WO2017161921A1 - 除草剂耐受性蛋白质、其编码基因及用途 - Google Patents

除草剂耐受性蛋白质、其编码基因及用途 Download PDF

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WO2017161921A1
WO2017161921A1 PCT/CN2016/109176 CN2016109176W WO2017161921A1 WO 2017161921 A1 WO2017161921 A1 WO 2017161921A1 CN 2016109176 W CN2016109176 W CN 2016109176W WO 2017161921 A1 WO2017161921 A1 WO 2017161921A1
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Prior art keywords
plant
herbicide
methyl
protein
bensulfuron
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PCT/CN2016/109176
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English (en)
French (fr)
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谢香庭
陶青
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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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 BR112018069243A priority Critical patent/BR112018069243A2/pt
Priority to DK16895265.3T priority patent/DK3425046T3/da
Priority to MX2018011581A priority patent/MX2018011581A/es
Priority to AU2016399292A priority patent/AU2016399292B2/en
Priority to ES16895265T priority patent/ES2902007T3/es
Priority to US16/086,298 priority patent/US10954528B2/en
Priority to CA3018255A priority patent/CA3018255C/en
Priority to EP16895265.3A priority patent/EP3425046B1/en
Publication of WO2017161921A1 publication Critical patent/WO2017161921A1/zh
Priority to ZA2018/06998A priority patent/ZA201806998B/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P15/00—Biocides for specific purposes not provided for in groups A01P1/00 - A01P13/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • 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
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/32—Ingredients for reducing the noxious effect of the active substances to organisms other than pests, e.g. toxicity reducing compositions, self-destructing compositions
    • 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
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
    • A01N47/28—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N<
    • A01N47/36—Ureas or thioureas containing the groups >N—CO—N< or >N—CS—N< containing the group >N—CO—N< directly attached to at least one heterocyclic ring; Thio analogues thereof
    • 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
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/46—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=C=S groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8275—Glyphosate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8278—Sulfonylurea
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)

Definitions

  • the present invention relates to a herbicide-tolerant protein, a gene encoding the same, and a use thereof, and more particularly to a protein, a gene encoding the same, and a use thereof, which are tolerant to a sulfonylurea herbicide.
  • 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.
  • growers can compensate for the weakness of glyphosate by tank mixing or other herbicides that control missing weeds, such as sulfonylurea weeding Agent.
  • Sulfonylurea herbicides have become the third largest herbicide after organophosphorus and acetamide herbicides. The annual global sales have reached more than US$3 billion. The annual application area of sulfonylurea herbicides in China has exceeded 2 million. The hectare is still expanding.
  • the invention provides a herbicide tolerance protein comprising:
  • the invention provides a herbicide tolerance gene comprising:
  • the invention provides an expression cassette comprising the aforementioned herbicide tolerance gene under the control of an operably linked regulatory sequence.
  • the present invention provides a recombinant vector comprising the aforementioned herbicide tolerance gene or the aforementioned expression cassette.
  • the invention provides a method of producing a herbicide-tolerant protein comprising:
  • the cells of the transgenic host organism are cultured under conditions that permit the production of a herbicide-tolerant protein
  • the transgenic host organism comprises a plant, animal, bacterium, yeast, baculovirus, nematode or algae.
  • the present invention provides a method of increasing tolerance to a range of herbicides comprising: the aforementioned herbicide tolerance protein or the herbicide-tolerant protein encoded by the aforementioned expression cassette in a plant and at least one a second protein different from the aforementioned herbicide-tolerant protein or the aforementioned herbicide-tolerant protein encoded by the expression cassette;
  • the second protein is 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, ammonium oxalate Phosphoacetyltransferase, alpha ketoglutarate-dependent dioxygenase, dicamba monooxygenase, 4-hydroxyphenylpyruvate dioxygenase, acetolactate synthase, cytochrome protein and/or protoplast Porphyrinogen oxidase.
  • the present invention provides a method of selecting a transformed plant cell, comprising: transforming a plurality of plant cells with the aforementioned herbicide tolerance gene or the aforementioned expression cassette, and allowing expression of the herbicide tolerance
  • the cell 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, the herbicide being a sulfonylurea herbicide;
  • the sulfonylurea herbicide is bensulfuron-methyl, mesulfuron-methyl, chloropyrazine, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, ethamsulfuron or Chlorsulfuron-methyl.
  • the present invention provides a method of controlling weeds comprising: applying an effective amount of a sulfonylurea herbicide to a field planting a plant of interest, the plant comprising the aforementioned herbicide tolerance gene or the aforementioned Expression cassette
  • the plant is a monocot or a dicot
  • the plant is corn, soybean, Arabidopsis, cotton, canola, rice, sorghum, wheat, barley, millet, sugar cane or oats;
  • the sulfonylurea herbicide is bensulfuron-methyl, sulfometuron, chlorpyrifossulfon, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, ethametsulfuron Or chlorsulfuron-methyl.
  • the present invention provides a method for protecting a plant from damage caused by a sulfonylurea herbicide, comprising: the aforementioned herbicide tolerance gene or the aforementioned expression cassette or the aforementioned recombinant vector Introducing the plant such that the introduced plant produces a herbicide-tolerant protein sufficient to protect it from the sulfonylurea herbicide damage;
  • the plant is a monocot or a dicot
  • the plant is corn, soybean, Arabidopsis, cotton, canola, rice, sorghum, wheat, barley, millet, sugar cane or oats;
  • the sulfonylurea herbicide is bensulfuron-methyl, sulfometuron, chlorpyrifossulfon, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, ethametsulfuron Or chlorsulfuron-methyl.
  • the present invention provides a method of controlling glyphosate-resistant weeds in a field of a glyphosate-tolerant plant, comprising: administering an effective amount of a sulfonyl group to a field planted with a glyphosate-tolerant plant a urea herbicide, the glyphosate-tolerant plant comprising the aforementioned herbicide tolerance gene or the aforementioned expression cassette;
  • the plant is a monocot or a dicot
  • the plant is corn, soybean, Arabidopsis, cotton, canola, rice, sorghum, wheat, barley, millet, sugar cane or oats;
  • the sulfonylurea herbicide is bensulfuron-methyl, sulfometuron, chlorpyrifossulfon, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, ethametsulfuron Or chlorsulfuron-methyl.
  • the present invention provides a method for imparting tolerance to a sulfonylurea herbicide of a plant, comprising: introducing the aforementioned herbicide tolerance gene or the aforementioned expression cassette or the aforementioned recombinant vector into a plant;
  • the plant is a monocot or a dicot
  • the plant is corn, soybean, Arabidopsis, cotton, canola, rice, sorghum, wheat, barley, millet, sugar cane or oats;
  • the sulfonylurea herbicide is bensulfuron-methyl, sulfometuron, chlorpyrifossulfon, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, ethametsulfuron Or chlorsulfuron-methyl.
  • the present invention provides a method of producing a plant resistant to a sulfonylurea herbicide, comprising introducing the aforementioned herbicide tolerance gene or the aforementioned expression cassette or the aforementioned recombinant vector into a genome of a plant;
  • the plant is a monocot or a dicot
  • the plant is corn, soybean, Arabidopsis, cotton, canola, rice, sorghum, wheat, barley, millet, sugar cane or oats;
  • the sulfonylurea herbicide is bensulfuron-methyl, sulfometuron, chlorpyrifossulfon, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, ethametsulfuron Or chlorsulfuron-methyl.
  • the present invention provides a method of culturing a plant resistant to a sulfonylurea herbicide, comprising: growing at least one plant propagule comprising a herbicide tolerance gene as described above in the genome of the plant propagule Or the aforementioned expression cassette;
  • the plant is a monocot or a dicot
  • the plant is corn, soybean, Arabidopsis, cotton, canola, rice, sorghum, wheat, barley, millet, sugar cane or oats;
  • the sulfonylurea herbicide is bensulfuron-methyl, sulfometuron, chlorpyrifossulfon, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, ethametsulfuron Or chlorsulfuron-methyl.
  • the present invention provides a planting system for controlling weed growth, comprising a sulfonylurea herbicide and a plant growth environment in which at least one plant of interest is contained, the plant comprising the aforementioned herbicide tolerance gene Or the aforementioned expression cassette;
  • the plant is a monocot or a dicot
  • the plant is corn, soybean, Arabidopsis, cotton, canola, rice, sorghum, wheat, barley, millet, sugar cane or oats;
  • the sulfonylurea herbicide is bensulfuron-methyl, sulfometuron, chlorpyrifossulfon, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, ethametsulfuron Or chlorsulfuron-methyl.
  • the present invention provides a planting system for controlling glyphosate-resistant weeds in a field of glyphosate-tolerant plants, comprising a sulfonylurea herbicide, a glyphosate herbicide, and planting at least one a field of the plant of interest, the glyphosate-tolerant plant comprising the aforementioned herbicide tolerance gene or the aforementioned expression cassette;
  • the plant is a monocot or a dicot
  • the plant is corn, soybean, Arabidopsis, cotton, canola, rice, sorghum, wheat, barley, millet, sugar cane or oats;
  • the sulfonylurea herbicide is bensulfuron-methyl, sulfometuron, chlorpyrifossulfon, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, ethametsulfuron Or chlorsulfuron-methyl.
  • the invention provides the use of a herbicide-tolerant protein-degrading sulfonylurea herbicide, the herbicide-tolerant protein comprising:
  • the sulfonylurea herbicide is bensulfuron-methyl, mesulfuron-methyl, chloropyrazine, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, ethamsulfuron or Chlorsulfuron-methyl.
  • introducing the herbicide tolerance 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, microprojectile bombardment, direct DNA uptake into protoplasts, electroporation or whisker silicon-mediated DNA introduction.
  • the sulfonylurea herbicide tolerance gene of the present invention and subsequent resistant crops provide excellent control of glyphosate resistant (or highly tolerant and successional) broadleaf weed species in crops select.
  • Sulfonylurea herbicides are broad-spectrum and potent broadleaf herbicides that can be used in both dicots and monocots. Providing greater crop tolerance provides excellent results for growers.
  • Sulfonylurea herbicide-tolerant transgenic dicots can also be more flexible in application time and dosage.
  • sulfonylurea herbicide tolerance traits Another use of sulfonylurea herbicide tolerance traits is that it can be used to prevent damage to normal sensitive crops such as sulfonylurea herbicide drift, volatilization, conversion (or other long-range movement phenomena), misuse, and destruction. .
  • the use of the SUM1 gene in plants provides protection against a broader spectrum of sulfonylurea herbicides, thereby increasing flexibility and controllable weed spectrum, providing drift to a full range of commercially available sulfonylurea herbicides or other Protection against long-distance sulfonylurea herbicide damage.
  • the SUM1 gene after genetic engineering for plant expression, has the property of allowing the use of sulfonylurea herbicides in plants in which the inherent tolerance is absent or insufficient to permit the use of these herbicides.
  • the SUM1 gene can provide protection against sulfonylurea herbicides in plants when natural tolerance is insufficient to allow selectivity. Plants containing only the SUM1 gene can now be treated in a continuous or tank mix with one, two or several sulfonylurea herbicides.
