WO2016127867A1 - 除草剂抗性蛋白质、其编码基因及用途 - Google Patents

除草剂抗性蛋白质、其编码基因及用途 Download PDF

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WO2016127867A1
WO2016127867A1 PCT/CN2016/073182 CN2016073182W WO2016127867A1 WO 2016127867 A1 WO2016127867 A1 WO 2016127867A1 CN 2016073182 W CN2016073182 W CN 2016073182W WO 2016127867 A1 WO2016127867 A1 WO 2016127867A1
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herbicide
protein
herbicide resistance
plant
plants
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PCT/CN2016/073182
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English (en)
French (fr)
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陶青
吴业春
牛晓广
谢香庭
庞洁
鲍晓明
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北京大北农科技集团股份有限公司
北京大北农生物技术有限公司
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Priority to CA2975773A priority Critical patent/CA2975773C/en
Priority to RU2017128762A priority patent/RU2681162C1/ru
Priority to KR1020177025455A priority patent/KR102054567B1/ko
Priority to MX2017010262A priority patent/MX2017010262A/es
Application filed by 北京大北农科技集团股份有限公司, 北京大北农生物技术有限公司 filed Critical 北京大北农科技集团股份有限公司
Priority to UAA201708603A priority patent/UA122220C2/uk
Priority to EP16748668.7A priority patent/EP3257936A4/en
Priority to BR112017017348-4A priority patent/BR112017017348B1/pt
Priority to NZ734705A priority patent/NZ734705A/en
Priority to AU2016218739A priority patent/AU2016218739B2/en
Priority to JP2017560856A priority patent/JP6486505B2/ja
Priority to US15/550,342 priority patent/US10655140B2/en
Publication of WO2016127867A1 publication Critical patent/WO2016127867A1/zh
Priority to PH12017501394A priority patent/PH12017501394A1/en
Priority to IL253967A priority patent/IL253967B/en
Priority to CONC2017/0009267A priority patent/CO2017009267A2/es

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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G13/00—Protecting plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
    • A01N57/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds
    • A01N57/20—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds containing acyclic or cycloaliphatic radicals
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    • 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)
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    • 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
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    • 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
    • C12N15/8275—Glyphosate
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • 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)
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
    • C12Y114/19002—Acyl-[acyl-carrier-protein] desaturase (1.14.19.2)

Definitions

  • the present invention relates to a herbicide resistance protein, a gene encoding the same, and a use thereof, in particular to a protein resistant to 2,4-D, a gene encoding the same, and use thereof.
  • Crops that are resistant to glyphosate such as corn, soybeans, cotton, sugar beets, wheat, and rice, have been developed. It is therefore possible to spray glyphosate on fields where glyphosate resistant crops are grown to control weeds without significantly damaging the crops.
  • Glyphosate has been used worldwide for more than 20 years, resulting in an over-reliance on glyphosate and glyphosate-tolerant crop technology and is naturally more tolerant or has developed for glyphosate in wild weed species. Plants that are glyphosate resistant have a high selection pressure applied. A few weeds have been reported to have developed resistance to glyphosate, including broadleaf weeds and grass weeds such as Swiss ryegrass, ryegrass, goosegrass, ragweed, small canopy, wild pond Artemisia and long leaves in front of the car.
  • weeds that are not agricultural problems before the widespread use of glyphosate-tolerant crops are becoming more prevalent and difficult to control with glyphosate-tolerant crops, which are mainly (but not only) difficult to control broadleaf weeds.
  • Appears such as genus, genus, genus tarax, and comfrey.
  • 2,4-dichlorophenoxyacetic acid 2,4-dichlorophenoxyacetic acid
  • 2,4-D has been used in agricultural and non-crop conditions for broad-spectrum broadleaf weed control for more than 65 years and remains one of the most widely used herbicides in the world.
  • a further limitation to the further use of 2,4-D is that its selectivity in dicots such as soybean or cotton is very low, so 2,4-D is generally not used (and generally not close) to sensitive dicot crops.
  • 2,4-D in gramineous crops is somewhat limited by the nature of crop damage that may occur.
  • the combination of 2,4-D and glyphosate has been used to provide stronger killing treatments before planting no-till soybeans and cotton, however, due to the sensitivity of these dicot species to 2,4-D, these extinctions Treatment must be carried out 14-30 days before planting.
  • 2,4-D is a phenoxy acid herbicide.
  • 2,4-D is used to selectively control broadleaf weeds in many monocot crops (such as corn, wheat, and rice) without seriously damaging the intended crop.
  • 2,4-D is a synthetic auxin derivative whose function is to make it normal The cytokine is homeostatically dysregulated and impedes the controlled growth of balance.
  • 2,4-D has different levels of selectivity for certain plants (eg dicots are more sensitive than gramineous plants).
  • the different metabolism of 2,4-D by different plants is an explanation for the different levels of selectivity. Plants typically metabolize 2,4-D slowly, so the different activities of the target site are more likely to explain the plant's different responses to 2,4-D. Plant metabolism of 2,4-D is generally achieved by two-step metabolism, typically hydroxylation followed by conjugation with amino acids or glucose.
  • TfdA catalyzes the conversion of 2,4-D acid to dichlorophenol (DCP) by an alpha ketoglutarate-dependent dioxygenase reaction.
  • DCP has almost no herbicide activity compared to 2,4-D.
  • TfdA is used in transgenic plants to import 2,4-D resistance into dicotyledonous plants (such as cotton and tobacco) that are typically sensitive to 2,4-D.
  • tfdA-type genes encoding proteins capable of degrading 2,4-D have been identified in the environment. Many homologs are similar to tfdA (amino acid identity > 85%) and have similar enzymatic activity as tfdA. However, not all proteins with domains such as TauD have 2,4-D degradation, and a large number of homologs have significantly lower identity (25-50%) than tfdA, but with alpha ketone An acid-dependent dioxygenase Fe +2 dioxygenase-related characteristic residue. Therefore, it is not clear what the substrate specificity of these different dioxygenases is.
  • rdpA from Sphingobium herbicidovorans. This enzyme has been shown to catalyze the first step in the mineralization of (R)-2,4-D propionic acid (and other (R)-phenoxypropionic acid) and 2,4-D (phenoxyacetic acid).
  • the present invention provides a herbicide resistance protein comprising:
  • the present invention provides a herbicide resistance gene comprising:
  • nucleotide sequence which hybridizes under stringent conditions to (a) a defined nucleotide sequence and which encodes a protein having an aryloxyalkanoate dioxygenase activity;
  • the stringent conditions may be hybridization in a solution of 6 x SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) at 65 ° C, followed by 2 x SSC, 0.1% SDS and 1 x SSC, The membrane was washed once for each 0.1% SDS.
  • 6 x SSC sodium citrate
  • SDS sodium dodecyl sulfate
  • the present invention also provides an expression cassette comprising the herbicide resistance gene under the control of an operably linked regulatory sequence.
  • the present invention also provides a recombinant vector comprising the herbicide resistance gene or the expression cassette.
  • the present invention also provides a method for producing a herbicide-resistant protein, comprising:
  • the cells of the transgenic host organism are cultured under conditions that permit the production of a herbicide resistant protein
  • the herbicide resistance protein is recovered.
  • the transgenic host organism comprises a plant, an animal, a bacterium, a yeast, a baculovirus, a nematode or an alga.
  • the plant is soybean, cotton, corn, rice, wheat, sugar beet or sugar cane.
  • the present invention also provides a method for increasing the range of tolerance to herbicides, comprising: treating the herbicide resistance protein or the herbicide resistance protein encoded by the expression cassette in a plant with at least one A second nucleotide different from the herbicide resistance protein or the herbicide resistance protein encoded by the expression cassette is expressed together.
  • the second nucleotide encodes a glyphosate resistant protein, a glufosinate resistant protein, a 4-hydroxyphenylpyruvate dioxygenase, an acetolactate synthase, a cytochrome protein or a protoplast Porphyrinogen oxidase.
  • expression of the 24DT22 herbicide resistance protein in a transgenic plant may be accompanied by expression of one or more glyphosate resistant proteins and/or glufosinate resistant proteins. Co-expression of such more than one herbicide resistance protein in the same transgenic plant can be achieved by genetic engineering to allow the plant to contain and express the desired gene.
  • one plant first parent
  • the second plant second parent
  • Resistant protein. Pass The first parent and the second parent are crossed to obtain progeny plants expressing all of the genes introduced into the first parent and the second parent.
  • the present invention also provides a method for selecting a transformed plant cell, comprising: transforming a plurality of plant cells with the herbicide resistance gene or the expression cassette, and allowing expression of the herbicide resistance
  • the gene or the transformed cell of the expression cassette is grown, and the cell is cultured at a herbicide concentration that kills untransformed cells or inhibits growth of untransformed cells, which is phenoxy auxin.
  • the present invention also provides a method for controlling weeds comprising: applying an effective amount of a herbicide to a field for growing a crop, the crop comprising the herbicide resistance gene or the expression cassette or the Recombinant vector.
  • the herbicide is phenoxy auxin.
  • the present invention also provides a method for protecting a plant from damage caused by a herbicide, comprising: introducing the herbicide resistance gene or the expression cassette or the recombinant vector into a plant, The introduced plants are rendered herbicide resistant proteins sufficient to protect them from herbicide damage.
  • the herbicide is phenoxy auxin or an aryloxyphenoxy alkanoate.
  • the plant is soybean, cotton, corn, rice, wheat, sugar beet or sugar cane.
  • the present invention also provides a method for controlling glyphosate-resistant weeds in a field of glyphosate-tolerant plants, comprising: applying an effective dose of a herbicide to a field planted with glyphosate-tolerant plants,
  • the glyphosate-tolerant plant comprises the herbicide resistance gene or the expression cassette or the recombinant vector.
  • the herbicide is phenoxy auxin.
  • the glyphosate-tolerant plant is a monocot or a dicot.
  • the present invention also provides a method for conferring 2,4-D herbicide resistance to a crop comprising: introducing the herbicide resistance gene or the expression cassette or the recombinant vector into a plant.
  • the plant is soybean, cotton, corn, rice, wheat, sugar beet or sugar cane.
  • the present invention also provides a use of a herbicide-resistant protein tolerant to a phenoxy auxin-based herbicide, the herbicide-resistant protein comprising:
  • introducing the herbicide resistance gene or the expression cassette or the recombinant vector into a plant in the present invention, introducing foreign DNA into a plant cell, and conventional transformation methods include, but are not limited to, Agrobacterium-mediated Transformation, microprojection bombardment, direct DNA uptake into protoplasts, electroporation or whisker silicon mediated DNA introduction.
  • the 2,4-D resistance gene and subsequent resistant crops of the present invention provide an excellent choice for controlling glyphosate resistant (or highly tolerant and successional) broadleaf weed species in crops.
  • 2,4-D is a broad-spectrum, relatively inexpensive and potent broadleaf herbicide that provides superior efficacy to growers if it provides greater crop tolerance in both dicotyledonous and monocotyledonous leaves.
  • 2,4-D resistant transgenic dicots also offer greater flexibility in application time and dosage.
  • Another use of the 2,4-D herbicide resistance trait is that it can be used to prevent damage to normal sensitive crops such as 2,4-D drift, volatilization, conversion (or other long-range movement phenomena), misuse, and destruction. .
  • active acids are usually prepared, but some are also commercially formulated into one of a variety of corresponding ester preparations. Since general plant esterases convert these esters into active acids in plants, these are also the same. It is considered to be a substrate for the 24DT22 enzyme in plants. Analogously, it may also be the corresponding organic or inorganic salt of the corresponding acid.
  • chiral propionic acid propionate or propionate herbicides, even if different CAS numbers may correspond to optically pure compounds, racemic (R, S) or optical purification is considered when naming these herbicides.
  • the (R or S) enantiomer is the same herbicide. Possible amounts can range from crop or non-crop applications alone or in combination with other herbicides.
  • the 24DT22 gene has the property of allowing the use of phenoxy auxin herbicides in plants after genetic engineering for plant expression, in which the inherent tolerance is absent or insufficient to permit the use of these herbicides.
  • the 24DT22 gene can provide protection against phenoxy auxin herbicides in plants when natural tolerance is insufficient to allow selectivity.
  • Plants containing only the 24DT22 gene can now be treated in a continuous or tank mix with one, two or several phenoxy auxin herbicides.
  • the amount of each phenoxy auxin herbicide used to control broad-spectrum dicotyledonous weeds ranges from 25 to 4000 g ae/ha, more typically from 100 to 2000 g ae/ha. Combining these different chemical classes and herbicides with different modes of action and ranges in the same field (continuous or tank mix) can provide control of most potential weeds that require herbicide control.
  • Glyphosate is widely used because it controls a very broad spectrum of broadleaf and grass weed species.
  • repeated use of glyphosate in glyphosate resistant crop and non-crop applications has (and will continue to be) selected to succeed weeds as naturally more tolerant species or glyphosate resistant biotypes.
  • Most herbicide resistance management strategies suggest the use of an effective amount of canned herbicide companion as a means of delaying the emergence of resistant weeds that provide control of the same species but with different modes of action.
  • Superimposing the 24DT22 gene with glyphosate tolerance traits (and/or other herbicide tolerance traits) can be achieved by allowing the selective use of glyphosate and phenoxy auxin (eg 2,4-D) for the same crop.
  • Glyphosate-resistant weed species in glyphosate-tolerant crops (broadleaf controlled by one or more phenoxy auxins) Control of weed species).
  • the use of these herbicides can be used simultaneously in a tank mix of two or more herbicides containing different modes of action, for individual use of individual herbicide compositions in continuous use (eg, before planting, before emergence or after emergence). (The interval used ranges from 2 hours to 3 months), or alternatively, at any time (from 7 months from planting to when harvesting crops (or for pre-harvest intervals for individual herbicides, the shortest) ))
  • Use a combination that represents any number of herbicides that can be applied to each compound category.
  • Flexibility in controlling broadleaf weeds is important, namely the time of use, the amount of individual herbicides, and the ability to control stubborn or resistant weeds.
  • the application of glyphosate in the crop with the glyphosate resistance gene/24DT22 gene can range from 250 to 2500 g ae/ha; the phenoxy auxin herbicide (one or more) can range from 25-4000 g ae/ Ha. The optimal combination of time for these applications depends on the specific conditions, species and environment.
  • Herbicide formulations such as ester, acid or salt formulations or soluble concentrates, emulsified concentrates or solvables
  • tank mix additives such as adjuvants or compatibilizers
  • Any chemical combination of any of the foregoing herbicides is within the scope of the invention.
  • glyphosate resistance eg resistant plants or Bacterial EPSPS, GOX, GAT
  • glufosinate resistance eg PAT, Bar
  • acetolactate synthase (ALS) inhibitory herbicide resistance eg imidazolinone, sulfonylurea, triazole pyrimidine, sulfanilide, Pyrimidine thiobenzoic acid and other chemical resistance genes such as AHAS, Csrl, SurA, etc.
  • bromoxynil resistant eg Bxn
  • HPPD 4-hydroxyphenylpyruvate dioxygenase
  • some other preferred ALS inhibitors include triazolopyrimidine sulfanilide (chlorosulfasalin, diclofenac, sulffenapyr, sulfazinamide, and pyrimidotriazole sulfonamide), Pyrimidine thiobenzoic acid and flucarbazone.
  • Some preferred HPPD inhibitors include mesotrione, isoxaflutole and sulcotrione.
  • Some preferred PPO inhibitors include propargylamine, flufenacetate, oxazolone, mesalil and diphenyl ethers (eg, acifluorfen, flufenazone, flufenacil and Oxyfluorfen).
  • the 24DT22 gene can be superimposed alone or in combination with other herbicide-tolerant crop characteristics. Superimposed with one or more other inputs (eg, insect resistance, fungal resistance or stress tolerance, etc.) or output (eg, increased yield, improved oil content, increased fiber quality, etc.).
  • the present invention can be used to provide a complete agronomic solution that flexibly and economically controls the ability of any number of agronomic pests and enhances crop quality.
  • the 24DT22 gene of the present invention is capable of degrading 2,4-D and is the basis for important herbicide tolerance to crop and selection marker characteristics.
  • the present invention allows transgenic expression to control the herbicide combination of almost all broadleaf weeds.
  • the 24DT22 gene can be used as an excellent herbicide tolerant to crop traits such as other herbicide tolerant crop traits (eg glyphosate resistance, glufosinate resistance, ALS inhibitors (eg imidazolinones, sulfonylureas, Triazolopyrimidine sulfonamides resistance, bromoxynil resistance, HPPD inhibitor resistance, PPO inhibitor resistance, etc.) and insect resistance traits (Cry1Ab, Cry1F, Vip3, other Bacillus thuringiensis proteins or non-spore A strain of Bacillus-derived insect resistance protein, etc.) is superimposed.
  • the 24DT22 gene can be used as a selection marker to aid in the selection of primary transformants of plants genetically engineered with another gene or gene population.
  • a phenoxy alkanoate group can be used to introduce a stable acid functional group into the herbicide.
  • the acidic group can be introduced into the phloem activity (the property required for the action of the herbicide) by "acid capture" so that the new herbicide can be integrated for the purpose of activity.
  • phloem activity the property required for the action of the herbicide
  • acid capture the property required for the action of the herbicide
  • the herbicide-tolerant crop traits of the present invention can be used in new combinations with other herbicide-tolerant crop traits including, but not limited to, glyphosate tolerance. These trait combinations produce new methods of controlling weed species due to newly acquired resistance or inherent tolerance to herbicides such as glyphosate.
