WO2017161914A1 - 除草剂耐受性蛋白质的用途 - Google Patents

除草剂耐受性蛋白质的用途 Download PDF

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Publication number
WO2017161914A1
WO2017161914A1 PCT/CN2016/108409 CN2016108409W WO2017161914A1 WO 2017161914 A1 WO2017161914 A1 WO 2017161914A1 CN 2016108409 W CN2016108409 W CN 2016108409W WO 2017161914 A1 WO2017161914 A1 WO 2017161914A1
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Prior art keywords
seq
nucleotide sequence
plant
glyphosate
alt
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PCT/CN2016/108409
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English (en)
French (fr)
Inventor
谢香庭
陶青
庞洁
丁德荣
鲍晓明
Original Assignee
北京大北农科技集团股份有限公司
北京大北农生物技术有限公司
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Application filed by 北京大北农科技集团股份有限公司, 北京大北农生物技术有限公司 filed Critical 北京大北农科技集团股份有限公司
Priority to US16/077,140 priority Critical patent/US20190029252A1/en
Priority to CA3014563A priority patent/CA3014563C/en
Priority to BR112018067529A priority patent/BR112018067529A2/pt
Priority to EP16895258.8A priority patent/EP3434778A4/en
Priority to MX2018010487A priority patent/MX2018010487A/es
Priority to AU2016399130A priority patent/AU2016399130B2/en
Publication of WO2017161914A1 publication Critical patent/WO2017161914A1/zh
Priority to ZA2018/05452A priority patent/ZA201805452B/en

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Definitions

  • the present invention relates to the use of a herbicide-tolerant protein, and more particularly to the use of a thifensulfuron hydrolase to degrade a sulfonate herbicide.
  • Crops that are resistant to glyphosate such as corn, soybeans, cotton, sugar beets, wheat, and rice, have been developed. It is therefore possible to spray glyphosate on fields where glyphosate resistant crops are grown to control weeds without significantly damaging the crops.
  • Glyphosate has been used worldwide for more than 20 years, resulting in an over-reliance on glyphosate and glyphosate-tolerant crop technology and is naturally more tolerant or has developed for glyphosate in wild weed species. Plants that are glyphosate resistant have a high selection pressure applied. A few weeds have been reported to have developed resistance to glyphosate, including broadleaf weeds and grass weeds such as Swiss ryegrass, ryegrass, goosegrass, ragweed, small canopy, wild pond Artemisia and long leaves in front of the car.
  • weeds that are not agricultural problems before the widespread use of glyphosate-tolerant crops are becoming more prevalent and difficult to control with glyphosate-tolerant crops, which are mainly (but not only) difficult to control broadleaf weeds.
  • Appears such as genus, genus, genus tarax, and comfrey.
  • growers can compensate for the weakness of glyphosate by tank mixing or other herbicides that control missing weeds, such as sulfonylurea weeding Agent.
  • Sulfonylurea herbicides have become the third largest herbicide after organophosphorus and acetamide herbicides. The annual global sales have reached more than US$3 billion. The annual application area of sulfonylurea herbicides in China has exceeded 2 million. The hectare is still expanding.
  • Sulfonylurea herbicides can be roughly classified into ester-containing and ester-free, and at least ten kinds of sulfonylurea herbicides having ester bonds and similar chemical structures are present.
  • thifensulfuron hydrolase has been identified to degrade thifensulfuron, but like thifensulfuron, fensulfuron is also a sulfonylurea herbicide containing ester bonds, and thifensulfuron hydrolase has not been found to be sulfonate. Herbicides are reported to be tolerant.
  • the tolerance range of the thifensulfuron hydrolase to the herbicide was increased.
  • the present invention provides a method of controlling weeds comprising applying a herbicide containing an effective amount of bensulfuron to a plant growth environment in which at least one transgenic plant is present, the transgenic plant comprising a coding in its genome
  • a herbicide containing an effective amount of bensulfuron to a plant growth environment in which at least one transgenic plant is present, the transgenic plant comprising a coding in its genome
  • the nucleotide sequence of the thifensulfuron hydrolase which has reduced plant damage and/or increased plant yield compared to other plants that do not have a nucleotide sequence encoding a thifensulfuron hydrolase.
  • the effective dose of bensulfuron is 9-144 g ai/ha.
  • transgenic plant is a monocot or a dicot.
  • the transgenic plant is corn, soybean, Arabidopsis, cotton, canola, rice, sorghum, wheat, barley, millet, sugar cane or oats.
  • the amino acid sequence of the thifensulfuron hydrolase has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 7.
  • nucleotide sequence of the thifensulfuron hydrolase has:
  • the transgenic plant may further comprise at least one second nucleotide different from the nucleotide sequence encoding the thifensulfuron hydrolase.
  • the second nucleotide encodes a selectable marker protein, a synthetic active protein, a degraded active protein, an antibiotic stress protein, an abiotic stress resistant protein, a male sterile protein, a protein that affects plant yield, and/or a protein that affects plant quality.
  • the second nucleotide encodes 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.
  • herbicides containing an effective amount of bensulfuron include glyphosate herbicides, glufosinate herbicides, auxin herbicides, grass herbicides, pre-emergence selective herbicides and/or post-germination Selective herbicide.
  • the present invention also provides a method of controlling glyphosate-tolerant weeds comprising applying an effective amount of a sulfonate herbicide and a glyphosate herbicide to a large planting of at least one transgenic plant
  • the field contains glyphosate-tolerant weeds or seeds thereof
  • the transgenic plant contains a nucleotide sequence encoding a thifensulfuron hydrolase and a nucleotide sequence encoding a glyphosate-tolerant protein in its genome.
  • Transgenic plants have reduced plant damage and/or increased plant yield compared to other plants that do not have a nucleotide sequence encoding a thifensulfuron hydrolase and/or a nucleotide sequence encoding a glyphosate-tolerant protein. .
  • the effective dose of bensulfuron is 9-144 g ai/ha.
  • the effective dose of glyphosate is 200-1600 g ae/ha.
  • transgenic plant is a monocot or a dicot.
  • the transgenic plant is corn, soybean, Arabidopsis, cotton, canola, rice, sorghum, wheat, barley, millet, sugar cane or oats.
  • the amino acid sequence of the thifensulfuron hydrolase has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 7.
  • nucleotide sequence of the thifensulfuron hydrolase has:
  • the glyphosate-tolerant protein includes 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase or glyphosate decarboxylase.
  • amino acid sequence of the glyphosate-tolerant protein has the amino acid sequence shown in SEQ ID NO: 10.
  • nucleotide sequence of the glyphosate-tolerant protein has:
  • the present invention also provides a planting system for controlling weed growth, comprising a bensulfuron-methyl herbicide and a plant growth environment in which at least one transgenic plant is present, and an effective dose of the bensulfuron-methyl herbicide is applied to
  • the transgenic plant comprises in its genome a nucleotide sequence encoding a thifensulfuron hydrolase, a transgenic plant and other plants not having a nucleotide sequence encoding a thifensulfuron hydrolase Compared to having reduced plant damage and/or having increased plant yield.
  • the effective dose of bensulfuron is 9-144 g ai/ha.
  • transgenic plant is a monocot or a dicot.
  • the transgenic plant is corn, soybean, Arabidopsis, cotton, canola, rice, sorghum, wheat, barley, millet, sugar cane or oats.
  • the amino acid sequence of the thifensulfuron hydrolase has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 7.
  • nucleotide sequence of the thifensulfuron hydrolase has:
  • the transgenic plant may further comprise at least one second nucleotide different from the nucleotide sequence encoding the thifensulfuron hydrolase.
  • the second nucleotide encodes a selectable marker protein, a synthetic active protein, a degraded active protein, an antibiotic stress protein, an abiotic stress resistant protein, a male sterile protein, a protein that affects plant yield, and/or a protein that affects plant quality.
  • the second nucleotide encodes 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, ammonium oxalate Phosphoacetyltransferase, alpha ketoglutarate-dependent dioxygenase, 4-hydroxyphenylpyruvate dioxygenase, acetolactate synthase, cytochrome protein and/or protoporphyrinogen oxidase.
  • herbicides containing a herbicidally effective amount of bensulfuron-methyl include glyphosate herbicides, glufosinate herbicides, auxin herbicides, grass herbicides, pre-emergence selective herbicides and/or germination. After selective herbicides.
  • the present invention also provides a planting system for controlling glyphosate-tolerant weeds, comprising a sulfonate herbicide, a glyphosate herbicide, and a field planted with at least one transgenic plant, the effective dose
  • the bensulfuron-methyl herbicide and the glyphosate herbicide are applied to a field in which at least one transgenic plant is grown, the field contains glyphosate-tolerant weeds or seeds thereof, and the transgenic plant contains a thiophenesulfuron-encoding in its genome.
  • nucleotide sequence of a hydrolase and a nucleotide sequence encoding a glyphosate-tolerant protein a transgenic plant and other nucleotide sequences not encoding a thifensulfuron hydrolase and/or a glyphosate-tolerant protein
  • the nucleotide sequence of the plant has reduced plant damage and/or increased plant yield compared to plants.
  • the effective dose of bensulfuron is 9-144 g ai/ha.
  • the effective dose of glyphosate is 200-1600 g ae/ha.
  • transgenic plant is a monocot or a dicot.
  • the transgenic plant is corn, soybean, Arabidopsis, cotton, canola, rice, sorghum, wheat, barley, millet, sugar cane or oats.
  • the amino acid sequence of the thifensulfuron hydrolase has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 7.
  • nucleotide sequence of the thifensulfuron hydrolase has:
  • the glyphosate-tolerant protein includes 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase or glyphosate decarboxylase.
  • amino acid sequence of the glyphosate-tolerant protein has the amino acid sequence shown in SEQ ID NO: 10.
  • nucleotide sequence of the glyphosate-tolerant protein has:
  • the present invention also provides a method for producing a plant resistant to a sulfonate herbicide, comprising introducing a nucleotide sequence encoding a thifensulfuron hydrolase into a genome of a plant, when an effective dose of benzenesulfon is contained.
  • the herbicide is applied to the field in which at least the plant is present, and the plant has reduced plant damage and/or increased plant yield compared to other plants that do not have a nucleotide sequence encoding a thifensulfuron hydrolase.
  • the present invention also provides a method for cultivating a plant resistant to a sulfonate herbicide, comprising:
  • the present invention also provides a method for protecting a plant from damage caused by a sulfonate herbicide, comprising applying a herbicide containing an effective amount of bensulfuron to a plant growth in which at least one transgenic plant is present.
  • the transgenic plant comprises a nucleotide sequence encoding a thifensulfuron hydrolase in its genome, and the transgenic plant has reduced plant damage and/or plants other than the nucleotide sequence encoding the thifensulfuron hydrolase. Or have increased plant yield.
  • the present invention also provides a method for degrading a bensulfuron-methyl herbicide by a thifensulfuron hydrolase, which comprises applying a herbicide containing an effective amount of bensulfuron to a plant growth environment in which at least one transgenic plant is present.
  • a transgenic plant comprising a nucleotide sequence encoding a thifensulfuron hydrolase in its genome, the transgenic plant having reduced plant damage and/or having a plant having no nucleotide sequence encoding a thifensulfuron hydrolase Increased plant yield.
  • the present invention also provides a use of a thifensulfuron hydrolase to degrade a sulfonate herbicide.
  • the use of thifensulfuron hydrolase to degrade a sulfonate herbicide comprises applying a herbicide comprising an effective amount of bensulfuron to a plant growth environment in which at least one transgenic plant is present, the transgenic plant comprising a thiophene in its genome
  • the nucleotide sequence of the sulfonate hydrolase, the transgenic plant has reduced plant damage and/or increased plant yield compared to other plants that do not have a nucleotide sequence encoding a thifensulfuron hydrolase.
  • the amino acid sequence of the thifensulfuron hydrolase has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 7.
  • nucleotide sequence of the thifensulfuron hydrolase has:
  • the transgenic plants are planted in the soil of the plant growth environment within 21 days of application of the herbicide.
  • the herbicide can be applied before, at the same time as, or after the planting of the transgenic plant.
  • the transgenic plants are planted in the soil within 12, 10, 7 or 3 days prior to application of the herbicide; the transgenic plants are planted in the soil within 12, 10, 7 or 3 days after application of the herbicide.
  • the herbicide can also be subjected to a second treatment of the transgenic plant, and the second treatment can be between the V1-V2 and V3-V4 stages, before flowering, during flowering, after flowering or at the time of seed formation.
  • tribenuron-methyl means 2-[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)-N-methylaminocarb.
  • Amidosulfonyl]methyl 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 means 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- 80g ai/ha or 65-75g ai/ha.
  • Dicotyledons in the present invention include, but are 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.
  • the dicotyledon refers to soybean, Arabidopsis, cotton or canola.
  • Monocotyledons in the present invention include, but are not limited to, corn, rice, sorghum, wheat, barley, rye, millet, sugar cane, oats or turfgrass.
  • monocotyledon refers to corn, rice, sorghum, wheat, barley, millet, sugar cane or oats.
  • the herbicide-tolerant protein is a thifensulfuron hydrolase as shown in SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 7 in the Sequence Listing.
  • the herbicide tolerance gene is a nucleotide sequence encoding a thifensulfuron hydrolase, such as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO in the sequence listing. : 8 and SEQ ID NO: 9.
  • the herbicide tolerance gene is used in plants, and may contain other elements in addition to the coding region of the thifensulfuron hydrolase, such as a coding selectable marker protein, a synthetic active protein, a decomposition active protein, an antibiotic stress protein, and an anti-non- A biotic-stressed protein, a male-sterile protein, a protein that affects plant yield, and/or a protein that affects plant quality, thereby obtaining a transgenic plant having both herbicide tolerance activity and other traits.
  • a coding selectable marker protein such as a coding selectable marker protein, a synthetic active protein, a decomposition active protein, an antibiotic stress protein, and an anti-non- A biotic-stressed protein, a male-sterile protein, a protein that affects plant yield, and/or a protein that affects plant quality, thereby obtaining a transgenic plant having both herbicide tolerance activity and other traits.
  • the antibiotic stress protein in the present invention means a protein which is resistant to stress exerted by other organisms, such as insect resistance protein, (virus, bacteria, fungus, nematode) disease resistance protein and the like.
  • the antibiotic stress protein in the present invention refers to a protein which is resistant to the stress applied by the external environment, such as a protein having tolerance to herbicides, drought, heat, cold, freezing, salt stress, oxidative stress and the like.
  • the protein which affects the quality of the plant in the present invention refers to a protein which affects the output trait of the plant, such as a protein which improves the quality and content of starch, oil, vitamins, and the like, and a protein which improves the fiber quality.
  • an expression cassette comprising a nucleotide sequence encoding a thifensulfuron hydrolase can also be expressed in a plant together with at least one protein encoding a herbicide tolerance gene, including but not limited to, 5 -enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate oxidoreductase (GOX), glyphosate-N-acetyltransferase (GAT), glyphosate decarboxylase, glufosinate acetyl Transferase (PAT), alpha ketoglutarate-dependent dioxygenase (AAD), dicamba monooxygenase (DMO), 4-hydroxyphenylpyruvate dioxygenase (HPPD), acetolactate synthase (ALS), cytochrome protein (P450) and/or protoporphyrinogen oxidase (Protox).
  • EPSPS 5 -enol
  • glyphosate means N-phosphonomethylglycine and a salt thereof
  • treatment with “glyphosate herbicide” means treatment with any herbicide preparation containing glyphosate.
  • Commercial formulations of glyphosate include, but are not limited to, (glyphosate as isopropylamine salt), (glyphosate as a potassium salt), DRY and (glyphosate as an amine salt), (glyphosate as a sodium salt) and (Glyphosate as trimethylsulfate).
  • the effective dose of glyphosate in the present invention means use at 200-1600 g ae/ha, including 250-1600 g ae/ha, 300-1600 g ae/ha, 500-1600 g ae/ha, 800-1500 g ae/ha, 1000- 1500g ae/ha or 1200-1500g ae/ha.
  • glufosinate also known as glufosinate
  • treatment with "glufosinate herbicide” means using any kind.
  • the herbicide formulation containing glufosinate is treated.
  • auxin herbicides of the present invention mimic or act as natural plant growth regulators known as auxins, which affect cell wall plasticity and nucleic acid metabolism, resulting in uncontrolled cell division and growth.
  • Symptoms of damage caused by auxin herbicides include upward bending or twisting of stems and stalks, cup-shaped or curled leaves, and abnormal leaf shapes and veins.
  • the auxin herbicides include, but are not limited to, a phenoxycarboxylic acid compound, a benzoic acid compound, a pyridinecarboxylic acid compound, a quinolinecarboxylic acid compound or a herbicide ethyl ester compound.
  • auxin herbicides are dicamba, 2,4-dichlorophenoxyacetic acid (2,4-D), (4-chloro-2-methylphenoxy)acetic acid (MCPA) and/or Or 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB).
  • Dicamba means 3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxybenzoic acid and its acids and salts.
  • the salts thereof include isopropylamine salt, diethylene glycol ammonium salt, dimethylamine salt, potassium salt and sodium salt.
  • Commercial preparations of dicamba include, but are not limited to, (as a DMA salt), (BASF, as DGA salt), VEL-58-CS-11TM and (BASF, as a DGA salt).
  • the grass herbicide of the present invention is not used for corn unless it has been tolerated by corn, and such tolerance can be provided by an alpha ketoglutarate-dependent dioxygenase (such as the AAD gene), but the herbicide includes Not limited to flupirin.
  • an alpha ketoglutarate-dependent dioxygenase such as the AAD gene
  • the pre-emergence selective herbicides of the present invention include, but are not limited to, acetanilide, acetochlor, acetolactate synthase inhibitor, dinitroaniline or protoporphyrinogen oxidase inhibitor.
  • the post-emergence selective herbicides of the present invention include, but are not limited to, nicosulfuron, rimsulfuron, 2,4-D, dicamba, acesulfame, and quizalofop.
  • the amount of herbicide applied in the present invention varies with soil structure, pH, organic content, tillage system and weed size, and is determined by looking at the appropriate herbicide application amount on the herbicide label.
  • the weeds controllable by the bensulfuron-methyl herbicide of the present invention include, but are not limited to, Amaranthus retroflexus, Aphis gossypii, Solanum nigrum, ramie, Willow locust, Sorrel, Oriental scorpion, Coilia, thrift , ⁇ , ⁇ , ⁇ , ⁇ , wolf grass, ⁇ , ⁇ , ⁇ , forgetful grass, citron, wenjing, thorn, needle, wormwood, feather leaf, wormwood, big leaf Sowing wormwood, Matricaria, thorn lettuce, sunflower, stalk flower, pig scorpion, scoparia, ragweed, maijiagong, Wang not staying, linseed, puffer mustard, wild mustard, white Mustard, water mustard, leeks, blue cabbage, sauerkraut, broccoli, big nest and leeks.
  • the weeds controlled by the glyphosate herbicide in the present invention include, but are not limited to, Big Spike, Amaranth, Wild Oat, Brome, Netweed, Valerian, Bluegrass, Foxtail, Callan, Purslane, Poria, Xanthium, ramie, medlar, plantain, sorghum, piglet and sedge.
  • the planting system 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.
  • Glyphosate is widely used because it controls a very broad spectrum of broadleaf and grass weed species.
  • repeated use of glyphosate in glyphosate resistant crop and non-crop applications has (and will continue to be) selected to succeed weeds as naturally more tolerant species or glyphosate resistant biotypes.
  • Most herbicide resistance management strategies recommend the use of effective amounts of multiple herbicides as a means of delaying the emergence of resistant weeds. Multiple herbicides provide control of the same species but have different modes of action.
  • glyphosate and bensulfuron-methyl selectively controls the control of glyphosate-resistant weed species (wideleaf weed species controlled by trisulfuron-methyl herbicides) in glyphosate-tolerant crops.
  • 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) ))
  • 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.
  • weed refers to a plant that competes with cultivated plants in a plant growth environment.
  • control and/or "control” in the present invention means that at least an effective amount of the bensulfuron-methyl herbicide is applied directly (for example by spraying) to the environment in which the plant grows, minimizing weed development and/or stopping growth.
  • the cultivated plants 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/or compared to non-transgenic wild-type plants and/or Or have increased plant yield. Attenuated plant damage, including but not limited to improved stem resistance, and/or increased kernel weight, and the like.
  • control and/or “control” effects of thifensulfuron hydrolase on weeds can exist independently and are not attenuated and/or disappeared by the presence of other substances that can "control” and/or “control” weeds.
  • any tissue of a transgenic plant containing a polynucleotide sequence encoding a thifensulfuron hydrolase
  • the thifensulfuron hydrolase and/or weed control can be A substance
  • the presence of another substance does not affect the "control” and / or “control” effects of thifensulfuron hydrolase on weeds, nor does it lead to "control” and / or “control” effects and / Or partially achieved by another substance, regardless of the thifensulfuron hydrolase.
  • expression of a thifensulfuron hydrolase in a transgenic plant can be accompanied by expression of one or more other herbicide-tolerant proteins. Co-expression of such more than one herbicide-tolerant protein in the same transgenic plant can be achieved by genetically engineering the plant to contain and express the desired gene.
  • one plant (the first parent) can express the thifensulfuron hydrolase by genetic engineering
  • the second plant (the second parent) can express other herbicide-tolerant proteins by genetic engineering.
  • 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 genome of a plant, plant tissue or plant cell in the present invention refers to any genetic material in a plant, plant tissue or plant cell, and includes the nucleus and plastid and mitochondrial genomes.
  • plant propagule in the present invention includes, but is not limited to, plant sexual propagules and plant asexual propagules.
  • Plant sexual propagules include, but are not limited to, plant seeds; plant asexual propagules refer to the vegetative organs of plants or a particular tissue that can produce new plants under ex vivo conditions; vegetative organs or a particular tissue including but not Limited to roots, stems and leaves, for example: plants with roots as vegetative propagules include strawberries and sweet potatoes; plants with stems as vegetative propagules include sugar cane and potatoes (tubers); plants with leaves as vegetative propagules include aloe vera And Begonia and so on.
  • “Resistance” in the present invention is heritable and allows plants to grow and multiply in the case where the herbicide is subjected to a general herbicide treatment for a given plant. As recognized by those skilled in the art, plants can be considered “resistant” even if the plants are significantly damaged by herbicide treatment.
  • the term “tolerance” or “tolerance” in the present invention is broader than the term “resistance” and includes “resistance” as well as the ability of a particular plant to have an increased resistance to herbicide-induced damage to various degrees. The same herbicide dose generally results in damage to the same genotype of wild type plants.
  • polynucleotides and/or nucleotides 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 includes The use of the polynucleotides and their complementary strands exemplified in the Sequence Listing.
  • a "coding strand” as commonly used in the art refers to a strand that binds to the antisense strand.
  • 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 nucleotide sequence of the thifensulfuron hydrolase 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 nucleotide sequence of the thifensulfuron hydrolase of the present invention is included in the present invention. These sequences are at least about 40%-50% homologous to the sequences of the invention, about 60%, 65% or 70% homologous, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93. Sequence homology of %, 94%, 95%, 96%, 97%, 98%, 99% or greater.
  • the invention provides functional proteins.
  • “Functional activity” (or “activity”) in the present invention means that the protein/enzyme (alone or in combination with other proteins) for use in the present invention has the ability to degrade or attenuate herbicide activity.
  • the plant producing the protein of the invention preferably produces an "effective amount" of the protein such that when the plant is treated with the herbicide, the level of protein expression is sufficient to give the plant complete or partial resistance to the herbicide (typically, unless otherwise stated). Or patience.
  • the herbicide can be used in an amount which normally kills the target plant, normal field amount and concentration.
  • the plant cells and plants of the invention are protected from growth inhibition or damage caused by herbicide treatment.
  • the transformed plants and plant cells of the invention preferably have resistance or tolerance to the bensulfuron-methyl herbicide, i.e., the transformed plants and plant cells are capable of growing in the presence of an effective amount of the bensulfuron-methyl herbicide.
  • genes and proteins of the present invention include not only specific exemplary sequences, but also portions and/or fragments that retain the herbicide tolerance activity profile of a particular exemplary protein (including internal and/or terminal deletions compared to full length proteins). ), variants, mutants, substitutions (proteins with alternative amino acids), chimeras and fusion proteins.
  • Variant or “variant” refers to a nucleotide sequence that encodes the same protein or an equivalent protein encoded with herbicide resistance activity.
  • Equivalent protein refers to a biologically active protein that has the same or substantially the same herbicide tolerance as the protein of the claims.
  • a “fragment” or “truncation” of a DNA molecule or protein sequence 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 foregoing sequences may vary, but is of sufficient length to ensure that the (encoding) protein is a herbicide tolerant protein.
  • a "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).
  • the amino acid sequence encoding the thifensulfuron hydrolase includes, but is not limited to, the sequence involved in the sequence listing of the present invention, and an amino acid sequence having a certain homology thereto is also included in the present invention. These sequences are typically more than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%, similar to the sequences of the present invention. Preferred polynucleotides and proteins of the invention may also be defined according to a more specific range of identity and/or similarity.
  • the sequence of the example of the present invention is 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% identity and/or similarity.
  • Regulatory sequences in the present invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the thifensulfuron hydrolase gene.
  • a "promoter expressible in a plant” in which a promoter is a promoter expressible in a plant means 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-induced expression pattern refers to when the plant is subjected to mechanical Or the wound caused by insect foraging, the expression of the coding sequence under the control of the promoter is significantly higher than that under 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.
  • a transit peptide (also known as a secretion signal sequence or a targeting sequence) directs the transgene product to a particular organelle or cell compartment.
  • the transit peptide can be heterologous, for example, using a sequence encoding a chloroplast transit peptide. Chloroplasts, either targeting the endoplasmic reticulum using the 'KDEL' retention sequence, or targeting the vacuole with the CTPP of the barley plant lectin gene.
  • the leader sequence includes, but is not limited to, a small RNA viral 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 immunity Ball protein heavy chain binding protein (BiP); non-translated 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 maize dwarf mosaic virus
  • BiP human immunity Ball protein heavy chain binding protein
  • AMV RNA4 alfalfa mosaic virus
  • TMV tobacco mosaic virus
  • Enhancers include, but are not limited to, cauliflower mosaic virus (CaMV) enhancer, Scrophulari mosaic virus (FMV) enhancer, carnation weathering ring virus (CERV) enhancer, cassava vein mosaic virus (CsVMV) enhancer, purple Jasmine mosaic virus (MMV) enhancer, night fragrant yellow leaf curl virus (CmYLCV) enhancer, Multan cotton leaf curl virus (CLCuMV), comfrey yellow mottle virus (CoYMV) and peanut chlorotic mosaic virus (PCLSV) enhancer.
  • CaMV cauliflower mosaic virus
  • FMV Scrophulari mosaic virus
  • CERV carnation weathering ring virus
  • CsVMVMV cassava vein mosaic virus
  • MMV purple Jasmine mosaic virus
  • CmYLCV night fragrant yellow leaf curl virus
  • CLCuMV Multan cotton leaf curl virus
  • CoYMV comfrey yellow mottle virus
  • PCLSV peanut chlorotic mosaic virus
  • introns include, but are not limited to, maize hsp70 introns, maize ubiquitin introns, Adh introns 1, sucrose synthase introns, or rice Actl introns.
  • introns include, but are not limited to, CAT-1 introns, pKANNIBAL introns, PIV2 introns, and "superubiquitin" introns.
  • 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, source Polyadenylation signal sequence of protease inhibitor II (pinII) gene, polyadenylation signal sequence derived from pea ssRUBISCO E9 gene, and polyadesoid derived from ⁇ -tubulin gene Glycosylation signal sequence.
  • NOS Agrobacterium tumefaciens nopaline synthase
  • pinII protease inhibitor II
  • pea ssRUBISCO E9 polyadesoid derived from ⁇ -tubulin gene Glycosylation signal sequence.
  • Effectively linked in the context of the invention denotes the association of nucleic acid sequences which allow one sequence to provide the function required for the linked sequence.
  • Effective ligation in the present invention may be the linking of a promoter to a sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter.
  • Effective ligation when a sequence of interest encodes a protein and is intended to obtain expression of the protein means that the promoter is ligated to the sequence in a manner that allows efficient translation of the resulting transcript.
  • the linker of the promoter to the coding sequence is a transcript fusion and it is desired to effect expression of the encoded protein, such ligation is made such that the first translation initiation codon in the resulting transcript is the start codon of the coding sequence.
  • the linkage of the promoter to the coding sequence is a translational fusion and it is desired to effect expression of the encoded protein, such linkage is made such that the first translation initiation codon and promoter contained in the 5' untranslated sequence Linked and linked such that the resulting translation product is in frame with the translational open reading frame encoding the desired protein.
  • Nucleic acid sequences that may be "operably linked” include, but are not limited to, sequences that provide for gene expression functions (ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, poly Adenylation site and/or transcription terminator), sequences that provide DNA transfer and/or integration functions (ie, T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), provide options Sexually functional sequences (ie, antibiotic resistance markers, biosynthetic genes), sequences that provide for the function of scoring markers, sequences that facilitate sequence manipulation in vitro or in vivo (ie, polylinker sequences, site-specific recombination sequences) and provision The sequence of the replication function (ie, the origin of replication of the bacteria, the autonomously replicating sequence, the centromeric sequence).
  • gene expression functions ie, gene expression elements such as promoters, 5' untranslated regions, introns, protein
  • the present invention confers new herbicide resistance traits on plants and does not observe adverse effects on phenotype including yield.
  • the plants of the present invention are tolerant to a general application level of at least one of the tested herbicides 2 x, 3 x, 4 x or 5 x. These levels of tolerance are within the scope of the invention. For example, predictable optimizations and further developments can be made to a variety of techniques known in the art to increase expression of a given gene.
  • the thifensulfuron hydrolase is resistant to the bensulfuron-methyl herbicide.
  • the plant of the present invention contains exogenous DNA in its genome, and the exogenous DNA comprises a nucleotide sequence encoding a thifensulfuron hydrolase by expressing an effective amount of the protein. Protect it from the threat of herbicides.
  • 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.
  • exogenous DNA is introduced into a plant, such as a gene encoding an thifensulfuron hydrolase or an expression cassette or a recombinant vector
  • conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, micro-launch bombardment Directly ingest DNA into protoplasts, electroporation or whisker silicon-mediated DNA introduction.
  • the present invention discloses for the first time that thifensulfuron hydrolase can exhibit high tolerance to bensulfuron-methyl herbicides, and thus has broad application prospects on plants.
  • the thifensulfuron hydrolase of the present invention is highly resistant to bensulfuron-methyl herbicide and can tolerate at least one-time field concentration.
  • Figure 1 is a flow chart showing the construction of a recombinant cloning vector DBN01-T containing an ALT nucleotide sequence for use of a herbicide-tolerant protein of the present invention
  • FIG. 2 is a flow chart showing the construction of a recombinant expression vector DBN100632 containing an ALT nucleotide sequence for use of a herbicide-tolerant protein of the present invention
  • Figure 3 is a schematic view showing the structure of a recombinant expression vector DBN100631 containing an ALT nucleotide sequence for use of a herbicide-tolerant protein of the present invention
  • Figure 4 is a diagram showing the effect of the transgenic Arabidopsis thaliana T1 plant on the tolerance of the bensulfuron-methyl herbicide to the herbicide-tolerant protein of the present invention
  • Figure 5 is a flow chart showing the construction of a recombinant expression vector DBN100828 containing an ALT nucleotide sequence for use of the herbicide-tolerant protein of the present invention
  • Figure 6 is a schematic view showing the structure of a recombinant expression vector DBN100827 containing an ALT nucleotide sequence for use of a herbicide-tolerant protein of the present invention
  • Figure 7 is a flow chart showing the construction of a recombinant cloning vector DBN05-T containing an ALT nucleotide sequence for use of a herbicide-tolerant protein of the present invention
  • Figure 8 is a flow chart showing the construction of a recombinant expression vector DBN100830 containing an ALT nucleotide sequence for use of a herbicide-tolerant protein of the present invention
  • Figure 9 is a schematic view showing the structure of a recombinant expression vector DBN100829 containing an ALT nucleotide sequence for use of a herbicide-tolerant protein of the present invention
  • Figure 10 is a diagram showing the effect of the transgenic maize T1 plant on the tolerance of the bensulfuron-methyl herbicide to the herbicide-tolerant protein of the present invention
  • Figure 11 is a graph showing the effect of transgenic soybean T1 plants on the tolerance of bensulfuron-methyl herbicides to the use of herbicide-tolerant proteins of the present invention.
  • ALT-1 thifensulfuron hydrolase-1
  • SEQ ID NO: 1 amino acids
  • ALT-1-01 nucleoside encoding the amino acid sequence corresponding to ALT-1
  • the acid sequence (1197 nucleotides) encodes the ALT-1-02 nucleotide sequence (1197 nucleotides) corresponding to the amino acid sequence of ALT-1, such as SEQ ID NO: 3 is shown in the sequence listing.
  • ALT-2 thifensulfuron hydrolase-2 (369 amino acids), as shown in SEQ ID NO: 4 in the Sequence Listing; ALT-2-01 nucleoside encoding the amino acid sequence corresponding to ALT-2
  • the acid sequence (1110 nucleotides) as shown in SEQ ID NO: 5 in the Sequence Listing, encodes the ALT-2-02 nucleotide sequence (1110 nucleotides) corresponding to the amino acid sequence of ALT-2, such as SEQ ID NO: 6 is shown in the sequence listing.
  • ALT-3 The amino acid sequence of thifensulfuron hydrolase-3 (ALT-3) (362 amino acids), as shown in SEQ ID NO: 7 in the Sequence Listing; ALT-3-01 nucleoside encoding the amino acid sequence corresponding to ALT-3
  • the amino acid sequence of the glyphosate-tolerant protein (455 amino acids), as shown in SEQ ID NO: 10 in the Sequence Listing; the EPSPS nucleotide sequence encoding the amino acid sequence corresponding to the glyphosate-tolerant protein (1368 Nucleotide) as shown in SEQ ID NO: 11 in the Sequence Listing.
  • ALT-1-01 nucleotide sequence (as shown in SEQ ID NO: 2 in the sequence listing), ALT-1-02 nucleotide sequence (as shown in SEQ ID NO: 3 in the sequence listing), ALT-2- 01 nucleotide sequence (as shown in SEQ ID NO: 5 in the Sequence Listing), ALT-2-02 nucleotide sequence (as shown in SEQ ID NO: 6 in the Sequence Listing), ALT-3-01 nucleotide a sequence (as shown in SEQ ID NO: 8 in the Sequence Listing), an ALT-3-02 nucleotide sequence (as shown in SEQ ID NO: 9 in the Sequence Listing), and an EPSPS nucleotide sequence (such as SEQ ID in the Sequence Listing) NO:11) synthesized by Nanjing Jinsrui Biotechnology Co., Ltd.; the 5' end of the synthesized ALT-1-01 nucleotide sequence (SEQ ID NO: 2) is also ligated with SpeI cleavage site,
  • the 5' end of the synthetic ALT-3-01 nucleotide sequence (SEQ ID NO: 8) is also ligated with a SpeI cleavage site, and the ALT-3-01 nucleotide sequence (SEQ ID NO: 8)
  • the 3' end is also ligated with a KasI cleavage site
  • the 5' end of the synthetic ALT-3-02 nucleotide sequence (SEQ ID NO: 9) is also ligated with a SpeI cleavage site, ALT-3-02 nucleoside
  • the 3' end of the acid sequence (SEQ ID NO: 9) is also ligated with a KasI cleavage site
  • the 5' end of the synthesized EP SPS nucleotide sequence (SEQ ID NO: 11) is also ligated with an NcoI cleavage site.
  • the 3' end of the EPSPS nucleotide sequence (SEQ ID NO: 11) is also ligated to the FspI cleavage site.
  • the synthetic ALT-1-01 nucleotide sequence was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the procedure was carried out according to the Promega product pGEM-T vector specification to obtain a recombinant cloning vector DBN01.
  • Figure 1 where Amp represents the ampicillin resistance gene; f1 represents the phage f1 The origin of replication; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; ALT-1-01 is the ALT-1-01 nucleotide sequence (SEQ ID NO: 2) ;MCS is a multiple cloning site).
  • the recombinant cloning vector DBN01-T was then transformed into E. coli T1 competent cells by heat shock method (Transgen, Beijing, China, CAT: CD501) under heat shock conditions: 50 ⁇ L E. coli T1 competent cells, 10 ⁇ L of plasmid DNA (recombinant) Cloning vector DBN01-T), water bath at 42 ° C for 30 seconds; shaking culture at 37 ° C for 1 hour (shake at 100 rpm), coated with IPTG (isopropylthio- ⁇ -D-galactoside) and X -gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside) ampicillin (100 mg/L) LB plate (tryptone 10 g/L, yeast extract 5 g/L, NaCl) 10 g/L, agar 15 g/L, adjusted to pH 7.5 with NaOH) and grown overnight.
  • heat shock method Transgen, Beijing, China, CAT: CD501
  • White colonies were picked and cultured in LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, ampicillin 100 mg/L, pH adjusted to 7.5 with NaOH) at 37 °C. overnight.
  • the plasmid was extracted by alkaline method: the bacterial solution was centrifuged at 12000 rpm for 1 min, the supernatant was removed, and the precipitated cells were pre-cooled with 100 ⁇ L of ice (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose.
  • the 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 37 ° C for 30 min; and stored at -20 ° C until use.
  • the extracted plasmid was identified by SpeI and KasI digestion, and the positive clone was verified by sequencing.
  • the result showed that the nucleotide sequence of ALT-1-01 inserted into the recombinant cloning vector DBN01-T was shown in SEQ ID NO: 2 in the sequence listing.
  • the synthetic ALT-2-01 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN02-T, wherein ALT-2-01 was ALT. -2-01 nucleotide sequence (SEQ ID NO: 5).
  • the ALT-2-01 nucleotide sequence was correctly inserted into the recombinant cloning vector DBN02-T by restriction enzyme digestion and sequencing.
  • the synthetic ALT-3-01 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN03-T, wherein ALT-3-01 was ALT. -3-01 nucleotide sequence (SEQ ID NO: 8).
  • the ALT-3-01 nucleotide sequence was correctly inserted into the recombinant cloning vector DBN03-T by restriction enzyme digestion and sequencing.
  • the synthesized EPSPS nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN04-T, wherein EPSPS was an EPSPS nucleotide sequence (SEQ ID NO: 11).
  • EPSPS was an EPSPS nucleotide sequence (SEQ ID NO: 11).
  • SEQ ID NO: 11 The correct insertion of the EPSPS nucleotide sequence in the recombinant cloning vector DBN04-T was confirmed by restriction enzyme digestion and sequencing.
  • Recombinant cloning vector DBN01-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases SpeI and KasI, respectively, and the ALT-1-01 nucleotide sequence was excised. The fragment was inserted between the SpeI and KasI sites of the expression vector DBNBC-01, and the vector was constructed by a conventional enzyme digestion method, which is well known to those skilled in the art, and constructed into a recombinant expression vector DBN100632 (localized to the cytoplasm).
  • the recombinant expression vector DBN100632 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 DBN100632), 42 ° C water bath for 30 seconds; 37 ° C oscillation Incubate for 1 hour (shake shake at 100 rpm); then in the presence of 50 mg / L spectinomycin (Spectinomycin) LB solid plate (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 15g / L, adjusted to pH 7.5 with NaOH) on the temperature of 37 ° C for 12 hours, picking white colonies It was cultured overnight at LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, spectinomycin 50 mg/L, pH adjusted to 7.5 with NaOH) at 37 °C.
  • the plasmid was extracted by an alkali method.
  • the extracted plasmid was digested with restriction endonucleases SpeI and KasI, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence between the SpeI and KasI sites of the recombinant expression vector DBN100632 was the SEQ ID in the sequence listing. NO: The nucleotide sequence shown in 2, the ALT-1-01 nucleotide sequence.
  • a recombinant expression vector DBN100631 (localized to chloroplast) containing the nucleotide sequence of ALT-1-01 was constructed, and its vector structure is shown in Fig.
  • vector skeleton pCAMBIA2301 (CAMBIA institution can provide Spec: Spectinomycin gene; RB: right border; prAtUbi10: Arabidopsis Ubiquitin 10 gene promoter (SEQ ID NO: 12); spAtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO: 17) ALT-1-01: ALT-1-01 nucleotide sequence (SEQ ID NO: 2); tNos: terminator of the nopaline synthase gene (SEQ ID NO: 13); prCaMV35S: cauliflower mosaic virus 35S Promoter (SEQ ID NO: 14); PAT: glufosinate acetyltransferase gene (SEQ ID NO: 15); tCaMV35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 16); LB: left border).
  • the positive clone was sequenced and verified. The result showed that the nucleotide sequence of ALT-1-01 inserted in the recombinant expression vector DBN100631 was the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing, namely ALT-1-01 nucleoside. The acid sequence is inserted correctly.
  • the ALT-2-01 nucleotide sequence excised from the SpeI and KasI recombinant cloning vector DBN02-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100634.
  • the nucleotide sequence in the recombinant expression vector DBN100634 was confirmed to be correctly inserted by the nucleotide sequence shown by SEQ ID NO: 5 in the sequence listing, that is, the nucleotide sequence of ALT-2-01.
  • the ALT-2-01 nucleotide sequence excised by SpeI and KasI recombinant cloning vector DBN02-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100633 (containing spAtCTP2, Located in the chloroplast).
  • the restriction enzyme digestion and sequencing confirmed that the nucleotide sequence in the recombinant expression vector DBN100633 contained the nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing, that is, the nucleotide sequence of ALT-2-01 was correctly inserted.
  • the ALT-3-01 nucleotide sequence excised from the SpeI and KasI recombinant cloning vector DBN03-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100636.
  • the restriction enzyme digestion and sequencing confirmed that the nucleotide sequence in the recombinant expression vector DBN100636 contained the nucleotide sequence shown in SEQ ID NO: 8 in the sequence listing, that is, the nucleotide sequence of ALT-3-01 was correctly inserted.
  • the ALT-3-01 nucleotide sequence excised by SpeI and KasI recombinant cloning vector DBN03-T was inserted into the expression vector DBNBC-01 to obtain a recombinant expression vector DBN100635 (containing spAtCTP2, Located in the chloroplast). Restriction and sequencing confirmed that the nucleotide sequence in the recombinant expression vector DBN100635 contained the nucleotide sequence shown in SEQ ID NO: 8 in the sequence listing, that is, the nucleotide sequence of ALT-3-01 was correctly inserted.
  • the recombinant expression vectors DBN100632, DBN100631, DBN100634, DBN100633, DBN100636 and DBN100635 which have been constructed correctly were transformed into Agrobacterium GV3101 by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L Agrobacterium GV3101, 3 ⁇ L plasmid DNA (recombinant expression vector); The cells were placed in liquid nitrogen for 10 minutes, and warmed at 37 ° C for 10 minutes. The transformed Agrobacterium GV3101 was inoculated into LB tubes and incubated at a temperature of 28 ° C and a rotation speed of 200 rpm for 2 hours, and applied to a 50 mg/L rifle.
  • Wild type Arabidopsis seeds were suspended in a 0.1% (w/v) agarose solution.
  • the suspended seeds were stored at 4 ° C for 2 days to complete the need for dormancy to ensure simultaneous seed germination.
  • the pretreated seeds were planted on a soil mixture and covered with a moisturizing hood for 7 days. Seeds were germinated and plants were grown in a greenhouse under constant temperature (22 ° C) constant humidity (40-50%) long day conditions (16 hours light / 8 hours dark) with a light intensity of 120-150 [mu]mol/m2 sec. Start irrigating the plants with Hoagland nutrient solution, then irrigate with deionized water to keep the soil moist but not soaked.
  • Arabidopsis thaliana was transformed using flower soaking.
  • One or more 15-30 mL precultures of YEP medium containing spectinomycin (50 mg/L) and rifampicin (10 mg/L) were inoculated with selected Agrobacterium colonies. The culture was incubated overnight at 28 ° C with constant shaking at 220 rpm.
  • Each preculture was used to inoculate two 500 mL cultures of YEP medium containing spectinomycin (50 mg/L) and rifampicin (10 mg/L) and the cultures were incubated overnight at 28 °C with constant shaking.
  • the cells were pelleted by centrifugation at about 8700 x g for 10 minutes at room temperature, and the resulting supernatant was discarded.
  • the cell pellet was gently resuspended in 500 mL of osmotic medium containing 1/2 x MS salt/B5 vitamin, 10% (w/v) sucrose, 0.044 ⁇ M benzylaminopurine (10 ⁇ L/L (1 mg/mL DMSO). In the stock solution)) and 300 ⁇ L/L Silvet L-77. Plants of about 1 month old were soaked in the medium for 15 seconds to ensure that the latest inflorescences were immersed. The sides of the plants were then placed downside and covered (transparent or opaque) for 24 hours, then washed with water and placed vertically. The plants were cultured at 22 ° C with a photoperiod of 16 hours light / 8 hours dark. Seeds were harvested after about 4 weeks of soaking.
  • Freshly harvested (ALT nucleotide sequence) T1 seeds were dried at room temperature for 7 days. Seeds were seeded in 26.5 x 51 cm germination trays, each receiving 200 mg T1 seeds (about 10,000 seeds), the seeds were previously suspended in 40 mL of 0.1% (w/v) agarose solution and stored at 4 ° C for 2 days to complete The need for dormancy is to ensure that seeds are germinated simultaneously.
  • the pretreated seeds (each 40 mL) were evenly planted on the soil mixture with a pipette and covered with a moisturizing hood for 4-5 days. The hood was removed 1 day prior to the initial transformant selection using glufosinate (selected co-transformed PAT gene) after emergence.
  • T1 was sprayed with a 0.2% solution of Liberty herbicide (200 g ai/L glufosinate) at a spray volume of 10 mL/disc (703 L/ha) after 7 days of planting (DAP) and again at 11 DAP using a DeVilbiss compressed air nozzle. Plants (coronal stage and 2-4 leaf stage, respectively) were provided to provide an effective amount of 280 g ai/ha of glufosinate per application. Surviving strains (plants that are actively growing) were identified 4-7 days after the last spraying, and transplanted into 7 cm x 7 cm square pots (3-5 per plate) prepared with horse manure and vermiculite, respectively.
  • Liberty herbicide 200 g ai/L glufosinate
  • the transplanted plants were covered with a moisturizing hood for 3-4 days and placed in a 22 ° C culture chamber as before or directly into the greenhouse. The hood was then removed and the plants were planted in the greenhouse at least 1 day prior to testing for the ability of the ALT gene to provide resistance to the sulfonate herbicide (22 ⁇ 5 ° C, 50 ⁇ 30% RH, 14 hours light: 10 hours dark, minimum 500 ⁇ E /m2s1 natural + supplement light).
  • the T1 transformants were first selected from the untransformed seed background using a glufosinate selection protocol. Approximately 40,000 T1 seeds were screened and 380 T1 positive transformants (PAT gene) were identified with a transformation efficiency of approximately 0.95%.
  • the recombinant expression vector DBN100632 was transformed into an Arabidopsis plant (At cytoplasmic ALT-1-01) which was transferred into the cytoplasm and transferred to the ALT-1-01 nucleotide sequence, and the recombinant expression vector DBN100631 was transformed into a chloroplast.
  • Arabidopsis thaliana plants (At chloroplast ALT-1-01) transformed into the ALT-1-01 nucleotide sequence; transformed recombinant expression vector DBN100634 is mapped to the cytoplasmic transfer of ALT-2-01 nucleotide sequence
  • Arabidopsis thaliana plants (At cytoplasmic ALT-2-01), transformed into recombinant expression vector DBN100633, are Arabidopsis plants (At chloroplast ALT-2-01) that are chloroplast-transferred into the ALT-2-01 nucleotide sequence.
  • the recombinant expression vector DBN100636 was transformed into an Arabidopsis thaliana plant (At cytoplasmic ALT-3-01) that was mapped to the cytoplasmic ALT-3-01 nucleotide sequence, and the recombinant expression vector DBN100635 was transformed into Chloroplasts of Arabidopsis plants (At chloroplast ALT-3-01) transferred into the ALT-3-01 nucleotide sequence.
  • T1 plants of At cytoplasmic ALT-1-01, T1 plants of At chloroplast ALT-1-01, T1 plants of At cytoplasmic ALT-2-01, T1 plants of At chloroplast ALT-2-01, At cytoplasm T1 plants of ALT-3-01, T1 plants of At chloroplast ALT-3-01, and wild-type Arabidopsis plants (14 days after sowing) were tested for herbicide tolerance in respectively.
  • T1 plants of At cytoplasmic ALT-1-01, T1 plants of At chloroplast ALT-1-01, T1 plants of At cytoplasmic ALT-2-01, T1 plants of At chloroplast ALT-2-01, and At cells T1 plants of ALT-3-01, T1 plants of At chloroplast ALT-3-01, and wild-type Arabidopsis plants were sprayed with tribenuron (18 g ai/ha, 1x field concentration) and a blank solvent (water). After 14 days of spraying, the plant resistance was counted: after 14 days, the growth condition and the blank solvent (water) were consistently classified as high resistant plants, and after 14 days, the height of the bolting was less than 1/2 of the blank solvent (water).
  • thifensulfuron hydrolase (ALT-1, ALT-2 and ALT-3) confer tolerance to the herbicides of Arabidopsis thaliana plants (some individual plants are not tolerant)
  • T1 generation plant insertion site is random, the expression level of the tolerance gene is different, showing a difference in tolerance levels); compared to the T1 plant of At cytoplasmic ALT-1-01, Atto cytoplasmic ALT-2-01 T1 plants and At cytoplasmic ALT-3-01 T1 plants, At chloroplast ALT-1-01 T1 plants, At chloroplast ALT-2-01 T1 plants and At chloroplast ALT- T1 plants of 3-01 were able to produce higher tolerant tolerant herbicides, indicating that the expression of thifensulfuron hydrolase (ALT-1, ALT-2 and ALT-3)
  • Thiosulfuron hydrolase may also be referred to as a sulfonylurea herbicide deesterase, which degrades a sulfonylurea herbicide having an ester bond (such as thifensulfuron) to a herbicide-free activity by hydrolyzing an ester bond.
  • a parent acid such that it does not degrade a sulfonylurea herbicide (such as nicosulfuron, chlorsulfuron, etc.) without an ester bond.
  • sulfonylurea herbicides having ester bonds and similar structures, such as bensulfuron-methyl, iodosulfuron-methyl, oxysulfuron-methyl, methyl disulfuron (methanesulfonate), pyrazosulfon Long, sulfometuron, chlorpyrifossulfonate and the like.
  • tribenuron 18 g ai/ha, 1x field concentration
  • blank solvent (water) spraying also using iodosulfuron (10g ai / ha, 1 times field concentration
  • Table 2 compares the responses of ALT-1, ALT-2, and ALT-3 inputs to thifensulfuron hydrolase activity to Arabidopsis thaliana T1 plants. Although all transformed Arabidopsis thaliana T1 plants were endowed with thifensulfuron hydrolase activity, in a given treatment (iodosulfuron, methyl disulfuron, and epoxisulfuron), all transformed immigrants None of the mustard T1 plants showed the ability to degrade the above sulfonylurea herbicides, and the degree of damage of all transformed Arabidopsis thaliana T1 plants (ALT-1, ALT-2 and ALT-3) and wild-type Arabidopsis plants There is no difference between them.
  • Table 2 fully illustrates that the results of Table 1 are unexpected. Although fensulfuron-methyl with thifensulfuron-methyl, iodosulfuron-methyl, methyldisulfuron-methyl and nonoxynsulfuron-methyl are all sulfonylurea herbicides with ester bonds and similar chemical structures, and the given treatment is comparable.
  • thifensulfuron hydrolase (ALT-1, ALT-2 and ALT-3) has been introduced and expressed at the expected level in plant individuals, whereas plants expressing thifensulfuron hydrolase are not It has the ability to degrade iodosulfuron-methyl, methyl disulfuron-methyl and epoxisulfuron, and it cannot protect itself from the above-mentioned sulfonylurea herbicides, and there is no difference in performance from wild-type plants. It is sufficient to confirm that the tolerance of thifensulfuron hydrolase (ALT-1, ALT-2 and ALT-3) to plants to terfensulfuron herbicide is unpredictable.
  • Recombinant cloning vector DBN01-T, DBN04-T and expression vector DBNBC-02 (vector backbone: pCAMBIA2301 (available from CAMBIA)) were digested with restriction endonucleases SpeI and KasI, NcoI and FspI, respectively, and the ALT was excised.
  • the -1-01 nucleotide sequence and the EPSPS nucleotide sequence fragment are inserted between the SpeI and KasI, NcoI and FspI sites of the expression vector DBNBC-02, respectively, and the construction of the vector by a conventional enzymatic cleavage method is known to those skilled in the art.
  • the recombinant expression vector DBN100828 was transformed into E. coli T1 competent cells by a heat shock method according to the method of 2 in the second embodiment, and the plasmid was extracted by an alkali method.
  • the extracted plasmid was digested with restriction endonucleases SpeI and KasI, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence between the SpeI and KasI sites of the recombinant expression vector DBN100828 was the SEQ ID in the sequence listing. NO: The nucleotide sequence shown in 2, the ALT-1-01 nucleotide sequence.
  • the recombinant expression vector DBN100827 (localized to chloroplast) containing the ALT-1-01 nucleotide sequence was constructed according to the above method for constructing the recombinant expression vector DBN100828, and its vector structure is shown in Fig.
  • vector skeleton pCAMBIA2301 (CAMBIA institution can provide Spec: Spectinomycin gene; RB: right border; prAtUbi10: Arabidopsis Ubiquitin 10 gene promoter (SEQ ID NO: 12); spAtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO: 17) ALT-1-01: ALT-1-01 nucleotide sequence (SEQ ID NO: 2); tNos: terminator of the nopaline synthase gene (SEQ ID NO: 13); prBrCBP: rapeseed eukaryotic elongation factor Gene 1 ⁇ (Tsf1) promoter (SEQ ID NO: 18); spAtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO: 17); EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 11); tPsE9: terminat
  • the positive clone was sequenced and verified. The result showed that the nucleotide sequence of ALT-1-01 inserted in the recombinant expression vector DBN100827 was the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing, namely ALT-1-01 nucleoside. The acid sequence is inserted correctly.
  • the ALT-2-01 nucleotide sequence and EPSPS nucleotide sequence excised by SpeI and KasI, NcoI and FspI recombinant cloning vectors DBN02-T and DBN04-T were inserted and expressed.
  • Vector DBNBC-02, recombinant expression vector DBN100826 was obtained.
  • the nucleotide sequence in the recombinant expression vector DBN100826 contains the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 11 in the sequence listing, namely the ALT-2-01 nucleotide sequence and EPSPS. The nucleotide sequence is inserted correctly.
  • the ALT-2-01 nucleotide sequence and EPSPS nucleotide sequence excised by SpeI and KasI, NcoI and FspI recombinant cloning vectors DBN02-T and DBN04-T were inserted and expressed.
  • Vector DBNBC-02, recombinant expression vector DBN100825 (containing spAtCTP2, localized to chloroplast) was obtained.
  • the nucleotide sequence in the recombinant expression vector DBN100825 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 5 and SEQ ID NO: 11 in the sequence listing, namely ALT-2-01 nucleotide sequence and EPSPS. The nucleotide sequence is inserted correctly.
  • the ALT-3-01 nucleotide sequence and EPSPS nucleotide sequence excised by SpeI and KasI, NcoI and FspI recombinant cloning vectors DBN03-T and DBN04-T were inserted and expressed.
  • Vector DBNBC-02, recombinant expression vector DBN100824 was obtained.
  • the nucleotide sequence in the recombinant expression vector DBN100824 contains the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 11 in the sequence listing, namely the ALT-3-01 nucleotide sequence and EPSPS. The nucleotide sequence is inserted correctly.
  • the ALT-3-01 nucleotide sequence and the EPSPS nucleotide sequence of the SpeI and KasI, NcoI and FspI digestion recombinant cloning vectors DBN03-T and DBN04-T were inserted and expressed.
  • Vector DBNBC-02, recombinant expression vector DBN100823 (containing spAtCTP2, localized to chloroplast) was obtained.
  • the nucleotide sequence in the recombinant expression vector DBN100823 was confirmed to be the nucleotide sequence shown by SEQ ID NO: 8 and SEQ ID NO: 11 in the sequence listing, ie, the ALT-3-01 nucleotide sequence and EPSPS. The nucleotide sequence is inserted correctly.
  • the recombinant expression vectors DBN100828, DBN100827, DBN100826, DBN100825, DBN100824 and DBN100823, which have been constructed correctly, were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 ⁇ L.
  • Agrobacterium LBA4404 3 ⁇ L of plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37 ° C warm water bath for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated in LB tube at a temperature of 28 ° C, 200 rpm Incubate for 2 hours, apply to LB plates containing 50 mg/L of rifampicin and 50 mg/L of spectinomycin until a positive monoclonal is grown, pick up the monoclonal culture and extract the plasmid, with restriction The dicer enzyme was digested and verified, and the results showed that the recombinant expression vectors DBN100828, DBN100827, DBN100826, DBN100825, DBN100824 and DBN100823 were completely correct.
  • the cotyledonary node tissue of the aseptically cultured soybean variety Zhonghuang 13 was co-cultured with the Agrobacterium of the second embodiment according to the conventional Agrobacterium infection method to construct the recombinant expression vector constructed in the first embodiment.
  • T-DNA in DBN100828, DBN100827, DBN100826, DBN100825, DBN100824 and DBN100823 (including the promoter sequence of Arabidopsis Ubiquitin10 gene, ALT-1-01 nucleotide sequence, ALT-2-01 nucleotide sequence, ALT- 3-01 nucleotide sequence, tNos terminator, rapeseed eukaryotic elongation factor gene 1 ⁇ promoter, Arabidopsis chloroplast transit peptide, 5-enolpyruvylshikimate-3-phosphate synthase gene, termination of pea RbcS gene
  • the sub-transformation into the soybean genome, the soybean recombinant plant (Gm cytoplasmic ALT-1-01) transformed into the ALT-1-01 nucleotide sequence which is mapped to the cytoplasm and transformed into the recombinant expression vector DBN100828 was obtained.
  • the recombinant expression vector DBN100827 is a soybean plant (Gm chloroplast ALT-1-01) which is mapped to the chloroplast and transferred to the ALT-1-01 nucleotide sequence; the recombinant expression vector DBN100826 is transformed into the cytoplasmic transfer ALT- 2-01 nucleotide sequence of soybean plants (Gm cytoplasmic ALT-2-01), transformed
  • the expression vector DBN100825 is a soybean plant (Gm chloroplast ALT-2-01) which is mapped to the chloroplast and transferred into the ALT-2-01 nucleotide sequence; the recombinant expression vector DBN100824 is transformed into the cytoplasmic transfer ALT- 3-01 nucleotide sequence of soybean plant (Gm cytoplasmic ALT-3-01), transformed into recombinant expression vector DBN100823, which is a chloroplast-transferred soybean plant (Gm chloroplast ALT) -3-01); At the same time, wild type soybean plants were used as controls.
  • 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 fresh green cotyledonary nodes are enlarged.
  • the hypocotyls were cut at 3-4 mm below the cotyledonary node, and the cotyledons were cut longitudinally to remove the top buds, lateral buds and seed roots.
  • the wound is treated at the cotyledonary node with the back of the scalpel, and the wounded cotyledonary node tissue is contacted with the Agrobacterium suspension, wherein the Agrobacterium can express the ALT-1-01 nucleotide sequence, the ALT-2-01 nucleotide sequence, Transfer of the ALT-3-01 nucleotide sequence to the wounded cotyledonary node tissue (step 1: Infection step)
  • Base MS salt 2.15g / L, B
  • Cotyledonary node tissue and Agrobacterium co-culture for a period of time (3 days) (Step 2: co-cultivation step).
  • cotyledonary node tissue is in solid after the infection step Medium (MS salt 4.3g / L, B5 vitamin, sucrose 20g / L, glucose 10g / L, 2-morpholine ethanesulfonic acid (MES) 4g / L, zeatin 2mg / L, agar 8g / L, pH5. 6) Upper culture.
  • MS salt 4.3g / L
  • B5 vitamin sucrose 20g / L
  • glucose 10g / L glucose 10g / L
  • zeatin 2mg / L agar 8g / L, pH5.
  • agar 8g / L pH5.
  • the medium is restored (B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonate) Acid (MES) 1g / L, sucrose 30 At least one of g/L, zeatin (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)
  • An antibiotic cephalosporin which inhibits the growth of Agrobacterium is not added, and a selection agent for the plant transformant is not added (step 3: recovery step).
  • the tissue block of the cotyledonary node regeneration is on a solid medium having an antibiotic but no selective agent.
  • the tissue blocks of the cotyledonary node regeneration are cultured on a medium containing a selective agent (glyphosate) and the grown transformed callus is selected (Step 4: Selecting step).
  • the cotyledonary node-regenerated tissue block is in a selective solid medium (B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/ L,6-benzyl adenine (6-BAP) 1 mg/L, agar 8 g/L, cephalosporin 150 mg/L, glutamic acid 100 mg/L, aspartic acid 100 mg/L, N-(phosphine carboxymethyl Incubation on the basis of glycine 0.25 mol/L, pH 5.6), resulting in selective growth of transformed cells.
  • B5 salt 3.1 g/L B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/ L,6-benzyl adenine (6-BAP) 1 mg/L, agar 8 g/L, cephalosporin 150 mg/L, glutamic acid 100 mg/L
  • the transformed cells are then regenerated into plants (step 5: regeneration step), preferably in the presence of a selection agent Culturing regenerated plants on the regeneration of cotyledonary node tissue growth on solid medium medium block (B5 B5 rooting medium and differentiation medium).
  • a selection agent Culturing regenerated plants on the regeneration of cotyledonary node tissue growth on solid medium medium block (B5 B5 rooting medium and differentiation medium).
  • the selected resistant tissue blocks were transferred to B5 differentiation medium (B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, zeatin (ZT) 1 mg/ L, agar 8g / L, cephalosporin 150mg / L, glutamic acid 50mg / L, aspartic acid 50mg / L, gibberellin 1mg / L, auxin 1mg / L, N- (phosphine carboxymethyl)
  • B5 differentiation medium B5 salt 3.1 g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1 g/L, sucrose 30 g/L, zeatin (ZT) 1 mg/ L, agar 8g / L, cephalosporin 150mg / L, glutamic acid 50mg / L, aspartic acid 50mg / L, gibberellin
  • B5 rooting medium B5 salt 3.1g/L, B5 vitamin, 2-morpholine ethanesulfonic acid (MES) 1g/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/ L, indole-3-butyric acid (IBA) 1 mg/L
  • MES 2-morpholine ethanesulfonic acid
  • IBA indole-3-butyric acid
  • Gm cytoplasmic ALT-1-01 soybean plants, Gm chloroplast ALT-1-01 soybean plants, Gm cytoplasmic ALT-2-01 soybean plants, Gm chloroplast ALT-2-01 soybean plants, Gm cells About 100 mg of the leaves of soybean plants of ALT-3-01 and Gm chloroplast ALT-3-01 were used as samples.
  • the genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the EPSPS gene was detected by Taqman probe fluorescent quantitative PCR. The copy number is used to determine the copy number of the ALT gene.
  • the wild type soybean plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was set to repeat 3 times and averaged.
  • the specific method for detecting the EPSPS gene copy number is as follows:
  • Step 11 Take Gm cytoplasmic ALT-1-01 soybean plant, Gm chloroplast ALT-1-01 soybean plant, Gm cytoplasmic ALT-2-01 soybean plant, Gm chloroplast ALT-2-01 soybean plant Soybean plants of Gm cytoplasmic ALT-3-01, soybean plants of Gm chloroplast ALT-3-01, and leaves of wild-type soybean plants were each 100 mg in a mortar, and homogenized by liquid nitrogen, respectively. 3 repetitions;
  • Step 12 Extract the genomic DNA of the above sample using Qiagen's DNeasy Plant Mini Kit, and refer to the product manual for the specific method;
  • Step 13 Determine the genomic DNA concentration of the above sample using NanoDrop 2000 (Thermo Scientific).
  • Step 14 adjusting the genomic DNA concentration of the above sample to the same concentration value, the concentration value ranges from 80 to 100 ng / ⁇ L;
  • Step 15 The Taqman probe real-time PCR method is used to identify the copy number of the sample, and the sample with the known copy number is used as a standard, and the sample of the wild type soybean plant is used as a control, and each sample is repeated for 3 times, and the average is taken. Value; the fluorescent PCR primers and probe sequences are:
  • Probe 1 ATGCAGGCGATGGGCGCCCGCATCCGTA as shown in SEQ ID NO: 22 in the Sequence Listing;
  • the PCR reaction system is:
  • the 50 ⁇ primer/probe mixture contained 45 ⁇ L of each primer at a concentration of 1 mM, 50 ⁇ L of probe at a concentration of 100 ⁇ M and 860 ⁇ L of 1 ⁇ TE buffer, and stored at 4° C. in an amber tube.
  • the PCR reaction conditions are:
  • Gm cytoplasmic ALT-1-01 soybean plant, Gm chloroplast ALT-1-01 soybean plant, Gm cytoplasmic ALT-2-01 soybean plant, Gm chloroplast ALT-2-01 soybean plant, Gm cytoplasm ALT-3-01 soybean plants, Gm chloroplast ALT-3-01 soybean plants and wild-type soybean plants (seedling stage) were tested for herbicide tolerance in the phenylsulfuron-methyl.
  • Gm cytoplasmic ALT-1-01 soybean plants, Gm chloroplast ALT-1-01 soybean plants, Gm cytoplasmic ALT-2-01 soybean plants, Gm chloroplast ALT-2-01 soybean plants, Gm cells Soybean plants of ALT-3-01, soybean plants of Gm chloroplast ALT-3-01, and wild-type soybean plants were sprayed with tribenuron (72 g ai/ha, 4 times field concentration) and a blank solvent (water). 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. .
  • Gm cytoplasmic ALT-1-01 soybean plants have 2 strains (S1 and S2), Gm Soybean plants with chloroplast ALT-1-01 total 2 strains (S3 and S4), Gm cytoplasmic ALT-2-01 soybean plants have 2 strains (S5 and S6), Gm chloroplast ALT-2-01 Soybean plants have 2 strains (S7 and S8), Gm cytoplasmic ALT-3-01 soybean plants have 2 strains (S9 and S10), and Gm chloroplast ALT-3-01 soybean plants have 2 strains. (S11 and S12), one strain of wild type soybean plants (CK1); 10-15 strains of each strain were tested. The results are shown in Table 3 and Figure 11.
  • thifensulfuron hydrolase (ALT-1, ALT-2, and ALT-3) confers high levels of bensulfuron-methyl herbicide tolerance to transgenic soybean plants; compared to Gm cytoplasmic ALT- 1-01 soybean plant, Gm cytoplasmic ALT-2-01 Soybean plants and Gm cytoplasmic ALT-3-01 soybean plants, Gm chloroplast ALT-1-01 soybean plants, Gm chloroplast ALT-2-01 soybean plants and Gm chloroplast ALT-3-01 soybean plants can be produced Higher tolerance of sulfonate herbicides, indicating that the expression of thifensulfuron hydrolase (ALT-1, ALT-2 and ALT-3) genes in chloroplasts enhances the tolerance of soybean plants to bensulfuron-methyl herbicides. Wild type soybean plants do not have tolerance to ben
  • Gm cytoplasmic ALT-1-01 soybean plant, Gm chloroplast ALT-1-01 soybean plant, Gm cytoplasmic ALT-2-01 soybean plant, Gm chloroplast ALT-2-01 soybean plant, Gm cytoplasm ALT-3-01 soybean plants, Gm chloroplast ALT-3-01 soybean plants and wild-type soybean plants (seedling stage) were tested for herbicide tolerance in glyphosate.
  • Gm cytoplasmic ALT-1-01 soybean plants, Gm chloroplast ALT-1-01 soybean plants, Gm cytoplasmic ALT-2-01 soybean plants, Gm chloroplast ALT-2-01 soybean plants, Gm cells Soybean plants of ALT-3-01, soybean plants of Gm chloroplast ALT-3-01, and two strains of wild-type soybean plants were selected, and 10-15 strains were selected from each strain for testing.
  • Herbicide victimization rate (%) ⁇ (number of affected plants ⁇ number of grades) / (total number of plants ⁇ highest level) ).
  • the symptoms of phytotoxicity are classified as shown in Table 5.
  • the synthetic ALT-1-02 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 a recombinant cloning vector DBN05.
  • the recombinant cloning vector DBN05-T was transformed into E. coli T1 competent cells by heat shock method according to the method of 1 in the second embodiment, and the plasmid was extracted by alkali method. The extracted plasmid was identified by SpeI and KasI digestion, and positive clones were obtained.
  • the sequencing results showed that the nucleotide sequence of ALT-1-02 inserted into the recombinant cloning vector DBN05-T was the nucleotide sequence shown by SEQ ID NO: 3 in the sequence listing, namely ALT-1-02 nucleotide. The sequence is inserted correctly.
  • the synthetic ALT-2-02 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN06-T, wherein ALT-2-02 was ALT. -2-02 nucleotide sequence (SEQ ID NO: 6).
  • the ALT-2-02 nucleotide sequence was correctly inserted into the recombinant cloning vector DBN06-T by restriction enzyme digestion and sequencing.
  • the synthetic ALT-3-02 nucleotide sequence was ligated into the cloning vector pGEM-T to obtain a recombinant cloning vector DBN07-T, wherein ALT-3-02 was ALT. -3-02 nucleotide sequence (SEQ ID NO: 9).
  • the ALT-3-02 nucleotide sequence was correctly inserted into the recombinant cloning vector DBN07-T by restriction enzyme digestion and sequencing.
  • Recombinant cloning vector DBN05-T and expression vector DBNBC-03 were digested with restriction endonucleases SpeI and KasI, respectively, and the ALT-1-02 nucleotide sequence was excised. The fragment was inserted between the SpeI and KasI sites of the expression vector DBNBC-03, and the construction of the vector by conventional enzymatic cleavage method is well known to those skilled in the art, and the recombinant expression vector DBN100830 (localized to the cytoplasm) was constructed.
  • the recombinant expression vector DBN100830 was transformed into E. coli T1 competent cells by a heat shock method according to the method of 2 in the second embodiment, and the plasmid was extracted by an alkali method.
  • the extracted plasmids were digested with restriction endonucleases SpeI and KasI, and the positive clones were sequenced.
  • the results showed that the nucleotide sequence between the SpeI and KasI sites of the recombinant expression vector DBN100830 was the SEQ ID in the sequence listing. NO: The nucleotide sequence shown by 3, the ALT-1-02 nucleotide sequence.
  • a recombinant expression vector DBN100829 (localized to chloroplast) containing the nucleotide sequence of ALT-1-02 was constructed, and its vector structure is shown in Fig.
  • vector skeleton pCAMBIA2301 (CAMBIA institution can provide Spec: spectinomycin gene; RB: right border; prUbi: maize Ubiquitin (ubiquitin) 1 gene promoter (SEQ ID NO: 23); spAtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO: 17); ALT-1-02: ALT-1-02 nucleotide sequence (SEQ ID NO: 3); tNos: terminator of the nopaline synthase gene (SEQ ID NO: 13); PMI: phosphomannose isomerase gene (SEQ ID NO: 24); LB: left border).
  • the positive clone was sequenced and verified.
  • nucleotide sequence of ALT-1-02 inserted in the recombinant expression vector DBN100829 was the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing, namely ALT-1-02 nucleoside. The acid sequence is inserted correctly.
  • the ALT-2-02 nucleotide sequence excised from the SpeI and KasI recombinant cloning vector DBN06-T was inserted into the expression vector DBNBC-03 to obtain a recombinant expression vector DBN100832. Restriction and sequencing confirmed that the nucleotide sequence in the recombinant expression vector DBN100832 contained the nucleotide sequence shown in SEQ ID NO: 6 in the sequence listing, that is, the nucleotide sequence of ALT-2-02 was correctly inserted.
  • the ALT-2-02 nucleotide sequence excised by SpeI and KasI recombinant cloning vector DBN06-T was inserted into the expression vector DBNBC-03 to obtain a recombinant expression vector DBN100831 (containing spAtCTP2, Located in the chloroplast).
  • the restriction enzyme digestion and sequencing confirmed that the nucleotide sequence in the recombinant expression vector DBN100831 contained the nucleotide sequence shown in SEQ ID NO: 6 in the sequence listing, that is, the nucleotide sequence of ALT-2-02 was correctly inserted.
  • the ALT-3-02 nucleotide sequence excised from the SpeI and KasI recombinant cloning vector DBN07-T was inserted into the expression vector DBNBC-03 to obtain a recombinant expression vector DBN100834.
  • the restriction enzyme digestion and sequencing confirmed that the nucleotide sequence in the recombinant expression vector DBN100834 contained the nucleotide sequence shown in SEQ ID NO: 9 in the sequence listing, that is, the nucleotide sequence of ALT-3-02 was correctly inserted.
  • the nucleotide sequence of ALT-3-02 excised by SpeI and KasI recombinant cloning vector DBN07-T was inserted into the expression vector DBNBC-03 to obtain a recombinant expression vector DBN100833 (containing spAtCTP2, Located in the chloroplast). Restriction and sequencing confirmed that the nucleotide sequence in the recombinant expression vector DBN100833 contained the nucleotide sequence shown in SEQ ID NO: 9 in the sequence listing, that is, the nucleotide sequence of ALT-3-02 was correctly inserted.
  • the recombinant expression vectors DBN100830, DBN100829, DBN100832, DBN100831, DBN100834 and DBN100833, 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.
  • the transformed Agrobacterium LBA4404 was inoculated into LB tubes and incubated at a temperature of 28 ° C and a rotation speed of 200 rpm for 2 hours, and applied to a 50 mg/L rifle.
  • a positive monoclonal was grown, and the monoclonal culture was picked and the plasmid was extracted, and the restriction endonuclease was used for restriction enzyme digestion.
  • the result showed that the recombinant expression vector DBN100830 The DBN100829, DBN100832, DBN100831, DBN100834, and DBN100833 structures are completely correct.
  • the immature embryo of the aseptically cultured maize variety 31 was co-cultured with the Agrobacterium of the ninth embodiment in accordance with the conventional Agrobacterium infection method to construct the recombinant expression of the ninth embodiment.
  • T-DNA in vectors DBN100830, DBN100829, DBN100832, DBN100831, DBN100834 and DBN100833 (including the promoter sequence of maize Ubiquitin1 gene, ALT-1-02 nucleotide sequence, ALT-2-02 nucleotide sequence, ALT-3) -02 nucleotide sequence, Arabidopsis thaliana chloroplast transit peptide, PMI gene and tNos terminator sequence) were transferred into the maize genome, and the transformed recombinant expression vector DBN100830 was obtained for cytoplasmic transfer into ALT-1- A nucleotide sequence of a maize plant (Zm cytoplasmic ALT-1-02) transformed into a recombinant expression vector
  • transformed recombinant expression vector DBN100832 is a maize plant (Zm cytoplasmic ALT-2-02) that is localized to the cytoplasmic transfer into the ALT-2-02 nucleotide sequence, and the recombinant expression vector DBN100831 is mapped to Chloroplasts of maize plants transferred to the ALT-2-02 nucleotide sequence (Zm chloroplast ALT-2-02)
  • the recombinant expression vector DBN100834 was transformed into a cytoplasmic maize plant (Zm cytoplasmic ALT-3-02) transfected with the ALT-3-02 nucleotide sequence, and the recombinant expression vector DBN100833 was transformed into a chloroplast-targeted transfer.
  • Maize plants with ALT-3-02 nucleotide sequence Zm chloroplast ALT-3-02
  • wild type maize plants were used as controls.
  • immature immature embryos are isolated from maize, and the immature embryos are contacted with Agrobacterium suspension, wherein Agrobacterium can ALT-1-02 nucleotide sequence, ALT-2- The 02 nucleotide sequence, the ALT-3-02 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, acetosyringone (AS) 100 mg/L) It was cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg/L, agar 8 g/L, pH 5.8). After this co-cultivation phase, there can be an optional "recovery" step.
  • the medium was restored (MS salt 4.3 g / L, MS vitamin, casein 300 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg /
  • At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium is present in L, plant gel 3 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step).
  • the immature embryos are cultured on a solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for the infected cells.
  • the inoculated immature embryos are cultured on a medium containing a selective agent (mannose) and the grown transformed callus is selected (step 4: selection step).
  • the immature embryo is screened in solid medium with selective agent (MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, mannose 12.5 g/L, 2,4-dichlorobenzene)
  • MS salt 4.3 g/L, MS vitamin, casein 300 mg/L, sucrose 30 g/L, mannose 12.5 g/L, 2,4-dichlorobenzene Incubation of oxyacetic acid (2,4-D) 1 mg/L, plant gel 3 g/L, pH 5.8) resulted in selective growth of transformed cells.
  • the callus regenerates the plant (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and MS rooting medium) Recycled plants.
  • the selected resistant callus was transferred to 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 3g / L, pH 5.8), culture differentiation at 25 ° C.
  • 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 3g / L, pH 5.8
  • MS rooting medium MS salt 2.15g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, indole-3-acetic acid 1mg/L, plant gel 3g/L, pH5 .8
  • culture at 25 ° C to a height of about 10 cm, and move to a greenhouse to grow to firmness. In the greenhouse, the cells were cultured at 28 ° C for 16 hours and then at 20
  • 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 4 CGATCTGCAGGTCGACGG as shown in SEQ ID NO: 26 in the Sequence Listing;
  • Probe 2 TCTCTTGCTAAGCTGGGAGCTCGATCC is shown as SEQ ID NO: 27 in the Sequence Listing.
  • ALT-1-02 nucleotide sequence the ALT-2-02 nucleotide sequence and the ALT-3-02 nucleotide sequence were integrated into the tested maize plants.
  • Zm cytoplasmic ALT-1-02 maize plant Zm chloroplast ALT-1-02 maize plant, Zm cytoplasmic ALT-2-02 corn plant, Zm chloroplast ALT-2-02 corn Plants, maize plants of Zm cytoplasmic ALT-3-02 and maize plants of Zm chloroplast ALT-3-02 obtained a single copy of the transgenic maize plants.
  • Maize plants of ALT-3-02 maize plants of Zm chloroplast ALT-3-02 and wild-type maize plants were sprayed with tribenuron (72 g ai/ha, 4 times field concentration) and blank solvent (water).
  • Zm cytoplasmic ALT-1-02 maize plants have 2 strains (S13 and S14), Zm chloroplast ALT-1-02 soybean plants have 2 strains (S15 and S16), Zm cytoplasmic ALT-2- There are 2 strains of maize plants in 02 (S17 and S18), 2 strains of soybean plants with Zm chloroplast ALT-2-02 (S19 and S20), and 2 maize plants with Zm cytoplasmic ALT-3-02. Strains (S21 and S22), Zm chloroplast ALT-3-02 soybean plants a total of 2 strains (S23 and S24), wild-type maize plants (CK2) a total of 1 strain; from each strain selected 10- 15 strains were tested. The results are shown in Table 4 and Figure 10.
  • thifensulfuron hydrolase (ALT-1, ALT-2, and ALT-3) confers high levels of bensulfuron-methyl herbicide tolerance in transgenic maize plants; compared to Zm cytoplasmic ALT- 1-02 maize plant, Zm cytoplasmic ALT-2-02 maize plant and Zm cytoplasmic ALT-3-02 maize plant, Zm chloroplast ALT-1-02 maize plant, Zm chloroplast ALT-2-02 Maize plants and Zm chloroplast ALT-3-02 maize plants were able to produce higher tolerant tolerant herbicides, indicating that the thifensulfuron hydrolase (ALT-1, ALT-2 and ALT-3) genes are localized to chloroplasts.
  • Medium expression can enhance the tolerance of corn plants to bensulfuron-methyl herbicides; wild
  • the present invention discloses for the first time that thifensulfuron hydrolase (ALT-1, ALT-2 and ALT-3) can exhibit high tolerance to bensulfuron-methyl herbicides and contains a thiophenesulfuron hydrolase.
  • thifensulfuron hydrolase ALT-1, ALT-2 and ALT-3
  • Arabidopsis plants, soybean plants and maize plants with nucleotide sequences are highly tolerant to bensulfuron-methyl herbicides and can tolerate at least one-fold field concentration, so they have broad application prospects on plants.

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Abstract

提供了一种控制杂草的方法，包括将编码噻吩磺隆水解酶的核苷酸序列转化至植物基因组中，所述转基因植物与对照植物相比具有减弱的植物损伤和/或增加的植物产量，再将含有效剂量苯磺隆的除草剂施加到所述转基因植物的生长环境中，由于噻吩磺隆水解酶对苯磺隆除草剂有较高的耐受性，从而实现控制杂草。

Description

除草剂耐受性蛋白质的用途 技术领域
本发明涉及一种除草剂耐受性蛋白质的用途，特别是涉及一种噻吩磺隆水解酶降解苯磺隆除草剂的用途。
背景技术
杂草可以迅速耗尽土壤中作物和其它目的植物所需要的有价值的养分。目前有许多类型的除草剂用于控制杂草，一种特别流行的除草剂是草甘膦。已经开发了对草甘膦具有抗性的作物，如玉米、大豆、棉花、甜菜、小麦和水稻等。因此可以对种植草甘膦抗性作物的田地喷洒草甘膦以控制杂草而不显著损害作物。
草甘膦已经在全球广泛使用超过20年，由此导致对草甘膦和草甘膦耐性作物技术的过度依赖，并在野生杂草物种中对草甘膦天然更具耐受性或已经发展出抗草甘膦活性的植物施加了高选择压。已报道有少数杂草已发展出对草甘膦的抗性，包括阔叶杂草和禾本科杂草，如瑞士黑麦草、多花黑麦草、牛筋草、豚草、小飞蓬、野塘蒿和长叶车前。此外，在广泛使用草甘膦耐性作物之前并不是农业问题的杂草也逐渐盛行，并且难于用草甘膦耐性作物控制，这些杂草主要与(但不仅与)难于控制的阔叶杂草一起出现，如苋属、藜属、蒲公英属和鸭跖草科物种。
在草甘膦抗性杂草或难于控制的杂草物种的地区，种植者可以通过罐混或换用能控制遗漏杂草的其它除草剂来弥补草甘膦的弱点，如磺酰脲类除草剂。磺酰脲类除草剂已经成为继有机磷、乙酰胺类除草剂后的第三大除草剂，全球年销售额达到30亿美元以上，我国磺酰脲类除草剂每年的应用面积已超过200万公顷，并仍呈扩大的趋势。
随着草甘膦抗性杂草的出现和磺酰脲类除草剂的扩大应用，需要对磺酰脲类除草剂敏感的目的植物中输入磺酰脲类除草剂耐受性。磺酰脲类除草剂可大致分为含酯键的和不含酯键的，其中含酯键且化学结构相近的磺酰脲类除草剂至少有十余种。仅已鉴定了噻吩磺隆水解酶可以降解噻吩磺隆，但和噻吩磺隆一样，苯磺隆也属于含酯键的磺酰脲类除草剂，而目前未发现噻吩磺隆水解酶对苯磺隆除草剂具有耐受性的报道。
发明内容
本发明的目的是提供一种除草剂耐受性蛋白质的用途，首次提供了将含有有效剂量苯磺隆的除草剂施加到存在至少一种表达噻吩磺隆水解酶的转基因植物的植物生长环境中以控制田间杂草生长的方法，增加了噻吩磺隆水解酶对除草剂的耐受范围。
为实现上述目的，本发明提供了一种控制杂草的方法，包括将含有有效剂量苯磺隆的除草剂施加到存在至少一种转基因植物的植物生长环境中，转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列，转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
进一步地，有效剂量苯磺隆为9-144g ai/ha。
更进一步地，转基因植物为单子叶植物或双子叶植物。
优选地，转基因植物为玉米、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，噻吩磺隆水解酶的氨基酸序列具有SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列。
优选地，噻吩磺隆水解酶的核苷酸序列具有：
(a)编码SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列的核苷酸序列；或
(b)SEQ ID NO:2或SEQ ID NO:3所示的核苷酸序列；或
(c)SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列；或
(d)SEQ ID NO:8或SEQ ID NO:9所示的核苷酸序列。
进一步地，转基因植物还可以包括至少一种不同于编码噻吩磺隆水解酶的核苷酸序列的第二种核苷酸。
第二种核苷酸编码选择标记蛋白质、合成活性蛋白质、分解活性蛋白质、抗生物胁迫蛋白质、抗非生物胁迫蛋白质、雄性不育蛋白质、影响植物产量的蛋白质和/或影响植物品质的蛋白质。
具体地，第二种核苷酸编码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。
可选择地，含有有效剂量苯磺隆的除草剂还包括草甘膦除草剂、草铵膦除草剂、植物生长素类除草剂、禾本科除草剂、发芽前选择性除草剂和/或发芽后选择性除草剂。
为实现上述目的，本发明还提供了一种控制草甘膦耐受性杂草的方法，包括将有效剂量的苯磺隆除草剂和草甘膦除草剂施加到种植至少一种转基因植物的大田中，大田中包含草甘膦耐受性杂草或其种子，转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列和编码草甘膦耐受性蛋白质的核苷酸序列，转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列和/或编码草甘膦耐受性蛋白质的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
进一步地，有效剂量苯磺隆为9-144g ai/ha。有效剂量草甘膦为200-1600g ae/ha。
更进一步地，转基因植物为单子叶植物或双子叶植物。
优选地，转基因植物为玉米、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，噻吩磺隆水解酶的氨基酸序列具有SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列。
优选地，噻吩磺隆水解酶的核苷酸序列具有：
(a)编码SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列的核苷酸序列；或
(b)SEQ ID NO:2或SEQ ID NO:3所示的核苷酸序列；或
(c)SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列；或
(d)SEQ ID NO:8或SEQ ID NO:9所示的核苷酸序列。
进一步地，草甘膦耐受性蛋白质包括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-乙酰转移酶或草甘膦脱羧酶。
具体地，草甘膦耐受性蛋白质的氨基酸序列具有SEQ ID NO:10所示的氨基酸序列。
优选地，草甘膦耐受性蛋白质的核苷酸序列具有：
(a)编码SEQ ID NO:10所示的氨基酸序列的核苷酸序列；或
(b)SEQ ID NO:11所示的核苷酸序列。
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，包括苯磺隆除草剂和存在至少一种转基因植物的植物生长环境，将含有有效剂量的苯磺隆除草剂施加到存在至少一种转基因植物的植物生长环境中，转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列，转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
进一步地，有效剂量苯磺隆为9-144g ai/ha。
更进一步地，转基因植物为单子叶植物或双子叶植物。
优选地，转基因植物为玉米、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，噻吩磺隆水解酶的氨基酸序列具有SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列。
优选地，噻吩磺隆水解酶的核苷酸序列具有：
(a)编码SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列的核苷酸序列；或
(b)SEQ ID NO:2或SEQ ID NO:3所示的核苷酸序列；或
(c)SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列；或
(d)SEQ ID NO:8或SEQ ID NO:9所示的核苷酸序列。
进一步地，转基因植物还可以包括至少一种不同于编码噻吩磺隆水解酶的核苷酸序列的第二种核苷酸。
第二种核苷酸编码选择标记蛋白质、合成活性蛋白质、分解活性蛋白质、抗生物胁迫蛋白质、抗非生物胁迫蛋白质、雄性不育蛋白质、影响植物产量的蛋白质和/或影响植物品质的蛋白质。
具体地，第二种核苷酸编码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、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。
可选择地，含有除草有效剂量苯磺隆的除草剂还包括草甘膦除草剂、草铵膦除草剂、植物生长素类除草剂、禾本科除草剂、发芽前选择性除草剂和/或发芽后选择性除草剂。
为实现上述目的，本发明还提供了一种控制草甘膦耐受性杂草的种植系统，包括苯磺隆除草剂、草甘膦除草剂和种植至少一种转基因植物的大田，将有效剂量的苯磺隆除草剂和草甘膦除草剂施加到种植至少一种转基因植物的大田中，大田中包含草甘膦耐受性杂草或其种子，转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列和编码草甘膦耐受性蛋白质的核苷酸序列，转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列和/或编码草甘膦耐受性蛋白质的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
进一步地，有效剂量苯磺隆为9-144g ai/ha。有效剂量草甘膦为200-1600g ae/ha。
更进一步地，转基因植物为单子叶植物或双子叶植物。
优选地，转基因植物为玉米、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，噻吩磺隆水解酶的氨基酸序列具有SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列。
优选地，噻吩磺隆水解酶的核苷酸序列具有：
(a)编码SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列的核苷酸序列；或
(b)SEQ ID NO:2或SEQ ID NO:3所示的核苷酸序列；或
(c)SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列；或
(d)SEQ ID NO:8或SEQ ID NO:9所示的核苷酸序列。
进一步地，草甘膦耐受性蛋白质包括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-乙酰转移酶或草甘膦脱羧酶。
具体地，草甘膦耐受性蛋白质的氨基酸序列具有SEQ ID NO:10所示的氨基酸序列。
优选地，草甘膦耐受性蛋白质的核苷酸序列具有：
(a)编码SEQ ID NO:10所示的氨基酸序列的核苷酸序列；或
(b)SEQ ID NO:11所示的核苷酸序列。
为实现上述目的，本发明还提供了一种产生耐受苯磺隆除草剂的植物的方法，包括向植物的基因组中引入编码噻吩磺隆水解酶的核苷酸序列，当含有有效剂量苯磺隆的除草剂施加到至少存在植物的大田中，植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
为实现上述目的，本发明还提供了一种培养耐受苯磺隆除草剂的植物的方法，包括：
种植至少一个植物繁殖体，植物繁殖体的基因组中包括编码噻吩磺隆水解酶的多核苷酸序列；
使植物繁殖体长成植株；
将含有有效剂量苯磺隆的除草剂施加到至少包含植株的植物生长环境中，收获与其他不具有编码噻吩磺隆水解酶的多核苷酸序列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。
为实现上述目的，本发明还提供了一种保护植物免受由苯磺隆除草剂引起的损伤的方法，包括将含有有效剂量苯磺隆的除草剂施加到存在至少一种转基因植物的植物生长环境中，转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列，转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
为实现上述目的，本发明还提供了一种噻吩磺隆水解酶降解苯磺隆除草剂的方法，包括将含有有效剂量苯磺隆的除草剂施加到存在至少一种转基因植物的植物生长环境中，转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列，转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
为实现上述目的，本发明还提供了一种噻吩磺隆水解酶降解苯磺隆除草剂的用途。
具体地，噻吩磺隆水解酶降解苯磺隆除草剂的用途包括将含有有效剂量苯磺隆的除草剂施加到存在至少一种转基因植物的植物生长环境中，转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列，转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
在上述技术方案的基础上，噻吩磺隆水解酶的氨基酸序列具有SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列。
优选地，噻吩磺隆水解酶的核苷酸序列具有：
(a)编码SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列的核苷酸序列；或
(b)SEQ ID NO:2或SEQ ID NO:3所示的核苷酸序列；或
(c)SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列；或
(d)SEQ ID NO:8或SEQ ID NO:9所示的核苷酸序列。
本发明中转基因植物在施用除草剂的21天内种植在植物生长环境的土壤中。可选择地，除草剂可以在转基因植物种植之前、同时或之后施用。具体地，转基因植物在施用除草剂之前12、10、7或3天内种植在土壤内；转基因植物在施用除草剂之后12、10、7或3天内种植在土壤内。除草剂还可以对转基因植物进行第二次处理，第二次处理可以为V1-V2和V3-V4阶段之间、开花前、开花时、开花后或种子形成时。
本发明中苯磺隆(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。
本发明中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，双子叶植物是指大豆、拟南芥、棉花或油菜。
本发明中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，单子叶植物是指玉米、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦。
本发明中，除草剂耐受性蛋白质为噻吩磺隆水解酶，如序列表中SEQ ID NO:1、SEQ ID NO:4和SEQ ID NO:7所示。除草剂耐受性基因为编码噻吩磺隆水解酶的核苷酸序列，如序列表中SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:8和SEQ ID NO:9所示。除草剂耐受性基因为用于植物，除了包含噻吩磺隆水解酶的编码区外，也可包含其他元件，例如编码选择标记蛋白质、合成活性蛋白质、分解活性蛋白质、抗生物胁迫蛋白质、抗非生物胁迫蛋白质、雄性不育蛋白质、影响植物产量的蛋白质和/或影响植物品质的蛋白质，从而获得既具有除草剂耐受性活性、又具有其它性状的转基因植物。
本发明中抗生物胁迫蛋白质是指抵抗由其他生物所施加的胁迫的蛋白质，如昆虫抗性蛋白质、(病毒、细菌、真菌、线虫)疾病抗性蛋白质等。
本发明中抗非生物胁迫蛋白质是指抵抗外界环境所施加的胁迫的蛋白质，如对除草剂、干旱、热、寒冷、冰冻、盐胁迫、氧化胁迫等具有耐性的蛋白质。
本发明中影响植物品质的蛋白质是指影响植物输出性状的蛋白质，如改进淀粉、油、维生素等质量和含量的蛋白质、提高纤维品质的蛋白质等。
此外，包含编码噻吩磺隆水解酶的核苷酸序列的表达盒在植物中还可以与至少一种编码除草剂耐受性基因的蛋白质一起表达，除草剂耐受性基因包括但不限于，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)。
本发明中“草甘膦”是指N-膦酰甲基甘氨酸和它的盐，用“草甘膦除草剂”处理是指使用任何一种含有草甘膦的除草剂制剂进行处理。草甘膦的商业制剂包括但不限于，
Figure PCTCN2016108409-appb-000001
(作为异丙胺盐的草甘膦)、
Figure PCTCN2016108409-appb-000002
(作为钾盐的草甘膦)、
Figure PCTCN2016108409-appb-000003
DRY和
Figure PCTCN2016108409-appb-000004
(作为胺盐的草甘膦)、
Figure PCTCN2016108409-appb-000005
(作为钠盐的草甘膦)和
Figure PCTCN2016108409-appb-000006
(作为三甲基硫盐的草甘膦)。
本发明中有效剂量草甘膦是指以200-1600g ae/ha使用，包括250-1600g ae/ha、300-1600g ae/ha、500-1600g ae/ha、800-1500g ae/ha、1000-1500g ae/ha或1200-1500g ae/ha。
本发明中“草铵膦”又名草丁膦，是指2-氨基-4-[羟基(甲基)膦酰基]丁酸铵，用“草铵膦除草剂”处理是指使用任何一种含有草铵膦的除草剂制剂进行处理。
本发明中植物生长素类除草剂模拟或如同称为生长素的天然植物生长调节剂起作用，其影响细胞壁可塑性和核酸代谢，从而导致不受控制的细胞分裂和生长。由植物生长素类除草剂引起的损伤症状包括茎和柄的偏上性弯曲或扭曲、叶成杯形或卷曲、以及异常的叶形状和叶脉。植物生长素类除草剂包括但不限于，苯氧基羧酸化合物、苯甲酸化合物、吡啶羧酸化合物、喹啉羧酸化合物或草除灵乙酯化合物。典型地，植物生长素类除草剂为麦草畏、2,4-二氯苯氧基乙酸(2,4-D)、(4-氯-2-甲基苯氧基)乙酸(MCPA)和/或4-(2,4-二氯苯氧基)丁酸(2,4-DB)。
本发明中“麦草畏”(Dicamba)是指3,6-二氯-邻-茴香酸或3,6-二氯-2-甲氧基苯甲酸及其酸和盐。其盐包括异丙胺盐、二甘醇铵盐、二甲胺盐、钾盐和钠盐。麦草畏的商业制剂包括但不限于，
Figure PCTCN2016108409-appb-000007
(作为DMA盐)、
Figure PCTCN2016108409-appb-000008
(BASF，作为DGA盐)、VEL-58-CS-11TM和
Figure PCTCN2016108409-appb-000009
(BASF，作为DGA盐)。
本发明中禾本科除草剂不用于玉米，除非玉米已经对其耐受，可以通过α酮戊二酸依赖性双加氧酶(如AAD基因)提供这样的耐受性，禾本科除草剂包括但不限于精吡氟禾草灵。
本发明中发芽前选择性除草剂包括但不限于，乙酰苯胺、乙草胺、乙酰乳酸合酶抑制剂、二硝基苯胺或原卟啉原氧化酶抑制剂。
本发明中发芽后选择性除草剂包括但不限于，烟嘧磺隆、砜嘧磺隆、2,4-D、麦草畏、乙羧氟草醚、精喹禾灵。
本发明中除草剂的施用量随土壤结构、pH值、有机物含量、耕作系统和杂草的大小而变化，并且通过查看除草剂标签上合适的除草剂施用量来确定。
本发明中苯磺隆除草剂可控制的杂草包括但不限于，反枝苋、凹头苋、龙葵、苘麻、柳叶刺蓼、酸模叶蓼、东方蓼、卷茎蓼、节蓼、藜、小藜、萹蓄、繁缕、狼把草、鬼针草、鸭跖草、离子草、勿忘草、香薷、问荆、水棘针、播娘蒿、羽叶播娘蒿、大叶播娘蒿、母菊属、刺叶莴苣、向日葵、鼬瓣花、猪秧秧、地肤、雀舌草、麦家公、王不留行、亚麻荠、波叶糖芥、野田芥、白芥、水芥菜、荠菜、遏蓝菜、猪毛菜、风花菜、大巢菜和苣荬菜等。
本发明中草甘膦除草剂可控制的杂草包括但不限于，大穗看麦娘、野燕麦、雀麦、网草、稗草、早熟禾、狗尾草、马唐、马齿苋、藜、苍耳、苘麻、蓼、车前草、繁缕、猪秧秧和莎草等。
本发明中种植系统是指植物、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。
草甘膦被广泛地使用，因为它控制非常广谱的阔叶和禾本科杂草物种。然而，在草甘膦耐性作物和非作物应用中重复使用草甘膦已经(而且仍将继续)选择使杂草演替为天然更具有耐性的物种或草甘膦抗性生物型。多数除草剂抗性管理策略建议使用有效用量的多种除草剂作为延缓出现抗性杂草的方法，多种除草剂提供对同一物种的控制，但具有不同的作用模式。将噻吩磺隆水解酶基因与草甘膦耐性性状(和/或其他除草剂耐性性状)叠加可通过允许对同一作物 选择性使用草甘膦和苯磺隆而实现对草甘膦耐性作物中草甘膦抗性杂草物种(被苯磺隆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。
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。
本发明中，杂草是指在植物生长环境中与耕种的植物竞争的植物。
本发明术语“控制”和/或“防治”是指至少将有效剂量的苯磺隆除草剂直接施用(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。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。噻吩磺隆水解酶对杂草的“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(含有编码噻吩磺隆水解酶的多核苷酸序列)的任何组织同时和/或不同步地，存在和/或产生，噻吩磺隆水解酶和/或可控制杂草的另一种物质，则另一种物质的存在既不影响噻吩磺隆水解酶对杂草的“控制”和/或“防治”作用，也不能导致“控制”和/或“防治”作用完全和/或部分由另一种物质实现，而与噻吩磺隆水解酶无关。
在本发明中，噻吩磺隆水解酶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噻吩磺隆水解酶，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。
本发明中的植物、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。植物有性繁殖体包括但不限于植物种子；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；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“抗性”是可遗传的，并允许植物在除草剂对给定植物进行一般除草剂有效处理的情况下生长和繁殖。正如本领域技术人员所认可的，即使植物受到除草剂处理的一定损伤程度明显，植物仍可被认为“抗性”。本发明中术语“耐性”或“耐受性”比术语“抗性”更广泛，并包括“抗性”，以及特定植物具有的抵抗除草剂诱导的各种程度损伤的提高的能力，而在同样的除草剂剂量下一般导致相同基因型野生型植物损伤。
本发明中的多核苷酸和/或核苷酸形成完整“基因”，在所需宿主细胞中编码蛋白质或多肽。本领域技术人员很容易认识到，可以将本发明的多核苷酸和/或核苷酸置于目的宿主中的调控序列控制下。
本领域技术人员所熟知的，DNA典型的以双链形式存在。在这种排列中，一条链与另一条链互补，反之亦然。由于DNA在植物中复制产生了DNA的其它互补链。这样，本发明包括对 序列表中示例的多核苷酸及其互补链的使用。本领域常使用的“编码链”指与反义链结合的链。为了在体内表达蛋白质，典型将DNA的一条链转录为一条mRNA的互补链，它作为模板翻译出蛋白质。mRNA实际上是从DNA的“反义”链转录的。“有义”或“编码”链有一系列密码子(密码子是三个核苷酸，一次读三个可以产生特定氨基酸)，其可作为开放阅读框(ORF)阅读来形成目的蛋白质或肽。本发明还包括与示例的DNA有相当功能的RNA。
本发明中核酸分子或其片段在严格条件下与本发明除草剂耐受性基因杂交。任何常规的核酸杂交或扩增方法都可以用于鉴定本发明除草剂耐受性基因的存在。核酸分子或其片段在一定情况下能够与其他核酸分子进行特异性杂交。本发明中，如果两个核酸分子能形成反平行的双链核酸结构，就可以说这两个核酸分子彼此间能够进行特异性杂交。如果两个核酸分子显示出完全的互补性，则称其中一个核酸分子是另一个核酸分子的“互补物”。本发明中，当一个核酸分子的每一个核苷酸都与另一个核酸分子的对应核苷酸互补时，则称这两个核酸分子显示出“完全互补性”。如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在至少常规的“低度严格”条件下退火且彼此结合，则称这两个核酸分子为“最低程度互补”。类似地，如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在常规的“高度严格”条件下退火且彼此结合，则称这两个核酸分子具有“互补性”。从完全互补性中偏离是可以允许的，只要这种偏离不完全阻止两个分子形成双链结构。为了使一个核酸分子能够作为引物或探针，仅需保证其在序列上具有充分的互补性，以使得在所采用的特定溶剂和盐浓度下能形成稳定的双链结构。
本发明中，基本同源的序列是一段核酸分子，该核酸分子在高度严格条件下能够和相匹配的另一段核酸分子的互补链发生特异性杂交。促进DNA杂交的适合的严格条件，例如，大约在45℃条件下用6.0×氯化钠/柠檬酸钠(SSC)处理，然后在50℃条件下用2.0×SSC洗涤，这些条件对本领域技术人员是公知的。例如，在洗涤步骤中的盐浓度可以选自低度严格条件的约2.0×SSC、50℃到高度严格条件的约0.2×SSC、50℃。此外，洗涤步骤中的温度条件可以从低度严格条件的室温约22℃，升高到高度严格条件的约65℃。温度条件和盐浓度可以都发生改变，也可以其中一个保持不变而另一个变量发生改变。优选地，本发明严格条件可为在6×SSC、0.5％SDS溶液中，在65℃下与本发明噻吩磺隆水解酶的核苷酸序列发生特异性杂交，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噻吩磺隆水解酶的核苷酸序列杂交的序列包括在本发明中。这些序列与本发明序列至少大约40％-50％同源，大约60％、65％或70％同源，甚至至少大约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更大的序列同源性。
本发明提供功能蛋白质。“功能活性”(或“活性”)在本发明中指本发明用途的蛋白质/酶(单独或与其它蛋白质组合)具有降解或减弱除草剂活性的能力。产生本发明蛋白质的植物优选产生“有效量”的蛋白质，从而在用除草剂处理植物时，蛋白质表达的水平足以给予植物对除草剂(若无特别说明则为一般用量)完全或部分的抗性或耐性。可以以通常杀死靶植物的用量、正常的大田用量和浓度使用除草剂。优选地，本发明的植物细胞和植物被保护免受除草剂处理引起的生长抑制或损伤。本发明的转化植物和植物细胞优选具有苯磺隆除草剂的抗性或耐性，即转化的植物和植物细胞能在有效量的苯磺隆除草剂存在下生长。
本发明中的基因和蛋白质不但包括特定的示例序列，还包括保存了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。“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。“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。“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 Letters 309：59-64)。
本发明中，编码噻吩磺隆水解酶的氨基酸序列包括但不限于本发明序列表中涉及的序列，与其具有一定同源性的氨基酸序列也包括在本发明中。这些序列与本发明序列类似性/相同性典型的大于60％，优选的大于75％，更优选的大于80％，甚至更优选的大于90％，并且可以大于95％。也可以根据更特定的相同性和/或类似性范围定义本发明的优选的多核苷酸和蛋白质。例如与本发明示例的序列有49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性和/或类似性。
本发明中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噻吩磺隆水解酶基因的调节序列。
启动子为植物中可表达的启动子，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。
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，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。
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。
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，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，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。
终止子可以为在植物中起作用的适合多聚腺苷酸化信号序列，包括但不限于，来源于农杆菌(Agrobacterium tumefaciens)胭脂碱合成酶(NOS)基因的多聚腺苷酸化信号序列、来源于蛋白酶抑制剂Ⅱ(pinⅡ)基因的多聚腺苷酸化信号序列、来源于豌豆ssRUBISCO E9基因的多聚腺苷酸化信号序列和来源于α-微管蛋白(α-tubulin)基因的多聚腺苷酸化信号序列。
本发明中“有效连接”表示核酸序列的联结，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“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：启动子与序列相连，相连的方式使得得到的转录物高效翻译。如果启动子与编码序列的连接是转录物融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得得到的转录物中第一翻译起始密码子是编码序列的起始密码子。备选地，如果启动子与编码序列的连接是翻译融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得5’非翻译序列中含有的第一翻译起始密码子与启动子相连结，并且连接方式使得得到的翻译产物与编码想要的蛋白的翻译开放读码框的关系是符合读码框的。可以“有效连接”的核酸序列包括但不限于：提供基因表达功能的序列(即基因表达元件，例如启动子、5’非翻译区域、内含子、蛋白编码区域、3’非翻译区域、聚腺苷化位点和/或转录终止子)、提供DNA转移和/或整合功能的序列(即T-DNA边界序列、位点特异性重组酶识别位点、整合酶识别位点)、提供选择性功能的序列(即抗生素抗性标记物、生物合成基因)、提供可计分标记物功能的序列、体外或体内协助序列操作的序列(即多接头序列、位点特异性重组序列)和提供复制功能的序列(即细菌的复制起点、自主复制序列、着丝粒序列)。
本发明可赋予植物新除草剂抗性性状，并且未观察到对表型包括产量的不良影响。本发明中植物能耐受住如至少一种受试除草剂2×、3×、4×或5×一般应用水平。这些耐性水平的提高在本发明的范围之内。例如可对本领域已知的多种技术进行可预见到的优化和进一步发展，以增加给定基因的表达。
本发明中噻吩磺隆水解酶对苯磺隆除草剂具有耐受性。本发明中的植物，在其基因组中含有外源DNA，外源DNA包含编码噻吩磺隆水解酶的核苷酸序列，通过表达有效量的该蛋白而 保护其免受除草剂的威胁。有效量是指未损伤的或轻微损伤的剂量。同时，植物在形态上应是正常的，且可在常规方法下培养以用于产物的消耗和/或生成。
植物材料中除草剂耐受性蛋白质的表达水平可通过本领域内所描述的多种方法进行检测，例如通过应用特异引物对组织内产生的编码除草剂耐受性蛋白质的mRNA进行定量，或直接特异性检测产生的除草剂耐受性蛋白质的量。
本发明中，将外源DNA导入植物，如将编码噻吩磺隆水解酶的基因或表达盒或重组载体导入植物细胞，常规的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。
本发明提供了一种除草剂耐受性蛋白质的用途，具有以下优点：
1、对除草剂耐受性广。本发明首次公开了噻吩磺隆水解酶可以对苯磺隆除草剂表现出较高的耐受性，因此在植物上应用前景广阔。
2、对除草剂耐受性强。本发明噻吩磺隆水解酶对苯磺隆除草剂的耐受性强，至少可以耐受1倍大田浓度。
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
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1为本发明除草剂耐受性蛋白质的用途的含有ALT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的用途的含有ALT核苷酸序列的重组表达载体DBN100632构建流程图；
图3为本发明除草剂耐受性蛋白质的用途的含有ALT核苷酸序列的重组表达载体DBN100631结构示意图；
图4为本发明除草剂耐受性蛋白质的用途的转基因拟南芥T1植株对苯磺隆除草剂耐受性效果图；
图5为本发明除草剂耐受性蛋白质的用途的含有ALT核苷酸序列的重组表达载体DBN100828构建流程图；
图6为本发明除草剂耐受性蛋白质的用途的含有ALT核苷酸序列的重组表达载体DBN100827结构示意图；
图7为本发明除草剂耐受性蛋白质的用途的含有ALT核苷酸序列的重组克隆载体DBN05-T构建流程图；
图8为本发明除草剂耐受性蛋白质的用途的含有ALT核苷酸序列的重组表达载体DBN100830构建流程图；
图9为本发明除草剂耐受性蛋白质的用途的含有ALT核苷酸序列的重组表达载体DBN100829结构示意图；
图10为本发明除草剂耐受性蛋白质的用途的转基因玉米T1植株对苯磺隆除草剂耐受性效果图；
图11为本发明除草剂耐受性蛋白质的用途的转基因大豆T1植株对苯磺隆除草剂耐受性效果图。
具体实施方式
下面通过具体实施例进一步说明本发明除草剂耐受性蛋白质的用途的技术方案。
第一实施例、ALT基因序列的获得和合成
1、获得ALT基因序列
噻吩磺隆水解酶-1(ALT-1)的氨基酸序列(398个氨基酸)，如序列表中SEQ ID NO:1所示；编码相应于ALT-1的氨基酸序列的ALT-1-01核苷酸序列(1197个核苷酸)，如序列表中SEQ ID NO:2所示，编码相应于ALT-1的氨基酸序列的ALT-1-02核苷酸序列(1197个核苷酸)，如序列表中SEQ ID NO:3所示。
噻吩磺隆水解酶-2(ALT-2)的氨基酸序列(369个氨基酸)，如序列表中SEQ ID NO:4所示；编码相应于ALT-2的氨基酸序列的ALT-2-01核苷酸序列(1110个核苷酸)，如序列表中SEQ ID NO:5所示，编码相应于ALT-2的氨基酸序列的ALT-2-02核苷酸序列(1110个核苷酸)，如序列表中SEQ ID NO:6所示。
噻吩磺隆水解酶-3(ALT-3)的氨基酸序列(362个氨基酸)，如序列表中SEQ ID NO:7所示；编码相应于ALT-3的氨基酸序列的ALT-3-01核苷酸序列(1089个核苷酸)，如序列表中SEQ ID NO:8所示，编码相应于ALT-3的氨基酸序列的ALT-3-02核苷酸序列(1089个核苷酸)，如序列表中SEQ ID NO:9所示。
2、获得EPSPS基因序列
草甘膦耐受性蛋白质的氨基酸序列(455个氨基酸)，如序列表中SEQ ID NO:10所示；编码相应于草甘膦耐受性蛋白质的氨基酸序列的EPSPS核苷酸序列(1368个核苷酸)，如序列表中SEQ ID NO:11所示。
3、合成上述核苷酸序列
ALT-1-01核苷酸序列(如序列表中SEQ ID NO:2所示)、ALT-1-02核苷酸序列(如序列表中SEQ ID NO:3所示)、ALT-2-01核苷酸序列(如序列表中SEQ ID NO:5所示)、ALT-2-02核苷酸序列(如序列表中SEQ ID NO:6所示)、ALT-3-01核苷酸序列(如序列表中SEQ ID NO:8所示)、ALT-3-02核苷酸序列(如序列表中SEQ ID NO:9所示)和EPSPS核苷酸序列(如序列表中SEQ ID NO:11所示)由南京金斯瑞生物科技有限公司合成；合成的ALT-1-01核苷酸序列(SEQ ID NO:2)的5’端还连接有SpeI酶切位点，ALT-1-01核苷酸序列(SEQ ID NO:2)的3’端还连接有KasI酶切位点；合成的ALT-1-02核苷酸序列(SEQ ID NO:3)的5’端还连接有SpeI酶切位点，ALT-1-02核苷酸序列(SEQ ID NO:3)的3’端还连接有KasI酶切位点；合成的ALT-2-01核苷酸序列(SEQ ID NO:5)的5’端还连接有SpeI酶切位点，ALT-2-01核苷酸序列(SEQ ID NO:5)的3’端还连接有KasI酶切位点；合成的ALT-2-02核苷酸序列(SEQ ID NO:6)的5’端还连接有SpeI酶切位点，ALT-2-02核苷酸序列(SEQ ID NO:6)的3’端还连接有KasI酶切位点；合成的ALT-3-01核苷酸序列(SEQ ID NO:8)的5’端还连接有SpeI酶切位点，ALT-3-01核苷酸序列(SEQ ID NO:8)的3’端还连接有KasI酶切位点；合成的ALT-3-02核苷酸序列(SEQ ID NO:9)的5’端还连接有SpeI酶切位点，ALT-3-02核苷酸序列(SEQ ID NO:9)的3’端还连接有KasI酶切位点；合成的EP SPS核苷酸序列(SEQ ID NO:11)的5’端还连接有NcoI酶切位点，EPSPS核苷酸序列(SEQ ID NO:11)的3’端还连接有FspI酶切位点。
第二实施例、拟南芥重组表达载体的构建
1、构建含有ALT核苷酸序列的拟南芥和大豆重组克隆载体
将合成的ALT-1-01核苷酸序列连入克隆载体pGEM-T(Promega，Madison，USA，CAT：A3600)上，操作步骤按Promega公司产品pGEM-T载体说明书进行，得到重组克隆载体DBN01-T，其构建流程如图1所示(其中，Amp表示氨苄青霉素抗性基因；f1表示噬菌体f1 的复制起点；LacZ为LacZ起始密码子；SP6为SP6RNA聚合酶启动子；T7为T7RNA聚合酶启动子；ALT-1-01为ALT-1-01核苷酸序列(SEQ ID NO:2)；MCS为多克隆位点)。
然后将重组克隆载体DBN01-T用热激方法转化大肠杆菌T1感受态细胞(Transgen，Beijing，China，CAT：CD501)，其热激条件为：50μL大肠杆菌T1感受态细胞、10μL质粒DNA(重组克隆载体DBN01-T)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)，在表面涂有IPTG(异丙基硫代-β-D-半乳糖苷)和X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)的氨苄青霉素(100mg/L)的LB平板(胰蛋白胨10g/L，酵母提取物5g/L，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SpeI和KasI酶切鉴定后，对阳性克隆进行测序验证，结果表明重组克隆载体DBN01-T中插入的ALT-1-01核苷酸序列为序列表中SEQ ID NO:2所示的核苷酸序列，即ALT-1-01核苷酸序列正确插入。
按照上述构建重组克隆载体DBN01-T的方法，将合成的ALT-2-01核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN02-T，其中，ALT-2-01为ALT-2-01核苷酸序列(SEQ ID NO:5)。酶切和测序验证重组克隆载体DBN02-T中ALT-2-01核苷酸序列正确插入。
按照上述构建重组克隆载体DBN01-T的方法，将合成的ALT-3-01核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN03-T，其中，ALT-3-01为ALT-3-01核苷酸序列(SEQ ID NO:8)。酶切和测序验证重组克隆载体DBN03-T中ALT-3-01核苷酸序列正确插入。
同时，按照上述构建重组克隆载体DBN01-T的方法，将合成的EPSPS核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN04-T，其中，EPSPS为EPSPS核苷酸序列(SEQ ID NO:11)。酶切和测序验证重组克隆载体DBN04-T中EPSPS核苷酸序列正确插入。
2、构建含有ALT核苷酸序列的拟南芥重组表达载体
用限制性内切酶SpeI和KasI分别酶切重组克隆载体DBN01-T和表达载体DBNBC-01(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的ALT-1-01核苷酸序列片段插到表达载体DBNBC-01的SpeI和KasI位点之间，利用常规的酶切方法构建载体是本领域技术人员所熟知的，构建成重组表达载体DBN100632(定位于胞质)，其构建流程如图2所示(Spec：壮观霉素基因；RB：右边界；prAtUbi10：拟南芥Ubiquitin(泛素)10基因启动子(SEQ ID NO:12)；ALT-1-01：ALT-1-01核苷酸序列(SEQ ID NO:2)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:13)；prCaMV35S：花椰菜花叶病毒35S启动子(SEQ ID NO:14)；PAT：草铵膦乙酰转移酶基因(SEQ ID NO:15)；tCaMV35S：花椰菜花叶病毒35S终止子(SEQ ID NO:16)；LB：左边界)。
将重组表达载体DBN100632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DBN100632)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)；然后在含50mg/L壮观霉素(Spectinomycin) 的LB固体平板(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂15g/L，用NaOH调pH至7.5)上于温度37℃条件下培养12小时，挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，壮观霉素50mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒。将提取的质粒用限制性内切酶SpeI和KasI酶切后鉴定，并将阳性克隆进行测序鉴定，结果表明重组表达载体DBN100632在SpeI和KasI位点间的核苷酸序列为序列表中SEQ ID NO:2所示核苷酸序列，即ALT-1-01核苷酸序列。
按照上述构建重组表达载体DBN100632的方法，构建含有ALT-1-01核苷酸序列的重组表达载体DBN100631(定位于叶绿体)，其载体结构如图3所示(载体骨架：pCAMBIA2301(CAMBIA机构可以提供)；Spec：壮观霉素基因；RB：右边界；prAtUbi10：拟南芥Ubiquitin(泛素)10基因启动子(SEQ ID NO:12)；spAtCTP2：拟南芥叶绿体转运肽(SEQ ID NO:17)；ALT-1-01：ALT-1-01核苷酸序列(SEQ ID NO:2)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:13)；prCaMV35S：花椰菜花叶病毒35S启动子(SEQ ID NO:14)；PAT：草铵膦乙酰转移酶基因(SEQ ID NO:15)；tCaMV35S：花椰菜花叶病毒35S终止子(SEQ ID NO:16)；LB：左边界)。对阳性克隆进行测序验证，结果表明重组表达载体DBN100631中插入的ALT-1-01核苷酸序列为序列表中SEQ ID NO:2所示的核苷酸序列，即ALT-1-01核苷酸序列正确插入。
按照上述构建重组表达载体DBN100632的方法，将SpeI和KasI酶切重组克隆载体DBN02-T切下的ALT-2-01核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100634。酶切和测序验证重组表达载体DBN100634中的核苷酸序列含有为序列表中SEQ ID NO:5所示核苷酸序列，即ALT-2-01核苷酸序列正确插入。
按照上述构建重组表达载体DBN100631的方法，将SpeI和KasI酶切重组克隆载体DBN02-T切下的ALT-2-01核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100633(含有spAtCTP2，定位于叶绿体)。酶切和测序验证重组表达载体DBN100633中的核苷酸序列含有为序列表中SEQ ID NO:5所示核苷酸序列，即ALT-2-01核苷酸序列正确插入。
按照上述构建重组表达载体DBN100632的方法，将SpeI和KasI酶切重组克隆载体DBN03-T切下的ALT-3-01核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100636。酶切和测序验证重组表达载体DBN100636中的核苷酸序列含有为序列表中SEQ ID NO:8所示核苷酸序列，即ALT-3-01核苷酸序列正确插入。
按照上述构建重组表达载体DBN100631的方法，将SpeI和KasI酶切重组克隆载体DBN03-T切下的ALT-3-01核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100635(含有spAtCTP2，定位于叶绿体)。酶切和测序验证重组表达载体DBN100635中的核苷酸序列含有为序列表中SEQ ID NO:8所示核苷酸序列，即ALT-3-01核苷酸序列正确插入。
第三实施例、转入ALT核苷酸序列的拟南芥植株的获得
1、重组表达载体转化农杆菌
对己经构建正确的重组表达载体DBN100632、DBN100631、DBN100634、DBN100633、DBN100636和DBN100635用液氮法转化到农杆菌GV3101中，其转化条件为：100μL农杆菌GV3101、3μL质粒DNA(重组表达载体)；置于液氮中10分钟，37℃温水浴10分钟；将转化后的农杆菌GV3101接种于LB试管中于温度28℃、转速为200rpm条件下培养2小时，涂于含50mg/L的利福平(Rifampicin)和50mg/L的壮观霉素的LB平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，用限制性内切酶进行酶切验证，结果表明重组表达载体DBN100632、DBN100631、DBN100634、DBN100633、DBN100636和DBN100635结构完全正确。
2、获得转基因拟南芥植株
将野生型拟南芥种子悬浮于0.1％(w/v)琼脂糖溶液中。将悬浮的种子在4℃下保存2天以完成对休眠的需要以保证种子同步萌发。用蛭石混合马粪土并用水地下灌溉至湿润，使土壤混合物排水24小时。将预处理后的种子种在土壤混合物上并用保湿罩覆盖7天。使种子萌发并在恒温(22℃)恒湿(40-50％)光强度为120-150μmol/m2秒的长日照条件(16小时光照/8小时黑暗)下在温室中培养植物。开始用霍格兰营养液灌溉植物，接着用去离子水灌溉，保持土壤潮湿但不湿透。
使用花浸泡法转化拟南芥。用选取的农杆菌菌落接种一份或多份15-30mL含壮观霉素(50mg/L)和利福平(10mg/L)的YEP培养液的预培养物。以220rpm将培养物在28℃恒速摇动孵育过夜。每个预培养物用于接种两份500mL含壮观霉素(50mg/L)和利福平(10mg/L)的YEP培养液的培养物并将培养物在28℃持续摇动孵育过夜。室温以约8700×g离心10分钟沉淀细胞，弃去得到的上清液。将细胞沉淀轻柔重悬于500mL渗透培养基中，渗透培养基含有1/2×MS盐/B5维生素、10％(w/v)蔗糖、0.044μM苄氨基嘌呤(10μL/L(1mg/mL DMSO中的原液))和300μL/L Silvet L-77。将约1月龄的植物在培养基中浸泡15秒，确保浸没最新的花序。接着将植物侧面放倒并覆盖(透明或不透明)24小时，接着用水洗涤并竖直放置。在22℃以16小时光照/8小时黑暗的光周期培养植物。浸泡约4周后收获种子。
将新收获的(ALT核苷酸序列)T1种子在室温干燥7天。将种子种在26.5×51cm萌发盘中，每盘接受200mgT1种子(约10000个种子)，种子事先已悬浮于40mL 0.1％(w/v)琼脂糖溶液并在4℃下保存2天以完成对休眠的需要以保证种子同步萌发。
用蛭石混合马粪土并用水地下灌溉至湿润，利用重力排水。用移液管将预处理后的种子(每个40mL)均匀地种在土壤混合物上，并用保湿罩覆盖4-5天。在使用出苗后喷洒草铵膦(选择共转化的PAT基因)进行最初转化体选择前1天移去罩。
在7个种植天数后(DAP)并于11DAP再次使用DeVilbiss压缩空气喷嘴以10mL/盘(703L/ha)的喷洒体积用Liberty除草剂(200g ai/L的草铵膦)的0.2％溶液喷洒T1植物(分别为子叶期和2-4叶期)，以提供每次应用280g ai/ha有效量的草铵膦。在最后喷洒后4-7天鉴定存活株(生长活跃的植物)，并分别移植到用马粪土和蛭石制备的7cmx7cm的方盆中(每盘3-5棵)。用保湿罩覆盖移植的植物3-4天，并如前置于22℃培养室中或直接移入温室。接着移去罩并在测试ALT基因提供苯磺隆除草剂抗性的能力之前至少1天将植物栽种到温室(22±5℃，50±30％RH，14小时光照：10小时黑暗，最小500μE/m2s1天然+补充光)。
第四实施例、转基因拟南芥植株的除草剂耐受性效果检测
首先使用草铵膦选择方案从未转化种子背景中选择T1转化体。筛选了约40000个T1种子中并鉴定了380株T1代阳性转化子(PAT基因)，约0.95％的转化效率。转化重组表达载体DBN100632的为定位于胞质的转入ALT-1-01核苷酸序列的拟南芥植株(At胞质ALT-1-01)，转化重组表达载体DBN100631的为定位于叶绿体的转入ALT-1-01核苷酸序列的拟南芥植株(At叶绿体ALT-1-01)；转化重组表达载体DBN100634的为定位于胞质的转入ALT-2-01核苷酸序列的拟南芥植株(At胞质ALT-2-01)，转化重组表达载体DBN100633的为定位于叶绿体的转入ALT-2-01核苷酸序列的拟南芥植株(At叶绿体ALT-2-01)；转化重组表达载体DBN100636的为定位于胞质的转入ALT-3-01核苷酸序列的拟南芥植株(At胞质ALT-3-01)，转化重组表达载体DBN100635的为定位于叶绿体的转入ALT-3-01核苷酸序列的拟南芥植株(At叶绿体ALT-3-01)。将At胞质ALT-1-01的T1植株、At叶绿体ALT-1-01的T1植株、At胞质ALT-2-01的T1植株、At叶绿体ALT-2-01的T1植株、At胞质ALT-3-01的T1植株、At叶绿体ALT-3-01的T1植株和野生型拟南芥植株(播种后14天)分别对苯磺隆进行除草剂耐受性效果检测。
分别将At胞质ALT-1-01的T1植株、At叶绿体ALT-1-01的T1植株、At胞质ALT-2-01的T1植株、At叶绿体ALT-2-01的T1植株、At胞质ALT-3-01的T1植株、At叶绿体ALT-3-01的T1植株和野生型拟南芥植株用苯磺隆(18g ai/ha，1倍大田浓度)和空白溶剂(水)喷洒。 喷施14天后统计植株抗性情况：14天后生长状况和空白溶剂(水)一致的划为高抗植株，14天后抽苔高度为低于1/2空白溶剂(水)抽苔高度的划为中抗植株，14天后仍不能抽苔的划为低抗植株，14天后死亡的划为不抗植株。由于每株拟南芥T1植株是独立的转化事件，可以预计在给定剂量内个体T1应答的显著差异。结果如表1和图4所示。
表1、转基因拟南芥T1植株对苯磺隆除草剂耐受性实验结果
Figure PCTCN2016108409-appb-000010
对于拟南芥，18g ai/ha苯磺隆除草剂是将敏感植物与具有平均抗性水平的植物区分开来的有效剂量。表1和图4的结果表明：噻吩磺隆水解酶(ALT-1、ALT-2和ALT-3)赋予个体拟南芥植物苯磺隆除草剂耐受性(有个别植株不具有耐受性的原因是由于T1代植物插入位点是随机的，因而耐受性基因的表达水平有差异，表现出耐受性水平的差异)；相比于At胞质ALT-1-01的T1植株、At胞质ALT-2-01的T1植株和At胞质ALT-3-01的T1植株，At叶绿体ALT-1-01的T1植株、At叶绿体ALT-2-01的T1植株和At叶绿体ALT-3-01的T1植株能够产生更高的苯磺隆除草剂耐受性，表明噻吩磺隆水解酶(ALT-1、ALT-2和ALT-3)基因定位于叶绿体中表达可以增强拟南芥植物对苯磺隆除草剂的耐受性；而野生型拟南芥植株则均不具有对苯磺隆除草剂的耐受性。
第五实施例、对于不同的磺酰脲类除草剂具有预料不到的技术效果
噻吩磺隆水解酶亦可称为磺酰脲类除草剂去酯酶，其通过水解酯键而使具有酯键的磺酰脲类除草剂(如噻吩磺隆等)降解为无除草剂活性的母酸类物质，因而其不能降解无酯键的磺酰脲类除草剂(如烟嘧磺隆、氯磺隆等)。现有技术中具有酯键且结构相近的磺酰脲类除草剂很多，如苯磺隆、碘甲磺隆、环氧嘧磺隆、甲基二磺隆(甲磺胺磺隆)、吡嘧磺隆、甲嘧磺隆、氯吡嘧磺隆等。
将第四实施例中At胞质ALT-1-01的T1植株、At叶绿体ALT-1-01的T1植株、At胞质ALT-2-01的T1植株、At叶绿体ALT-2-01的T1植株、At胞质ALT-3-01的T1植株、At叶绿体ALT-3-01的T1植株和野生型拟南芥植株除了用苯磺隆(18g ai/ha，1倍大田浓度)和空白溶剂(水)喷洒外，也分别用碘甲磺隆(10g ai/ha，1倍大田浓度)、甲基二磺隆(14g ai/ha，1倍大 田浓度)和环氧嘧磺隆(60g ai/ha，1倍大田浓度)喷洒。喷施14天后统计植株抗性情况：14天后生长状况和空白溶剂(水)一致的划为高抗植株，14天后抽苔高度为低于1/2空白溶剂(水)抽苔高度的划为中抗植株，14天后仍不能抽苔的划为低抗植株，14天后死亡的划为不抗植株。由于每株拟南芥T1植株是独立的转化事件，可以预计在给定剂量内个体T1应答的显著差异。结果如表2和图4所示。
表2、转基因拟南芥T1植株对磺酰脲类除草剂耐受性实验结果
Figure PCTCN2016108409-appb-000011
表2比较了ALT-1、ALT-2和ALT-3输入噻吩磺隆水解酶活性到拟南芥T1植株的应答。虽然所有转化的拟南芥T1植株被赋予了噻吩磺隆水解酶活性，但是在给定的处理(碘甲磺隆、甲基二磺隆和环氧嘧磺隆)中，所有转化的拟南芥T1植株均未表现出具有降解上述磺酰脲类除草剂的能力，所有转化的拟南芥T1植株(ALT-1、ALT-2和ALT-3)的损伤程度和野生型拟南芥植株之间无任何差异。
表2充分说明表1的结果是预料不到的。虽然苯磺隆与噻吩磺隆、碘甲磺隆、甲基二磺隆和环氧嘧磺隆均为具有酯键且化学结构相近的磺酰脲类除草剂，且给定的处理也是具有可比性的(1倍大田浓度)，同时噻吩磺隆水解酶(ALT-1、ALT-2和ALT-3)在植物个体中已以预期水平输入并表达，然而表达噻吩磺隆水解酶的植物不具有降解碘甲磺隆、甲基二磺隆和环氧嘧磺隆的能力，也不能保护其自身免受上述磺酰脲类除草剂的损伤，与野生型植株的表现无任何差异，这些数据足以证实：噻吩磺隆水解酶(ALT-1、ALT-2和ALT-3)赋予植物对苯磺隆除草剂耐受性是难以预料的。
第六实施例、大豆重组表达载体的构建及重组表达载体转化农杆菌
1、构建含有ALT核苷酸序列的大豆重组表达载体
用限制性内切酶SpeI和KasI、NcoI和FspI分别酶切重组克隆载体DBN01-T、DBN04-T和表达载体DBNBC-02(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的ALT-1-01核苷酸序列和EPSPS核苷酸序列片段分别插到表达载体DBNBC-02的SpeI和KasI、NcoI和FspI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DBN100828(定位于胞质)，其构建流程如图5所示(Spec：壮观霉素基因；RB：右边界；prAtUbi10：拟南芥Ubiquitin(泛素)10基因启动子(SEQ ID NO:12)；ALT-1-01：ALT-1-01核苷酸序列(SEQ ID NO:2)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:13)；prBrCBP：油菜真核延长因子基因1α(Tsf1)启动子(SEQ ID NO:18)；spAtCTP2：拟南芥叶绿体转运肽(SEQ ID NO:17)；EPSPS：5-烯醇丙酮酸莽草酸-3-磷酸合酶基因(SEQ ID NO:11)；tPsE9：豌豆RbcS基因的终止子(SEQ ID NO:19)；LB：左边界)。
按照第二实施例中2的方法将重组表达载体DBN100828用热激方法转化大肠杆菌T1感受态细胞，并碱法提取其质粒。将提取的质粒用限制性内切酶SpeI和KasI酶切后鉴定，并将阳性克隆进行测序鉴定，结果表明重组表达载体DBN100828在SpeI和KasI位点间的核苷酸序列为序列表中SEQ ID NO:2所示核苷酸序列，即ALT-1-01核苷酸序列。
按照上述构建重组表达载体DBN100828的方法，构建含有ALT-1-01核苷酸序列的重组表达载体DBN100827(定位于叶绿体)，其载体结构如图6所示(载体骨架：pCAMBIA2301(CAMBIA机构可以提供)；Spec：壮观霉素基因；RB：右边界；prAtUbi10：拟南芥Ubiquitin(泛素)10基因启动子(SEQ ID NO:12)；spAtCTP2：拟南芥叶绿体转运肽(SEQ ID NO:17)；ALT-1-01：ALT-1-01核苷酸序列(SEQ ID NO:2)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:13)；prBrCBP：油菜真核延长因子基因1α(Tsf1)启动子(SEQ ID NO:18)；spAtCTP2：拟南芥叶绿体转运肽(SEQ ID NO:17)；EPSPS：5-烯醇丙酮酸莽草酸-3-磷酸合酶基因(SEQ ID NO:11)；tPsE9：豌豆RbcS基因的终止子(SEQ ID NO:19)；LB：左边界)。对阳性克隆进行测序验证，结果表明重组表达载体DBN100827中插入的ALT-1-01核苷酸序列为序列表中SEQ ID NO:2所示的核苷酸序列，即ALT-1-01核苷酸序列正确插入。
按照上述构建重组表达载体DBN100828的方法，将SpeI和KasI、NcoI和FspI酶切重组克隆载体DBN02-T和DBN04-T切下的ALT-2-01核苷酸序列和EPSPS核苷酸序列插入表达载体DBNBC-02，得到重组表达载体DBN100826。酶切和测序验证重组表达载体DBN100826中的核苷酸序列含有为序列表中SEQ ID NO:5和SEQ ID NO:11所示核苷酸序列，即ALT-2-01核苷酸序列和EPSPS核苷酸序列正确插入。
按照上述构建重组表达载体DBN100827的方法，将SpeI和KasI、NcoI和FspI酶切重组克隆载体DBN02-T和DBN04-T切下的ALT-2-01核苷酸序列和EPSPS核苷酸序列插入表达载体DBNBC-02，得到重组表达载体DBN100825(含有spAtCTP2，定位于叶绿体)。酶切和测序验证重组表达载体DBN100825中的核苷酸序列含有为序列表中SEQ ID NO:5和SEQ ID NO:11所示核苷酸序列，即ALT-2-01核苷酸序列和EPSPS核苷酸序列正确插入。
按照上述构建重组表达载体DBN100828的方法，将SpeI和KasI、NcoI和FspI酶切重组克隆载体DBN03-T和DBN04-T切下的ALT-3-01核苷酸序列和EPSPS核苷酸序列插入表达载体DBNBC-02，得到重组表达载体DBN100824。酶切和测序验证重组表达载体DBN100824中的核苷酸序列含有为序列表中SEQ ID NO:8和SEQ ID NO:11所示核苷酸序列，即ALT-3-01核苷酸序列和EPSPS核苷酸序列正确插入。
按照上述构建重组表达载体DBN100827的方法，将SpeI和KasI、NcoI和FspI酶切重组克隆载体DBN03-T和DBN04-T切下的ALT-3-01核苷酸序列和EPSPS核苷酸序列插入表达载体DBNBC-02，得到重组表达载体DBN100823(含有spAtCTP2，定位于叶绿体)。酶切和测序验证重组表达载体DBN100823中的核苷酸序列含有为序列表中SEQ ID NO:8和SEQ ID NO:11所示核苷酸序列，即ALT-3-01核苷酸序列和EPSPS核苷酸序列正确插入。
2、重组表达载体转化农杆菌
对己经构建正确的重组表达载体DBN100828、DBN100827、DBN100826、DBN100825、DBN100824和DBN100823用液氮法转化到农杆菌LBA4404(Invitrgen，Chicago，USA，CAT：18313-015)中，其转化条件为：100μL农杆菌LBA4404、3μL质粒DNA(重组表达载体)；置于液氮中10分钟，37℃温水浴10分钟；将转化后的农杆菌LBA4404接种于LB试管中于温度28℃、转速为200rpm条件下培养2小时，涂于含50mg/L的利福平(Rifampicin)和50mg/L的壮观霉素的LB平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，用限制性内切酶进行酶切验证，结果表明重组表达载体DBN100828、DBN100827、DBN100826、DBN100825、DBN100824和DBN100823结构完全正确。
第七实施例、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的子叶节组织与第六实施例中2的农杆菌共培养，以将第六实施例中1构建的重组表达载体DBN100828、DBN100827、DBN100826、DBN100825、DBN100824和DBN100823中的T-DNA(包括拟南芥Ubiquitin10基因的启动子序列、ALT-1-01核苷酸序列、ALT-2-01核苷酸序列、ALT-3-01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，获得了转化重组表达载体DBN100828的为定位于胞质的转入ALT-1-01核苷酸序列的大豆植株(Gm胞质ALT-1-01)，转化重组表达载体DBN100827的为定位于叶绿体的转入ALT-1-01核苷酸序列的大豆植株(Gm叶绿体ALT-1-01)；转化重组表达载体DBN100826的为定位于胞质的转入ALT-2-01核苷酸序列的大豆植株(Gm胞质ALT-2-01)，转化重组表达载体DBN100825的为定位于叶绿体的转入ALT-2-01核苷酸序列的大豆植株(Gm叶绿体ALT-2-01)；转化重组表达载体DBN100824的为定位于胞质的转入ALT-3-01核苷酸序列的大豆植株(Gm胞质ALT-3-01)，转化重组表达载体DBN100823的为定位于叶绿体的转入ALT-3-01核苷酸序列的大豆植株(Gm叶绿体ALT-3-01)；同时以野生型大豆植株作为对照。
对于农杆菌介导的大豆转化，简要地，将成熟的大豆种子在大豆萌发培养基(B5盐3.1g/L，B5维他命，蔗糖20g/L，琼脂8g/L，pH5.6)中进行萌发，将种子接种于萌发培养基上，按以下条件培养：温度25±1℃；光周期(光/暗)为16/8h。萌发4-6天后取鲜绿的子叶节处膨大的大 豆无菌苗，在子叶节下3-4毫米处切去下胚轴，纵向切开子叶，去顶芽、侧芽和种子根。用解剖刀的刀背在子叶节处进行创伤，用农杆菌悬浮液接触创伤过的子叶节组织，其中农杆菌能够将ALT-1-01核苷酸序列、ALT-2-01核苷酸序列、ALT-3-01核苷酸序列传递至创伤过的子叶节组织(步骤1：侵染步骤)在此步骤中，子叶节组织优选地浸入农杆菌悬浮液(OD660＝0.5-0.8，侵染培养基(MS盐2.15g/L、B5维他命、蔗糖20g/L、葡萄糖10g/L、乙酰丁香酮(AS)40mg/L、2-吗啉乙磺酸(MES)4g/L、玉米素(ZT)2mg/L，pH5.3)中以启动接种。子叶节组织与农杆菌共培养一段时期(3天)(步骤2：共培养步骤)。优选地，子叶节组织在侵染步骤后在固体培养基(MS盐4.3g/L、B5维他命、蔗糖20g/L、葡萄糖10g/L、2-吗啉乙磺酸(MES)4g/L、玉米素2mg/L、琼脂8g/L，pH5.6)上培养。在此共培养阶段后，可以有一个选择性的“恢复”步骤。在“恢复”步骤中，恢复培养基(B5盐3.1g/L、B5维他命、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、玉米素(ZT)2mg/L、琼脂8g/L，头孢霉素150mg/L，谷氨酸100mg/L，天冬氨酸100mg/L，pH5.6)中至少存在一种己知抑制农杆菌生长的抗生素(头孢霉素)，不添加植物转化体的选择剂(步骤3：恢复步骤)。优选地，子叶节再生的组织块在有抗生素但没有选择剂的固体培养基上培养，以消除农杆菌并为侵染细胞提供恢复期。接着，子叶节再生的组织块在含选择剂(草甘膦)的培养基上培养并选择生长着的转化愈伤组织(步骤4：选择步骤)。优选地，子叶节再生的组织块在有选择剂的筛选固体培养基(B5盐3.1g/L、B5维他命、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、6-苄基腺嘌呤(6-BAP)1mg/L、琼脂8g/L，头孢霉素150mg/L，谷氨酸100mg/L，天冬氨酸100mg/L，N-(膦羧甲基)甘氨酸0.25mol/L，pH5.6)上培养，导致转化的细胞选择性生长。然后，转化的细胞再生成植物(步骤5：再生步骤)，优选地，在含选择剂的培养基上生长的子叶节再生的组织块在固体培养基(B5分化培养基和B5生根培养基)上培养以再生植物。
筛选得到的抗性组织块转移到B5分化培养基(B5盐3.1g/L、B5维他命、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、玉米素(ZT)1mg/L、琼脂8g/L、头孢霉素150mg/L、谷氨酸50mg/L、天冬氨酸50mg/L、赤霉素1mg/L、生长素1mg/L、N-(膦羧甲基)甘氨酸0.25mol/L，pH5.6)上，25℃下培养分化。分化出来的小苗转移到B5生根培养基(B5盐3.1g/L、B5维他命、2-吗啉乙磺酸(MES)1g/L、蔗糖30g/L、琼脂8g/L、头孢霉素150mg/L、吲哚-3-丁酸(IBA)1mg/L)，在生根培养上，25℃下培养至约10cm高，移至温室培养至结实。在温室中，每天于26℃下培养16小时，再于20℃下培养8小时。
2、用TaqMan验证转基因大豆植株
分别取Gm胞质ALT-1-01的大豆植株、Gm叶绿体ALT-1-01的大豆植株、Gm胞质ALT-2-01的大豆植株、Gm叶绿体ALT-2-01的大豆植株、Gm胞质ALT-3-01的大豆植株和Gm叶绿体ALT-3-01的大豆植株的叶片约100mg作为样品，用Qiagen的DNeasy Plant Maxi Kit提取其基因组DNA，通过Taqman探针荧光定量PCR方法检测EPSPS基因拷贝数以确定ALT基因的拷贝数。同时以野生型大豆植株作为对照，按照上述方法进行检测分析。实验设3次重复，取平均值。
检测EPSPS基因拷贝数的具体方法如下：
步骤11、分别取Gm胞质ALT-1-01的大豆植株、Gm叶绿体ALT-1-01的大豆植株、Gm胞质ALT-2-01的大豆植株、Gm叶绿体ALT-2-01的大豆植株、Gm胞质ALT-3-01的大豆植株、Gm叶绿体ALT-3-01的大豆植株和野生型大豆植株的叶片各100mg，分别在研钵中用液氮研成匀浆，每个样品取3个重复；
步骤12、使用Qiagen的DNeasy Plant Mini Kit提取上述样品的基因组DNA，具体方法参考其产品说明书；
步骤13、用NanoDrop 2000(Thermo Scientific)测定上述样品的基因组DNA浓度；
步骤14、调整上述样品的基因组DNA浓度至同一浓度值，浓度值的范围为80-100ng/μL；
步骤15、采用Taqman探针荧光定量PCR方法鉴定样品的拷贝数，以经过鉴定已知拷贝数的样品作为标准品，以野生型大豆植株的样品作为对照，每个样品3个重复，取其平均值；荧光定量PCR引物和探针序列分别是：
以下引物和探针用来检测EPSPS基因序列：
引物1：CTGGAAGGCGAGGACGTCATCAATA如序列表中SEQ ID NO:20所示；
引物2：TGGCGGCATTGCCGAAATCGAG如序列表中SEQ ID NO:21所示；
探针1：ATGCAGGCGATGGGCGCCCGCATCCGTA如序列表中SEQ ID NO:22所示；
PCR反应体系为：
Figure PCTCN2016108409-appb-000012
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 PCTCN2016108409-appb-000013
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ALT-1-01核苷酸序列、ALT-2-01核苷酸序列和ALT-3-01核苷酸序列均己整合到所检测的大豆植株的染色体组中，而且Gm胞质ALT-1-01的大豆植株、Gm叶绿体ALT-1-01的大豆植株、Gm胞质ALT-2-01的大豆植株、Gm叶绿体ALT-2-01的大豆植株、Gm胞质ALT-3-01的大豆植株和Gm叶绿体ALT-3-01的大豆植株均获得了单拷贝的转基因大豆植株。
第八实施例、转基因大豆植株的除草剂耐受性效果检测
1、苯磺隆耐受性
将Gm胞质ALT-1-01的大豆植株、Gm叶绿体ALT-1-01的大豆植株、Gm胞质ALT-2-01的大豆植株、Gm叶绿体ALT-2-01的大豆植株、Gm胞质ALT-3-01的大豆植株、Gm叶绿体ALT-3-01的大豆植株和野生型大豆植株(幼苗期)分别对苯磺隆进行除草剂耐受性效果检测。
分别取Gm胞质ALT-1-01的大豆植株、Gm叶绿体ALT-1-01的大豆植株、Gm胞质ALT-2-01的大豆植株、Gm叶绿体ALT-2-01的大豆植株、Gm胞质ALT-3-01的大豆植株、Gm叶绿体ALT-3-01的大豆植株和野生型大豆植株，用苯磺隆(72g ai/ha，4倍大田浓度)和空白溶剂(水)喷洒。分别在喷施后3天(3DAT)、7天(7DAT)、14天(14DAT)及21天(21DAT)后，根据叶片卷曲程度和生长点损伤程度来统计每株植株受除草剂的损伤程度：以叶片平整如未处理植株、生长点完好无损为0％；叶脉局部变褐且新叶畸形、植株生长较慢为50％；叶脉发紫至整株死亡且生长点变褐干枯为100％。Gm胞质ALT-1-01的大豆植株共2个株系(S1和S2)，Gm 叶绿体ALT-1-01的大豆植株共2个株系(S3和S4)，Gm胞质ALT-2-01的大豆植株共2个株系(S5和S6)，Gm叶绿体ALT-2-01的大豆植株共2个株系(S7和S8)，Gm胞质ALT-3-01的大豆植株共2个株系(S9和S10)，Gm叶绿体ALT-3-01的大豆植株共2个株系(S11和S12)，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3和图11所示。
表3、转基因大豆T1植株除草剂耐受性实验结果
Figure PCTCN2016108409-appb-000014
对于大豆，72g ai/ha苯磺隆除草剂是将敏感植物与具有平均抗性水平的植物区分开来的有效剂量。表3和图11的结果表明：噻吩磺隆水解酶(ALT-1、ALT-2和ALT-3)赋予转基因大豆植物高水平苯磺隆除草剂耐受性；相比于Gm胞质ALT-1-01的大豆植株、Gm胞质ALT-2-01 的大豆植株和Gm胞质ALT-3-01的大豆植株，Gm叶绿体ALT-1-01的大豆植株、Gm叶绿体ALT-2-01的大豆植株和Gm叶绿体ALT-3-01的大豆植株能够产生更高的苯磺隆除草剂耐受性，表明噻吩磺隆水解酶(ALT-1、ALT-2和ALT-3)基因定位于叶绿体中表达可以增强大豆植物对苯磺隆除草剂的耐受性；而野生型大豆植株则不具有对苯磺隆除草剂的耐受性。
2、草甘膦耐受性
将Gm胞质ALT-1-01的大豆植株、Gm叶绿体ALT-1-01的大豆植株、Gm胞质ALT-2-01的大豆植株、Gm叶绿体ALT-2-01的大豆植株、Gm胞质ALT-3-01的大豆植株、Gm叶绿体ALT-3-01的大豆植株和野生型大豆植株(幼苗期)分别对草甘膦进行除草剂耐受性效果检测。
分别取Gm胞质ALT-1-01的大豆植株、Gm叶绿体ALT-1-01的大豆植株、Gm胞质ALT-2-01的大豆植株、Gm叶绿体ALT-2-01的大豆植株、Gm胞质ALT-3-01的大豆植株、Gm叶绿体ALT-3-01的大豆植株和野生型大豆植株各2个株系，从每个株系选10-15株进行测试。用草甘膦(840g ae/ha，1倍大田浓度)和空白溶剂(水)喷洒。在喷施14天(14DAT)后，根据药害症状来统计每株植株的除草剂受害率：除草剂受害率(％)＝∑(同级受害株数×级别数)/(总株数×最高级别)。药害症状分级如表5所示。
表5、草甘膦除草剂对大豆药害程度的分级标准
药害级别 症状描述
1 生长正常，无任何受害症状
2 轻微药害，药害少于10％
3 中等药害，以后能恢复
4 药害较重，难以恢复
5 药害严重，不能恢复
结果表明：Gm胞质ALT-1-01的大豆植株、Gm叶绿体ALT-1-01的大豆植株、Gm胞质ALT-2-01的大豆植株、Gm叶绿体ALT-2-01的大豆植株、Gm胞质ALT-3-01的大豆植株和Gm叶绿体ALT-3-01的大豆植株的草甘膦除草剂受害率基本为0％，而野生型大豆植株(CK1)的草甘膦除草剂受害率高达90％以上；由此，Gm胞质ALT-1-01的大豆植株、Gm叶绿体ALT-1-01的大豆植株、Gm胞质ALT-2-01的大豆植株、Gm叶绿体ALT-2-01的大豆植株、Gm胞质ALT-3-01的大豆植株和Gm叶绿体ALT-3-01的大豆植株具有良好的草甘膦除草剂耐受性。
第九实施例、玉米重组表达载体的构建
1、构建含有ALT核苷酸序列的玉米重组克隆载体
将合成的ALT-1-02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DBN05-T，其构建流程如图7所示(其中，Amp表示氨苄青霉素抗性基因；f1表示噬菌体f1的复制起点；LacZ为LacZ起始密码子；SP6为SP6RNA聚合酶启动子；T7为T7RNA聚合酶启动子；ALT-1-02为ALT-1-02核苷酸序列(SEQ ID NO:3)；MCS为多克隆位点)。
按照第二实施例中1的方法将重组克隆载体DBN05-T用热激方法转化大肠杆菌T1感受态细胞，并用碱法提取其质粒，提取的质粒经SpeI和KasI酶切鉴定后，对阳性克隆进行测序验证，结果表明重组克隆载体DBN05-T中插入的ALT-1-02核苷酸序列为序列表中SEQ ID NO:3所示的核苷酸序列，即ALT-1-02核苷酸序列正确插入。
按照上述构建重组克隆载体DBN05-T的方法，将合成的ALT-2-02核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN06-T，其中，ALT-2-02为ALT-2-02核苷酸序列(SEQ ID NO:6)。酶切和测序验证重组克隆载体DBN06-T中ALT-2-02核苷酸序列正确插入。
按照上述构建重组克隆载体DBN05-T的方法，将合成的ALT-3-02核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN07-T，其中，ALT-3-02为ALT-3-02核苷酸序列(SEQ ID NO:9)。酶切和测序验证重组克隆载体DBN07-T中ALT-3-02核苷酸序列正确插入。
2、构建含有ALT核苷酸序列的玉米重组表达载体
用限制性内切酶SpeI和KasI分别酶切重组克隆载体DBN05-T和表达载体DBNBC-03(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的ALT-1-02核苷酸序列片段插到表达载体DBNBC-03的SpeI和KasI位点之间，利用常规的酶切方法构建载体是本领域技术人员所熟知的，构建成重组表达载体DBN100830(定位于胞质)，其构建流程如图8所示(Spec：壮观霉素基因；RB：右边界；prUbi：玉米Ubiquitin(泛素)1基因启动子(SEQ ID NO:23)；ALT-1-02：ALT-1-02核苷酸序列(SEQ ID NO:3)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:13)；PMI：磷酸甘露糖异构酶基因(SEQ ID NO:24)；LB：左边界)。
按照第二实施例中2的方法将重组表达载体DBN100830用热激方法转化大肠杆菌T1感受态细胞，并碱法提取其质粒。将提取的质粒用限制性内切酶SpeI和KasI酶切后鉴定，并将阳性克隆进行测序鉴定，结果表明重组表达载体DBN100830在SpeI和KasI位点间的核苷酸序列为序列表中SEQ ID NO:3所示核苷酸序列，即ALT-1-02核苷酸序列。
按照上述构建重组表达载体DBN100830的方法，构建含有ALT-1-02核苷酸序列的重组表达载体DBN100829(定位于叶绿体)，其载体结构如图9所示(载体骨架：pCAMBIA2301(CAMBIA机构可以提供)；Spec：壮观霉素基因；RB：右边界；prUbi：玉米Ubiquitin(泛素)1基因启动子(SEQ ID NO:23)；spAtCTP2：拟南芥叶绿体转运肽(SEQ ID NO:17)；ALT-1-02：ALT-1-02核苷酸序列(SEQ ID NO:3)；tNos：胭脂碱合成酶基因的终止子(SEQ ID NO:13)；PMI：磷酸甘露糖异构酶基因(SEQ ID NO:24)；LB：左边界)。对阳性克隆进行测序验证，结果表明重组表达载体DBN100829中插入的ALT-1-02核苷酸序列为序列表中SEQ ID NO:3所示的核苷酸序列，即ALT-1-02核苷酸序列正确插入。
按照上述构建重组表达载体DBN100830的方法，将SpeI和KasI酶切重组克隆载体DBN06-T切下的ALT-2-02核苷酸序列插入表达载体DBNBC-03，得到重组表达载体DBN100832。酶切和测序验证重组表达载体DBN100832中的核苷酸序列含有为序列表中SEQ ID NO:6所示核苷酸序列，即ALT-2-02核苷酸序列正确插入。
按照上述构建重组表达载体DBN100829的方法，将SpeI和KasI酶切重组克隆载体DBN06-T切下的ALT-2-02核苷酸序列插入表达载体DBNBC-03，得到重组表达载体DBN100831(含有spAtCTP2，定位于叶绿体)。酶切和测序验证重组表达载体DBN100831中的核苷酸序列含有为序列表中SEQ ID NO:6所示核苷酸序列，即ALT-2-02核苷酸序列正确插入。
按照上述构建重组表达载体DBN100830的方法，将SpeI和KasI酶切重组克隆载体DBN07-T切下的ALT-3-02核苷酸序列插入表达载体DBNBC-03，得到重组表达载体DBN100834。酶切和测序验证重组表达载体DBN100834中的核苷酸序列含有为序列表中SEQ ID NO:9所示核苷酸序列，即ALT-3-02核苷酸序列正确插入。
按照上述构建重组表达载体DBN100829的方法，将SpeI和KasI酶切重组克隆载体DBN07-T切下的ALT-3-02核苷酸序列插入表达载体DBNBC-03，得到重组表达载体DBN100833(含有spAtCTP2，定位于叶绿体)。酶切和测序验证重组表达载体DBN100833中的核苷酸序列含有为序列表中SEQ ID NO:9所示核苷酸序列，即ALT-3-02核苷酸序列正确插入。
3、玉米重组表达载体转化农杆菌
对己经构建正确的重组表达载体DBN100830、DBN100829、DBN100832、DBN100831、DBN100834和DBN100833用液氮法转化到农杆菌LBA4404(Invitrgen，Chicago，USA，CAT：18313-015)中，其转化条件为：100μL农杆菌LBA4404、3μL质粒DNA(重组表达载体)； 置于液氮中10分钟，37℃温水浴10分钟；将转化后的农杆菌LBA4404接种于LB试管中于温度28℃、转速为200rpm条件下培养2小时，涂于含50mg/L的利福平(Rifampicin)和50mg/L的壮观霉素的LB平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，用限制性内切酶进行酶切验证，结果表明重组表达载体DBN100830、DBN100829、DBN100832、DBN100831、DBN100834和DBN100833结构完全正确。
第十实施例、转基因玉米植株的获得和验证
按照常规采用的农杆菌侵染法，将无菌培养的玉米品种综31(Z31)的幼胚与第九实施例中3的农杆菌共培养，以将第九实施例中2构建的重组表达载体DBN100830、DBN100829、DBN100832、DBN100831、DBN100834和DBN100833中的T-DNA(包括玉米Ubiquitin1基因的启动子序列、ALT-1-02核苷酸序列、ALT-2-02核苷酸序列、ALT-3-02核苷酸序列、拟南芥叶绿体转运肽、PMI基因和tNos终止子序列)转入到玉米染色体组中，获得了转化重组表达载体DBN100830的为定位于胞质的转入ALT-1-02核苷酸序列的玉米植株(Zm胞质ALT-1-02)，转化重组表达载体DBN100829的为定位于叶绿体的转入ALT-1-02核苷酸序列的玉米植株(Zm叶绿体ALT-1-02)；转化重组表达载体DBN100832的为定位于胞质的转入ALT-2-02核苷酸序列的玉米植株(Zm胞质ALT-2-02)，转化重组表达载体DBN100831的为定位于叶绿体的转入ALT-2-02核苷酸序列的玉米植株(Zm叶绿体ALT-2-02)；转化重组表达载体DBN100834的为定位于胞质的转入ALT-3-02核苷酸序列的玉米植株(Zm胞质ALT-3-02)，转化重组表达载体DBN100833的为定位于叶绿体的转入ALT-3-02核苷酸序列的玉米植株(Zm叶绿体ALT-3-02)；同时以野生型玉米植株作为对照。