  • each sulfonylurea herbicide used to control broad-spectrum dicotyledonous weeds ranges from 7.5 to 150 g ai/ha, more typically from 20 to 50 g ai/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.
  • SUM1 gene with glyphosate tolerance traits can achieve glyphosate in glyphosate-tolerant crops by allowing selective use of glyphosate and sulfonylurea herbicides for the same crop Control of phosphine-resistant weed species (wideleaf weed species controlled by one or more sulfonylurea herbicides).
  • 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) ))
  • 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/SUM1 gene may range from 200 to 1600 gae/ha; the sulfonylurea herbicide(s) may range from 7.5 to 150 g ai/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.
  • the following resistance traits can be superimposed, alone or in multiple combinations, to provide the ability to effectively control or prevent weed succession against any of the aforementioned classes of herbicides: specifically 5- Enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate oxidoreductase (GOX), glyphosate-N-acetyltransferase (GAT), glyphosate decarboxylase, glufosinate acetyltransferase Enzyme (PAT), alpha ketoglutarate-dependent dioxygenase (AAD), dicamba monooxygenase (DMO), 4-hydroxyphenylpyruvate dioxygenase (HPPD), acetolactate synthase ( ALS), cytochrome protein (P450) and/or protoporphyrinogen oxidase (Protox).
  • EPSPS 5- Enolpyruvylshikimate-3-phosphate synthase
  • the SUM1 gene can be superimposed alone or in combination with other herbicide-tolerant crop characteristics followed by one or more other inputs (eg insect resistance, fungal resistance or stress tolerance, etc.) or output (eg increased yield, improvement) The amount of oil, increased fiber quality, etc.) superimposed on the traits.
  • 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.
  • SUM1 gene of the present invention to degrade sulfonylurea herbicides 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 SUM1 gene can be used as an excellent herbicide tolerant to crop traits and, for example, other herbicide-tolerant crop traits (eg glyphosate resistance, glufosinate resistance, other ALS inhibitors (eg imidazolinones, triazolopyrimidines) Sulfonamide resistance, bromoxynil resistance, HPPD inhibitor resistance, PPO inhibitor resistance, etc.) and insect resistance traits (Cry1Ab, Cry1F, Vip3, other Bacillus thuringiensis proteins or non-Bacillus origin) Insect resistance proteins, etc.) are superimposed.
  • the SUM1 gene can be used as a selection marker to assist in the selection of primary transformants of plants genetically engineered with another gene or gene population.
  • 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 can be applied to a variety of plants including, but not limited to, alfalfa, kidney bean, broccoli, kale, carrot, celery, cotton, cucumber, eggplant, lettuce, melon, pea, pepper, zucchini, radish, rape , spinach, soybean, pumpkin, tomato, Arabidopsis or watermelon; preferably, the dicot refers to soybean, Arabidopsis, tobacco, cotton or canola.
  • the monocot plants include, but are not limited to, corn, rice, sorghum, wheat, barley, rye, millet, sugar cane, oat or turfgrass; preferably, the monocot refers to corn, rice, sorghum, wheat, barley , millet, sugar cane or oatmeal.
  • the SUM1 gene of the present invention can be more actively used in grass crops with moderate tolerance, and the improved tolerance obtained by the trait will provide the grower with the ability to use these herbicides in a more effective amount and for a wider application time without The possibility of crop damage risk.
  • the planting system referred to in the present invention refers to a plant, which exhibits any herbicide tolerance and/or a combination of herbicide treatments available at different stages of plant development, to produce plants that are highly productive and/or attenuate damage.
  • the weed refers to a plant that competes with the cultivated plant of interest in a plant growth environment.
  • control and/or "control” in the present invention means that at least an effective amount of a sulfonylurea herbicide is applied directly (for example by spraying) to the environment in which the plant is grown to minimize weed development and/or to stop growth.
  • the cultivated plant of interest should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product; preferably, with reduced plant damage and compared to non-transgenic wild-type plants and / or have increased plant yield.
  • the reduced plant damage includes, but is not limited to, improved stem resistance, and/or increased kernel weight, and the like.
  • control and/or “control” effects of the protein SUM1 on weeds can exist independently and are not attenuated and/or disappeared by the presence of other substances that can "control” and/or “control” weeds.
  • any tissue of the transgenic plant (containing the SUM1 gene) is present and/or produced simultaneously and/or asynchronously, the herbicide tolerance protein SUM1 and/or another substance that can control weeds,
  • the presence of the other substance neither affects the "control” and/or “control” effect of the herbicide-tolerant protein SUM1 on weeds, nor does it lead to the "control” and/or “control” effects Complete and/or partial realization by the other substance is independent of the herbicide tolerance protein SUM1.
  • 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.
  • Plant propagules as used in the present invention include, but are not limited to, plant sexual propagules and plant asexual propagules.
  • the plant sexual propagule includes, but is not limited to, a plant seed; the plant asexual propagule refers to a vegetative organ of a plant body or a special tissue which can produce a new plant under ex vivo conditions; the vegetative organ or a certain Specific tissues include, but are not limited to, roots, stems and leaves, for example: plants with roots as vegetative propagules including strawberries and sweet potatoes; plants with stems as vegetative propagules including sugar cane and potatoes (tubers), etc.; leaves as asexual Plants of the propagule include aloe vera and begonia.
  • 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” or “tolerance” in the present invention is broader than the term “resistance” and includes “resistance” as well as the ability of a particular plant to have an increased resistance to herbicide-induced damage to various degrees. The same herbicide dose 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 invention also includes RNA that is functionally equivalent to the exemplified DNA.
  • the nucleic acid molecule or fragment thereof of the present invention hybridizes under stringent conditions to the herbicide tolerance gene of the present invention. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the herbicide tolerance 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 acids if two nucleic acids The two nucleic acid molecules are said to be “complementary” when they are capable of hybridizing to each other with sufficient stability to anneal under conventional "highly stringent” conditions and 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. Both the temperature conditions and the salt concentration can be changed, or one of them remains unchanged while the other variable changes.
  • the stringent conditions of the present invention may be specific hybridization with the SUM1 gene of the present invention at 65 ° C in a 6 ⁇ SSC, 0.5% SDS solution, followed by 2 ⁇ SSC, 0.1% SDS and 1 ⁇ SSC, The membrane was washed once for each 0.1% SDS.
  • sequences having herbicide tolerance activity and hybridizing under stringent conditions to the SUM1 gene of the present invention 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 sulfonylurea herbicides, i.e., transformed plants and plant cells can be grown in the presence of an effective amount of a sulfonylurea 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 tolerance activity characteristics of the proteins of the specific examples (including in comparison with full length proteins). / 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)
  • the length of the aforementioned sequence may vary, but is of sufficient length to ensure that the (encoding) protein is a herbicide tolerant protein.
  • substantially identical sequence refers to an amino acid substitution, deletion, addition or insertion but is not substantially Sequences that affect herbicide tolerance activity also include fragments that retain herbicide tolerance 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, deVos 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).
  • 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 SUM1 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); non-translated leader sequence of the coat protein mRNA of alfalfa mosaic virus (AMVRNA4); 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
  • AMVRNA4 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 may be a suitable polyadenylation signal sequence that functions in plants, including but not limited to, a polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene, a polyadenylation signal sequence derived from the protease inhibitor II (pinII) gene, a polyadenylation signal sequence derived from the pea ssRUBISCOE9 gene, and a polyadesoid derived from the ⁇ -tubulin gene Glycosylation signal sequence.
  • NOS Agrobacterium tumefaciens nopaline synthase
  • pinII protease inhibitor II
  • pea ssRUBISCOE9 a polyadesoid derived from the ⁇ -tubulin gene Glycosylation signal sequence.
  • 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-tolerant protein is a SUM1 amino acid sequence as shown in SEQ ID NO: 1 in the Sequence Listing.
  • the herbicide tolerance gene is a SUM1 nucleotide sequence, such as a sequence The SEQ ID NO: 2 and SEQ ID NO: 3 are shown in the table.
  • the herbicide tolerance gene can be used in plants, and can contain other elements besides the coding region of the protein encoded by the SUM1 nucleotide sequence, 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 herbicide-tolerant protein SUM1 of the present invention is resistant to most sulfonylurea herbicides.
  • the plant of the present invention contains exogenous DNA in its genome, and the exogenous DNA comprises a SUM1 nucleotide sequence which is protected from 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-tolerant 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-tolerant protein produced in the tissue, or directly The amount of herbicide-tolerant protein produced is specifically detected.
  • the present invention provides a herbicide-tolerant protein, a gene encoding the same, and use thereof, and has the following advantages:
  • the herbicide-tolerant protein SUM1 of the present invention is highly resistant to sulfonylurea herbicides and can withstand a 4-fold field concentration.
  • the herbicide-tolerant protein SUM1 of the present invention can exhibit high resistance to various sulfonylurea 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 SUM1 nucleotide sequence of a herbicide tolerance protein, a gene encoding the same, and a use thereof;
  • FIG. 2 is a flow chart showing the construction of a recombinant expression vector DBN100996 containing a SUM1 nucleotide sequence of the herbicide tolerance protein, the gene encoding the same, and the use thereof;
  • FIG. 3 is a schematic diagram showing the structure of a recombinant expression vector DBN100996N1 containing the control sequence 1 of the herbicide tolerance protein, the coding gene thereof and the use thereof;
  • 4-1 and 4-2 are diagrams showing the effect of the herbicide-tolerant protein, the gene encoding the same, and the use of the transgenic Arabidopsis thaliana T1 plant against sulfonylurea herbicides;
  • Figure 5 is a flow chart showing the construction of a recombinant expression vector DBN130028 containing the SUM1 nucleotide sequence of the herbicide tolerance protein, the coding gene thereof and the use thereof;
  • Figure 6 is a schematic view showing the structure of a recombinant expression vector DBN130028N1 containing the control sequence 1 of the herbicide tolerance protein, the coding gene thereof and the use thereof;
  • Figure 7 is a flow chart showing the construction of a recombinant cloning vector DBN02-T containing a SUM1 nucleotide sequence of the herbicide tolerance protein, the gene encoding the same, and the use thereof;
  • Figure 8 is a flow chart showing the construction of a recombinant expression vector DBN130035 containing the SUM1 nucleotide sequence of the herbicide tolerance protein, the gene encoding the same, and the use thereof;
  • Figure 9 is a schematic view showing the structure of a recombinant expression vector DBN130035N1 containing the control sequence 1 of the herbicide tolerance protein, the gene encoding the same, and the use thereof.
  • herbicide-tolerant protein of the present invention is further illustrated by specific examples.
  • SUM1-01 nucleotide sequence (as shown in SEQ ID NO: 2 in the Sequence Listing) and the SUM1-02 nucleotide sequence (as shown in SEQ ID NO: 3 in the Sequence Listing) are manufactured by Nanjing Kingsray Biotechnology Co., Ltd.
  • the synthesized SUM1-1-01 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.
  • 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 overnight at 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. .
  • the plasmid was extracted by alkali method: the bacterial solution was centrifuged at 12000 rpm for 1 min, the supernatant was removed, and the precipitated cells were precooled with 100 ⁇ L of ice.
