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WO2024230371A1 - 除草剂耐受性基因及其使用方法 - Google Patents

除草剂耐受性基因及其使用方法 Download PDF

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Publication number
WO2024230371A1
WO2024230371A1 PCT/CN2024/085723 CN2024085723W WO2024230371A1 WO 2024230371 A1 WO2024230371 A1 WO 2024230371A1 CN 2024085723 W CN2024085723 W CN 2024085723W WO 2024230371 A1 WO2024230371 A1 WO 2024230371A1
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Prior art keywords
plant
dna molecule
recombinant dna
herbicide
transgenic
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French (fr)
Inventor
莫苏东
张俊杰
刘桂智
李华荣
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Qingdao Kingagroot Seed Science Co Ltd
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Qingdao Kingagroot Seed Science Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
    • C12Y113/12019—2-Oxuglutarate dioxygenase (ethylene-forming) (1.13.12.19)

Definitions

  • 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.
  • transgenic traits produced using biotechnology methods.
  • Heterologous genes also referred to as transgenics
  • the expression of transgenes in plants imparts desired traits to plants, such as herbicide tolerance.
  • transgenic herbicide tolerance traits include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance.
  • glyphosate tolerance glyphosate tolerance
  • glufosinate tolerance glufosinate tolerance
  • dicamba tolerance dicamba tolerance
  • 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 ⁇ -ketoglutarate-dependent dioxygenases, RdpA (R-2,4-dichloropropionate dioxygenase) and SdpA (S-2,4-dichloropropionate dioxygenase).
  • RdpA R-2,4-dichloropropionate dioxygenase
  • SdpA S-2,4-dichloropropionate dioxygenase
  • 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.
  • 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.
  • 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,
  • the recombinant DNA molecule is operably linked to a heterologous promoter functional in plant cells.
  • 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.
  • it further comprises a DNA molecule encoding a chloroplast transit peptide operably linked to the recombinant DNA molecule.
  • the present invention provides a plant, seed, plant tissue, plant part or cell, which comprises the recombinant DNA molecule.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • engineered proteins refers to non-natural DNA, proteins or organisms that are not usually found in nature and produced by human intervention.
  • Engineerered 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.
  • 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.
  • 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.
  • 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.
  • wild-type means naturally occurring.
  • a wild-type DNA molecule wild-type polypeptide
  • 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.
  • control means an experimental control designed for comparison purposes.
  • 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).
  • 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,
  • 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.
  • 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).
  • transgenic means comprising a transgene
  • a transgenic plant refers to a plant comprising a transgene in its genome
  • a transgenic trait refers to a characteristic or phenotype transmitted or conferred by the presence of a transgene incorporated into the plant genome.
  • 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.
  • heterologous refers to the relationship between two or more substances that are derived from different sources and are therefore not generally related in nature.
  • 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.
  • 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.
  • 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.
  • transgenic expression means 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).
  • mRNA messenger RNA
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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
  • 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,
  • percent 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
  • MUSCLE version 3.6
  • 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.
  • the engineered proteins provided herein provide novel proteins having one or more altered protein characteristics relative to wild-type proteins found in nature.
  • 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.
  • 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.
  • 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.
  • zygosity 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.
  • 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.
  • 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:
  • Benzoic acids dicamba, fenthion, chlorpyrifos, naphthamide, etc.
  • 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.
  • 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
  • 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;
  • R represents independently unsubstituted C1-C14 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C12 cycloalkyl or phenyl
  • 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;
  • 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 Ph
  • 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.
  • the compounds of the general formula I and I-1 are both in R configuration (the carbon atom at the * is a chiral center).
  • 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);
  • 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);
  • A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, Q represents methyl, X represents O, and M represents tetrahydrofuran-2-ylmethyl
  • R configuration i.e. compound C
  • 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:
  • Aryloxyphenoxypropionic acid quizalofop-p-butyl, clodinafop-butyl, cyhalofop-butyl, diclofop-butyl, fenoxaprop-butyl, fluazifop-butyl, fluazifop-butyl, methomyl, oxadiazine, quinazolin, etc.
  • Cyclohexenone cypermethrin, clethodim, cypermethrin, butyclothiocarb, cypermethrin, sethoxydim, pyraclostrobin, trimethoprim-butyl, etc.
  • the common name of the active compound 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.
  • 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.
  • 2,4-D or 2,4-D butyric acid salts such as sodium salt, potassium salt, dimethylammonium salt, triethanolammonium salt
  • 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.
  • 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).
  • two applications such as pre-planting application and post-emergence application or pre-emergence application and post-emergence application
  • three applications such as pre-planting application, pre-emergence application and post-emergence application or pre-emergence application and two post-emergence applications.
  • “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.
  • 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.
  • 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.
  • hybridization means to cultivate two independent plants to produce progeny plants.Therefore, two transgenic plants can hybridize to produce the progeny containing transgenic traits.
  • 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
  • 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.
  • 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 can be used together with any breeding method generally known in the art.
  • 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.
  • 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.
  • 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
  • analyzing the phenotype of the whole plant 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
  • 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.
  • a plant genotype lacking the desired transgenic trait may be referred to as an untransformed genotype, line, inbred plant, or hybrid.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the wild type showed obvious drug damage, while the transgenic plantlets containing the M7 protein encoding gene grew normally.
  • 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
  • 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.
  • 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.
  • 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.