  • the scope of the invention includes a novel method of controlling weeds using a herbicide wherein the tolerance to the herbicide is produced by the enzyme in the transgenic crop.
  • the invention is applicable to a variety of plants such as Arabidopsis thaliana, tobacco, soybean, cotton, rice, corn and alfalfa.
  • the invention is also applicable to a variety of other monocotyledons (such as forage grasses or turfgrass) and dicotyledonous crops (such as alfalfa, clover, arbor species, etc.).
  • 2,4-D or other 24DT22 substrates
  • the genome of a plant, plant tissue or plant cell as referred to in the present invention refers to any genetic material within a plant, plant tissue or plant cell, and includes the nucleus and plastid and mitochondrial genomes.
  • the "resistance” described in the present invention is heritable and allows the plants to grow and multiply in the case where the herbicide is effectively treated with a general herbicide for a given plant. As recognized by those skilled in the art, plants can be considered “resistant” even if the plants are significantly damaged by herbicide treatment.
  • the term “tolerance” in the present invention is broader than the term “resistance” and includes “resistance”, as well as the ability of certain plants to have increased resistance to herbicide-induced damage to various degrees, while in the same weeding The dose of the agent generally results in damage to the same genotype of wild type plants.
  • polynucleotides and/or nucleotides described herein form a complete "gene" encoding a protein or polypeptide in a desired host cell.
  • polynucleotides and/or nucleotides of the invention can be placed under the control of regulatory sequences in a host of interest.
  • DNA typically exists in a double stranded form. In this arrangement, one chain is complementary to the other and vice versa. Since DNA is replicated in plants, other complementary strands of DNA are produced. Thus, the invention encompasses the use of the polynucleotides exemplified in the Sequence Listing and their complementary strands.
  • a "coding strand” as commonly used in the art refers to a strand that binds to the antisense strand.
  • To express a protein in vivo one strand of DNA is typically transcribed into a complementary strand of mRNA that is used as a template to translate the protein. mRNA is actually transcribed from the "antisense" strand of DNA.
  • a “sense” or “encoding” strand has a series of codons (codons are three nucleotides, three reads at a time to produce a particular amino acid), which can be read as an open reading frame (ORF) to form a protein or peptide of interest.
  • the present invention also includes RNA and PNA (peptide nucleic acid) having comparable functions to the exemplified DNA.
  • the nucleic acid molecule or fragment thereof of the present invention hybridizes under stringent conditions to the herbicide resistance gene of the present invention. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the herbicide resistance gene of the present invention.
  • a nucleic acid molecule or fragment thereof is capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. In the present invention, if two nucleic acid molecules can form an anti-parallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules are capable of specifically hybridizing each other. If two nucleic acid molecules exhibit complete complementarity, one of the nucleic acid molecules is said to be the "complement" of the other nucleic acid molecule.
  • nucleic acid molecules when each nucleotide of one nucleic acid molecule is complementary to a corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to exhibit "complete complementarity".
  • Two nucleic acid molecules are said to be “minimally complementary” if they are capable of hybridizing to one another with sufficient stability such that they anneal under at least conventional "low stringency” conditions and bind to each other.
  • two nucleic acid molecules are said to be “complementary” if they are capable of hybridizing to one another with sufficient stability such that they anneal under conventional "highly stringent” conditions and bind to each other.
  • Deviation from complete complementarity is permissible as long as such deviation does not completely prevent the two molecules from forming a double-stranded structure.
  • a nucleic acid molecule In order for a nucleic acid molecule to function as a primer or probe, it is only necessary to ensure that it is sufficiently complementary in sequence to allow for the formation of a stable double-stranded structure at the particular solvent and salt concentration employed.
  • a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing to a complementary strand of another matched nucleic acid molecule under highly stringent conditions.
  • Suitable stringent conditions for promoting DNA hybridization for example, treatment with 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by washing with 2.0 x SSC at 50 ° C, these conditions are known to those skilled in the art. It is well known.
  • the salt concentration in the washing step can be selected from about 2.0 x SSC under low stringency conditions, 50 ° C to about 0.2 x SSC, 50 ° C under highly stringent conditions.
  • the temperature conditions in the washing step can be raised from a low temperature strict room temperature of about 22 ° C to about 65 ° C under highly stringent conditions. Temperature conditions and The salt concentration can all change, or one of them can remain unchanged while the other variable changes.
  • the stringent conditions of the present invention may be specific hybridization with SEQ ID NO: 1 at 65 ° C in 6 x SSC, 0.5% SDS solution, followed by 2 x SSC, 0.1% SDS and 1 x SSC. 0.1% SDS was washed once each time.
  • sequences having herbicide tolerance activity and hybridizing to the sequence 1 of the present invention under stringent conditions are included in the present invention. These sequences are at least about 40%-50% homologous to the sequences of the invention, about 60%, 65% or 70% homologous, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93. Sequence homology of %, 94%, 95%, 96%, 97%, 98%, 99% or greater.
  • the invention provides functional proteins.
  • “Functional activity” (or “activity”) in the present invention means that the protein/enzyme (alone or in combination with other proteins) for use in the present invention has the ability to degrade or attenuate herbicide activity.
  • the plant producing the protein of the invention preferably produces an "effective amount" of the protein such that when the plant is treated with the herbicide, the level of protein expression is sufficient to give the plant complete or partial resistance to the herbicide (typically, unless otherwise stated). Or patience.
  • the herbicide can be used in an amount which normally kills the target plant, normal field amount and concentration.
  • the plant cells and plants of the invention are protected from growth inhibition or damage caused by herbicide treatment.
  • the transformed plants and plant cells of the invention preferably have resistance or tolerance to 2,4-D herbicides, i.e., transformed plants and plant cells can be grown in the presence of an effective amount of a 2,4-D herbicide.
  • genes and proteins described in the present invention include not only specific exemplary sequences, but also portions and/or fragments that retain the herbicide resistance activity characteristics of the proteins of the specific examples (including compared to the full length protein and/or Or terminal deletions, variants, mutants, substitutions (proteins with alternative amino acids), chimeras and fusion proteins.
  • the "variant” or “variant” refers to a nucleotide sequence that encodes the same protein or an equivalent protein encoded with herbicide resistance activity.
  • the "equivalent protein” refers to a biologically active protein having the same or substantially the same herbicide tolerance as the protein of the claims.
  • a “fragment” or “truncated” sequence of a DNA molecule or protein sequence as used in the present invention refers to a portion of the original DNA or protein sequence (nucleotide or amino acid) involved or an artificially engineered form thereof (eg, a sequence suitable for plant expression)
  • an artificially engineered form thereof eg, a sequence suitable for plant expression
  • the inclusion of adjacent fragments and internal and/or terminal deletions compared to full length molecules, the length of the foregoing sequences may vary, but is of sufficient length to ensure that the (encoding) protein is a herbicide resistant protein. In some cases (especially expression in plants), it may be advantageous to use a truncated gene encoding a truncated protein.
  • Preferred truncated genes generally encode 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 of full-length proteins. , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%.
  • substantially identical sequence refers to a sequence that has an amino acid substitution, deletion, addition or insertion but does not substantially affect herbicide resistance activity, and also includes fragments that retain herbicide resistance activity.
  • Substitution, deletion or addition of an amino acid sequence in the present invention is a conventional technique in the art, and it is preferred that such an amino acid change is: a small change in properties, that is, a conservative amino acid substitution that does not significantly affect the folding and/or activity of the protein; a small deletion, Typically a deletion of about 1-30 amino acids; a small amino or carboxy terminal extension, such as a methionine residue at the amino terminus; and a small linker peptide, for example about 20-25 residues in length.
  • conservative substitutions are substitutions occurring within the following amino acid groups: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, asparagine, hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small molecules Amino acids (such as glycine, alanine, serine, threonine, and methionine). Those amino acid substitutions that generally do not alter a particular activity are well known in the art and have been described, for example, by N. Neurath and R. L.
  • substitutions can occur outside of the regions that are important for molecular function and still produce active polypeptides.
  • amino acids from the polypeptides of the invention that are essential for their activity and are therefore selected for unsubstitution they can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells). , 1989, Science 244: 1081-1085).
  • site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells). , 1989, Science 244: 1081-1085).
  • the latter technique introduces a mutation at each positively charged residue in the molecule, and detects the herbicide resistance activity of the resulting mutant molecule, thereby determining an amino acid residue important for the activity of the molecule.
  • the substrate-enzyme interaction site can also be determined by analysis of its three-dimensional structure, which can be determined by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, eg, de Vos et al., 1992, Science 255). : 306-312; Smith et al, 1992, J. Mol. Biol 224: 899-904; Wlodaver et al, 1992, FEBS Letters 309: 59-64).
  • amino acid sequence having a certain homology with the amino acid sequence shown in SEQ ID NO: 2 is also included in the present invention.
  • sequences are typically more than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%, similar to the sequences of the present invention.
  • Preferred polynucleotides and proteins of the invention may also be defined according to a more specific range of identity and/or similarity.
  • the sequence of the example of the present invention is 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity and/or similarity.
  • Regulatory sequences of the invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the 24DT22 gene.
  • the promoter is a promoter expressible in a plant
  • the "promoter expressible in a plant” refers to a promoter which ensures expression of a coding sequence linked thereto in a plant cell.
  • a promoter expressible in a plant can be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, the 35S promoter derived from cauliflower mosaic virus, the maize Ubi promoter, the promoter of the rice GOS2 gene, and the like.
  • a promoter expressible in a plant may be a tissue-specific promoter, ie the promoter directs the expression level of the coding sequence in some tissues of the plant, such as in green tissue, to be higher than other tissues of the plant (through conventional The RNA assay is performed), such as the PEP carboxylase promoter.
  • a promoter expressible in a plant can be a wound-inducible promoter.
  • a wound-inducible promoter or a promoter that directs a wound-inducible expression pattern means that when the plant is subjected to mechanical or wounding by insect foraging, the expression of the coding sequence under the control of the promoter is significantly improved compared to normal growth conditions.
  • wound-inducible promoters include, but are not limited to, promoters of protease inhibitory genes (pinI and pinII) and maize protease inhibitory genes (MPI) of potato and tomato.
  • the transit peptide (also known as a secretion signal sequence or targeting sequence) directs the transgene product to a particular organelle or cell compartment, and for the receptor protein, the transit peptide can be heterologous, for example, using a coding chloroplast transporter
  • the peptide sequence targets the chloroplast, or targets the endoplasmic reticulum using the 'KDEL' retention sequence, or the CTPP-targeted vacuole using the barley plant lectin gene.
  • the leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a potato virus group leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin protein heavy chain binding protein (BiP); untranslated leader sequence of the coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader sequence.
  • EMCV leader sequence 5' non-coding region of encephalomyocarditis virus
  • a potato virus group leader sequence such as a MDMV (maize dwarf mosaic virus) leader sequence
  • MDMV human immunoglobulin protein heavy chain binding protein
  • AdMV alfalfa mosaic virus
  • TMV tobacco mosaic virus
  • the enhancer includes, but is not limited to, a cauliflower mosaic virus (CaMV) enhancer, a figwort mosaic virus (FMV) enhancer, a carnation weathering ring virus (CERV) enhancer, and a cassava vein mosaic virus (CsVMV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • CERV carnation weathering ring virus
  • CsVMV cassava vein mosaic virus
  • MMV Purple Jasmine Mosaic Virus
  • MMV Yellow Jasmine Mosaic Virus
  • CmYLCV Night fragrant yellow leaf curl virus
  • CLCuMV Multan cotton leaf curl virus
  • CoYMV Acanthus yellow mottle virus
  • PCLSV peanut chlorotic line flower Leaf virus
  • the introns include, but are not limited to, maize hsp70 introns, maize ubiquitin introns, Adh introns 1, sucrose synthase introns, or rice Actl introns.
  • the introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "super ubiquitin" intron.
  • the terminator can be a suitable polyadenylation signal sequence that functions in plants, including but, it is not limited to a polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene, a polyadenylation signal sequence derived from a protease inhibitor II (pin II) gene, and a source thereof.
  • NOS Agrobacterium tumefaciens nopaline synthase
  • pin II protease inhibitor II
  • operably linked refers to the joining of nucleic acid sequences that allow one sequence to provide the function required for the linked sequence.
  • the "operably linked” in the present invention may be such that the promoter is ligated to the sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter.
  • Effective ligation when a sequence of interest encodes a protein and is intended to obtain expression of the protein means that the promoter is ligated to the sequence in a manner that allows efficient translation of the resulting transcript.
  • the linker of the promoter to the coding sequence is a transcript fusion and it is desired to effect expression of the encoded protein, such ligation is made such that the first translation initiation codon in the resulting transcript is the start codon of the coding sequence.
  • the linkage of the promoter to the coding sequence is a translational fusion and it is desired to effect expression of the encoded protein, such linkage is made such that the first translation initiation codon and promoter contained in the 5' untranslated sequence Linked and linked such that the resulting translation product is in frame with the translational open reading frame encoding the desired protein.
  • Nucleic acid sequences that may be "operably linked” include, but are not limited to, sequences that provide for gene expression functions (ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, poly Adenylation site and/or transcription terminator), sequences that provide DNA transfer and/or integration functions (ie, T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), provide options Sexually functional sequences (ie, antibiotic resistance markers, biosynthetic genes), sequences that provide for the function of scoring markers, sequences that facilitate sequence manipulation in vitro or in vivo (ie, polylinker sequences, site-specific recombination sequences) and provision The sequence of the replication function (ie, the origin of replication of the bacteria, the autonomously replicating sequence, the centromeric sequence).
  • gene expression functions ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein
  • the present invention confers new herbicide resistance traits on plants and does not observe adverse effects on phenotype including yield.
  • the plants of the present invention are tolerant to a general application level of at least one of the tested herbicides 2 x, 3 x, 4 x or 5 x. These levels of tolerance are within the scope of the invention. For example, predictable optimizations and further developments can be made to a variety of techniques known in the art to increase expression of a given gene.
  • the herbicide resistance protein is a 24DT22 amino acid sequence as shown in SEQ ID NO: 2 in the Sequence Listing.
  • the herbicide resistance gene is a 24DT22 nucleotide sequence as shown in SEQ ID NO: 1 in the Sequence Listing.
  • the herbicide resistance gene is for use in a plant, and may comprise, in addition to the coding region of a protein encoded by the 24DT22 nucleotide sequence, other elements, such as a coding region encoding a transit peptide, a protein encoding a selectable marker, or conferring The coding region of an insect-resistant protein.
  • the 24DT22 herbicide resistance protein of the present invention is resistant to most phenoxy auxin herbicides.
  • the plant of the present invention contains exogenous DNA in its genome, and the exogenous DNA comprises The 24DT22 nucleotide sequence protects against the herbicide by expressing an effective amount of the protein.
  • An effective amount refers to a dose that is undamaged or slightly damaged.
  • the plants should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product.
  • the expression level of the herbicide resistance protein in the plant material can be detected by various methods described in the art, for example, by using a specific primer to quantify the mRNA encoding the herbicide resistance protein produced in the tissue, or directly specific The amount of herbicide resistant protein produced is detected.
  • the present invention provides a herbicide resistance protein, a gene encoding the same and use thereof, and has the following advantages:
  • the herbicide resistance protein 24DT22 of the invention is highly resistant to herbicides, especially against phenoxy auxin herbicides, especially 2,4-D.
  • the herbicide resistance protein 24DT22 protein of the invention can exhibit high resistance to various phenoxy auxin herbicides, and thus has broad application prospects on plants.
  • FIG. 1 is a flow chart showing the construction of a recombinant cloning vector DBN01-T containing a 24DT22 nucleotide sequence of a herbicide resistance protein, a gene encoding the same, and a use thereof;
  • FIG. 2 is a flow chart showing the construction of a recombinant expression vector DBN100301 containing a 24DT22 nucleotide sequence of a herbicide resistance protein, a gene encoding the same, and a use thereof;
  • FIG. 3 is a flow chart showing the construction of a recombinant expression vector DBN100301N containing a control sequence of a herbicide resistance protein, a coding gene thereof and use thereof;
  • Figure 4 is a diagram showing the herbicide resistance effect of the transgenic Arabidopsis T 1 plant of the herbicide resistance protein, the gene encoding the same, and the use thereof;
  • Figure 5 is a diagram showing the herbicide resistance effect of the herbicide-resistant protein, the gene encoding the same, and the use of the transgenic soybean T 1 plant of the present invention
  • Figure 6 is a flow chart showing the construction of a recombinant expression vector DBN100764 containing a 24DT22 nucleotide sequence of the herbicide resistance protein, the coding gene thereof and the use thereof;
  • Figure 7 is a flow chart showing the construction of a recombinant expression vector DBN100764N containing a control sequence of the herbicide resistance protein, its coding gene and use thereof.
  • the herbicide-resistant protein of the present invention the gene encoding the same, and the use of the same are described below by way of specific examples.
  • Amino acid sequence of the 24DT22 herbicide resistance protein (292 amino acids) as shown in SEQ ID NO: 2 in the Sequence Listing; obtaining an amino acid sequence encoding a herbicide resistance protein corresponding to the 24DT22 according to a plant preference codon (292)
  • the nucleotide sequence (879 nucleotides) of the amino acid is shown as SEQ ID NO: 1 in the Sequence Listing.
