[go: up one dir, main page]

CN118931929A - Herbicide tolerance genes and methods of use thereof - Google Patents

Herbicide tolerance genes and methods of use thereof Download PDF

Info

Publication number
CN118931929A
CN118931929A CN202410397339.3A CN202410397339A CN118931929A CN 118931929 A CN118931929 A CN 118931929A CN 202410397339 A CN202410397339 A CN 202410397339A CN 118931929 A CN118931929 A CN 118931929A
Authority
CN
China
Prior art keywords
plant
dna molecule
recombinant dna
herbicide
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202410397339.3A
Other languages
Chinese (zh)
Inventor
请求不公布姓名
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qingdao Qingyuan Seed Science Co ltd
Original Assignee
Qingdao Qingyuan Seed Science Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qingdao Qingyuan Seed Science Co ltd filed Critical Qingdao Qingyuan Seed Science Co ltd
Publication of CN118931929A publication Critical patent/CN118931929A/en
Pending legal-status Critical Current

Links

Classifications

    • 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)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physiology (AREA)
  • Environmental Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Botany (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

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

Description

Herbicide tolerance genes and methods of use thereof
Technical Field
The present invention relates generally to the field of biotechnology. More particularly, the present invention relates to recombinant DNA molecules encoding enzymes that degrade synthetic hormonal and/or ACCase inhibitor herbicides. The invention also relates to transgenic plants, parts, seeds, cells and plant parts comprising the recombinant DNA molecules, and methods of using the same.
Background
Crop production typically utilizes transgenic traits produced using biotechnology methods. Heterologous genes (also known as transgenes) are introduced into plants to produce transgenic traits. Expression of the transgene in plants imparts desirable traits, such as herbicide tolerance, to the plant. Examples of transgenic herbicide tolerance traits include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance. With the increasing number of weed species that are resistant to the most commonly used herbicides, new herbicide tolerance traits are needed in the field. Herbicides of particular interest are synthetic hormonal herbicides. Synthetic hormonal herbicides provide control of a range of glyphosate resistant weeds, resulting in traits that confer tolerance to these herbicides, particularly for use in crop systems in combination with other herbicide tolerance traits.
The herbicide-eating sphingolipid (Sphingobium herbicidovorans) strain MH isolated from a soil sample degraded by 2, 4-D propionic acid (dichloroprop) was identified as an ether linkage capable of cleaving the various phenoxy alkanoic acid (phyenoxyalkanoic acid) herbicides, thereby utilizing this as the sole carbon and energy source for its growth (HPE Kohler, journal of Industrial Microbiology & Biotechnology (1999) 23:336-340). Catabolism of herbicides is performed by two different enantioselective alpha-ketoglutarate dependent dioxygenases RdpA (R-2, 4-drop propionate dioxygenase) and SdpA (S-2, 4-drop propionate dioxygenase). (A Westendorf, et al, microbiological Research (2002) 157:317-322; westendorf, et al, actaBiotechnologica (2003) 23 (1): 3-17). RdpA have been derived from the herbicides Sphingomonas (GenBank accession AF516752 (DNA) and AAM90965 (protein)) and from the herbicides Deuteromycetes (Delftia acidovorans) (GenBank accession NG_036924 (DNA) and YP_009083283 (protein)) (TA 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). The use of protein engineering techniques to improve RdpA enzyme activity to produce proteins for transgenic plants would allow for 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 ：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 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.
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 a nucleic acid sequence encoding the same amino acid sequence as the indicated sequence due to the degeneracy of the genetic code.
In a specific embodiment, wherein the recombinant DNA molecule is operably linked to a heterologous promoter functional in a plant cell.
In another embodiment, wherein the recombinant DNA molecule is further operably linked to a DNA molecule encoding a chloroplast transit peptide.
The invention also provides a DNA construct comprising a heterologous promoter functional in a plant cell operably linked to said 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 embodiment, wherein the DNA construct is present in the genome of the transgenic plant.
The present invention provides a plant, seed, plant tissue, plant part or cell comprising said 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 and ACCase inhibitor herbicides.
The invention also provides a plant, seed, plant tissue, plant part or cell comprising said DNA construct.
The invention also provides a plant, seed, plant tissue, plant part or cell comprising a polypeptide encoded by said recombinant DNA molecule.
The present invention also provides a polypeptide having at least 98% or at least 99% identity ：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 to an amino acid sequence selected from the group consisting of seq id no.
In a specific embodiment, wherein 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, said method comprising expressing said polypeptide in said plant, seed, cell or plant part.
In a specific embodiment, wherein the plant, seed, cell or plant part comprises a DNA construct comprising a heterologous promoter functional in a plant cell operably linked to a recombinant DNA molecule comprising a polypeptide encoding said polypeptide.
In another specific embodiment, wherein the plant, seed, cell or plant part 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 producing a herbicide tolerant transgenic plant comprising transforming a plant cell or tissue with said recombinant DNA molecule or said DNA construct, and regenerating a herbicide tolerant transgenic plant from said transformed plant cell or tissue.
In a specific embodiment, wherein 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 growing area, said method comprising contacting a plant growing area comprising a plant or seed comprising said recombinant DNA molecule and being tolerant to said at least one herbicide selected from the group consisting of a synthetic hormonal herbicide, an ACCase inhibitor herbicide.