对于农杆菌介导的玉米转化，简要地，从玉米中分离未成熟的幼胚，用农杆菌悬浮液接触幼胚，其中农杆菌能够将ALT-1-02核苷酸序列、ALT-2-02核苷酸序列、ALT-3-02核苷酸序列传递至幼胚之一的至少一个细胞(步骤1：侵染步骤)。在此步骤中，幼胚优选地浸入农杆菌悬浮液(OD660＝0.4-0.6，侵染培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖68.5g/L、葡萄糖36g/L、乙酰丁香酮(AS)40mg/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L，pH5.3))中以启动接种。幼胚与农杆菌共培养一段时期(3天)(步骤2：共培养步骤)。优选地，幼胚在侵染步骤后在固体培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖20g/L、葡萄糖10g/L、乙酰丁香酮(AS)100mg/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、琼脂8g/L，pH5.8)上培养。在此共培养阶段后，可以有一个选择性的“恢复”步骤。在“恢复”步骤中，恢复培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、植物凝胶3g/L，pH5.8)中至少存在一种己知抑制农杆菌生长的抗生素(头孢霉素)，不添加植物转化体的选择剂(步骤3：恢复步骤)。优选地，幼胚在有抗生素但没有选择剂的固体培养基上培养，以消除农杆菌并为侵染细胞提供恢复期。接着，接种的幼胚在含选择剂(甘露糖)的培养基上培养并选择生长着的转化愈伤组织(步骤4：选择步骤)。优选地，幼胚在有选择剂的筛选固体培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、甘露糖12.5g/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、植物凝胶3g/L，pH5.8)上培养，导致转化的细胞选择性生长。然后，愈伤组织再生成植物(步骤5：再生步骤)，优选地，在含选择剂的培养基上生长的愈伤组织在固体培养基(MS分化培养基和MS生根培养基)上培养以再生植物。
筛选得到的抗性愈伤组织转移到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MS生根培养基(MS盐2.15g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、吲哚-3-乙酸1mg/L、植物凝胶3g/L，pH5.8)上，25℃下培养至约10cm高，移至温室培养至结实。在温室中，每天于28℃下培养16小时，再于20℃下培养8小时。
2、用TaqMan验证转基因玉米植株
按照第七实施例中2用TaqMan验证转基因大豆植株的方法，对Zm胞质ALT-1-02的玉米植株、Zm叶绿体ALT-1-02的玉米植株、Zm胞质ALT-2-02的玉米植株、Zm叶绿体ALT-2-02的玉米植株、Zm胞质ALT-3-02的玉米植株和Zm叶绿体ALT-3-02的玉米植株进行检测分析。通过Taqman探针荧光定量PCR方法检测PMI基因拷贝数以确定ALT基因的拷贝数。同时以野生型玉米植株作为对照，按照上述方法进行检测分析。实验设3次重复，取平均值。
以下引物和探针用来检测PMI基因序列：
引物3：GCTGTAAGAGCTTACTGAAAAAATTAACA如序列表中SEQ ID NO:25所示；
引物4：CGATCTGCAGGTCGACGG如序列表中SEQ ID NO:26所示；
探针2：TCTCTTGCTAAGCTGGGAGCTCGATCC如序列表中SEQ ID NO:27所示。
通过分析PMI基因拷贝数的实验结果，进而证实ALT-1-02核苷酸序列、ALT-2-02核苷酸序列和ALT-3-02核苷酸序列均己整合到所检测的玉米植株的染色体组中，而且Zm胞质ALT-1-02的玉米植株、Zm叶绿体ALT-1-02的玉米植株、Zm胞质ALT-2-02的玉米植株、Zm叶绿体ALT-2-02的玉米植株、Zm胞质ALT-3-02的玉米植株和Zm叶绿体ALT-3-02的玉米植株均获得了单拷贝的转基因玉米植株。
第十一实施例、转基因玉米植株的除草剂耐受性效果检测
将Zm胞质ALT-1-02的玉米植株、Zm叶绿体ALT-1-02的玉米植株、Zm胞质ALT-2-02的玉米植株、Zm叶绿体ALT-2-02的玉米植株、Zm胞质ALT-3-02的玉米植株、Zm叶绿体ALT-3-02的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Zm胞质ALT-1-02的玉米植株、Zm叶绿体ALT-1-02的玉米植株、Zm胞质ALT-2-02的玉米植株、Zm叶绿体ALT-2-02的玉米植株、Zm胞质ALT-3-02的玉米植株、Zm叶绿体ALT-3-02的玉米植株和野生型玉米植株，用苯磺隆(72g ai/ha，4倍大田浓度)和空白溶剂(水)喷洒。分别在喷施后3天(3DAT)、7天(7DAT)、14天(14DAT)及21天(21DAT)后，根据植株的生长状况来统计每株植株受除草剂的损伤程度：以与未处理植株生长状况相当的为0％；叶片局部褪绿发黄但基本不影响植株正常生长的为50％；整株发紫濒临死亡的为100％。Zm胞质ALT-1-02的玉米植株共2个株系(S13和S14)，Zm叶绿体ALT-1-02的大豆植株共2个株系(S15和S16)，Zm胞质ALT-2-02的玉米植株共2个株系(S17和S18)，Zm叶绿体ALT-2-02的大豆植株共2个株系(S19和S20)，Zm胞质ALT-3-02的玉米植株共2个株系(S21和S22)，Zm叶绿体ALT-3-02的大豆植株共2个株系(S23和S24)，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4和图10所示。
表4、转基因玉米T1植株除草剂耐受性实验结果
Figure PCTCN2016108409-appb-000015
Figure PCTCN2016108409-appb-000016
对于玉米，72g ai/ha苯磺隆除草剂是将敏感植物与具有平均抗性水平的植物区分开来的有效剂量。表4和图10的结果表明：噻吩磺隆水解酶(ALT-1、ALT-2和ALT-3)赋予转基因玉米植物高水平苯磺隆除草剂耐受性；相比于Zm胞质ALT-1-02的玉米植株、Zm胞质ALT-2-02的玉米植株和Zm胞质ALT-3-02的玉米植株，Zm叶绿体ALT-1-02的玉米植株、Zm叶绿体ALT-2-02的玉米植株和Zm叶绿体ALT-3-02的玉米植株能够产生更高的苯磺隆除草剂耐受性，表明噻吩磺隆水解酶(ALT-1、ALT-2和ALT-3)基因定位于叶绿体中表达可以增强玉米植物对苯磺隆除草剂的耐受性；而野生型玉米植株则不具有对苯磺隆除草剂的耐受性。
综上，本发明首次公开了噻吩磺隆水解酶(ALT-1、ALT-2和ALT-3)可以对苯磺隆除草剂表现出较高的耐受性，且含有编码噻吩磺隆水解酶核苷酸序列的拟南芥植株、大豆植株和玉米植株对苯磺隆除草剂的耐受性强，至少可以耐受1倍大田浓度，因此在植物上应用前景广阔。
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。

Claims (48)

  1. 一种控制杂草的方法，其特征在于，包括将含有有效剂量苯磺隆的除草剂施加到存在至少一种转基因植物的植物生长环境中，所述转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列，所述转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
  2. 根据权利要求1所述控制杂草的方法，其特征在于，所述有效剂量苯磺隆为9-144g ai/ha。
  3. 根据权利要求1或2所述控制杂草的方法，其特征在于，所述转基因植物为单子叶植物或双子叶植物。
  4. 根据权利要求3所述控制杂草的方法，其特征在于，所述转基因植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦。
  5. 根据权利要求1-4任一项所述控制杂草的方法，其特征在于，所述噻吩磺隆水解酶的氨基酸序列具有SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列。
  6. 根据权利要求5所述控制杂草的方法，其特征在于，所述噻吩磺隆水解酶的核苷酸序列具有：
    (a)编码SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列的核苷酸序列；或
    (b)SEQ ID NO:2或SEQ ID NO:3所示的核苷酸序列；或
    (c)SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列；或
    (d)SEQ ID NO:8或SEQ ID NO:9所示的核苷酸序列。
  7. 根据权利要求1至6任一项所述控制杂草的方法，其特征在于，所述转基因植物还可以包括至少一种不同于编码所述噻吩磺隆水解酶的核苷酸序列的第二种核苷酸。
  8. 根据权利要求7所述控制杂草的方法，其特征在于，所述第二种核苷酸编码选择标记蛋白质、合成活性蛋白质、分解活性蛋白质、抗生物胁迫蛋白质、抗非生物胁迫蛋白质、雄性不育蛋白质、影响植物产量的蛋白质和/或影响植物品质的蛋白质。
  9. 根据权利要求8所述控制杂草的方法，其特征在于，所述第二种核苷酸编码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。
  10. 根据权利要求1-9任一项所述控制杂草的方法，其特征在于，所述含有有效剂量苯磺隆的除草剂还包括草甘膦除草剂、草铵膦除草剂、植物生长素类除草剂、禾本科除草剂、发芽前选择性除草剂和/或发芽后选择性除草剂。
  11. 一种控制草甘膦耐受性杂草的方法，其特征在于，包括将有效剂量的苯磺隆除草剂和草甘膦除草剂施加到种植至少一种转基因植物的大田中，所述大田中包含草甘膦耐受性杂草或其种子，所述转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列和编码草甘膦耐受性蛋白质的核苷酸序列，所述转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列和/或编码草甘膦耐受性蛋白质的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
  12. 根据权利要求11所述控制草甘膦耐受性杂草的方法，其特征在于，所述有效剂量苯磺隆为9-144g ai/ha。
  13. 根据权利要求11或12所述控制草甘膦耐受性杂草的方法，其特征在于，所述有效剂量草甘膦为200-1600g ae/ha。
  14. 根据权利要求11-13任一项所述控制草甘膦耐受性杂草的方法，其特征在于，所述转基因植物为单子叶植物或双子叶植物。
  15. 根据权利要求14所述控制草甘膦耐受性杂草的方法，其特征在于，所述转基因植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦。
  16. 根据权利要求11-15任一项所述控制草甘膦耐受性杂草的方法，其特征在于，所述噻吩磺隆水解酶的氨基酸序列具有SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列。
  17. 根据权利要求16所述控制草甘膦耐受性杂草的方法，其特征在于，所述噻吩磺隆水解酶的核苷酸序列具有：
    (a)编码SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列的核苷酸序列；或
    (b)SEQ ID NO:2或SEQ ID NO:3所示的核苷酸序列；或
    (c)SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列；或
    (d)SEQ ID NO:8或SEQ ID NO:9所示的核苷酸序列。
  18. 根据权利要求11-17任一项所述控制草甘膦耐受性杂草的方法，其特征在于，所述草甘膦耐受性蛋白质包括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-乙酰转移酶或草甘膦脱羧酶。
  19. 根据权利要求18所述控制草甘膦耐受性杂草的方法，其特征在于，所述草甘膦耐受性蛋白质的氨基酸序列具有SEQ ID NO:10所示的氨基酸序列。
  20. 根据权利要求19所述控制草甘膦耐受性杂草的方法，其特征在于，所述草甘膦耐受性蛋白质的核苷酸序列具有：
    (a)编码SEQ ID NO:10所示的氨基酸序列的核苷酸序列；或
    (b)SEQ ID NO:11所示的核苷酸序列。
  21. 一种控制杂草生长的种植系统，其特征在于，包括苯磺隆除草剂和存在至少一种转基因植物的植物生长环境，将含有有效剂量的所述苯磺隆除草剂施加到所述存在至少一种转基因植物的植物生长环境中，所述转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列，所述转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
  22. 根据权利要求21所述控制杂草生长的种植系统，其特征在于，所述有效剂量苯磺隆为9-144g ai/ha。
  23. 根据权利要求21或22所述控制杂草生长的种植系统，其特征在于，所述转基因植物为单子叶植物或双子叶植物。
  24. 根据权利要求3所述控制杂草生长的种植系统，其特征在于，所述转基因植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦。
  25. 根据权利要求21-24任一项所述控制杂草生长的种植系统，其特征在于，所述噻吩磺隆水解酶的氨基酸序列具有SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列。
  26. 根据权利要求25所述控制杂草生长的种植系统，其特征在于，所述噻吩磺隆水解酶的核苷酸序列具有：
    (a)编码SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列的核苷酸序列；或
    (b)SEQ ID NO:2或SEQ ID NO:3所示的核苷酸序列；或
    (c)SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列；或
    (d)SEQ ID NO:8或SEQ ID NO:9所示的核苷酸序列。
  27. 根据权利要求21至26任一项所述控制杂草生长的种植系统，其特征在于，所述转基因植物还可以包括至少一种不同于编码所述噻吩磺隆水解酶的核苷酸序列的第二种核苷酸。
  28. 根据权利要求27所述控制杂草生长的种植系统，其特征在于，所述第二种核苷酸编码选择标记蛋白质、合成活性蛋白质、分解活性蛋白质、抗生物胁迫蛋白质、抗非生物胁迫蛋白质、雄性不育蛋白质、影响植物产量的蛋白质和/或影响植物品质的蛋白质。
  29. 根据权利要求28所述控制杂草生长的种植系统，其特征在于，所述第二种核苷酸编码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、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。
  30. 根据权利要求21-29任一项所述控制杂草生长的种植系统，其特征在于，所述含有除草有效剂量苯磺隆的除草剂还包括草甘膦除草剂、草铵膦除草剂、植物生长素类除草剂、禾本科除草剂、发芽前选择性除草剂和/或发芽后选择性除草剂。
  31. 一种控制草甘膦耐受性杂草的种植系统，其特征在于，包括苯磺隆除草剂、草甘膦除草剂和种植至少一种转基因植物的大田，将有效剂量的所述苯磺隆除草剂和所述草甘膦除草剂施加到所述种植至少一种转基因植物的大田中，所述大田中包含草甘膦耐受性杂草或其种子，所述转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列和编码草甘膦耐受性蛋白质的核苷酸序列，所述转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列和/或编码草甘膦耐受性蛋白质的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
  32. 根据权利要求31所述控制草甘膦耐受性杂草的种植系统，其特征在于，所述有效剂量苯磺隆为9-144gai/ha。
  33. 根据权利要求31或32所述控制草甘膦耐受性杂草的种植系统，其特征在于，所述有效剂量草甘膦为200-1600gae/ha。
  34. 根据权利要求31-33任一项所述控制草甘膦耐受性杂草的种植系统，其特征在于，所述转基因植物为单子叶植物或双子叶植物。
  35. 根据权利要求34所述控制草甘膦耐受性杂草的种植系统，其特征在于，所述转基因植物为玉米、大豆、拟南芥、棉花、油菜、水稻、高粱、小麦、大麦、粟、甘蔗或燕麦。
  36. 根据权利要求31-35任一项所述控制草甘膦耐受性杂草的种植系统，其特征在于，所述噻吩磺隆水解酶的氨基酸序列具有SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列。
  37. 根据权利要求36所述控制草甘膦耐受性杂草的种植系统，其特征在于，所述噻吩磺隆水解酶的核苷酸序列具有：
    (a)编码SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列的核苷酸序列；或
    (b)SEQ ID NO:2或SEQ ID NO:3所示的核苷酸序列；或
    (c)SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列；或
    (d)SEQ ID NO:8或SEQ ID NO:9所示的核苷酸序列。
  38. 根据权利要求31-37任一项所述控制草甘膦耐受性杂草的种植系统，其特征在于，所述草甘膦耐受性蛋白质包括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-乙酰转移酶或草甘膦脱羧酶。
  39. 根据权利要求38所述控制草甘膦耐受性杂草的种植系统，其特征在于，所述草甘膦耐受性蛋白质的氨基酸序列具有SEQ ID NO:10所示的氨基酸序列。
  40. 根据权利要求39所述控制草甘膦耐受性杂草的种植系统，其特征在于，所述草甘膦耐受性蛋白质的核苷酸序列具有：
    (a)编码SEQ ID NO:10所示的氨基酸序列的核苷酸序列；或
    (b)SEQ ID NO:11所示的核苷酸序列。
  41. 一种产生耐受苯磺隆除草剂的植物的方法，其特征在于，包括向植物的基因组中引入编码噻吩磺隆水解酶的核苷酸序列，当含有有效剂量苯磺隆的除草剂施加到至少存在所述植物的大田中，所述植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
  42. 一种培养耐受苯磺隆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编码噻吩磺隆水解酶的多核苷酸序列；
    使所述植物繁殖体长成植株；
    将含有有效剂量苯磺隆的除草剂施加到至少包含所述植株的植物生长环境中，收获与其他不具有编码噻吩磺隆水解酶的多核苷酸序列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。
  43. 一种保护植物免受由苯磺隆除草剂引起的损伤的方法，其特征在于，包括将含有有效剂量苯磺隆的除草剂施加到存在至少一种转基因植物的植物生长环境中，所述转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列，所述转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
  44. 一种噻吩磺隆水解酶降解苯磺隆除草剂的方法，其特征在于，包括将含有有效剂量苯磺隆的除草剂施加到存在至少一种转基因植物的植物生长环境中，所述转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列，所述转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
  45. 一种噻吩磺隆水解酶降解苯磺隆除草剂的用途。
  46. 根据权利要求45所述噻吩磺隆水解酶降解苯磺隆除草剂的用途，其特征在于，所述噻吩磺隆水解酶降解苯磺隆除草剂的用途包括将含有有效剂量苯磺隆的除草剂施加到存在至少一种转基因植物的植物生长环境中，所述转基因植物在其基因组中包含编码噻吩磺隆水解酶的核苷酸序列，所述转基因植物与其他不具有编码噻吩磺隆水解酶的核苷酸序列的植物相比具有减弱的植物损伤和/或具有增加的植物产量。
  47. 根据权利要求41-46任一项所述方法或用途，其特征在于，所述噻吩磺隆水解酶的氨基酸序列具有SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列。
  48. 根据权利要求47所述方法或用途，其特征在于，所述噻吩磺隆水解酶的核苷酸序列具有：
    (a)编码SEQ ID NO:1、SEQ ID NO:4或SEQ ID NO:7所示的氨基酸序列的核苷酸序列；或
    (b)SEQ ID NO:2或SEQ ID NO:3所示的核苷酸序列；或
    (c)SEQ ID NO:5或SEQ ID NO:6所示的核苷酸序列；或
    (d)SEQ ID NO:8或SEQ ID NO:9所示的核苷酸序列。
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CN107099548B (zh) * 2017-05-09 2020-11-03 北京大北农生物技术有限公司 提高大豆转化效率的方法
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CN110100686A (zh) * 2019-06-19 2019-08-09 福建省农业科学院亚热带农业研究所（福建省农业科学院蔗麻研究中心） 一种果蔗防倒伏和保护蔗茎茎秆颜色的栽培方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102286501A (zh) * 2011-07-25 2011-12-21 南京农业大学 噻吩磺隆水解酶基因tsmE及其应用
CN105746255A (zh) * 2016-03-22 2016-07-13 北京大北农科技集团股份有限公司 除草剂耐受性蛋白质的用途

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9624927D0 (en) * 1996-11-29 1997-01-15 Oxford Glycosciences Uk Ltd Gels and their use
CN103484531B (zh) * 2005-07-01 2015-10-21 巴斯福股份公司 抗除草剂的向日葵植物、编码抗除草剂的乙酰羟酸合酶大亚基蛋白的多核苷酸和使用方法
WO2008141154A2 (en) * 2007-05-09 2008-11-20 Dow Agrosciences Llc Novel herbicide resistance genes
CN101451143A (zh) * 2008-12-25 2009-06-10 浙江大学 一种抗除草剂基因及其应用
CN107418969A (zh) * 2010-03-17 2017-12-01 巴斯夫农业化学产品公司 耐受除草剂的植物
RU2648155C2 (ru) * 2012-05-08 2018-03-22 Монсанто Текнолоджи Ллс Объект кукурузы mon 87411
CN103013939B (zh) * 2012-12-25 2015-01-21 北京大北农科技集团股份有限公司 除草剂抗性蛋白质、其编码基因及用途
CN103013938B (zh) * 2012-12-25 2014-12-17 北京大北农科技集团股份有限公司 除草剂抗性蛋白质、其编码基因及用途
AU2014369229A1 (en) * 2013-12-18 2016-06-09 BASF Agro B.V. Plants having increased tolerance to herbicides
CN104611307B (zh) * 2015-02-13 2018-04-27 北京大北农科技集团股份有限公司 除草剂抗性蛋白质、其编码基因及用途
CN104611308B (zh) * 2015-02-13 2019-01-11 北京大北农科技集团股份有限公司 除草剂抗性蛋白质、其编码基因及用途
US9617247B1 (en) * 2015-12-01 2017-04-11 Rotam Agrochem International Company Limited Form of halosulfuron-methyl, a process for its preparation and use of the same
US9643936B1 (en) * 2015-12-01 2017-05-09 Rotam Agrochem International Company Limited Form of tribenuron-methyl, a process for its preparation and use of the same
CN105766992B (zh) * 2016-03-22 2018-06-22 北京大北农科技集团股份有限公司 除草剂耐受性蛋白质的用途
CN105724139B (zh) * 2016-03-22 2018-10-30 北京大北农科技集团股份有限公司 除草剂耐受性蛋白质的用途
CN105802933B (zh) * 2016-03-22 2020-05-05 北京大北农科技集团股份有限公司 除草剂耐受性蛋白质、其编码基因及用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102286501A (zh) * 2011-07-25 2011-12-21 南京农业大学 噻吩磺隆水解酶基因tsmE及其应用
CN105746255A (zh) * 2016-03-22 2016-07-13 北京大北农科技集团股份有限公司 除草剂耐受性蛋白质的用途

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CUNNINGHAM; WELLS, SCIENCE, vol. 244, 1989, pages 1081 - 1085
DE VOS ET AL., SCIENCE, vol. 255, 1992, pages 306 - 312
HUANG, XING ET AL.: "Separation Identification and Degradation Characteristics of Thifensulfuron-Methyl Degradation Bacteria FLX", CHINA ENVIRONMENTAL SCIENCE, vol. 26, no. 2, 28 February 2006 (2006-02-28), pages 214 - 218, XP009511711, ISSN: 1000-6923 *
LI, HAITAO ET AL.: "Generation and Characterization of Tribenuron-Methyl Herbicide-Resistant Rapeseed (Brasscia Napus) for Hybrid Seed Production Using Chemically Induced Male Sterility", THEORETICAL AND APPLIED GENETICS, vol. 128, no. 1, 31 January 2015 (2015-01-31), pages 107 - 118, XP035417356, ISSN: 0040-5752 *
N. NEURATH; R. L. HILL: "Protein", 1979, ACADEMIC PRESS
See also references of EP3434778A4
SMITH ET AL., J. MOL. BIOL, vol. 224, 1992, pages 899 - 904
WLODAVER ET AL., FEBS LETTERS, vol. 309, 1992, pages 59 - 64

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