WO2019153952A1 - 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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WO2019153952A1
WO2019153952A1 PCT/CN2018/124916 CN2018124916W WO2019153952A1 WO 2019153952 A1 WO2019153952 A1 WO 2019153952A1 CN 2018124916 W CN2018124916 W CN 2018124916W WO 2019153952 A1 WO2019153952 A1 WO 2019153952A1
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seq
herbicide
amino acid
plant
acid sequence
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PCT/CN2018/124916
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English (en)
French (fr)
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何健
刘斌
彭乾
陶青
肖翔
鲍晓明
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北京大北农生物技术有限公司
南京农业大学
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Priority to US16/967,815 priority Critical patent/US20210324404A1/en
Priority to BR112020015958-1A priority patent/BR112020015958A2/pt
Publication of WO2019153952A1 publication Critical patent/WO2019153952A1/zh

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    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • 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
    • A01P13/00—Herbicides; Algicides
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • 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
    • 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 to glyphosate in wild weed species or Plants that have developed glyphosate-resistant activity exert a high selection pressure.
  • 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.
  • Leaf weeds occur together, such as Amaranthus, Chenopodium, Taraxacum, and Commelinaceae species.
  • 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 present invention provides a herbicide-tolerant protein comprising:
  • herbicide tolerance protein comprises:
  • the amino acid sequence in (a) further has an arginine substitution at position 80 of SEQ ID NO: 1 and/or an alanine substitution at position 81 and/or an arginine substitution at position 182;
  • the amino acid sequence in (b) further has an arginine substitution at position 44 of SEQ ID NO: 19 and/or an alanine substitution at position 45 and/or an arginine substitution at position 146;
  • the amino acid sequence in (c) further has an arginine substitution at position 44 of SEQ ID NO: 35 and/or an alanine substitution at position 45 and/or an arginine substitution at position 146;
  • amino acid sequence in (i) (d) further has an arginine substitution at position 35 of SEQ ID NO: 51 and/or has an alanine substitution at position 36 and/or an arginine substitution at position 137;
  • herbicide tolerance protein comprises:
  • (n) has the amino acid sequence shown in SEQ ID NO: 55, SEQ ID NO: 59 or SEQ ID NO: 63.
  • the present invention also provides a herbicide tolerance gene comprising:
  • the present invention also provides an expression cassette comprising the herbicide tolerance gene under the control of an operably linked regulatory sequence.
  • the present invention also provides a recombinant vector comprising the herbicide tolerance gene or the expression cassette.
  • the present invention also provides a method for 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 herbicide tolerant protein is recovered.
  • the transgenic host organism comprises a plant, an animal, a bacterium, a yeast, a baculovirus, a nematode or an alga.
  • the present invention also provides a method for increasing the range of tolerance to herbicides, comprising: treating the herbicide-tolerant protein or the herbicide-tolerant protein encoded by the expression cassette at The plant is expressed together with at least one second protein different from the herbicide tolerance protein or the herbicide tolerance 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.
  • Expression of the herbicide-tolerant protein of the present invention in a transgenic plant can be accompanied by expression of one or more other herbicide (e.g., glyphosate or glufosinate)-tolerant proteins. Co-expression of such more than one herbicide-tolerant protein in the same transgenic plant can be achieved by genetic engineering to allow the plant to contain and express the desired gene.
  • one plant (first parent) can express the herbicide tolerance protein of the present invention by genetic engineering operation
  • the second plant (second parent) can express other herbicides (such as glyphosate) by genetic engineering operation.
  • glufosinate glufosinate
  • Progeny plants expressing all of the genes introduced into the first parent and the second parent are obtained by hybridization of the first parent and the second parent.
  • the present invention also provides a method of selecting a transformed plant cell, comprising: transforming a plurality of plant cells with the herbicide tolerance gene or the expression cassette, and allowing expression of the herbicide
  • the tolerance gene or transformed cells of the expression cassette are grown, and the cells are cultured at a herbicide concentration that kills untransformed cells or inhibits growth of untransformed cells, the herbicide being a sulfonylurea herbicide.
  • the present invention also provides a method for controlling weeds comprising: applying an effective amount of a sulfonylurea herbicide to a field of planting a plant of interest, the plant comprising the herbicide tolerance gene or The expression cassette.
  • the present invention also provides a method for protecting a plant from damage caused by a sulfonylurea herbicide, comprising: the herbicide tolerance gene or the expression cassette or the The recombinant vector is introduced into the plant such that the introduced plant produces a herbicide-tolerant protein that is sufficiently protected from the sulfonylurea herbicide.
  • the present invention also provides a method for controlling glyphosate-resistant weeds in a field of glyphosate-tolerant plants, comprising: administering an effective dose to a field planted with glyphosate-tolerant plants A sulfonylurea herbicide comprising the herbicide tolerance gene or the expression cassette.
  • the present invention also provides a method for imparting tolerance to a sulfonylurea herbicide of a plant, comprising: introducing the herbicide tolerance gene or the expression cassette or the recombinant vector into a plant.
  • the present invention also provides a method for producing a plant resistant to a sulfonylurea herbicide, comprising introducing the herbicide tolerance gene or the expression cassette or the recombinant into a genome of a plant. Carrier.
  • the present invention also provides a method of cultivating a plant resistant to a sulfonylurea herbicide, comprising:
  • 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, chlorpyrifossulfuron, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, Amphetsulfuron or chlorsulfuron.
  • the present invention also 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 herbicide tolerance Gene or the expression cassette.
  • the present invention also 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 A field of a plant of interest, the glyphosate-tolerant plant comprising the herbicide tolerance gene or the 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, chlorpyrifossulfuron, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, Amphetsulfuron or chlorsulfuron.
  • the present invention also provides the use of a herbicide-tolerant protein-degrading sulfonylurea herbicide, the herbicide-tolerant protein comprising:
  • herbicide tolerance protein comprises:
  • the amino acid sequence in (1) further has an arginine substitution at position 80 of SEQ ID NO: 1 and/or an alanine substitution at position 81 and/or an arginine substitution at position 182;
  • the amino acid sequence in (2) further has an arginine substitution at position 44 of SEQ ID NO: 19 and/or an alanine substitution at position 45 and/or an arginine substitution at position 146;
  • the amino acid sequence in (3) further has an arginine substitution at position 44 of SEQ ID NO: 35 and/or an alanine substitution at position 45 and/or an arginine substitution at position 146;
  • the amino acid sequence in (4) further has an arginine substitution at position 35 of SEQ ID NO: 51 and/or has an alanine substitution at position 131 and/or a proline substitution at position 133;
  • herbicide tolerance protein comprises:
  • the sulfonylurea herbicide is bensulfuron-methyl, mesulfuron-methyl, chloropyrazine, pyrazosulfuron, thifensulfuron, bensulfuron-methyl, metsulfuron-methyl, ethamsulfuron or Chlorsulfuron-methyl.
  • “Sulfometuron-methyl” means methyl 2-(4,6-dimethylpyrimidin-2-ylcarbamoylaminosulfonyl)benzoate as a white solid.
  • Commonly used dosage forms are 10% mesulfuron-methyl wettable powder, 10% aqueous suspension of mesulfuron-methyl (also known as dry suspension or dry suspension).
  • Commercial formulations of sulfometuron include, but are not limited to, Oust, Mori.
  • the effective dose of sulfometuron in the present invention is used at 9-120 g ai/ha, including 10-100 g ai/ha, 15-90 g ai/ha, 20-80 g ai/ha, 25-70 g ai/ha. 30-60g ai/ha or 40-50g ai/ha.
  • Tribenuron-methyl means 2-[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)-N-methyl Methyl carbamate sulfonyl]benzoate as a white solid.
  • Commonly used dosage forms are 10% bensulfuron-methyl wettable powder, 75% bensulfuron-methyl dispersible granules (also known as dry suspension or dry suspension).
  • Commercial formulations of bensulfuron include, but are not limited to, listings, broadleaf nets.
  • the effective dose of bensulfuron in the present invention is used at 9-144 g ai/ha, including 15-120 g ai/ha, 30-110 g ai/ha, 40-100 g ai/ha, 50-90 g ai/ha, 60-80 g ai/ha or 65-75 g ai/ha.
  • introducing the herbicide tolerance gene of the present invention or the expression cassette or the recombinant vector into a plant, in the present invention, introducing foreign DNA into a plant cell, and conventional transformation methods include, but are not limited to, Agrobacterium-mediated Transformation, microprojection bombardment, direct DNA uptake into protoplasts, electroporation or whisker silicon mediated DNA introduction.
  • the herbicide tolerance gene of the present invention and subsequent resistant crops provide an excellent selection for controlling glyphosate resistant (or highly tolerant and successional) broadleaf weed species in crops.
  • Sulfonylurea herbicides are broad-spectrum and potent broadleaf herbicides that provide superior efficacy to growers if they provide greater crop tolerance in both dicots and monocots.
  • Sulfonylurea herbicide-tolerant transgenic dicots can also be more flexible in application time and dosage.
  • Another use of sulfonylurea herbicide tolerance traits is that it can be used to prevent sulfonylurea herbicide drift, volatilization, conversion (or other long-range movement phenomena), misuse, destruction, etc. Damage.
  • the use of the herbicide tolerance gene of the present invention in plants provides protection against a broader spectrum of sulfonylurea herbicides, thereby increasing flexibility and controllable weed spectrum, providing a full range of commercially available sulfonyl groups.
  • the herbicide tolerance gene of the present invention has the property of allowing the use of a sulfonylurea herbicide in plants after genetic engineering for plant expression, in which the inherent tolerance is absent or insufficient. These herbicides are allowed. Furthermore, the herbicide tolerance gene of the present invention may provide protection against sulfonylurea herbicides in plants when natural tolerance is insufficient to allow selectivity. Plants containing only the herbicide tolerance gene of the present invention 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-tolerant crops and non-crop applications has (and will continue to be) selected to make weeds a 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.
  • glyphosate tolerance traits can be achieved by allowing selective use of glyphosate and sulfonylureas for the same crop
  • the control of glyphosate-resistant weed species a broadleaf 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/herbicide tolerance gene of the present invention may range from 200 to 1600 g ae/ha; the sulfonylurea herbicide (one or more) may follow the ai/ha from 7.5-150g. 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 (PAT), alpha Ketoglutarate-dependent dioxygenase (AAD), dicamba monooxygenase (DMO), 4-hydroxyphenylpyruvate dioxygenase (HPPD), acetolactate synthase (ALS), cytochromes Protein (P450) and/or protoporphyrinogen oxidase (Protox).
  • EPSPS 5-enolpyruvylshikimate 3-phosphate synthase
  • GOX
  • the herbicide tolerance gene of the present invention may be superimposed alone or in combination with other herbicide-tolerant crop characteristics with one or more other inputs (eg, insect resistance, fungal resistance, or stress tolerance, etc.) ) or output (such as increased yield, improved oil content, improved fiber quality, etc.) trait overlay.
  • the present invention can be used to provide a complete agronomic solution that flexibly and economically controls the ability of any number of agronomic pests and enhances crop quality.
  • the herbicide tolerance gene of the present invention is capable of degrading sulfonylurea herbicides and is the basis for important herbicide tolerance to crop and selection marker characteristics.
  • the present invention allows transgenic expression to control the herbicide combination of almost all broadleaf weeds.
  • the herbicide tolerance gene of the present invention can be used as an excellent herbicide tolerant crop traits and, for example, other herbicide tolerant crop traits (such as glyphosate resistance, glufosinate resistance, other ALS inhibitors (such as imidazole) Linoleone, triazolopyrimidine sulfonamide resistance, bromoxynil resistance, HPPD inhibitor resistance, PPO inhibitor resistance, etc.) and insect resistance traits (Cry1Ab, Cry1F, Vip3, other Bacillus thuringiensis) Protein or non-Bacillus derived insect resistance proteins, etc.) are superimposed.
  • the herbicide tolerance gene of the present invention 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 result in 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 herbicide tolerance gene of the present invention can be more actively used in grassy crops with moderate tolerance, whereby the improved tolerance obtained by the trait will provide the grower with a more effective dosage and a wider range. The time of application to use these herbicides without the risk of crop damage.
  • 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 herbicide-tolerant proteins of the present invention on weeds can exist independently without the presence of other substances that can "control” and/or “control” weeds. Weaken and / or disappear.
  • any tissue of a transgenic plant containing the herbicide tolerance gene of the invention
  • the herbicide tolerance protein of the invention and/or controllable Another substance of weeds the presence of said other substance does not affect the "control” and/or "control” effect of the herbicide-tolerant protein of the present invention on weeds, nor does it result in said
  • the "control" and/or “control” effect is achieved entirely and / or partially by the other substance, regardless of the herbicide-tolerant protein of the present invention.
  • 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); Plants that are asexually propagated, including 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” 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, while in the same herbicide The dose generally results in damage to the same genotype of wild type plants.
  • polynucleotides and/or nucleotides of the invention 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 acid molecules are said to be “complementary” if they are capable of hybridizing to one another with sufficient stability such that they anneal under conventional "highly stringent” conditions and bind to each other.
  • Deviation from complete complementarity is permissible as long as such deviation does not completely prevent the two molecules from forming a double-stranded structure.
  • a nucleic acid molecule In order for a nucleic acid molecule to function as a primer or probe, it is only necessary to ensure that it is sufficiently complementary in sequence to allow for the formation of a stable double-stranded structure at the particular solvent and salt concentration employed.
  • a substantially homologous sequence is a nucleic acid molecule that is capable of specifically hybridizing to a complementary strand of another matched nucleic acid molecule under highly stringent conditions.
  • Suitable stringent conditions for promoting DNA hybridization for example, treatment with 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by washing with 2.0 x SSC at 50 ° C, these conditions are known to those skilled in the art. It is well known.
  • the salt concentration in the washing step can be selected from about 2.0 x SSC under low stringency conditions, 50 ° C to about 0.2 x SSC, 50 ° C under highly stringent conditions.
  • the temperature conditions in the washing step can be raised from a low temperature strict room temperature of about 22 ° C to about 65 ° C under highly stringent conditions. 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 herbicide tolerance gene of the present invention at 65 ° C in a 6 ⁇ SSC, 0.5% SDS solution, followed by 2 ⁇ SSC, 0.1%. The membrane was washed once with SDS and 1 x SSC and 0.1% SDS.
  • sequences having herbicide tolerance activity and hybridizing under stringent conditions to the herbicide tolerance 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 the activity of the sulfonylurea herbicide.
  • the plant producing the herbicide-tolerant protein of the present 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 administer the sulfonylurea herbicide to the plant (if not specified otherwise) It is in general dosage) full or partial resistance or tolerance.
  • 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 comparisons with full length proteins and/or Or terminal deletions, variants, mutants, substitutions (proteins with alternative amino acids), chimeras and fusion proteins.
  • the "variant” or “variant” refers to a nucleotide sequence that encodes the same protein or an equivalent protein encoded with herbicide resistance activity.
  • the "equivalent protein” refers to a biologically active protein having the same or substantially the same herbicide tolerance as the protein of the claims.
  • a “fragment” or “truncated” sequence of a DNA molecule or protein sequence as used in the present invention refers to a portion of the original DNA or protein sequence (nucleotide or amino acid) involved or an artificially engineered form thereof (eg, a sequence suitable for plant expression)
  • 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 a sequence that has an amino acid substitution, deletion, addition or insertion but does not substantially affect herbicide tolerance activity, and also includes 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, de Vos et al., 1992, Science 255). : 306-312; Smith et al, 1992, J. Mol. Biol 224: 899-904; Wlodaver et al, 1992, FEBS Letters 309: 59-64).
  • 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 herbicide tolerance genes of the invention.
  • 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 (pin I and pin II) and maize protease inhibitory genes (MPI) of potato and tomato.
  • the transit peptide (also known as a secretion signal sequence or targeting sequence) directs the transgene product to a particular organelle or cell compartment, and for the receptor protein, the transit peptide can be heterologous, for example, using a coding chloroplast transporter
  • the peptide sequence targets the chloroplast, or targets the endoplasmic reticulum using the 'KDEL' retention sequence, or the CTPP-targeted vacuole using the barley plant lectin gene.
  • the leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a potato virus group leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin protein heavy chain binding protein (BiP); untranslated leader sequence of the coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader sequence.
  • EMCV leader sequence 5' non-coding region of encephalomyocarditis virus
  • a potato virus group leader sequence such as a MDMV (maize dwarf mosaic virus) leader sequence
  • MDMV human immunoglobulin protein heavy chain binding protein
  • AdMV alfalfa mosaic virus
  • TMV tobacco mosaic virus
  • the enhancer includes, but is not limited to, a cauliflower mosaic virus (CaMV) enhancer, a figwort mosaic virus (FMV) enhancer, a carnation weathering ring virus (CERV) enhancer, and a cassava vein mosaic virus (CsVMV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • CERV carnation weathering ring virus
  • CsVMV cassava vein mosaic virus
  • MMV Purple Jasmine Mosaic Virus
  • MMV Yellow Jasmine Mosaic Virus
  • CmYLCV Night fragrant yellow leaf curl virus
  • CLCuMV Multan cotton leaf curl virus
  • CoYMV Acanthus yellow mottle virus
  • PCLSV peanut chlorotic line flower Leaf virus
  • the introns include, but are not limited to, maize hsp70 introns, maize ubiquitin introns, Adh introns 1, sucrose synthase introns, or rice Actl introns.