Compared with wild RdpA protein, the engineering protein not only maintains the resistance of original p-phenoxy carboxylic acid and other synthetic hormone herbicides and/or ACCase inhibitor herbicides, but also increases the resistance of pyridyloxy acid herbicides, and widens the resistance spectrum of herbicides.
Detailed Description
The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in practicing the present invention. Unless otherwise indicated, terms are to be construed according to conventional usage by those of ordinary skill 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 normally found in nature and are produced by human intervention. An "engineered protein" is a protein whose polypeptide sequence is envisaged 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 the 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 herein are enzymes having oxygenase activity. As used herein, the term "oxygenase activity" means the ability to oxidize a substrate by transferring oxygen from molecular oxygen to the substrate, a byproduct or an intermediate. The oxygenase activity of the engineered proteins provided by the invention can inactivate one or more of the synthetic hormone herbicides and/or 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 is pre-existing in nature. Wild-type versions of polypeptides, proteins or DNA molecules may be suitable for comparison with engineered proteins or genes. Wild-type versions of the protein or DNA molecule may be used as controls in experiments.
As used herein, "control" means an experimental control designed for comparison purposes. For example, the control plant in the transgenic plant assay is a plant of the same type as the experimental plant (i.e., the plant it is to be tested for) but without the transgenic insert, recombinant DNA molecule or DNA construct of the experimental plant. Examples of control plants suitable for comparison with transgenic corn plants are non-transgenic LH244 corn (U.S. patent No. 6,252,148) and examples of control plants suitable for comparison with transgenic soybean plants are non-transgenic a3555 soybean (U.S. patent No.7,700,846).
As used herein, the term "recombinant" refers to non-natural DNA, polypeptides or proteins that are the result of genetic engineering and thus are not normally found in nature and are produced by human intervention. A "recombinant DNA molecule" is a DNA molecule, e.g., a DNA molecule encoding an engineered protein, that comprises a DNA sequence that does not occur in nature and is thus the result of human intervention. Another example is a DNA molecule consisting of a combination of at least two DNA molecules heterologous to each other, such as a DNA molecule encoding a protein and an operably linked heterologous promoter. Examples of recombinant DNA molecules are DNA molecules ：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 comprising at least one sequence selected from the group consisting of.
A "recombinant polypeptide" or "recombinant protein" is a polypeptide or protein, e.g., an engineered protein, that comprises an amino acid sequence that does not occur in nature and is thus the result of human intervention.
The term "transgene" refers to a DNA molecule that is artificially incorporated into the genome of an organism as a result of human intervention (e.g., by plant transformation methods). As used herein, the term "transgenic" means a plant comprising a transgene, e.g., a "transgenic plant" refers to a plant comprising a transgene in its genome, and a "transgenic trait" refers to a characteristic or phenotype that is transmitted or conferred by the presence of a transgene incorporated into the plant genome. As a result of this genomic change, the transgenic plant is a plant that is significantly different from the relevant wild type plant, and the transgenic trait is a trait not naturally found in the wild type plant. The transgenic plants of the invention comprise the recombinant DNA molecules and engineered proteins provided by the invention.
As used herein, the term "heterologous" refers to a relationship between two or more substances that originate from different sources and are therefore not normally associated in nature. For example, a recombinant DNA molecule encoding a protein is heterologous with respect to an operably linked promoter if such a combination is not normally present in nature. Furthermore, when a particular recombinant DNA molecule does not naturally occur in the particular cell or organism, it may be heterologous with respect to the cell or organism into which it is inserted.
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 the sequence encoding the protein or the sequence encoding the polypeptide are generally determined by a translation initiation codon at the 5 '-end and a translation termination codon at the 3' -end. The protein-encoding molecule or the polypeptide-encoding molecule may comprise a DNA sequence encoding a protein or a polypeptide sequence. As used herein, "transgene expression," "expression transgene," "protein expression," "polypeptide expression," "expression protein," and "expression of a polypeptide" mean the production of a protein or polypeptide by the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into a polypeptide chain (which may ultimately fold into a protein). The DNA molecule encoding a protein or the DNA molecule encoding a polypeptide may be operably linked to a heterologous promoter in a DNA construct for expression of the protein or polypeptide in a cell transformed with the 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 another DNA molecule. The operably linked DNA molecules may be part of a single continuous molecule and may or may not be contiguous. For example, a promoter is operably linked to a DNA molecule encoding a protein or a DNA molecule encoding a polypeptide in a DNA construct, wherein the two DNA molecules are arranged such that the promoter can affect expression of the transgene.