  • Solution I 25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose, pH 8.0
  • solution II 0.2 M NaOH, 1% SDS (sodium dodecyl sulfate) was added.
  • the tube was inverted 4 times, mixed, placed on ice for 3-5 min; 150 ⁇ L of ice-cold solution III (3M potassium acetate, 5 M acetic acid) was added, and immediately mixed well, placed on ice for 5-10 min; at a temperature of 4 ° C, 12000 rpm After centrifugation for 5 min, add 2 volumes of absolute ethanol to the supernatant, mix and let stand for 5 min at room temperature; centrifuge at 5 ° C, 12000 rpm for 5 min, discard the supernatant, and use a concentration (V/V) of 70 for precipitation.
  • ice-cold solution III 3M potassium acetate, 5 M acetic acid
  • % ethanol was washed and air-dried; 30 ⁇ L of RNase (20 ⁇ g/mL) of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was added to dissolve the precipitate; the water was digested at a temperature of 37 ° C for 30 min; the temperature was maintained at -20 ° C. spare.
  • RNase 20 ⁇ g/mL
  • TE 10 mM Tris-HCl, 1 mM EDTA, pH 8.0
  • the positive clone was sequenced and verified. The result showed that the SUM1-01 nucleotide sequence inserted into the recombinant cloning vector DBN01-T was the nucleus shown by SEQ ID NO: 2 in the sequence listing. The nucleotide sequence, the SUM1-01 nucleotide sequence, was correctly inserted.
  • Recombinant cloning vector DBN01-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases SpeI and KasI, respectively, and the cut SUM1-01 nucleotide sequence fragment was inserted. Between the SpeI and KasI sites of the expression 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 DBN100996 is constructed, and the construction process thereof is shown in FIG.
  • the recombinant expression vector DBN100996 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 DBN100996), 42 ° C water bath for 30 seconds; 37 ° C oscillation Incubate for 1 hour (shake at 100 rpm); then LB solid plate containing 50 mg/L Spectinomycin (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L) Incubate with NaOH to pH 7.5) for 12 hours at 37 °C, pick white colonies, in LB liquid medium (trypsin 10g / L, yeast extract 5g / L, NaCl10g / L, spectinomycin) 50 mg/L, adjusted to pH 7.5 with NaOH, and incubated at 37 ° C overnight.
  • 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 DBN100996 was SEQ ID NO: The nucleotide sequence shown in 2, the SUM1-01 nucleotide sequence.
  • the recombinant cloning vector DBN01-T containing the SUM1 nucleotide sequence was constructed according to the method of 1 in the present Example, and the control sequence 1 (SEQ ID NO: 9) and the control sequence 2 (SEQ ID NO: 10) were used to construct the control sequence 1 containing Recombinant cloning vector DBN01R1-T and recombinant cloning vector DBN01R2-T containing control sequence 2. The positive clones were sequenced and verified.
  • control sequence 1 inserted into the recombinant cloning vector DBN01R1-T was the nucleotide sequence shown in SEQ ID NO: 9 in the sequence listing, and the control sequence 2 inserted in the recombinant cloning vector DBN01R2-T was sequenced.
  • the nucleotide sequence shown by SEQ ID NO: 10 in the list, that is, the control sequence was correctly inserted.
  • a recombinant expression vector containing the control sequence 1 was constructed using the control sequence 1 by the method of constructing the recombinant expression vector DBN100996 containing the SUM1 nucleotide sequence as described in Example 2 DBN100996N1, its vector structure is shown in Figure 3 (vector backbone: pCAMBIA2301 (available from CAMBIA); Spec: spectinomycin gene; RB: right border; prAtUbi10: Arabidopsis Ubiquitin 10 gene promoter (SEQIDNO) :4); mN1: control sequence 1 (SEQ ID NO: 9); tNos: terminator of the nopaline synthase gene (SEQ ID NO: 5); prCaMV35S: cauliflower mosaic virus 35S promoter (SEQ ID NO: 6); PAT: ammonium oxalate Phosphoacetyltransferase gene (SEQ ID NO: 7); tCaMV35S: cauliflower mosaic virus 35S terminator (SEQ
  • control sequence 1 inserted in the recombinant expression vector DBN100996N1 was the nucleotide sequence shown in SEQ ID NO: 9 in the sequence listing, that is, the control sequence 1 was correctly inserted.
  • the recombinant expression vector DBN100996N2 containing the control sequence 2 was constructed using the control sequence 2 according to the above method of constructing the recombinant expression vector DBN100996N1 containing the control sequence 1.
  • the positive clone was verified by sequencing, and the result showed that the control sequence 2 inserted in the recombinant expression vector DBN100996N2 was the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing, that is, the control sequence 2 was correctly inserted.
  • the results showed that the recombinant expression vectors DBN100996, DBN100996N1
  • 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. Seeds were germinated and plants were grown in a greenhouse under constant temperature (22 ° C) constant humidity (40-50%) long day conditions (16 hours light / 8 hours dark) with a light intensity of 120-150 [mu]mol/m2 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 medium containing spectinomycin (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 spectinomycin (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 (1 mg/) Stock solution in mL DMSO)) and 300 ⁇ L/LSilvet 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.
  • Freshly harvested (SUM1 nucleotide sequence and control sequence) T1 seeds were dried at room temperature for 7 days. The seeds were planted in a 26.5 x 51 cm germination tray, each receiving 200 mg T1 seeds (about 10,000 seeds). The seeds were previously suspended in 40 mL of 0.1% (w/v) agarose solution and stored at 4 °C for 2 days to complete the need for dormancy to ensure 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.
  • T1 plants were sprayed with a 0.2% solution of Liberty herbicide (200 gai/L glufosinate) at a spray volume of 10 mL/disc (703 L/ha) after 7 days of planting (DAP) and again at 11 DAP using a DeVilbiss compressed air nozzle. (Cotyledon stage and 2-4 leaf stage, respectively) to provide an effective amount of glufosinate of 280 gai/ha 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 for the ability of the SUM1 gene to provide sulfonylurea herbicide tolerance (22 ⁇ 5 ° C, 50 ⁇ 30% RH, 14 hours light: 10 hours dark, Minimum 500 ⁇ E/m2s1 natural + supplemental light).
  • the T1 transformants were first selected from the untransformed seed background using a glufosinate selection protocol. Approximately 40,000 T1 seeds were screened and 380 T1 positive transformants (PAT gene) were identified with a transformation efficiency of approximately 0.95%.
  • Arabidopsis thaliana T1 plants transferred to the SUM1-01 nucleotide sequence, Arabidopsis thaliana T1 plants transferred to control sequence 1, Arabidopsis thaliana T1 plants transferred to control sequence 2, and wild-type Arabidopsis plants (after sowing) The tolerance effect of the sulfonylurea herbicide was tested separately for 18 days.
  • Arabidopsis thaliana T1 plants transferred to the SUM1-01 nucleotide sequence, Arabidopsis thaliana T1 plants transferred to control sequence 1, Arabidopsis thaliana T1 plants transferred to control sequence 2, and wild-type Arabidopsis plants were used, respectively.
  • a 1x field concentration of a sulfonylurea herbicide is an effective dose to distinguish sensitive plants from plants having an average level of resistance.
  • Table 1 and Figure 4 indicate that the herbicide-tolerant protein SUM1 confers tolerance to sulfonylurea herbicides in Arabidopsis plants (the reason why individual plants are not tolerated is due to T1 plant insertion)
  • the loci are random, and thus the expression levels of the tolerance genes are different, showing differences in tolerance levels); for any sulfonylurea herbicide, compared to Arabidopsis thaliana transferred to control sequence 1.
  • Recombinant cloning vector DBN01-T and expression vector DBNBC-02 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases SpeI and KasI, respectively, and the cut SUM1-01 nucleotide sequence fragments were respectively Insertion into the expression vector DBNBC-02 between the SpeI and KasI sites, the construction of the vector by conventional enzymatic digestion method is well known to those skilled in the art, and the recombinant expression vector DBN130028 is constructed, and the construction process thereof is shown in FIG.
  • prAtUbi10 Arabidopsis Ubiquitin 10 gene promoter (SEQ ID NO: 4); SUM1-01: SUM1-01 nucleotide sequence (SEQ ID NO: 2); tNos: Terminator of the nopaline synthase gene (SEQ ID NO: 5); prBrCBP: rapeseed eukaryotic elongation factor gene 1 ⁇ (Tsf1) promoter (SEQ ID NO: 11); spAtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO: 12); EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 13); tPsE9: terminator of pea RbcS gene (SEQ ID NO: 14); LB: left border).
  • the recombinant expression vector DBN130028 was transformed into E. coli T1 competent cells by a heat shock method according to the method of 2 in the second embodiment, and 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 DBN130028 was SEQ ID NO: The nucleotide sequence shown in 2, the SUM1-01 nucleotide sequence.
  • the recombinant expression vector DBN130028N1 containing the control sequence 1 was constructed using the control sequence 1 according to the method of constructing the recombinant expression vector DBN130028 containing the SUM1 nucleotide sequence according to the first embodiment of the present invention, and the vector structure thereof is shown in Fig.
  • pCAMBIA2301 vector skeleton : pCAMBIA2301 (available by CAMBIA); Spec: spectinomycin gene; RB: right border; prAtUbi10: Arabidopsis Ubiquitin 10 gene promoter (SEQ ID NO: 4); mN1: control sequence 1 (SEQ ID NO: 9) tNos: terminator of the nopaline synthase gene (SEQ ID NO: 5); prBrCBP: rapeseed eukaryotic elongation factor gene 1 ⁇ (Tsf1) promoter (SEQ ID NO: 11); spAtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO: 12) EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 13); tPsE9: terminator of pea RbcS gene (SEQ ID NO: 14); LB: left border).
  • control sequence 1 inserted in the recombinant expression vector DBN130028N1 was the nucleotide sequence shown in SEQ ID NO: 9 in the sequence listing, that is, the control sequence 1 was correctly inserted.
  • the recombinant expression vector DBN130028N2 containing the control sequence 2 was constructed using the control sequence 2 according to the above method of constructing the recombinant expression vector DBN130028N1 containing the control sequence 1.
  • the positive clone was verified by sequencing, and the result showed that the control sequence 2 inserted in the recombinant expression vector DBN130028N2 was the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing, that is, the control sequence 2 was correctly inserted.
  • the recombinant expression vectors DBN130028, DBN130028N1 and DBN130028N2, 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 plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, heated at 37 ° C for 10 minutes; after transformation Agrobacterium tumefaciens LBA4404 was inoculated in LB tubes and incubated at a temperature of 28 ° C and 200 rpm for 2 hours, and applied to LB plates containing 50 mg/L of rifampicin and 50 mg/L of spectinomycin until long.
  • Agrobacterium LBA4404 Invitrgen, Chicago, USA, CAT: 18313-015
  • the cotyledonary node tissue of the aseptically cultured soybean variety Zhonghuang 13 was co-cultured with the Agrobacterium described in the third embodiment in accordance with the conventional Agrobacterium infection method to construct the first and second embodiments in the fifth embodiment.