  • the introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "super ubiquitin" intron.
  • the terminator 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 (pin II) gene, a polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and a gene derived from the ⁇ -tubulin gene. Polyadenylation signal sequence.
  • NOS Agrobacterium tumefaciens nopaline synthase
  • 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 8 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 of the present invention may be a protein having the amino acid sequence of SEQ ID NO: 1, and it has an alanine substitution at least at position 176 of SEQ ID NO: 1 and/or has a ⁇ at position 178.
  • the amino acid substitution is exemplified by SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 15 in the Sequence Listing.
  • the herbicide tolerance gene of the present invention may be a gene encoding the above herbicide tolerance protein, and examples are SEQ ID NO: 8-10, SEQ ID NO: 12-14 and SEQ ID NO: 16 in the Sequence Listing. 18 is shown.
  • the herbicide-tolerant protein of the present invention may be a protein having the amino acid sequence of SEQ ID NO: 19, and which has an alanine substitution at least at position 140 of SEQ ID NO: 19 and/or has a ⁇ position at position 142
  • the amino acid substitution is exemplified by SEQ ID NO: 23, SEQ ID NO: 27 or SEQ ID NO: 31 in the Sequence Listing.
  • the herbicide tolerance gene of the present invention may be a gene encoding the above herbicide tolerance protein, and examples are SEQ ID NO: 24-26, SEQ ID NO: 28-30 and SEQ ID NO: 32 in the Sequence Listing. 34 is shown.
  • the herbicide-tolerant protein of the present invention may be a protein having the amino acid sequence of SEQ ID NO: 35, and it has an alanine substitution at least at position 140 of SEQ ID NO: 35 and/or has a ⁇ position at position 142
  • the amino acid substitution is exemplified by SEQ ID NO: 39, SEQ ID NO: 43 or SEQ ID NO: 47 in the Sequence Listing.
  • the herbicide tolerance gene of the present invention may be a gene encoding the herbicide tolerance protein described above, and examples are SEQ ID NO: 40-42, SEQ ID NO: 44-46 and SEQ ID NO: 48 in the Sequence Listing. 50 is shown.
  • the herbicide-tolerant protein of the present invention may be a protein having the amino acid sequence of SEQ ID NO: 51, and it has an alanine substitution at at position 131 of SEQ ID NO: 51 and/or has a purine at position 133.
  • the amino acid substitution is exemplified by SEQ ID NO: 55, SEQ ID NO: 59 or SEQ ID NO: 63 in the Sequence Listing.
  • the herbicide tolerance gene of the present invention may be a gene encoding the above herbicide tolerance protein, and examples are SEQ ID NO: 56-58, SEQ ID NO: 60-62 and SEQ ID NO: 64 in the Sequence Listing. 66 is shown.
  • the herbicide tolerance gene of the present invention can be used in plants, and can comprise, in addition to the coding region comprising the herbicide tolerance protein of the present invention, other elements, such as a coding region encoding a transit peptide, encoding a selectable marker.
  • a coding region encoding a transit peptide, encoding a selectable marker.
  • the herbicide-tolerant proteins of the present invention are tolerant to most sulfonylurea herbicides.
  • the plant of the present invention contains exogenous DNA in its genome, the exogenous DNA comprising the herbicide tolerance gene of the present invention, which is protected from the sulfonylurea herbicide by expressing an effective amount of the protein. Threat.
  • 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 of the present invention is highly resistant to sulfonylurea herbicides, in particular, it can withstand 8-fold field concentration of bensulfuron-methyl.
  • the herbicide-tolerant protein of the present invention has a broad application prospect in plants.
  • FIG. 1 is a flow chart showing the construction of a recombinant cloning vector DBN01-T containing the ALT02M1-01 nucleotide sequence of the herbicide tolerance protein, the gene encoding the same, and the use thereof;
  • FIG. 2 is a flow chart showing the construction of a recombinant expression vector DBN100825 containing the ALT02M1-01 nucleotide sequence of the herbicide tolerance protein, the gene encoding the same, and the use thereof;
  • Figure 3 is a schematic diagram showing the structure of a control recombinant expression vector DBN100828N of the herbicide tolerance protein, the coding gene thereof and the use thereof;
  • A is an ALT02-01 transgenic soybean plant
  • B is an ALT02M1-01 transgenic soybean.
  • Figure 5 is a flow chart showing the construction of a recombinant cloning vector DBN02-T containing the ALT02M1-02 nucleotide sequence of the herbicide tolerance protein, the gene encoding the same, and the use thereof;
  • Figure 6 is a flow chart showing the construction of a recombinant expression vector DBN100833 containing the ALT02M1-02 nucleotide sequence of the herbicide tolerance protein, the coding gene thereof and the use thereof;
  • Figure 7 is a schematic view showing the structure of a control recombinant expression vector DBN100830N of the herbicide tolerance protein, the coding gene thereof and the use thereof;
  • FIG 8 herbicide tolerance proteins of the present invention, and the use of the gene encoding the maize Transgenic plants T 1 of FIG acid resistance effect; wherein A is ALT02-02 transgenic corn plants; B is transgenic maize ALT02M1-02 Plant; C is ALT02M2-02 transgenic maize plant; D is ALT02M3-02 transgenic maize plant; E is control corn plant; F is wild type corn plant.
  • herbicide-tolerant protein of the present invention is further illustrated by specific examples.
  • the nucleotide sequence (1197 nucleotides) of the ALT01 gene is synthesized, as shown in SEQ ID NO: 2 in the Sequence Listing, which encodes the ALT01 protein (398 amino acids) as shown in SEQ ID NO: 1 of the Sequence Listing.
  • the nucleotide sequence of the synthesized ALT01 gene (SEQ ID NO: 2) is ligated with a Spe I cleavage site at the 5' end and a Kas I cleavage site at the 3' end.
  • ALT01-01 nucleotide sequence encoding an amino acid sequence corresponding to the ALT01 according to a soybean-preferred codon, as shown in SEQ ID NO: 3 in the Sequence Listing, obtaining a coding according to the maize-preferred codon corresponding to the herbicidal
  • the ALT01-02 nucleotide sequence of the amino acid sequence of the agent-tolerant protein ALT01 is shown in SEQ ID NO: 4 in the Sequence Listing.
  • ALT01 gene was amplified by PCR, it was cloned into the vector pGEM-T according to the procedure of the Promega product pGEM-T vector (Promega, Madison, USA, CAT: A3600), and then the above-mentioned linked
  • the product was introduced into Escherichia coli DH5 ⁇ as a template, and error-prone PCR was carried out using primer 1 and primer 2, so that the ALT01 gene was mutated due to random base mismatch.
  • the primer and error-prone PCR reaction system are as follows:
  • Primer 1 atggaaaccgataaaaaaccg, as shown in SEQ ID NO: 5 in the Sequence Listing;
  • Primer 2 tcagctttcgttctgatctaag, as shown in SEQ ID NO: 6 in the Sequence Listing;
  • the error-prone PCR reaction system (total volume 50 ⁇ L) is:
  • the concentration of the plasmid DNA template was 1-10 ng/ ⁇ L, the concentration of the primer 1 was 10 ⁇ M, the concentration of the primer 2 was 10 ⁇ M, and it was stored in an amber tube at 4 °C.
  • the above error-prone PCR product was transformed into the p-sulfuron-sensitive Escherichia coli DH10B ilvG + by heat shock at 42 ° C to construct a random mutation library of ALT01 gene.
  • the transformation product in the above mutant library was inoculated to a screening medium (glucose 5 g/L, ampicillin 100 mg/L, proline 200 mg/L, leucine 200 mg/L, (containing glucose) at a concentration of 200 mg/L.
  • a screening medium glucose 5 g/L, ampicillin 100 mg/L, proline 200 mg/L, leucine 200 mg/L, (containing glucose) at a concentration of 200 mg/L.
  • NH4 glucose 5 g/L
  • MgSO 4 ⁇ 7H 2 O 200mg/L CaCl 2 ⁇ 2H 2 O 10mg/L
  • FeSO 4 ⁇ 7H 2 O1mg/L Na 2 HPO 4 ⁇ 12H 2 O 1.5g/L
  • KH 2 PO 4 1.5 g/L KH 2 PO 4 1.5 g/L
  • the above-mentioned mutant library is subjected to high-throughput screening using the principle described above, and the above-mentioned screening culture containing the concentration of 200 mg/L of fensulfuron is isolated.
  • Escherichia coli DH10B ilvG + which can still grow on the basis to obtain a resistance gene.
  • ALT01M1 three resistance genes of ALT01 mutation were obtained, which were named ALT01M1, ALT01M2 and ALT01M3 genes respectively.
  • the 527th nucleotide sequence of ALT01M1 was mutated from the original g to c, resulting in the 176th amino acid sequence.
  • the glycine mutation is alanine; the ALT01M2 nucleotide sequence positions 532 and 533 are mutated from the original tc to gt, resulting in the amino acid sequence of position 178 being mutated from the original serine to proline; the ALT01M3 nucleoside
  • the 239-242 acid sequence was mutated from the original cata to gagc, and the 527-544 was mutated from the original gaaactccagtaaagaag to caaacgtcagtaaagaaa, resulting in amino acid sequence 80-81 from the original proline and tyrosine mutations. It is arginine and alanine, and positions 176, 178 and 182 are mutated from the original glycine, serine and glycine to alanine, valine and arginine.
  • the amino acid sequence of the herbicide tolerance protein ALT01M1 encodes the ALT01M1 nucleotide sequence corresponding to the amino acid sequence of the herbicide tolerance protein ALT01M1, as in the sequence listing ID NO: 8; obtaining an ALT01M1-01 nucleotide sequence encoding an amino acid sequence corresponding to the herbicide tolerance protein ALT01M1 according to a soybean preference codon, as shown in SEQ ID NO: 9 in the sequence listing,
  • the ALT01M1-02 nucleotide sequence encoding the amino acid sequence corresponding to the herbicide-tolerant protein ALT01M1 was obtained based on the maize-preferred codon, as shown in SEQ ID NO: 10 in the Sequence Listing.
  • the amino acid sequence of the herbicide tolerance protein ALT01M2 encodes the ALT01M2 nucleotide sequence corresponding to the amino acid sequence of the herbicide tolerance protein ALT01M2, as in the sequence listing ID NO: 12; obtaining an ALT01M2-01 nucleotide sequence encoding an amino acid sequence corresponding to the herbicide tolerance protein ALT01M2 according to a soybean preference codon, as shown in SEQ ID NO: 13 in the sequence listing,
  • the ALT01M2-02 nucleotide sequence encoding the amino acid sequence corresponding to the herbicide tolerance protein ALT01M2 was obtained based on the maize preference codon as shown in SEQ ID NO: 14 of the Sequence Listing.
  • the amino acid sequence of ALT02 (369 amino acids), as shown in SEQ ID NO: 19 in the Sequence Listing; the ALT02 nucleotide sequence (1110 nucleotides) corresponding to the amino acid sequence of ALT02, as in the sequence listing ID NO: 20; obtains the ALT02-01 nucleotide sequence encoding the amino acid sequence corresponding to the ALT02 according to the soybean preference codon, as shown in SEQ ID NO: 21 in the Sequence Listing, based on the maize preferred codon
  • the ALT02-02 nucleotide sequence encoding the amino acid sequence corresponding to the herbicide tolerance protein ALT02 was obtained as shown in SEQ ID NO: 22 of the Sequence Listing.
  • the herbicide tolerance protein ALT02M1 is mutated from the original glycine to alanine at position 140 of the amino acid sequence of the ALT02, and the amino acid sequence of the ALT02M1 is as shown in SEQ ID NO: 23 in the sequence listing, and the coding corresponds to the The ALT02M1 nucleotide sequence of the amino acid sequence of the herbicide tolerance protein ALT02M1, as shown in SEQ ID NO: 24 of the Sequence Listing; obtaining an amino acid corresponding to the herbicide tolerance protein ALT02M1 according to the soybean preference codon
  • the ALT02M1-01 nucleotide sequence of the sequence as shown in SEQ ID NO: 25 of the Sequence Listing, obtains the ALT02M1-02 nucleoside encoding the amino acid sequence corresponding to the herbicide tolerance protein ALT02M1 according to the maize preference codon.
  • the acid sequence is shown as SEQ ID NO: 26 in the Sequence Listing.
  • the herbicide tolerance protein ALT02M2 is the 142th amino acid sequence of the ALT02, which is mutated from the original serine to proline.
  • the amino acid sequence of the ALT02M2 is shown in SEQ ID NO: 27 in the sequence listing, and the code corresponds to the The ALT02M2 nucleotide sequence of the amino acid sequence of the herbicide tolerance protein ALT02M2, as shown in SEQ ID NO: 28 in the Sequence Listing; obtaining an amino acid corresponding to the herbicide tolerance protein ALT02M2 according to the soybean preference codon
  • the acid sequence is shown as SEQ ID NO: 30 in the Sequence Listing.
  • the herbicide tolerance protein ALT02M3 is mutated from the original proline and tyrosine to arginine and alanine at positions 44-45 of the ALT02 amino acid sequence, and the 140th, 142th and 146th positions are from the original Glycine, serine and glycine are mutated to alanine, valine and arginine, and the amino acid sequence of ALT02M3 is represented by SEQ ID NO: 31 in the sequence listing, and encodes a corresponding to the herbicide tolerance protein ALT02M3.
  • ALT02M3 nucleotide sequence of the amino acid sequence is shown as SEQ ID NO: 32 in the Sequence Listing; ALT02M3-01 nucleotide encoding the amino acid sequence corresponding to the herbicide tolerance protein ALT02M3 is obtained based on the soybean preference codon a sequence, as shown in SEQ ID NO: 33 of the Sequence Listing, obtains the ALT02M3-02 nucleotide sequence encoding the amino acid sequence corresponding to the herbicide tolerance protein ALT02M3 according to the maize preference codon, as in the sequence listing ID NO: 34 is shown.
  • the amino acid sequence of ALT03 (362 amino acids), as shown in SEQ ID NO: 35 in the Sequence Listing; the ALT03 nucleotide sequence (1089 nucleotides) corresponding to the amino acid sequence of ALT03, as in the sequence listing ID NO: 36; obtains the ALT03-01 nucleotide sequence encoding the amino acid sequence corresponding to the ALT03 according to the soybean preference codon, as shown in SEQ ID NO: 37 in the Sequence Listing, based on the maize preferred codon
  • the ALT03-02 nucleotide sequence encoding the amino acid sequence corresponding to the herbicide tolerance protein ALT03 is obtained as shown in SEQ ID NO: 38 of the Sequence Listing.
  • the herbicide tolerance protein ALT03M1 is mutated from the original glycine to alanine at position 140 of the amino acid sequence of the ALT03, and the amino acid sequence of the ALT03M1 is represented by SEQ ID NO: 39 in the sequence listing, and the coding corresponds to the The ALT03M1 nucleotide sequence of the amino acid sequence of the herbicide tolerance protein ALT03M1, as shown in SEQ ID NO: 40 in the Sequence Listing; obtaining an amino acid corresponding to the herbicide tolerance protein ALT03M1 according to the soybean preference codon
  • the acid sequence is shown as SEQ ID NO: 42 in the Sequence Listing.
  • the herbicide tolerance protein ALT03M2 is the 142th amino acid sequence of the ALT03, which is mutated from the original serine to proline.
  • the amino acid sequence of the ALT03M2 is shown in SEQ ID NO: 43 in the sequence listing, and the coding corresponds to the The ALT03M2 nucleotide sequence of the amino acid sequence of the herbicide tolerance protein ALT03M2, as shown in SEQ ID NO: 44 in the Sequence Listing; obtaining an amino acid corresponding to the herbicide tolerance protein ALT03M2 according to the soybean preference codon
  • the acid sequence is shown as SEQ ID NO: 46 in the Sequence Listing.
  • the herbicide tolerance protein ALT03M3 is mutated from the original proline and tyrosine to arginine and alanine at positions 44-45 of the ALT03 amino acid sequence, and the 140th, 142th and 146th positions are from the original Glycine, serine and glycine are mutated to alanine, valine and arginine, and the amino acid sequence of ALT03M3 is represented by SEQ ID NO: 47 in the sequence listing, and corresponds to the herbicide tolerance protein ALT03M3.
  • ALT03M3 nucleotide sequence of the amino acid sequence is shown as SEQ ID NO: 48 in the Sequence Listing; ALT03M3-01 nucleotide encoding the amino acid sequence corresponding to the herbicide tolerance protein ALT03M3 is obtained according to the soybean preference codon The sequence, as shown in SEQ ID NO: 49 of the Sequence Listing, obtains the ALT03M3-02 nucleotide sequence encoding the amino acid sequence corresponding to the herbicide tolerance protein ALT03M3 according to the maize preference codon, as in the sequence listing ID NO: 50 is shown.