As used herein, a "DNA construct" is a recombinant DNA molecule comprising two or more heterologous DNA sequences. The DNA constructs are suitable for transgene expression and may be included in vectors and plasmids. The DNA construct may be used in a vector for transformation (i.e., introduction of heterologous DNA into a host cell) to produce transgenic plants and cells, and thus may also be included in plasmid DNA or genomic DNA of the transgenic plant, seed, cell, or plant part. As used herein, "vector" means any recombinant DNA molecule that can be used for plant transformation purposes. The recombinant DNA molecule as set forth in the sequence listing may be inserted into a vector, for example, as part of a construct having the recombinant DNA molecule operably linked to a promoter that functions in a plant to drive expression of an engineered protein encoded by the recombinant DNA molecule. Methods for constructing DNA constructs and vectors are well known in the art. The components of the DNA construct or vector comprising the DNA construct generally include, but are not limited to, one or more of the following: suitable promoters for expression of the operably linked DNA, operably linked non-human DNA molecules encoding the protein, and the 3 'untranslated region (3' -UTR). Promoters suitable for use in the practice of the present invention include promoters that function in plants to express operably linked polynucleotides. Such promoters are diverse and well known in the art and include inducible, viral, synthetic, constitutive, time regulated, spatially regulated and/or space time 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 constructs of the present invention may comprise CTP molecules operably linked to the DNA molecules encoding proteins provided herein. CTPs suitable for use in practicing the present invention include those used to facilitate the intracellular localization of engineered protein molecules. By promoting protein localization within cells, CTPs can increase the accumulation of engineered proteins, protect them from proteolytic degradation, enhance herbicide tolerance levels, and thereby reduce the level of injury after herbicide application. CTP molecules for use in the present invention are known in the art and include, but are not limited to, arabidopsis thaliana EPSPS CTP (Klee et al, 1987), petunia EPSPS CTP (della-Ciopa et al, 1986), maize cab-m7 signal sequence (Becker et al, 1992; PCT WO 97/41228) and pea glutathione reductase signal sequence (Creissen et al, 1991; PCT WO 97/41228).
The recombinant DNA molecules of the invention may 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 recombinant-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 ：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 to any of the recombinant DNA molecules or engineered protein sequences provided herein, e.g., to a recombinant DNA molecule comprising a sequence selected from the group consisting of.
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 complement) as compared to a test ("subject") sequence (or its complement) when optimally aligned with the two sequences (with the appropriate nucleotide or amino acid insertions, deletions, or gaps of less than 20% of the total of the reference sequence within the window of comparison). The optimal sequence alignment for the alignment window is well known to those skilled in the art and can be performed by the following means: such as the local homology algorithms of Smith and Waterman, the homology alignment algorithms of Needleman and Wunsch, the similarity search methods of Pearson and Lipman, and are implemented by computerized implementations of these algorithms, such as using default parameters asWisconsinGAP, BESTFIT, FASTA and TFASTA available to (Accelrys Inc., san Diego, calif.), MEGAlign (DNAStar, inc.,1228S.park St., madison, wis. 53715) and a portion of MUSCLE (version 3.6) (RCEdgar, nucleic ACIDS RESEARCH (2004) 32 (5): 1792-1797). The "identity score" of an aligned segment of a test sequence and a 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 segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. Percent sequence identity is expressed as the identity score multiplied by 100. The comparison of one or more sequences may be for the full length sequence or a portion thereof, or for a longer sequence.
Engineered proteins can be produced by altering (i.e., modifying) wild-type proteins to produce novel proteins having a novel combination of useful protein characteristics (e.g., altered Vmax, km, substrate specificity, substrate selectivity, and protein stability). The modification may be made at a specific amino acid position in the protein and may be substitution of an amino acid found at that position in nature (i.e., in wild-type proteins) with a different amino acid. The amino acid sequence of the wild-type protein RdpA suitable for protein engineering is shown as SEQ ID NO. 1. An engineered protein is designed that has at least about 92% sequence identity ：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 to an amino acid sequence selected from the group consisting of seq id nos 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 or improved protein stability against one or more herbicides, as compared to a similar wild-type protein or any combination of such characteristics. In one embodiment, the invention provides engineered proteins and recombinant DNA molecules encoding the same that 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 ：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 to an engineered protein sequence selected from the group consisting of seq id nos. Amino acid mutations can be made as single amino acid substitutions in a protein or in combination with one or more other mutations (e.g., one or more other amino acid substitutions, deletions, or additions). Mutations may be made as described herein or by any other method known to those of skill in the art.
Transgenic plants
One aspect of the invention includes transgenic plant cells, transgenic plant tissues, transgenic plants and transgenic seeds comprising the recombinant DNA molecules and engineered proteins provided herein. These cells, tissues, plants and seeds comprising the recombinant DNA molecules and the engineered proteins exhibit herbicide tolerance to one or more of synthetic hormonal herbicides, ACCase inhibitor herbicides.
Suitable methods for transforming host plant cells for use in the present invention include virtually any method by which DNA can be introduced into a cell (e.g., wherein a recombinant DNA construct is stably integrated into a plant chromosome) and are known in the art. An exemplary and widely used method for introducing recombinant DNA constructs into plants is the agrobacterium transformation system, which is well known to those skilled in the art. Transgenic plants can be regenerated from transformed plant cells by plant cell culture methods. Transgenic plants homozygous for the transgene (i.e., two allelic copies of the transgene) can be produced by self-pollination (selfing) a transgenic plant comprising a single transgenic allele with itself (e.g., an R0 plant) to produce an R1 seed. One quarter of the R1 seeds produced will be homozygous for the transgene. Plants grown from germinated R1 seeds are typically tested for zygosity using SNP assays, DNA sequencing, or thermal amplification assays that allow differentiation between heterozygotes and homozygotes, referred to as zygosity assays.