  • T-DNA in recombinant expression vectors DBN130028, DBN130028N1 and DBN130028N2 including the promoter sequence of Arabidopsis Ubiquitin10 gene, SUM1-01 nucleotide sequence, control sequence 1, control sequence 2, tNos terminator, eukaryotic elongation of rapeseed
  • the factor 1 ⁇ promoter, the Arabidopsis thaliana chloroplast transit peptide, the 5-enolpyruvylshikimate-3-phosphate synthase gene, and the terminator of the pea RbcS gene were transferred into the soybean genome and transferred to SUM1- A soybean plant having a nucleotide sequence of 01, a soybean plant transferred to the control sequence 1, and a soybean plant transferred to the control sequence 2; and a wild type soybean plant 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 Trauma is performed at the cotyledonary node with the scalpel's blade back, and the wounded cotyledonary node tissue is contacted with the Agrobacterium suspension, wherein Agrobacterium can deliver the SUM1-01 nucleotide sequence to the wounded cotyledonary node tissue (Step 1 : Invasion step)
  • infecting medium MS salt 2.15 g/L, B5 vitamin, sucrose 20 g/L, glucose 10 g/ L
  • Step 2 Cotyledonary node tissue and Agrobacterium Co-cultivation for a period of time (3 days)
  • the cotyledonary node tissue is in solid medium (MS salt 4.3 g/L, B5 vitamin, sucrose 20 g/L, glucose 10 g after the infection step) /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 after the infection step
  • MES 2-morpholine ethanesulfonic acid
  • zeatin 2mg / L agar 8g / L, pH 5.6
  • Step 3 restore the medium (B5 salt 3.1g / L, B5 vitamins, 2-morpholine ethanesulfonic acid (MES) 1g / L, sucrose 30g / L, zein (ZT) 2mg / L, agar 8g / L, cephalosporin
  • MES 2-morpholine ethanesulfonic acid
  • ZT zein
  • cephalosporin There is at least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium (150 mg/L, glutamic acid 100 mg/L, aspartic acid 100 mg/L, pH 5.6), and no plant transformant is added.
  • Agent Step 3: Recovery step).
  • the cotyledonary node-regenerated tissue block is cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells.
  • cotyledonary node regeneration The tissue block 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 in the selection of a screening solid.
  • the transformed cells then regenerate the plants (step 5: regeneration step), preferably the cotyledonary node-regenerated tissue grown on the medium containing the selection agent Block in solid medium (B5 differentiation culture
  • B5 differentiation culture The nutrient and B5 rooting medium were cultured to regenerate the plants.
  • 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, auxin 1mg/L, N-(phosphine carboxymethyl
  • 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
  • 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
  • the probe fluorescent quantitative PCR method detects the EPSPS gene copy number to determine the copy number of the SUM1 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 EPSPS gene copy number is as follows:
  • Step 11 The soybean plants transformed with the SUM1-01 nucleotide sequence, the soybean plants transferred to the control sequence 1, the soybean plants transferred to the control sequence 2, and the leaves of the wild-type soybean plants were each 100 mg, respectively, in a mortar. The mixture was homogenized with liquid nitrogen, and each sample was taken in 3 replicates;
  • Step 12 using Qiagen's DNeasyPlantMiniKit to extract the genomic DNA of the above sample, the specific method refers to the product specification;
  • Step 13 Determine the genomic DNA concentration of the sample by using NanoDrop2000 (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 ATGCAGGCGATGGGCGCCCGCATCCGTA as shown in SEQ ID NO: 17 in the Sequence Listing;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mixture contained 45 ⁇ L of each primer at a concentration of 1 mM, 50 ⁇ L of a 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 SUM1-01 nucleotide sequence, soybean plants transferred to the control sequence 1, soybean plants transferred to the control sequence 2, and wild-type soybean plants (seedling stage) were respectively weeded with the sulfonylurea herbicide Agent tolerance effect detection.
  • Soybean plants transformed with the SUM1-01 nucleotide sequence soybean plants transferred to the control sequence 1, soybean plants transferred to the control sequence 2, and wild-type soybean plants, respectively, were treated with tribenuron-methyl (72 gai/ha, 4 times). Daejeon concentration), sulfometuron (120gai/ha, 4 times field concentration), chloropyrazine (34gai/ha, 1 times field concentration), pyrazosulfuron (25gai/ha, 1 times field concentration), Thiosulfuron-methyl (120gai/ha, 4x field concentration), bensulfuron-methyl (120gai/ha, 4x field concentration), metsulfuron-methyl (30gai/ha, 4x field concentration), ethamsulfuron (60gai/) Ha, 4 times field concentration), chlorsulfuron (60 gai/ha, 4 times field concentration) and blank solvent (water) spray.
  • Daejeon concentration sulfometuron (120gai
  • the degree of damage of herbicides per plant was calculated according to the degree of leaf curl and the degree of growth point damage.
  • the leaves are flat as untreated plants, and the growth point is intact at 0%; the veins are partially browned and the new leaves are deformed, and the plant growth is slower than 50%; the veins are purple to the whole plant and the growth point becomes brown and dry.
  • Two soybean lines (S1 and S2) transferred to the SUM1-01 nucleotide sequence, and two soybean lines (S3 and S4) transferred to the control sequence 1 were transferred to the soybean plant of the control sequence 2.
  • a total of 2 strains (S5 and S6) and 1 strain of wild-type soybean plants (CK1) were selected; 10-15 strains were selected from each strain for testing. The results are shown in Table 2.
  • SUM1-02 is the SUM1-02 nucleotide sequence (SEQ ID NO: 3); MCS is the multiple cloning site).
  • the recombinant cloning vector DBN01-T was transformed into E. coli T1 competent cells by a heat shock method according to the method of the first embodiment, and 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 of the recombinant cloning vector DBN02-T between SpeI and KasI was in the sequence listing.
  • Recombinant cloning vector DBN02-T and expression vector DBNBC-03 were digested with restriction endonucleases SpeI and KasI, respectively, and the cut SUM1-02 nucleotide sequence fragment was inserted. Between the SpeI and KasI sites of the expression vector DBNBC-03, the construction of the vector by conventional enzymatic cleavage method is well known to those skilled in the art, and the recombinant expression vector DBN130035 is constructed, and the construction process thereof is shown in Fig.
  • the recombinant expression vector DBN130035 was transformed into E. coli T1 competent cells by a heat shock method according to the method of 2 in the second embodiment, and 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 DBN130035 was SEQ ID NO: The nucleotide sequence shown in 3, the SUM1-02 nucleotide sequence.
  • the recombinant expression vector DBN130035N1 containing the control sequence 1 was constructed by using the control sequence 1 according to the method of constructing the recombinant expression vector DBN130035 containing the SUM1 nucleotide sequence as described in Example 2, and the vector structure thereof is shown in Fig.
  • vector skeleton pCAMBIA2301 (available by the CAMBIA facility); Spec: spectinomycin gene; RB: right border; prUbi: maize Ubiquitin (ubiquitin) 1 gene promoter (SEQ ID NO: 18); mN1: control sequence 1 (SEQ ID NO: 9); tNos: terminator of the nopaline synthase gene (SEQ ID NO: 5); PMI: phosphomannose isomerase gene (SEQ ID NO: 19); LB: left border).
  • control sequence 1 inserted in the recombinant expression vector DBN130035N1 was the nucleotide sequence shown by SEQ ID NO: 9 in the sequence listing, that is, the control sequence 1 was correctly inserted.
  • the recombinant expression vector DBN130035N2 containing the control sequence 2 was constructed using the control sequence 2 according to the above method of constructing the recombinant expression vector DBN130035N1 containing the control sequence 1.
  • the positive clone was verified by sequencing, and the result showed that the control sequence 2 inserted in the recombinant expression vector DBN130035N2 was the nucleotide sequence shown by SEQ ID NO: 10 in the sequence listing, that is, the control sequence 2 was correctly inserted.
  • the recombinant expression vectors DBN130035, DBN130035N1 and DBN130035N2, 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 plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37 ° C warm water bath for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated in LB tube and incubated at a temperature of 28 ° C, 200 rpm for 2 hours, applied to On LB plates containing 50 mg/L of rifampicin and 50 mg/L of spectinomycin until positive monoclonals were grown, monoclonal cultures were picked and plasmids were extracted and digested with restriction endonucleases. The results showed that the recombinant expression vectors DBN130035, DBN13
  • T-DNA including promoter sequence of maize Ubiquitin1 gene, SUM1-02 nucleotide sequence, control sequence 1, control sequence 2, PMI gene and tNos terminator sequence
  • DBN130035, DBN130035N1 and DBN130035N2 Into the maize genome, a maize plant transformed with the SUM1-02 nucleotide sequence, a maize plant transferred to the control sequence 1 and a maize plant transferred to the control sequence 2 were obtained; and the wild type maize plant was used as a control.
  • immature immature embryos are isolated from maize, and the immature embryos are contacted with an Agrobacterium suspension, wherein Agrobacterium can transfer the SUM1-02 nucleotide sequence to one of the young embryos.
  • At least one cell 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.
  • the maize plants transformed with the SUM1-02 nucleotide sequence, the maize plants transferred to the control sequence 1 and the maize plants transferred to the control sequence 2 were detected and analyzed.
  • the copy number of the PMI gene was detected by Taqman probe real-time PCR to determine the copy number of the SUM1 gene.
  • the wild type corn 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.
  • Primer 3 GCTGTAAGAGCTTACTGAAAAAATTAACA as shown in SEQ ID NO: 20 in the sequence listing;
  • Primer 4 CGATCTGCAGGTCGACGG as shown in SEQ ID NO: 21 in the Sequence Listing;
  • Probe 2 TCTCTTGCTAAGCTGGGAGCTCGATCC is shown as SEQ ID NO: 22 in the Sequence Listing.
  • Daejeon concentration sulfometuron (120
  • the herbicide-tolerant protein SUM1 of the present invention can exhibit high tolerance to a sulfonylurea herbicide and contains a SUM1-01 core encoding the herbicide-tolerant protein SUM1.
  • the nucleotide sequence and the SUM1-02 nucleotide sequence are particularly suitable for expression in plants due to the use of plant-preferred codons, and Arabidopsis plants transferred to the SUM1-01 nucleotide sequence are transferred to the SUM1-01 nucleus.
  • Soybean plants with a nucleotide sequence and a maize plant transformed with the SUM1-02 nucleotide sequence are highly resistant to sulfonylurea herbicides and can tolerate a 4-fold field concentration, and thus have broad application prospects on plants.