  • the 24DT22 nucleotide sequence (as shown in SEQ ID NO: 1 in the Sequence Listing) was synthesized by Nanjing Kingsray Biotechnology Co., Ltd.; the 5' of the 24DT22 nucleotide sequence (SEQ ID NO: 1) synthesized The SpeI restriction site is also ligated to the end, and the 3' end of the 24DT22 nucleotide sequence (SEQ ID NO: 1) is also ligated with a KasI cleavage site.
  • the synthetic 24DT22 nucleotide sequence was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the procedure was carried out according to the Promega product pGEM-T vector specification to obtain a recombinant cloning vector DBN01-T.
  • FIG. 1 wherein Amp represents the ampicillin resistance gene; f1 represents the origin of replication of phage f1; LacZ is the LacZ initiation codon; SP6 is the SP6 RNA polymerase promoter; and T7 is the T7 RNA polymerase promoter; 24DT22 is the 24DT22 nucleotide sequence (SEQ ID NO: 1); MCS is the multiple cloning site).
  • the recombinant cloning vector DBN01-T was then transformed into E. coli T1 competent cells by heat shock method (Transgen, Beijing, China, CAT: CD501) under heat shock conditions: 50 ⁇ L E. coli T1 competent cells, 10 ⁇ L of plasmid DNA (recombinant) Cloning vector DBN01-T), water bath at 42 ° C for 30 seconds; shaking culture at 37 ° C for 1 hour (shake at 100 rpm), coated with IPTG (isopropylthio- ⁇ -D-galactoside) and X -gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) ampicillin (100 mg/L) in LB plate (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g) /L, agar 15 g/L, adjusted to pH 7.5 with NaOH) and grown overnight.
  • heat shock method Transgen, Beijing, China, CAT: CD501
  • White colonies were picked and cultured in LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, ampicillin 100 mg/L, pH adjusted to 7.5 with NaOH) at 37 °C. overnight.
  • the plasmid was extracted by alkaline method: the bacterial solution was centrifuged at 12000 rpm for 1 min, the supernatant was removed, and the precipitated cells were pre-cooled with 100 ⁇ L of ice (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose.
  • the 24DT22 nucleotide sequence inserted into the recombinant cloning vector DBN01-T was the nucleot represented by SEQ ID NO: 1 in the sequence listing.
  • the nucleotide sequence, the 24DT22 nucleotide sequence, was inserted correctly.
  • the recombinant cloning vector DBN01-T and the expression vector DBNBC-01 were digested with restriction endonucleases SpeI and KasI, respectively, and the excised 24DT22 nucleotide sequence fragment was inserted into the expression. Between the SpeI and KasI sites of the vector DBNBC-01, the construction of the vector by conventional enzymatic cleavage method is well known to those skilled in the art, and the recombinant expression vector DBN100301 is constructed, and the construction process thereof is shown in Fig.
  • the recombinant expression vector DBN100301 was transformed into E. coli T1 competent cells by heat shock method.
  • the heat shock conditions were: 50 ⁇ L E. coli T1 competent cells, 10 ⁇ L of plasmid DNA (recombinant expression vector DBN100301), 42 ° C water bath for 30 seconds; 37 ° C oscillation Incubate for 1 hour (shake shake at 100 rpm); then LB solid plate containing 50 mg/L kanamycin (trypeptin 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g) /L, adjust the pH to 7.5 with NaOH and incubate at 37 °C for 12 hours, pick white colonies, in LB liquid medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, Kanamycin 50 mg/L was adjusted to pH 7.5 with NaOH and incubated overnight at 37 °C.
  • the plasmid was extracted by an alkali method.
  • the extracted plasmid was digested with restriction endonucleases SpeI and KasI, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence between the SpeI and KasI sites of the recombinant expression vector DBN100301 was SEQ ID in the sequence listing. NO: The nucleotide sequence shown in 1, which is the 24DT22 nucleotide sequence.
  • a recombinant cloning vector DBN01R-T containing a control sequence was constructed using the control sequence (SEQ ID NO: 8) according to the method of constructing the recombinant cloning vector DBN01-T containing the nucleotide sequence of 24DT22 according to the second embodiment of the present invention. .
  • the positive clone was subjected to sequencing verification, and the result showed that the control sequence inserted into the recombinant cloning vector DBN01R-T was the nucleotide sequence shown by SEQ ID NO: 8 in the sequence listing, that is, the control sequence was correctly inserted.
  • a method for constructing a recombinant expression vector DBN100301 containing a 24DT22 nucleotide sequence according to the second embodiment of the present invention 2 using a control sequence to construct a recombinant expression vector containing a control sequence DBN100301N, its vector structure is shown in Figure 3 (vector backbone: pCAMBIA2301 (available from CAMBIA); Kan: kanamycin gene; RB: right border; AtUbi10: Arabidopsis Ubiquitin 10 gene promoter ( SEQ ID NO: 3); mN: control sequence (SEQ ID NO: 8); Nos: terminator of the nopaline synthase gene (SEQ ID NO: 4); prCaMV35S: cauliflower mosaic virus 35S promoter (SEQ ID NO) :5); PAT: glufosinate acetyltransferase gene (SEQ ID NO: 6); tCaMV35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 7); LB
  • control sequence inserted into the recombinant expression vector DBN100301N was the nucleotide sequence shown by SEQ ID NO: 8 in the sequence listing, that is, the control sequence was correctly inserted.
  • the recombinant expression vectors DBN100301 and DBN100301N (control sequences) which have been constructed correctly were transformed into Agrobacterium GV3101 by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L Agrobacterium GV3101, 3 ⁇ L plasmid DNA (recombinant expression vector); In 10 minutes, a 37 ° C warm water bath for 10 minutes; the transformed Agrobacterium GV3101 was inoculated in LB tubes and incubated at a temperature of 28 ° C, 200 rpm for 2 hours, and applied to 50 mg / L of rifampicin (Rifampicin) And 50mg/L kanamycin on the LB plate until the positive monoclonal growth, pick the monoclonal culture and extract the plasmid, digest the DBN100301 with the restriction enzymes SmaI and PstI, and then perform the enzyme digestion verification.
  • 100 ⁇ L Agrobacterium GV3101, 3 ⁇ L plasmid DNA recomb
  • the endonuclease SmaI and BglI were digested with DBN100301N (control sequence) and then digested with the enzyme. The results showed that the recombinant expression vectors DBN100301 and DBN100301N (control sequences) were completely correct.
  • Wild type Arabidopsis seeds were suspended in a 0.1% (w/v) agarose solution.
  • the suspended seeds were stored at 4 ° C for 2 days to complete the need for dormancy to ensure simultaneous seed germination.
  • the pretreated seeds were planted on a soil mixture and covered with a moisturizing hood for 7 days.
  • the seeds were germinated and the plants were grown in a greenhouse under constant temperature (22 ° C) constant humidity (40-50%) long light conditions (16 hours light / 8 hours dark) with a light intensity of 120-150 ⁇ mol/m 2 sec. Start irrigating the plants with Hoagland nutrient solution, then irrigate with deionized water to keep the soil moist but not soaked.
  • Arabidopsis thaliana was transformed using flower soaking.
  • One or more 15-30 mL precultures of YEP broth containing kanamycin (50 mg/L) and rifampicin (10 mg/L) were inoculated with selected Agrobacterium colonies. The culture was incubated overnight at 28 ° C with constant shaking at 220 rpm.
  • Each preculture was used to inoculate two 500 mL cultures of YEP medium containing kanamycin (50 mg/L) and rifampicin (10 mg/L) and the cultures were incubated overnight at 28 °C with constant shaking.
  • the cells were pelleted by centrifugation at about 8700 x g for 10 minutes at room temperature, and the resulting supernatant was discarded.
  • the cell pellet was gently resuspended in 500 mL osmotic medium containing 1/2 x MS salt / B5 vitamin, 10% (w/v) sucrose, 0.044 ⁇ M benzylaminopurine (10 ⁇ L/L (stock solution in 1 mg/mL DMSO)) and 300 ⁇ L/L Silvet L-77. Plants of about 1 month old were soaked in the medium for 15 seconds to ensure that the latest inflorescences were immersed. The sides of the plants were then placed downside and covered (transparent or opaque) for 24 hours, then washed with water and placed vertically. The plants were cultured at 22 ° C with a photoperiod of 16 hours light / 8 hours dark. Seeds were harvested after about 4 weeks of soaking.
  • T 1 seeds were dried at room temperature for 7 days.
  • the seeds were planted in 26.5 ⁇ 51cm germination pots, seed receiving 200mgT 1 (about 10,000 seeds) per plate, the seed has been previously suspended in 40mL 0.1% (w / v) agarose solution and stored at 4 °C 2 days In order to complete the need for dormancy to ensure the simultaneous seed germination.
  • the pretreated seeds (each 40 mL) were evenly planted on the soil mixture with a pipette and covered with a moisturizing hood for 4-5 days. The hood was removed 1 day prior to the initial transformant selection using glufosinate (selected co-transformed PAT gene) after emergence.
  • a 0.2% solution of Liberty herbicide 200 g ai/L glufosinate was sprayed with a spray volume of 10 mL/disc (703 L/ha). 1 plant (coronal stage and 2-4 leaf stage, respectively) to provide an effective amount of 280 g ai/ha of glufosinate per application.
  • Surviving strains plants that are actively growing were identified 4-7 days after the last spraying, and transplanted into 7 cm x 7 cm square pots (3-5 per plate) prepared with horse manure and vermiculite, respectively.
  • the transplanted plants were covered with a moisturizing hood for 3-4 days and placed in a 22 ° C culture chamber as before or directly into the greenhouse. The hood was then removed and the plants were planted in the greenhouse at least 1 day prior to testing the ability of the 24DT22 gene to provide phenoxy auxin herbicide resistance (22 ⁇ 5 ° C, 50 ⁇ 30% RH, 14 hours light: 10 hours dark, Minimum 500 ⁇ E/m 2 s 1 natural + supplemental light).
  • the first Arabidopsis transformation was carried out using the 24DT22 gene. First, using the glufosinate selection scheme selected from untransformed T 1 seed background transformants. Screening of about 20,000 seeds T 1 and T 1 314 identified positive transformants substituting (PAT gene), about 1.6% of conversion efficiency.
  • the nucleotide sequence into 24DT22 T 1 Arabidopsis plants Arabidopsis control sequences into T 1 plants and wild-type Arabidopsis plants (18 days after sowing) were 2,4-D dimethylammonium of Salt and methyltetrachloride were tested for herbicide resistance.
  • the nucleotide sequences were transferred 24DT22 T 1 Arabidopsis plants Arabidopsis control sequences into T 1 plants and wild-type Arabidopsis plants are salts with dimethyl 2,4-D (560g ae / Ha, 1 times field concentration), xylene tetrachloride (560 g ae/ha, 1 time field concentration) and blank solvent (water) spray. Plant resistance was measured after spraying for 7 days and 14 days: after 7 days, the growth condition and the blank solvent (water) were consistently classified as high-resistance plants, and after 7 days, the rosettes of the rosettes were classified as medium-resistant plants, and the leaves could not be bolted after 14 days.
  • T 1 Arabidopsis plants per plant is an independent transformation event, significant differences can be expected within a given dose for the individual T 1 responses. The results are shown in Table 1 and Figure 4.
  • the recombinant expression vectors DBN100301 and DBN100301N (control sequences) which have been constructed correctly were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L Agrobacterium LBA4404, 3 ⁇ L of plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, and warmed at 37 ° C for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated into LB tubes and incubated at a temperature of 28 ° C and a rotation speed of 200 rpm for 2 hours.
  • the cotyledonary node tissue of the aseptically cultured soybean variety Zhonghuang 13 was co-cultured with the Agrobacterium described in Example 1 in accordance with the conventional Agrobacterium infestation method to construct the 2 and 3 in the second embodiment.
  • Recombinant expression vectors DBN100301 and DBN100301N T-DNA including Arabidopsis thaliana
  • Promoter sequence of Ubiquitin10 gene, 24DT22 nucleotide sequence, control sequence, Nos terminator, cauliflower mosaic virus 35S promoter, glufosinate acetyltransferase gene, cauliflower mosaic virus 35S terminator transferred to soybean genome
  • wild type soybean plants were used as a control.
  • soybean germination medium B5 salt 3.1 g/L, B5 vitamin, sucrose 20 g/L, agar 8 g/L, pH 5.6.
  • the seeds were inoculated on a germination medium and cultured under the following conditions: temperature 25 ⁇ 1 ° C; photoperiod (light/dark) was 16/8 h.
  • photoperiod light/dark
  • the soybean sterile seedlings of the fresh green cotyledon nodes were taken, the hypocotyls were cut at 3-4 mm below the cotyledonary nodes, and the cotyledons were cut longitudinally to remove the top buds, lateral buds and seed roots.
  • Step 1 Invasion Dyeing step
  • infecting medium MS salt 2.15 g/L, B5 vitamin, sucrose 20 g/L, glucose 10 g/L
  • AS acetosyringone
  • MES 2-morpholine ethanesulfonic acid
  • ZT zeatin
  • Step 2 Cotyledonary node tissue and Agrobacterium Culture for a period of time (3 days)
  • the cotyledonary node tissue is in solid medium after the infection step (MS salt 4.3 g/L, B5 vitamin, sucrose 20 g/L, glucose 10 g/ L, 2-morpholine ethanesulfonic acid (MES) 4g / L, zeatin 2mg / L, agar 8g / L, pH 5.6) culture.
  • MS salt 4.3 g/L, B5 vitamin, sucrose 20 g/L, glucose 10 g/ L, 2-morpholine ethanesulfonic acid (MES) 4g / L, zeatin 2mg / L, agar 8g / L, pH 5.6
  • step 3 restore the medium (B5 salt 3.1g / L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1g / L, sucrose 30g / L, zeatin (ZT) 2mg / L , agar 8g/L, cephalosporin
  • MES 2-morpholine ethanesulfonic acid
  • ZT zeatin
  • agar 8g/L agar 8g/L
  • cephalosporin At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium is present in the peptide 150 mg/L, glutamic acid 100 mg/L, aspartic acid 100 mg/L, pH 5.6, without the addition of plant transformants.
  • Selector step 3: recovery step).
  • the cotyledonary node-regenerated tissue mass is cultured on a solid medium with antibiotics but no selective agent to eliminate Agrobacterium and provide a recovery period for the infected cells.
  • the regenerated tissue mass is cultured on a medium containing a selective agent (glyphosate) and the grown transformed callus is selected (step 4: selection step).
  • the tissue block of the cotyledonary node regeneration is screened with a selective agent Solid medium (B5 salt 3.1g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1g/L, sucrose 30g/L, 6-benzyl adenine (6-BAP) 1mg/L, agar 8g /L, cephalosporin 150 mg / L, glutamic acid 100 mg / L, aspartic acid 100 mg / L, glufosinate 6 mg / L, pH 5.6) culture, resulting in selective growth of transformed cells.
  • transformation Cell regenerating plants step 5: regeneration step
  • the selected resistant tissue blocks were transferred to the B5 differentiation medium (B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, zeatin (ZT)) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, The auxin 1 mg/L, glufosinate 6 mg/L, pH 5.6) were cultured and differentiated at 25 °C.
  • B5 differentiation medium B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, zeatin (ZT)
  • MES 2-morpholine ethanesulfonic acid
  • ZT zeatin
  • cephalosporin 150mg/L gluta
  • the differentiated seedlings were transferred to the B5 rooting medium (B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, agar 8 g/L, cephalosporin) 150 mg/L, indole-3-butyric acid (IBA) 1 mg/L), cultured in rooting culture at 25 ° C to a height of about 10 cm, and transferred to a greenhouse for cultivation to firmness. In the greenhouse, the cells were cultured at 26 ° C for 16 hours and then at 20 ° C for 8 hours.
  • B5 rooting medium B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, agar 8 g/L, cephalosporin
  • IBA indole-3-butyric acid
  • soybean plants transferred to the 24DT22 nucleotide sequence and soybean plants transferred to the control nucleotide sequence were used as samples, and the genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the Taqman probe fluorescent quantitative PCR method was used. The PAT gene copy number was detected to determine the copy number of the 24DT22 gene.
  • the wild type soybean plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was set to repeat 3 times and averaged.
  • the specific method for detecting the copy number of the PAT gene is as follows:
  • Step 11 Take 100 mg of soybean plants transferred to the 24DT22 nucleotide sequence, soybean plants transferred to the control sequence, and wild type soybean plants, respectively, and homogenize them with liquid nitrogen in a mortar, and take each sample. 3 repetitions;
  • Step 12 Extract the genomic DNA of the above sample using Qiagen's DNeasy Plant Mini Kit, and refer to the product manual for the specific method;
  • Step 13 Determine the genomic DNA concentration of the above sample using NanoDrop 2000 (Thermo Scientific).
  • Step 14 adjusting the genomic DNA concentration of the above sample to the same concentration value, the concentration value ranges from 80 to 100 ng / ⁇ L;
  • Step 15 The Taqman probe real-time PCR method is used to identify the copy number of the sample, and the sample with the known copy number is used as a standard, and the sample of the wild type soybean plant is used as a control, and each sample is repeated for 3 times, and the average is taken. Value; the fluorescent PCR primers and probe sequences are:
  • Probe 1 CTTACGCTGGGCCCTGGAAGGCTAG as shown in SEQ ID NO: 13 in the Sequence Listing;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mix contained 45 ⁇ L of each primer at a concentration of 1 mM, 50 ⁇ L of probe at a concentration of 100 ⁇ M and 860 ⁇ L of 1 ⁇ TE buffer, and was stored in an amber tube at 4°C.