The plants, seeds, plant parts, plant tissues and cells provided by the 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 salts and ester derivatives thereof.
In the present invention, a "synthetic hormone herbicide" is a substance having herbicidal activity itself or a substance used in combination with other herbicides and/or additives capable of changing the effect thereof, which belongs to a phytohormone-interfering herbicide, and is well known in the art, and includes, for example, at least one of the following active ingredients or derivatives thereof:
(1) Pyridine carboxylic acids (Pyridine carboxylic acids): picloram, fluroxypyr (fluroxypyr), isooctyl fluroxypyr, aminopyralid, clopyralid, triclopyr (triclopyr), fluroxypyr, pyridyloxy acid compounds of formula (I), salts, ester derivatives thereof, and the like;
(2) Benzoic acids (Benzoic acids): dicamba, oxaziclomefone, oxadiazon, and the like;
(3) Phenoxy carboxylic acids (Phenoxycarboxylic acids): 2, 4-dichlorophenoxyacetic acid (2, 4-D), 2, 4-dichlorophenoxybutyric acid (2, 4-D butyric acid), 2,4-D isopropyl acid, chloroformyl oxamide, dimethyltetrachloro isopropyl acid, dimethyltetrachloro butyric acid, and the like;
(4) Quinoline carboxylic acids (Quinoline carboxylic acids): quinclorac, clorac, and the like;
(5) Other: benazolin, and the like.
The pyridyloxy acid compound shown in the formula I and salts and ester derivatives thereof,
Wherein A, B each independently represents halogen, C1-C6 alkyl, halo 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, hydroxyC 1-C6 alkyl, C1-C6 alkoxyC 1-C2 alkyl, cyanoC 1-C2 alkyl, C1-C6 alkylamino C1-C2 alkyl, benzyl, naphthyl, furyl, thienyl, thiazolyl, pyridyl, pyrimidinyl, and optionally substituted C1-C6 alkyl Phenyl unsubstituted or substituted by at least one of C1-C6 alkyl, halo 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 metal salt, ammonium salt NH 4 +, primary amine salt RNH 2, secondary amine salt (R) 2 NH, tertiary amine salt (R) 3 N, quaternary amine salt (R) 4N+, morpholine salt, piperidine salt, pyridine salt, aminopropyl morpholine salt, jeff amine D-230 salt, 2,4, 6-tris (dimethylaminomethyl) phenol and sodium hydroxide salt, C1-C14 alkyl sulfonium salt, C1-C14 alkyl sulfoxonium salt, C1-C14 alkyl phosphonium salt;
Wherein R each independently represents unsubstituted C1-C14 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C12 cycloalkyl or phenyl, and C1-C14 alkyl is optionally substituted with 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, cyanoC 1-C2 alkyl, nitroC 1-C2 alkyl, C1-C6 alkoxyC 1-C2 alkyl, C2-C6 alkoxycarbonylC 1-C2 alkyl, - (C1-C2 alkyl) -Z, Tetrahydrofuranyl, pyridinyl, naphthyl, furanyl, thienyl,Unsubstituted or C1-C6 alkyl substitutedPhenyl which is unsubstituted or substituted by C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkylamino, halogen or C1-C6 alkoxy;
Z represents Tetrahydrofuranyl, pyridyl,Thienyl, furyl, naphthyl, phenyl which is unsubstituted or substituted by at least one of C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, cyano and halogen;
R 3 independently of one another represents C1-C6 alkyl;
R 4、R5、R6 independently represents hydrogen, C1-C6 alkyl, C1-C6 alkoxycarbonyl;
r' represents hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl.
In one embodiment, compounds I and I-1 of the general formula are both in the R configuration (the carbon atom is the chiral center). In another embodiment, compound I of the general formula wherein a represents chloro, B represents chloro, C represents fluoro, Y represents amino, Q represents methyl, and is in the R configuration (where the carbon atom is the chiral center) (i.e., compound a); in the general formula compound I-1, A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, Q represents methyl, X represents O, M represents methyl, and the R configuration (the carbon atom is chiral center) (namely, compound B); or in the general formula compound I-1, A represents chlorine, B represents chlorine, C represents fluorine, Y represents amino, Q represents methyl, X represents O, M represents tetrahydrofuran-2-ylmethylAnd is in the R configuration (i.e., compound C) (. Where the carbon atom is the chiral center).
In the present invention, the term "ACCase inhibitor-type herbicide" means a herbicide targeting acetyl-coa carboxylase, and is well known in the art, and includes, for example, at least one of the following active ingredients or derivatives thereof:
(1) Aryloxy-phenoxy-propionic acids: quizalofop-p-ethyl, clodinafop-propargyl, cyhalofop-butyl, fenoxaprop-p-ethyl, haloxyfop-butyl, oxazophos, oxaziclomefone, quizalofop-butyl and the like;
(2) Cyclohexenones: gramineae, clethodim clomazone, fenpropidone buprofezin, sethoxydim, pyrone, triclopyr, and the like;
(3) Benzopyridines: pinoxaden, and the like.
In the context of the present specification, if the abbreviated form of the generic name of the active compound is used, all customary derivatives, such as esters and salts, as well as isomers, in particular optical isomers, in particular one or more commercially available forms, are included in each case. If the generic name denotes esters or salts, all other customary derivatives are also included in each case, such as other esters and salts, free acids and neutral compounds, as well as isomers, in particular optical isomers, in particular one or more commercially available forms. The chemical name given to a compound means at least one compound covered by a common name, generally the 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 butyrate such as sodium salt, potassium salt, dimethylammonium salt, triethanolamine salt, isopropylamine salt, choline, etc., and 2,4-D or 2,4-D butyrate such as methyl ester, ethyl ester, butyl ester, isooctyl ester, etc.; the dimethyltetrachloro derivatives include, but are not limited to: sodium, potassium, ammonium, isopropyl, etc., and methyl, ethyl, isooctyl, ethyl, etc.
Herbicides can be applied to a plant growing area comprising plants and seeds provided herein as a method of controlling weeds. Plants and seeds provided herein comprise herbicide tolerance traits and are thus tolerant to the application of one or more synthetic hormone herbicides, ACCase inhibitor herbicides. In applying the herbicide, the plant growing area may or may not include weed plants.