  • the representative test results are shown in Figure 3.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

提供了编码降解合成激素类除草剂和/或ACCase抑制剂类除草剂的酶的重组DNA分子。还提供了含有重组DNA分子的转基因植物、部分、种子、细胞和植物部分，以及使用它们的方法。

Description

除草剂耐受性基因及其使用方法 技术领域
本发明总体上涉及生物技术领域。更具体地，本发明涉及编码降解合成激素类和/或ACCase抑制剂类除草剂的酶的重组DNA分子。本发明还涉及含有重组DNA分子的转基因植物、部分、种子、细胞和植物部分，以及使用它们的方法。
背景技术
农作物生产通常利用使用生物技术方法产生的转基因性状。将异源基因(也称为转基因)引入植物中以产生转基因性状。转基因在植物中的表达赋予植物以期望的性状，如除草剂耐受性。转基因除草剂耐受性性状的实例包括草甘膦耐受性、草铵膦耐受性和麦草畏耐受性。随着对最常用的除草剂有抗性的杂草物种的增加，在所述领域中需要新的除草剂耐受性性状。特别感兴趣的除草剂是合成激素类除草剂。合成激素类除草剂提供对一系列抗草甘膦杂草的控制，从而产生赋予这些除草剂耐受性的特别用于与其他除草剂耐受性性状组合的作物系统中的性状。
从2,4-滴丙酸(dichloroprop)降解土壤样品分离的食除草剂鞘脂菌(Sphingobium herbicidovorans)菌株MH被鉴定为能够裂解各种苯氧基烷酸(phyenoxyalkanoic acid)除草剂的醚键，从而利用此作为其生长的唯一碳源和能量来源(HPE Kohler,Journal of Industrial Microbiology&Biotechnology(1999)23:336-340)。除草剂的分解代谢通过两种不同的对映选择性α-酮戊二酸依赖性双加氧酶RdpA(R-2,4-滴丙酸双加氧酶)和SdpA(S-2,4-滴丙酸双加氧酶)进行。(A Westendorf,等人,Microbiological Research(2002)157:317-322；Westendorf,等人,ActaBiotechnologica(2003)23(1):3-17)。RdpA已自食除草剂鞘脂菌(GenBank登录AF516752(DNA)和AAM90965(蛋白质))和食酸代尔夫特菌(Delftia acidovorans)(GenBank登录NG_036924(DNA)和YP_009083283(蛋白质))(TA Mueller,等人,Applied and Environmental Microbiology(2004)70(10):6066-6075)。RdpA和SdpA基因已经用于植物转化以赋予作物以除草剂耐受性(TR Wright,等人,Proceedings of the National Academy of Sciences USA,(2010)107(47):20240-5)。使用蛋白质工程化技术改进RdpA酶的活性以产生用于转基因植物的蛋白质将允许更高的除草剂施加率，从而改进转基因作物安全性和杂草控制措施。
发明简述
本发明提供一种重组DNA分子，其包含编码与选自由以下组成的组的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的多肽的核酸序列：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或137。
在一个具体实施方式中，所述核酸序列选自由以下组成的组：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或167，以及因遗传密码的简并性而与所示序列编码相同氨基酸序列的核酸序列。
在一个具体实施方式中，其中所述重组DNA分子可操作地连接至在植物细胞中有功能的异源启动子。
在另一个具体实施方式中，其中所述重组DNA分子进一步可操作地连接至编码叶绿体转运肽的DNA分子。
本发明还提供一种DNA构建体，其包含可操作地连接至所述的重组DNA分子的在植物细胞中有功能的异源启动子。
在一个具体实施方式中，其还包含可操作地连接至所述重组DNA分子的编码叶绿体转运肽的DNA分子。
在另一个具体实施方式中，其中所述DNA构建体存在于转基因植物的基因组中。
本发明提供一种植物、种子、植物组织、植物部分或细胞，其包含所述的重组DNA分子。
在一个具体实施方式中，其中所述植物、种子、植物组织、植物部分或细胞包含对至少一种选自由以下组成的组的除草剂的耐受性：合成激素类除草剂、ACCase抑制剂类除草剂。
本发明还提供一种植物、种子、植物组织、植物部分或细胞，其包含所述的DNA构建体。
本发明还提供一种植物、种子、植物组织、植物部分或细胞，其包含由所述的重组DNA分子编码的多肽。
本发明还提供一种多肽，其与选自由以下组成的组的氨基酸序列具有至少98％或至少99％同一性：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或137。
在一个具体实施方式中，其中所述多肽对至少一种选自由以下组成的组的除草剂具有加氧酶活性：合成激素类除草剂、ACCase抑制剂类除草剂。
本发明还提供一种用于赋予植物、种子、细胞或植物部分以除草剂耐受性的方法，所述方法包括在所述植物、种子、细胞或植物部分中表达所述的多肽。
在一个具体实施方式中，其中所述植物、种子、细胞或植物部分包含DNA构建体，所述DNA构建体包含可操作地连接至重组DNA分子的在植物细胞中有功能的异源启动子，所述重组DNA分子包含编码所述的多肽。
在另一个具体实施方式中，其中所述植物、种子、细胞或植物部分包含对至少一种选自由以下组成的组的除草剂的耐受性：合成激素类除草剂、ACCase抑制剂类除草剂。
本发明还提供一种用于产生除草剂耐受性转基因植物的方法，所述方法包括用所述的重组DNA分子或所述的DNA构建体转化植物细胞或组织，和从所述转化的植物细胞或组织再生除草剂耐受性转基因植物。
