CN105724140B - The purposes of herbicide tolerant protein - Google Patents

The purposes of herbicide tolerant protein Download PDF

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Publication number
CN105724140B
CN105724140B CN201610165041.5A CN201610165041A CN105724140B CN 105724140 B CN105724140 B CN 105724140B CN 201610165041 A CN201610165041 A CN 201610165041A CN 105724140 B CN105724140 B CN 105724140B
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seq
nucleotide sequence
glyphosate
herbicide
plant
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CN105724140A (en
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谢香庭
丁德荣
鲍晓明
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Beijing Dabeinong Biotechnology Co Ltd
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Beijing Dbn Biotech Co Ltd
Beijing Dabeinong Technology Group Co Ltd
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G13/00—Protecting plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract

The present invention relates to a kind of purposes of herbicide tolerant protein, the method of the control weeds includes that the herbicide containing effective dose pyrazosulfuron is applied to there are in the plant growth environment of at least one genetically modified plants, the genetically modified plants include the nucleotide sequence for encoding thifensulfuronmethyl hydrolase in its genome, and the genetically modified plants with the plant injury weakened and/or have increased plant products compared with other do not have the plant of the nucleotide sequence of coding thifensulfuronmethyl hydrolase.Present invention firstly discloses thifensulfuronmethyl hydrolases, and higher tolerance can be shown to pyrazosulfuron herbicide, and the plant containing coding thifensulfuronmethyl hydrolase nucleotide sequence is strong to the tolerance of pyrazosulfuron herbicide, it can be at least resistant to 1 times of crop field concentration, therefore had a extensive future on plant.

Description

The purposes of herbicide tolerant protein
Technical field
The present invention relates to a kind of purposes of herbicide tolerant protein, drop more particularly to a kind of thifensulfuronmethyl hydrolase Solve the purposes of pyrazosulfuron herbicide.
Background technique
Weeds can valuable nutrient required for crop and other purpose plants in exhausted soil rapidly.Have perhaps at present For the herbicide of polymorphic type for controlling weeds, a kind of especially popular herbicide is glyphosate.It has developed and has had to glyphosate Resistant crop, such as corn and soybean, cotton, beet, wheat and rice.It therefore can be to plantation glyphosate resistance crop Field sprinkling glyphosate to control weeds without significantly damaging crop.
Glyphosate is widely used in the whole world more than 20 years, thus causes to glyphosate and glyphosate tolerant crop technology Depend on unduly, and glyphosate is naturally had more in wild weed species and tolerance or has been developed that resistance glyphosate is active Plant is applied with high selection pressure.It has been reported that the resistance to glyphosate, including broadleaf weeds and grass has been developed in a small number of weeds Section weeds, such as wimmera ryegrass, Itanlian rye, herba eleusines indicae, artemisiifolia, Horseweed Herb, wild pool wormwood artemisia and ribwort.In addition, wide It is general using be not before glyphosate tolerant crop agricultural problem weeds it is also gradually prevailing, and be difficult to be made with glyphosate-tolerant Object control, these weeds mainly are difficult to occur together with the broadleaf weeds controlled with (but not only with), such as Amaranthus, Chenopodium, dandelion Belong to and Commelianaceae species.
In glyphosate-resistant weeds or it is difficult to the area of weed species controlled, grower can be mixed by tank or uses energy instead Control omits other herbicides of weeds to make up the weakness of glyphosate, such as sulfonylurea herbicide.Sulfonylurea herbicide is Through becoming after the third-largest herbicide organic phosphorus, after acetyl herbicide, global annual sales amount reaches 3,000,000,000 dollars or more, I The annual application area of state's sulfonylurea herbicide is still in widened trend more than 2,000,000 hectares.
With the appearance of glyphosate-resistant weeds and the expansion application of sulfonylurea herbicide, need to sulfonylurea herbicide Sulfonylurea herbicide tolerance is inputted in the purpose plant of agent sensitivity.Sulfonylurea herbicide can be roughly divided into the sum containing ester bond Without ester bond, wherein containing sulfonylurea herbicide at least more than ten similar in ester bond and chemical structure.Thiophene is only identified The grand hydrolase of pheno sulphur can degrade thifensulfuronmethyl, but as thifensulfuronmethyl, pyrazosulfuron also belongs to the sulfonylurea containing ester bond Herbicide, and do not find that thifensulfuronmethyl hydrolase has the report of tolerance to pyrazosulfuron herbicide at present.
Summary of the invention
The object of the present invention is to provide a kind of purposes of herbicide tolerant protein, and effective agent will be contained for the first time by providing Amount pyrazosulfuron herbicide be applied to there are it is at least one expression thifensulfuronmethyl hydrolase genetically modified plants plant growth Method in environment to control weeds in field growth, increases thifensulfuronmethyl hydrolase to the tolerance range of herbicide.
To achieve the above object, the present invention provides a kind of methods for controlling weeds, including will to contain effective dose pyrrole phonetic The grand herbicide of sulphur is applied to there are in the plant growth environment of at least one genetically modified plants, and the genetically modified plants are in its base Because of the nucleotide sequence comprising coding thifensulfuronmethyl hydrolase in group, the genetically modified plants do not have coding thiophene sulphur with other The plant of the nucleotide sequence of grand hydrolase is compared with the plant injury weakened and/or has increased plant products.
Further, the effective dose pyrazosulfuron is 9-50g ai/ha.
Further, the genetically modified plants are monocotyledon or dicotyledon.
Preferably, the genetically modified plants be corn and soybean, it is arabidopsis, cotton, rape, rice, sorghum, wheat, big Wheat, grain, sugarcane or oat.
Based on the above technical solution, the amino acid sequence of the thifensulfuronmethyl hydrolase have SEQ ID NO:1, Amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:7.
Preferably, the nucleotide sequence of the thifensulfuronmethyl hydrolase includes
(a) nucleotides sequence of amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7 is encoded Column；Or
(b) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(c) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(d) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
Further, the genetically modified plants can also include at least one different from encoding the thifensulfuronmethyl hydrolase Nucleotide sequence second of nucleotide.
Second of nucleotide coding selected marker protein, synthesizing activity protein, degrading activity protein, antibiosis Object stress protein matter, resisting abiotic stress protein, male sterility protein, the protein for influencing plant products and/or influence The protein of plant quality.
Specifically, second of nucleotide coding 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate be also The double oxygenations of protoenzyme, glyphosate-N-acetyl transferase, glyphosate decarboxylase, glufosinate-ammonium transacetylase, alpha Ketoglutarate dependence Enzyme, dicamba monooxygenase enzyme, 4- hydroxyphenyl pyravate dioxygenase, acetolactate synthase, cytochromes proteinoid and/or original Protoporphyrinogen oxidase.
Selectively, the herbicide containing effective dose pyrazosulfuron further includes that glyphosate herbicidal, glufosinate-ammonium are removed Before careless agent, plant auxins herbicide, gramineous herbicide, germination selective herbicide and/or germination after selective herbicidal Agent.
To achieve the above object, the present invention also provides a kind of methods for controlling glyphosate tolerant weeds, including will have The pyrazosulfuron herbicide and glyphosate herbicidal for imitating dosage are applied to the big Tanaka for planting at least one genetically modified plants, described Big Tanaka includes glyphosate tolerant weeds or its seed, and the genetically modified plants include coding thifensulfuronmethyl in its genome The nucleotide sequence of the nucleotide sequence of hydrolase and coding glyphosate tolerant protein, the genetically modified plants and other not The plant of the nucleotide sequence of nucleotide sequence and/or coding glyphosate tolerant protein with coding thifensulfuronmethyl hydrolase Object is compared with the plant injury weakened and/or has increased plant products.
Further, the effective dose pyrazosulfuron is 9-50g ai/ha.The effective dose glyphosate is 200- 1600g ae/ha。
Further, the genetically modified plants are monocotyledon or dicotyledon.
Preferably, the genetically modified plants be corn and soybean, it is arabidopsis, cotton, rape, rice, sorghum, wheat, big Wheat, grain, sugarcane or oat.