Herbicide application may be tank mixed sequentially with one, two or a combination of several synthetic hormonal herbicides, ACCase inhibitor herbicides or any other compatible herbicide. A herbicide or a combination of two or more herbicides or multiple applications alone may be used in the area containing the transgenic plants of the invention during the growing season for controlling a broad spectrum of dicotyledonous weeds, monocotyledonous weeds, or both, for example, two applications (such as pre-planting and post-emergence applications or pre-emergence and post-emergence applications) or three applications (such as pre-planting, pre-emergence and post-emergence applications or pre-emergence and two post-emergence applications).
As used herein, "resistance," "herbicide resistance," "tolerance," or "herbicide tolerance" refers to the ability of a plant, seed, plant tissue, plant part, or cell to resist the toxic effects of one or more herbicides. 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 to a suitable control. For example, herbicide tolerance can be indicated by applying the herbicide to a plant comprising a recombinant DNA molecule encoding a protein capable of conferring herbicide tolerance (test plant) and a plant not comprising a recombinant DNA molecule encoding a protein capable of conferring herbicide tolerance (control plant), and then comparing the plant damage of the two plants, wherein herbicide tolerance of the test plant is indicated by a reduced damage rate compared to the damage rate of the control plant. Herbicide tolerant plants, seeds, plant tissues, plant parts or cells show reduced response to the toxic effects of herbicides when compared to control plants, seeds, plant tissues, plant parts or cells. As used herein, a "herbicide tolerance trait" is a transgenic trait that imparts improved herbicide tolerance to a plant as compared to a wild-type plant or a control plant.
Transgenic plants, progeny, seeds, plant cells and plant parts of the invention may also contain one or more additional transgenic traits. Additional transgenic traits can be introduced by crossing a plant containing a transgene comprising a recombinant DNA molecule provided herein with another plant containing an additional transgenic trait. As used herein, "crossing" means growing two separate plants to produce a progeny plant. Thus, two transgenic plants can be crossed to produce progeny that contain the transgenic trait. As used herein, "progeny" means the progeny of any passage of a parent plant, and transgenic progeny comprise the DNA construct provided by the invention and inherited from at least one parent plant. Alternatively, the additional transgenic trait can be introduced by co-transforming the DNA construct of the additional transgenic trait with a DNA construct comprising the recombinant DNA molecule provided herein (e.g., wherein all of the DNA construct is presented as part of the same vector for plant transformation) or by inserting the additional trait into a transgenic plant comprising the DNA construct provided herein or vice versa (e.g., by using any method of plant transformation with respect to a transgenic plant or plant cell). 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 of skill in the art; for example, the United States Department of Agriculture (USDA) animal and plant health inspection Agency (APHIS) provides a list of such traits and can be found on their website www.aphis.usda.gov.
Transgenic plants and progeny comprising the transgenic traits provided herein can be used with any cultivation method generally known in the art. In plant lines comprising two or more transgenic traits, the transgenic traits may be independently isolated, linked, or a combination of both in plant lines comprising three or more transgenic traits. Backcrossing with parent plants and outcrossing with non-transgenic plants, as well as asexual propagation, are also contemplated. Descriptions of cultivation methods commonly used for different traits and crops are well known to those skilled in the art. To confirm the presence of the transgene in a particular plant or seed, a variety of assays can be performed. Such assays include, for example, molecular biological assays such as southern and northern blots, PCR and DNA sequencing; biochemical assays, such as for example the detection of the presence of protein products by immunological methods (ELISA and western blot) or by enzymatic function; 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 plant genotype is achieved as a result of the backcross transformation process. The genotype of a plant in which a transgenic trait has been introgressed may be referred to as a backcross transformed genotype, line, inbred plant or hybrid. Similarly, a plant genotype lacking a 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.
Drawings
FIG. 1 results of a partial engineered protein to 2,4-D reaction rate assay.
FIG. 2 effect of treatment of T0 transgenic maize plants expressing M7 protein with 10 g/mu of Compound C.
FIG. 3 effect of treatment of T1 transgenic maize plants expressing M1, M7, M11, M12, M13, M14, M16, M18, M19, M23, M24, M25, M26 proteins with 150 g/mu of Compound C.
FIG. 4 effect of treatment of T1 transgenic soybean plants expressing M19 and M13 proteins with 10 g/mu of Compound C.
DESCRIPTION OF THE SEQUENCES
Detailed Description
Example one, initial protein engineering and enzyme analysis
Sequence blast is carried out on RdpA protein sequences in NCBI database, 9 protein sequences with different sources and different sequence similarity are respectively selected from output results, and the combined sequence comparison results are combined, and RdpA is respectively subjected to large fragment recombination with the protein sequences with different sources by using methods such as Golden Gate Shuffling and the like, so that more than 8700 unique engineering proteins and recombinant DNA molecules for encoding the same are generated for further analysis and characterization. Because of the large number of requirements for testing the engineered proteins produced and testing and comparing the enzymatic activity of each protein, high throughput bacterial protein expression and screening systems have been developed for rapid analysis using crude bacterial products.