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Abstract

一种除草剂耐受性蛋白质SUM1、其编码基因及用途，所述除草剂耐受性蛋白质包括：(a)具有SEQ ID NO:1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噻吩磺隆水解酶活性的由(a)衍生的蛋白质。所述除草剂耐受性蛋白质可以对多种磺酰脲类除草剂表现出较高的抗性，可以耐受4倍大田浓度，因此在植物上应用前景广阔。

Description

除草剂耐受性蛋白质、其编码基因及用途
相关申请的交叉引用
本申请要求2016年3月22日提交的第CN105802933A号中国发明专利申请的优先权，所述申请以引用的方式整体并入本文。
技术领域
本发明涉及一种除草剂耐受性蛋白质、其编码基因及用途，特别是涉及一种对磺酰脲类除草剂具有耐受性的蛋白质、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，如磺酰脲类除草剂。磺酰脲类除草剂已经成为继有机磷、乙酰胺类除草剂后的第三大除草剂，全球年销售额达到30亿美元以上，我国磺酰脲类除草剂每年的应用面积已超过200万公顷，并仍呈扩大的趋势。
随着草甘膦抗性杂草的出现和磺酰脲类除草剂的扩大应用，需要对磺酰脲类除草剂敏感的目的植物中输入磺酰脲类除草剂耐受性。目前未发现除草剂耐受性蛋白质SUM1对磺酰脲类除草剂具有耐受性的报道。
发明内容
本发明的目的是提供一种除草剂耐受性蛋白质、其编码基因及用途，所述除草剂耐受性蛋白质SUM1在植物中对磺酰脲类除草剂具有较高的耐受性。
为实现上述目的，本发明采用了如下的技术方案：
一方面，本发明提供了一种除草剂耐受性蛋白质，包括：
(a)具有SEQIDNO:1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噻吩磺隆水解酶活性的由(a)衍生的蛋白质。
另一方面，本发明还提供了一种除草剂耐受性基因，包括：
(a)编码前述除草剂耐受性蛋白质的核苷酸序列；或
(b)具有SEQIDNO:2所示的核苷酸序列；或
(c)具有SEQIDNO:3所示的核苷酸序列。
又一方面，本发明提供了一种表达盒，包含在有效连接的调控序列调控下的前述的除草剂耐受性基因。
再一方面，本发明提供了一种包含前述的除草剂耐受性基因或前述的表达盒的重组载体。
另一方面，本发明还提供了一种产生除草剂耐受性蛋白质的方法，包括：
获得包含前述的除草剂耐受性基因或前述的表达盒的转基因宿主生物的细胞；
在允许产生除草剂耐受性蛋白质的条件下培养所述转基因宿主生物的细胞；
回收所述除草剂耐受性蛋白质；
优选地，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。
又一方面，本发明提供了一种增加耐受除草剂范围的方法，包括：将前述的除草剂耐受性蛋白质或前述的表达盒编码的除草剂耐受性蛋白质在植物中与至少一种不同于前述的除草剂耐受性蛋白质或前述的表达盒编码的除草剂耐受性蛋白质的第二种蛋白质一起表达；
优选地，所述第二种蛋白质为5-烯醇丙酮酰莽草酸-3-磷酸合酶、草甘膦氧化还原酶、草甘膦-N-乙酰转移酶、草甘膦脱羧酶、草铵膦乙酰转移酶、α酮戊二酸依赖性双加氧酶、麦草畏单加氧酶、4-羟苯基丙酮酸双加氧酶、乙酰乳酸合酶、细胞色素类蛋白质和/或原卟啉原氧化酶。
再一方面，本发明提供了一种选择转化的植物细胞的方法，包括：用前述的除草剂耐受性基因或前述的表达盒转化多个植物细胞，并在允许表达所述除草剂耐受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，所述除草剂为磺酰脲类除草剂；
优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
另一方面，本发明还提供了一种控制杂草的方法，包括：对种植目的植物的大田施用有效剂量的磺酰脲类除草剂，所述植物包含前述的除草剂耐受性基因或前述的表达盒；
优选地，所述植物为单子叶植物或双子叶植物；
更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
又一方面，本发明提供了一种用于保护植物免受由磺酰脲类除草剂引起的损伤的方法，包括：将前述的除草剂耐受性基因或前述的表达盒或前述的重组载体导入植物，使导入后的植物产生足够保护其免受磺酰脲类除草剂损害量的除草剂耐受性蛋白质；
优选地，所述植物为单子叶植物或双子叶植物；
更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
再一方面，本发明提供了一种控制草甘膦耐受性植物的大田中草甘膦抗性杂草的方法，包括：对种植草甘膦耐受性植物的大田施用有效剂量的磺酰脲类除草剂，所述草甘膦耐受性植物包含前述的除草剂耐受性基因或前述的表达盒；
优选地，所述植物为单子叶植物或双子叶植物；
更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
另一方面，本发明还提供了一种赋予植物磺酰脲类除草剂耐受性的方法，包括：将前述的除草剂耐受性基因或前述的表达盒或前述的重组载体导入植物；
优选地，所述植物为单子叶植物或双子叶植物；
更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
又一方面，本发明提供了一种产生耐受磺酰脲类除草剂的植物的方法，包括向植物的基因组中引入前述的除草剂耐受性基因或前述的表达盒或前述的重组载体；
优选地，所述植物为单子叶植物或双子叶植物；
更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
再一方面，本发明提供了一种培养耐受磺酰脲类除草剂的植物的方法，包括：种植至少一个植物繁殖体，所述植物繁殖体的基因组中包括前述的除草剂耐受性基因或前述的表达盒；
使所述植物繁殖体长成植株；
将有效剂量的磺酰脲类除草剂施加到至少包含所述植株的植物生长环境中，收获与其他不具有前述的除草剂耐受性基因或前述的表达盒的植株相比具有减弱的植物损伤和/或具有增加的植物产量的植株；
优选地，所述植物为单子叶植物或双子叶植物；
更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
另一方面，本发明还提供了一种控制杂草生长的种植系统，包括磺酰脲类除草剂和存在至少一种目的植物的植物生长环境，所述植物包含前述的除草剂耐受性基因或前述的表达盒；
优选地，所述植物为单子叶植物或双子叶植物；
更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
又一方面，本发明提供了一种控制草甘膦耐受性植物的大田中草甘膦抗性杂草的种植系统，包括磺酰脲类除草剂、草甘膦除草剂和种植至少一种目的植物的大田，所述草甘膦耐受性植物包含前述的除草剂耐受性基因或前述的表达盒；
优选地，所述植物为单子叶植物或双子叶植物；
更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
再一方面，本发明提供了一种除草剂耐受性蛋白质降解磺酰脲类除草剂的用途，所述除草剂耐受性蛋白质包括：
(a)具有SEQIDNO:1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噻吩磺隆水解酶活性的由(a)衍生的蛋白质；
优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
将所述的除草剂耐受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。
本发明所述的磺酰脲类除草剂耐受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。磺酰脲类除草剂是广谱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。磺酰脲类除草剂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。磺酰脲类除草剂耐性性状的另一用途是它可用于预防磺酰脲类除草剂漂移、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。在植物中使用SUM1基因可以提供对更广谱的磺酰脲类除草剂的防护，从而提高灵活性和可控制的杂草谱，提供对全范围市售磺酰脲类除草剂的漂移或其它远距离磺酰脲类除草剂损伤的防护。
现已鉴定了SUM1基因在遗传改造用于植物表达后具有允许在植物中使用磺酰脲类除草剂的特性，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，SUM1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磺酰脲类除草剂的防护。现在可以连续或罐混地与一种、两种或若干磺酰脲类除草剂的组合处理仅含SUM1基因的植物。用于控制广谱双子叶杂草的每种磺酰脲类除草剂的用量范围从7.5至150g ai/ha，更通常从20至50g ai/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SUM1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磺酰脲类除草剂而实现对草甘膦耐性作物中草甘膦抗性杂草物种(被一种或多种磺酰脲类除草剂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/SUM1基因叠加的草甘膦应用范围可以从200至1600gae/ha；磺酰脲类除草剂(一种或多种)可按照从7.5-150g ai/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：具体可以为5- 烯醇丙酮酰莽草酸-3-磷酸合酶(EPSPS)、草甘膦氧化还原酶(GOX)、草甘膦-N-乙酰转移酶(GAT)、草甘膦脱羧酶、草铵膦乙酰转移酶(PAT)、α酮戊二酸依赖性双加氧酶(AAD)、麦草畏单加氧酶(DMO)、4-羟苯基丙酮酸双加氧酶(HPPD)、乙酰乳酸合酶(ALS)、细胞色素类蛋白质(P450)和/或原卟啉原氧化酶(Protox)。
此外，可以将SUM1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SUM1基因能降解磺酰脲类除草剂，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。SUM1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、其它ALS抑制剂(如咪唑啉酮类、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，SUM1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。
本发明的除草剂耐受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受性)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，所述双子叶植物包括但不限于苜蓿、菜豆、花椰菜、甘蓝、胡萝卜、芹菜、棉花、黄瓜、茄子、莴苣、甜瓜、豌豆、胡椒、西葫芦、萝卜、油菜、菠菜、大豆、南瓜、番茄、拟南芥或西瓜；优选地，所述双子叶植物是指大豆、拟南芥、烟草、棉花或油菜。所述单子叶植物包括但不限于玉米、水稻、高粱、小麦、大麦、黑麦、粟、甘蔗、燕麦或草坪草；优选地，所述单子叶植物是指玉米、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦。本发明SUM1基因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。
本发明中所述种植系统是指植物、其显示的任一种除草剂耐受性和/或在植物发育的不同阶段可用的除草剂处理的组合，产生高产和/或减弱损伤的植物。
本发明中，所述杂草是指在植物生长环境中与耕种的目的植物竞争的植物。
本发明术语“控制”和/或“防治”是指至少将有效剂量的磺酰脲类除草剂直接施用(例如通过喷雾)到植物生长环境中，使杂草发育最小化和/或停止生长。同时，耕种的目的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；优选地，与非转基因的野生型植株相比具有减弱的植物损伤和/或具有增加的植物产量。所述具有减弱的植物损伤，具体表现包括但不限于改善的茎秆抗性、和/或提高的籽粒重量等。所述除草剂耐受性 蛋白质SUM1对杂草的“控制”和/或“防治”作用是可以独立存在的，不因其它可“控制”和/或“防治”杂草的物质的存在而减弱和/或消失。具体地，转基因植物(含有SUM1基因)的任何组织同时和/或不同步地，存在和/或产生，所述除草剂耐受性蛋白质SUM1和/或可控制杂草的另一种物质，则所述另一种物质的存在既不影响所述除草剂耐受性蛋白质SUM1对杂草的“控制”和/或“防治”作用，也不能导致所述“控制”和/或“防治”作用完全和/或部分由所述另一种物质实现，而与所述除草剂耐受性蛋白质SUM1无关。
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。
本发明中所述的“植物繁殖体”包括但不限于植物有性繁殖体和植物无性繁殖体。所述植物有性繁殖体包括但不限于植物种子；所述植物无性繁殖体是指植物体的营养器官或某种特殊组织，其可以在离体条件下产生新植株；所述营养器官或某种特殊组织包括但不限于根、茎和叶，例如：以根为无性繁殖体的植物包括草莓和甘薯等；以茎为无性繁殖体的植物包括甘蔗和马铃薯(块茎)等；以叶为无性繁殖体的植物包括芦荟和秋海棠等。
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”或“耐受性”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。
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耐受性基因杂交。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耐受性基因的存在。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℃下与本发明SUM1基因发生特异性杂交，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SUM1基因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磺酰脲类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磺酰脲类除草剂存在下生长。