Based on the above technical solution, the amino acid sequence of the thifensulfuronmethyl hydrolase have SEQ ID NO:1, Amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:7.
Preferably, the nucleotide sequence of the thifensulfuronmethyl hydrolase includes
(a) nucleotides sequence of amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7 is encoded Column；Or
(b) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(c) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(d) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
Further, the glyphosate tolerant protein include 5- enol pyruvylshikimate -3- phosphate synthase, grass it is sweet Phosphine oxidoreducing enzyme, glyphosate-N-acetyl transferase or glyphosate decarboxylase.
Specifically, the amino acid sequence of the glyphosate tolerant protein has amino acid shown in SEQ ID NO:10 Sequence.
Preferably, the nucleotide sequence of the glyphosate tolerant protein includes
(a) nucleotide sequence of amino acid sequence shown in SEQ ID NO:10 is encoded；Or
(b) nucleotide sequence shown in SEQ ID NO:11.
To achieve the above object, the present invention also provides a kind of implant systems for controlling weed growth, including pyrazosulfuron Herbicide and there are the plant growth environments of at least one genetically modified plants, by the pyrazosulfuron weeding containing effective dose Agent is applied to described there are in the plant growth environment of at least one genetically modified plants, and the genetically modified plants are in its genome Nucleotide sequence comprising encoding thifensulfuronmethyl hydrolase, the genetically modified plants do not have coding thifensulfuronmethyl hydrolysis with other The plant of the nucleotide sequence of enzyme is compared with the plant injury weakened and/or has increased plant products.
Further, the effective dose pyrazosulfuron is 9-50g ai/ha.
Further, the genetically modified plants are monocotyledon or dicotyledon.
Preferably, the genetically modified plants be corn and soybean, it is arabidopsis, cotton, rape, rice, sorghum, wheat, big Wheat, grain, sugarcane or oat.
Based on the above technical solution, the amino acid sequence of the thifensulfuronmethyl hydrolase have SEQ ID NO:1, Amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:7.
Preferably, the nucleotide sequence of the thifensulfuronmethyl hydrolase includes
(a) nucleotides sequence of amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7 is encoded Column；Or
(b) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(c) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(d) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
Further, the genetically modified plants can also include at least one different from encoding the thifensulfuronmethyl hydrolase Nucleotide sequence second of nucleotide.
Second of nucleotide coding selected marker protein, synthesizing activity protein, degrading activity protein, antibiosis Object stress protein matter, resisting abiotic stress protein, male sterility protein, the protein for influencing plant products and/or influence The protein of plant quality.
Specifically, second of nucleotide coding 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate be also The double oxygenations of protoenzyme, glyphosate-N-acetyl transferase, glyphosate decarboxylase, glufosinate-ammonium transacetylase, alpha Ketoglutarate dependence Enzyme, 4- hydroxyphenyl pyravate dioxygenase, acetolactate synthase, cytochromes proteinoid and/or proporphyrinogen oxidase.
Selectively, the herbicide containing weeding effective dose pyrazosulfuron further includes glyphosate herbicidal, careless ammonium Before phosphine herbicide, plant auxins herbicide, gramineous herbicide, germination selective herbicide and/or germination after selectivity Herbicide.
To achieve the above object, the present invention also provides a kind of implant systems for controlling glyphosate tolerant weeds, including The crop field of pyrazosulfuron herbicide, glyphosate herbicidal and at least one genetically modified plants of plantation, by the pyrrole of effective dose Sulfometuron Methyl herbicide and the glyphosate herbicidal are applied to the big Tanaka of at least one genetically modified plants of plantation, described big Tanaka includes glyphosate tolerant weeds or its seed, and the genetically modified plants include coding thifensulfuronmethyl water in its genome The nucleotide sequence of enzyme and the nucleotide sequence of coding glyphosate tolerant protein are solved, the genetically modified plants do not have with other There is the nucleotide sequence of coding thifensulfuronmethyl hydrolase and/or encodes the plant of the nucleotide sequence of glyphosate tolerant protein Compared to the plant injury weakened and/or with increased plant products.
Further, the effective dose pyrazosulfuron is 9-50g ai/ha.The effective dose glyphosate is 200- 1600g ae/ha。
Further, the genetically modified plants are monocotyledon or dicotyledon.
Preferably, the genetically modified plants be corn and soybean, it is arabidopsis, cotton, rape, rice, sorghum, wheat, big Wheat, grain, sugarcane or oat.
Based on the above technical solution, the amino acid sequence of the thifensulfuronmethyl hydrolase have SEQ ID NO:1, Amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:7.
Preferably, the nucleotide sequence of the thifensulfuronmethyl hydrolase includes
(a) nucleotides sequence of amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7 is encoded Column；Or
(b) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(c) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(d) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
Further, the glyphosate tolerant protein include 5- enol pyruvylshikimate -3- phosphate synthase, grass it is sweet Phosphine oxidoreducing enzyme, glyphosate-N-acetyl transferase or glyphosate decarboxylase.
Specifically, the amino acid sequence of the glyphosate tolerant protein has amino acid shown in SEQ ID NO:10 Sequence.
Preferably, the nucleotide sequence of the glyphosate tolerant protein includes
(a) nucleotide sequence of amino acid sequence shown in SEQ ID NO:10 is encoded；Or
(b) nucleotide sequence shown in SEQ ID NO:11.
To achieve the above object, a kind of method that the plant of pyrazosulfuron herbicide is resistant to the present invention also provides generation, Including in the genome to plant introduce coding thifensulfuronmethyl hydrolase nucleotide sequence, when contain effective dose pyrazosulfuron Herbicide be applied to the big Tanaka that at least there is the plant, the plant and other do not have coding thifensulfuronmethyl hydrolase The plant of nucleotide sequence compare with the plant injury weakened and/or with increased plant products.
To achieve the above object, a kind of method that the plant of pyrazosulfuron herbicide is resistant to the present invention also provides culture, Include:
At least one propagulum is planted, includes coding thifensulfuronmethyl hydrolase in the genome of the propagulum Polynucleotide sequence；
The propagulum is set to grow up to plant；
Herbicide containing effective dose pyrazosulfuron is applied in the plant growth environment including at least the plant, Harvesting has the plant injury weakened compared with the plant of other polynucleotide sequences for not having coding thifensulfuronmethyl hydrolase And/or the plant with increased plant products.
To achieve the above object, the present invention also provides one kind to protect the plants from the damage as caused by pyrazosulfuron herbicide The method of wound, including being applied to the herbicide containing effective dose pyrazosulfuron, there are the plants of at least one genetically modified plants In growing environment, the genetically modified plants include the nucleotide sequence for encoding thifensulfuronmethyl hydrolase in its genome, described Genetically modified plants have the plant weakened compared with other do not have the plant of the nucleotide sequence of coding thifensulfuronmethyl hydrolase Damage and/or have increased plant products.
To achieve the above object, the present invention also provides a kind of sides of thifensulfuronmethyl hydrolase pyrazosulfuron herbicide Method, including the herbicide containing effective dose pyrazosulfuron to be applied to the plant growth ring there are at least one genetically modified plants In border, the genetically modified plants include the nucleotide sequence for encoding thifensulfuronmethyl hydrolase, the transgenosis in its genome Plant with other do not have coding thifensulfuronmethyl hydrolase nucleotide sequences plant compare with weaken plant injury with/ Or there are increased plant products.
To achieve the above object, the present invention also provides a kind of use of thifensulfuronmethyl hydrolase pyrazosulfuron herbicide On the way.
Specifically, the purposes of the thifensulfuronmethyl hydrolase pyrazosulfuron herbicide includes that will contain effective dose pyrrole The herbicide of Sulfometuron Methyl is applied to there are in the plant growth environment of at least one genetically modified plants, and the genetically modified plants are at it Nucleotide sequence comprising coding thifensulfuronmethyl hydrolase in genome, the genetically modified plants do not have coding thiophene with other The plant of the nucleotide sequence of the grand hydrolase of sulphur is compared with the plant injury weakened and/or has increased plant products.
Based on the above technical solution, the amino acid sequence of the thifensulfuronmethyl hydrolase have SEQ ID NO:1, Amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:7.
Preferably, the nucleotide sequence of the thifensulfuronmethyl hydrolase includes
(a) nucleotides sequence of amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7 is encoded Column；Or
(b) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(c) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(d) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
Heretofore described genetically modified plants are planted in the plant growth environment in 21 days for applying the herbicide Soil in.Selectively, the herbicide can be applied prior to, concurrently with, or after genetically modified plants are planted.Specifically, institute Genetically modified plants are stated to be planted in soil in 12,10,7 or 3 days before applying the herbicide；The genetically modified plants are being applied With being planted in soil in after the herbicide 12,10,7 or 3 days.The herbicide can also to the genetically modified plants into Row second is handled, second of processing can between V1-V2 the and V3-V4 stage, before blooming, when blooming, Post flowering or When Seed Development.
Heretofore described pyrazosulfuron (Pyrazosulfuron-ethyl) refers to 5- [(4,6- dimethoxy -2- pyrimidine Base) amino carbonyl amino sulfonyl] -1- methylpyrazole -4- carboxylic acid, ethyl ester is white solid.Common formulations are 10% wettable Pulvis, the commercial formulation of pyrazosulfuron include but is not limited to that careless jinx, Mercury, Han Lexing, grass go out star, careless prestige, Tadalafil and one It is gram net.
Heretofore described effective dose pyrazosulfuron refers to be used with 9-50g ai/ha, including 10-50g ai/ha, 12-48g ai/ha, 15-45g ai/ha, 18-40g ai/ha, 20-35g ai/ha or 25-30g ai/ha.
Heretofore described dicotyledon includes but is not limited to clover, Kidney bean, cauliflower, wild cabbage, carrot, celery, cotton Flower, cucumber, eggplant, lettuce, muskmelon, pea, pepper, cucurbita pepo, radish, rape, spinach, soybean, pumpkin, tomato, arabidopsis or Watermelon.Preferably, the dicotyledon refers to soybean, arabidopsis, cotton or rape.
Heretofore described monocotyledon includes but is not limited to corn, rice, sorghum, wheat, barley, rye, grain, sweet Sugarcane, oat or turfgrass.Preferably, the monocotyledon refers to corn, rice, sorghum, wheat, barley, grain, sugarcane or swallow Wheat.
In the present invention, the herbicide tolerant protein is thifensulfuronmethyl hydrolase, such as SEQ ID NO in sequence table: 1, shown in SEQ ID NO:4 and SEQ ID NO:7.The herbicide tolerance gene is the nucleosides for encoding thifensulfuronmethyl hydrolase Acid sequence, such as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 in sequence table With shown in SEQ ID NO:9.The herbicide tolerance gene is for plant, in addition to the coding comprising thifensulfuronmethyl hydrolase It also may include other elements outside area, such as encoding selectable markers protein, synthesizing activity protein, degrading activity protein, anti- Biotic protein, resisting abiotic stress protein, male sterility protein, the protein and/or shadow for influencing plant products The protein of plant quality is rung, to obtain not only with herbicide tolerant activity but also with the genetically modified plants of other characters.
Heretofore described biotic protein refers to the protein for resisting the stress applied by other biological, such as Insect resistance proteins matter, (virus, bacterium, fungi, nematode) disease resistance protein etc..
Heretofore described resisting abiotic stress protein refers to the protein for resisting the stress that external environment is applied, such as Has indefatigable protein to herbicide, arid, heat, cold, frost, salt stress, oxidative stress etc..
The heretofore described protein for influencing plant quality refers to the protein for influencing plant output character, such as improves and forms sediment The protein of the quality such as powder, oil, vitamin and content, the protein for improving fiber quality etc..
In addition, the expression cassette of the nucleotide sequence comprising encoding thifensulfuronmethyl hydrolase can also be at least one in plant The protein of kind encoding herbicide-tolerant gene is expressed together, and the herbicide tolerance gene includes but is not limited to 5- alkene Alcohol pyruvoyl shikimic acid -3- phosphate synthase (EPSPS), glyphosate oxidoreductase (GOX), glyphosate-N-acetyl transferase (GAT), glyphosate decarboxylase, glufosinate-ammonium transacetylase (PAT), alpha Ketoglutarate dependence dioxygenase (AAD), dicamba Monooxygenase (DMO), 4- hydroxyphenyl pyravate dioxygenase (HPPD), acetolactate synthase (ALS), cytochromes proteinoid (P450) and/or proporphyrinogen oxidase (Protox).
Heretofore described " glyphosate " refers to the salt of N- phosphonomethylglycine and it, is handled with " glyphosate herbicidal " Refer to and is handled using any one containing the herbicide formulations of glyphosate.The commercial formulation of glyphosate includes but is not limited to,(glyphosate as isopropyl amine salt),(grass as sylvite is sweet by WEATHERMAX Phosphine),DRY and(glyphosate as amine salt),GEOFORCE (as The glyphosate of sodium salt) and(glyphosate as trimethyl sulfosalt).
Heretofore described effective dose glyphosate refers to 200-1600g ae/ha use, including 250-1600g ae/ Ha, 300-1600g ae/ha, 500-1600g ae/ha, 800-1500g ae/ha, 1000-1500g ae/ha or 1200- 1500g ae/ha。
Heretofore described " glufosinate-ammonium " also known as glufosinate refer to 2- amino -4- [hydroxyl (methyl) phosphono] butyric acid ammonium, Referred to " glufosinate-ammonium herbicide " processing and is handled using any one containing the herbicide formulations of glufosinate-ammonium.
The simulation of plant auxins herbicide or the natural plants growth regulator such as referred to as auxin rise in the present invention Effect influences cell wall plasticity and nucleic acid metabolism, so as to cause uncontrolled cell division and growth.By plant growth Injury symptoms caused by plain class herbicide include the epinasty bending or distortion, leaf cup-shaped or curling and exception of stem and handle Leaf shape and vein.Plant auxins herbicide includes but is not limited to, phenoxy carboxylic acid compounds, benzoic acid compounds, Pyridineacarboxylicaacidacompound, quinoline carboxylic acid compound or benazolinethyl compound.Typically, plant auxins herbicide is wheat Careless fear, 2,4 dichloro benzene ethoxyacetic acid (2,4-D), (4- chloro-2-methyl phenoxy group) acetic acid (MCPA) and/or 4- (2,4- dichloro Phenoxy group) butyric acid (2,4-DB).
Heretofore described " dicamba " (Dicamba) refers to the chloro- o- anisic acid of 3,6- bis- or the chloro- 2- methoxy of 3,6- bis- Yl benzoic acid and its acid and salt.Its salt includes isopropyl amine salt, diethylene glycol (DEG) ammonium salt, dimethylamine salt, sylvite and sodium salt.The quotient of dicamba Industry preparation includes but is not limited to,(as DMA salt),(BASF, as DGA salt), VEL-58-CS- 11TMWith(BASF, as DGA salt).
Heretofore described gramineous herbicide is not used in corn, unless corn is resistant to it, can pass through α ketone penta Tolerance diacid dependence dioxygenase (such as AAD gene) offer, the gramineous herbicide include but is not limited to essence Fluazifop.
Selective herbicide includes but is not limited to before heretofore described germination, and antifebrin, Acetochlor, acetolactic acid close Enzyme inhibitor, dinitroaniline or proporphyrinogen oxidase inhibitor.
Selective herbicide includes but is not limited to after heretofore described germination, nicosulfuron, rimsulfuron, 2,4-D, wheat Careless fear, fluoroglycofen-ethyl, Quizalotop-ethyl.
The amount of application of herbicide is with soil texture, pH value, content of organics, cultivating system and the size of weeds in the present invention And change, and determined by checking on herbicide label suitable herbicide application amount.
The heretofore described controllable weeds of pyrazosulfuron herbicide include but is not limited to barnyard grass, rice Leersia Sw, water Sha It is grass, difformed galingale herb, Monochoria vaginalis, needle spikesedge herb, flat stalk Fischer grass, Japan's Fischer grass, bur beggar-ticks, Monochoria korsakowii, alisma canaliculatum, rhizoma alismatis, short kind Aunt, angustifolia arrowhead herb, carp intestines, bog pondweed, mexicana, firefly Lin, duckweed, duckweed, floating life water starwort, Chinese celery, small najas marina and three calyx ditches are numerous Thread etc..
The heretofore described controllable weeds of glyphosate herbicidal include but is not limited to big fringe amur foxtail, wild avena sativa, sparrow Wheat, net grass, barnyard grass, annual bluegrass, herba setariae viridis, herba digitariae, purslane, Chenopodiaceae, Siberian cocklebur, piemarker, knotweed, Asiatic plantain, herba stellariae mediae, Herba Galii Teneri and Sha Grass etc..
Heretofore described implant system refers to plant, any herbicide tolerant of its display and/or sends out in plant The combination of the available herbicide treatment of the different phase educated generates high yield and/or weakens the plant of damage.
Glyphosate is widely used, because it controls the very broad-leaved of wide spectrum and gramineae weed species.However, in grass Glyphosate is reused in sweet phosphine tolerable crop and the application of non-crop (and will continue) to select to make weeds succession day Right more indefatigable species or glyphosate resistance bion.Most Herbicid resistant management strategy suggestions use effective dose For a variety of herbicides as the method for resistant weed occur is delayed, a variety of herbicides provide the control to same species, but have There is different binding modes.By thifensulfuronmethyl hydrolase gene and glyphosate tolerance trait (and/or other herbicide tolerances Shape) superposition can be by allowing to same crop-selective to realize using glyphosate and pyrazosulfuron in glyphosate tolerant crop The control of glyphosate-resistant weeds species (the broadleaf weeds species controlled by pyrazosulfuron herbicide).The application of these herbicides Can be in the tank mixture of two or more herbicides containing different role mode while using, continuous use (such as Before plantation, before emergence or after emergence) in the exclusive use of single herbicidal composition (the interval time range used was from 2 hours By 3 months), or it is alternatively possible at any time (when out of long-term cropping 7 months to harvesting crops (or for individually removing Careless agent is harvest space before, takes most short person)) it can be using the combination of the arbitrary number herbicide of every kind of chemical combination classification using representative.
Herbicide formulations (such as ester, acid or salt formula or solvable concentrating agents, emulsion concentrate or can solution body) and tank is mixed adds Add agent (such as adjuvant or compatilizer) that the combined Weeds distribution of given herbicide or one or more herbicides can be significantly affected. Any chemical combination of any aforementioned herbicides is within the scope of the present invention.
In the present invention, the weeds refer to the plant in plant growth environment with the plant competition of cultivation.
Term " control " of the present invention and/or " prevention and treatment ", which refer to, at least directly applies the pyrazosulfuron herbicide of effective dose (such as by spraying) makes weeds development minimize and/or stop growing into plant growth environment.Meanwhile the plant of cultivation It should be morphologically normal, and can cultivate under conventional approaches with the consumption and/or generation for product；Preferably, with it is non- The WT lines of transgenosis are compared with the plant injury weakened and/or have increased plant products.It is described that there is decrease Plant injury, specific manifestation includes but is not limited to improved stalk resistance, and/or the kernel weight of raising etc..The thiophene The grand hydrolase of sulphur is to " control " and/or " prevention and treatment " of weeds effect can be self-existent, " can not control " because other and/or The presence of the substance of " prevention and treatment " weeds and weaken and/or disappear.Specifically, genetically modified plants (contain coding thifensulfuronmethyl hydrolysis The polynucleotide sequence of enzyme) any tissue simultaneously and/or asynchronously, exist and/or generate, thifensulfuronmethyl hydrolase and/ Or another substance of controllable weeds, then the presence of another substance neither influences thifensulfuronmethyl hydrolase to weeds " control " and/or " prevention and treatment " effect can not cause " control " and/or " prevention and treatment " effect completely and/or part is by described Another substance realization, and it is unrelated with thifensulfuronmethyl hydrolase.
In the present invention, expression of the thifensulfuronmethyl hydrolase in a kind of genetically modified plants can be along with one or more The expression of other herbicide tolerant protein.This is more than that a kind of herbicide tolerant protein is planted in same strain transgenosis Co-expressing in object can make plant include and express required gene to realize by genetic engineering.In addition, a kind of plant ( 1 parent) thifensulfuronmethyl hydrolase can be expressed by genetic engineering procedure, second of plant (the 2nd parent) can pass through heredity Engineering operation expresses other herbicide tolerant protein.Expression, which is obtained, by the 1st parent and the 2nd parents introduces the 1st parent The progeny plants of all genes of this and the 2nd parent.
The genome of heretofore described plant, plant tissue or plant cell, refers to plant, plant tissue or plant Intracellular any inhereditary material, and including nucleus and plastid and mitochondrial genomes.
Heretofore described " propagulum " includes but is not limited to plant tannins and plant vegetative propagule. The plant tannins include but is not limited to vegetable seeds；The plant vegetative propagule refers to the nutrition organs of plant Or certain particular tissues, new plant can be generated in vitro；The nutrition organs or certain particular tissues include but It is not limited to root, stem and leaf, such as: it include strawberry and sweet potato etc. by the plant of vegetative propagule of root；Using stem as vegetative propagule Plant include sugarcane and potato (stem tuber) etc.；It include aloe and begonia etc. by the plant of vegetative propagule of leaf.
Heretofore described " resistance " is heritable, and plant is allowed to carry out general weeding to given plant in herbicide Agent is grown in the case where being effectively treated and breeding.As those skilled in the art are approved, though plant by herbicide at Certain degree of injury of reason is obvious, and plant can still be considered " resistance ".Term " patience " or " tolerance " compare term in the present invention " resistance " more extensively, and mentioning of damaging of the various degree for the resistances herbicide induction having including " resistance " and specified plant High ability, and the damage of phase homogenic type wild-type plant is generally resulted under same doses.
Heretofore described polynucleotides and/or nucleotide form complete " gene ", encode in required host cell Protein or polypeptide.Those skilled in the art are it is readily appreciated that polynucleotides and/or nucleotide of the invention can be placed in Under regulating and controlling sequence control in purpose host.
Well-known to those skilled in the art, DNA typically exists with double-stranded form.In this arrangement, chain with Another chain complementation, vice versa.Other complementary strands of DNA are produced since DNA is replicated in plant.In this way, packet of the present invention Include the use to polynucleotides exemplary in sequence table and its complementary strand." coding strand " that this field is often used refers to be chained with antisense The chain of conjunction.In order to express protein in vivo, a chain of DNA is transcribed into the complementary strand of a mRNA by typical case, it is as mould Plate translates protein.MRNA is actually to transcribe from " antisense " chain of DNA." ariyoshi " or " coding " chain has a series of passwords Son (codon is three nucleotide, and primary reading three can produce specific amino acids), can be used as open reading frame (ORF) and reads It reads to form target protein or peptide.The invention also includes the RNA for having suitable function with exemplary DNA.
Nucleic acid molecule of the present invention or its segment hybridize with herbicide tolerance gene of the present invention under strict conditions.It is any Conventional nucleic acid hybridization or amplification method may be used to identify the presence of herbicide tolerance gene of the present invention.Nucleic acid molecules or Its segment can carry out specific hybrid with other nucleic acid molecules in any case.In the present invention, if two nucleic acid molecules Antiparallel double-strandednucleic acid structure can be formed, so that it may say that the two nucleic acid molecules are able to carry out specific hybrid to each other.Such as Two nucleic acid molecules of fruit show complete complementarity, then claiming one of nucleic acid molecules is that another nucleic acid molecules is " complementary Object ".In the present invention, when each nucleotide of a nucleic acid molecules is complementary with the corresponding nucleotide of another nucleic acid molecules When, then claim the two nucleic acid molecules to show " complete complementarity ".If two nucleic acid molecules can be with enough stability phases Mutual cross then claims the two nucleic acid molecules to make them anneal and be bonded to each other under the conditions of at least conventional " low stringent " For " minimum level is complementary ".Similarly, if two nucleic acid molecules can be with enough stability phase mutual crosses to make them It anneals and is bonded to each other under the conditions of conventional " height is stringent ", then claim the two nucleic acid molecules that there is " complementarity ".From complete Deviateing in complementarity can permit, as long as this deviation not exclusively prevents two molecules from forming duplex structure.In order to make one A nucleic acid molecules can be used as primer or probe, it is only necessary to it is adequately complementary guarantee that it has in sequence, so that being adopted Stable duplex structure can be formed under specific solvent and salinity.
In the present invention, substantially homologous sequence is one section of nucleic acid molecules, which can under high stringency Specific hybrid occurs with the complementary strand of another section of nucleic acid molecules to match.Promote the suitable stringent condition of DNA hybridization, example Such as, it is about handled under the conditions of 45 DEG C with 6.0 × sodium chloride/sodium citrate (SSC), then with 2.0 × SSC under the conditions of 50 DEG C Washing, these conditions are well known to those skilled in the art.For example, the salinity in washing step can be selected from low tight About 2.0 × SSC of glazing bar part, 50 DEG C to high stringency of about 0.2 × SSC, 50 DEG C.In addition, the temperature in washing step Condition can be increased to about 65 DEG C of high stringency from about 22 DEG C of room temperature of Low stringency conditions.Temperature condition and salt are dense Degree can all change, can also one of them remain unchanged and another variable changes.Preferably, of the present invention Stringent condition can in 6 × SSC, 0.5%SDS solution, at 65 DEG C with the nucleotides sequence of thifensulfuronmethyl hydrolase of the present invention Specific hybrid occurs for column, is then respectively washed film 1 time with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS.
Therefore, there is herbicide tolerant activity and the under strict conditions nucleotide with thifensulfuronmethyl hydrolase of the present invention The sequence of sequence hybridization is included in the invention.These sequences and sequence of the present invention at least about 40%-50% are homologous, about 60%, 65% or 70% is homologous, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or bigger sequence homology.
The present invention provides functional protein." functional activity " (or " activity ") refers to the albumen of purposes of the present invention in the present invention Matter/enzyme (combining individually or with other oroteins) has the ability degraded or weaken herbicidal activity.Generate present protein Plant preferably generate the protein of " effective quantity ", so that the level of protein expression is enough when with herbicide treatment plant Give plant the resistance or patience complete or partial to herbicide (being then unless otherwise noted general dosage).It can be usually to kill Dosage, normal crop field dosage and the concentration of dead target plant use herbicide.Preferably, plant cell of the invention and plant quilt It is protected from growth inhibition caused by herbicide treatment or damage.Conversion plant of the invention and plant cell preferably have pyrrole phonetic The resistance or patience of the grand herbicide of sulphur, that is, the plant converted and plant cell can be in the presence of a effective amount of pyrazosulfuron herbicides Growth.
Heretofore described gene and protein not only includes specific exemplary sequence, further include save it is described specific The part of the herbicide tolerant living features of exemplary protein and/segment (including compared with full length protein and/or Terminal deletion), variant, mutant, the substituent protein of amino acid (have substitution), chimera and fusion protein.It is described " to become Body " or " variation " refer to that the same albumen of coding or coding have the nucleotide sequence of the equivalent protein of herbicide resistance activity.It is described " equivalent protein " refers to the egg for the bioactivity for having identical or essentially identical herbicide tolerant with the albumen of claim It is white.
The original DNA or egg that " segment " or " truncation " of heretofore described DNA molecular or protein sequence refers to A part of Bai Xulie (nucleotide or amino acid) or its artificial reconstructed form (such as the sequence for being suitble to plant expression), aforementioned sequence Variation may be present in the length of column, but length is enough to ensure that (coding) protein is herbicide tolerant protein.
Due to the Feng Yuxing of genetic codon, a variety of different DNA sequence dnas can encode identical amino acid sequence.It generates These encode the alternative DNA sequence dna of identical or essentially identical albumen just in the technical level of those skilled in the art.This A little different DNA sequence dnas are included within the scope of the invention." substantially the same " sequence, which refers to, to be had amino acid substitution, lacks It loses, add or be inserted into but substantially do not influence the active sequence of herbicide tolerant, also include retaining herbicide tolerant activity Segment.
Replacing, missing or adding for amino acid sequence is the ordinary skill in the art in the present invention, preferably this amino acid Variation are as follows: small characteristic changing, i.e., the folding and/or active conserved amino acid for not significantly affecting albumen replace；Small missing, The missing of normally about 1-30 amino acid；Small amino or c-terminus extend, such as aminoterminal extends a methionine residues； Small link peptide, for example, about 20-25 residue are long.
The example of conservative substitution is the substitution occurred in following amino acid group: basic amino acid (such as arginine, lysine And histidine), it is acidic amino acid (such as glutamic acid and aspartic acid), polar amino acid (such as glutamine, asparagine), hydrophobic Acidic amino acid (such as leucine, isoleucine and valine), ArAA (such as phenylalanine, tryptophan and tyrosine), with And small molecule amino acid (such as glycine, alanine, serine, threonine and methionine).Do not change given activity usually Those amino acid substitutions are well-known in the art, and by for example, N.Neurath and R.L.Hill are 1979 It is described in " Protein " published by year new york academic publishing house (Academic Press).The most common exchange has 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 and Asp/Gly and their opposite exchanges.
For a person skilled in the art it should be evident that this substitution can play an important role to molecular function Region except occur, and still generate active peptides.For by polypeptide of the invention, activity is required and therefore selects not Substituted amino acid residue can reflect according to methods known in the art, such as direct mutagenesis or alanine scanning mutagenesis Determine (such as referring to Cunningham and Wells, 1989, Science244:1081-1085).Latter technique is each in the molecule Mutation, the herbicide resistance activity of detection gained mutating molecule, so that it is determined that the molecule are introduced at a positively charged residue Important amino acid residue for activity.Substrate-enzyme interacting site can also be measured by the analysis of its three-dimensional structure, This three-dimensional structure can be measured by technologies such as nuclear magnetic resonance spectroscopy, crystallography or photoaffinity labeling (referring to, such as de Vos, 1992, Science 255:306-312；Smith etc., 1992, J.Mol.Biol 224:899-904；Wlodaver etc., 1992, FEBS Letters 309:59-64).
In the present invention, the amino acid sequence of coding thifensulfuronmethyl hydrolase includes but is not limited to involved in sequence table of the present invention Sequence, be also included in the present invention with its amino acid sequence with certain homology.These sequences and sequence class of the present invention It is typically larger than 60%, preferably greater than 75%, more preferably greater than 80% like property/phase same sex, is even more preferably greater than 90%, and 95% can be greater than.Can also according to particularly the phase same sex and/or similarity range define it is of the invention preferred Polynucleotides and protein.Such as have 49% with the exemplary sequence of the present invention, 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% The phase same sex and/or similarity.
Heretofore described regulating and controlling sequence include but is not limited to promoter, transit peptides, terminator, enhancer, leader sequence, Introne and other adjusting sequences for being operably connected to the thifensulfuronmethyl hydrolase gene.
The promoter is effable promoter in plant, and " the effable promoter in plant " refers to and ensure The promoter that coded sequence connected to it is expressed in plant cell.Effable promoter can be composing type in plant Promoter.The example for instructing the promoter of constitutive expression in plant includes but is not limited to, from cauliflower mosaic virus 35S promoter, corn Ubi promoter, promoter of rice GOS2 gene etc..Alternatively, effable promoter can in plant For the promoter of organizing specific, i.e. the promoter table that such as instructs coded sequence in chlorenchyma in some tissues of plant Up to horizontal its hetero-organization (can be tested and be measured by conventional RNA) for being higher than plant, such as PEP carboxylase promoter.Alternatively, Effable promoter can be wound-induced promoter in plant.Wound-induced promoter or the expression pattern for instructing wound-induced Promoter when referring to the wound caused by plant is subjected to machinery or is gnawed by insect, the table of the coded sequence under promoter regulation It is significantly increased up under the conditions of compared with normal growth.The example of wound-induced promoter includes but is not limited to potato and tomato Protease suppressor (pin I and pin II) and zein enzyme suppressor (MPI) promoter.
The transit peptides (also known as secretory signal sequence or targeting sequencing) are to instruct transgene product to specific organelle Or cellular compartment, for receptor protein, the transit peptides can be it is heterologous, for example, utilizing encoding chloroplast transit peptide Sequence targets chloroplaset, perhaps utilizes ' KDEL ' to retain sequence targeting endoplasmic reticulum or utilizes barley plants agglutinin gene CTPP targets vacuole.
The leader sequence is including but not limited to picornavirus leader sequence, such as EMCV leader sequence (encephalomyo-carditis disease Malicious 5 ' noncoding regions)；Potyvirus leaders, such as MDMV (Maize Dwarf Mosaic Virus) leader sequence；Human immunity Globular protein heavy-chain binding protein matter (BiP)；The coat protein mRNA's of alfalfa mosaic virus does not translate leader sequence (AMV RNA4)；Tobacco mosaic virus (TMV) (TMV) leader sequence.
The enhancer is including but not limited to cauliflower mosaic virus (CaMV) enhancer, figwort mosaic virus (FMV) increase Hadron, carnation weathering circovirus virus (CERV) enhancer, cassava vein mosaic virus (CsVMV) enhancer, Mirabilis jalapa mosaic virus (MMV) enhancer, dama de noche tomato yellow leaf curl China virus (CmYLCV) enhancer, Cotton leaf curl Multan virus (CLCuMV), duck plantar Straw colour mottle virus (CoYMV) and peanut chlorisis streak mosaic virus (PCLSV) enhancer.
For monocotyledon application, the introne is including but not limited to corn hsp70 introne, corn are general Plain introne, Adh introne 1, crose synthase intron or rice Act1 introne.For dicotyledon application, institute Introne is stated including but not limited to CAT-1 introne, pKANNIBAL introne, PIV2 introne and " super ubiquitin " include Son.
The terminator can be the suitable polyadenylation signal sequence to work in plant, including but unlimited In from the Polyadenylation of Agrobacterium (Agrobacterium tumefaciens) rouge alkali synthetase (NOS) gene Signal sequence, derives from pea at the polyadenylation signal sequence for deriving from protease-inhibitor Ⅱ (pin II) gene The polyadenylation signal sequence of ssRUBISCO E9 gene and the poly for deriving from alpha-tubulin (α-tubulin) gene Polyadenylation signal sequence.
Heretofore described " effectively connection " indicates the connection of nucleic acid sequence, described to be coupled so that a sequence can provide pair The function of being needed for linked sequence.Described in the present invention " effectively connection " can be by promoter and interested sequence phase Even, so that the transcription of the interested sequence is controlled and regulates and controls by the promoter.When interested sequential coding albumen and " effectively connection " indicates when going for the expression of the albumen: promoter is connected with the sequence, and connected mode to obtain Transcript efficient translation.If promoter and the connection of coded sequence are that transcript merges and wants to realize the albumen of coding Expression when, such connection is manufactured, so that the first translation initiation codon is the starting of coded sequence in obtained transcript Codon.Alternatively, if it is the table for the albumen that realization coding was merged and wanted in translation that promoter is with the connection of coded sequence Up to when, manufacture such connection so that the first translation initiation codon contained in 5 ' non-translated sequences is connected with promoter, And the relationship of the translation opening code-reading frame for the albumen that the translation product that connection type makes and coding are wanted is to meet reading Code frame.The nucleic acid sequence that " can effectively connect " includes but is not limited to: providing sequence (the i.e. gene expression of gene expression function Element, for example, promoter, 5 ' untranslated regions, introne, protein encoding regions, 3 ' untranslated regions, poly- putative adenylylation site and/ Or transcription terminator), provide DNA transfer and/or integration function sequence (i.e. T-DNA border sequence, locus specificity recombinase Recognition site integrates enzyme recognition site), provide selectivity function sequence (i.e. antibiotic resistance markers, biosynthesis base Cause), provide can score marker function sequence, in vitro or in vivo assist series of operations sequence (i.e. polylinker sequence, site Specific recombination sites) and provide copy function sequence (the i.e. replication orgin, autonomously replicating sequence of bacterium, centromere sequence Column).
The present invention can assign plant novel herbicide resistance character, and do not observe include to phenotype yield bad shadow It rings.In the present invention plant be resistant to as at least one tested herbicide 2 ×, 3 ×, 4 × or 5 × be normally applied level.These The raising of tolerant levels is within the scope of the present invention.Such as multiple technologies known in the art can be carried out forseeable excellent Change and further develop, to increase the expression of given gene.
Thifensulfuronmethyl hydrolase has tolerance to pyrazosulfuron herbicide in the present invention.Plant in the present invention, at it Contain exogenous DNA in genome, the exogenous DNA includes the nucleotide sequence of coding thifensulfuronmethyl hydrolase, is had by expression The albumen of effect amount and the threat for protecteding from herbicide.Effective quantity refers to unmarred or slight damage dosage.Meanwhile Plant should be morphologically normal, and can cultivate under conventional approaches with the consumption and/or generation for product.
The expression of herbicide tolerant protein can pass through described a variety of methods in the art in vegetable material It is detected, such as is determined by the mRNA of the encoding herbicide-tolerant protein generated in application primer pair tissue Amount, or directly specific detection generate herbicide tolerant protein amount.
In the present invention, by Exogenous DNA transfered plant, as described in will encode the gene of thifensulfuronmethyl hydrolase or expression cassette or Recombinant vector imports plant cell, and conventional method for transformation includes but is not limited to that Agrobacterium-medialed transformation, micro transmitting are banged The DNA importing hit, directly mediate DNA intake protoplast, electroporation or silicon whisker.
The present invention provides a kind of purposes of herbicide tolerant protein, have the advantage that
1, wide to herbicide tolerant.Present invention firstly discloses thifensulfuronmethyl hydrolases can be to pyrazosulfuron herbicide Higher tolerance is shown, therefore is had a extensive future on plant.
2, strong to herbicide tolerant.Thifensulfuronmethyl hydrolase of the present invention is strong to the tolerance of pyrazosulfuron herbicide, until It can be resistant to 1 times of crop field concentration less.
Below by drawings and examples, technical scheme of the present invention will be described in further detail.
Detailed description of the invention
Fig. 1 is the recombinant cloning vector containing ALT nucleotide sequence of the purposes of herbicide tolerant protein of the present invention DBN01-T constructs flow chart；
Fig. 2 is the recombinant expression carrier containing ALT nucleotide sequence of the purposes of herbicide tolerant protein of the present invention DBN100632 constructs flow chart；
Fig. 3 is the recombinant expression carrier containing ALT nucleotide sequence of the purposes of herbicide tolerant protein of the present invention DBN100631 structural schematic diagram；
Fig. 4 is the transgenic arabidopsis T of the purposes of herbicide tolerant protein of the present invention1Plant pair pyrazosulfuron weeding Agent toleragenic effects figure；
Fig. 5 is the recombinant expression carrier containing ALT nucleotide sequence of the purposes of herbicide tolerant protein of the present invention DBN100828 constructs flow chart；
Fig. 6 is the recombinant expression carrier containing ALT nucleotide sequence of the purposes of herbicide tolerant protein of the present invention DBN100827 structural schematic diagram；
Fig. 7 is the recombinant cloning vector containing ALT nucleotide sequence of the purposes of herbicide tolerant protein of the present invention DBN05-T constructs flow chart；
Fig. 8 is the recombinant expression carrier containing ALT nucleotide sequence of the purposes of herbicide tolerant protein of the present invention DBN100830 constructs flow chart；
Fig. 9 is the recombinant expression carrier containing ALT nucleotide sequence of the purposes of herbicide tolerant protein of the present invention DBN100829 structural schematic diagram.
Specific embodiment
The technical solution of the purposes of herbicide tolerant protein of the present invention is further illustrated below by specific embodiment.
The acquisition and synthesis of first embodiment, ALT gene order
1, ALT gene order is obtained
The amino acid sequence (398 amino acid) of thifensulfuronmethyl hydrolase -1 (ALT-1), such as SEQ ID NO in sequence table: Shown in 1；Coding corresponds to the ALT-1-01 nucleotide sequence (1197 nucleotide) of the amino acid sequence of the ALT-1, such as sequence In list shown in SEQ ID NO:2, ALT-1-02 nucleotide sequence of the coding corresponding to the amino acid sequence of the ALT-1 (1197 nucleotide), as shown in SEQ ID NO:3 in sequence table.
The amino acid sequence (369 amino acid) of thifensulfuronmethyl hydrolase -2 (ALT-2), such as SEQ ID NO in sequence table: Shown in 4；Coding corresponds to the ALT-2-01 nucleotide sequence (1110 nucleotide) of the amino acid sequence of the ALT-2, such as sequence In list shown in SEQ ID NO:5, ALT-2-02 nucleotide sequence of the coding corresponding to the amino acid sequence of the ALT-2 (1110 nucleotide), as shown in SEQ ID NO:6 in sequence table.
The amino acid sequence (362 amino acid) of thifensulfuronmethyl hydrolase -3 (ALT-3), such as SEQ ID NO in sequence table: Shown in 7；Coding corresponds to the ALT-3-01 nucleotide sequence (1089 nucleotide) of the amino acid sequence of the ALT-3, such as sequence In list shown in SEQ ID NO:8, ALT-3-02 nucleotide sequence of the coding corresponding to the amino acid sequence of the ALT-3 (1089 nucleotide), as shown in SEQ ID NO:9 in sequence table.
2, EPSPS gene order is obtained
The amino acid sequence (455 amino acid) of glyphosate tolerant protein, such as SEQ ID NO:10 institute in sequence table Show；EPSPS nucleotide sequence (1368 nucleosides of the coding corresponding to the amino acid sequence of the glyphosate tolerant protein Acid), as shown in SEQ ID NO:11 in sequence table.
3, above-mentioned nucleotide sequence is synthesized
The ALT-1-01 nucleotide sequence (as shown in SEQ ID NO:2 in sequence table), the ALT-1-02 nucleotide Sequence (as shown in SEQ ID NO:3 in sequence table), the ALT-2-01 nucleotide sequence (SEQ ID NO:5 in such as sequence table It is shown), the ALT-2-02 nucleotide sequence (as shown in SEQ ID NO:6 in sequence table), the ALT-3-01 nucleotides sequence (as shown in SEQ ID NO:8 in sequence table), the ALT-3-02 nucleotide sequence are arranged (such as SEQ ID NO:9 institute in sequence table Show) and the EPSPS nucleotide sequence (as shown in SEQ ID NO:11 in sequence table) it is limited by Nanjing Jin Sirui biotechnology Company's synthesis；5 ' ends of the ALT-1-01 nucleotide sequence (SEQ ID NO:2) of synthesis are also connected with SpeI restriction enzyme site, 3 ' ends of the ALT-1-01 nucleotide sequence (SEQ ID NO:2) are also connected with KasI restriction enzyme site；The ALT- of synthesis 5 ' ends of 1-02 nucleotide sequence (SEQ ID NO:3) are also connected with SpeI restriction enzyme site, the ALT-1-02 nucleotide sequence 3 ' the ends of (SEQ ID NO:3) are also connected with KasI restriction enzyme site；ALT-2-01 nucleotide sequence (the SEQ ID of synthesis NO:5 5 ' ends) are also connected with SpeI restriction enzyme site, and 3 ' ends of the ALT-2-01 nucleotide sequence (SEQ ID NO:5) also connect It is connected to KasI restriction enzyme site；5 ' ends of the ALT-2-02 nucleotide sequence (SEQ ID NO:6) of synthesis are also connected with SpeI 3 ' ends of restriction enzyme site, the ALT-2-02 nucleotide sequence (SEQ ID NO:6) are also connected with KasI restriction enzyme site；Synthesis 5 ' ends of the ALT-3-01 nucleotide sequence (SEQ ID NO:8) are also connected with SpeI restriction enzyme site, the ALT-3-01 core 3 ' ends of nucleotide sequence (SEQ ID NO:8) are also connected with KasI restriction enzyme site；The ALT-3-02 nucleotide sequence of synthesis 5 ' the ends of (SEQ ID NO:9) are also connected with SpeI restriction enzyme site, the ALT-3-02 nucleotide sequence (SEQ ID NO:9) 3 ' ends are also connected with KasI restriction enzyme site；5 ' ends of the EPSPS nucleotide sequence (SEQ ID NO:11) of synthesis are also connected with There is NcoI restriction enzyme site, 3 ' ends of the EPSPS nucleotide sequence (SEQ ID NO:11) are also connected with FspI restriction enzyme site.
The building of second embodiment, arabidopsis recombinant expression carrier
1, the arabidopsis containing ALT nucleotide sequence and soybean recombinant cloning vector are constructed
By the ALT-1-01 nucleotide sequence of synthesis be connected into cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600 on), operating procedure is carried out by Promega Products pGEM-T carrier specification, obtains recombinant cloning vector DBN01- T, (wherein, Amp indicates ampicillin resistance gene to building process as shown in Figure 1；F1 indicates that the duplication of bacteriophage f1 rises Point；LacZ is LacZ initiation codon；SP6 is SP6RNA polymerase promoter；T7 is t7 rna polymerase promoter；ALT-1- 01 is ALT-1-01 nucleotide sequence (SEQ ID NO:2)；MCS is multiple cloning sites).
Then by recombinant cloning vector DBN01-T with heat shock method convert Escherichia coli T1 competent cell (Transgen, Beijing, China, CAT:CD501), hot shock condition are as follows: 50 μ L Escherichia coli T1 competent cells, 10 μ L Plasmid DNA (weight Group cloning vector DBN01-T), 42 DEG C water-bath 30 seconds；37 DEG C shaken cultivation 1 hour (shaking table shakes under 100rpm revolving speed), in table Face is coated with the ammonia of IPTG (isopropylthio-β-D-galactoside) and X-gal (the chloro- 3- indoles-β-D- galactoside of the bromo- 4- of 5-) Parasiticin (100mg/L) LB plate (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L, Overnight with NaOH tune pH to growth on 7.5).Picking white colony, in LB liquid medium, (tryptone 10g/L, yeast are extracted Object 5g/L, NaCl 10g/L, ampicillin 100mg/L, with NaOH tune pH to being cultivated under the conditions of 37 DEG C of temperature in 7.5) Night.Its plasmid of alkalinity extraction: being centrifuged 1min under 12000rpm revolving speed for bacterium solution, removes supernatant, and precipitating thallus is pre- with 100 μ L ice Cold solution I (25mM Tris-HCl, 10mM EDTA (ethylenediamine tetra-acetic acid), 50mM glucose, pH8.0) suspends；It is added 200 The solution II (0.2M NaOH, 1%SDS (lauryl sodium sulfate)) that μ L is newly prepared, pipe is overturned 4 times, and mixing is set on ice 3-5min；The ice-cold solution III of 150 μ L (3M potassium acetate, 5M acetic acid) is added, mixes well immediately, places 5-10min on ice； It is centrifuged 5min under the conditions of 4 DEG C of temperature, revolving speed 12000rpm, 2 times of volume dehydrated alcohols, room temperature after mixing are added in supernatant Place 5min；It is centrifuged 5min under the conditions of 4 DEG C of temperature, revolving speed 12000rpm, abandons supernatant, precipitating is 70% with concentration (V/V) Ethanol washing after dry；It is molten that TE (10mM Tris-HCl, 1mM EDTA, pH8.0) of the 30 μ L containing RNase (20 μ g/mL) is added Solution precipitating；The water-bath 30min at 37 DEG C of temperature digests RNA；It is saved backup in -20 DEG C of temperature.