The genes encoding each engineered protein were cloned into bacterial expression vectors containing a histidine tag (His-tag) at the C-terminus to achieve high-throughput protein expression. The vector was transformed into escherichia coli (ESCHERICHIA COLI) (e.coli) and bacterial expression of the engineered protein was induced. The E.coli cultures were selected and incubated overnight in centrifuge tubes with the addition of substrate and IPTG, or with the addition of substrate alone, and the cultures were centrifuged the next day to pellet the bacteria. Alternatively, E.coli cultures were selected and incubated overnight in centrifuge tubes, and the following day after substrate addition reaction centrifuged to pellet the bacteria. The reaction supernatant was pipetted into a 96-well plate to measure the oxygenase activity of the engineered protein (i.e., its enzymatic activity) by endpoint colorimetric measurement of absorbance at 510nm from 4-aminoantipyrine and potassium ferricyanide to detect phenol product, and by high performance liquid chromatography to detect substrate elimination and product yield. The activity of the proteins was compared by calculating the conversion rate, and some of the results are shown in Table 1.
Conversion = (initial substrate peak area-post-reaction substrate peak area)/initial substrate peak area 100%
TABLE 1 conversion of the individual mutants
Note that: reaction condition 1: culturing the bacteria overnight, and adding a substrate compound A and IPTG to react overnight;
Reaction condition 2: bacteria were cultured overnight and reacted overnight with 8-fold doses of substrate compound a added to reaction condition 1;
reaction condition 3: bacteria were cultured overnight and the following day was allowed to react for 1h with 8-fold doses of substrate compound a added to reaction condition 1.
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 with 33 engineered proteins from table 2. Protein purification protein extract purity was assessed by SDS-PAGE analysis using conventional Ni column affinity chromatography, protein concentration was determined by BCA method, enzyme activity was determined, reference was made to purified wild type enzyme, and enzyme kinetics of protein was determined using 0,10, 20, 50, 100, 200, 500, 1000uM of Compound A.
TABLE 2 determination of engineered proteins
Note that: N/D represents too low an enzyme activity to measure its enzymatic kinetic parameters.
Table 2 shows Km, vmax, kcat, kcat/Km measured for 33 proteins with Compound A as substrate. The enzymatic kinetic parameters of these 33 engineered proteins indicate that the enzymatic activity of the proteins, km and Kcat, can be significantly enhanced by protein engineering.
In addition, the catalytic activity on 2,4-DP/2,4-D was measured by the same method as described above, and some representative data are shown in Table 3 and FIG. 1.
TABLE 3 determination of the enzymatic Activity of partially engineered proteins on 2,4-DP (2, 4-D-isopropyl acid)
Table 3 shows Km, vmax, kcat, kcat/Km measured for the 4 proteins of which compound 2,4-DP is the substrate. The enzyme kinetic parameters of these 4 engineered proteins indicate that the measured engineered proteins maintain or even significantly increase activity on 2,4-DP compared to RdpA wild-type enzyme.
FIG. 1 shows the maximum reaction rate (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 measured engineered proteins maintain or even significantly increase 2,4-D activity compared to RdpA wild-type enzyme.
Example two expression of engineered proteins in maize
The engineered proteins were selected for corn transformation and plant analysis. The DNA construct is transformed into maize by agrobacterium tumefaciens and standard methods known in the art.
The transformed T0 generation transgenic plantlet and non-transgenic receptor plant expressing the engineering protein are cultured in a greenhouse. The T0 generation transgenic plantlet and the wild corn plant under the strict control condition are respectively sprayed with 10 g/mu of compound C for testing. As shown in FIG. 2, the wild type showed obvious phytotoxicity, while transgenic plantlets containing the M7 protein encoding gene were grown normally. In addition, T0 transgenic plantlets expressing other engineered proteins of the invention (e.g., M1, M11, M12, M13, M14, M16, M18, M19, M23, M24, M25, M26) can also grow normally. Thus, compared with wild corn, the transgenic corn containing the engineering protein coding gene can have better drug resistance under the treatment condition of 10 g/mu of compound C.
And (3) growing the T0 generation transgenic resistant plants after spraying the pesticide in a greenhouse, and collecting the T1 generation corn plant seeds generated by all the T0 generation transgenic resistant plants. The T1 generation seeds were sown, and 0, 90, 120 and 150 g/mu of compound C was sprayed at about the two-leaf one-heart growth stage, and the degree of resistance of the plants was evaluated by recording after the spraying treatment. After 150 g/mu of compound C11DAT is applied, compared with wild type plants, transgenic corn plants containing the engineered protein encoding gene of the invention show better drug resistance, which indicates that the tolerance dose of the T1 generation transgenic corn plants expressing the engineered protein to compound C is at least 150 g/mu. Representative test results are shown in FIG. 3.
Example III expression of engineered proteins in soybeans
The engineered proteins were selected for soybean transformation and plant analysis. The DNA construct is transformed into soybean by agrobacterium tumefaciens and standard methods known in the art.
The transformed T0 generation transgenic plantlet is grown in a greenhouse, and T1 generation seeds of the plantlet which are identified to be positive by the transgene are collected. Sowing T1 generation seeds, spraying 10 g/mu of compound C on T1 generation plantlets, and recording and evaluating the resistance degree of the plants after 12DAT is sprayed. Compared with the wild type plants which are not viable due to serious phytotoxicity, the transgenic soybean plants containing the genes encoding the engineered proteins (such as M13, M16, M19, M23 and M24) show better drug resistance, and representative test results are shown in FIG. 4.
Meanwhile, a plurality of tests show that the recombinant DNA molecules are introduced into arabidopsis thaliana, brachypodium distachyon and other mode plants, so that the corresponding level of drug resistance of synthetic hormone herbicides and/or ACCase inhibitor herbicides is improved. It is known that the transgenic plant can generate corresponding resistance characters to other plants, such as grain crops, bean crops, oil crops, fiber crops, fruit crops, rhizome crops, vegetable crops, flower crops, medicinal crops, raw material crops, pasture crops, sugar crops, beverage crops, lawn plants, tree crops, nut crops and the like, and has good industrial value.
Finally, it should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