  • the amino acid sequence of ALT04 (350 amino acids), as shown in SEQ ID NO: 51 in the Sequence Listing; the ALT04 nucleotide sequence (1053 nucleotides) corresponding to the amino acid sequence of ALT04, as in the sequence listing ID NO: 52; obtains the ALT04-01 nucleotide sequence encoding the amino acid sequence corresponding to the ALT04 according to the soybean preference codon, as shown in SEQ ID NO: 53 in the Sequence Listing, based on the maize preferred codon
  • the ALT04-02 nucleotide sequence encoding the amino acid sequence corresponding to the herbicide tolerance protein ALT04 was obtained as shown in SEQ ID NO: 54 of the Sequence Listing.
  • the herbicide tolerance protein ALT04M1 is the 131st amino acid sequence of the ALT04, which is mutated from the original glycine to alanine, and the amino acid sequence of the ALT04M1 is shown in SEQ ID NO: 55 in the sequence listing, and the coding corresponds to the The ALT04M1 nucleotide sequence of the amino acid sequence of the herbicide tolerance protein ALT04M1, as shown in SEQ ID NO: 56 in the Sequence Listing; obtaining an amino acid corresponding to the herbicide tolerance protein ALT04M1 according to the soybean preference codon
  • the acid sequence is shown as SEQ ID NO: 58 in the Sequence Listing.
  • the herbicide tolerance protein ALT04M2 is the 133th amino acid sequence of the ALT04, which is mutated from the original serine to proline.
  • the amino acid sequence of the ALT04M2 is shown in SEQ ID NO: 59 in the sequence listing, and the coding corresponds to the The ALT04M2 nucleotide sequence of the amino acid sequence of the herbicide tolerance protein ALT04M2, as shown in SEQ ID NO: 60 in the Sequence Listing; obtaining an amino acid corresponding to the herbicide tolerance protein ALT04M2 according to the soybean preference codon
  • the ALT04M2-01 nucleotide sequence of the sequence as shown in SEQ ID NO: 61 of the Sequence Listing, obtains the ALT04M2-02 nucleoside encoding the amino acid sequence corresponding to the herbicide tolerance protein ALT04M2 according to the maize preference codon.
  • the acid sequence is shown as SEQ ID NO: 62 in the Sequence Listing.
  • the herbicide tolerance protein ALT04M3 is mutated from the original proline and tyrosine to arginine and alanine at positions 35-36 of the ALT04 amino acid sequence, and positions 131, 133 and 137 are from the original Glycine, serine and glycine are mutated to alanine, valine and arginine, and the amino acid sequence of ALT04M3 is shown as SEQ ID NO: 63 in the Sequence Listing, encoding the herbicide tolerance protein ALT04M3.
  • ALT04M3 nucleotide sequence of the amino acid sequence is shown as SEQ ID NO: 64 in the Sequence Listing; ALT04M3-01 nucleotide encoding the amino acid sequence corresponding to the herbicide tolerance protein ALT04M3 is obtained according to the soybean preference codon The sequence, as shown in SEQ ID NO: 65 of the Sequence Listing, obtains the ALT04M3-02 nucleotide sequence encoding the amino acid sequence corresponding to the herbicide tolerance protein ALT04M3 according to the maize preference codon, as in the sequence listing ID NO: 66 is shown.
  • Primer 3 tgcaga catatg gaaaccgataaaaaac (underlined as Nde I restriction site), as shown in SEQ ID NO: 67 in the Sequence Listing;
  • Primer 4 cccaagctt ctagctttcgttctgatctaagccgtgc (underlined as Hind III restriction site), as shown in SEQ ID NO: 68 in the Sequence Listing;
  • the ALT01M1 gene (terminal Nde I and Hind III restriction sites) was amplified using the following PCR amplification system:
  • the PCR reaction conditions were: denaturation at 98 ° C for 1 min; then entering the following cycles: denaturation at 98 ° C for 15 s, annealing at 55 ° C for 15 s, extension at 72 ° C for 1 min for a total of 29 cycles; finally extending at 72 ° C for 10 min, cooling to room temperature.
  • the ALT01M2 nucleotide sequence, the ALT01M3 nucleotide sequence, the ALT01 nucleotide sequence, the ALT03M1 nucleotide sequence, and the ALT03M2 nucleotide of the Nde I and Hind III restriction sites are amplified according to the above PCR amplification method.
  • ALT03M3 nucleotide sequence ALT03 nucleotide sequence, ALT04M1 nucleotide sequence, ALT04M2 nucleotide sequence, ALT04M3 nucleotide sequence and ALT04 nucleotide sequence; synthesis of ALT02M1 nucleotide sequence, ALT02M2 nucleotide sequence ALT02M3 nucleotide sequence, ALT02 nucleotide sequence (ends contain Nde I and Hind III restriction sites, respectively).
  • PCR amplification products were digested with restriction endonucleases Nde I and Hind III, respectively (the nucleotide sequence of ALT01M1, ALT01M2 nucleotide sequence, ALT01M3 nucleotide containing Nde I and Hind III restriction sites) Sequence, ALT01 nucleotide sequence, ALT02M1 nucleotide sequence, ALT02M2 nucleotide sequence, ALT02M3 nucleotide sequence, ALT02 nucleotide sequence, ALT03M1 nucleotide sequence, ALT03M2 nucleotide sequence, ALT03M3 nucleotide sequence, ALT03 nucleotide sequence, ALT04M1 nucleotide sequence, ALT04M2 nucleotide sequence, ALT04M3 nucleotide sequence and ALT04 nucleotide sequence) and bacterial expression vector pET-30a(+), and the above-mentioned gene fragments are respectively cut
  • the recombinant microorganisms BL21 (ALT01M1), BL21 (ALT01M2), BL21 (ALT01M3), BL21 (ALT01), BL21 (ALT02M1), BL21 (ALT02M2), BL21 (ALT02M3), BL21 (ALT02), BL21 (ALT03M1), BL21 (ALT03M2), BL21 (ALT03M3), BL21 (ALT03), BL21 (ALT04M1), BL21 (ALT04M2), BL21 (ALT04M3), and BL21 (ALT04) in 100 mL of LB medium (tryptone 10 g/L, yeast extract) 5g / L, NaCl 10g / L, ampicillin 100mg / L, adjusted to pH 7.5 with NaOH) to a concentration of OD 600nm 0.6-0.8, added 0.4 mM isopropyl thiogalactoside (IPTG ), induced at a temperature of 16 ° C for 20
  • the cells were collected, and the cells were resuspended in 20 mL Tris-HCl buffer (100 mM, pH 8.0), sonicated (X0-900D ultrasonic processor ultrasonic processor, 30% intensity) for 10 min, then centrifuged, and the supernatant was collected for nickel ion.
  • the obtained herbicide-tolerant protein was purified by affinity chromatography column, and the purification result was detected by SDS-PAGE protein electrophoresis. The band size was consistent with the theoretically predicted band size.
  • Enzyme active reaction system (1 mL): containing 0.2 ⁇ g of the reaction enzyme (the above herbicide tolerance proteins ALT01M1, ALT01M2, ALT01M3, ALT01, ALT02M1, ALT02M2, ALT02M3, ALT02, ALT03M1, ALT03M2, ALT03M3, ALT03, ALT04M1, ALT04M2, ALT04M3 and ALT04), 0.2 mM thifensulfuron (methopersulfuron, chlorsulfuron, bensulfuron-methyl, sulfometuron or tribenuron), buffer system is 50 mM phosphate buffer (pH7) .0), the reaction was carried out in a water bath at a temperature of 30 ° C for 20 min, and each reaction was started by adding a reaction enzyme, and the reaction was terminated with 1 mL of dichloromethane, and the organic phase was dehydrated with anhydrous sodium sulfate.
  • the reaction enzyme the above herb
  • LC-MS liquid chromatography-mass spectrometry
  • the primary ion mass spectrometry conditions are: ion detection mode is multi-reactive ion detection; ion polarity is negative ion; ionization mode is electrospray ionization; capillary voltage is 4000 volts; dry gas temperature is 330 ° C, flow rate is 10 L/min, atomization The gas pressure is 35 psi, the collision voltage is 135 volts, and the mass scanning range is 300-500 m/z.
  • the conditions of the secondary ion mass spectrometry were as follows: the collision voltage was 90 volts; the mass scanning range was 30-400 m/z, and other conditions were the same as those of the primary ion mass spectrometry.
  • the above experimental results indicate that the degradation efficiency of the purified herbicide tolerance protein ALT01M1 to tribenuron, bensulfuron-methyl and thifensulfuron is ALT01 compared with the herbicide-tolerant protein ALT01, respectively. 1.7, 2.3 and 3.3 times; the degradation efficiency of the herbicide-tolerant protein ALT01M2 to benzepure, bensulfuron-methyl and thifensulfuron was 6.0, 1.4 and 3.9 times of ALT01, respectively; The degradation efficiency of the herbicide-tolerant protein ALT01M3 to fensulfuron, metsulfuron-methyl and chlorsulfuron-methyl was 1.9, 2.1 and 14.2 times of ALT01, respectively.
  • the degradation efficiency of the purified herbicide-tolerant protein ALT02M1 to benzosulfuron, bensulfuron-methyl and thifensulfuron was 1.7, 2.3 and 3.3 of ALT02, respectively.
  • the degradation efficiency of the herbicide-tolerant protein ALT02M2 to tribenuron, bensulfuron-methyl and thifensulfuron was 5.9, 1.4 and 3.9 times of ALT02, respectively; the herbicide tolerance after purification
  • the degradation efficiencies of the protein ALT02M3 on tribenuron, metsulfuron-methyl and chlorsulfuron-methyl were 1.8, 2.1 and 14.2 times of ALT02, respectively.
  • the degradation efficiency of the herbicide-tolerant protein ALT03M1 to tribenuron, bensulfuron-methyl and thifensulfuron was 1.5, 2.1 and 3.0 of ALT03, respectively.
  • the degradation efficiency of the herbicide-tolerant protein ALT03M2 to tribenuron, bensulfuron-methyl and thifensulfuron was 5.4, 1.3 and 3.5 times, respectively, of ALT03; the herbicide tolerance after purification
  • the degradation efficiencies of the protein ALT03M3 on tribenuron, metsulfuron-methyl and chlorsulfuron-methyl were 1.6, 1.9 and 13.0 times of ALT03, respectively.
  • the degradation efficiency of the herbicide-tolerant protein ALT04M1 to tribenuron, bensulfuron-methyl and thifensulfuron was 1.5, 1.9 and 2.8 of ALT04, respectively.
  • the degradation efficiency of the herbicide-tolerant protein ALT04M2 to tribenuron, bensulfuron-methyl and thifensulfuron was 5.1, 1.2 and 3.3 times of ALT04, respectively; the herbicide tolerance after purification
  • the degradation efficiencies of the protein ALT04M3 for bensulfuron-methyl, metsulfuron-methyl and chlorsulfuron-methyl were 1.6, 1.8 and 12.4 times that of ALT04, respectively.
  • the amino acid sequence of the herbicide tolerance protein ALT01 is mutated from alanine to alanine at position 176 and/or from serine to valine at position 178 to enhance the mutant gene (such as the ALT01M1).
  • the amino acid sequence of the herbicide tolerance protein ALT02 (or ALT03) is mutated from alanine to alanine at position 140 and/or from serine to proline at position 142 to enhance the mutant gene (such as the ALT02M1).
  • ALT02M2, ALT02M3, ALT03M1, ALT03M2 or ALT03M3 genes The ability of the ALT02M2, ALT02M3, ALT03M1, ALT03M2 or ALT03M3 genes to degrade sulfonylurea herbicides, especially fensulfuron.
  • the 131th amino acid sequence of the herbicide tolerance protein ALT04 is mutated from alanine to alanine and/or 133 from serine to proline to increase the mutant gene (such as the ALT04M1, ALT04M2 or ALT04M3 gene).
  • the ALT02M1-01 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-T.
  • ALT02M1-01 is the ALT02M1-01 nucleotide sequence (SEQ ID NO: 25); MCS is the multiple cloning site).
  • the recombinant cloning vector DBN01-T was then transformed into E. coli T1 competent cells by heat shock method (Transgen, Beijing, China, CAT: CD501) under heat shock conditions: 50 ⁇ L E. coli T1 competent cells, 10 ⁇ L of plasmid DNA (recombinant)
  • the cloning vector DBN01-T was incubated at 42 ° C for 30 s; shaken at 37 ° C for 1 h (shake at 100 rpm), coated with IPTG (isopropylthio- ⁇ -D-galactoside) and X-gal.
  • LB plate of ampicillin 100 mg/L (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/ L, agar 15 g / L, adjusted to pH 7.5 with NaOH) overnight growth.
  • White colonies were picked and cultured in LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, ampicillin 100 mg/L, pH adjusted to 7.5 with NaOH) at 37 °C. overnight.
  • the plasmid was extracted by alkaline method: the bacterial solution was centrifuged at 12000 rpm for 1 min, the supernatant was removed, and the precipitated cells were pre-cooled with 100 ⁇ L of ice (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose).
  • the TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was dissolved in the precipitate; the RNA was digested in a water bath at a temperature of 37 ° C for 30 min; and stored at a temperature of -20 ° C until use.
  • the positive clone was sequenced and verified.
  • the result showed that the ALT02M1-01 nucleotide sequence inserted into the recombinant cloning vector DBN01-T was SEQ ID NO: 25 in the sequence listing.
  • Recombinant cloning vector DBN01-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases Spe I and Kas I, respectively, and the ALT02M1-01 nucleotide sequence was excised. The fragment was inserted between the Spe I and Kas I sites of the expression vector DBNBC-01, and the vector was constructed by a conventional restriction enzyme digestion method. The recombinant expression vector DBN100825 was constructed.
  • the recombinant expression vector DBN100825 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 DBN100825), 42 ° C water bath for 30 s; 37 ° C shaking culture 1h (shake shake at 100 rpm); then in LB solid plate containing 50 mg / L spectinomycin (Spectinomycin) (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15g / L, The pH was adjusted to 7.5 with NaOH and incubated at 37 °C for 12 h, and white colonies were picked up in LB liquid medium (trypeptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, spectinomycin 50 mg).
  • the plasmid was extracted by an alkali method.
  • the extracted plasmids were digested with restriction endonucleases Spe I and Kas I, and the positive clones were sequenced and identified.
  • the results showed that the nucleotide sequence of the recombinant expression vector DBN100825 between Spe I and Kas I sites was sequenced.
  • the recombinant expression vector DBN100826 containing the ALT02M2-01 nucleotide sequence, the recombinant expression vector DBN100827 containing the ALT02M3-01 nucleotide sequence, and the ALT02 containing ALT02M3-01 nucleotide sequence were constructed according to the above method for constructing the recombinant expression vector DBN100825 containing the nucleotide sequence of ALT02M1-01.
  • the positive clones were sequenced and verified.
  • nucleotide sequences of ALT02M2-01, ALT02M3-01 and ALT02-01 inserted into the recombinant expression vectors DBN100825, DBN100826, DBN100827 and DBN100828 were SEQ ID NO: 29 and SEQ ID in the sequence listing, respectively.
  • the nucleotide sequence shown by NO:33 and SEQ ID NO: 21, that is, the ALT02M2-01 nucleotide sequence, the ALT02M3-01 nucleotide sequence, and the ALT02-01 nucleotide sequence were correctly inserted.
  • the recombinant expression vector DBN100828N was constructed according to the above method for constructing the recombinant expression vector DBN100825 containing the nucleotide sequence of ALT02M1-01, and its vector structure is shown in Fig. 3 (vector skeleton: pCAMBIA2301 (available by CAMBIA); Spec: Specimen Gene; RB: right border; prBrCBP: rapeseed eukaryotic elongation factor gene 1 ⁇ (Tsf1) promoter (SEQ ID NO: 71); spAtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO: 72); EPSPS: 5- Enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 73); tPsE9: terminator of pea RbcS gene (SEQ ID NO: 74); LB: left border).
  • the positive clones were sequenced and verified, and the results showed that
  • the recombinant expression vectors DBN100825, DBN100826, DBN100827, DBN100828 and DBN100828N, which have been constructed correctly, were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L Agrobacterium LBA4404 3 ⁇ L of plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 min, 37 ° C warm water bath for 10 min; the transformed Agrobacterium LBA4404 was inoculated in LB tube and incubated at a temperature of 28 ° C, 200 rpm for 2 h, 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 vector
  • 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 infestation method to reconstitute the construct of the second embodiment.