在一个具体实施方式中，其中所述除草剂耐受性转基因植物包含对至少一种选自由以下组成的组的除草剂的耐受性：合成激素类除草剂、ACCase抑制剂类除草剂。
本发明还提供一种用于控制植物生长区域中的杂草的方法，所述方法包括使包括植物或种子的植物生长区域与至少一种选自由合成激素类除草剂、ACCase抑制剂类除草剂组成的组的除草剂接触，所述植物或种子包含所述的重组DNA分子且耐受所述至少一种除草剂。
本发明所述的工程化蛋白质与野生型RdpA蛋白相比，不但保留了原始对苯氧羧酸类等合成激素类除草剂和/或ACCase抑制剂类除草剂的抗性，还增加了对吡啶基氧基酸类除草剂的抗性，拓宽了对除草剂的抗性谱。
发明详述
提供以下定义和方法以更好地限定本发明并指导本领域的普通技术人员实施本发明。除非另外说明，否则术语应根据相关领域的普通技术人员的常规用法来理解。
工程化蛋白质和重组DNA分子
本发明提供新颖的工程化蛋白质和编码它们的重组DNA分子。如本文所用，术语“工程化的”是指通常不会在自然界中发现并且通过人为干预产生的非天然DNA、蛋白质或生物体。“工程化蛋白质”是在实验室中使用一种或多种蛋白质工程化技术，如使用定点诱变的蛋白质设计和使用随机诱变和DNA改组的定向进化来设想和创建其多肽序列的蛋白质。例如，工程化蛋白质可相对于野生型蛋白质的编码序列具有一个或多个缺失、插入或取代，并且每个缺失、插入或取代可由一个或多个氨基酸组成。工程化蛋白质的实例在本文提供为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和137。
本发明提供的工程化蛋白质是具有加氧酶活性的酶。如本文所用，术语“加氧酶活性”意指通过将氧从分子氧转移至底物、副产物或中间物来氧化底物的能力。本发明提供的工程化蛋白的加氧酶活性可使合成激素类除草剂和/或ACCase抑制剂类除草剂中的一种或多种失活。
如本文所用，“野生型”意指天然存在的。如本文所用，“野生型DNA分子”、“野生型多肽”或“野生型蛋白质”是天然存在的DNA分子、多肽或蛋白质，即在自然界中预先存在的DNA分子、多肽或蛋白质。多肽、蛋白质或DNA分子的野生型型式可适用于与工程化蛋白质或基因进行比较。蛋白质或DNA分子的野生型型式可适用作实验中的对照。
如本文所用，“对照”意指为比较目的而设计的实验对照。例如，转基因植物分析中的对照植物是与实验植物(即其待测试的植物)相同类型但不含实验植物的转基因插入片段、重组DNA分子或DNA构建体的植物。适用于与转基因玉米植物比较的对照植物的实例是非转基因LH244玉米(美国专利号6,252,148)并且适用于与转基因大豆植物比较的对照植物的实例是非转基因A3555大豆(美国专利No.7,700,846)。
如本文所用，术语“重组”是指为遗传工程化的结果并且因此通常不会在自然界中发现并且通过人为干预产生的非天然DNA、多肽或蛋白质。“重组DNA分子”是包含不天然存在的并且因此是人为干预的结果的DNA序列的DNA分子，例如编码工程化蛋白质的DNA分子。 另一个实例是由至少两个彼此异源的DNA分子(如编码蛋白质的DNA分子和可操作地连接的异源启动子)的组合组成的DNA分子。重组DNA分子的实例是包含至少一个选自以下的序列的DNA分子：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和167。
“重组多肽”或“重组蛋白质”是包含不天然存在的氨基酸序列并且因此是人为干预的结果的多肽或蛋白质，例如工程化蛋白质。
术语“转基因”是指作为人为干预(如通过植物转化方法)的结果人工并入生物体基因组中的DNA分子。如本文所用，术语“转基因的”意指包含转基因，例如“转基因植物”是指在其基因组中包含转基因的植物，“转基因性状”是指由并入植物基因组中的转基因的存在传递或赋予的特征或表型。作为这种基因组改变的结果，所述转基因植物是与相关的野生型植物明显不同的植物，并且转基因性状是未在野生型植物中天然发现的性状。本发明的转基因植物包含通过本发明提供的重组DNA分子和工程化蛋白质。
如本文所用，术语“异源”是指源自不同来源且因此在自然界中通常不相关的两种或更多种物质之间的关系。例如，编码蛋白质的重组DNA分子相对于可操作地连接的启动子是异源的，如果这种组合在自然界中通常不存在。此外，当特定重组DNA分子不天然存在于所述特定细胞或生物体中时，其可相对于其所插入的细胞或生物体是异源的。
如本文所用，术语“编码蛋白质的DNA分子”或“编码多肽的DNA分子”是指包含编码蛋白质或多肽的核苷酸序列的DNA分子。“编码蛋白质的序列”或“编码多肽的序列”意指编码蛋白质或多肽的DNA序列。“序列”意指核苷酸或氨基酸的顺序排列。编码蛋白质的序列或编码多肽的序列的边界通常由5'-末端的翻译起始密码子和3'-末端的翻译终止密码子决定。编码蛋白质的分子或编码多肽的分子可包含编码蛋白质或多肽序列的DNA序列。如本文所用，“转基因表达”、“表达转基因”、“蛋白质表达”、“多肽表达”、“表达蛋白质”和“表达多肽”意指通过将DNA分子转录成信使RNA(mRNA)并且将mRNA翻译成多肽链(其可最终折叠成蛋白质)的过程产生蛋白质或多肽。编码蛋白质的DNA分子或编码多肽的DNA分子可以可操作地连接至DNA构建体中的异源启动子，以用于在用重组DNA分子转化的细胞中表达蛋白质或多肽。如本文所用，“可操作地连接”是指以使得一个DNA分子可影响另一个DNA分子的功能的方式连接的两个DNA分子。可操作连接的DNA分子可以是单个连续分子的一部分，并且可以是或可以不是相邻的。例如，启动子与DNA构建体中的编码蛋白质的DNA分子或编码多肽的DNA分子可操作地连接，其中两个DNA分子被排列成使得所述启动子可影响转基因的表达。