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。1. 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. 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 IDNO: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。2. The recombinant DNA molecule of claim 1, wherein the nucleic acid sequence is selected from the group consisting of: SEQ IDNO: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 sequences shown due to the degeneracy of the genetic code. 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。3. 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. 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。4. A DNA construct comprising a heterologous promoter functional in a plant cell that is operably linked to a recombinant DNA molecule as described in any one of claims 1 to 3; preferably, further comprising a DNA molecule encoding a chloroplast transit peptide that is operably linked to the recombinant DNA molecule; preferably, the DNA construct is present in the genome of a transgenic plant. 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。5. A plant, seed, plant tissue, plant part or cell comprising the recombinant DNA molecule of any one of claims 1 to 3, the DNA construct of claim 4, or a polypeptide encoded by the recombinant DNA molecule of 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. 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。6. 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 a synthetic hormone herbicide, an ACCase inhibitor herbicide. 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。7. A method for conferring herbicide tolerance to a plant, seed, cell or plant part, the method comprising expressing in the plant, seed, cell or plant part a polypeptide as claimed in claim 6; preferably, the plant, seed, cell or plant part comprises a DNA construct comprising a heterologous promoter functional in a plant cell operably linked to a recombinant DNA molecule comprising a polypeptide encoding as claimed in claim 6. 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。8. 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. 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。9. 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. 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。10. 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.
CN202410397339.3A 2023-05-10 2024-04-03 Herbicide tolerance genes and methods of use thereof Pending CN118931929A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202310525401 2023-05-10
CN2023105254018 2023-05-10