  • T-DNA in the expression vectors DBN100825, DBN100826, DBN100827, DBN100828 and DBN100828N (including the promoter sequence of the Arabidopsis Ubiquitin10 gene, ALT02M1-01 nucleotide sequence, ALT02M2-01 nucleotide sequence, ALT02M3-01 nucleotide Sequence, ALT02-01 nucleotide sequence, tNos terminator, rapeseed eukaryotic elongation factor gene 1 ⁇ promoter, Arabidopsis chloroplast transit peptide, 5-enolpyruvylshikimate-3-phosphate synthase gene, pea RbcS gene
  • the terminator was transferred to the soybean genome, and a soybean plant transformed with the nucleotide sequence of ALT02M1-01, a soybean plant transformed with the nucleotide sequence of ALT02M2-01, and a nucleotide sequence of ALT02M3-01 were obtained. Soybean
  • 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.
  • the soybean sterile seedlings of the fresh green cotyledonary nodes were taken, and 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.
  • the wound was treated at the cotyledonary node with the scalpel's blade back, and the wounded cotyledonary node tissue was contacted with the Agrobacterium suspension, wherein the Agrobacterium was able to bind the ALT02M1-01 nucleotide sequence (ALT02M2-01 nucleotide sequence, ALT02M3- Delivery of the 01 nucleotide sequence or the ALT02-01 nucleotide sequence to the wounded cotyledonary node tissue (step 1: Infection step)
  • Cotyledonary node tissue and Agrobacterium co-culture for a period of time (3 days) (step 2: co-cultivation step).
  • cotyledonary node tissue in After the infection step, culture on solid medium (MS salt 4.3 g / L, B5 vitamin, sucrose 20 g / L, glucose 10 g / L, MES 4 g / L, ZT 2 mg / L, agar 8 g / L, pH 5.6)
  • there is an optional “recovery” step In the “recovery” step, the medium is restored (B5 salt 3.1 g/L, B5 vitamins, MES 1 g/L, sucrose 30 g/L).
  • At least one antibiotic known to inhibit the growth of Agrobacterium exists in ZT 2 mg/L, agar 8 g/L, cephalosporin 150 mg/L, glutamic acid 100 mg/L, aspartic acid 100 mg/L, pH 5.6. (Cefosporin), no selection of plant transformants (step 3: recovery step).
  • the cotyledonary node-regenerated tissue pieces are cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium A recovery period is provided for the infected cells.
  • the cotyledonary node-regenerated tissue pieces are cultured on a medium containing a selection agent (glyphosate) and the grown transformed callus is selected (step 4: selection step).
  • the cotyledonary node regenerated tissue block was selected in solid medium with selective agent (B5 salt 3.1g/L, B5 vitamin, MES 1g/L, sucrose 30g/L, 6-benzyl adenine (6-BAP) 1mg/L , agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, N-(phosphocarboxymethyl)glycine 0.25mol/L, pH5.6), resulting in The transformed cells are selectively grown. Then, the transformed cells regenerate the plants (step 5: regeneration step), preferably, cotyledons grown on the medium containing the selection agent Regenerated tissue blocks on a solid medium (B5 B5 differentiation medium and rooting medium) to regenerate the plants.
  • selective agent B5 salt 3.1g/L, B5 vitamin, MES 1g/L, sucrose 30g/L, 6-benzyl adenine (6-BAP) 1mg/L ,
  • the selected resistant tissue blocks were transferred to the B5 differentiation medium (B5 salt 3.1 g/L, B5 vitamin, MES 1 g/L, sucrose 30 g/L, ZT 1 mg/L, agar 8 g/L, cephalosporin 150 mg).
  • B5 differentiation medium B5 salt 3.1 g/L, B5 vitamin, MES 1 g/L, sucrose 30 g/L, ZT 1 mg/L, agar 8 g/L, cephalosporin 150 mg.
  • /L glutamic acid 50mg / L, aspartic acid 50mg / L, gibberellin 1mg / L, auxin 1mg / L, N- (phosphine carboxymethyl) glycine 0.25mol / L, pH 5.6
  • the culture was differentiated at 25 °C.
  • the differentiated seedlings were transferred to the B5 rooting medium (B5 salt 3.1 g/L, B5 vitamin, MES 1 g/L, sucrose 30 g/L, agar 8 g/L, cephalosporin 150 mg/L, ⁇ -3- Butyric acid (IBA) 1 mg/L) was 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, culture was carried out at 26 ° C for 16 h every day and then at 20 ° C for 8 h.
  • B5 rooting medium B5 salt 3.1 g/L, B5 vitamin, MES 1 g/L, sucrose 30 g/L, agar 8 g/L, cephalosporin 150 mg/L, ⁇ -3- Butyric acid (IBA) 1 mg/L
  • Soybean plants transformed with ALT02M1-01 nucleotide sequence soybean plants transferred to ALT02M2-01 nucleotide sequence, soybean plants transferred to ALT02M3-01 nucleotide sequence, and transferred to ALT02-01 nucleotide sequence
  • Approximately 100 mg of the leaves of the soybean plants and the control soybean plants were used as samples, and the genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the EPSPS gene copy number was detected by Taqman probe fluorescent quantitative PCR to determine the copy number of the target 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 21 Soybean plants transformed with ALT02M1-01 nucleotide sequence, soybean plants transferred to ALT02M2-01 nucleotide sequence, soybean plants transferred to ALT02M3-01 nucleotide sequence, and transferred to ALT02-01 nucleus 100 mg of the leaves of the soybean plant, the control soybean plant and the wild type soybean plant of the nucleotide sequence, respectively, were homogenized by liquid nitrogen in a mortar, and each sample was taken in 3 replicates;
  • Step 22 Extract the genomic DNA of the above sample using Qiagen's DNeasy Plant Mini Kit, and refer to the product specification for the specific method;
  • Step 23 Determine the genomic DNA concentration of the above sample by NanoDrop 2000 (Thermo Scientific).
  • Step 24 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 25 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:
  • Primer 5 ctggaaggcgaggacgtcatcaata is shown in SEQ ID NO: 75 in the Sequence Listing;
  • Primer 6 tggcggcattgccgaaatcgag is shown in SEQ ID NO: 76 in the Sequence Listing;
  • Probe 1 atgcaggcgatgggcgcccgcatccgta as shown in SEQ ID NO: 77 in the Sequence Listing;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mix contained 45 ⁇ L of each primer at a concentration of 1 mM, 50 ⁇ L of probe at a concentration of 100 ⁇ M and 860 ⁇ L of 1 ⁇ TE buffer, and was stored in an amber tube at 4°C.
  • the PCR reaction conditions are:
  • ALT02M1-01 nucleotide sequence, ALT02M2-01 nucleotide sequence, ALT02M3-01 nucleotide sequence and ALT02-01 nucleotide sequence have been integrated into the detected Soybean plants that have been transferred to the ALT02M1-01 nucleotide sequence, soybean plants that have been transferred to the ALT02M2-01 nucleotide sequence, soybean plants that have been transferred to the ALT02M3-01 nucleotide sequence, and transferred to ALT02 in the genome of soybean plants.
  • Both the soybean sequence of the -99 nucleotide sequence and the control soybean plant obtained a single copy of the transgenic soybean plant.
  • Soybean plants transformed with ALT02M1-01 nucleotide sequence soybean plants transferred to ALT02M2-01 nucleotide sequence, soybean plants transferred to ALT02M3-01 nucleotide sequence, and transferred to ALT02-01 nucleotide sequence
  • Soybean plants, control soybean plants and wild-type soybean plants were sprayed with tribenuron (144 g ai/ha, 8 times field concentration) and blank solvent (water), respectively. After 3 days (3DAT), 7 days (7DAT), 14 days (14DAT) and 21 days (21DAT) after spraying, 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. .
  • a total of 3 strains (S1, S2 and S3) of soybean plants transferred to the ALT02M1-01 nucleotide sequence were transferred to soybean plants with ALT02M2-01 nucleotide sequence (S4, S5 and S6).
  • a total of 2 strains (S13 and S14) of soybean plants and 1 strain of wild type soybean plants (CK1) were tested; 10-15 strains were selected from each strain for testing. The results are shown in Table 2 and Figure 4.
  • the synthetic ALT02M1-02 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 the recombinant cloning vector DBN02-T.
  • the construction process is shown in Figure 5 (wherein Amp represents the ampicillin resistance gene; f1 represents the origin of replication of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; and T7 is initiated by T7 RNA polymerase).
  • ALT02M1-02 is the ALT02M1-02 nucleotide sequence (SEQ ID NO: 26); MCS is the multiple cloning site).
  • the recombinant cloning vector DBN01-T was transformed into E. coli T1 competent cells by heat shock 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 Spe I and Kas I, and the positive clones were sequenced and identified.
  • the results showed that the nucleotide sequence of the recombinant cloning vector DBN02-T between Spe I and Kas I sites was confirmed.
  • the recombinant cloning vector DBN02-T and the expression vector DBNBC-02 were digested with restriction endonucleases Spe I and Kas I, respectively, and the ALT02M1-02 nucleotide sequence was excised. The fragment was inserted between the Spe I and Kas I sites of the expression vector DBNBC-02, and the vector was constructed by a conventional restriction enzyme digestion method.
  • the recombinant expression vector DBN100833 was constructed as shown in Fig. 6.
  • the recombinant expression vector DBN100833 was transformed into E. coli T1 competent cells by a heat shock method according to the method of 2 in the fourth embodiment, and the plasmid was extracted by an alkali method.
  • the extracted plasmid was digested with restriction endonucleases Spe I and Kas I, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence of the recombinant expression vector DBN100833 was between Spe I and Kas I.
  • the recombinant expression vector DBN100832 containing the ALT02M2-02 nucleotide sequence, the recombinant expression vector DBN100831 containing the nucleotide sequence of ALT02M3-02, and the ALT02 containing ALT02M3-02 nucleotide sequence were constructed according to the above method for constructing the recombinant expression vector DBN100833 containing the nucleotide sequence of ALT02M1-02. -02 Nucleotide sequence recombinant expression vector DBN100830. The positive clones were sequenced and verified.
  • nucleotide sequences of ALT02M2-02, ALT02M3-02 and ALT02-02 inserted into the recombinant expression vectors DBN100832, DBN100831 and DBN100830 were SEQ ID NO: 30 and SEQ ID NO in the sequence listing, respectively.
  • the nucleotide sequence shown in 34 and SEQ ID NO: 22, ie, the ALT02M2-02 nucleotide sequence, the ALT02M3-02 nucleotide sequence, and the ALT02-02 nucleotide sequence were correctly inserted.
  • the recombinant expression vector DBN100830N was constructed according to the above method for constructing the recombinant expression vector DBN100833 containing the nucleotide sequence of ALT02M1-02, and the vector structure thereof was as shown in Fig. 7 (vector skeleton: pCAMBIA2301 (available by CAMBIA mechanism); Spec: Specimen mold Gene; RB: right border; prUbi: maize ubiquitin 1 gene promoter (SEQ ID NO: 78); PMI: phosphomannose isomerase gene (SEQ ID NO: 79); tNos: nopaline synthesis Terminator of the enzyme gene (SEQ ID NO: 70); LB: left border).
  • the positive clones were sequenced and verified, and the results showed that the control recombinant expression vector DBN100830N was constructed correctly.
  • the recombinant expression vectors DBN100833, DBN100832, DBN100831, DBN100830 and DBN100830N which have been constructed correctly, were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L Agrobacterium LBA4404 3 ⁇ L of plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 min, 37 ° C warm water bath for 10 min; the transformed Agrobacterium LBA4404 was inoculated in LB tube and incubated at a temperature of 28 ° C, 200 rpm for 2 h, 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 D
  • T-DNA in recombinant expression vectors DBN100833, DBN100832, DBN100831, DBN100830 and DBN100830N (including the promoter sequence of maize Ubiquitin1 gene, ALT02M1-02 nucleotide sequence, ALT02M2-02 nucleotide sequence, ALT02M3-02 nucleotide sequence) , ALT02-02 nucleotide sequence, PMI gene and tNos terminator sequence) were transferred into the maize genome, and the maize plant transferred to the ALT02M1-02 nucleotide sequence was obtained, and the nucleotide sequence of ALT02M2-02 was transferred.
  • immature immature embryos are isolated from maize, and the immature embryos are contacted with Agrobacterium suspension, wherein Agrobacterium is capable of expressing the ALT02M1-02 nucleotide sequence (ALT02M2-02 nuclear
  • Agrobacterium is capable of expressing the ALT02M1-02 nucleotide sequence (ALT02M2-02 nuclear
  • the nucleotide sequence, the ALT02M3-02 nucleotide sequence or the ALT02-02 nucleotide sequence is delivered to at least one cell of one of the young embryos (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, AS 100 mg/L, 2,4-D) Incubate on 1 mg/L, agar 8 g/L, pH 5.8).
  • the medium was recovered (MS salt 4.3 g / L, MS vitamin, casein 300 mg / L, sucrose 30 g / L, 2,4-D 1 mg / L, plant gel 3 g / L, pH 5.
  • 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-D 1 mg/ L, plant gel 3g / L, pH 5.8) culture, resulting 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, culture was carried out at 28 ° C for 16 h every day and then at 20 ° C for 8 h.
  • TaqMan was used to verify the transgenic soybean plants, and the maize plants transformed into the ALT02M1-02 nucleotide sequence, the maize plants transferred to the ALT02M2-02 nucleotide sequence, and the ALT02M3-02 nucleoside were transferred.
  • the acid sequence of the maize plants, the maize plants transferred to ALT02-02, and the control corn plants were subjected to detection analysis.
  • the copy number of the PMI gene was detected by Taqman probe real-time PCR to determine the copy number of the gene of interest.
  • 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 7 gctgtaagagcttactgaaaaattaaca as shown in SEQ ID NO: 80 in the Sequence Listing;
  • Primer 8 cgatctgcaggtcgacgg is shown in SEQ ID NO: 81 in the Sequence Listing;
  • Probe 2 tctcttgctaagctgggagctcgatcc is shown as SEQ ID NO: 82 in the Sequence Listing.
  • the ALT02M1-02 nucleotide sequence, the ALT02M2-02 nucleotide sequence, the ALT02M3-02 nucleotide sequence and the ALT02-02 nucleotide sequence were integrated into the detected Maize plants in the genome of maize plants, and transferred to the ALT02M1-02 nucleotide sequence of maize plants, maize plants transferred to the ALT02M2-02 nucleotide sequence, maize plants transferred to the ALT02M3-02 nucleotide sequence, and transferred to ALT02 Both the -02 nucleotide sequence of the maize plants and the control maize plants obtained a single copy of the transgenic maize plants.
  • Maize plants, control corn plants and wild-type maize plants (V3-V4 period) were tested for herbicide tolerance in respectively.
  • the amino acid sequence of the herbicide tolerance protein ALT01 of the present invention is mutated from glycine to alanine at the 176th position and/or from serine to valine at position 178 (for example, the herbicide is tolerant)
  • the protein ALT01M1, ALT01M2 or ALT01M3 can exhibit higher tolerance to sulfonylurea herbicides, especially fensulfuron;
  • the amino acid sequence of the herbicide tolerance protein ALT02 (or ALT03) is 140th.
  • Sulfonylurea can be mutated from a glycine to an alanine and/or a 142th position from a serine to a proline (eg, the herbicide tolerance protein ALT02M1, ALT02M2, ALT02M3, ALT03M1, ALT03M2 or ALT03M3)
  • the herbicide exhibits a high tolerance, in particular fensulfuron; the amino acid sequence of the herbicide-tolerant protein ALT04 is mutated from alanine to alanine at the 131st position and/or from serine to 133 at the 133th position.
  • the sulfonylurea herbicide may exhibit higher tolerance, particularly bensulfuron-methyl, when the herbicide (for example, the herbicide tolerance protein ALT04M1, ALT04M2 or ALT04M3).
  • the coding gene of the above herbicide-tolerant protein is particularly suitable for expression in plants due to the use of plant preference codons, and soybean plants and corn plants transferred to the above herbicide-tolerant proteins are herbicidal to sulfonylureas.
  • the agent is highly tolerant, especially the bensulfuron-methyl which can withstand 8 times the field concentration, so it has a promising application on plants.