  • the PCR reaction conditions are:
  • Soybean plants transferred to the 24DT22 nucleotide sequence, soybean plants transferred to the control sequence, and wild-type soybean plants (seedling stage) were herbicide-resistant to 2,4-D dimethylammonium salt and dimethyltetrachloride, respectively. Detection.
  • 6HAT 2 days
  • 7DAT 7 days
  • 14DAT 14 days
  • 2240 g ae/ha 2,4-D and methotetrachloroethylene are effective doses that distinguish sensitive plants from plants with average resistance levels.
  • the results in Table 2 and FIG. 5 show: 24DT22 transgenic soybean plants genes that confer high level herbicide resistance, especially phenoxy auxin herbicides; and wild type soybean plants and control sequences into soybean plants T 1 is not It has phenoxy auxin herbicide resistance.
  • the recombinant cloning vector DBN01-T and the expression vector DBNBC-02 were digested with restriction endonucleases SpeI and KasI, respectively, and the excised 24DT22 nucleotide sequence fragment was inserted into the expression.
  • the construction of vectors between the SpeI and KasI sites of the vector DBNBC-02 by conventional enzymatic cleavage methods is well known to those skilled in the art, and the SpeI and KasI cleavage sites in the expression vector DBNBC-02 are also subjected to conventional enzyme digestion.
  • the recombinant expression vector DBN100764 was constructed by the method.
  • the recombinant expression vector DBN100764 was transformed into E. coli T1 competent cells by heat shock method, and the heat shock conditions were: 50 ⁇ L of E. coli T1 competent cells, 10 ⁇ L of plasmid DNA (recombinant expression vector DBN100764), 42 ° C water bath for 30 seconds; 37 ° C oscillation Incubate for 1 hour (shake shake at 100 rpm); then LB solid plate (pancreatic egg) containing 50 mg/L kanamycin 10 g / L of white peony, 5 g / L of yeast extract, 10 g / L of NaCl, 15 g / L of agar, adjusted to pH 7.5 with NaOH), incubated at 37 ° C for 12 hours, picking white colonies, and culturing in LB liquid
  • the base tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, spectinomycin 50 mg/L, pH adjusted to 7.5 with NaOH was culture
  • the plasmid was extracted by an alkali method.
  • the extracted plasmids were digested with restriction endonucleases SpeI and KasI, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence between the SpeI and KasI sites of the recombinant expression vector DBN100764 was the SEQ ID in the sequence listing. NO: The nucleotide sequence shown in 1, which is the 24DT22 nucleotide sequence.
  • a recombinant cloning vector DBN02R-T containing a control sequence was constructed using the control sequence (SEQ ID NO: 8) according to the method of constructing the recombinant cloning vector DBN01-T containing the nucleotide sequence of 24DT22 according to the second embodiment of the present invention. .
  • the positive clone was verified by sequencing, and the result showed that the control sequence inserted into the recombinant cloning vector DBN02R-T was the nucleotide sequence shown by SEQ ID NO: 8 in the sequence listing, that is, the control sequence was correctly inserted.
  • the recombinant expression vector DBN100764N containing the control sequence was constructed using the control sequence, and the vector structure thereof is shown in Fig.
  • vector skeleton pCAMBIA2301 (available by CAMBIA); Kan: kanamycin gene; RB: right border; Ubi: maize Ubiquitin 1 gene promoter (SEQ ID NO: 9); mN: control sequence (SEQ ID NO) :8); Nos: terminator of the nopaline synthase gene (SEQ ID NO: 4); PMI: phosphomannose isomerase gene (SEQ ID NO: 10); LB: left border).
  • control sequence inserted in the recombinant expression vector DBN100764N was the nucleotide sequence shown in SEQ ID NO: 8 in the sequence listing, that is, the control sequence was correctly inserted.
  • the recombinant expression vectors DBN100764 and DBN100764N (control sequences) which have been constructed correctly were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L Agrobacterium LBA4404, 3 ⁇ L of plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, and warmed at 37 ° C for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated into LB tubes and incubated at a temperature of 28 ° C and a rotation speed of 200 rpm for 2 hours.
  • the sterile cultured corn variety 31 (Z31) is young Embryos were co-cultured with Agrobacterium described in 3 of the seventh embodiment to activate T-DNA (including the promoter of maize Ubiquitin1 gene) in the recombinant expression vectors DBN100764 and DBN100764N (control sequences) constructed in 1 and 2 of the seventh embodiment.
  • Subsequence, 24DT22 nucleotide sequence, control sequence, PMI gene and Nos terminator sequence were transferred into the maize genome to obtain a maize plant transformed into the 24DT22 nucleotide sequence and a maize plant transformed into the control sequence; Wild type corn plants were used as controls.
  • immature immature embryos are isolated from maize, and the immature embryos are contacted with an Agrobacterium suspension, wherein Agrobacterium can deliver the 24DT22 nucleotide sequence to at least one of the young embryos.
  • Cells (Step 1: Infection step).
  • the immature embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step).
  • the immature embryo is in solid medium after the infection step (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 20 g/L, glucose 10 g/L, acetosyringone (AS) 100 mg/L) It was cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8). After this co-cultivation phase, there can be an optional "recovery" step.
  • the medium was restored (MS salt 4.3 g / L, MS vitamin, casein 300 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg /
  • At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium is present in L, plant gel 3 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step).
  • the immature embryos are cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells.
  • the inoculated immature embryos are cultured on a medium containing a selective agent (mannose) and the grown transformed callus is selected (step 4: selection step).
  • the immature embryo is screened in solid medium with selective agent (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, mannose 12.5 g/L, 2,4-dichlorobenzene)
  • MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, mannose 12.5 g/L, 2,4-dichlorobenzene Incubation of oxyacetic acid (2,4-D) 1 mg/L, plant gel 3 g/L, pH 5.8) resulted in selective growth of transformed cells.
  • the callus regenerates the plant (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and MS rooting medium) Recycled plants.
  • the selected resistant callus was transferred to the MS differentiation medium (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, 6-benzyl adenine 2 mg/L, mannose) 5 g/L, plant gel 3 g/L, pH 5.8), cultured and differentiated at 25 °C.
  • the differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, indole-3-acetic acid 1 mg/L, plant gel 3 g/L) , pH 5.8), cultured at 25 ° C to a height of about 10 cm, moved to a greenhouse to grow to firm. In the greenhouse, the cells were cultured at 28 ° C for 16 hours and then at 20 ° C for 8 hours.
  • TaqMan was used to verify the maize plants that were transferred into the 24DT22 nucleotide sequence.
  • the specific method for detecting the copy number of the PMI gene is as follows:
  • Step 31 Each of the maize plants transformed into the 24DT22 nucleotide sequence, the maize plants transferred to the control sequence, and the leaves of the wild-type maize plant were each 100 mg, and respectively ground in a mortar with liquid nitrogen, each sample was taken. 3 repetitions;
  • Step 32 using Qiagen's DNeasy Plant Mini Kit to extract the genomic DNA of the above sample, the specific method refers to the product specification;
  • Step 33 measuring the genomic DNA concentration of the above sample by NanoDrop 2000 (Thermo Scientific).
  • Step 34 adjusting the genomic DNA concentration of the above sample to the same concentration value, the concentration value ranges from 80 to 100 ng / ⁇ L;
  • Step 35 The copy number of the sample is identified by Taqman probe real-time PCR, and the sample with the known copy number is used as a standard, and the sample of the wild type corn plant is used as a control, and each sample has 3 replicates, and the average is taken. Value; the fluorescent PCR primers and probe sequences are:
  • Primer 4 CGATCTGCAGGTCGACGG as shown in SEQ ID NO: 15 in the Sequence Listing;
  • Probe 2 TCTCTTGCTAAGCTGGGAGCTCGATCC as shown in SEQ ID NO: 16 in the Sequence Listing;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mix contained 45 ⁇ L of each primer at a concentration of 1 mM, 50 ⁇ L of probe at a concentration of 100 ⁇ M and 860 ⁇ L of 1 ⁇ TE buffer, and was stored in an amber tube at 4°C.
  • the PCR reaction conditions are:
  • maize plants, soybean plants, and Arabidopsis plants that have been transferred to the 24DT22 nucleotide sequence have high herbicide resistance.
  • the 24DT22 herbicide resistance gene of the invention adopts a plant preference codon, so that the herbicide resistance gene of the invention is particularly suitable for expression in plants, and the 24DT22 herbicide resistance protein of the invention has broad resistance to herbicides, especially phenoxy growth. Herbicide.

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Abstract

提供了一种除草剂抗性蛋白质、其编码基因及用途。除草剂抗性蛋白质包括：(a)具有SEQ ID NO:2所示的氨基酸序列组成的蛋白质；或(b)在(a)中的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸，且具有除草剂抗性活性的由(a)衍生的蛋白质。该除草剂抗性蛋白质对苯氧基生长素除草剂具有特别好的抗性。

Description

除草剂抗性蛋白质、其编码基因及用途 技术领域
本发明涉及一种除草剂抗性蛋白质、其编码基因及用途，特别是涉及一种对2,4-D具有抗性的蛋白质、其编码基因及用途。
背景技术
杂草可以迅速耗尽土壤中作物和其它目的植物所需要的有价值的养分。目前有许多类型的除草剂用于控制杂草，一种特别流行的除草剂是草甘膦。已经开发了对草甘膦具有抗性的作物，如玉米、大豆、棉花、甜菜、小麦和水稻等。因此可以对种植草甘膦抗性作物的田地喷洒草甘膦以控制杂草而不显著损害作物。
草甘膦已经在全球广泛使用超过20年，由此导致对草甘膦和草甘膦耐性作物技术的过度依赖，并在野生杂草物种中对草甘膦天然更具耐受性或已经发展出抗草甘膦活性的植物施加了高选择压。已报道有少数杂草已发展出对草甘膦的抗性，包括阔叶杂草和禾本科杂草，如瑞士黑麦草、多花黑麦草、牛筋草、豚草、小飞蓬、野塘蒿和长叶车前。此外，在广泛使用草甘膦耐性作物之前并不是农业问题的杂草也逐渐盛行，并且难于用草甘膦耐性作物控制，这些杂草主要与(但不仅与)难于控制的阔叶杂草一起出现，如苋属、藜属、蒲公英属和鸭跖草科物种。
在草甘膦抗性杂草或难于控制的杂草物种的地区，种植者可以通过罐混或换用能控制遗漏杂草的其它除草剂来弥补草甘膦的弱点。在多数情况下控制阔叶杂草的一种流行且有效的罐混伴侣为2,4-二氯苯氧乙酸(2,4-D)。2,4-D已经在农业和非作物条件下用于广谱阔叶杂草控制超过65年，仍是全球最广泛使用的除草剂之一。对进一步使用2,4-D的限制在于它在双子叶植物(如大豆或棉花)中的选择性非常低，因此2,4-D一般不用于(且一般不靠近)敏感性双子叶作物。此外，2,4-D在禾本科作物中的用途在某种程度上受限于可能出现的作物损伤的性质。2,4-D和草甘膦的组合已经用于在种植免耕大豆和棉花之前提供更强的灭生处理，然而，由于这些双子叶物种对2,4-D的敏感性，这些灭生处理必须在种植前14-30天进行。
和MCPA、2-甲-4-氯丙酸和2,4-D丙酸一样，2,4-D是苯氧酸类除草剂。2,4-D用于在许多单子叶作物(如玉米、小麦和水稻)中选择性控制阔叶杂草而不严重损伤目的作物。2,4-D是合成的植物生长素衍生物，其作用为使正常 的细胞激素内稳态失调，并阻碍平衡的受控生长。
2,4-D对某些植物具有不同水平的选择性(如双子叶植物比禾本科植物更敏感)。不同植物对2,4-D的不同代谢是不同水平选择性的一种解释。通常植物缓慢代谢2,4-D，因此靶位点的不同活性更可能解释植物对2,4-D不同的应答。2,4-D的植物代谢一般通过两步代谢实现，一般是羟基化后接着与氨基酸或葡萄糖缀合。
随着时间的发展，微生物种群已经发展出降解此特定外来物的有效的替代途径，所述途径引起2,4-D的完全矿化。对微生物连续应用除草剂可用来选择能利用除草剂作为碳源用于生长(从而使其在土壤中具有竞争优势)的微生物。因为这个原因，目前将2,4-D配制为具有相对短的土壤半衰期，并且对其后的作物没有遇到明显的遗留效应。这促进了2,4-D的除草剂应用。
已经广泛研究了其降解2,4-D能力的一种生物是真养雷氏菌(Ralstonia eutropha)。编码矿化途径中的第一个酶促步骤的基因为tfdA。TfdA通过α酮戊二酸依赖性双加氧酶反应催化2,4-D酸转化成二氯苯酚(DCP)。DCP与2,4-D相比几乎不具有除草剂活性。TfdA在转基因植物中用于向通常对2,4-D敏感的双子叶植物(如棉花和烟草)中输入2,4-D抗性。
已在环境中鉴定了大量编码能降解2,4-D的蛋白质的tfdA型基因。许多同系物与tfdA类似(氨基酸同一性>85％)并具有与tfdA相似的酶活性。然而，不是所有具有TauD等结构域的蛋白质都具有2,4-D降解功能，并且有大量同系物与tfdA具有显著更低的同一性(25-50％)，但却具有与α酮戊二酸依赖性双加氧酶Fe+2双加氧酶相关的特征残基。因此这些不同的双加氧酶的底物特异性是什么并不明确。与tfdA具有低同源性(氨基酸同一性28％)的独特实例是来自Sphingobium herbicidovorans的rdpA。已经显示此酶催化(R)-2,4-D丙酸(和其它(R)-苯氧丙酸)以及2,4-D(苯氧乙酸)矿化的第一步。
随着草甘膦抗性杂草的出现和2,4-D除草剂的扩大应用，需要对2,4-D敏感的目的植物中输入2,4-D抗性。目前未发现24DT22除草剂抗性蛋白在植物中的表达水平和对除草剂的耐受性报道。
发明内容
本发明的目的是提供一种除草剂抗性蛋白质、其编码基因及用途，本发明旨在提供一种新的24DT22基因，所述24DT22蛋白在植物中对除草剂具有较高的耐受性。
为实现上述目的，本发明提供了一种除草剂抗性蛋白质，包括：
(a)具有SEQ ID NO:2所示的氨基酸序列组成的蛋白质；或
(b)在(a)中的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨 基酸且具有芳氧基链烷酸酯双加氧酶活性的由(a)衍生的蛋白质。
为实现上述目的，本发明提供了一种除草剂抗性基因，包括：
(a)编码所述除草剂抗性蛋白质的核苷酸序列；或
(b)在严格条件下与(a)限定的核苷酸序列杂交且编码具有芳氧基链烷酸酯双加氧酶活性的蛋白质的核苷酸序列；或
(c)具有SEQ ID NO:1所示的核苷酸序列。
所述严格条件可为在6×SSC(柠檬酸钠)、0.5％SDS(十二烷基硫酸钠)溶液中，在65℃下杂交，然后用2×SSC、0.1％SDS和1×SSC、0.1％SDS各洗膜1次。
为实现上述目的，本发明还提供了一种表达盒，包含在有效连接的调控序列调控下的所述除草剂抗性基因。
为实现上述目的，本发明还提供了一种包含所述除草剂抗性基因或所述表达盒的重组载体。
为实现上述目的，本发明还提供了一种产生除草剂抗性蛋白质的方法，包括：
获得包含所述除草剂抗性基因或所述表达盒的转基因宿主生物的细胞；
在允许产生除草剂抗性蛋白质的条件下培养所述转基因宿主生物的细胞；
回收所述除草剂抗性蛋白质。
进一步地，所述转基因宿主生物包括植物、动物、细菌、酵母、杆状病毒、线虫或藻类。
优选地，所述植物为大豆、棉花、玉米、水稻、小麦、甜菜或甘蔗。
为实现上述目的，本发明还提供了一种增加耐受除草剂范围的方法，包括：将所述除草剂抗性蛋白质或所述表达盒编码的除草剂抗性蛋白质在植物中与至少一种不同于所述除草剂抗性蛋白质或所述表达盒编码的除草剂抗性蛋白质的第二种核苷酸一起表达。
进一步地，所述第二种核苷酸编码草甘膦抗性蛋白质、草铵膦抗性蛋白质、4-羟苯基丙酮酸双加氧酶、乙酰乳酸合酶、细胞色素类蛋白质或原卟啉原氧化酶。
在本发明中，24DT22除草剂抗性蛋白质在一种转基因植物中的表达可以伴随着一个或多个草甘膦抗性蛋白质和/或草铵膦抗性蛋白质的表达。这种超过一种的除草剂抗性蛋白质在同一株转基因植物中共同表达可以通过遗传工程使植物包含并表达所需的基因来实现。另外，一种植物(第1亲本)可以通过遗传工程操作表达24DT22除草剂抗性蛋白质，第二种植物(第2亲本)可以通过遗传工程操作表达草甘膦抗性蛋白质和/或草铵膦抗性蛋白质。通过 第1亲本和第2亲本杂交获得表达引入第1亲本和第2亲本的所有基因的后代植物。
为实现上述目的，本发明还提供了一种选择转化的植物细胞的方法，包括：用所述除草剂抗性基因或所述表达盒转化多个植物细胞，并在允许表达所述除草剂抗性基因或所述表达盒的转化细胞生长，而杀死未转化细胞或抑制未转化细胞生长的除草剂浓度下培养所述细胞，所述除草剂为苯氧基生长素。
为实现上述目的，本发明还提供了一种控制杂草的方法，包括：对种植作物的大田施用有效剂量的除草剂，所述作物包含所述除草剂抗性基因或所述表达盒或所述重组载体。
优选地，所述除草剂为苯氧基生长素。
为实现上述目的，本发明还提供了一种用于保护植物免受由除草剂引起的损伤的方法，包括：将所述除草剂抗性基因或所述表达盒或所述重组载体导入植物，使导入后的植物产生足够保护其免受除草剂损害量的除草剂抗性蛋白质。
优选地，所述除草剂为苯氧基生长素或芳氧基苯氧链烷酸酯。所述植物为大豆、棉花、玉米、水稻、小麦、甜菜或甘蔗。
为实现上述目的，本发明还提供了一种控制草甘膦耐性植物的大田中草甘膦抗性杂草的方法，包括：对种植草甘膦耐性植物的大田施用有效剂量的除草剂，所述草甘膦耐性植物包含所述除草剂抗性基因或所述表达盒或所述重组载体。
优选地，所述除草剂为苯氧基生长素。所述草甘膦耐性植物为单子叶植物或双子叶植物。
为实现上述目的，本发明还提供了一种赋予作物2,4-D除草剂抗性的方法，包括：将所述除草剂抗性基因或所述表达盒或所述重组载体导入植物。
优选地，所述植物为大豆、棉花、玉米、水稻、小麦、甜菜或甘蔗。
为实现上述目的，本发明还提供了一种除草剂抗性蛋白质耐受苯氧基生长素类除草剂的用途，所述除草剂抗性蛋白质包括：
(a)具有SEQ ID NO:2所示的氨基酸序列组成的蛋白质；或
(b)在(a)中的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸且具有芳氧基链烷酸酯双加氧酶活性的由(a)衍生的蛋白质。
将所述的除草剂抗性基因或所述的表达盒或所述的重组载体导入植物，在本发明中为将外源DNA导入植物细胞，常规转化方法包括但不限于，农杆菌介导的转化、微量发射轰击、直接将DNA摄入原生质体、电穿孔或晶须硅介导的DNA导入。
本发明所述的2,4-D抗性基因及其后的抗性作物提供用于在作物中控制草甘膦抗性(或高耐性和演替的)阔叶杂草物种的优良选择。2,4-D是广谱、相对便宜且强力的阔叶除草剂，如果在双子叶和单子叶中同样能提供更强的作物耐受性，则可为种植者提供优良的效用。2,4-D耐性转基因双子叶植物还可在应用时间和用量上具有更高的灵活性。2,4-D除草剂耐性性状的另一用途是它可用于预防2,4-D漂移、挥发、转化(或其它远距离的移动现象)、误用、破坏等对正常敏感性作物的损害。已经广泛使用不同苯氧基生长素组合的多种混合物来处理不同地区特定的杂草谱和环境条件。在植物中使用24DT22基因可以提供对更广谱的苯氧基生长素除草剂的防护，从而提高灵活性和可控制的杂草谱，提供对全范围市售苯氧基生长素的漂移或其它远距离苯氧基除草剂损伤的防护。
对于苯氧基生长素除草剂通常制成活性酸，但也有一些商品化配制为多种相应酯制剂之一，由于一般的植物酯酶在植物中将这些酯转换成活性酸，因此这些也同样认为是在植物中24DT22酶的底物。类似的还可以是相应酸的相应有机或无机盐。当表示手性丙酸、丙酸盐或丙酸酯除草剂时，即使不同的CAS号可能对应于光学纯的化合物，在命名这些除草剂时仍认为外消旋(R，S)或光学纯化的(R或S)对映体是同一除草剂。可能的用量范围可以是作物或非作物用途中单独处理或与其他除草剂组合。
现已鉴定了24DT22基因在遗传改造用于植物表达后具有允许在植物中使用苯氧基生长素除草剂的特性，所述植物中固有耐性不存在或不足以允许使用这些除草剂。此外，24DT22基因可以在天然耐性不足以允许选择性时在植物中提供对苯氧基生长素除草剂的防护。现在可以连续或罐混地与一种、两种或若干苯氧基生长素除草剂的组合处理仅含24DT22基因的植物。用于控制广谱双子叶杂草的每种苯氧基生长素除草剂的用量范围从25至4000g ae/ha，更通常从100至2000g ae/ha。在同一大田里(连续或罐混组合地)组合这些不同化学类别和具有不同作用模式和范围的除草剂可以提供对大多数需要除草剂控制的潜在杂草的控制。
草甘膦被广泛地使用，因为它控制非常广谱的阔叶和禾本科杂草物种。然而，在草甘膦耐性作物和非作物应用中重复使用草甘膦已经(而且仍将继续)选择使杂草演替为天然更具有耐性的物种或草甘膦抗性生物型。多数除草剂抗性管理策略建议使用有效用量的罐混除草剂伴侣作为延缓出现抗性杂草的方法，所述除草剂伴侣提供对同一物种的控制，但具有不同的作用模式。将24DT22基因与草甘膦耐性性状(和/或其他除草剂耐性性状)叠加可通过允许对同一作物选择性使用草甘膦和苯氧基生长素(如2,4-D)而实现对草甘膦耐性作物中草甘膦抗性杂草物种(被一种或多种苯氧基生长素控制的阔叶 杂草物种)的控制。这些除草剂的应用可以是在含有不同作用模式的两种或更多除草剂的罐混合物中同时使用、在连续使用(如种植前、出苗前或出苗后)中单个除草剂组合物的单独使用(使用的间隔时间范围从2小时到3个月)，或者备选地，可以在任何时间(从种植作物7个月内到收获作物时(或对于单个除草剂为收获前间隔，取最短者))使用代表可应用每种化合类别的任意数目除草剂的组合。
在控制阔叶杂草中具有灵活性是很重要的，即使用时间、单个除草剂用量和控制顽固或抗性杂草的能力。作物中与草甘膦抗性基因/24DT22基因叠加的草甘膦应用范围可以从250至2500g ae/ha；苯氧基生长素除草剂(一种或多种)可按照从25-4000g ae/ha。这些应用的时间的最佳组合取决于具体的条件、物种和环境。
除草剂制剂(如酯、酸或盐配方或可溶浓缩剂、乳化浓缩剂或可溶液体)和罐混添加剂(如佐剂或相容剂)可显著影响给定的除草剂或一种或多种除草剂的组合的杂草控制。任意前述除草剂的任意化学组合均在本发明的范围内。
本领域技术人员所熟知的，两种或更多作用模式的组合在提高受控杂草谱和/或天然更具耐性物种或抗性杂草物种上的益处还可扩展到通过人工(转基因或非转基因)在作物中产生除草甘膦耐性作物外的除草剂耐性的化学品。事实上，可以单独或以多重组合叠加编码以下抗性的性状以提供有效控制或防止杂草演替对任意前述类别的除草剂的抗性的能力：草甘膦抗性(如抗性植物或细菌EPSPS、GOX、GAT)、草铵膦抗性(如PAT、Bar)、乙酰乳酸合酶(ALS)抑制性除草剂抗性(如咪唑啉酮、磺酰脲、三唑嘧啶、磺苯胺、嘧啶硫代苯甲酸和其它化学品抗性基因如AHAS、Csrl、SurA等)、溴草腈抗性(如Bxn)、对HPPD(4-羟苯基丙酮酸双加氧酶)酶抑制剂的抗性、对八氢番茄红素去饱和酶(PDS)抑制剂的抗性、对光系统Ⅱ抑制性除草剂的抗性(如psbA)、对光系统Ⅰ抑制性除草剂的抗性、对原卟啉原氧化酶Ⅸ(PPO)抑制性除草剂抗性(如PPO-1)、对苯脲除草剂的抗性(如CYP76B1)、二氯甲氧苯酸降解酶等等。
关于其他除草剂，一些其它优选的ALS抑制剂包括三唑嘧啶磺苯胺(氯酯磺草胺、双氯磺草胺、唑嘧磺草胺、磺草唑胺和嘧啶并三唑类磺胺)、嘧啶硫代苯甲酸和氟唑磺隆。一些优选的HPPD抑制剂包括甲基磺草酮、异恶唑草酮和磺草酮。一些优选的PPO抑制剂包括丙炔氟草胺、氟丙嘧草酯、唑草酮、甲磺草胺和二苯醚(如三氟羧草醚、氟磺胺草醚、乳氟禾草灵和乙氧氟草醚)。
此外，可以将24DT22基因单独或与其它除草剂耐受作物特征叠加后再 与一种或多种其它输入(如昆虫抗性、真菌抗性或胁迫耐性等)或输出(如提高的产量、改进的油量、提高的纤维品质等)性状叠加。因此，本发明可用于提供以灵活且经济地控制任何数目的农学害虫的能力和提高作物品质的完整农学解决方案。
本发明24DT22基因能降解2,4-D，是重要的除草剂耐受作物和选择标记物特征可能性的基础。
本发明可进行转基因表达，可以控制几乎所有阔叶杂草的除草剂组合。24DT22基因可作为优秀的除草剂耐受作物性状与例如其它除草剂耐受作物性状(如草甘膦抗性、草铵膦抗性、ALS抑制剂(如咪唑啉酮类、磺酰脲类、三唑并嘧啶磺酰胺类)抗性、溴草腈抗性、HPPD抑制剂抗性、PPO抑制剂抗性等)和昆虫抗性性状(Cry1Ab、Cry1F、Vip3、其它苏云金芽孢杆菌蛋白质或非芽孢杆菌属来源的昆虫抗性蛋白等)叠加。此外，24DT22基因可作为选择标记物辅助选择用另一个基因或基因群遗传改造的植物的原代转化体。
苯氧基链烷酸酯基团可用于将稳定的酸官能团引入除草剂。酸性基团可通过“酸捕获”输入韧皮部活性(除草剂作用所需的属性)，从而可以为了活性目的而整合进新除草剂。存在很多可能为24DT22底物的市售和实验性除草剂。因此，使用本发明基因还可以得到对其它除草剂的耐性。
本发明的除草剂耐性作物性状可用在与其它除草剂耐性作物性状(包括但不限于草甘膦耐性)的新组合中。由于对除草剂(如草甘膦)的新获得的抗性或固有的耐性，这些性状组合产生控制杂草物种的新方法。因此，除了除草剂耐性作物性状，本发明的范围包括使用除草剂控制杂草的新方法，其中通过转基因作物中的所述酶产生对所述除草剂的耐性。
本发明可应用于多种植物中，如拟南芥、烟草、大豆、棉花、稻、玉米和芸薹。本发明还可用于多种其它单子叶(如牧草禾本科或草坪草禾本科)和双子叶作物(如苜蓿、三叶草、乔木物种等)。类似的，2,4-D(或其它24DT22底物)可更积极地用于耐性适中的禾本科作物中，由此性状得到的提高的耐性将为种植者提供能以更有效的用量和更广的施用时间来使用这些除草剂而无作物损伤风险的可能性。
本发明中所述的植物、植物组织或植物细胞的基因组，是指植物、植物组织或植物细胞内的任何遗传物质，且包括细胞核和质体和线粒体基因组。
本发明中所述“抗性”是可遗传的，并允许植物在除草剂对给定植物进行一般除草剂有效处理的情况下生长和繁殖。正如本领域技术人员所认可的，即使植物受到除草剂处理的一定损伤程度明显，植物仍可被认为“抗性”。本发明中术语“耐性”比术语“抗性”更广泛，并包括“抗性”，以及特定植物具有的抵抗除草剂诱导的各种程度损伤的提高的能力，而在同样的除草 剂剂量下一般导致相同基因型野生型植物损伤。
本发明中所述的多核苷酸和/或核苷酸形成完整“基因”，在所需宿主细胞中编码蛋白质或多肽。本领域技术人员很容易认识到，可以将本发明的多核苷酸和/或核苷酸置于目的宿主中的调控序列控制下。
本领域技术人员所熟知的，DNA典型的以双链形式存在。在这种排列中，一条链与另一条链互补，反之亦然。由于DNA在植物中复制产生了DNA的其它互补链。这样，本发明包括对序列表中示例的多核苷酸及其互补链的使用。本领域常使用的“编码链”指与反义链结合的链。为了在体内表达蛋白质，典型将DNA的一条链转录为一条mRNA的互补链，它作为模板翻译出蛋白质。mRNA实际上是从DNA的“反义”链转录的。“有义”或“编码”链有一系列密码子(密码子是三个核苷酸，一次读三个可以产生特定氨基酸)，其可作为开放阅读框(ORF)阅读来形成目的蛋白质或肽。本发明还包括与示例的DNA有相当功能的RNA和PNA(肽核酸)。
本发明中核酸分子或其片段在严格条件下与本发明除草剂抗性基因杂交。任何常规的核酸杂交或扩增方法都可以用于鉴定本发明除草剂抗性基因的存在。核酸分子或其片段在一定情况下能够与其他核酸分子进行特异性杂交。本发明中，如果两个核酸分子能形成反平行的双链核酸结构，就可以说这两个核酸分子彼此间能够进行特异性杂交。如果两个核酸分子显示出完全的互补性，则称其中一个核酸分子是另一个核酸分子的“互补物”。本发明中，当一个核酸分子的每一个核苷酸都与另一个核酸分子的对应核苷酸互补时，则称这两个核酸分子显示出“完全互补性”。如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在至少常规的“低度严格”条件下退火且彼此结合，则称这两个核酸分子为“最低程度互补”。类似地，如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在常规的“高度严格”条件下退火且彼此结合，则称这两个核酸分子具有“互补性”。从完全互补性中偏离是可以允许的，只要这种偏离不完全阻止两个分子形成双链结构。为了使一个核酸分子能够作为引物或探针，仅需保证其在序列上具有充分的互补性，以使得在所采用的特定溶剂和盐浓度下能形成稳定的双链结构。
本发明中，基本同源的序列是一段核酸分子，该核酸分子在高度严格条件下能够和相匹配的另一段核酸分子的互补链发生特异性杂交。促进DNA杂交的适合的严格条件，例如，大约在45℃条件下用6.0×氯化钠/柠檬酸钠(SSC)处理，然后在50℃条件下用2.0×SSC洗涤，这些条件对本领域技术人员是公知的。例如，在洗涤步骤中的盐浓度可以选自低度严格条件的约2.0×SSC、50℃到高度严格条件的约0.2×SSC、50℃。此外，洗涤步骤中的温度条件可以从低度严格条件的室温约22℃，升高到高度严格条件的约65℃。温度条件和 盐浓度可以都发生改变，也可以其中一个保持不变而另一个变量发生改变。优选地，本发明所述严格条件可为在6×SSC、0.5％SDS溶液中，在65℃下与SEQ ID NO:1发生特异性杂交，然后用2×SSC、0.1％SDS和1×SSC、0.1％SDS各洗膜1次。
因此，具有除草剂耐受性活性并在严格条件下与本发明序列1杂交的序列包括在本发明中。这些序列与本发明序列至少大约40％-50％同源，大约60％、65％或70％同源，甚至至少大约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更大的序列同源性。
本发明提供功能蛋白质。“功能活性”(或“活性”)在本发明中指本发明用途的蛋白质/酶(单独或与其它蛋白质组合)具有降解或减弱除草剂活性的能力。产生本发明蛋白质的植物优选产生“有效量”的蛋白质，从而在用除草剂处理植物时，蛋白质表达的水平足以给予植物对除草剂(若无特别说明则为一般用量)完全或部分的抗性或耐性。可以以通常杀死靶植物的用量、正常的大田用量和浓度使用除草剂。优选地，本发明的植物细胞和植物被保护免受除草剂处理引起的生长抑制或损伤。本发明的转化植物和植物细胞优选具有2,4-D除草剂的抗性或耐性，即转化的植物和植物细胞能在有效量的2,4-D除草剂存在下生长。
本发明中所述的基因和蛋白质不但包括特定的示例序列，还包括保存了所述特定示例的蛋白质的除草剂抗性活性特征的部分和/片段(包括与全长蛋白质相比在内和/或末端缺失)、变体、突变体、取代物(有替代氨基酸的蛋白质)、嵌合体和融合蛋白。所述“变体”或“变异”是指编码同一蛋白或编码有除草剂抗性活性的等价蛋白的核苷酸序列。所述“等价蛋白”是指与权利要求的蛋白具有相同或基本相同的除草剂耐受性的生物活性的蛋白。
本发明中所述的DNA分子或蛋白序列的“片段”或“截短”是指涉及的原始DNA或蛋白序列(核苷酸或氨基酸)的一部分或其人工改造形式(例如适合植物表达的序列)，包括临近片段和与全长分子相比内部和/或末端的缺失，前述序列的长度可存在变化，但长度足以确保(编码)蛋白质为除草剂抗性蛋白质。在一些情况下(特别是植物中的表达)，使用编码截短蛋白质的截短基因可能是有利的。优选的截短基因一般编码全长蛋白质的40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98或99％。
由于遗传密码子的丰余性，多种不同的DNA序列可以编码相同的氨基酸序列。产生这些编码相同或基本相同的蛋白的可替代DNA序列正在本领域技 术人员的技术水平内。这些不同的DNA序列包括在本发明的范围内。所述“基本上相同的”序列是指有氨基酸取代、缺失、添加或插入但实质上不影响除草剂抗性活性的序列，亦包括保留除草剂抗性活性的片段。
本发明中氨基酸序列的取代、缺失或添加是本领域的常规技术，优选这种氨基酸变化为：小的特性改变，即不显著影响蛋白的折叠和/或活性的保守氨基酸取代；小的缺失，通常约1-30个氨基酸的缺失；小的氨基或羧基端延伸，例如氨基端延伸一个甲硫氨酸残基；小的连接肽，例如约20-25个残基长。
保守取代的实例是在下列氨基酸组内发生的取代：碱性氨基酸(如精氨酸、赖氨酸和组氨酸)、酸性氨基酸(如谷氨酸和天冬氨酸)、极性氨基酸(如谷氨酰胺、天冬酰胺)、疏水性氨基酸(如亮氨酸、异亮氨酸和缬氨酸)、芳香氨基酸(如苯丙氨酸、色氨酸和酪氨酸)，以及小分子氨基酸(如甘氨酸、丙氨酸、丝氨酸、苏氨酸和甲硫氨酸)。通常不改变特定活性的那些氨基酸取代在本领域内是众所周知的，并且已由，例如，N.Neurath和R.L.Hill在1979年纽约学术出版社(Academic Press)出版的《Protein》中进行了描述。最常见的互换有Ala/Ser，Val/Ile，Asp/Glu，Thu/Ser，Ala/Thr，Ser/Asn，Ala/Val，Ser/Gly，Tyr/Phe，Ala/Pro，Lys/Arg，Asp/Asn，Leu/Ile，Leu/Val，Ala/Glu和Asp/Gly，以及它们相反的互换。
对于本领域的技术人员而言显而易见地，这种取代可以在对分子功能起重要作用的区域之外发生，而且仍产生活性多肽。对于由本发明的多肽，其活性必需的并因此选择不被取代的氨基酸残基，可以根据本领域已知的方法，如定点诱变或丙氨酸扫描诱变进行鉴定(如参见，Cunningham和Wells，1989，Science 244：1081-1085)。后一技术是在分子中每一个带正电荷的残基处引入突变，检测所得突变分子的除草剂抗性活性，从而确定对该分子活性而言重要的氨基酸残基。底物-酶相互作用位点也可以通过其三维结构的分析来测定，这种三维结构可由核磁共振分析、结晶学或光亲和标记等技术测定(参见，如de Vos等，1992，Science 255：306-312；Smith等，1992，J.Mol.Biol224:899-904；Wlodaver等，1992，FEBS Letters 309：59-64)。
因此，与序列2所示的氨基酸序列具有一定同源性的氨基酸序列也包括在本发明中。这些序列与本发明序列类似性/相同性典型的大于60％，优选的大于75％，更优选的大于80％，甚至更优选的大于90％，并且可以大于95％。也可以根据更特定的相同性和/或类似性范围定义本发明的优选的多核苷酸和蛋白质。例如与本发明示例的序列有49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、 91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性和/或类似性。
本发明中所述调控序列包括但不限于启动子、转运肽、终止子，增强子，前导序列，内含子以及其它可操作地连接到所述24DT22基因的调节序列。
所述启动子为植物中可表达的启动子，所述的“植物中可表达的启动子”是指确保与其连接的编码序列在植物细胞内进行表达的启动子。植物中可表达的启动子可为组成型启动子。指导植物内组成型表达的启动子的示例包括但不限于，来源于花椰菜花叶病毒的35S启动子、玉米Ubi启动子、水稻GOS2基因的启动子等。备选地，植物中可表达的启动子可为组织特异的启动子，即该启动子在植物的一些组织内如在绿色组织中指导编码序列的表达水平高于植物的其他组织(可通过常规RNA试验进行测定)，如PEP羧化酶启动子。备选地，植物中可表达的启动子可为创伤诱导启动子。创伤诱导启动子或指导创伤诱导的表达模式的启动子是指当植物经受机械或由昆虫啃食引起的创伤时，启动子调控下的编码序列的表达较正常生长条件下有显著提高。创伤诱导启动子的示例包括但不限于，马铃薯和西红柿的蛋白酶抑制基因(pinⅠ和pinⅡ)和玉米蛋白酶抑制基因(MPI)的启动子。
所述转运肽(又称分泌信号序列或导向序列)是指导转基因产物到特定的细胞器或细胞区室，对受体蛋白质来说，所述转运肽可以是异源的，例如，利用编码叶绿体转运肽序列靶向叶绿体，或者利用‘KDEL’保留序列靶向内质网，或者利用大麦植物凝集素基因的CTPP靶向液泡。
所述前导序列包含但不限于，小RNA病毒前导序列，如EMCV前导序列(脑心肌炎病毒5’非编码区)；马铃薯Y病毒组前导序列，如MDMV(玉米矮缩花叶病毒)前导序列；人类免疫球蛋白质重链结合蛋白质(BiP)；苜蓿花叶病毒的外壳蛋白质mRNA的不翻译前导序列(AMV RNA4)；烟草花叶病毒(TMV)前导序列。
所述增强子包含但不限于，花椰菜花叶病毒(CaMV)增强子、玄参花叶病毒(FMV)增强子、康乃馨风化环病毒(CERV)增强子、木薯脉花叶病毒(CsVMV)增强子、紫茉莉花叶病毒(MMV)增强子、夜香树黄化曲叶病毒(CmYLCV)增强子、木尔坦棉花曲叶病毒(CLCuMV)、鸭跖草黄斑驳病毒(CoYMV)和花生褪绿线条花叶病毒(PCLSV)增强子。
对于单子叶植物应用而言，所述内含子包含但不限于，玉米hsp70内含子、玉米泛素内含子、Adh内含子1、蔗糖合酶内含子或水稻Act1内含子。对于双子叶植物应用而言，所述内含子包含但不限于，CAT-1内含子、pKANNIBAL内含子、PIV2内含子和“超级泛素”内含子。
所述终止子可以为在植物中起作用的适合多聚腺苷酸化信号序列，包括 但不限于，来源于农杆菌(Agrobacterium tumefaciens)胭脂碱合成酶(NOS)基因的多聚腺苷酸化信号序列、来源于蛋白酶抑制剂Ⅱ(pinⅡ)基因的多聚腺苷酸化信号序列、来源于豌豆ssRUBISCO E9基因的多聚腺苷酸化信号序列和来源于α-微管蛋白(α-tubulin)基因的多聚腺苷酸化信号序列。
本发明中所述“有效连接”表示核酸序列的联结，所述联结使得一条序列可提供对相连序列来说需要的功能。在本发明中所述“有效连接”可以为将启动子与感兴趣的序列相连，使得该感兴趣的序列的转录受到该启动子控制和调控。当感兴趣的序列编码蛋白并且想要获得该蛋白的表达时“有效连接”表示：启动子与所述序列相连，相连的方式使得得到的转录物高效翻译。如果启动子与编码序列的连接是转录物融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得得到的转录物中第一翻译起始密码子是编码序列的起始密码子。备选地，如果启动子与编码序列的连接是翻译融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得5’非翻译序列中含有的第一翻译起始密码子与启动子相连结，并且连接方式使得得到的翻译产物与编码想要的蛋白的翻译开放读码框的关系是符合读码框的。可以“有效连接”的核酸序列包括但不限于：提供基因表达功能的序列(即基因表达元件，例如启动子、5’非翻译区域、内含子、蛋白编码区域、3’非翻译区域、聚腺苷化位点和/或转录终止子)、提供DNA转移和/或整合功能的序列(即T-DNA边界序列、位点特异性重组酶识别位点、整合酶识别位点)、提供选择性功能的序列(即抗生素抗性标记物、生物合成基因)、提供可计分标记物功能的序列、体外或体内协助序列操作的序列(即多接头序列、位点特异性重组序列)和提供复制功能的序列(即细菌的复制起点、自主复制序列、着丝粒序列)。
本发明可赋予植物新除草剂抗性性状，并且未观察到对表型包括产量的不良影响。本发明中植物能耐受住如至少一种受试除草剂2×、3×、4×或5×一般应用水平。这些耐性水平的提高在本发明的范围之内。例如可对本领域已知的多种技术进行可预见到的优化和进一步发展，以增加给定基因的表达。
本发明中，所述除草剂抗性蛋白质为24DT22氨基酸序列，如序列表中SEQ ID NO:2所示。所述除草剂抗性基因为24DT22核苷酸序列，如序列表中SEQ ID NO:1所示。所述除草剂抗性基因为用于植物，除了包含由24DT22核苷酸序列编码的蛋白质的编码区外，也可包含其他元件，例如编码转运肽的编码区、编码选择性标记的蛋白质或赋予昆虫抗性的蛋白质的编码区。
本发明中24DT22除草剂抗性蛋白质对大多数苯氧基生长素除草剂具有耐性。本发明中的植物，在其基因组中含有外源DNA，所述外源DNA包含 24DT22核苷酸序列，通过表达有效量的该蛋白而保护其免受除草剂的威胁。有效量是指未损伤的或轻微损伤的剂量。同时，植物在形态上应是正常的，且可在常规方法下培养以用于产物的消耗和/或生成。
植物材料中除草剂抗性蛋白质的表达水平可通过本领域内所描述的多种方法进行检测，例如通过应用特异引物对组织内产生的编码除草剂抗性蛋白质的mRNA进行定量，或直接特异性检测产生的除草剂抗性蛋白质的量。
本发明提供了一种除草剂抗性蛋白质、其编码基因及用途，具有以下优点：
1、对除草剂抗性强。本发明除草剂抗性蛋白质24DT22对除草剂的抗性强，尤其是针对苯氧基生长素除草剂，特别是2,4-D。
2、对除草剂抗性广。本发明除草剂抗性蛋白质24DT22蛋白可以对多种苯氧基植物生长素除草剂表现出较高的抗性，因此在植物上应用前景广阔。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图说明
图1为本发明除草剂抗性蛋白质、其编码基因及用途的含有24DT22核苷酸序列的重组克隆载体DBN01-T构建流程图；
图2为本发明除草剂抗性蛋白质、其编码基因及用途的含有24DT22核苷酸序列的重组表达载体DBN100301构建流程图；
图3为本发明除草剂抗性蛋白质、其编码基因及用途的含有对照序列的重组表达载体DBN100301N构建流程图；
图4为本发明除草剂抗性蛋白质、其编码基因及用途的转基因拟南芥T1植株除草剂抗性效果图；
图5为本发明除草剂抗性蛋白质、其编码基因及用途的转基因大豆T1植株除草剂抗性效果图；
图6为本发明除草剂抗性蛋白质、其编码基因及用途的含有24DT22核苷酸序列的重组表达载体DBN100764构建流程图；
图7为本发明除草剂抗性蛋白质、其编码基因及用途的含有对照序列的重组表达载体DBN100764N构建流程图。
具体实施方式
下面通过具体实施例进一步说明本发明除草剂抗性蛋白质、其编码基因及用途的技术方案。
第一实施例、24DT22基因序列的获得和合成
1、获得24DT22基因序列
24DT22除草剂抗性蛋白质的氨基酸序列(292个氨基酸)，如序列表中SEQ ID NO:2所示；依据植物偏好性密码子获得编码相应于所述24DT22除草剂抗性蛋白质的氨基酸序列(292个氨基酸)的核苷酸序列(879个核苷酸)，如序列表中SEQ ID NO:1所示。
2、合成上述24DT22核苷酸序列
所述24DT22核苷酸序列(如序列表中SEQ ID NO:1所示)由南京金斯瑞生物科技有限公司合成；合成的所述24DT22核苷酸序列(SEQ ID NO:1)的5’端还连接有SpeI酶切位点，所述24DT22核苷酸序列(SEQ ID NO:1)的3’端还连接有KasI酶切位点。
第二实施例、拟南芥和大豆重组表达载体的构建
1、构建含有24DT22核苷酸序列的重组克隆载体DBN01-T
将合成的24DT22核苷酸序列连入克隆载体pGEM-T(Promega，Madison，USA，CAT：A3600)上，操作步骤按Promega公司产品pGEM-T载体说明书进行，得到重组克隆载体DBN01-T，其构建流程如图1所示(其中，Amp表示氨苄青霉素抗性基因；f1表示噬菌体f1的复制起点；LacZ为LacZ起始密码子；SP6为SP6RNA聚合酶启动子；T7为T7RNA聚合酶启动子；24DT22为24DT22核苷酸序列(SEQ ID NO:1)；MCS为多克隆位点)。
然后将重组克隆载体DBN01-T用热激方法转化大肠杆菌T1感受态细胞(Transgen，Beijing，China，CAT：CD501)，其热激条件为：50μL大肠杆菌T1感受态细胞、10μL质粒DNA(重组克隆载体DBN01-T)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)，在表面涂有IPTG(异丙基硫代-β-D-半乳糖苷)和X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)的氨苄青霉素(100mg/L)的LB平板(胰蛋白胨10g/L，酵母提取物5g/L，NaCl10g/L，琼脂15g/L，用NaOH调pH至7.5)上生长过夜。挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，氨苄青霉素100mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒：将菌液在12000rpm转速下离心1min，去上清液，沉淀菌体用100μL冰预冷的溶液I(25mM Tris-HCl，10mM EDTA(乙二胺四乙酸)，50mM葡萄糖，pH8.0)悬浮；加入200μL新配制的溶液II(0.2M NaOH，1％SDS(十二烷基硫酸钠))，将管子颠倒4次，混合，置冰上3-5min；加入150μL冰冷的溶液III(3M醋酸钾，5M醋酸)，立即充分混匀，冰上放置5-10min；于温度4℃、转速12000rpm条件下离心5min，在上清液中加入2倍体积无水乙醇，混匀后室温放置5min；于温度4℃、转速12000rpm条件下离心5min，弃上清液，沉淀用浓度(V/V)为70％的乙醇洗涤后晾干；加入30μL含RNase(20μg/mL)的TE(10mM Tris-HCl，1mM EDTA，pH8.0)溶解沉淀；于温 度37℃下水浴30min，消化RNA；于温度-20℃保存备用。
提取的质粒经SpeI和KasI酶切鉴定后，对阳性克隆进行测序验证，结果表明重组克隆载体DBN01-T中插入的所述24DT22核苷酸序列为序列表中SEQ ID NO:1所示的核苷酸序列，即24DT22核苷酸序列正确插入。
2、构建含有24DT22核苷酸序列的拟南芥和大豆重组表达载体DBN100301
用限制性内切酶SpeI和KasI分别酶切重组克隆载体DBN01-T和表达载体DBNBC-01(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的24DT22核苷酸序列片段插到表达载体DBNBC-01的SpeI和KasI位点之间，利用常规的酶切方法构建载体是本领域技术人员所熟知的，构建成重组表达载体DBN100301，其构建流程如图2所示(Kan：卡那霉素基因；RB：右边界；AtUbi10：拟南芥Ubiquitin(泛素)10基因启动子(SEQ ID NO:3)；24DT22：24DT22核苷酸序列(SEQ ID NO:1)；Nos：胭脂碱合成酶基因的终止子(SEQ ID NO:4)；prCaMV35S：花椰菜花叶病毒35S启动子(SEQ ID NO:5)；PAT：草丁膦乙酰转移酶基因(SEQ ID NO:6)；tCaMV35S：花椰菜花叶病毒35S终止子(SEQ ID NO:7)；LB：左边界)。
将重组表达载体DBN100301用热激方法转化大肠杆菌T1感受态细胞，其热激条件为：50μL大肠杆菌T1感受态细胞、10μL质粒DNA(重组表达载体DBN100301)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)；然后在含50mg/L卡那霉素(Kanamycin)的LB固体平板(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂15g/L，用NaOH调pH至7.5)上于温度37℃条件下培养12小时，挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，卡那霉素50mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒。将提取的质粒用限制性内切酶SpeI和KasI酶切后鉴定，并将阳性克隆进行测序鉴定，结果表明重组表达载体DBN100301在SpeI和KasI位点间的核苷酸序列为序列表中SEQ ID NO:1所示核苷酸序列，即24DT22核苷酸序列。
3、构建含有对照序列的拟南芥和大豆重组表达载体DBN100301N
按照本发明第二实施例中1所述的构建含有24DT22核苷酸序列的重组克隆载体DBN01-T的方法，利用对照序列(SEQ ID NO:8)构建含有对照序列的重组克隆载体DBN01R-T。对阳性克隆进行测序验证，结果表明重组克隆载体DBN01R-T中插入的对照序列为序列表中SEQ ID NO:8所示的核苷酸序列，即对照序列正确插入。
按照本发明第二实施例中2所述的构建含有24DT22核苷酸序列的重组表达载体DBN100301的方法，利用对照序列构建含有对照序列的重组表达载体 DBN100301N，其载体结构如图3所示(载体骨架：pCAMBIA2301(CAMBIA机构可以提供)；Kan：卡那霉素基因；RB：右边界；AtUbi10：拟南芥Ubiquitin(泛素)10基因启动子(SEQ ID NO:3)；mN：对照序列(SEQ ID NO:8)；Nos：胭脂碱合成酶基因的终止子(SEQ ID NO:4)；prCaMV35S：花椰菜花叶病毒35S启动子(SEQ ID NO:5)；PAT：草丁膦乙酰转移酶基因(SEQ ID NO:6)；tCaMV35S：花椰菜花叶病毒35S终止子(SEQ ID NO:7)；LB：左边界)。对阳性克隆进行测序验证，结果表明重组表达载体DBN100301N中插入的对照序列为序列表中SEQ ID NO:8所示的核苷酸序列，即对照序列正确插入。
第三实施例、转入24DT21核苷酸序列的拟南芥植株的获得
1、重组表达载体转化农杆菌
对己经构建正确的重组表达载体DBN100301和DBN100301N(对照序列)用液氮法转化到农杆菌GV3101中，其转化条件为：100μL农杆菌GV3101、3μL质粒DNA(重组表达载体)；置于液氮中10分钟，37℃温水浴10分钟；将转化后的农杆菌GV3101接种于LB试管中于温度28℃、转速为200rpm条件下培养2小时，涂于含50mg/L的利福平(Rifampicin)和50mg/L的卡那霉素的LB平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，用限制性内切酶SmaI和PstI酶切DBN100301后进行酶切验证，用限制性内切酶SmaI和BglI酶切DBN100301N(对照序列)后进行酶切验证，结果表明重组表达载体DBN100301和DBN100301N(对照序列)结构完全正确。
2、获得转基因拟南芥植株
将野生型拟南芥种子悬浮于0.1％(w/v)琼脂糖溶液中。将悬浮的种子在4℃下保存2天以完成对休眠的需要以保证种子同步萌发。用蛭石混合马粪土并用水地下灌溉至湿润，使土壤混合物排水24小时。将预处理后的种子种在土壤混合物上并用保湿罩覆盖7天。使种子萌发并在恒温(22℃)恒湿(40-50％)光强度为120-150μmol/m2秒的长日照条件(16小时光照/8小时黑暗)下在温室中培养植物。开始用霍格兰营养液灌溉植物，接着用去离子水灌溉，保持土壤潮湿但不湿透。
使用花浸泡法转化拟南芥。用选取的农杆菌菌落接种一份或多份15-30mL含卡那霉素(50mg/L)和利福平(10mg/L)的YEP培养液的预培养物。以220rpm将培养物在28℃恒速摇动孵育过夜。每个预培养物用于接种两份500mL含卡那霉素(50mg/L)和利福平(10mg/L)的YEP培养液的培养物并将培养物在28℃持续摇动孵育过夜。室温以约8700×g离心10分钟沉淀细胞，弃去得到的上清液。将细胞沉淀轻柔重悬于500mL渗透培养基中，所述渗透培养基含有1/2×MS盐/B5维生素、10％(w/v)蔗糖、0.044μM苄氨基嘌呤 (10μL/L(1mg/mL DMSO中的原液))和300μL/L Silvet L-77。将约1月龄的植物在培养基中浸泡15秒，确保浸没最新的花序。接着将植物侧面放倒并覆盖(透明或不透明)24小时，接着用水洗涤并竖直放置。在22℃以16小时光照/8小时黑暗的光周期培养植物。浸泡约4周后收获种子。
将新收获的(24DT22核苷酸序列和对照序列)T1种子在室温干燥7天。将种子种在26.5×51cm萌发盘中，每盘接受200mgT1种子(约10000个种子)，所述种子事先已悬浮于40mL 0.1％(w/v)琼脂糖溶液并在4℃下保存2天以完成对休眠的需要以保证种子同步萌发。
用蛭石混合马粪土并用水地下灌溉至湿润，利用重力排水。用移液管将预处理后的种子(每个40mL)均匀地种在土壤混合物上，并用保湿罩覆盖4-5天。在使用出苗后喷洒草铵膦(选择共转化的PAT基因)进行最初转化体选择前1天移去罩。
在7个种植天数后(DAP)并于11DAP再次使用DeVilbiss压缩空气喷嘴以10mL/盘(703L/ha)的喷洒体积用Liberty除草剂(200g ai/L的草铵膦)的0.2％溶液喷洒T1植物(分别为子叶期和2-4叶期)，以提供每次应用280g ai/ha有效量的草铵膦。在最后喷洒后4-7天鉴定存活株(生长活跃的植物)，并分别移植到用马粪土和蛭石制备的7cmx7cm的方盆中(每盘3-5棵)。用保湿罩覆盖移植的植物3-4天，并如前置于22℃培养室中或直接移入温室。接着移去罩并在测试24DT22基因提供苯氧基生长素除草剂抗性的能力之前至少1天将植物栽种到温室(22±5℃，50±30％RH，14小时光照：10小时黑暗，最小500μE/m2s1天然+补充光)。
第四实施例、转基因拟南芥植株的除草剂抗性效果检测
用24DT22基因进行首次拟南芥转化。首先使用草铵膦选择方案从未转化种子背景中选择T1转化体。筛选了约20000个T1种子中并鉴定了314株T1代阳性转化子(PAT基因)，约1.6％的转化效率。将转入24DT22核苷酸序列的拟南芥T1植株、转入对照序列的拟南芥T1植株和野生型拟南芥植株(播种后18天)分别对2,4-D二甲铵盐和二甲四氯进行除草剂抗性效果检测。
分别将转入24DT22核苷酸序列的拟南芥T1植株、转入对照序列的拟南芥T1植株和野生型拟南芥植株分别用2,4-D二甲铵盐(560g ae/ha，1倍大田浓度)、二甲四氯(560g ae/ha，1倍大田浓度)和空白溶剂(水)喷洒。喷施7天和14天后统计植株抗性情况：7天后生长状况和空白溶剂(水)一致的划为高抗植株，7天后有莲座叶卷曲的划为中抗植株，14天后仍不能抽苔的划为低抗植株，14天后死亡的划为不抗植株。由于每株拟南芥T1植株是独立的转化事件，可以预计在给定剂量内个体T1应答的显著差异。结果如表1和图4所示。
表1、转基因拟南芥T1植株除草剂抗性实验结果
Figure PCTCN2016073182-appb-000001
对于拟南芥，560g ae/ha 2,4-D和二甲四氯是将敏感植物与具有平均抗性水平的植物区分开来的有效剂量。表1和图4的结果表明：24DT22基因赋予个体拟南芥植物除草剂抗性(只有部分植株具有抗性的原因是由于T1代植物插入位点是随机的，因而抗性基因的表达水平有差异，表现出抗性水平的差异)，尤其是苯氧基生长素除草剂；而野生型拟南芥植株和转入对照序列的拟南芥T1植株则均不具有苯氧基生长素除草剂抗性。
第五实施例、转基因大豆植株的获得及验证
1、重组表达载体转化农杆菌
对己经构建正确的重组表达载体DBN100301和DBN100301N(对照序列)用液氮法转化到农杆菌LBA4404(Invitrgen，Chicago，USA，CAT：18313-015)中，其转化条件为：100μL农杆菌LBA4404、3μL质粒DNA(重组表达载体)；置于液氮中10分钟，37℃温水浴10分钟；将转化后的农杆菌LBA4404接种于LB试管中于温度28℃、转速为200rpm条件下培养2小时，涂于含50mg/L的利福平(Rifampicin)和50mg/L的卡那霉素的LB平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，用限制性内切酶SmaI和PstI酶切DBN100301后进行酶切验证，用限制性内切酶SmaI和BglI酶切DBN100301N(对照序列)后进行酶切验证，结果表明重组表达载体DBN100301和DBN100301N(对照序列)结构完全正确。
2、获得转基因大豆植株
按照常规采用的农杆菌侵染法，将无菌培养的大豆品种中黄13的子叶节组织与本实施例中1所述的农杆菌共培养，以将第二实施例中2和3构建的重组表达载体DBN100301和DBN100301N中的T-DNA(包括拟南芥 Ubiquitin10基因的启动子序列、24DT22核苷酸序列、对照序列、Nos终止子、花椰菜花叶病毒35S启动子、草丁膦乙酰转移酶基因、花椰菜花叶病毒35S终止子)转入到大豆染色体组中，获得了转入24DT22和对照核苷酸序列的大豆植株；同时以野生型大豆植株作为对照。
对于农杆菌介导的大豆转化，简要地，将成熟的大豆种子在大豆萌发培养基(B5盐3.1g/L，B5维他命，蔗糖20g/L，琼脂8g/L，pH5.6)中进行萌发，将种子接种于萌发培养基上，按以下条件培养：温度25±1℃；光周期(光/暗)为16/8h。萌发4-6天后取鲜绿的子叶节处膨大的大豆无菌苗，在子叶节下3-4毫米处切去下胚轴，纵向切开子叶，去顶芽、侧芽和种子根。用解剖刀的刀背在子叶节处进行创伤，用农杆菌悬浮液接触创伤过的子叶节组织，其中农杆菌能够将所述24DT22核苷酸序列传递至创伤过的子叶节组织(步骤1：侵染步骤)在此步骤中，子叶节组织优选地浸入农杆菌悬浮液(OD660＝0.5-0.8，侵染培养基(MS盐2.15g/L、B5维他命、蔗糖20g/L、葡萄糖10g/L、乙酰丁香酮(AS)40mg/L、2-吗啉乙磺酸(MES)4g/L、玉米素(ZT)2mg/L，pH5.3)中以启动接种。子叶节组织与农杆菌共培养一段时期(3天)(步骤2：共培养步骤)。优选地，子叶节组织在侵染步骤后在固体培养基(MS盐4.3g/L、B5维他命、蔗糖20g/L、葡萄糖10g/L、2-吗啉乙磺酸(MES)4g/L、玉米素2mg/L、琼脂8g/L，pH5.6)上培养。在此共培养阶段后，可以有一个选择性的“恢复”步骤。在“恢复”步骤中，恢复培养基(B5盐3.1g/L、B5维他命、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、玉米素(ZT)2mg/L、琼脂8g/L，头孢霉素150mg/L，谷氨酸100mg/L，天冬氨酸100mg/L，pH5.6)中至少存在一种己知抑制农杆菌生长的抗生素(头孢霉素)，不添加植物转化体的选择剂(步骤3：恢复步骤)。优选地，子叶节再生的组织块在有抗生素但没有选择剂的固体培养基上培养，以消除农杆菌并为侵染细胞提供恢复期。接着，子叶节再生的组织块在含选择剂(草丁膦)的培养基上培养并选择生长着的转化愈伤组织(步骤4：选择步骤)。优选地，子叶节再生的组织块在有选择剂的筛选固体培养基(B5盐3.1g/L、B5维他命、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、6-苄基腺嘌呤(6-BAP)1mg/L、琼脂8g/L，头孢霉素150mg/L，谷氨酸100mg/L，天冬氨酸100mg/L，草丁膦6mg/L，pH5.6)上培养，导致转化的细胞选择性生长。然后，转化的细胞再生成植物(步骤5：再生步骤)，优选地，在含选择剂的培养基上生长的子叶节再生的组织块在固体培养基(B5分化培养基和B5生根培养基)上培养以再生植物。
筛选得到的抗性组织块转移到所述B5分化培养基(B5盐3.1g/L、B5维他命、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、玉米素(ZT)1mg/L、琼脂8g/L、头孢霉素150mg/L、谷氨酸50mg/L、天冬氨酸50mg/L、赤霉素1mg/L、 生长素1mg/L、草丁膦6mg/L，pH5.6)上，25℃下培养分化。分化出来的小苗转移到所述B5生根培养基(B5盐3.1g/L、B5维他命、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、琼脂8g/L、头孢霉素150mg/L、吲哚-3-丁酸(IBA)1mg/L)，在生根培养上，25℃下培养至约10cm高，移至温室培养至结实。在温室中，每天于26℃下培养16小时，再于20℃下培养8小时。
3、用TaqMan验证转基因大豆植株
分别取转入24DT22核苷酸序列的大豆植株和转入对照核苷酸序列的大豆植株的叶片约100mg作为样品，用Qiagen的DNeasy Plant Maxi Kit提取其基因组DNA，通过Taqman探针荧光定量PCR方法检测PAT基因拷贝数以确定24DT22基因的拷贝数。同时以野生型大豆植株作为对照，按照上述方法进行检测分析。实验设3次重复，取平均值。
检测PAT基因拷贝数的具体方法如下：
步骤11、分别取转入24DT22核苷酸序列的大豆植株、转入对照序列的大豆植株和野生型大豆植株的叶片各100mg，分别在研钵中用液氮研成匀浆，每个样品取3个重复；
步骤12、使用Qiagen的DNeasy Plant Mini Kit提取上述样品的基因组DNA，具体方法参考其产品说明书；
步骤13、用NanoDrop 2000(Thermo Scientific)测定上述样品的基因组DNA浓度；
步骤14、调整上述样品的基因组DNA浓度至同一浓度值，所述浓度值的范围为80-100ng/μL；
步骤15、采用Taqman探针荧光定量PCR方法鉴定样品的拷贝数，以经过鉴定已知拷贝数的样品作为标准品，以野生型大豆植株的样品作为对照，每个样品3个重复，取其平均值；荧光定量PCR引物和探针序列分别是：
以下引物和探针用来检测PAT核苷酸序列：
引物1：GAGGGTGTTGTGGCTGGTATTG如序列表中SEQ ID NO:11所示；
引物2：TCTCAACTGTCCAATCGTAAGCG如序列表中SEQ ID NO:12所示；
探针1：CTTACGCTGGGCCCTGGAAGGCTAG如序列表中SEQ ID NO:13所示；
PCR反应体系为：
Figure PCTCN2016073182-appb-000002
Figure PCTCN2016073182-appb-000003
所述50×引物/探针混合物包含1mM浓度的每种引物各45μL，100μM浓度的探针50μL和860μL 1×TE缓冲液，并且在4℃，贮藏在琥珀试管中。
PCR反应条件为：
Figure PCTCN2016073182-appb-000004
利用SDS2.3软件(Applied Biosystems)分析数据。
通过分析PAT基因拷贝数的实验结果，进而证实24DT22核苷酸序列均己整合到所检测的大豆植株的染色体组中，而且转入24DT22核苷酸序列的大豆植株和转入对照序列的大豆植株均获得了单拷贝的转基因大豆植株。
第六实施例、转基因大豆植株的除草剂抗性效果检测
将转入24DT22核苷酸序列的大豆植株、转入对照序列的大豆植株和野生型大豆植株(幼苗期)分别对2,4-D二甲铵盐和二甲四氯进行除草剂抗性效果检测。
分别取转入24DT22核苷酸序列的大豆植株、转入对照序列的大豆植株和野生型大豆植株，并分别用2,4-D二甲铵盐(2240g ae/ha，4倍大田浓度)、二甲四氯(2240g ae/ha，4倍大田浓度)和空白溶剂(水)喷洒。分别在喷施后6小时(6HAT)、2天(2DAT)、7天(7DAT)及14天(14DAT)后，根据叶片卷曲程度和生长点损伤程度来统计每株植株受除草剂的损伤程度：以叶片平整如野生型、生长点完好无损为0％；叶片卷曲萎蔫且生长点死亡为100％。转入24DT22核苷酸序列的共3个株系(S1、S2和S3)，转入对照序列的共2个株系(S4和S5)，野生型的(CK1)共1个株系；从每个株系选10-15株进行测试。结果如表2及图5所示。
表2、转基因大豆T1植株除草剂抗性实验结果
Figure PCTCN2016073182-appb-000005
Figure PCTCN2016073182-appb-000006
对于大豆，2240g ae/ha 2,4-D和二甲四氯是将敏感植物与具有平均抗性水平的植物区分开来的有效剂量。表2和图5的结果表明：24DT22基因赋予转基因大豆植物高水平除草剂抗性，尤其是苯氧基生长素除草剂；而野生型大豆植株和转入对照序列的大豆T1植株则均不具有苯氧基生长素除草剂抗性。
第七实施例、玉米重组表达载体的构建及重组表达载体转化农杆菌
1、构建含有24DT22核苷酸序列的玉米重组表达载体DBN100764
用限制性内切酶SpeI和KasI分别酶切重组克隆载体DBN01-T和表达载体DBNBC-02(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的24DT22核苷酸序列片段插到表达载体DBNBC-02的SpeI和KasI位点之间，利用常规的酶切方法构建载体是本领域技术人员所熟知的，表达载体DBNBC-02中的SpeI和KasI酶切位点也是利用常规的酶切方法引入的，构建成重组表达载体DBN100764，其构建流程如图6所示(Kan：卡那霉素基因；RB：右边界；Ubi：玉米Ubiquitin(泛素)1基因启动子(SEQ ID NO:9)；24DT22：24DT22核苷酸序列(SEQ ID NO:1)；Nos：胭脂碱合成酶基因的终止子(SEQ ID NO:4)；PMI：磷酸甘露糖异构酶基因(SEQ ID NO:10)；LB：左边界)。
将重组表达载体DBN100764用热激方法转化大肠杆菌T1感受态细胞，其热激条件为：50μL大肠杆菌T1感受态细胞、10μL质粒DNA(重组表达载体DBN100764)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)；然后在含50mg/L卡那霉素(Kanamycin)的LB固体平板(胰蛋 白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂15g/L，用NaOH调pH至7.5)上于温度37℃条件下培养12小时，挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，壮观霉素50mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒。将提取的质粒用限制性内切酶SpeI和KasI酶切后鉴定，并将阳性克隆进行测序鉴定，结果表明重组表达载体DBN100764在SpeI和KasI位点间的核苷酸序列为序列表中SEQ ID NO:1所示核苷酸序列，即24DT22核苷酸序列。
2、构建含有对照序列的玉米重组表达载体DBN100764N
按照本发明第二实施例中1所述的构建含有24DT22核苷酸序列的重组克隆载体DBN01-T的方法，利用对照序列(SEQ ID NO:8)构建含有对照序列的重组克隆载体DBN02R-T。对阳性克隆进行测序验证，结果表明重组克隆载体DBN02R-T中插入的对照序列为序列表中SEQ ID NO:8所示的核苷酸序列，即对照序列正确插入。
按照本发明本实施例中1所述的构建含有24DT22核苷酸序列的重组表达载体DBN100764的方法，利用对照序列构建含有对照序列的重组表达载体DBN100764N，其载体结构如图7所示(载体骨架：pCAMBIA2301(CAMBIA机构可以提供)；Kan：卡那霉素基因；RB：右边界；Ubi：玉米Ubiquitin(泛素)1基因启动子(SEQ ID NO:9)；mN：对照序列(SEQ ID NO:8)；Nos：胭脂碱合成酶基因的终止子(SEQ ID NO:4)；PMI：磷酸甘露糖异构酶基因(SEQ ID NO:10)；LB：左边界)。对阳性克隆进行测序验证，结果表明重组表达载体DBN100764N中插入的对照序列为序列表中SEQ ID NO:8所示的核苷酸序列，即对照序列正确插入。
3、玉米重组表达载体转化农杆菌
对己经构建正确的重组表达载体DBN100764和DBN100764N(对照序列)用液氮法转化到农杆菌LBA4404(Invitrgen，Chicago，USA，CAT：18313-015)中，其转化条件为：100μL农杆菌LBA4404、3μL质粒DNA(重组表达载体)；置于液氮中10分钟，37℃温水浴10分钟；将转化后的农杆菌LBA4404接种于LB试管中于温度28℃、转速为200rpm条件下培养2小时，涂于含50mg/L的利福平(Rifampicin)和50mg/L的卡那霉素的LB平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，用限制性内切酶SmaI和EcoRV酶切DBN100764后进行酶切验证，用限制性内切酶StyI和BglI酶切DBN100764N(对照序列)后进行酶切验证，结果表明重组表达载体DBN100764和DBN100764N(对照序列)结构完全正确。
第八实施例、转入24DT22核苷酸序列的玉米植株的获得及验证
按照常规采用的农杆菌侵染法，将无菌培养的玉米品种综31(Z31)的幼 胚与第七实施例中3所述的农杆菌共培养，以将第七实施例中1和2构建的重组表达载体DBN100764和DBN100764N(对照序列)中的T-DNA(包括玉米Ubiquitin1基因的启动子序列、24DT22核苷酸序列、对照序列、PMI基因和Nos终止子序列)转入到玉米染色体组中，获得了转入24DT22核苷酸序列的玉米植株和转入对照序列的玉米植株；同时以野生型玉米植株作为对照。
对于农杆菌介导的玉米转化，简要地，从玉米中分离未成熟的幼胚，用农杆菌悬浮液接触幼胚，其中农杆菌能够将24DT22核苷酸序列传递至幼胚之一的至少一个细胞(步骤1：侵染步骤)。在此步骤中，幼胚优选地浸入农杆菌悬浮液(OD660＝0.4-0.6，侵染培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖68.5g/L、葡萄糖36g/L、乙酰丁香酮(AS)40mg/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L，pH5.3))中以启动接种。幼胚与农杆菌共培养一段时期(3天)(步骤2：共培养步骤)。优选地，幼胚在侵染步骤后在固体培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖20g/L、葡萄糖10g/L、乙酰丁香酮(AS)100mg/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、琼脂8g/L，pH5.8)上培养。在此共培养阶段后，可以有一个选择性的“恢复”步骤。在“恢复”步骤中，恢复培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、植物凝胶3g/L，pH5.8)中至少存在一种己知抑制农杆菌生长的抗生素(头孢霉素)，不添加植物转化体的选择剂(步骤3：恢复步骤)。优选地，幼胚在有抗生素但没有选择剂的固体培养基上培养，以消除农杆菌并为侵染细胞提供恢复期。接着，接种的幼胚在含选择剂(甘露糖)的培养基上培养并选择生长着的转化愈伤组织(步骤4：选择步骤)。优选地，幼胚在有选择剂的筛选固体培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、甘露糖12.5g/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、植物凝胶3g/L，pH5.8)上培养，导致转化的细胞选择性生长。然后，愈伤组织再生成植物(步骤5：再生步骤)，优选地，在含选择剂的培养基上生长的愈伤组织在固体培养基(MS分化培养基和MS生根培养基)上培养以再生植物。
筛选得到的抗性愈伤组织转移到所述MS分化培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、6-苄基腺嘌呤2mg/L、甘露糖5g/L、植物凝胶3g/L，pH5.8)上，25℃下培养分化。分化出来的小苗转移到所述MS生根培养基(MS盐2.15g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、吲哚-3-乙酸1mg/L、植物凝胶3g/L，pH5.8)上，25℃下培养至约10cm高，移至温室培养至结实。在温室中，每天于28℃下培养16小时，再于20℃下培养8小时。
2、用TaqMan验证转入24DT22核苷酸序列的玉米植株
分别取转入24DT22核苷酸序列的玉米植株和转入对照序列的玉米植株的叶片约100mg作为样品，用Qiagen的DNeasy Plant Maxi Kit提取其基因组DNA，通过Taqman探针荧光定量PCR方法检测PMI基因拷贝数以确定24DT22基因的拷贝数。同时以野生型玉米植株作为对照，按照上述方法进行检测分析。实验设3次重复，取平均值。
检测PMI基因拷贝数的具体方法如下：
步骤31、分别取转入24DT22核苷酸序列的玉米植株、转入对照序列的玉米植株和野生型玉米植株的叶片各100mg，分别在研钵中用液氮研成匀浆，每个样品取3个重复；
步骤32、使用Qiagen的DNeasy Plant Mini Kit提取上述样品的基因组DNA，具体方法参考其产品说明书；
步骤33、用NanoDrop 2000(Thermo Scientific)测定上述样品的基因组DNA浓度；
步骤34、调整上述样品的基因组DNA浓度至同一浓度值，所述浓度值的范围为80-100ng/μL；
步骤35、采用Taqman探针荧光定量PCR方法鉴定样品的拷贝数，以经过鉴定已知拷贝数的样品作为标准品，以野生型玉米植株的样品作为对照，每个样品3个重复，取其平均值；荧光定量PCR引物和探针序列分别是：
以下引物和探针用来检测PMI核苷酸序列：
引物3：GCTGTAAGAGCTTACTGAAAAAATTAACA如序列表中SEQ ID NO:14所示；
引物4：CGATCTGCAGGTCGACGG如序列表中SEQ ID NO:15所示；
探针2：TCTCTTGCTAAGCTGGGAGCTCGATCC如序列表中SEQ ID NO:16所示；
PCR反应体系为：
Figure PCTCN2016073182-appb-000007
所述50×引物/探针混合物包含1mM浓度的每种引物各45μL，100μM浓度的探针50μL和860μL 1×TE缓冲液，并且在4℃，贮藏在琥珀试管中。
PCR反应条件为：
Figure PCTCN2016073182-appb-000008
Figure PCTCN2016073182-appb-000009
利用SDS2.3软件(Applied Biosystems)分析数据。
通过分析PMI基因拷贝数的实验结果，进而证实24DT22核苷酸序列均己整合到所检测的玉米植株的染色体组中，而且转入24DT22核苷酸序列的玉米植株和转入对照序列的玉米植株均获得了单拷贝的转基因玉米植株。
第九实施例、转基因玉米植株的除草剂抗性效果检测
将转入24DT22核苷酸序列的玉米植株、转入对照序列的玉米植株和野生型玉米植株(V3-V4时期)分别对2,4-D二甲铵盐和二甲四氯进行除草剂抗性效果检测。
分别取转入24DT22核苷酸序列的玉米植株、转入对照序列的玉米植株和野生型玉米植株，并分别用2,4-D二甲铵盐(8960g ae/ha，16倍大田浓度)、二甲四氯(8960g ae/ha，16倍大田浓度)和空白溶剂(水)喷洒。喷施21天后统计支撑根发育情况。转入24DT22核苷酸序列的共3个株系(S6、S7和S8)，转入对照序列的共2个株系(S9和S10)，野生型的(CK2)共1个株系；从每个株系选10-15株进行测试。结果如表3所示。
表3、转基因玉米T1植株除草剂抗性实验结果
Figure PCTCN2016073182-appb-000010
Figure PCTCN2016073182-appb-000011
表3的结果表明：24DT22基因赋予转基因玉米植物除草剂的高水平抗性，尤其是苯氧基生长素除草剂(由于单子叶植物本身对苯氧基生长素除草剂具有一定抗性，因而表现出高水平抗性)；而野生型玉米植株和转入对照序列的玉米植株则均不具有高水平的苯氧基生长素除草剂抗性。
综上所述，转入24DT22核苷酸序列的玉米植株、大豆植株和拟南芥植株都具有较高除草剂抗性能力。本发明24DT22除草剂抗性基因采用植物的偏好密码子，使得本发明除草剂抗性基因特别适合在植物中表达，本发明24DT22除草剂抗性蛋白质对除草剂抗性广，尤其苯氧基生长素除草剂。
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 (32)

  1. 一种除草剂抗性蛋白质，其特征在于，包括：
    (a)具有SEQ ID NO:2所示的氨基酸序列组成的蛋白质；或
    (b)在(a)中的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸且具有芳氧基链烷酸酯双加氧酶活性的蛋白质。
  2. 一种除草剂抗性基因，其特征在于，包括：
    (a)编码权利要求1所述的除草剂抗性蛋白质的核苷酸序列；或
    (b)在严格条件下与(a)限定的核苷酸序列杂交且编码具有芳氧基链烷酸酯双加氧酶活性的蛋白质的核苷酸序列；或
    (c)具有SEQ ID NO:1所示的核苷酸序列。
  3. 一种表达盒，其特征在于，包含调控序列和权利要求2所述的除草剂抗性基因。
  4. 一种重组载体，其特征在于，包含权利要求2所述的除草剂抗性基因或权利要求3所述的表达盒。
  5. 一种产生除草剂抗性蛋白质的方法，其特征在于，包括以下步骤：
    获得包含权利要求2所述的除草剂抗性基因或权利要求3所述的表达盒的转基因宿主生物的细胞；
    在允许产生除草剂抗性蛋白质的条件下培养所述转基因宿主生物的细胞；
    回收所述除草剂抗性蛋白质。
  6. 根据权利要求5所述的方法，其特征在于，所述转基因宿主生物包括植物、动物、细菌、酵母、杆状病毒、线虫或藻类。
  7. 根据权利要求6所述的方法，其特征在于，所述植物为大豆、棉花、玉米、水稻、小麦、甜菜或甘蔗。
  8. 一种增加除草剂耐受范围的方法，其特征在于，包括：将权利要求1所述的除草剂抗性蛋白质或权利要求3所述表达盒编码的除草剂抗性蛋白质在植物中与第二种核苷酸编码的蛋白质一起表达，所述第二种核苷酸编码至少一种不同于权利要求1所述除草剂抗性蛋白质或权利要求3所述表达盒编码的除草剂抗性蛋白质的蛋白质。
  9. 根据权利要求8所述的方法，其特征在于，所述第二种核苷酸编码草甘膦抗性蛋白质、草铵膦抗性蛋白质、4-羟苯基丙酮酸双加氧酶、乙酰乳酸合酶、细胞色素类蛋白质或原卟啉原氧化酶。
  10. 一种转化的植物细胞的选择方法，其特征在于，包括：用权利要求2所述的除草剂抗性基因或权利要求3所述的表达盒转化多个植物细胞，并在 允许表达所述除草剂抗性基因或所述表达盒的转化细胞生长，而杀死未转化细胞或抑制未转化细胞生长的除草剂浓度下培养所述细胞，所述除草剂为苯氧基生长素。
  11. 一种控制杂草的方法，其特征在于，包括：对种植作物的大田施用有效剂量的除草剂，所述作物包含权利要求2所述的除草剂抗性基因或权利要求3所述的表达盒或权利要求4所述的重组载体。
  12. 根据权利要求11所述的方法，其特征在于，所述除草剂为苯氧基生长素。
  13. 一种用于保护植物免受由除草剂引起的损伤的方法，其特征在于，包括：将权利要求2所述的除草剂抗性基因或权利要求3所述的表达盒或权利要求4所述的重组载体导入植物。
  14. 根据权利要求13所述的方法，其特征在于，所述除草剂为苯氧基生长素。
  15. 根据权利要求13或14所述的方法，其特征在于，所述植物为大豆、棉花、玉米、水稻、小麦、甜菜或甘蔗。
  16. 一种控制草甘膦耐性植物的大田中草甘膦抗性杂草的方法，其特征在于，包括：对种植草甘膦耐性植物的大田施用有效剂量的除草剂，所述草甘膦耐性植物包含权利要求2所述的除草剂抗性基因或权利要求3所述的表达盒或权利要求4所述的重组载体。
  17. 根据权利要求16所述的方法，其特征在于，所述除草剂为苯氧基生长素。
  18. 根据权利要求16或17所述的方法，其特征在于，所述草甘膦耐性植物为单子叶植物或双子叶植物。
  19. 一种赋予作物2,4-D除草剂抗性的方法，其特征在于，包括：将权利要求2所述的除草剂抗性基因或权利要求3所述的表达盒或权利要求4所述的重组载体导入所述作物。
  20. 根据权利要求19所述的方法，其特征在于，所述作物为大豆、棉花、玉米、水稻、小麦、甜菜或甘蔗。
  21. 一种除草剂抗性蛋白质耐受苯氧基生长素类除草剂的用途，其特征在于，所述除草剂抗性蛋白质包括：
    (a)具有SEQ ID NO:2所示的氨基酸序列组成的蛋白质；或
    (b)在(a)中的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸且具有芳氧基链烷酸酯双加氧酶活性的蛋白质。
  22. 一种如权利要求1所述的除草剂抗性蛋白质在控制杂草、保护植物免受由除草剂引起的损伤方面的应用。
  23. 根据权利要求22所述的应用，其特征在于，所述控制杂草的应用包括：对种植作物的大田施用有效剂量的除草剂，所述作物包含权利要求2所述的除草剂抗性基因或权利要求3所述的表达盒或权利要求4所述的重组载体。
  24. 根据权利要求23所述的应用，其特征在于，所述除草剂为苯氧基生长素。
  25. 根据权利要求22所述的应用，其特征在于，所述保护植物免受由除草剂引起的损伤的应用包括：将权利要求2所述的除草剂抗性基因或权利要求3所述的表达盒或权利要求4所述的重组载体导入植物。
  26. 根据权利要求25所述的应用，其特征在于，所述除草剂为苯氧基生长素。
  27. 根据权利要求25或26所述的应用，其特征在于，所述植物为大豆、棉花、玉米、水稻、小麦、甜菜或甘蔗。
  28. 根据权利要求22所述的应用，其特征在于，所述控制杂草的应用为控制草甘膦耐性植物的大田中草甘膦抗性杂草，包括：对种植草甘膦耐性植物的大田施用有效剂量的除草剂，所述草甘膦耐性植物包含权利要求2所述的除草剂抗性基因或权利要求3所述的表达盒或权利要求4所述的重组载体。
  29. 根据权利要求28所述的应用，其特征在于，所述除草剂为苯氧基生长素。
  30. 根据权利要求28或29所述的应用，其特征在于，所述草甘膦耐性植物为单子叶植物或双子叶植物。
  31. 根据权利要求22所述的应用，其特征在于，所述保护植物免受由除草剂引起的损伤的应用包括赋予作物2,4-D除草剂抗性。
  32. 根据权利要求31所述的应用，其特征在于，所述作物为大豆、棉花、玉米、水稻、小麦、甜菜或甘蔗。
PCT/CN2016/073182 2015-02-13 2016-02-02 除草剂抗性蛋白质、其编码基因及用途 WO2016127867A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103361316A (zh) * 2005-10-28 2013-10-23 美国陶氏益农公司 新除草剂抗性基因
CN103826444A (zh) * 2011-07-26 2014-05-28 陶氏益农公司 大豆事件pDAB9582.814.19.1和pDAB4468.04.16.1的昆虫抗性和除草剂耐受性育种叠加
CN104611308A (zh) * 2015-02-13 2015-05-13 北京大北农科技集团股份有限公司 除草剂抗性蛋白质、其编码基因及用途

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Publication number Priority date Publication date Assignee Title
JP5907657B2 (ja) 2007-05-09 2016-04-26 ダウ アグロサイエンシィズ エルエルシー 新規な除草剤抵抗性遺伝子
RU2551781C2 (ru) 2008-07-31 2015-05-27 Англо Нетерлендс Грейн Бв Устойчивые к гербицидам растения подсолнечника
US9540656B2 (en) 2010-12-03 2017-01-10 Dow Agrosciences Llc Stacked herbicide tolerance event 8291.45.36.2, related transgenic soybean lines, and detection thereof
CN103060279B (zh) * 2012-12-25 2014-08-13 北京大北农科技集团股份有限公司 除草剂抗性蛋白质、其编码基因及用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103361316A (zh) * 2005-10-28 2013-10-23 美国陶氏益农公司 新除草剂抗性基因
CN103826444A (zh) * 2011-07-26 2014-05-28 陶氏益农公司 大豆事件pDAB9582.814.19.1和pDAB4468.04.16.1的昆虫抗性和除草剂耐受性育种叠加
CN104611308A (zh) * 2015-02-13 2015-05-13 北京大北农科技集团股份有限公司 除草剂抗性蛋白质、其编码基因及用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3257936A4 *

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