Publications (1)

Publication Number Publication Date
CN118931929A true CN118931929A (en) 2024-11-12

Family

ID=93349140

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202410397339.3A Pending CN118931929A (en) 2023-05-10 2024-04-03 Herbicide tolerance genes and methods of use thereof

Country Status (2)

Country Link
CN (1) CN118931929A (en)
WO (1) WO2024230371A1 (en)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10260456A1 (en) * 2002-12-18 2004-07-08 Ufz-Umweltforschungszentrum Leipzig-Halle Gmbh Mutants of the Delftia acidovorans MC1 bacterial strain and their use in the production of high-purity R enantiomers of 2-phenoxypropionic acid derivatives
PL2308977T5 (en) * 2004-04-30 2017-10-31 Dow Agrosciences Llc Novel herbicide resistance gene
AP2017009831A0 (en) * 2014-10-15 2017-03-31 Monsanto Technology Llc Herbicide tolerance genes and methods of use thereof
CN118147174A (en) * 2020-08-06 2024-06-07 青岛清原种子科学有限公司 Herbicide resistance protein, gene and use
WO2023160362A1 (en) * 2022-02-25 2023-08-31 青岛清原化合物有限公司 Herbicide-tolerant genes and method for using same

Also Published As

Publication number Publication date
WO2024230371A1 (en) 2024-11-14

Similar Documents

Publication Publication Date Title
US12049636B2 (en) Herbicide tolerance genes and methods of use thereof
JP2021506232A (en) Methods and compositions for PPO herbicide resistance
JP2019523004A (en) Methods and compositions for gene expression in plants
IL291089A (en) Hydroxyphenylpyruvate mutant dioxygenase polypeptide, its encoding gene and its use
Mushtaq et al. Isolation of biotic stress resistance genes from cotton (Gossypium arboreum) and their analysis in model plant tobacco (Nicotiana tabacum) for resistance against cotton leaf curl disease complex
CN116694654A (en) Herbicide tolerance genes and methods of use thereof
US11060106B2 (en) Cytoplasmic male sterility gene ORF147 of pigeonpea, and uses thereof
CN118931929A (en) Herbicide tolerance genes and methods of use thereof
Yang et al. Modifying Myxococcus xanthus protoporphyrinogen oxidase to plant codon usage and high level of oxyfluorfen resistance in transgenic rice
CN118345098B (en) A corn ACCase mutant gene with herbicide resistance and its application
EP4450617A1 (en) Mutated hydroxyphenylpyruvate dioxygenase polypeptide, and coding gene and use thereof
US20240415123A1 (en) Methods and compositions for icafolin herbicide tolerance
AU2022456077A1 (en) Use of protoporphyrinogen oxidase
CN118591285A (en) Triketide dioxygenase variants for herbicide tolerance
CN119570752A (en) Active polypeptide of protoporphyrinogen oxidase and application thereof
Vogt et al. Cloning and characterization of apple lipoxygenase (LOX) genes putatively involved in the formation of fruit flavour volatiles
BR112017006910B1 (en) RECOMBINANT DNA MOLECULE, DNA CONSTRUCTION, POLYPEPTIDE, AND METHODS FOR CONFERRING HERBICIDES TOLERANCE TO A PLANT, SEED, CELL, OR PART OF A PLANT, TO PRODUCE A TRANSGENIC HERBICIDE TOLERANT PLANT, AND TO CONTROL WEEDS IN A GROWING AREA PLANT

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination