule provided by the present invention. CTPs suitable for practicing the present invention include those for promoting the localization of engineered protein molecules in cells. By promoting the localization of proteins in cells, CTP can increase the accumulation of engineered proteins, protect them from proteolytic degradation, enhance herbicide tolerance levels, and thereby reduce the level of damage after 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), corn 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).

The recombinant DNA molecules of the present invention can be synthesized and modified in whole or in part by methods known in the art, particularly where it is desired to provide sequences suitable for DNA manipulation (e.g., restriction enzyme recognition sites or recombination-gene cloning sites), plant preferred sequences (e.g., plant codon usage or Kozak consensus sequences), or sequences suitable for DNA construct design (e.g., spacer or linker sequences). The present invention includes recombinant DNA molecules and engineered proteins having at least about 80% (percent) 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 any of the recombinant DNA molecules or engineered protein sequences provided herein, such as with a recombinant DNA molecule comprising a sequence selected from the group consisting of: SEQ ID NO:2,3,4,6,7,8,10,11,12,14,15,16,18,19,20,22,23,24,26,27,28,30,31,32,34,35,36,38,39,40,42,43,44,46,47,48,50,51,52,54,55,56, 58, 59, 60, 62, 63, 64, 66, 67, 68, 70, 71, 72, 74, 75, 76, 78, 79, 80, 82, 83, 84, 86, 87, 8 8, 90, 91, 92, 94, 95, 96, 98, 99, 100, 102, 103, 104, 106, 107, 108, 110, 111, 112, 114, 115, 116, 118, 119, 120, 122, 123, 124, 126, 127, 128, 130, 131 ,132,134,135,136,138,139,140,142,143,144,145,146,147,148,149,150,151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 and 167.

As used herein, the term "percent sequence identity" or "% sequence identity" refers to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of a reference ("query") sequence (or its complementary strand) compared to a test ("subject") sequence (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide or amino acid insertions, deletions or gaps totaling less than 20% of those of the reference sequence within the comparison window). Optimal sequence alignment for aligning a comparison window is well known to those skilled in the art and can be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and by computerized implementations of these algorithms, such as the Optimal Alignment Algorithm (AAL) using default parameters. Wisconsin (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) available as part of GAP, BESTFIT, FASTA and TFASTA. The "identity score" of the aligned fragment of the test sequence and the reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence fragment, i.e., the entire reference sequence or a smaller limited portion of the reference sequence. The percentage of sequence identity is expressed as the identity score multiplied by 100. The comparison of one or more sequences can be for the full-length sequence or a portion thereof, or for longer sequences.

Engineered proteins can be produced by altering (i.e., modifying) wild-type proteins to produce new proteins with novel combinations of useful protein characteristics (e.g., altered Vmax, Km, substrate specificity, substrate selectivity, and protein stability). The modification can be made at a specific amino acid position in the protein, and can be a substitution of the amino acid found at the position in nature (i.e., in the wild-type protein) with a different amino acid. The amino acid sequence of the wild-type protein RdpA suitable for protein engineering is shown in SEQ ID NO: 1. An engineered protein is designed that has at least about 92% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133 and 137, and comprises at least one of these amino acid mutations. Thus, the engineered proteins provided herein provide novel proteins having 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 against one or more herbicides or improved protein stability, compared to a similar wild-type protein or any combination of such characteristics. In one embodiment, the present invention provides engineered proteins and recombinant DNA molecules encoding the same, which have 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: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133 and 137. Amino acid mutations can be performed as single amino acid substitutions in the protein or in combination with one or more other mutations (such as one or more other amino acid substitutions, deletions or additions). Mutations can be performed as described herein or by any other method known to those skilled in the art.

Transgenic plants

One aspect of the present invention comprises transgenic plant cells, transgenic plant tissues, transgenic plants and transgenic seeds comprising recombinant DNA molecules and engineered proteins provided by the invention. These cells, tissues, plants and seeds comprising recombinant DNA molecules and engineered proteins show one or more herbicide tolerance in synthetic hormone herbicides, ACCase inhibitor herbicides.