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Abstract

本发明涉及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包括：(a)具有SEQ ID NO：1所示的氨基酸序列，且其至少在SEQ ID NO：1第176位具有丙氨酸替换和/或第178位具有缬氨酸替换；或(b)具有SEQ ID NO：3所示的氨基酸序列；或(c)具有SEQ ID NO：5所示的氨基酸序列；或(d)具有SEQ ID NO：7所示的氨基酸序列；或(e)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。本发明所述除草剂耐受性蛋白质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 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受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受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受性作物控制，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(Amaranthus)、藜属(Chenopodium)、蒲公英属(Taraxacum)和鸭跖草科(Commelinaceae)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，需要更多能够降解磺酰脲类除草剂的基因并将其输入对磺酰脲类除草剂敏感的目的植物中以增加该植物对磺酰脲类除草剂的耐受性。
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能够较好的降解磺酰脲类除草剂，并使得转入所述除草剂耐受 性蛋白质编码基因的植物对磺酰脲类除草剂具有较高的耐受性。
为实现上述目的，本发明提供了一种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，且其至少在SEQ ID NO：1第176位具有丙氨酸替换和/或第178位具有缬氨酸替换；或
(b)具有SEQ ID NO：19所示的氨基酸序列，且其至少在SEQ ID NO：19第140位具有丙氨酸替换和/或第142位具有缬氨酸替换；或
(c)具有SEQ ID NO：35所示的氨基酸序列，且其至少在SEQ ID NO：35第140位具有丙氨酸替换和/或第142位具有缬氨酸替换；或
(d)具有SEQ ID NO：51所示的氨基酸序列，且其至少在SEQ ID NO：51第131位具有丙氨酸替换和/或第133位具有缬氨酸替换；或
(e)在(a)-(d)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)-(d)衍生的蛋白质。
进一步地，所述除草剂耐受性蛋白质包括：
(f)(a)中的氨基酸序列在SEQ ID NO：1第80位还具有精氨酸替换和/或第81位具有丙氨酸替换和/或第182位具有精氨酸替换；或
(g)(b)中的氨基酸序列在SEQ ID NO：19第44位还具有精氨酸替换和/或第45位具有丙氨酸替换和/或第146位具有精氨酸替换；或
(h)(c)中的氨基酸序列在SEQ ID NO：35第44位还具有精氨酸替换和/或第45位具有丙氨酸替换和/或第146位具有精氨酸替换；或
(i)(d)中的氨基酸序列在SEQ ID NO：51第35位还具有精氨酸替换和/或第36位具有丙氨酸替换和/或第137位具有精氨酸替换；或
(j)在(f)-(i)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)-(d)衍生的蛋白质。
更进一步地，所述除草剂耐受性蛋白质包括：
(k)具有SEQ ID NO：7、SEQ ID NO：11或SEQ ID NO：15所示的氨基酸序列；或
(l)具有SEQ ID NO：23、SEQ ID NO：27或SEQ ID NO：31所示的氨基酸序列；或
(m)具有SEQ ID NO：39、SEQ ID NO：43或SEQ ID NO：47所示的氨基酸序列；或
(n)具有SEQ ID NO：55、SEQ ID NO：59或SEQ ID NO：63所示的氨基酸序 列。
为实现上述目的，本发明还提供了一种除草剂耐受性基因，包括：
(o)编码(a)-(n)所述除草剂耐受性蛋白质的核苷酸序列；或
(p)具有SEQ ID NO：8、9、10、12、13、14、16、17或18所示的核苷酸序列；或
(q)具有SEQ ID NO：24、25、26、28、29、30、32、33或34所示的核苷酸序列；或
(r)具有SEQ ID NO：40、41、42、44、45、46、48、49或50所示的核苷酸序列。
为实现上述目的，本发明还提供了一种表达盒，其特征在于，包含在有效连接的调控序列调控下的所述除草剂耐受性基因。
为实现上述目的，本发明还提供了一种包含所述除草剂耐受性基因或所述表达盒的重组载体。
为实现上述目的，本发明还提供了一种产生除草剂耐受性蛋白质的方法，包括：
获得包含所述除草剂耐受性基因或所述表达盒的转基因宿主生物的细胞；
在允许产生除草剂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。
进一步地，所述转基因宿主生物包括植物、动物、细菌、酵母、杆状病毒、线虫或藻类。
为实现上述目的，本发明还提供了一种增加耐受除草剂范围的方法，其特征在于，包括：将所述除草剂耐受性蛋白质或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所述除草剂耐受性蛋白质或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。
进一步地，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。
本发明所述除草剂耐受性蛋白质在一种转基因植物中的表达可以伴随着一个或多个其它除草剂(如草甘膦或草铵膦)耐受性蛋白质的表达。这种超过一种的除草剂耐受性蛋白质在同一株转基因植物中共同表达可以通过遗传工程使植物包 含并表达所需的基因来实现。另外，一种植物(第1亲本)可以通过遗传工程操作表达本发明所述除草剂耐受性蛋白质，第二种植物(第2亲本)可以通过遗传工程操作表达其它除草剂(如草甘膦或草铵膦)耐受性蛋白质。通过第1亲本和第2亲本杂交获得表达引入第1亲本和第2亲本的所有基因的后代植物。
为实现上述目的，本发明还提供了一种选择转化的植物细胞的方法，包括：用所述除草剂耐受性基因或所述表达盒转化多个植物细胞，并在允许表达所述除草剂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。
为实现上述目的，本发明还提供了一种控制杂草的方法，包括：对种植目的植物的大田施用有效剂量的磺酰脲类除草剂，所述植物包含所述除草剂耐受性基因或所述表达盒。
为实现上述目的，本发明还提供了一种用于保护植物免受由磺酰脲类除草剂引起的损伤的方法，包括：将所述除草剂耐受性基因或所述表达盒或所述重组载体导入植物，使导入后的植物产生足够保护其免受磺酰脲类除草剂损害量的除草剂耐受性蛋白质。
为实现上述目的，本发明还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所述除草剂耐受性基因或所述表达盒。
为实现上述目的，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：将所述除草剂耐受性基因或所述表达盒或所述重组载体导入植物。
为实现上述目的，本发明还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所述除草剂耐受性基因或所述表达盒或所述重组载体。
为实现上述目的，本发明还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所述除草剂耐受性基因或所述表达盒；
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，收获与其他不具有所述除草剂耐受性基因或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。
进一步地，所述植物为单子叶植物或双子叶植物。
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。
在上述技术方案的基础上，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。
为实现上述目的，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，所述植物包含所述除草剂耐受性基因或所述表达盒。
为实现上述目的，本发明还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所述除草剂耐受性基因或所述表达盒。
进一步地，所述植物为单子叶植物或双子叶植物。
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。
在上述技术方案的基础上，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。
为实现上述目的，本发明还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：
(1)具有SEQ ID NO：1所示的氨基酸序列，且其至少在SEQ ID NO：1第176位具有丙氨酸替换和/或第178位具有缬氨酸替换；或
(2)具有SEQ ID NO：19所示的氨基酸序列，且其至少在SEQ ID NO：19第140位具有丙氨酸替换和/或第142位具有缬氨酸替换；或
(3)具有SEQ ID NO：35所示的氨基酸序列，且其至少在SEQ ID NO：35第140位具有丙氨酸替换和/或第142位具有缬氨酸替换；或
(4)具有SEQ ID NO：51所示的氨基酸序列，且其至少在SEQ ID NO：51第131位具有丙氨酸替换和/或第133位具有缬氨酸替换；或
(5)在(1)-(4)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(1)-(4)衍生的蛋白质。
进一步地，所述除草剂耐受性蛋白质包括：
(6)(1)中的氨基酸序列在SEQ ID NO：1第80位还具有精氨酸替换和/或第81位具有丙氨酸替换和/或第182位具有精氨酸替换；或
(7)(2)中的氨基酸序列在SEQ ID NO：19第44位还具有精氨酸替换和/或第45位具有丙氨酸替换和/或第146位具有精氨酸替换；或
(8)(3)中的氨基酸序列在SEQ ID NO：35第44位还具有精氨酸替换和/或第45位具有丙氨酸替换和/或第146位具有精氨酸替换；或
(9)(4)中的氨基酸序列在SEQ ID NO：51第35位还具有精氨酸替换和/或第131位具有丙氨酸替换和/或第133位具有缬氨酸替换；或
(10)在(6)-(9)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(6)-(9)衍生的蛋白质。
更进一步地，所述除草剂耐受性蛋白质包括：
(11)具有SEQ ID NO：7、SEQ ID NO：11或SEQ ID NO：15所示的氨基酸序列；或
(12)具有SEQ ID NO：23、SEQ ID NO：27或SEQ ID NO：31所示的氨基酸序列；或
(13)具有SEQ ID NO：39、SEQ ID NO：43或SEQ ID NO：47所示的氨基酸序列；或
(14)具有SEQ ID NO：55、SEQ ID NO：59或SEQ ID NO：63所示的氨基酸序列。
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。
本发明中所述甲嘧磺隆(Sulfometuron-methyl)是指2-(4，6-二甲基嘧啶-2-基氨基甲酰氨基磺酰基)苯甲酸甲酯，为白色固体。常用剂型为10％甲嘧磺隆可湿性粉剂、10％甲嘧磺隆水悬剂(也称为干燥悬浮剂或干悬浮剂)。甲嘧磺隆的商业制剂包括但不限于，Oust、森草净。
本发明中所述有效剂量甲嘧磺隆是指以9-120g ai/ha使用，包括10-100g ai/ha、15-90g ai/ha、20-80g ai/ha、25-70g ai/ha、30-60g ai/ha或40-50g ai/ha。
本发明中所述苯磺隆(Tribenuron-methyl)是指2-[N-(4-甲氧基-6-甲基-1，3，5-三嗪-2-基)-N-甲基氨基甲酰胺基磺酰基]苯甲酸甲酯，为白色固体。常用剂型为10％苯磺隆可湿性粉剂、75％苯磺隆水分散粒剂(也称为干燥悬浮剂或干悬浮剂)。苯磺隆的商业制剂包括但不限于，巨星、阔叶净。
本发明中所述有效剂量苯磺隆是指以9-144g ai/ha使用，包括15-120g ai/ha、30-110g ai/ha、40-100g ai/ha、50-90g ai/ha、60-80g ai/ha或65-75g ai/ha。
将本发明所述除草剂耐受性基因或所述表达盒或所述重组载体导入植物，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(或高耐受性和演替的)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。磺酰脲类除草剂耐受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。磺酰脲类除草剂耐受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。在植物中使用本发明所述除草剂耐受性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。
现已鉴定了本发明所述除草剂耐受性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，所述植物中固有耐受性不存在或不足以允许使用这些除草剂。此外，本发明所述除草剂耐受性基因可以在天然耐受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本发明所述除草剂耐受性基因的植物。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，在草甘膦耐受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受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。将本发明所述除草剂耐受性基因与草甘膦耐受性性状(和/或其他除草剂耐受性性状)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受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/本发明所述除草剂耐受性基因叠加的草甘膦应用范围可以从200至1600g ae/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/或天然更具耐受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受性作物外的除草剂耐受性的化学品。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，可以将本发明所述除草剂耐受性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、真菌抗性或胁迫耐受性等)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。
本发明所述除草剂耐受性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。本发明所述除草剂耐受性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，本发明所述除草剂耐受性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受性作物性状(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受性，这些性状组合产生控制杂草物种的新方法。因此，除了除草剂耐受性作物性状，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受性。
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。本发明所述除草剂耐受性基因可更积极地用于耐受性适中的禾本科作物中，由此性状得到的提高的耐受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。本发明所述除草剂耐受性蛋白质对杂草的“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(含有本发明所述除草剂耐受性基因)的任何组织同时和/或不同步地，存在和/或产生，本发明所述除草剂耐受 性蛋白质和/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本发明所述除草剂耐受性蛋白质对杂草的“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，而与本发明所述除草剂耐受性蛋白质无关。
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所述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所述除草剂耐受性基 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℃下与本发明所述除草剂耐受性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所述除草剂耐受性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)具有降解或减弱磺酰脲类除草剂活性的能力。产生本发明所述除草剂耐受性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磺酰脲类除草剂(若无特别说明则为一般用量)完全或部分的抗性或耐受性。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受性，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(Academic Press)出版的《Protein》中进行了描述。最常见的互换有Ala/Ser，Val/Ile，Asp/Glu，Thu/Ser，Ala/Thr，Ser/Asn，Ala/Val，Ser/Gly，Tyr/Phe，Ala/Pro，Lys/Arg，Asp/Asn， Leu/Ile，Leu/Val，Ala/Glu和Asp/Gly，以及它们相反的互换。
对于本领域的技术人员而言显而易见地，这种取代可以在对分子功能起重要作用的区域之外发生，而且仍产生活性多肽。对于由本发明的多肽，其活性必需的并因此选择不被取代的氨基酸残基，可以根据本领域已知的方法，如定点诱变或丙氨酸扫描诱变进行鉴定(如参见，Cunningham和Wells，1989，Science 244：1081-1085)。后一技术是在分子中每一个带正电荷的残基处引入突变，检测所得突变分子的除草剂抗性活性，从而确定对该分子活性而言重要的氨基酸残基。底物-酶相互作用位点也可以通过其三维结构的分析来测定，这种三维结构可由核磁共振分析、结晶学或光亲和标记等技术测定(参见，如de Vos等，1992，Science 255：306-312；Smith等，1992，J.Mol.Biol 224：899-904；Wlodaver等，1992，FEBS Letters309：59-64)。
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本发明所述除草剂耐受性基因的调节序列。
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(pin I和pin II)和玉米蛋白酶抑制基因(MPI)的启动子。
所述转运肽(又称分泌信号序列或导向序列)是指导转基因产物到特定的细胞器或细胞区室，对受体蛋白质来说，所述转运肽可以是异源的，例如，利用编码叶绿体转运肽序列靶向叶绿体，或者利用‘KDEL’保留序列靶向内质网，或者利用大麦植物凝集素基因的CTPP靶向液泡。
所述前导序列包含但不限于，小RNA病毒前导序列，如EMCV前导序列(脑 心肌炎病毒5’非编码区)；马铃薯Y病毒组前导序列，如MDMV(玉米矮缩花叶病毒)前导序列；人类免疫球蛋白质重链结合蛋白质(BiP)；苜蓿花叶病毒的外壳蛋白质mRNA的不翻译前导序列(AMV RNA4)；烟草花叶病毒(TMV)前导序列。
所述增强子包含但不限于，花椰菜花叶病毒(CaMV)增强子、玄参花叶病毒(FMV)增强子、康乃馨风化环病毒(CERV)增强子、木薯脉花叶病毒(CsVMV)增强子、紫茉莉花叶病毒(MMV)增强子、夜香树黄化曲叶病毒(CmYLCV)增强子、木尔坦棉花曲叶病毒(CLCuMV)、鸭跖草黄斑驳病毒(CoYMV)和花生褪绿线条花叶病毒(PCLSV)增强子。
对于单子叶植物应用而言，所述内含子包含但不限于，玉米hsp70内含子、玉米泛素内含子、Adh内含子1、蔗糖合酶内含子或水稻Act1内含子。对于双子叶植物应用而言，所述内含子包含但不限于，CAT-1内含子、pKANNIBAL内含子、PIV2内含子和“超级泛素”内含子。
所述终止子可以为在植物中起作用的适合多聚腺苷酸化信号序列，包括但不限于，来源于农杆菌(Agrobacterium tumefaciens)胭脂碱合成酶(NOS)基因的多聚腺苷酸化信号序列、来源于蛋白酶抑制剂II(pin II)基因的多聚腺苷酸化信号序列、来源于豌豆ssRUBISCO E9基因的多聚腺苷酸化信号序列和来源于α-微管蛋白(α-tubulin)基因的多聚腺苷酸化信号序列。
本发明中所述“有效连接”表示核酸序列的联结，所述联结使得一条序列可提供对相连序列来说需要的功能。在本发明中所述“有效连接”可以为将启动子与感兴趣的序列相连，使得该感兴趣的序列的转录受到该启动子控制和调控。当感兴趣的序列编码蛋白并且想要获得该蛋白的表达时“有效连接”表示：启动子与所述序列相连，相连的方式使得得到的转录物高效翻译。如果启动子与编码序列的连接是转录物融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得得到的转录物中第一翻译起始密码子是编码序列的起始密码子。备选地，如果启动子与编码序列的连接是翻译融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得5’非翻译序列中含有的第一翻译起始密码子与启动子相连结，并且连接方式使得得到的翻译产物与编码想要的蛋白的翻译开放读码框的关系是符合读码框的。可以“有效连接”的核酸序列包括但不限于：提供基因表达功能的序列(即基因表达元件，例如启动子、5’非翻译区域、内含子、蛋白编码区域、3’非翻译区域、聚腺苷化位点和/或转录终止子)、提供DNA转移和/或整合功能的序列(即 T-DNA边界序列、位点特异性重组酶识别位点、整合酶识别位点)、提供选择性功能的序列(即抗生素抗性标记物、生物合成基因)、提供可计分标记物功能的序列、体外或体内协助序列操作的序列(即多接头序列、位点特异性重组序列)和提供复制功能的序列(即细菌的复制起点、自主复制序列、着丝粒序列)。
本发明可赋予植物新除草剂抗性性状，并且未观察到对表型包括产量的不良影响。本发明中植物能耐受住如至少一种受试除草剂2×、3×、4×或8×一般应用水平。这些耐受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。
本发明所述除草剂耐受性蛋白质可以为具有SEQ ID NO：1所示氨基酸序列的蛋白质，且其至少在SEQ ID NO：1第176位具有丙氨酸替换和/或第178位具有缬氨酸替换，示例如序列表中SEQ ID NO：7、SEQ ID NO：11或SEQ ID NO：15所示。本发明所述除草剂耐受性基因可以为上述除草剂耐受性蛋白质的编码基因，示例如序列表中SEQ ID NO：8-10、SEQ ID NO：12-14和SEQ ID NO：16-18所示。
本发明所述除草剂耐受性蛋白质可以为具有SEQ ID NO：19所示氨基酸序列的蛋白质，且其至少在SEQ ID NO：19第140位具有丙氨酸替换和/或第142位具有缬氨酸替换，示例如序列表中SEQ ID NO：23、SEQ ID NO：27或SEQ ID NO：31所示。本发明所述除草剂耐受性基因可以为上述除草剂耐受性蛋白质的编码基因，示例如序列表中SEQ ID NO：24-26、SEQ ID NO：28-30和SEQ ID NO：32-34所示。
本发明所述除草剂耐受性蛋白质可以为具有SEQ ID NO：35所示氨基酸序列的蛋白质，且其至少在SEQ ID NO：35第140位具有丙氨酸替换和/或第142位具有缬氨酸替换，示例如序列表中SEQ ID NO：39、SEQ ID NO：43或SEQ ID NO：47所示。本发明所述除草剂耐受性基因可以为上述除草剂耐受性蛋白质的编码基因，示例如序列表中SEQ ID NO：40-42、SEQ ID NO：44-46和SEQ ID NO：48-50所示。
本发明所述除草剂耐受性蛋白质可以为具有SEQ ID NO：51所示氨基酸序列的蛋白质，且其至少在SEQ ID NO：51第131位具有丙氨酸替换和/或第133位具有缬氨酸替换，示例如序列表中SEQ ID NO：55、SEQ ID NO：59或SEQ ID NO：63所示。本发明所述除草剂耐受性基因可以为上述除草剂耐受性蛋白质的编码基因，示例如序列表中SEQ ID NO：56-58、SEQ ID NO：60-62和SEQ ID NO：64-66所示。
本发明所述除草剂耐受性基因可用于植物，除了包含本发明所述除草剂耐受性蛋白质的编码区外，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。
本发明所述除草剂耐受性蛋白质对大多数磺酰脲类除草剂具有耐受性。本发明中的植物，在其基因组中含有外源DNA，所述外源DNA包含本发明所述除草剂耐受性基因，通过表达有效量的该蛋白而保护其免受磺酰脲类除草剂的威胁。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、本发明除草剂耐受性蛋白质对磺酰脲类除草剂的耐受性强，特别是可以耐受8倍大田浓度的苯磺隆。
2、本发明除草剂耐受性蛋白质在植物上应用前景广阔。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图说明
图1为本发明除草剂耐受性蛋白质、其编码基因及用途的含有ALT02M1-01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ALT02M1-01核苷酸序列的重组表达载体DBN100825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对照重组表达载体DBN100828N结构示意图；
图4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大豆T 1植株对苯磺酸耐受性效果图；其中A为ALT02-01转基因大豆植株；B为ALT02M1-01转基因大豆植株；C为ALT02M2-01转基因大豆植株；D为ALT02M3-01转基因大豆植株；E为对照大豆植株；F为野生型大豆植株；
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ALT02M1-02核苷酸序列的重组克隆载体DBN02-T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ALT02M1-02核苷酸序列的重组表达载体DBN100833构建流程图；
图7为本发明除草剂耐受性蛋白质、其编码基因及用途的对照重组表达载体DBN100830N结构示意图；
图8为本发明除草剂耐受性蛋白质、其编码基因及用途的转基因玉米T 1植株对苯磺酸耐受性效果图；其中A为ALT02-02转基因玉米植株；B为ALT02M1-02转基因玉米植株；C为ALT02M2-02转基因玉米植株；D为ALT02M3-02转基因玉米植株；E为对照玉米植株；F为野生型玉米植株。
具体实施方式
下面通过具体实施例进一步说明本发明除草剂耐受性蛋白质、其编码基因及用途的技术方案。
第一实施例、ALT基因的突变和筛选
1、合成ALT基因
合成所述ALT01基因的核苷酸序列(1197个核苷酸)，如序列表中SEQ ID NO：2所示，其编码ALT01蛋白(398个氨基酸)，如序列表中SEQ ID NO：1所示；合成的所述ALT01基因的核苷酸序列(SEQ ID NO：2)的5’端连接有Spe I酶切位点，3’端连接有Kas I酶切位点。依据大豆偏好性密码子获得编码相应于所述ALT01的氨基酸序列的ALT01-01核苷酸序列，如序列表中SEQ ID NO：3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ALT01的氨基酸序列的ALT01-02核苷酸序列，如序列表中SEQ ID NO：4所示。
2、构建ALT01基因突变文库
将上述合成的ALT01基因经PCR扩增后，按照Promega公司产品pGEM-T载体(Promega，Madison，USA，CAT：A3600)说明书的操作步骤，克隆到载体pGEM-T上，然后将上述连接后的产物导入大肠杆菌DH5α作为模板，用引物1和引物2进行易错PCR，使得ALT01基因由于碱基随机错配而发生突变。引物和易错PCR反应体系如下：
引物1：atggaaaccgataaaaaaaccg，如序列表中SEQ ID NO：5所示；
引物2：tcagctttcgttctgatctaag，如序列表中SEQ ID NO：6所示；
易错PCR反应体系(总体积50μL)为：
Figure PCTCN2018124916-appb-000001
Figure PCTCN2018124916-appb-000002
所述质粒DNA模板的浓度为1-10ng/μL，所述引物1的浓度为10μM，所述引物2的浓度为10μM，并且在4℃，贮藏在琥珀试管中。
易错PCR反应条件为：
Figure PCTCN2018124916-appb-000003
将上述易错PCR产物，温度42℃下热击转化到对苯磺隆敏感的大肠杆菌DH10B ilvG +中，构建ALT01基因随机突变文库。
3、对ALT01基因突变文库进行筛选
将上述突变文库中的转化产物接种于含有浓度为200mg/L苯磺隆的筛选培养基(葡萄糖5g/L、氨苄青霉素100mg/L、缬氨酸200mg/L、亮氨酸200mg/L、(NH4) 28O 4 2g/L、MgSO 4·7H 2O 200mg/L、CaCl 2·2H 2O 10mg/L、FeSO 4·7H 2O1mg/L、Na 2HPO 4·12H 2O 1.5g/L和KH 2PO 4 1.5g/L)上，在温度37℃下培养24h。
鉴于抗性基因能够将苯磺隆转化为对细菌无毒的苯磺酸，因而利用所述原理对上述突变文库进行高通量筛选，分离在上述含有浓度为200mg/L苯磺隆的筛选培养基上仍能生长的大肠杆菌DH10B ilvG +以获得抗性基因。
4、获得突变的抗性基因
测序结果表明获得三个ALT01突变的抗性基因，分别命名为ALT01M1、ALT01M2和ALT01M3基因，所述ALT01M1核苷酸序列第527位由原来的g突变为c，导致其氨基酸序列第176位由原来的甘氨酸突变为丙氨酸；所述ALT01M2核苷酸序列第532和533位由原来的tc突变为gt，导致其氨基酸序列第178位由原来的丝氨酸突变为缬氨酸；所述ALT01M3核苷酸序列第239-242位由原来的cata依次突变为gagc，且第527-544位由原来的gaaactccagtaaagaag突变为caaacgtcagtaaagaaa，导致其氨基酸序列第80-81位由原来的脯氨酸和酪氨酸突变为精氨酸和丙氨酸，且第176、178和182位由原来的甘氨酸、丝氨酸和甘氨酸突变为丙氨酸、缬氨酸和精氨酸。
除草剂耐受性蛋白质ALT01M1的氨基酸序列，如序列表中SEQ ID NO：7所示，编码相应于所述除草剂耐受性蛋白质ALT01M1的氨基酸序列的ALT01M1核苷酸序列，如序列表中SEQ ID NO：8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ALT01M1的氨基酸序列的ALT01M1-01核苷酸序列，如序列表中SEQ ID NO：9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ALT01M1的氨基酸序列的ALT01M1-02核苷酸序列，如序列表中SEQ ID NO：10所示。
除草剂耐受性蛋白质ALT01M2的氨基酸序列，如序列表中SEQ ID NO：11所示，编码相应于所述除草剂耐受性蛋白质ALT01M2的氨基酸序列的ALT01M2核苷酸序列，如序列表中SEQ ID NO：12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ALT01M2的氨基酸序列的ALT01M2-01核苷酸序列，如序列表中SEQ ID NO：13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ALT01M2的氨基酸序列的ALT01M2-02核苷酸序列，如序列表中SEQ ID NO：14所示。
除草剂耐受性蛋白质ALT01M3的氨基酸序列，如序列表中SEQ ID NO：15所示，编码相应于所述除草剂耐受性蛋白质ALT01M3的氨基酸序列的ALT01M3核苷酸序列，如序列表中SEQ ID NO：16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ALT01M3的氨基酸序列的ALT01M3-01核苷酸序列，如序列表中SEQ ID NO：17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ALT01M3的氨基酸序列的ALT01M3-02核苷酸序列，如序列表中SEQ ID NO：18所示。
合成下述核苷酸序列：
ALT02的氨基酸序列(369个氨基酸)，如序列表中SEQ ID NO：19所示；编码相应于所述ALT02的氨基酸序列的ALT02核苷酸序列(1110个核苷酸)，如序列表中SEQ ID NO：20所示；依据大豆偏好性密码子获得编码相应于所述ALT02的氨基酸序列的ALT02-01核苷酸序列，如序列表中SEQ ID NO：21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ALT02的氨基酸序列的ALT02-02核苷酸序列，如序列表中SEQ ID NO：22所示。
除草剂耐受性蛋白质ALT02M1为所述ALT02的氨基酸序列第140位由原来的甘氨酸突变为丙氨酸，所述ALT02M1的氨基酸序列如序列表中SEQ ID NO：23所示，编码相应于所述除草剂耐受性蛋白质ALT02M1的氨基酸序列的ALT02M1 核苷酸序列，如序列表中SEQ ID NO：24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ALT02M1的氨基酸序列的ALT02M1-01核苷酸序列，如序列表中SEQ ID NO：25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ALT02M1的氨基酸序列的ALT02M1-02核苷酸序列，如序列表中SEQ ID NO：26所示。
除草剂耐受性蛋白质ALT02M2为所述ALT02的氨基酸序列第142位由原来的丝氨酸突变为缬氨酸，所述ALT02M2的氨基酸序列如序列表中SEQ ID NO：27所示，编码相应于所述除草剂耐受性蛋白质ALT02M2的氨基酸序列的ALT02M2核苷酸序列，如序列表中SEQ ID NO：28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ALT02M2的氨基酸序列的ALT02M2-01核苷酸序列，如序列表中SEQ ID NO：29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ALT02M2的氨基酸序列的ALT02M2-02核苷酸序列，如序列表中SEQ ID NO：30所示。
除草剂耐受性蛋白质ALT02M3为所述ALT02的氨基酸序列第44-45位由原来的脯氨酸和酪氨酸突变为精氨酸和丙氨酸，且第140、142和146位由原来的甘氨酸、丝氨酸和甘氨酸突变为丙氨酸、缬氨酸和精氨酸，所述ALT02M3的氨基酸序列如序列表中SEQ ID NO：31所示，编码相应于所述除草剂耐受性蛋白质ALT02M3的氨基酸序列的ALT02M3核苷酸序列，如序列表中SEQ ID NO：32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ALT02M3的氨基酸序列的ALT02M3-01核苷酸序列，如序列表中SEQ ID NO：33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ALT02M3的氨基酸序列的ALT02M3-02核苷酸序列，如序列表中SEQ ID NO：34所示。
ALT03的氨基酸序列(362个氨基酸)，如序列表中SEQ ID NO：35所示；编码相应于所述ALT03的氨基酸序列的ALT03核苷酸序列(1089个核苷酸)，如序列表中SEQ ID NO：36所示；依据大豆偏好性密码子获得编码相应于所述ALT03的氨基酸序列的ALT03-01核苷酸序列，如序列表中SEQ ID NO：37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ALT03的氨基酸序列的ALT03-02核苷酸序列，如序列表中SEQ ID NO：38所示。
除草剂耐受性蛋白质ALT03M1为所述ALT03的氨基酸序列第140位由原来的甘氨酸突变为丙氨酸，所述ALT03M1的氨基酸序列如序列表中SEQ ID NO：39所示，编码相应于所述除草剂耐受性蛋白质ALT03M1的氨基酸序列的ALT03M1 核苷酸序列，如序列表中SEQ ID NO：40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ALT03M1的氨基酸序列的ALT03M1-01核苷酸序列，如序列表中SEQ ID NO：41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ALT03M1的氨基酸序列的ALT03M1-02核苷酸序列，如序列表中SEQ ID NO：42所示。
除草剂耐受性蛋白质ALT03M2为所述ALT03的氨基酸序列第142位由原来的丝氨酸突变为缬氨酸，所述ALT03M2的氨基酸序列如序列表中SEQ ID NO：43所示，编码相应于所述除草剂耐受性蛋白质ALT03M2的氨基酸序列的ALT03M2核苷酸序列，如序列表中SEQ ID NO：44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ALT03M2的氨基酸序列的ALT03M2-01核苷酸序列，如序列表中SEQ ID NO：45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ALT03M2的氨基酸序列的ALT03M2-02核苷酸序列，如序列表中SEQ ID NO：46所示。
除草剂耐受性蛋白质ALT03M3为所述ALT03的氨基酸序列第44-45位由原来的脯氨酸和酪氨酸突变为精氨酸和丙氨酸，且第140、142和146位由原来的甘氨酸、丝氨酸和甘氨酸突变为丙氨酸、缬氨酸和精氨酸，所述ALT03M3的氨基酸序列如序列表中SEQ ID NO：47所示，编码相应于所述除草剂耐受性蛋白质ALT03M3的氨基酸序列的ALT03M3核苷酸序列，如序列表中SEQ ID NO：48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ALT03M3的氨基酸序列的ALT03M3-01核苷酸序列，如序列表中SEQ ID NO：49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ALT03M3的氨基酸序列的ALT03M3-02核苷酸序列，如序列表中SEQ ID NO：50所示。
ALT04的氨基酸序列(350个氨基酸)，如序列表中SEQ ID NO：51所示；编码相应于所述ALT04的氨基酸序列的ALT04核苷酸序列(1053个核苷酸)，如序列表中SEQ ID NO：52所示；依据大豆偏好性密码子获得编码相应于所述ALT04的氨基酸序列的ALT04-01核苷酸序列，如序列表中SEQ ID NO：53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ALT04的氨基酸序列的ALT04-02核苷酸序列，如序列表中SEQ ID NO：54所示。
除草剂耐受性蛋白质ALT04M1为所述ALT04的氨基酸序列第131位由原来的甘氨酸突变为丙氨酸，所述ALT04M1的氨基酸序列如序列表中SEQ ID NO：55所示，编码相应于所述除草剂耐受性蛋白质ALT04M1的氨基酸序列的ALT04M1 核苷酸序列，如序列表中SEQ ID NO：56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ALT04M1的氨基酸序列的ALT04M1-01核苷酸序列，如序列表中SEQ ID NO：57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ALT04M1的氨基酸序列的ALT04M1-02核苷酸序列，如序列表中SEQ ID NO：58所示。
除草剂耐受性蛋白质ALT04M2为所述ALT04的氨基酸序列第133位由原来的丝氨酸突变为缬氨酸，所述ALT04M2的氨基酸序列如序列表中SEQ ID NO：59所示，编码相应于所述除草剂耐受性蛋白质ALT04M2的氨基酸序列的ALT04M2核苷酸序列，如序列表中SEQ ID NO：60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ALT04M2的氨基酸序列的ALT04M2-01核苷酸序列，如序列表中SEQ ID NO：61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ALT04M2的氨基酸序列的ALT04M2-02核苷酸序列，如序列表中SEQ ID NO：62所示。
除草剂耐受性蛋白质ALT04M3为所述ALT04的氨基酸序列第35-36位由原来的脯氨酸和酪氨酸突变为精氨酸和丙氨酸，且第131、133和137位由原来的甘氨酸、丝氨酸和甘氨酸突变为丙氨酸、缬氨酸和精氨酸，所述ALT04M3的氨基酸序列如序列表中SEQ ID NO：63所示，编码相应于所述除草剂耐受性蛋白质ALT04M3的氨基酸序列的ALT04M3核苷酸序列，如序列表中SEQ ID NO：64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ALT04M3的氨基酸序列的ALT04M3-01核苷酸序列，如序列表中SEQ ID NO：65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ALT04M3的氨基酸序列的ALT04M3-02核苷酸序列，如序列表中SEQ ID NO：66所示。
第二实施例、蛋白表达及纯化
1、基因的PCR扩增
设计一对引物：
引物3：tgcaga catatggaaaccgataaaaaaac(下划线为Nde I酶切位点)，如序列表中SEQ ID NO：67所示；
引物4： cccaagcttctagctttcgttctgatctaagccgtgc(下划线为Hind III酶切位点)，如序列表中SEQ ID NO：68所示；
用下述PCR扩增体系扩增所述ALT01M1基因(末端含Nde I和Hind III酶切位点)：
Figure PCTCN2018124916-appb-000004
PCR反应条件为：98℃变性1min；然后进入下列循环：98℃变性15s，55℃退火15s，72℃延伸1min，共29个循环；最后72℃延伸10min，冷却至室温。
按照上述PCR扩增方法扩增末端含Nde I和Hind III酶切位点的所述ALT01M2核苷酸序列、ALT01M3核苷酸序列、ALT01核苷酸序列、ALT03M1核苷酸序列、ALT03M2核苷酸序列、ALT03M3核苷酸序列、ALT03核苷酸序列、ALT04M1核苷酸序列、ALT04M2核苷酸序列、ALT04M3核苷酸序列和ALT04核苷酸序列；合成ALT02M1核苷酸序列、ALT02M2核苷酸序列、ALT02M3核苷酸序列、ALT02核苷酸序列(末端均分别含Nde I和Hind III酶切位点)。
2、细菌表达载体的构建和重组微生物获得
用限制性内切酶Nde I和Hind III分别酶切上述PCR扩增产物(末端含Nde I和Hind III酶切位点的所述ALT01M1核苷酸序列、ALT01M2核苷酸序列、ALT01M3核苷酸序列、ALT01核苷酸序列、ALT02M1核苷酸序列、ALT02M2核苷酸序列、ALT02M3核苷酸序列、ALT02核苷酸序列、ALT03M1核苷酸序列、ALT03M2核苷酸序列、ALT03M3核苷酸序列、ALT03核苷酸序列、ALT04M1核苷酸序列、ALT04M2核苷酸序列、ALT04M3核苷酸序列和ALT04核苷酸序列)和细菌表达载体pET-30a(+)，将切下的上述基因片段分别与酶切后的细菌表达载体pET-30a(+)进行酶连，将酶连产物分别转化到表达宿主菌BL21(DE3)，获得重组微生物BL21(ALT01M1)、BL21(ALT01M2)、BL21(ALT01M3)、BL21(ALT01)、BL21(ALT02M1)、BL21(ALT02M2)、BL21(ALT02M3)、BL21(ALT02)、BL21(ALT03M1)、BL21(ALT03M2)、BL21(ALT03M3)、BL21(ALT03)、BL21(ALT04M1)、BL21(ALT04M2)、BL21(ALT04M3)和BL21(ALT04)。
3、除草剂耐受性蛋白质在大肠杆菌中的表达及纯化
所述重组微生物BL21(ALT01M1)、BL21(ALT01M2)、BL21(ALT01M3)、BL21(ALT01)、BL21(ALT02M1)、BL21(ALT02M2)、BL21(ALT02M3)、BL21(ALT02)、BL21(ALT03M1)、BL21(ALT03M2)、BL21(ALT03M3)、BL21(ALT03)、BL21(ALT04M1)、BL21(ALT04M2)、BL21(ALT04M3)和BL21(ALT04)分别在100mL的LB培养基(胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L、氨苄青霉素100mg/L，用NaOH调pH至7.5)中培养至浓度为OD 600nm＝0.6-0.8，加入浓度为0.4mM的异丙基硫代半乳糖苷(IPTG)，在温度16℃下诱导20h。离心，收集菌体，用20mL Tris-HCl buffer(100mM，pH 8.0)重悬菌体，超声破碎(X0-900D ultrasonic processor ultrasonic processor，30％intensity)10min，然后离心，收集上清，用镍离子亲和层析柱分别对获得的上述除草剂耐受性蛋白质进行纯化，用SDS-PAGE蛋白电泳检测纯化结果，条带大小和理论预测的条带大小一致。
第三实施例、测定除草剂耐受性蛋白质的酶活力
酶活反应体系(1mL)：含有0.2μg反应酶(上述纯化获得的所述除草剂耐受性蛋白质ALT01M1、ALT01M2、ALT01M3、ALT01、ALT02M1、ALT02M2、ALT02M3、ALT02、ALT03M1、ALT03M2、ALT03M3、ALT03、ALT04M1、ALT04M2、ALT04M3和ALT04)、0.2mM噻吩磺隆(甲磺隆、氯嘧磺隆、苄嘧磺隆、甲嘧磺隆或苯磺隆)，缓冲体系为浓度50mM的磷酸缓冲液(pH7.0)，温度30℃下在水浴锅中反应20min，每个反应以加入反应酶开始计时，用1mL二氯甲烷终止反应，分层后有机相经无水硫酸钠脱水。
将上述脱水后的反应液用氮气吹干后，加入1mL甲醇过滤，取20μL滤液进行液相色谱-质谱联用(LC-MS)，高效液相色谱(HPLC)条件为：流动相为甲醇∶水(80∶20，V/V)，Zorbax XDB-C18色谱柱(3.5μm，2.1×50mm，Agilent，USA)，柱温为室温，紫外检测器，测定波长为255nm，进样量为20μL，流速为0.25mL/min。一级离子质谱条件为：离子检测方式为多反应离子检测；离子极性为负离子；离子化方式为电喷雾离子化；毛细管电压为4000伏；干燥气温度330℃，流速10L/min，雾化气压力35psi，碰撞电压135伏；质量扫描范围300-500m/z。二级离子质谱条件为：碰撞电压90伏；质量扫描范围30-400m/z，其它条件与一级离子质谱条件相同。经LC-MS鉴定，噻吩磺隆的代谢产物为噻磺酸，甲磺隆、氯嘧磺隆、苄嘧磺隆、甲嘧磺隆或苯磺隆的代谢产物为其对应的磺酸。高效液相色谱(HPLC)检测代谢产物噻磺酸的生成量。一个酶活力单位定义为：在pH7.0、 温度30℃条件下1min内降解噻吩磺隆(甲磺隆、氯嘧磺隆、苄嘧磺隆、甲嘧磺隆或苯磺隆)催化减少1μmol噻吩磺隆(甲磺隆、氯嘧磺隆、苄嘧磺隆、甲嘧磺隆或苯磺隆)所需要酶的量，以U表示，实验结果如表1所示。
表1、除草剂耐受性蛋白质降解磺酰脲类除草剂的实验结果
Figure PCTCN2018124916-appb-000005
上述实验结果表明：与所述除草剂耐受性蛋白质ALT01相比，纯化后的所述除草剂耐受性蛋白质ALT01M1对苯磺隆、苄嘧磺隆和噻吩磺隆的降解效率分别为ALT01的1.7、2.3和3.3倍；纯化后的所述除草剂耐受性蛋白质ALT01M2对苯磺隆、苄嘧磺隆和噻吩磺隆降解效率分别为ALT01的6.0、1.4和3.9倍；纯化后的所述除草剂耐受性蛋白质ALT01M3对苯磺隆、甲磺隆和氯嘧磺隆的降解效率分别为ALT01的1.9、2.1和14.2倍。
与所述除草剂耐受性蛋白质ALT02相比，纯化后的所述除草剂耐受性蛋白质ALT02M1对苯磺隆、苄嘧磺隆和噻吩磺隆的降解效率分别为ALT02的1.7、2.3和3.3倍；纯化后的所述除草剂耐受性蛋白质ALT02M2对苯磺隆、苄嘧磺隆和噻 吩磺隆降解效率分别为ALT02的5.9、1.4和3.9倍；纯化后的所述除草剂耐受性蛋白质ALT02M3对苯磺隆、甲磺隆和氯嘧磺隆的降解效率分别为ALT02的1.8、2.1和14.2倍。
与所述除草剂耐受性蛋白质ALT03相比，纯化后的所述除草剂耐受性蛋白质ALT03M1对苯磺隆、苄嘧磺隆和噻吩磺隆的降解效率分别为ALT03的1.5、2.1和3.0倍；纯化后的所述除草剂耐受性蛋白质ALT03M2对苯磺隆、苄嘧磺隆和噻吩磺隆降解效率分别为ALT03的5.4、1.3和3.5倍；纯化后的所述除草剂耐受性蛋白质ALT03M3对苯磺隆、甲磺隆和氯嘧磺隆的降解效率分别为ALT03的1.6、1.9和13.0倍。
与所述除草剂耐受性蛋白质ALT04相比，纯化后的所述除草剂耐受性蛋白质ALT04M1对苯磺隆、苄嘧磺隆和噻吩磺隆的降解效率分别为ALT04的1.5、1.9和2.8倍；纯化后的所述除草剂耐受性蛋白质ALT04M2对苯磺隆、苄嘧磺隆和噻吩磺隆降解效率分别为ALT04的5.1、1.2和3.3倍；纯化后的所述除草剂耐受性蛋白质ALT04M3对苯磺隆、甲磺隆和氯嘧磺隆的降解效率分别为ALT04的1.6、1.8和12.4倍。
由此可见，所述除草剂耐受性蛋白质ALT01的氨基酸序列第176位由甘氨酸突变为丙氨酸和/或第178位由丝氨酸突变为缬氨酸均能提升突变基因(如所述ALT01M1、ALT01M2或ALT01M3基因)对磺酰脲类除草剂的降解能力，特别是苯磺隆。所述除草剂耐受性蛋白质ALT02(或ALT03)的氨基酸序列第140位由甘氨酸突变为丙氨酸和/或第142位由丝氨酸突变为缬氨酸均能提升突变基因(如所述ALT02M1、ALT02M2、ALT02M3、ALT03M1、ALT03M2或ALT03M3基因)对磺酰脲类除草剂的降解能力，特别是苯磺隆。所述除草剂耐受性蛋白质ALT04的氨基酸序列第131位由甘氨酸突变为丙氨酸和/或第133位由丝氨酸突变为缬氨酸均能提升突变基因(如所述ALT04M1、ALT04M2或ALT04M3基因)对磺酰脲类除草剂的降解能力，特别是苯磺隆。
第四实施例、大豆重组表达载体的构建
1、构建含有ALT02M1-01核苷酸序列的大豆重组克隆载体
将所述ALT02M1-01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；ALT02M1-01为ALT02M1-01核苷酸序列(SEQ ID NO：25)；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30s；37℃振荡培养1h(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、NaCl 10g/L、琼脂15g/L，用NaOH调pH至7.5)上生长过夜。挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L、氨苄青霉素100mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒：将菌液在12000rpm转速下离心1min，去上清液，沉淀菌体用100μL冰预冷的溶液I(25mM Tris-HCl、10mM EDTA(乙二胺四乙酸)、50mM葡萄糖，pH8.0)悬浮；加入200μL新配制的溶液II(0.2M NaOH、1％SDS(十二烷基硫酸钠))，将管子颠倒4次，混合，置冰上3-5min；加入150μL冰冷的溶液III(3M醋酸钾、5M醋酸)，立即充分混匀，冰上放置5-10min；于温度4℃、转速12000rpm条件下离心5min，在上清液中加入2倍体积无水乙醇，混匀后室温放置5min；于温度4℃、转速12000rpm条件下离心5min，弃上清液，沉淀用浓度(V/V)为70％的乙醇洗涤后晾干；加入30μL含RNase(20μg/mL)的TE(10mM Tris-HCl、1mM EDTA，pH8.0)溶解沉淀；于温度37℃下水浴30min，消化RNA；于温度-20℃保存备用。
提取的质粒经Spe I和Kas I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ALT02M1-01核苷酸序列为序列表中SEQ ID NO：25所示的核苷酸序列，即ALT02M1-01核苷酸序列正确插入。
2、构建含有ALT02M1-01核苷酸序列的大豆重组表达载体
用限制性内切酶Spe I和Kas I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ALT02M1-01核苷酸序列片段插到表达载体DBNBC-01的Spe I和Kas I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DBN100825，其构建流程如图2所示(Spec：壮观霉素基因；RB：右边界；prAtUbi10：拟南芥泛素(Ubiquitin)10基因启动子(SEQ ID NO：69)；ALT02M1-01：ALT02M1-01核苷酸序列(SEQ ID NO：25)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO：70)； prBrCBP：油菜真核延长因子基因1α(Tsf1)启动子(SEQ ID NO：71)；spAtCTP2：拟南芥叶绿体转运肽(SEQ ID NO：72)；EPSPS：5-烯醇丙酮酸莽草酸-3-磷酸合酶基因(SEQ ID NO：73)；tPsE9：豌豆RbcS基因的终止子(SEQ ID NO：74)；LB：左边界)。
将重组表达载体DBN100825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DBN100825)，42℃水浴30s；37℃振荡培养1h(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、NaCl 10g/L、琼脂15g/L，用NaOH调pH至7.5)上于温度37℃条件下培养12h，挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L、酵母提取物5g/L、NaCl 10g/L、壮观霉素50mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒。将提取的质粒用限制性内切酶Spe I和Kas I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DBN100825在Spe I和Kas I位点间的核苷酸序列为序列表中SEQ ID NO：25所示核苷酸序列，即ALT02M1-01核苷酸序列。
按照上述构建含有ALT02M1-01核苷酸序列的重组表达载体DBN100825的方法，构建含有ALT02M2-01核苷酸序列的重组表达载体DBN100826、含有ALT02M3-01核苷酸序列的重组表达载体DBN100827和含有ALT02-01核苷酸序列的重组表达载体DBN100828。对阳性克隆进行测序验证，结果表明重组表达载体DBN100825、DBN100826、DBN100827和DBN100828中插入的ALT02M2-01、ALT02M3-01和ALT02-01核苷酸序列分别为序列表中SEQ ID NO：29、SEQ ID NO：33和SEQ ID NO：21所示的核苷酸序列，即ALT02M2-01核苷酸序列、ALT02M3-01核苷酸序列和ALT02-01核苷酸序列正确插入。
按照上述构建含有ALT02M1-01核苷酸序列的重组表达载体DBN100825的方法，构建对照重组表达载体DBN100828N，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；prBrCBP：油菜真核延长因子基因1α(Tsf1)启动子(SEQ ID NO：71)；spAtCTP2：拟南芥叶绿体转运肽(SEQ ID NO：72)；EPSPS：5-烯醇丙酮酸莽草酸-3-磷酸合酶基因(SEQ ID NO：73)；tPsE9：豌豆RbcS基因的终止子(SEQ ID NO：74)；LB：左边界)。对阳性克隆进行测序验证，结果表明对照重组表达载体DBN100828N构建正确。
3、重组表达载体转化农杆菌
对已经构建正确的重组表达载体DBN100825、DBN100826、DBN100827、DBN100828和DBN100828N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10min，37℃温水浴10min；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2h，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DBN100825、DBN100826、DBN100827、DBN100828和DBN100828N结构完全正确。
第五实施例、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的子叶节组织与第四实施例中3所述的农杆菌共培养，以将第四实施例中2构建的重组表达载体DBN100825、DBN100826、DBN100827、DBN100828和DBN100828N中的T-DNA(包括拟南芥Ubiquitin10基因的启动子序列、ALT02M1-01核苷酸序列、ALT02M2-01核苷酸序列、ALT02M3-01核苷酸序列、ALT02-01核苷酸序列、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ALT02M1-01核苷酸序列的大豆植株、转入ALT02M2-01核苷酸序列的大豆植株、转入ALT02M3-01核苷酸序列的大豆植株和转入ALT02-01核苷酸序列的大豆植株；同时以转入对照重组表达载体DBN100828N中的T-DNA的对照大豆植株和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3-4mm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ALT02M1-01核苷酸序列(ALT02M2-01核苷酸序列、ALT02M3-01核苷酸序列或ALT02-01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(OD 660＝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、MES 4g/L、ZT 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、MES 1g/L、蔗糖30g/L、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)中至少存在一种已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、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、MES 1g/L、蔗糖30g/L、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、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16h，再于20℃下培养8h。
2、用TaqMan验证转基因大豆植株
分别取转入ALT02M1-01核苷酸序列的大豆植株、转入ALT02M2-01核苷酸序列的大豆植株、转入ALT02M3-01核苷酸序列的大豆植株、转入ALT02-01核苷酸序列的大豆植株和对照大豆植株的叶片约100mg作为样品，用Qiagen的DNeasy  Plant Maxi Kit提取其基因组DNA，通过Taqman探针荧光定量PCR方法检测EPSPS基因拷贝数以确定目的基因的拷贝数。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21、分别取转入ALT02M1-01核苷酸序列的大豆植株、转入ALT02M2-01核苷酸序列的大豆植株、转入ALT02M3-01核苷酸序列的大豆植株、转入ALT02-01核苷酸序列的大豆植株、对照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22、使用Qiagen的DNeasy Plant Mini Kit提取上述样品的基因组DNA，具体方法参考其产品说明书；
步骤23、用NanoDrop 2000(Thermo Scientific)测定上述样品的基因组DNA浓度；
步骤24、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25、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5：ctggaaggcgaggacgtcatcaata如序列表中SEQ ID NO：75所示；
引物6：tggcggcattgccgaaatcgag如序列表中SEQ ID NO：76所示；
探针1：atgcaggcgatgggcgcccgcatccgta如序列表中SEQ ID NO：77所示；
PCR反应体系为：
Figure PCTCN2018124916-appb-000006
所述50×引物/探针混合物包含1mM浓度的每种引物各45μL，100μM浓度的探针50μL和860μL 1×TE缓冲液，并且在4℃，贮藏在琥珀试管中。
PCR反应条件为：
Figure PCTCN2018124916-appb-000007
Figure PCTCN2018124916-appb-000008
利用SDS2.3软件(Applied Biosystems)分析数据。
通过分析EPSPS基因拷贝数的实验结果，进而证实ALT02M1-01核苷酸序列、ALT02M2-01核苷酸序列、ALT02M3-01核苷酸序列和ALT02-01核苷酸序列均已整合到所检测的大豆植株的染色体组中，而且转入ALT02M1-01核苷酸序列的大豆植株、转入ALT02M2-01核苷酸序列的大豆植株、转入ALT02M3-01核苷酸序列的大豆植株、转入ALT02-01核苷酸序列的大豆植株和对照大豆植株均获得了单拷贝的转基因大豆植株。
第六实施例、转基因大豆植株的除草剂耐受性效果检测
将转入ALT02M1-01核苷酸序列的大豆植株、转入ALT02M2-01核苷酸序列的大豆植株、转入ALT02M3-01核苷酸序列的大豆植株、转入ALT02-01核苷酸序列的大豆植株、对照大豆植株和野生型大豆植株(幼苗期V3-V4)分别对苯磺隆进行除草剂耐受性效果检测。
分别取转入ALT02M1-01核苷酸序列的大豆植株、转入ALT02M2-01核苷酸序列的大豆植株、转入ALT02M3-01核苷酸序列的大豆植株、转入ALT02-01核苷酸序列的大豆植株、对照大豆植株和野生型大豆植株，分别用苯磺隆(144g ai/ha，8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ALT02M1-01核苷酸序列的大豆植株共3个株系(S1、S2和S3)，转入ALT02M2-01核苷酸序列的大豆植株共3个株系(S4、S5和S6)，转入ALT02M3-01核苷酸序列的大豆植株共3个株系(S7、S8和S9)，转入ALT02-01核苷酸序列的大豆植株共3个株系(S10、S11和S12)，对照大豆植株共2个株系(S13和S14)，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和图4所示。
表2、转基因大豆T 1植株除草剂耐受性实验结果
Figure PCTCN2018124916-appb-000009
Figure PCTCN2018124916-appb-000010
对于大豆，8倍大田浓度的苯磺隆是高压力处理的有效剂量。表2和图4的结果表明：所述除草剂耐受性蛋白质ALT02M1-01、ALT02M2-01、ALT02M3-01和ALT02-01均能赋予转基因大豆植物对苯磺酸的耐受性；相比于转入ALT02-01核苷酸序列的大豆植株，转入ALT02M1-01核苷酸序列的大豆植株、转入ALT02M2-01核苷酸序列的大豆植株和转入ALT02M3-01核苷酸序列的大豆植株对苯磺酸均具有显著增高的耐受性；而对照大豆植株和野生型大豆植株则对苯磺酸不具有耐受性。
第七实施例、玉米重组表达载体的构建
1、构建含有ALT02M1-02核苷酸序列的玉米重组克隆载体
将合成的ALT02M1-02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5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；ALT02M1-02为ALT02M1-02核苷酸序列(SEQ ID NO：26)；MCS为多克隆位点)。
按照第四实施例中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Spe I和Kas I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Spe I和Kas I位点间的核苷酸序列为序列表中SEQ ID NO：26所示核苷酸序列，即ALT02M1-02核苷酸序列。
2、构建含有ALT02M1-02核苷酸序列的玉米重组表达载体
用限制性内切酶Spe I和Kas I分别酶切重组克隆载体DBN02-T和表达载体DBNBC-02(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的ALT02M1-02核苷酸序列片段插到表达载体DBNBC-02的Spe I和Kas I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DBN100833，其构建流程如图6所示(Spec：壮观霉素基因；RB：右边界；prUbi：玉米泛素(Ubiquitin)1基因启动子(SEQ ID NO：78)；ALT02M1-02：ALT02M1-02核苷酸序列(SEQ ID NO：26)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO：70)；PMI：磷酸甘露糖异构酶基因(SEQ ID NO：79)；LB：左边界)。
按照第四实施例中2的方法将重组表达载体DBN100833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Spe I和 Kas I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DBN100833在Spe I和Kas I位点间的核苷酸序列为序列表中SEQ ID NO：26所示核苷酸序列，即ALT02M1-02核苷酸序列。
按照上述构建含有ALT02M1-02核苷酸序列的重组表达载体DBN100833的方法，构建含有ALT02M2-02核苷酸序列的重组表达载体DBN100832、含有ALT02M3-02核苷酸序列的重组表达载体DBN100831和含有ALT02-02核苷酸序列的重组表达载体DBN100830。对阳性克隆进行测序验证，结果表明重组表达载体DBN100832、DBN100831和DBN100830中插入的ALT02M2-02、ALT02M3-02和ALT02-02核苷酸序列分别为序列表中SEQ ID NO：30、SEQ ID NO：34和SEQ ID NO：22所示的核苷酸序列，即ALT02M2-02核苷酸序列、ALT02M3-02核苷酸序列和ALT02-02核苷酸序列正确插入。
按照上述构建含有ALT02M1-02核苷酸序列的重组表达载体DBN100833的方法，构建对照重组表达载体DBN100830N，其载体结构如图7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：玉米泛素(Ubiquitin)1基因启动子(SEQ ID NO：78)；PMI：磷酸甘露糖异构酶基因(SEQ ID NO：79)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO：70)；LB：左边界)。对阳性克隆进行测序验证，结果表明对照重组表达载体DBN100830N构建正确。
3、玉米重组表达载体转化农杆菌
对已经构建正确的重组表达载体DBN100833、DBN100832、DBN100831、DBN100830和DBN100830N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10min，37℃温水浴10min；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2h，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DBN100833、DBN100832、DBN100831、DBN100830和DBN100830N结构完全正确。
第八实施例、转基因玉米植株的获得和验证
1、获得转基因玉米植株
按照常规采用的农杆菌侵染法，将无菌培养的玉米品种综31(Z31)的幼胚与第七实施例中3所述的农杆菌共培养，以将第七实施例中2构建的重组表达载体DBN100833、DBN100832、DBN100831、DBN100830和DBN100830N中的T-DNA(包括玉米Ubiquitin1基因的启动子序列、ALT02M1-02核苷酸序列、ALT02M2-02核苷酸序列、ALT02M3-02核苷酸序列、ALT02-02核苷酸序列、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ALT02M1-02核苷酸序列的玉米植株、转入ALT02M2-02核苷酸序列的玉米植株、转入ALT02M3-02核苷酸序列的玉米植株和转入ALT02-02的玉米植株；同时以转入对照重组表达载体DBN100830N中的T-DNA的对照玉米植株和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，其中农杆菌能够将所述ALT02M1-02核苷酸序列(ALT02M2-02核苷酸序列、ALT02M3-02核苷酸序列或ALT02-02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(OD 660＝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、AS100mg/L、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-D 1mg/L、植物凝胶3g/L，pH5.8)中至少存在一种已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-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16h，再于20℃下培养8h。
2、用TaqMan验证转基因玉米植株
按照第五实施例中2用TaqMan验证转基因大豆植株的方法，对转入ALT02M1-02核苷酸序列的玉米植株、转入ALT02M2-02核苷酸序列的玉米植株、转入ALT02M3-02核苷酸序列的玉米植株、转入ALT02-02的玉米植株和对照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基因拷贝数以确定目的基因的拷贝数。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7：gctgtaagagcttactgaaaaaattaaca如序列表中SEQ ID NO：80所示；
引物8：cgatctgcaggtcgacgg如序列表中SEQ ID NO：81所示；
探针2：tctcttgctaagctgggagctcgatcc如序列表中SEQ ID NO：82所示。
通过分析PMI基因拷贝数的实验结果，进而证实ALT02M1-02核苷酸序列、ALT02M2-02核苷酸序列、ALT02M3-02核苷酸序列和ALT02-02核苷酸序列均已整合到所检测的玉米植株的染色体组中，而且转入ALT02M1-02核苷酸序列的玉米植株、转入ALT02M2-02核苷酸序列的玉米植株、转入ALT02M3-02核苷酸序列的玉米植株、转入ALT02-02核苷酸序列的玉米植株和对照玉米植株均获得了单拷贝的转基因玉米植株。
第九实施例、转基因玉米植株的除草剂耐受性效果检测
将转入ALT02M1-02核苷酸序列的玉米植株、转入ALT02M2-02核苷酸序列的玉米植株、转入ALT02M3-02核苷酸序列的玉米植株、转入ALT02-02核苷酸序列的玉米植株、对照玉米植株和野生型玉米植株(V3-V4时期)分别对苯磺隆进行除草剂耐受性效果检测。
分别取转入ALT02M1-02核苷酸序列的玉米植株、转入ALT02M2-02核苷酸序列的玉米植株、转入ALT02M3-02核苷酸序列的玉米植株、转入ALT02-02核苷酸序列的玉米植株、对照玉米植株和野生型玉米植株，分别用苯磺隆(144g ai/ha， 8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ALT02M1-02核苷酸序列的玉米植株共3个株系(S15、S16和S17)，转入ALT02M2-02核苷酸序列的玉米植株共3个株系(S18、S19和S20)，转入ALT02M3-02核苷酸序列的玉米植株共3个株系(S21、S22和S23)，转入ALT02-02核苷酸序列的玉米植株共3个株系(S24、S25和S26)，对照玉米植株共2个株系(S27和S28)，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和图8所示。
表3、转基因玉米T 1植株除草剂耐受性实验结果
Figure PCTCN2018124916-appb-000011
Figure PCTCN2018124916-appb-000012
对于玉米，8倍大田浓度的苯磺隆是高压力处理的有效剂量。表3和图8的结果表明：所述除草剂耐受性蛋白质ALT02M1-02、ALT02M2-02、ALT02M3-02和ALT02-02均能赋予转基因玉米植物对苯磺酸的耐受性；相比于转入ALT02-02核苷酸序列的玉米植株，转入ALT02M1-02核苷酸序列的玉米植株、转入ALT02M2-02核苷酸序列的玉米植株和转入ALT02M3-02核苷酸序列的玉米植株对苯磺酸均具有显著增高的耐受性；而对照玉米植株和野生型玉米植株则对苯磺酸不具有耐受性。
综上所述，本发明所述除草剂耐受性蛋白质ALT01的氨基酸序列第176位由甘氨酸突变为丙氨酸和/或第178位由丝氨酸突变为缬氨酸(例如所述除草剂耐受性蛋白质ALT01M1、ALT01M2或ALT01M3)时可以对磺酰脲类除草剂表现出较高的耐受性，特别是苯磺隆；所述除草剂耐受性蛋白质ALT02(或ALT03)的氨基酸序列第140位由甘氨酸突变为丙氨酸和/或第142位由丝氨酸突变为缬氨酸(例如所述除草剂耐受性蛋白质ALT02M1、ALT02M2、ALT02M3、ALT03M1、ALT03M2或ALT03M3)时可以对磺酰脲类除草剂表现出较高的耐受性，特别是苯磺隆；所述除草剂耐受性蛋白质ALT04的氨基酸序列第131位由甘氨酸突变为丙氨酸和/或第133位由丝氨酸突变为缬氨酸(例如所述除草剂耐受性蛋白质ALT04M1、ALT04M2或ALT04M3)时可以对磺酰脲类除草剂表现出较高的耐受性，特别是苯磺隆。且上述除草剂耐受性蛋白质的编码基因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，转入上述除草剂耐受性蛋白质的大豆植株和玉米植株对磺酰脲类除草剂的耐受性强，特别是可以耐受8倍大田浓度的苯磺隆，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。

Claims (15)

  1. 一种除草剂耐受性蛋白质，其特征在于，包括：
    (a)具有SEQ ID NO：1所示的氨基酸序列，且其至少在SEQ ID NO：1第176位具有丙氨酸替换和/或第178位具有缬氨酸替换；或
    (b)具有SEQ ID NO：19所示的氨基酸序列，且其至少在SEQ ID NO：19第140位具有丙氨酸替换和/或第142位具有缬氨酸替换；或
    (c)具有SEQ ID NO：35所示的氨基酸序列，且其至少在SEQ ID NO：35第140位具有丙氨酸替换和/或第142位具有缬氨酸替换；或
    (d)具有SEQ ID NO：51所示的氨基酸序列，且其至少在SEQ ID NO：51第131位具有丙氨酸替换和/或第133位具有缬氨酸替换；或
    (e)在(a)-(d)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)-(d)衍生的蛋白质；
    优选地，所述除草剂耐受性蛋白质包括：
    (f)(a)中的氨基酸序列在SEQ ID NO：1第80位还具有精氨酸替换和/或第81位具有丙氨酸替换和/或第182位具有精氨酸替换；或
    (g)(b)中的氨基酸序列在SEQ ID NO：19第44位还具有精氨酸替换和/或第45位具有丙氨酸替换和/或第146位具有精氨酸替换；或
    (h)(c)中的氨基酸序列在SEQ ID NO：35第44位还具有精氨酸替换和/或第45位具有丙氨酸替换和/或第146位具有精氨酸替换；或
    (i)(d)中的氨基酸序列在SEQ ID NO：51第35位还具有精氨酸替换和/或第36位具有丙氨酸替换和/或第137位具有缬氨酸替换；或
    (j)在(f)-(i)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)-(d)衍生的蛋白质；
    优选地，所述除草剂耐受性蛋白质包括：
    (k)具有SEQ ID NO：7、SEQ ID NO：11或SEQ ID NO：15所示的氨基酸序列；或
    (1)具有SEQ ID NO：23、SEQ ID NO：27或SEQ ID NO：31所示的氨基酸序列；或
    (m)具有SEQ ID NO：39、SEQ ID NO：43或SEQ ID NO：47所示的氨基酸序列；或
    (n)具有SEQ ID NO：55、SEQ ID NO：59或SEQ ID NO：63所示的氨基酸序列。
  2. 一种除草剂耐受性基因，其特征在于，包括：
    (o)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
    (p)具有SEQ ID NO：8、9、10、12、13、14、16、17或18所示的核苷酸序列；或
    (q)具有SEQ ID NO：24、25、26、28、29、30、32、33或34所示的核苷酸序列；或
    (r)具有SEQ ID NO：40、41、42、44、45、46、48、49或50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。
  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，所述控制杂草的方法包括：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，所述杂草为草甘膦抗性杂草。
  9. 一种用于保护植物免受由磺酰脲类除草剂引起的损伤或赋予植物磺酰脲类除草剂耐受性或产生耐受磺酰脲类除草剂的植物的方法，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。
  10. 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。
  11. 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，所述控制杂草生长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草甘膦耐受性植物的大田，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，所述杂草为草甘膦抗性杂草。
  12. 根据权利要求8-11任一项所述方法或所述种植系统，其特征在于，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。
  13. 根据权利要求7-12任一项所述方法或所述种植系统，其特征在于，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：
    (1)具有SEQ ID NO：1所示的氨基酸序列，且其至少在SEQ ID NO：1第176位具有丙氨酸替换和/或第178位具有缬氨酸替换；或
    (2)具有SEQ ID NO：19所示的氨基酸序列，且其至少在SEQ ID NO：19第140位具有丙氨酸替换和/或第142位具有缬氨酸替换；或
    (3)具有SEQ ID NO：35所示的氨基酸序列，且其至少在SEQ ID NO：35第140位具有丙氨酸替换和/或第142位具有缬氨酸替换；或
    (4)具有SEQ ID NO：51所示的氨基酸序列，且其至少在SEQ ID NO：51第131位具有丙氨酸替换和/或第133位具有缬氨酸替换；或
    (5)在(1)-(4)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(1)-(4)衍生的蛋白质；
    优选地，所述除草剂耐受性蛋白质包括：
    (6)(1)中的氨基酸序列在SEQ ID NO：1第80位还具有精氨酸替换和/或第81位具有丙氨酸替换和/或第182位具有精氨酸替换；或
    (7)(2)中的氨基酸序列在SEQ ID NO：19第44位还具有精氨酸替换和/或第45位具有丙氨酸替换和/或第146位具有精氨酸替换；或
    (8)(3)中的氨基酸序列在SEQ ID NO：35第44位还具有精氨酸替换和/或第45位具有丙氨酸替换和/或第146位具有精氨酸替换；或
    (9)(4)中的氨基酸序列在SEQ ID NO：51第35位还具有精氨酸替换和/或第36位具有丙氨酸替换和/或第137位具有精氨酸替换；或
    (10)在(6)-(9)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(6)-(9)衍生的蛋白质；
    优选地，所述除草剂耐受性蛋白质包括：
    (11)具有SEQ ID NO：7、SEQ ID NO：11或SEQ ID NO：15所示的氨基酸序列；或
    (12)具有SEQ ID NO：23、SEQ ID NO：27或SEQ ID NO：31所示的氨基酸序列；或
    (13)具有SEQ ID NO：39、SEQ ID NO：43或SEQ ID NO：47所示的氨基酸序列；或
    (14)具有SEQ ID NO：55、SEQ ID NO：59或SEQ ID NO：63所示的氨基酸序列。
  15. 根据权利要求14所述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。
PCT/CN2018/124916 2018-02-07 2018-12-28 除草剂耐受性蛋白质、其编码基因及用途 WO2019153952A1 (zh)

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