如本文所用，“DNA构建体”是包含两个或更多个异源DNA序列的重组DNA分子。DNA构建体适用于转基因表达，并且可包含在载体和质粒中。DNA构建体可出于转化(即将异源DNA引入宿主细胞中)的目的用于载体中以便产生转基因植物和细胞，并且因此也可包含在转基因植物、种子、细胞或植物部分的质粒DNA或基因组DNA中。如本文所用，“载体”意指可用于植物转化目的的任何重组DNA分子。如序列表中所示的重组DNA分子可例如作为构建体的一部分插入载体中，所述构建体具有可操作地连接至启动子的重组DNA分子，所述启动子在植物中起作用以驱动由所述重组DNA分子编码的工程化蛋白质的表达。用于构建DNA构建体和载体的方法是本领域中熟知的。DNA构建体或包含DNA构建体的载体的组分通常包括但不限于以下中的一种或多种：用于表达可操作地连接的DNA的合适启动子、可操作地连接的编码蛋白质非人DNA分子和3'非翻译区(3’-UTR)。适用于实践本发明的启动子包括在植物中起作用以表达可操作地连接的多核苷酸的启动子。此类启动子是多种多样的且是本领域中熟知的，并且包括诱导型的、病毒的、合成的、组成型、时间调控型的、空间调控型的和/或时空调控型的。另外的任选组分包括但不限于以下元件中的一个或多个：5'-UTR、增强子、前导序列、顺式作用元件、内含子、叶绿体转运肽(CTP)和一个或多个选择性标记转基因。
本发明的DNA构建体可包含可操作地连接至本发明提供的编码蛋白质的DNA分子的CTP分子。适用于实践本发明的CTP包括用于促进工程化蛋白分子在细胞内定位的那些。通过促进细胞内的蛋白质定位，CTP可增加工程化蛋白质的积累，保护其免受蛋白水解降解，增强除草剂耐受性水平，并且由此降低除草剂施加后的损伤水平。用于本发明的CTP分子是本领域中已知的，包括但不限于拟南芥EPSPS CTP(Klee等人,1987)、矮牵牛EPSPS CTP(della-Cioppa等人,1986)、玉米cab-m7信号序列(Becker等人,1992；PCT WO 97/41228)和豌豆谷胱甘肽还原酶信号序列(Creissen等人,1991；PCT WO 97/41228)。
本发明的重组DNA分子可通过本领域中已知的方法完全或部分地合成和修饰，特别是在期望提供适用于DNA操作的序列(如限制性酶识别位点或重组-基因克隆位点)、植物优选序列(如植物密码子使用或Kozak共有序列)或适用于DNA构建体设计的序列(如间隔区或接头序列)的情况下。本发明包括重组DNA分子和工程化蛋白质，所述重组DNA分子和工程化蛋白质与本文提供的重组DNA分子或工程化蛋白质序列中的任一个，例如与包含选自由以下组成的组的序列的重组DNA分子具有至少约80％(百分比)序列同一性、约85％序列同一性、约90％序列同一性、约91％序列同一性、约92％序列同一性、约93％序列同一性、约94％序列同一性、约95％序列同一性、约96％序列同一性、约97％序列同一性、约98％序列同一性和约99％序列同一性：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和167。
如本文所用，术语“百分比序列同一性”或“％序列同一性”是指在最佳比对两个序列时(在比较窗内具有总计少于参考序列的20％的适当核苷酸或氨基酸插入、缺失或空位)，与测试(“主题”)序列(或其互补链)相比，参考(“查询”)序列(或其互补链)的线性多核苷酸或多肽序列中的相同核苷酸或氨基酸的百分比。用于比对比较窗口的最佳序列比对是本领域的技术人员所熟知的并且可由以下工具实施：如Smith和Waterman的局部同源性算法、Needleman和Wunsch的同源性比对算法、Pearson和Lipman的相似性搜索方法，并且由这些算法的计算机化实现方式来实施，如使用默认参数的作为Wisconsin(Accelrys Inc.,San Diego,CA)、MEGAlign(DNAStar,Inc.,1228S.Park St.,Madison,Wis.53715)和MUSCLE(3.6版)(RCEdgar,Nucleic Acids Research(2004)32(5):1792-1797)的一部分可获得的GAP、BESTFIT、FASTA和TFASTA。测试序列和参考序列的比对片段的“同一性分数”是由两个比对序列共享的相同组分的数目除以参考序列片段中组分的总数，即整个参考序列或参考序列的较小限定部分。序列同一性百分比表示为同一性分数乘以100。一个或多个序列的比较可以是针对全长序列或其一部分，或针对更长的序列。
可通过改变(即修饰)野生型蛋白质以产生具有有用蛋白质特征(如改变的Vmax、Km、底物特异性、底物选择性和蛋白质稳定性)的新颖组合的新蛋白质来产生工程化蛋白质。修饰可在蛋白质中的特定氨基酸位置进行，并且可以是在自然界(即在野生型蛋白质中)中在所述位置发现的氨基酸被不同的氨基酸取代。适用于蛋白质工程化的野生型蛋白质RdpA的氨基酸序列如SEQ ID NO:1所示。设计工程化蛋白质，所述工程化蛋白质与选自由以下组成的组的氨基酸序列具有至少约92％序列同一性：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和137，并且包含这些氨基酸突变中的至少一种。因此，本发明提供的工程化蛋白质提供相对于在自然界中发现的野生型蛋白质具有一种或多种改变的蛋白质特征的新蛋白质。在本发明的一个实施方案中，与类似的野生型蛋白质或此类特征的任何组合相比，工程化蛋白质具有改变的蛋白质特征，如针对一种或多种除草剂的改进的或降低的活性或改进的蛋白质稳定性。在一个实施方案中，本发明提供工程化蛋白质和编码其的重组DNA分子，其与选自由以下组成的组的工程化蛋白质序列具有至少约80％序列同一性、约85％序列同一性、约90％序列同一性、约91％序列同一性、约92％序列同一性、约93％序列同一性、约94％序列同一性、约95％序列同一性、约96％序列同一性、约97％序列同一性、约98％序列同一性和约99％序列同一性：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和137。氨基酸突变可作为蛋白质中的单个氨基酸取代或与一种或多种其它突变(如一个或多个其它氨基酸取代、缺失或添加)的组合进行。可如本文所描述或通过本领域的技术人员已知的任何其它方法进行突变。
转基因植物
本发明的一个方面包括包含本发明提供的重组DNA分子和工程化蛋白的转基因植物细胞、转基因植物组织、转基因植物和转基因种子。包含重组DNA分子和工程化蛋白质的这些细胞、组织、植物和种子显示对合成激素类除草剂、ACCase抑制剂类除草剂中的一种或多种的除草剂耐受性。
用于转化用于本发明的宿主植物细胞的合适方法实际上包括可将DNA引入细胞(例如，其中重组DNA构建体稳定整合至植物染色体中)的任何方法并且在本领域中是已知的。用于将重组DNA构建体引入植物中的示例性和广泛使用的方法是土壤杆菌属转化系统，其是本领域的技术人员熟知的。转基因植物可通过植物细胞培养的方法从转化的植物细胞再生。关于转基因纯合的转基因植物(即，转基因的两个等位基因拷贝)可通过将包含单个转基因等位基因的转基因植物与其自身(例如R0植物)自花授粉(自交)以产生R1种子。所产生的R1种子的四分之一对于转基因将是纯合的。通常使用SNP测定、DNA测序或允许杂合子与纯合子之间的区别的热扩增测定来测试从发芽的R1种子生长的植物的接合性，称为接合性测定。
本发明提供的植物、种子、植物部分、植物组织和细胞显示对合成激素类除草剂、ACCase抑制剂类除草剂中的一种或多种的除草剂耐受性，尤其是显示对如式Ⅰ所示的吡啶基氧基酸类化合物及其盐、酯衍生物的除草剂耐受性。
在本发明中，“合成激素类除草剂”是本身有除草活性的物质或者与能改变其效果的其他除草剂和/或添加剂合用的物质，其属于植物激素干扰型除草剂，在本领域中是熟知的，例如包括以下有效成分或其衍生物中的至少一种：
(1)吡啶羧酸类(Pyridine carboxylic acids)：氨氯吡啶酸(毒莠定)、氯氟吡氧乙酸(氟草烟)、氯氟吡氧乙酸异辛酯、氯氨吡啶酸、二氯吡啶酸、三氯吡氧乙酸(绿草定)、氯氟吡啶酯、氟氯吡啶酯、如式Ⅰ所示的吡啶基氧基酸类化合物及其盐、酯衍生物等；
(2)苯甲酸类(Benzoic acids)：麦草畏、草灭平、草芽畏、萘草胺等；
(3)苯氧羧酸类(Phenoxycarboxylic acids)：2,4-二氯苯氧乙酸(2,4-D)、2,4-二氯苯氧丁酸(2,4-D丁酸)、2,4-D异丙酸、氯甲酰草胺、二甲四氯、二甲四氯异丙酸、二甲四氯丁酸等；
(4)喹啉羧酸类(Quinoline carboxylic acids)：二氯喹啉酸、氯甲喹啉酸等；
(5)其他：草除灵等。
所述如式Ⅰ所示的吡啶基氧基酸类化合物及其盐、酯衍生物，
其中，A、B分别独立地代表卤素、C1-C6烷基、卤代C1-C6烷基、C3-C6环烷基；
C代表氢、卤素、C1-C6烷基、卤代C1-C6烷基；
Q代表C1-C6烷基、卤代C1-C6烷基、C3-C6环烷基、C2-C6烯基、C2-C6炔基、卤素、氰基、氨基、硝基、甲酰基、C1-C6烷氧基、C1-C6烷硫基、C1-C6烷氧羰基、羟基C1-C6烷基、C1-C6烷氧基C1-C2烷基、氰基C1-C2烷基、C1-C6烷氨基C1-C2烷基、苄基、萘基、呋喃基、噻吩基、噻唑基、吡啶基、嘧啶基，以及未取代或被C1-C6烷基取代的 未取代或被C1-C6烷基、卤代C1-C6烷基、卤素和C1-C6烷氧基中的至少一个基团所取代的苯基；
Y代表氨基、C1-C6烷基氨基、C1-C6烷基羰基氨基、苯基羰基氨基、苄基氨基、未取代或卤代C1-C6烷基取代的呋喃基亚甲基氨基；
所述盐为金属盐、铵盐NH4 +、伯胺盐RNH2、仲胺盐(R)2NH、叔胺盐(R)3N、季胺盐(R)4N+、吗啉盐、哌啶盐、吡啶盐、氨基丙基吗啉盐、Jeff胺D-230盐、2,4,6-三(二甲基氨基甲基)苯酚和氢氧化钠的盐、C1-C14烷基锍盐、C1-C14烷基氧化锍盐、C1-C14烷基鏻盐、C1-C14烷醇鏻盐；
其中，R分别独立地代表未取代的C1-C14烷基、C2-C12烯基、C2-C12炔基、C3-C12环烷基或苯基，以及C1-C14烷基任选地被一个或多个以下基团取代：卤素、羟基、C1-C6烷氧基、C1-C6烷硫基、羟基C1-C6烷氧基、氨基、C1-C6烷基氨基、氨基C1-C6烷基氨基、苯基；
所述酯为其中，X代表O或S；
M代表C1-C18烷基、卤代C1-C8烷基、C3-C6环烷基、C2-C6烯基、卤代C2-C6烯基、C2-C6炔基、C1-C6烷氧基、C1-C6烷氧羰基、C1-C6烷基磺酰基、氰基C1-C2烷基、硝基C1-C2烷基、C1-C6烷氧基C1-C2烷基、C1-C6烷氧羰基C1-C2烷基、C2-C6烯氧基羰基C1-C2烷基、-(C1-C2烷基)-Z、四氢呋喃基、吡啶基、萘基、呋喃基、噻吩基、以及未取代或C1-C6烷基取代的未取代或被C1-C6烷基、卤代C1-C6烷基、C1-C6烷基氨基、卤素或C1-C6烷氧基取代的苯基；
Z代表四氢呋喃基、吡啶基、噻吩基、呋喃基、萘基，以及未取代或被C1-C6烷基、C1-C6烷氧基、卤代C1-C6烷基、氰基和卤素中的至少一个基团取代的苯基；
R3分别独立地代表C1-C6烷基；
R4、R5、R6分别独立地代表氢、C1-C6烷基、C1-C6烷氧基羰基；
R’代表氢、C1-C6烷基、卤代C1-C6烷基。
在一个实施方案中，所述通式化合物I和I-1均为R构型(*处碳原子为手性中心)。在另一个实施方案中，所述通式化合物I中A代表氯、B代表氯、C代表氟、Y代表氨基、Q代表甲基，且为R构型(*处碳原子为手性中心)(即化合物A)；所述通式化合物I-1中A代表氯、B代表氯、C代表氟、Y代表氨基、Q代表甲基，X代表O，M代表甲基，且为R构型(*处碳原子为手性中心)(即化合物B)；或者所述通式化合物I-1中A代表氯、B代表氯、C代表氟、Y代表氨基、Q代表甲基，X代表O，M代表四氢呋喃-2-基甲基且为R构型(即化合物C)(*处碳原子为手性中心)。
在本发明中，“ACCase抑制剂类除草剂”是指以乙酰辅酶A羧化酶为作用靶标的除草剂，在本领域中是熟知的，例如包括以下有效成分或其衍生物中的至少一种：
(1)芳氧苯氧丙酸类：精喹禾灵、炔草酯、氰氟草酯、禾草灵、精噁唑禾草灵、精吡氟禾草灵、高效氟吡禾草灵、噁唑酰草胺、噁草酸、喹禾糠酯等；
(2)环己烯酮类：禾草灭、烯草酮、环苯草酮、丁苯草酮、噻草酮、烯禾啶、吡喃草酮、三甲苯草酮等；
(3)苯并吡啶类：唑啉草酯等。
在本说明书的上下文中，如果使用活性化合物的通用名称的缩写形式，则在每种情况下包括所有的常规衍生物，例如酯和盐，以及异构体，特别是光学异构体，特别是一种或多种市售形式。如果通用名称表示酯或盐，则在每种情况下还包括所有其他的常规衍生物，例如其他的酯和盐、游离酸和中性化合物，以及异构体，特别是光学异构体，特别是一种或多种市售形式。给出的化合物的化学名称表示至少一种被通用名称涵盖的化合物，通常是优选的化合物。例如，2,4-D或2,4-D丁酸衍生物包含但不限于：2,4-D或2,4-D丁酸盐如钠盐、钾盐、二甲铵盐、三乙醇铵盐、异丙胺盐、胆碱等，以及2,4-D或2,4-D丁酸酯如甲酯、乙酯、丁酯、异辛酯等；二甲四氯衍生物包含但不限于：二甲四氯钠盐、钾盐、二甲铵盐、异丙胺盐等，以及二甲四氯甲酯、乙酯、异辛酯、乙硫酯等。
除草剂可施加至包含本发明提供的植物和种子的植物生长区域作为控制杂草的方法。本发明提供的植物和种子包含除草剂耐受性性状，且因此耐受一种或多种合成激素类除草剂、ACCase抑制剂类除草剂的施加。在施加除草剂时，植物生长区可包括或可不包括杂草植物。
除草剂施加可依次与几种合成激素类除草剂、ACCase抑制剂类除草剂或任何其它相容性除草剂中的一种、两种或组合槽混。一种除草剂或两种或更多种除草剂组合或单独的多次施加可在生长季节内用于包含本发明的转基因植物的区域以用于控制广谱双子叶杂草、单子叶杂草或两者，例如，两次施加(如种植前施加和芽后施加或芽前施加和芽后施加)或三次施加(如种植前施加、芽前施加和芽后施加或芽前施加和两次芽后施加)。
如本文所用，“抗性”、“除草剂抗性”、“耐受性”或“除草剂耐受性”意指植物、种子、植物组织、植物部分或细胞抵抗一种或多种除草剂的毒性作用的能力。植物、种子、植物组织、植物部分或细胞的除草剂耐受性可通过将植物、种子、植物组织、植物部分或细胞与合适的对照进行比较来测量。例如，除草剂耐受性可通过将除草剂施加至包含编码能够赋予除草剂耐受性的蛋白质的重组DNA分子的植物(测试植物)和不包含编码能够赋予除草剂耐受性的蛋白质的重组DNA分子的植物(对照植物)，且然后比较两种植物的植物损伤，其中测试植物的除草剂耐受性通过与对照植物的损伤率相比减少的损伤率指示。当与对照植物、种子、植物组织、植物部分或细胞相比时，除草剂耐受性植物、种子、植物组织、植物部分或细胞显示对除草剂的毒性作用的反应降低。如本文所用，“除草剂耐受性性状”是与野生型植物或对照植物相比赋予植物改善的除草剂耐受性的转基因性状。
本发明的转基因植物、子代、种子、植物细胞和植物部分还可含有一种或多种另外的转基因性状。可通过使含有包含本发明提供的重组DNA分子的转基因的植物与含有另外的转基因性状的另一种植物杂交来引入另外的转基因性状。如本文所用，“杂交”意指培育两种单独的植物以产生子代植物。因此，两种转基因植物可杂交以产生含有转基因性状的子代。如本文所用，“子代”意指亲本植物的任何传代的后代，并且转基因子代包含由本发明提供并且从至少一种亲本植物遗传的DNA构建体。或者，可通过用包含本发明提供的重组DNA分子的DNA构建体共转化所述另外的转基因性状的DNA构建体(例如，其中所有的DNA构建体呈现为用于植物转化的同一载体的部分)或通过将另外的性状插入包含本发明提供的DNA构建体的转基因植物中或反之亦然(例如，通过使用关于转基因植物或植物细胞的植物转化的任何方法)来引入另外的转基因性状。此类另外的转基因性状包括但不限于增加的昆虫抗性、增加的水利用效率、增加的产量性能、增加的抗旱性、增加的种子质量、改进的营养品质、杂交种种子生产和除草剂耐受性，其中性状是相对于野生型植物或对照植物测量的。此类另外的转基因性状是本领域的技术人员已知的；例如，美国农业部(USDA)动物和植物健康检查局(APHIS)提供了此类性状的列表，并且可在它们的网站www.aphis.usda.gov上找到。
含有本发明提供的转基因性状的转基因植物和子代可与本领域中通常已知的任何培育方法一起使用。在包含两种或更多种转基因性状的植物系中，转基因性状可在包含三种或更多种转基因性状的植物系中独立地分离、连接或两者的组合。还考虑与亲本植物的回交和与非转基因植物的异交，以及无性繁殖。通常用于不同性状和作物的培育方法的描述是本领域的技术人员熟知的。为了证实转基因在特定植物或种子中的存在，可进行多种测定。此类测定包括例如分子生物学测定，如DNA印迹和RNA印迹、PCR和DNA测序；生物化学测定，如例如通过免疫学方法(ELISA和蛋白质印迹)或通过酶功能检测蛋白质产物的存在；植物部分测定，如叶或根测定；以及还有通过分析整株植物的表型。
作为回交转化过程的结果实现转基因性状向植物基因型的基因渗入。其中已经基因渗入转基因性状的植物基因型可称为回交转化的基因型、系、近交植物或杂交种。类似地，缺乏所需转基因性状的植物基因型可称为未转化的基因型、系、近交植物或杂交种。
如本文所用，术语“包含”是指“包括但不限于”。
附图说明
图1部分工程化蛋白质对2,4-D反应速率测定结果。
图2用10g/亩化合物C处理表达M7蛋白的T0代转基因玉米植株的效果。
图3用150g/亩化合物C处理表达M1、M7、M11、M12、M13、M14、M16、M18、M19、M23、M24、M25、M26蛋白的T1代转基因玉米植株的效果。
图4用10g/亩化合物C处理表达M19和M13蛋白的T1代转基因大豆植株的效果。
序列说明




具体实施方式
实施例一、初始蛋白质工程化和酶分析
将RdpA蛋白序列在NCBI数据库中进行序列blast，从输出结果中分别选择了9条不同来源、不同序列相似度的蛋白序列，结合序列比对结果，使用Golden Gate Shuffling等方法，将RdpA分别与不同来源的蛋白序列进行大片段重组，产生超过8700种独特的工程化蛋白质和编码它们的重组DNA分子以用于进一步分析和表征。由于大量用于测试所产生的工程化蛋白质以及测试和比较每种蛋白质的酶活性的需要，开发了高通量细菌蛋白质表达和筛选系统以用于使用粗细菌产物进行快速分析。
将编码每种工程化蛋白质的基因克隆至C-末端含有组氨酸标签(His-标签)的细菌表达载体中来实现高通量蛋白表达。将载体转化大肠埃希氏菌(Escherichia coli)(E.coli)，并且诱导工程化蛋白质的细菌表达。挑选大肠埃希氏菌培养物在离心管中过夜培养，同时添加底物和 IPTG，或者只添加底物，第二天将培养物离心以沉淀细菌。或者挑选大肠埃希氏菌培养物在离心管中过夜培养，第二天添加底物反应后离心以沉淀细菌。吸取反应液上清至96孔板通过终点比色测量来自4-氨基安替比林和铁氰化钾在510nm下的吸光度检测酚产物、以及通过高效液相色谱检测底物消减量和产物产生量来测量工程化蛋白的加氧酶活性(即，其酶活性)。通过计算转化率来比较蛋白质的活性，部分结果如表1所示。
转化率＝(初始底物峰面积-反应后底物峰面积)/初始底物峰面积*100％
表1.各个突变体的转化率

注：反应条件1：过夜培养细菌，并加入底物化合物A和IPTG反应过夜；
反应条件2：过夜培养细菌，并加入反应条件1中8倍剂量的底物化合物A反应过夜；
反应条件3：过夜培养细菌，第二日加入反应条件1中8倍剂量的底物化合物A反应1h。
基于高通量液相测定系统的结果，选择具有代表性的工程化蛋白质进行蛋白纯化。用来自表2的33种工程化蛋白质进行进一步蛋白质表征，如Km、Vmax和Kcat。蛋白纯化使用常规Ni柱亲和层析，通过SDS-PAGE分析评估蛋白质提取物纯度，通过BCA法测定蛋白质浓度后进行酶活性测定，对照是纯化的野生型酶，使用0、10、20、50、100、200、500、1000uM的化合物A进行蛋白质的酶动力学测定。
表2.工程化蛋白质的测定结果


注：N/D代表酶活性过低，无法测定其酶动力学参数。
表2显示出对于化合物A为底物的33种蛋白质测量的Km、Vmax、Kcat、Kcat/Km。这33种工程化蛋白质的酶动力学参数表明，可通过蛋白质工程化显著提高蛋白质的酶活性，即Km和Kcat。
另外，利用上述相同方法测定对2,4-DP/2,4-D的催化活性，部分代表性数据如表3和图1所示。
表3.部分工程化蛋白质对2,4-DP(2,4-D异丙酸)酶活测定结果
表3显示出对于化合物2，4-DP为底物的4种蛋白质测量的Km、Vmax、Kcat、Kcat/Km。这4种工程化蛋白质的酶动力学参数表明，所测工程化蛋白质相比较RdpA野生型酶维持甚至明显提高了对2,4-DP的活性。
图1显示出对于化合物2,4-D为底物的19种蛋白质测量的最大反应速率(单位为abs/1000min)。这19种工程化蛋白质的反应速率表明，所测工程化蛋白质相比较RdpA野生型酶对2,4-D活性维持甚至明显提高。
实施例二、玉米中工程化蛋白质的表达
选择工程化蛋白质用于玉米转化和植物分析。DNA构建体通过根癌土壤杆菌和本领域中已知的标准方法进行玉米转化。
将转化后的表达工程化蛋白的T0代转基因小植株和非转基因受体植株在温室中培养。对严格对照条件下的T0代转基因小植株和野生型玉米植株分别喷药10g/亩化合物C进行测试。如图2所示，野生型表现出明显药害，而含M7蛋白编码基因的转基因小植株可正常生长。另外，表达本发明其他工程化蛋白(如M1、M11、M12、M13、M14、M16、M18、M19、M23、M24、M25、M26)的T0代转基因小植株同样可正常生长。由此得出含本发明所述工程化蛋白编码基因的转基因玉米相比较野生型玉米可在10g/亩化合物C处理条件下有更优的 抗药性。
令喷药后的T0代转基因抗性植株在温室中生长，收取所有T0代转基因抗性植株产生的T1代玉米植物种子。播种T1代种子，在大约两叶一心生长阶段分别用0、90、120、150g/亩化合物C喷洒，喷洒处理后记录评价植物的抗性程度。施加150g/亩化合物C11DAT后，相比较野生型植物，含本发明所述工程化蛋白编码基因的转基因玉米植物显示出了更优的耐药性，这表明表达工程化蛋白质的T1代转基因玉米植物对化合物C的耐受剂量至少为150g/亩。其中，代表性测试结果如图3所示。
实施例三、大豆中工程化蛋白质的表达
选择工程化蛋白质用于大豆转化和植物分析。DNA构建体通过根癌土壤杆菌和本领域中已知的标准方法进行大豆转化。
将转化后的T0代转基因小植株在温室中生长，收取经过转基因鉴定阳性的小植株的T1代种子。播种T1代种子，T1代小植株分别用10g/亩化合物C喷洒，喷洒处理12DAT后记录评价植物的抗性程度。相较于野生型植物因药害严重无法存活，含本发明所述工程化蛋白(如M13、M16、M19、M23、M24)编码基因的转基因大豆植物显示出了更优的耐药性，代表性测试结果如图4所示。
同时经过很多测试发现，将本发明所述重组DNA分子导入拟南芥、二穗短柄草等模式植物中，都产生了对合成激素类除草剂和/或ACCase抑制剂类除草剂相应水平的耐药性提升。由此可知，将其转基因到其他植物，如粮食作物、豆类作物、油料作物、纤维作物、水果类作物、根茎类作物、蔬菜类作物、花卉作物、药用作物、原料作物、牧草作物、糖料作物、饮料作物、草坪植物、树木作物、坚果作物等，也会产生相应的抗性性状，具有良好的产业价值。
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。

Claims (10)

  1. 一种重组DNA分子，其包含编码与选自由以下组成的组的氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的多肽的核酸序列：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或137。
  2. 如权利要求1所述的重组DNA分子，其中所述核酸序列选自由以下组成的组：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或167，以及因遗传密码的简并性而与所示序列编码相同氨基酸序列的核酸序列。
  3. 如权利要求1或2所述的重组DNA分子，其中所述重组DNA分子可操作地连接至在植物细胞中有功能的异源启动子；优选地，所述重组DNA分子进一步可操作地连接至编码叶绿体转运肽的DNA分子。
  4. 一种DNA构建体，其包含可操作地连接至如权利要求1-3任意一项所述的重组DNA分子的在植物细胞中有功能的异源启动子；优选地，还包含可操作地连接至所述重组DNA分子的编码叶绿体转运肽的DNA分子；优选地，所述DNA构建体存在于转基因植物的基因组中。
  5. 一种植物、种子、植物组织、植物部分或细胞，其包含如权利要求1-3任意一项所述的重组DNA分子，如权利要求4所述的DNA构建体，或由如权利要求1-3所述的重组DNA分子编码的多肽；优选地，所述植物、种子、植物组织、植物部分或细胞包含对至少一种选自由以下组成的组的除草剂的耐受性：合成激素类除草剂、ACCase抑制剂类除草剂。