Suitable methods for transforming host plant cells for the present invention actually include any method that can introduce DNA into cells (for example, wherein the recombinant DNA construct is stably integrated into the plant chromosome) and are known in the art. The exemplary and widely used method for introducing the recombinant DNA construct into plants is the Agrobacterium transformation system, which is well known to those skilled in the art. Transgenic plants can be regenerated from the transformed plant cells by the method for plant cell culture. About the homozygous transgenic plant (that is, two allele copies of transgenic) of transgenic plants can be by self-pollination (selfing) of the transgenic plant comprising a single transgenic allele with itself (for example R0 plant) to produce R1 seeds. One quarter of the R1 seeds produced will be homozygous for transgenic. Usually using SNP determination, DNA sequencing or allowing the heat amplification determination of the difference between heterozygote and homozygote to test the zygosity of the plant grown from the R1 seeds of germination, is called zygosity determination.

The plants, seeds, plant parts, plant tissues and cells provided by the present invention show herbicide tolerance to one or more of synthetic hormone herbicides and ACCase inhibitor herbicides, especially to pyridyloxy acid compounds as shown in formula I and their salts and ester derivatives.

In the present invention, "synthetic hormone herbicides" are substances that have herbicidal activity themselves or are used in combination with other herbicides and/or additives that can change their effects. They belong to plant hormone interfering herbicides and are well known in the art. For example, they include at least one of the following active ingredients or their derivatives:

(1) Pyridine carboxylic acids: amiloride, fluroxypyr, fluroxypyr ethyl ester, aminopyralid, clopyralid, triclopyr, fluroxypyr ester, fluroxypyr ester, pyridinyloxy acid compounds as shown in formula I and their salts and ester derivatives;

(2) Benzoic acids: dicamba, fenthion, chlorpyrifos, naphthamide, etc.

(3) Phenoxycarboxylic acids: 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-dichlorophenoxybutyric acid (2,4-D butyric acid), 2,4-D isopropionic acid, chloramic acid, dimethyltetrachlorobenzene, dimethyltetrachloroisopropionic acid, dimethyltetrachlorobutyric acid, etc.

(4) Quinoline carboxylic acids: dichloroquinoline acid, clomaquinoline acid, etc.

(5) Others: herbicide, etc.

The pyridyloxy acid compound and its salt and ester derivatives as shown in formula I,

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

C represents hydrogen, halogen, C1-C6 alkyl, halogenated C1-C6 alkyl;

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, furyl, thienyl, thiazolyl, pyridyl, pyrimidyl, and unsubstituted or substituted C1-C6 alkyl Phenyl which is unsubstituted or substituted by at least one of C1-C6 alkyl, halogenated C1-C6 alkyl, halogen and C1-C6 alkoxy;

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

The salt is a metal salt, an ammonium salt NH ₄ ⁺ , a primary amine salt RNH ₂ , a secondary amine salt (R) ₂ NH, a tertiary amine salt (R) ₃ N, a quaternary amine salt (R) ₄ N ⁺ , a morpholine salt, a piperidine salt, a pyridine salt, an aminopropylmorpholine salt, a Jeff amine D-230 salt, a salt of 2,4,6-tris(dimethylaminomethyl)phenol and sodium hydroxide, a C1-C14 alkyl sulfonium salt, a C1-C14 alkylsulfonium oxide salt, a C1-C14 alkylphosphonium salt, a C1-C14 alkoxidephosphonium salt;

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

The ester is Wherein, X represents O or S;

M represents C1-C18 alkyl, halogenated C1-C8 alkyl, C3-C6 cycloalkyl, C2-C6 alkenyl, halogenated 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 alkenyloxycarbonyl C1-C2 alkyl, -(C1-C2 alkyl)-Z, Tetrahydrofuranyl, pyridyl, naphthyl, furanyl, thienyl, and unsubstituted or C1-C6 alkyl-substituted Phenyl which is unsubstituted or substituted by C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkylamino, halogen or C1-C6 alkoxy;

Z stands for Tetrahydrofuranyl, pyridinyl, Thiphenyl, furyl, naphthyl, and phenyl which is unsubstituted or substituted with at least one of C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, cyano and halogen;

R ₃ each independently represents a C1-C6 alkyl group;

R ₄ , R ₅ , and R ₆ independently represent hydrogen, C1-C6 alkyl, or C1-C6 alkoxycarbonyl;

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

In one embodiment, the compounds of the general formula I and I-1 are both in R configuration (the carbon atom at the * is a chiral center). In another embodiment, in the compound of the general formula I, A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, Q represents methyl, and is in R configuration (the carbon atom at the * is a chiral center) (i.e., compound A); in the compound of the general formula 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 is in R configuration (the carbon atom at the * is a chiral center) (i.e., compound B); or in the compound of the general formula 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 And it is of R configuration (i.e. compound C) (the carbon atom at the * is the chiral center).