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耐受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)，前述序列的长度可存在变化，但长度足以确保(编码)蛋白质为除草剂耐受性蛋白质。
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 影响除草剂耐受性活性的序列，亦包括保留除草剂耐受性活性的片段。
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(AcademicPress)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，Science244：1081-1085)。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deVos等，1992，Science255：306-312；Smith等，1992，J.Mol.Biol224:899-904；Wlodaver等，1992，FEBSLetters309：59-64)。
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SUM1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(AMVRNA4)；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(Agrobacteriumtumefaciens)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ssRUBISCOE9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，所述除草剂耐受性蛋白质为SUM1氨基酸序列，如序列表中SEQIDNO:1所示。所述除草剂耐受性基因为SUM1核苷酸序列，如序列 表中SEQIDNO:2和SEQIDNO:3所示。所述除草剂耐受性基因可用于植物，除了包含由SUM1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。
本发明中除草剂耐受性蛋白质SUM1对大多数磺酰脲类除草剂具有耐性。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SUM1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。
植物材料中除草剂耐受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耐受性蛋白质的mRNA进行定量，或直接特异性检测产生的除草剂耐受性蛋白质的量。
本发明提供了一种除草剂耐受性蛋白质、其编码基因及用途，具有以下优点：
1、对除草剂耐受性强。本发明除草剂耐受性蛋白质SUM1对磺酰脲类除草剂的耐受性强，可以耐受4倍大田浓度。
2、对除草剂耐受性广。本发明除草剂耐受性蛋白质SUM1可以对多种磺酰脲类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。
附图的简要说明
图1为本发明除草剂耐受性蛋白质、其编码基因及用途的含有SUM1核苷酸序列的重组克隆载体DBN01-T构建流程图；
图2为本发明除草剂耐受性蛋白质、其编码基因及用途的含有SUM1核苷酸序列的重组表达载体DBN100996构建流程图；
图3为本发明除草剂耐受性蛋白质、其编码基因及用途的含有对照序列1的重组表达载体DBN100996N1结构示意图；
图4-1和图4-2为本发明除草剂耐受性蛋白质、其编码基因及用途的转基因拟南芥T1植株对磺酰脲类除草剂耐受性效果图；
图5为本发明除草剂耐受性蛋白质、其编码基因及用途的含有SUM1核苷酸序列的重组表达载体DBN130028构建流程图；
图6为本发明除草剂耐受性蛋白质、其编码基因及用途的含有对照序列1的重组表达载体DBN130028N1结构示意图；
图7为本发明除草剂耐受性蛋白质、其编码基因及用途的含有SUM1核苷酸序列的重组克隆载体DBN02-T构建流程图；
图8为本发明除草剂耐受性蛋白质、其编码基因及用途的含有SUM1核苷酸序列的重组表达载体DBN130035构建流程图；
图9为本发明除草剂耐受性蛋白质、其编码基因及用途的含有对照序列1的重组表达载体DBN130035N1结构示意图。
实施发明的最佳方式
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实 施例仅仅是用于更详细具体地说明之用，而不应理解为用于以任何形式限制本发明。
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚，在上下文中，如果未特别说明，本发明所用材料和操作方法是本领域公知的。
下面通过具体实施例进一步说明本发明除草剂耐受性蛋白质、其编码基因及用途的技术方案。
第一实施例：SUM1基因序列的获得和合成
1、获得SUM1基因序列
除草剂耐受性蛋白质SUM1的氨基酸序列(350个氨基酸)，如序列表中SEQIDNO:1所示；依据大豆偏好性密码子获得编码相应于所述除草剂耐受性蛋白质SUM1的氨基酸序列的SUM1-01核苷酸序列(1053个核苷酸)，如序列表中SEQIDNO:2所示，依据玉米偏好性密码子获得编码相应于所述除草剂耐受性蛋白质SUM1的氨基酸序列的SUM1-02核苷酸序列(1053个核苷酸)，如序列表中SEQIDNO:3所示。
2、合成上述核苷酸序列
所述SUM1-01核苷酸序列(如序列表中SEQIDNO:2所示)和所述SUM1-02核苷酸序列(如序列表中SEQIDNO:3所示)由南京金斯瑞生物科技有限公司合成；合成的所述SUM1-01核苷酸序列(SEQIDNO:2)的5’端还连接有SpeI酶切位点，所述SUM1-01核苷酸序列(SEQIDNO:2)的3’端还连接有KasI酶切位点；合成的所述SUM1-02核苷酸序列(SEQIDNO:3)的5’端还连接有SpeI酶切位点，所述SUM1-02核苷酸序列(SEQIDNO:3)的3’端还连接有KasI酶切位点。
第二实施例：拟南芥重组表达载体的构建
1、构建含有SUM1核苷酸序列的拟南芥和大豆重组克隆载体
将合成的SUM1-1-01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；SUM1-01为SUM1-01核苷酸序列(SEQIDNO:2)；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，NaCl10g/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(25mMTris-HCl，10mMEDTA(乙二胺四乙酸)，50mM葡萄糖，pH8.0)悬浮；加入200μL新配制的溶液II(0.2MNaOH，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(10mMTris-HCl，1mMEDTA，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SUM1-01核苷酸序列为序列表中SEQIDNO:2所示的核苷酸序列，即SUM1-01核苷酸序列正确插入。
2、构建含有SUM1核苷酸序列的拟南芥重组表达载体
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SUM1-01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DBN100996，其构建流程如图2所示(Spec：壮观霉素基因；RB：右边界；prAtUbi10：拟南芥Ubiquitin(泛素)10基因启动子(SEQIDNO:4)；SUM1-01：SUM1-01核苷酸序列(SEQIDNO:2)；tNos：胭脂碱合成酶基因的终止子(SEQIDNO:5)；prCaMV35S：花椰菜花叶病毒35S启动子(SEQIDNO:6)；PAT：草铵膦乙酰转移酶基因(SEQIDNO:7)；tCaMV35S：花椰菜花叶病毒35S终止子(SEQIDNO:8)；LB：左边界)。
将重组表达载体DBN100996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DBN100996)，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壮观霉素(Spectinomycin)的LB固体平板(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，NaCl10g/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DBN100996在SpeI和KasI位点间的核苷酸序列为序列表中SEQIDNO:2所示核苷酸序列，即SUM1-01核苷酸序列。
3、构建含有对照序列的拟南芥重组表达载体
按照本实施例中1所述的构建含有SUM1核苷酸序列的重组克隆载体DBN01-T的方法，利用对照序列1(SEQIDNO:9)和对照序列2(SEQIDNO:10)构建含有对照序列1的重组克隆载体DBN01R1-T和含有对照序列2的重组克隆载体DBN01R2-T。对阳性克隆进行测序验证，结果表明重组克隆载体DBN01R1-T中插入的对照序列1为序列表中SEQIDNO:9所示的核苷酸序列，重组克隆载体DBN01R2-T中插入的对照序列2为序列表中SEQIDNO:10所示的核苷酸序列，即对照序列正确插入。
按照本实施例中2所述的构建含有SUM1核苷酸序列的重组表达载体DBN100996的方法，利用对照序列1构建含有对照序列1的重组表达载体 DBN100996N1，其载体结构如图3所示(载体骨架：pCAMBIA2301(CAMBIA机构可以提供)；Spec：壮观霉素基因；RB：右边界；prAtUbi10：拟南芥Ubiquitin(泛素)10基因启动子(SEQIDNO:4)；mN1：对照序列1(SEQIDNO:9)；tNos：胭脂碱合成酶基因的终止子(SEQIDNO:5)；prCaMV35S：花椰菜花叶病毒35S启动子(SEQIDNO:6)；PAT：草铵膦乙酰转移酶基因(SEQIDNO:7)；tCaMV35S：花椰菜花叶病毒35S终止子(SEQIDNO:8)；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DBN100996N1中插入的对照序列1为序列表中SEQIDNO:9所示的核苷酸序列，即对照序列1正确插入。
按照上述构建含有对照序列1的重组表达载体DBN100996N1的方法，利用对照序列2构建含有对照序列2的重组表达载体DBN100996N2。对阳性克隆进行测序验证，结果表明重组表达载体DBN100996N2中插入的对照序列2为序列表中SEQIDNO:10所示的核苷酸序列，即对照序列2正确插入。
第三实施例：转入SUM1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DBN100996、DBN100996N1和DBN100996N2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的壮观霉素的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，用限制性内切酶进行酶切验证，结果表明重组表达载体DBN100996、DBN100996N1和DBN100996N2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含壮观霉素(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含壮观霉素(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/mLDMSO中的原液))和300μL/LSilvetL-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(SUM1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40mL0.1％(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(200gai/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280gai/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SUM1基因提供磺酰脲类除草剂耐受性的能力之前至少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耐受性效果检测
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40000个T1种子中并鉴定了380株T1代阳性转化子(PAT基因)，约0.95％的转化效率。将转入SUM1-01核苷酸序列的拟南芥T1植株、转入对照序列1的拟南芥T1植株、转入对照序列2的拟南芥T1植株和野生型拟南芥植株(播种后18天)分别对磺酰脲类除草剂进行耐受性效果检测。
分别将转入SUM1-01核苷酸序列的拟南芥T1植株、转入对照序列1的拟南芥T1植株、转入对照序列2的拟南芥T1植株和野生型拟南芥植株分别用苯磺隆(18gai/ha，1倍大田浓度)、甲嘧磺隆(30gai/ha，1倍大田浓度)、氯吡嘧磺隆(34gai/ha，1倍大田浓度)、吡嘧磺隆(25gai/ha，1倍大田浓度)、噻吩磺隆(30gai/ha，1倍大田浓度)、苄嘧磺隆(30gai/ha，1倍大田浓度)、甲磺隆(7.5gai/ha，1倍大田浓度)、胺苯磺隆(15gai/ha，1倍大田浓度)、氯嘧磺隆(15gai/ha，1倍大田浓度)和空白溶剂(水)喷洒。喷施14天后统计植株抗性情况：14天后生长状况和空白溶剂(水)一致的划为高抗植株，14天后抽苔高度为低于1/2空白溶剂(水)抽苔高度的划为中抗植株，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植株对磺酰脲类除草剂耐受性实验结果
Figure PCTCN2016109176-appb-000001
Figure PCTCN2016109176-appb-000002
对于拟南芥，1倍大田浓度的磺酰脲类除草剂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：所述除草剂耐受性蛋白质SUM1赋予个体拟南芥植物磺酰脲类除草剂耐受性(有个别植株不具有耐受性的原因是由于T1代植物插入位点是随机的，因而耐受性基因的表达水平有差异，表现出耐受性水平的差异)；对于任意一种磺酰脲类除草剂，相比于转入对照序列1的拟南芥T1植株和转入对照序列2的拟南 芥T1植株，转入SUM1-01核苷酸序列的拟南芥T1植株对磺酰脲类除草剂均具有显著增高的耐受性；而野生型拟南芥植株则不具有对磺酰脲类除草剂的耐受性。
第五实施例：大豆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SUM1核苷酸序列的大豆重组表达载体
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SUM1-01核苷酸序列片段分别插到表达载体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，构建成重组表达载体DBN130028，其构建流程如图5所示(Spec：壮观霉素基因；RB：右边界；prAtUbi10：拟南芥Ubiquitin(泛素)10基因启动子(SEQIDNO:4)；SUM1-01：SUM1-01核苷酸序列(SEQIDNO:2)；tNos：胭脂碱合成酶基因的终止子(SEQIDNO:5)；prBrCBP：油菜真核延长因子基因1α(Tsf1)启动子(SEQIDNO:11)；spAtCTP2：拟南芥叶绿体转运肽(SEQIDNO:12)；EPSPS：5-烯醇丙酮酸莽草酸-3-磷酸合酶基因(SEQIDNO:13)；tPsE9：豌豆RbcS基因的终止子(SEQIDNO:14)；LB：左边界)。
按照第二实施例中2的方法将重组表达载体DBN130028用热激方法转化大肠杆菌T1感受态细胞，并碱法提取其质粒。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DBN130028在SpeI和KasI位点间的核苷酸序列为序列表中SEQIDNO:2所示核苷酸序列，即SUM1-01核苷酸序列。
2、构建含有对照序列的大豆重组表达载体
按照本实施例中1所述的构建含有SUM1核苷酸序列的重组表达载体DBN130028的方法，利用对照序列1构建含有对照序列1的重组表达载体DBN130028N1，其载体结构如图6所示(载体骨架：pCAMBIA2301(CAMBIA机构可以提供)；Spec：壮观霉素基因；RB：右边界；prAtUbi10：拟南芥Ubiquitin(泛素)10基因启动子(SEQIDNO:4)；mN1：对照序列1(SEQIDNO:9)；tNos：胭脂碱合成酶基因的终止子(SEQIDNO:5)；prBrCBP：油菜真核延长因子基因1α(Tsf1)启动子(SEQIDNO:11)；spAtCTP2：拟南芥叶绿体转运肽(SEQIDNO:12)；EPSPS：5-烯醇丙酮酸莽草酸-3-磷酸合酶基因(SEQIDNO:13)；tPsE9：豌豆RbcS基因的终止子(SEQIDNO:14)；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DBN130028N1中插入的对照序列1为序列表中SEQIDNO:9所示的核苷酸序列，即对照序列1正确插入。
按照上述构建含有对照序列1的重组表达载体DBN130028N1的方法，利用对照序列2构建含有对照序列2的重组表达载体DBN130028N2。对阳性克隆进行测序验证，结果表明重组表达载体DBN130028N2中插入的对照序列2为序列表中SEQIDNO:10所示的核苷酸序列，即对照序列2正确插入。
3、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DBN130028、DBN130028N1和DBN130028N2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的壮观霉素的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，用限制性内切酶进行酶切验证，结果表明重组表达载体DBN130028、DBN130028N1和DBN130028N2结构完全正确。
第六实施例：转基因大豆植株的获得和验证
1、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的子叶节组织与第五实施例中3所述的农杆菌共培养，以将第五实施例中1和2构建的重组表达载体DBN130028、DBN130028N1和DBN130028N2中的T-DNA(包括拟南芥Ubiquitin10基因的启动子序列、SUM1-01核苷酸序列、对照序列1、对照序列2、tNos终止子、油菜真核延长因子基因1α启动子、拟南芥叶绿体转运肽、5-烯醇丙酮酸莽草酸-3-磷酸合酶基因、豌豆RbcS基因的终止子)转入到大豆染色体组中，获得了转入SUM1-01核苷酸序列的大豆植株、转入对照序列1的大豆植株和转入对照序列2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SUM1-01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(草甘膦)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，N-(膦羧甲基)甘氨酸0.25mol/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、N-(膦羧甲基)甘氨酸0.25mol/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。
2、用TaqMan验证转基因大豆植株
分别取转入SUM1-01核苷酸序列的大豆植株、转入对照序列1的大豆植株和转入对照序列2的大豆植株的叶片约100mg作为样品，用Qiagen的DNeasyPlantMaxiKit提取其基因组DNA，通过Taqman探针荧光定量PCR方法检测EPSPS基因拷贝数以确定SUM1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EPSPS基因拷贝数的具体方法如下：
步骤11、分别取转入SUM1-01核苷酸序列的大豆植株、转入对照序列1的大豆植株、转入对照序列2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DNeasyPlantMiniKit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NanoDrop2000(ThermoScientific)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EPSPS基因序列：
引物1：CTGGAAGGCGAGGACGTCATCAATA如序列表中SEQIDNO:15所示；
引物2：TGGCGGCATTGCCGAAATCGAG如序列表中SEQIDNO:16所示；
探针1：ATGCAGGCGATGGGCGCCCGCATCCGTA如序列表中SEQIDNO:17所示；
PCR反应体系为：
Figure PCTCN2016109176-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860μL1×TE缓冲液，并且在4℃，贮藏在琥珀试管中。
PCR反应条件为：
Figure PCTCN2016109176-appb-000004
利用SDS2.3软件(AppliedBiosystems)分析数据。
通过分析EPSPS基因拷贝数的实验结果，进而证实SUM1-01核苷酸序列、对照序列1和对照序列2均己整合到所检测的大豆植株的染色体组中，而且转入SUM1-01核苷酸序列的大豆植株、转入对照序列1的大豆植株和转入对照序列2的大豆植株均获得了单拷贝的转基因大豆植株。
第七实施例、转基因大豆植株的除草剂耐受性效果检测
将转入SUM1-01核苷酸序列的大豆植株、转入对照序列1的大豆植株、转入对照序列2的大豆植株和野生型大豆植株(幼苗期)分别对磺酰脲类除草剂进行除草剂耐受性效果检测。
分别取转入SUM1-01核苷酸序列的大豆植株、转入对照序列1的大豆植株、转入对照序列2的大豆植株和野生型大豆植株，分别用苯磺隆(72gai/ha，4倍大田浓度)、甲嘧磺隆(120gai/ha，4倍大田浓度)、氯吡嘧磺隆(34gai/ha，1倍大田浓度)、吡嘧磺隆(25gai/ha，1倍大田浓度)、噻吩磺隆(120gai/ha，4倍大田浓度)、苄嘧磺隆(120gai/ha，4倍大田浓度)、甲磺隆(30gai/ha，4倍大田浓度)、胺苯磺隆(60gai/ha，4倍大田浓度)、氯嘧磺隆(60gai/ha，4倍大田浓度)和空白溶剂(水)喷洒。分别在喷施后3天(3DAT)、7天(7DAT)、14天(14DAT)及21天(21DAT)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：以叶片平整如未处理植株、生长点完好无损为0％；叶脉局部变褐且新叶畸形、植株生长较慢为50％；叶脉发紫至整株死亡且生长点变褐干枯为100％。转入SUM1-01核苷酸序列的大豆植株共2个株系(S1和S2)，转入对照序列1的大豆植株共2个株系(S3和S4)，转入对照序列2的大豆植株共2个株系(S5和S6)，野生型大豆植株(CK1)共1个株系；从每个株系选10-15株进行测试。结果如表2所示。
表2、转基因大豆T1植株除草剂耐受性实验结果
Figure PCTCN2016109176-appb-000005
Figure PCTCN2016109176-appb-000006
Figure PCTCN2016109176-appb-000007
Figure PCTCN2016109176-appb-000008
对于大豆，4倍大田浓度的多数磺酰脲类除草剂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的结果表明：所述除草剂耐受性蛋白质SUM1赋予转基因大豆植物对磺酰脲类除草剂的耐受性；对于任意一种磺酰脲类除草剂，相比于转入对照序列1的大豆植株和转入对照序列2的大豆植株，转入SUM1-01核苷酸序列的大豆植株对磺酰脲类除草剂均具有显著增高的耐受性；而野生型大豆植株则对多数磺酰脲类除草剂不具耐受性。
第八实施例：玉米重组表达载体的构建
1、构建含有SUM1核苷酸序列的玉米重组克隆载体
将合成的SUM1-02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DBN02-T，其构建流程如图7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；SUM1-02为SUM1-02核苷酸序列(SEQIDNO:3)；MCS为多克隆位点)。
按照第二实施例中1的方法将重组克隆载体DBN01-T用热激方法转化大肠杆菌T1感受态细胞，并碱法提取其质粒。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，结果表明重组克隆载体DBN02-T在SpeI和KasI位点间的核苷酸序列为序列表中SEQIDNO:3所示核苷酸序列，即SUM1-02核苷酸序列。
2、构建含有SUM1核苷酸序列的玉米重组表达载体
用限制性内切酶SpeI和KasI分别酶切重组克隆载体DBN02-T和表达载体DBNBC-03(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的SUM1-02核苷酸序列片段插到表达载体DBNBC-03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DBN130035，其构建流程如图8所示(Spec：壮观霉素基因；RB： 右边界；prUbi：玉米Ubiquitin(泛素)1基因启动子(SEQIDNO:18)；SUM1-02：SUM1-02核苷酸序列(SEQIDNO:3)；tNos：胭脂碱合成酶基因的终止子(SEQIDNO:5)；PMI：磷酸甘露糖异构酶基因(SEQIDNO:19)；LB：左边界)。
按照第二实施例中2的方法将重组表达载体DBN130035用热激方法转化大肠杆菌T1感受态细胞，并碱法提取其质粒。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DBN130035在SpeI和KasI位点间的核苷酸序列为序列表中SEQIDNO:3所示核苷酸序列，即SUM1-02核苷酸序列。
3、构建含有对照序列的玉米重组表达载体
按照本实施例中2所述的构建含有SUM1核苷酸序列的重组表达载体DBN130035的方法，利用对照序列1构建含有对照序列1的重组表达载体DBN130035N1，其载体结构如图9所示(载体骨架：pCAMBIA2301(CAMBIA机构可以提供)；Spec：壮观霉素基因；RB：右边界；prUbi：玉米Ubiquitin(泛素)1基因启动子(SEQIDNO:18)；mN1：对照序列1(SEQIDNO:9)；tNos：胭脂碱合成酶基因的终止子(SEQIDNO:5)；PMI：磷酸甘露糖异构酶基因(SEQIDNO:19)；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DBN130035N1中插入的对照序列1为序列表中SEQIDNO:9所示的核苷酸序列，即对照序列1正确插入。
按照上述构建含有对照序列1的重组表达载体DBN130035N1的方法，利用对照序列2构建含有对照序列2的重组表达载体DBN130035N2。对阳性克隆进行测序验证，结果表明重组表达载体DBN130035N2中插入的对照序列2为序列表中SEQIDNO:10所示的核苷酸序列，即对照序列2正确插入。
4、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DBN130035、DBN130035N1和DBN130035N2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的壮观霉素的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，用限制性内切酶进行酶切验证，结果表明重组表达载体DBN130035、DBN130035N1和DBN130035N2结构完全正确。
第九实施例：转基因玉米植株的获得和验证
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)的幼胚与第八实施例中4所述的农杆菌共培养，以将第八实施例中2和3构建的重组表达载体DBN130035、DBN130035N1和DBN130035N2中的T-DNA(包括玉米Ubiquitin1基因的启动子序列、SUM1-02核苷酸序列、对照序列1、对照序列2、PMI基因和tNos终止子序列)转入到玉米染色体组中，获得了转入SUM1-02核苷酸序列的玉米植株、转入对照序列1的玉米植株和转入对照序列2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SUM1-02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验证转基因玉米植株
按照第六实施例中2用TaqMan验证转基因大豆植株的方法，对转入SUM1-02核苷酸序列的玉米植株、转入对照序列1的玉米植株和转入对照序列2的玉米植株进行检测分析。通过Taqman探针荧光定量PCR方法检测PMI基因拷贝数以确定SUM1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。
以下引物和探针用来检测PMI基因序列：
引物3：GCTGTAAGAGCTTACTGAAAAAATTAACA如序列表中SEQIDNO:20所示；
引物4：CGATCTGCAGGTCGACGG如序列表中SEQIDNO:21所示；
探针2：TCTCTTGCTAAGCTGGGAGCTCGATCC如序列表中SEQIDNO:22所示。
通过分析PMI基因拷贝数的实验结果，进而证实SUM1-02核苷酸序列、对照序列1和对照序列2均己整合到所检测的玉米植株的染色体组中，而且转入SUM1-02核苷酸序列的玉米植株、转入对照序列1的玉米植株和转入对照序列2的玉米植株均获得了单拷贝的转基因玉米植株。
第十实施例：转基因玉米植株的除草剂耐受性效果检测
将转入SUM1-02核苷酸序列的玉米植株、转入对照序列1的玉米植株、转入对照序列2的玉米植株和野生型玉米植株(V3-V4时期)分别对磺酰脲类除草剂进行除草剂耐受性效果检测。
分别取转入SUM1-02核苷酸序列的玉米植株、转入对照序列1的玉米植株、转入对照序列2的玉米植株和野生型玉米植株，分别用苯磺隆(72gai/ha，4倍大田浓度)、甲嘧磺隆(120gai/ha，4倍大田浓度)、氯吡嘧磺隆(136gai/ha，4倍大田浓度)、吡嘧磺隆(100gai/ha，4倍大田浓度)、噻吩磺隆(120gai/ha，4倍大田浓度)、苄嘧磺隆(120gai/ha，4倍大田浓度)、甲磺隆(30gai/ha，4倍大田浓度)、胺苯磺隆(60gai/ha，4倍大田浓度)、氯嘧磺隆(60gai/ha，4倍大田浓度)和空白溶剂(水)喷洒。分别在喷施后3天(3DAT)、7天(7DAT)、14天(14DAT)及21天(21DAT)后，根据植株的生长状况来统计每株植株受除草剂的损伤程度：以与未处理植株生长状况相当的为0％；叶片局部褪绿发黄但基本不影响植株正常生长的为50％；整株发紫濒临死亡的为100％。转入SUM1-02核苷酸序列的玉米植株共2个株系(S7和S8)，转入对照序列1的玉米植株共2个株系(S9和S10)，转入对照序列2的玉米植株共2个株系(S11和S12)，野生型玉米植株(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植株除草剂耐受性实验结果
Figure PCTCN2016109176-appb-000009
Figure PCTCN2016109176-appb-000010
Figure PCTCN2016109176-appb-000011
Figure PCTCN2016109176-appb-000012
对于玉米，4倍大田浓度的多数磺酰脲类除草剂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3的结果表明：所述除草剂耐受性蛋白质SUM1赋予转基因玉米植物对磺酰脲类除草剂的耐受性；对于任意一种磺酰脲类除草剂，相比于转入对照序列1的玉米植株和转入对照序列2的玉米植株，转入SUM1-02核苷酸序列的玉米植株对磺酰脲类除草剂均具有显著增高的耐受性；而野生型玉米植株则对多数磺酰脲类除草剂不具耐受性。
综上所述，本发明所述除草剂耐受性蛋白质SUM1可以对磺酰脲类除草剂表现出较高的耐受性，且含有编码所述除草剂耐受性蛋白质SUM1的SUM1-01核苷酸序列和SUM1-02核苷酸序列由于采用了植物的偏好密码子，使得其特别适合在植物中表达，转入SUM1-01核苷酸序列的拟南芥植株、转入SUM1-01核苷酸序列的大豆植株和转入SUM1-02核苷酸序列的玉米植株对磺酰脲类除草剂的耐受性强，可以耐受4倍大田浓度，因此在植物上应用前景广阔。
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。
Figure PCTCN2016109176-appb-000013
Figure PCTCN2016109176-appb-000014
Figure PCTCN2016109176-appb-000015
Figure PCTCN2016109176-appb-000016
Figure PCTCN2016109176-appb-000017
Figure PCTCN2016109176-appb-000018
Figure PCTCN2016109176-appb-000019
Figure PCTCN2016109176-appb-000020
Figure PCTCN2016109176-appb-000021
Figure PCTCN2016109176-appb-000022
Figure PCTCN2016109176-appb-000023
Figure PCTCN2016109176-appb-000024
Figure PCTCN2016109176-appb-000025
Figure PCTCN2016109176-appb-000026
Figure PCTCN2016109176-appb-000027

Claims (16)

  1. 一种除草剂耐受性蛋白质，其特征在于，包括：
    (a)具有SEQIDNO:1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噻吩磺隆水解酶活性的由(a)衍生的蛋白质。
  2. 一种除草剂耐受性基因，其特征在于，包括：
    (a)编码权利要求1所述除草剂耐受性蛋白质的核苷酸序列；或
    (b)具有SEQIDNO:2所示的核苷酸序列；或
    (c)具有SEQIDNO:3所示的核苷酸序列。
  3. 一种表达盒，其特征在于，包含在有效连接的调控序列调控下的权利要求2所述除草剂耐受性基因。
  4. 一种包含权利要求2所述除草剂耐受性基因或权利要求3所述表达盒的重组载体。
  5. 一种产生除草剂耐受性蛋白质的方法，其特征在于，包括：
    获得包含权利要求2所述除草剂耐受性基因或权利要求3所述表达盒的转基因宿主生物的细胞；
    在允许产生除草剂耐受性蛋白质的条件下培养所述转基因宿主生物的细胞；
    回收所述除草剂耐受性蛋白质；
    优选地，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。
  6. 一种增加耐受除草剂范围的方法，其特征在于，包括：将权利要求1所述除草剂耐受性蛋白质或权利要求3所述表达盒编码的除草剂耐受性蛋白质在植物中与至少一种不同于权利要求1所述除草剂耐受性蛋白质或权利要求3所述表达盒编码的除草剂耐受性蛋白质的第二种蛋白质一起表达；
    优选地，所述第二种蛋白质为5-烯醇丙酮酰莽草酸-3-磷酸合酶、草甘膦氧化还原酶、草甘膦-N-乙酰转移酶、草甘膦脱羧酶、草铵膦乙酰转移酶、α酮戊二酸依赖性双加氧酶、麦草畏单加氧酶、4-羟苯基丙酮酸双加氧酶、乙酰乳酸合酶、细胞色素类蛋白质和/或原卟啉原氧化酶。
  7. 一种选择转化的植物细胞的方法，其特征在于，包括：用权利要求2所述除草剂耐受性基因或权利要求3所述表达盒转化多个植物细胞，并在允许表达所述除草剂耐受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，所述除草剂为磺酰脲类除草剂；
    优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡 嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
  8. 一种控制杂草的方法，其特征在于，包括：对种植目的植物的大田施用有效剂量的磺酰脲类除草剂，所述植物包含权利要求2所述除草剂耐受性基因或权利要求3所述表达盒；
    优选地，所述植物为单子叶植物或双子叶植物；
    更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
    进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
  9. 一种用于保护植物免受由磺酰脲类除草剂引起的损伤的方法，其特征在于，包括：将权利要求2所述除草剂耐受性基因或权利要求3所述表达盒或权利要求4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磺酰脲类除草剂损害量的除草剂耐受性蛋白质；
    优选地，所述植物为单子叶植物或双子叶植物；
    更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
    进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
  10. 一种控制草甘膦耐受性植物的大田中草甘膦抗性杂草的方法，其特征在于，包括：对种植草甘膦耐受性植物的大田施用有效剂量的磺酰脲类除草剂，所述草甘膦耐受性植物包含权利要求2所述除草剂耐受性基因或权利要求3所述表达盒；
    优选地，所述植物为单子叶植物或双子叶植物；
    更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
    进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
  11. 一种赋予植物磺酰脲类除草剂耐受性的方法，其特征在于，包括：将权利要求2所述除草剂耐受性基因或权利要求3所述表达盒或权利要求4所述重组载体导入植物；
    优选地，所述植物为单子叶植物或双子叶植物；
    更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
    进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
  12. 一种产生耐受磺酰脲类除草剂的植物的方法，其特征在于，包括向植物的基因组中引入权利要求2所述除草剂耐受性基因或权利要求3所述表达盒或权利要求4所述重组载体；
    优选地，所述植物为单子叶植物或双子叶植物；
    更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
    进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
  13. 一种培养耐受磺酰脲类除草剂的植物的方法，其特征在于，包括：
    种植至少一个植物繁殖体，所述植物繁殖体的基因组中包括权利要求2所述除草剂耐受性基因或权利要求3所述表达盒；
    使所述植物繁殖体长成植株；
    将有效剂量的磺酰脲类除草剂施加到至少包含所述植株的植物生长环境中，收获与其他不具有权利要求2所述除草剂耐受性基因或权利要求3所述表达盒的植株相比具有减弱的植物损伤和/或具有增加的植物产量的植株；
    优选地，所述植物为单子叶植物或双子叶植物；
    更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
    进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
  14. 一种控制杂草生长的种植系统，其特征在于，包括磺酰脲类除草剂和存在至少一种目的植物的植物生长环境，所述植物包含权利要求2所述除草剂耐受性基因或权利要求3所述表达盒；
    优选地，所述植物为单子叶植物或双子叶植物；
    更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
    进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
  15. 一种控制草甘膦耐受性植物的大田中草甘膦抗性杂草的种植系统，其特征在于，包括磺酰脲类除草剂、草甘膦除草剂和种植至少一种目的植物的大田，所述草甘膦耐受性植物包含权利要求2所述除草剂耐受性基因或权利要求3所述表达盒；
    优选地，所述植物为单子叶植物或双子叶植物；
    更优选地，所述植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦；
    进一步优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
  16. 一种除草剂耐受性蛋白质降解磺酰脲类除草剂的用途，其特征在于，所述除草剂耐受性蛋白质包括：
    (a)具有SEQIDNO:1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噻吩磺隆水解酶活性的由(a)衍生的蛋白质；
    优选地，所述磺酰脲类除草剂为苯磺隆、甲嘧磺隆、氯吡嘧磺隆、吡嘧磺隆、噻吩磺隆、苄嘧磺隆、甲磺隆、胺苯磺隆或氯嘧磺隆。
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EP3425046A4 (en) 2019-08-28
CA3018255A1 (en) 2017-09-28
US20190106705A1 (en) 2019-04-11
DK3425046T3 (da) 2022-01-03
AU2016399292A1 (en) 2018-11-15
AR107385A1 (es) 2018-04-25
EP3425046B1 (en) 2021-12-01
CN105802933B (zh) 2020-05-05
ES2902007T3 (es) 2022-03-24
US10954528B2 (en) 2021-03-23
ZA201806998B (en) 2020-01-29

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