  6. 一种多肽，其与选自由以下组成的组的氨基酸序列具有至少98％或至少99％同一性：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或137；优选地，所述多肽对至少一种选自由以下组成的组的除草剂具有加氧酶活性：合成激素类除草剂、ACCase抑制剂类除草剂。
  7. 一种用于赋予植物、种子、细胞或植物部分以除草剂耐受性的方法，所述方法包括在所述植物、种子、细胞或植物部分中表达如权利要求6所述的多肽；优选地，所述植物、种子、细胞或植物部分包含DNA构建体，所述DNA构建体包含可操作地连接至重组DNA分子的在植物细胞中有功能的异源启动子，所述重组DNA分子包含编码如权利要求6所述的多肽。
  8. 如权利要求7所述的方法，其中所述植物、种子、细胞或植物部分包含对至少一种选自由以下组成的组的除草剂的耐受性：合成激素类除草剂、ACCase抑制剂类除草剂。
  9. 一种用于产生除草剂耐受性转基因植物的方法，所述方法包括用如权利要求1-3任意一项所述的重组DNA分子或如权利要求4所述的DNA构建体转化植物细胞或组织，和从所述转化的植物细胞或组织再生除草剂耐受性转基因植物；优选地，所述除草剂耐受性转基因植物包含对至少一种选自由以下组成的组的除草剂的耐受性：合成激素类除草剂、ACCase抑制剂类除草剂。
  10. 一种用于控制植物生长区域中的杂草的方法，所述方法包括使包括植物或种子的植物生长区域与至少一种选自由合成激素类除草剂、ACCase抑制剂类除草剂组成的组的除草剂接触，所述植物或种子包含如权利要求1-3任意一项所述的重组DNA分子且耐受所述至少一种除草剂。
PCT/CN2024/085723 2023-05-10 2024-04-03 除草剂耐受性基因及其使用方法 Pending WO2024230371A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1576194B1 (de) * 2002-12-18 2006-05-31 Ufz-Umweltforschungszentrum Leipzig-Halle Gmbh Bakterienstämme delftia acidovorans mc1-r, ihre herstellung sowie ihre verwendung zur produktion der r-enantiomeren von 2-phenoxypropionsäure-derivaten
CN1984558A (zh) * 2004-04-30 2007-06-20 美国陶氏益农公司 新除草剂抗性基因
CN106793761A (zh) * 2014-10-15 2017-05-31 孟山都技术公司 除草剂耐受性基因及其使用方法
CN114085849A (zh) * 2020-08-06 2022-02-25 青岛清原化合物有限公司 除草剂抗性蛋白、基因和用途
CN116694654A (zh) * 2022-02-25 2023-09-05 青岛清原种子科学有限公司 除草剂耐受性基因及其使用方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1576194B1 (de) * 2002-12-18 2006-05-31 Ufz-Umweltforschungszentrum Leipzig-Halle Gmbh Bakterienstämme delftia acidovorans mc1-r, ihre herstellung sowie ihre verwendung zur produktion der r-enantiomeren von 2-phenoxypropionsäure-derivaten
CN1984558A (zh) * 2004-04-30 2007-06-20 美国陶氏益农公司 新除草剂抗性基因
CN106793761A (zh) * 2014-10-15 2017-05-31 孟山都技术公司 除草剂耐受性基因及其使用方法
CN114085849A (zh) * 2020-08-06 2022-02-25 青岛清原化合物有限公司 除草剂抗性蛋白、基因和用途
CN116694654A (zh) * 2022-02-25 2023-09-05 青岛清原种子科学有限公司 除草剂耐受性基因及其使用方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Master's thesis", 6 March 2017, ZHEJIANG UNIVERSITY, CN, article DONGDONG ZHANG: "Enantioselective Degradation and Enantiomerization of the Chiral Dichlorprop Herbicide in Soils", pages: 1 - 96, XP093236786 *
MÜLLER TINA A., FLEISCHMANN THOMAS, VAN DER MEER JAN ROELOF, KOHLER HANS-PETER E.: "Purification and Characterization of Two Enantioselective α-Ketoglutarate-Dependent Dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 72, no. 7, 1 July 2006 (2006-07-01), US , pages 4853 - 4861, XP093236779, ISSN: 0099-2240, DOI: 10.1128/AEM.02758-05 *

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