In the present invention, "ACCase inhibitor herbicides" refer to herbicides that target acetyl-CoA carboxylase, which are well known in the art, and include, for example, at least one of the following active ingredients or their derivatives:

(1) Aryloxyphenoxypropionic acid: quizalofop-p-butyl, clodinafop-butyl, cyhalofop-butyl, diclofop-butyl, fenoxaprop-butyl, fluazifop-butyl, fluazifop-butyl, methomyl, oxadiazine, quinazolin, etc.

(2) Cyclohexenone: cypermethrin, clethodim, cypermethrin, butyclothiocarb, cypermethrin, sethoxydim, pyraclostrobin, trimethoprim-butyl, etc.

(3) Benzopyridines: pinoxazoline, etc.

In the context of the present specification, if the abbreviation of the common name of the active compound is used, all customary derivatives, such as esters and salts, and isomers, in particular optical isomers, in particular one or more commercially available forms are included in each case. If the common name denotes an ester or a salt, all other customary derivatives, such as other esters and salts, free acids and neutral compounds, and isomers, in particular optical isomers, in particular one or more commercially available forms are also included in each case. The chemical name of a compound given denotes at least one compound covered by the common name, generally a preferred compound. For example, 2,4-D or 2,4-D butyric acid derivatives include but are not limited to: 2,4-D or 2,4-D butyric acid salts such as sodium salt, potassium salt, dimethylammonium salt, triethanolammonium salt, isopropylamine salt, choline, etc., and 2,4-D or 2,4-D butyric acid esters such as methyl ester, ethyl ester, butyl ester, isooctyl ester, etc.; dimethyltetrachloro derivatives include but are not limited to: dimethyltetrachloro sodium salt, potassium salt, dimethylammonium salt, isopropylamine salt, etc., and dimethyltetrachloromethyl ester, ethyl ester, isooctyl ester, ethylthioester, etc.

Weed killer herbicide can be applied to the plant growth area comprising plant and seed provided by the invention as a method for controlling weeds. Plant and seed provided by the invention comprise a weed killer herbicide tolerance trait, and therefore tolerate the application of one or more synthetic hormone herbicides, ACCase inhibitor herbicides. When applying weed killer herbicide, the plant growth area may or may not include weed plants.

The herbicide application can be tank mixed sequentially with one, two or a combination of several synthetic hormone herbicides, ACCase inhibitor herbicides or any other compatible herbicides. Multiple applications of one herbicide or two or more herbicides in combination or alone can be used in the growing season in an area containing the transgenic plants of the present invention for controlling a broad spectrum of dicotyledonous weeds, monocotyledonous weeds or both, for example, two applications (such as pre-planting application and post-emergence application or pre-emergence application and post-emergence application) or three applications (such as pre-planting application, pre-emergence application and post-emergence application or pre-emergence application and two post-emergence applications).

As used herein, "resistance", "herbicide resistance", "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. The herbicide tolerance of a plant, seed, plant tissue, plant part or cell can be measured by comparing the plant, seed, plant tissue, plant part or cell with a suitable control. For example, herbicide tolerance can be measured by applying a herbicide to a plant (test plant) comprising a recombinant DNA molecule encoding a protein capable of conferring herbicide tolerance and a plant (control plant) not comprising a recombinant DNA molecule encoding a protein capable of conferring herbicide tolerance, and then comparing the plant damage of the two plants, wherein the herbicide tolerance of the test plant is indicated by a reduced injury rate compared to the injury rate of the control plant. When compared to a control plant, seed, plant tissue, plant part or cell, a herbicide tolerant plant, seed, plant tissue, plant part or cell shows a reduced response to the toxic effects of the herbicide. As used herein, a "herbicide tolerance trait" is a transgenic trait that confers improved herbicide tolerance to a plant compared to wild-type plants or control plants.

Transgenic plant of the present invention, progeny, seed, vegetable cell and plant part also can contain one or more other transgenic traits.Can introduce other transgenic traits by making the transgenic plant containing recombinant DNA molecule provided by the invention and another plant hybrid containing other transgenic traits.As used herein, "hybridization" means to cultivate two independent plants to produce progeny plants.Therefore, two transgenic plants can hybridize to produce the progeny containing transgenic traits.As used herein, "progeny" means the offspring of any passage of parental plant, and the transgenic progeny comprises the DNA construct provided by the present invention and from at least one parental plant inheritance.Or, can by using the DNA construct co-transformation of the other transgenic traits described in the DNA construct comprising recombinant DNA molecule provided by the invention (for example, wherein all DNA constructs are presented as the part of the same vector for plant transformation) or by inserting other traits into the transgenic plant comprising DNA construct provided by the invention or vice versa (for example, by using any method for plant transformation about transgenic plant or vegetable cell) introduce other transgenic traits. 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 wild-type plants or control plants. Such additional transgenic traits are known to those skilled in the art; for example, the U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) provides a list of such traits and can be found on their website www.aphis.usda.gov.

Transgenic plants and progeny containing transgenic traits provided by the invention can be used together with any breeding method generally known in the art. In a plant line comprising two or more transgenic traits, transgenic traits can be separated, connected or a combination of the two independently in a plant line comprising three or more transgenic traits. Backcrossing with parental plants and outcrossing with non-transgenic plants, and asexual reproduction are also considered. The description of the breeding method generally used for different traits and crops is well known to those skilled in the art. In order to confirm the presence of transgenic in a particular plant or seed, multiple assays can be performed. Such assays include, for example, molecular biological assays, such as southern blotting and northern blotting, PCR and DNA sequencing; biochemical assays, such as, for example, by immunological methods (ELISA and Western blotting) or by the presence of enzyme function detection protein products; plant part assays, such as leaf or root assays; and also by analyzing the phenotype of the whole plant.

Introgression of the transgenic trait into the plant genotype is achieved as a result of the backcross transformation process. The plant genotype into which the transgenic trait has been introgressed may be referred to as a backcross transformed genotype, line, inbred plant, or hybrid. Similarly, a plant genotype lacking the desired transgenic trait may be referred to as an untransformed genotype, line, inbred plant, or hybrid.

As used herein, the term "comprising" means "including but not limited to".

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Results of the reaction rate determination of some engineered proteins towards 2,4-D.

FIG. 2 shows the effect of treating T0 transgenic corn plants expressing M7 protein with 10 g/mu of compound C.

Figure 3 shows the effect of treating T1 transgenic corn plants expressing M1, M7, M11, M12, M13, M14, M16, M18, M19, M23, M24, M25, and M26 proteins with 150 g/mu of compound C.

FIG. 4 shows the effect of treating T1 transgenic soybean plants expressing M19 and M13 proteins with 10 g/mu of compound C.

Sequence Description

DETAILED DESCRIPTION

Example 1. Initial protein engineering and enzyme analysis

The RdpA protein sequence was sequence blasted in the NCBI database, and 9 protein sequences of different sources and different sequence similarities were selected from the output results. Combined with the sequence alignment results, Golden Gate Shuffling and other methods were used to recombinant RdpA with protein sequences from different sources for large fragments, generating more than 8,700 unique engineered proteins and recombinant DNA molecules encoding them for further analysis and characterization. Due to the need to test a large number of engineered proteins produced and to test and compare the enzymatic activity of each protein, a high-throughput bacterial protein expression and screening system was developed for rapid analysis using crude bacterial products.

The gene encoding each engineered protein was cloned into a bacterial expression vector containing a histidine tag (His-tag) at the C-terminus to achieve high-throughput protein expression. The vector was transformed into Escherichia coli (E. coli) and bacterial expression of the engineered protein was induced. An E. coli culture was selected and grown overnight in a centrifuge tube while adding substrate and IPTG, or only add substrate, and centrifuge the culture the next day to precipitate bacteria. Alternatively, select Escherichia coli culture and culture it overnight in a centrifuge tube, add substrate and react the next day, and centrifuge to precipitate bacteria. The supernatant of the reaction solution is drawn into a 96-well plate, and the phenol product is detected by end-point colorimetric measurement of 4-aminoantipyrine and potassium ferrocyanide at 510nm, and the oxygenase activity (i.e., its enzyme activity) of the engineered protein is measured by high-performance liquid chromatography to detect the amount of substrate reduction and product production. The activity of the protein is compared by calculating the conversion rate, and some of the results are shown in Table 1.

Conversion rate = (initial substrate peak area - post-reaction substrate peak area) / initial substrate peak area * 100%

Table 1. Transformation rate of each mutant

Note: Reaction condition 1: Culture bacteria overnight, and add substrate compound A and IPTG to react overnight;

Reaction condition 2: Culture the bacteria overnight and add 8 times the dosage of substrate compound A in reaction condition 1 to react overnight;

Reaction condition 3: Culture the bacteria overnight, and add 8 times the dosage of substrate compound A in reaction condition 1 on the second day for 1 hour.

Based on the results of the high-throughput liquid phase assay system, representative engineered proteins were selected for protein purification. Further protein characterization, such as Km, Vmax and Kcat, was performed using 33 engineered proteins from Table 2. Protein purification used conventional Ni column affinity chromatography, protein extract purity was assessed by SDS-PAGE analysis, and enzyme activity was determined after protein concentration was determined by the BCA method. The control was the purified wild-type enzyme, and the enzyme kinetics of the protein was determined using 0, 10, 20, 50, 100, 200, 500, 1000uM of compound A.

Table 2. Test results of engineered proteins


Note: N/D means the enzyme activity is too low to determine its enzyme kinetic parameters.

Table 2 shows the Km, Vmax, Kcat, Kcat/Km measured for 33 proteins with Compound A as substrate. The enzyme kinetic parameters of these 33 engineered proteins indicate that the enzyme activity, i.e., Km and Kcat, of proteins can be significantly improved by protein engineering.

In addition, the catalytic activity towards 2,4-DP/2,4-D was determined using the same method as above, and some representative data are shown in Table 3 and Figure 1 .

Table 3. Results of the enzyme activity assay of some engineered proteins against 2,4-DP (2,4-D-isopropanoate)

Table 3 shows the Km, Vmax, Kcat, and Kcat/Km measured for the four proteins with compound 2,4-DP as substrate. The enzyme kinetic parameters of the four engineered proteins indicate that the measured engineered proteins maintain or even significantly improve the activity towards 2,4-DP compared to the wild-type RdpA enzyme.

Figure 1 shows the maximum reaction rates (in abs/1000 min) measured for 19 proteins with compound 2,4-D as substrate. The reaction rates of these 19 engineered proteins indicate that the tested engineered proteins maintain or even significantly improve the 2,4-D activity compared to the wild-type RdpA enzyme.

Example 2: Expression of engineered proteins in corn

Engineered proteins were selected for maize transformation and plant analysis. DNA constructs were transformed into maize using Agrobacterium tumefaciens and standard methods known in the art.

The transformed T0 transgenic plantlets expressing the engineered protein and the non-transgenic recipient plants were cultured in a greenhouse. The T0 transgenic plantlets and wild-type corn plants under strict control conditions were sprayed with 10g/mu of compound C for testing. As shown in Figure 2, the wild type showed obvious drug damage, while the transgenic plantlets containing the M7 protein encoding gene grew normally. In addition, the T0 transgenic plantlets expressing other engineered proteins of the present invention (such as M1, M11, M12, M13, M14, M16, M18, M19, M23, M24, M25, and M26) can also grow normally. It can be concluded that the transgenic corn containing the engineered protein encoding gene of the present invention can have better performance under the treatment conditions of 10g/mu compound C than the wild-type corn. Drug resistance.

The T0 transgenic resistant plants after spraying were grown in a greenhouse, and the T1 corn plant seeds produced by all T0 transgenic resistant plants were collected. T1 seeds were sown and sprayed with 0, 90, 120, and 150 g/mu of compound C at the approximately two-leaf and one-heart growth stage, and the resistance of the plants was recorded and evaluated after the spraying treatment. After applying 150 g/mu of compound C11 DAT, the transgenic corn plants containing the engineered protein encoding gene of the present invention showed better drug resistance than wild-type plants, indicating that the tolerance dose of the T1 transgenic corn plants expressing the engineered protein to compound C is at least 150 g/mu. Among them, the representative test results are shown in Figure 3.

Example 3: Expression of engineered proteins in soybeans

Engineered proteins were selected for soybean transformation and plant analysis.DNA constructs were transformed into soybean using Agrobacterium tumefaciens and standard methods known in the art.

The transformed T0 transgenic plantlets were grown in a greenhouse, and T1 seeds of the plantlets that were positive for transgenic identification were collected. T1 seeds were sown, and T1 plantlets were sprayed with 10 g/mu of compound C, and the resistance of the plants was recorded and evaluated 12 DAT after the spraying treatment. Compared with wild-type plants that could not survive due to severe drug damage, transgenic soybean plants containing genes encoding the engineered proteins (such as M13, M16, M19, M23, and M24) of the present invention showed better drug resistance, and representative test results are shown in Figure 4.

At the same time, after many tests, it was found that the recombinant DNA molecules of the present invention were introduced into model plants such as Arabidopsis thaliana and Brachypodium distachyon, and the corresponding levels of resistance to synthetic hormone herbicides and/or ACCase inhibitor herbicides were improved. It can be seen that it can be genetically modified into other plants, such as food crops, legume crops, oil crops, fiber crops, fruit crops, root crops, vegetable crops, flower crops, medicinal crops, raw material crops, forage crops, sugar crops, beverage crops, lawn plants, tree crops, nut crops, etc., and corresponding resistance traits will also be produced, which has good industrial value.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

A recombinant DNA molecule comprising a nucleic acid sequence encoding a polypeptide having 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% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133 or 137. 63,64,66,67,68,70,71,72,74,75,76,78,79,80,82,83,84,86,87,88,90,91,92,94,95,96,98,99 ,100,102,103,104,106,107,108,110,111,112,114,115,116,118,119,120,122,123,124,126,127,128,130,131,132,134,135,136,138,139, 140, 142, 1 43, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 or 167, and nucleic acid sequences that encode the same amino acid sequence as the sequences shown due to the degeneracy of the genetic code. The recombinant DNA molecule of claim 1 or 2, wherein the recombinant DNA molecule is operably linked to a heterologous promoter functional in a plant cell; preferably, the recombinant DNA molecule is further operably linked to a DNA molecule encoding a chloroplast transit peptide. A DNA construct comprising a heterologous promoter functional in plant cells operably linked to the recombinant DNA molecule as described in any one of claims 1 to 3; preferably, further comprising a DNA molecule encoding a chloroplast transit peptide operably linked to the recombinant DNA molecule; preferably, the DNA construct is present in the genome of a transgenic plant. A plant, seed, plant tissue, plant part or cell comprising the recombinant DNA molecule according to any one of claims 1 to 3, the DNA construct according to claim 4, or a polypeptide encoded by the recombinant DNA molecule according to claims 1 to 3; preferably, the plant, seed, plant tissue, plant part or cell comprises tolerance to at least one herbicide selected from the group consisting of: synthetic hormone herbicides, ACCase inhibitor herbicides. A polypeptide having at least 98% or at least 99% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133 or 137; preferably, the polypeptide has oxygenase activity against at least one herbicide selected from the group consisting of: synthetic hormone herbicides, ACCase inhibitor herbicides. A method for conferring herbicide tolerance to plants, seeds, cells or plant parts, the method comprising expressing the polypeptide of claim 6 in the plants, seeds, cells or plant parts; preferably, the plants, seeds, cells or plant parts comprise a DNA construct comprising a heterologous promoter functional in plant cells operably linked to a recombinant DNA molecule, the recombinant DNA molecule comprising a polypeptide encoding claim 6. The method of claim 7, wherein the plant, seed, cell or plant part comprises tolerance to at least one herbicide selected from the group consisting of: synthetic hormone herbicides, ACCase inhibitor herbicides. A method for producing a herbicide-tolerant transgenic plant, the method comprising transforming a plant cell or tissue with a recombinant DNA molecule as described in any one of claims 1 to 3 or a DNA construct as described in claim 4, and regenerating a herbicide-tolerant transgenic plant from the transformed plant cell or tissue; preferably, the herbicide-tolerant transgenic plant comprises tolerance to at least one herbicide selected from the group consisting of: synthetic hormone herbicides, ACCase inhibitor herbicides. A method for controlling weeds in a plant growing area, the method comprising contacting a plant growing area including plants or seeds with at least one herbicide selected from the group consisting of synthetic hormone herbicides and ACCase inhibitor herbicides, the plants or seeds comprising the recombinant DNA molecule of any one of claims 1 to 3 and being tolerant to the at least one herbicide.