The plasmid of extraction carries out sequence verification after SpeI and KasI digestion identification, to positive colony, the results showed that recombination The ALT-1-01 nucleotides sequence being inserted into cloning vector DBN01-T is classified as nucleosides shown in SEQ ID NO:2 in sequence table Acid sequence, i.e. ALT-1-01 nucleotide sequence are correctly inserted into.
According to the method for above-mentioned building recombinant cloning vector DBN01-T, by the ALT-2-01 nucleotide sequence of synthesis It is connected on cloning vector pGEM-T, obtains recombinant cloning vector DBN02-T, wherein ALT-2-01 is ALT-2-01 nucleotides sequence It arranges (SEQ ID NO:5).ALT-2-01 nucleotide sequence described in digestion and sequence verification recombinant cloning vector DBN02-T is correct Insertion.
According to the method for above-mentioned building recombinant cloning vector DBN01-T, by the ALT-3-01 nucleotide sequence of synthesis It is connected on cloning vector pGEM-T, obtains recombinant cloning vector DBN03-T, wherein ALT-3-01 is ALT-3-01 nucleotides sequence It arranges (SEQ ID NO:8).ALT-3-01 nucleotide sequence described in digestion and sequence verification recombinant cloning vector DBN03-T is correct Insertion.
Meanwhile according to the method for above-mentioned building recombinant cloning vector DBN01-T, by the EPSPS nucleotides sequence of synthesis Column are connected on cloning vector pGEM-T, obtain recombinant cloning vector DBN04-T, wherein EPSPS is EPSPS nucleotide sequence (SEQ ID NO:11).EPSPS nucleotide sequence described in digestion and sequence verification recombinant cloning vector DBN04-T is correctly inserted Enter.
2, the arabidopsis recombinant expression carrier containing ALT nucleotide sequence is constructed
Digestion recombinant cloning vector DBN01-T and expression vector DBNBC-01 is distinguished with restriction enzyme SpeI and KasI (carrier framework: pCAMBIA2301 (CAMBIA mechanism can provide)), the ALT-1-01 nucleotide sequence fragment cut is inserted into It is those skilled in the art using conventional enzymatic cleavage methods carrier construction between the site SpeI and KasI of expression vector DBNBC-01 Member known to, be built into recombinant expression carrier DBN100632 (being positioned at cytoplasm), building process as shown in Figure 2 (Spec: Spectinomycin gene；RB: right margin；PrAtUbi10: arabidopsis Ubiquitin (ubiquitin) 10 gene promoter (SEQ ID NO: 12)；ALT-1-01:ALT-1-01 nucleotide sequence (SEQ ID NO:2)；TNos: the terminator of rouge alkali synthetase gene (SEQ ID NO:13)；PrCaMV35S: cauliflower mosaic virus 35 S promoter (SEQ ID NO:14)；PAT: glufosinate-ammonium acetyl Transferase gene (SEQ ID NO:15)；TCaMV35S: cauliflower mosaic virus 35S terminator (SEQ ID NO:16)；LB: left Boundary).
Recombinant expression carrier DBN100632 heat shock method is converted into Escherichia coli T1 competent cell, hot shock condition Are as follows: 50 μ L Escherichia coli T1 competent cells, 10 μ L Plasmid DNA (recombinant expression carrier DBN100632), 42 DEG C water-bath 30 seconds； 37 DEG C shaken cultivation 1 hour (shaking table shakes under 100rpm revolving speed)；Then at spectinomycin containing 50mg/L (Spectinomycin) LB solid plate (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L, extremely with NaOH tune pH 7.5) it is cultivated 12 hours under the conditions of 37 DEG C of temperature on, picking white colony, in LB liquid medium (tryptone 10g/L, ferment Female extract 5g/L, NaCl 10g/L, spectinomycin 50mg/L, with NaOH tune pH to being cultivated under the conditions of 37 DEG C of temperature in 7.5) Overnight.Its plasmid of alkalinity extraction.It will be identified after restriction enzyme SpeI and the KasI digestion of the plasmid of extraction, and by positive gram It is grand to carry out sequencing identification, the results showed that nucleotides sequence of the recombinant expression carrier DBN100632 between the site SpeI and KasI is classified as Nucleotide sequence shown in SEQ ID NO:2, i.e. ALT-1-01 nucleotide sequence in sequence table.
According to the method for above-mentioned building recombinant expression carrier DBN100632, building contains ALT-1-01 nucleotide sequence Recombinant expression carrier DBN100631 (is positioned at chloroplaset), carrier structure (carrier framework: pCAMBIA2301 as shown in Figure 3 (CAMBIA mechanism can provide)；Spec: spectinomycin gene；RB: right margin；PrAtUbi10: arabidopsis Ubiquitin is (general Element) 10 gene promoters (SEQ ID NO:12)；SpAtCTP2: arabidopsis chloroplast transit peptides (SEQ ID NO:17)；ALT- 1-01:ALT-1-01 nucleotide sequence (SEQ ID NO:2)；TNos: rouge alkali synthetase gene terminator (SEQ ID NO: 13)；PrCaMV35S: cauliflower mosaic virus 35 S promoter (SEQ ID NO:14)；PAT: glufosinate-ammonium acetyl transferase gene (SEQ ID NO:15)；TCaMV35S: cauliflower mosaic virus 35S terminator (SEQ ID NO:16)；LB: left margin).To sun Property clone carry out sequence verification, the results showed that the ALT-1-01 nucleotides sequence being inserted into recombinant expression carrier DBN100631 is classified as Nucleotide sequence shown in SEQ ID NO:2, i.e. ALT-1-01 nucleotide sequence are correctly inserted into sequence table.
According to the method for above-mentioned building recombinant expression carrier DBN100632, by SpeI and KasI digestion recombinant cloning vector The ALT-2-01 nucleotide sequence that DBN02-T is cut is inserted into expression vector DBNBC-01, obtains recombinant expression carrier DBN100634.Nucleotide sequence in digestion and sequence verification recombinant expression carrier DBN100634 contains for SEQ in sequence table Nucleotide sequence shown in ID NO:5, i.e. ALT-2-01 nucleotide sequence are correctly inserted into.
According to the method for above-mentioned building recombinant expression carrier DBN100631, by SpeI and KasI digestion recombinant cloning vector The ALT-2-01 nucleotide sequence that DBN02-T is cut is inserted into expression vector DBNBC-01, obtains recombinant expression carrier DBN100633 (containing spAtCTP2, is positioned at chloroplaset).In digestion and sequence verification recombinant expression carrier DBN100633 Nucleotide sequence contains correctly to be inserted for nucleotide sequence shown in SEQ ID NO:5, i.e. ALT-2-01 nucleotide sequence in sequence table Enter.
According to the method for above-mentioned building recombinant expression carrier DBN100632, by SpeI and KasI digestion recombinant cloning vector The ALT-3-01 nucleotide sequence that DBN03-T is cut is inserted into expression vector DBNBC-01, obtains recombinant expression carrier DBN100636.Nucleotide sequence in digestion and sequence verification recombinant expression carrier DBN100636 contains for SEQ in sequence table Nucleotide sequence shown in ID NO:8, i.e. ALT-3-01 nucleotide sequence are correctly inserted into.
According to the method for above-mentioned building recombinant expression carrier DBN100631, by SpeI and KasI digestion recombinant cloning vector The ALT-3-01 nucleotide sequence that DBN03-T is cut is inserted into expression vector DBNBC-01, obtains recombinant expression carrier DBN100635 (containing spAtCTP2, is positioned at chloroplaset).In digestion and sequence verification recombinant expression carrier DBN100635 Nucleotide sequence contains correctly to be inserted for nucleotide sequence shown in SEQ ID NO:8, i.e. ALT-3-01 nucleotide sequence in sequence table Enter.
3rd embodiment, be transferred to ALT nucleotide sequence Arabidopsis plant acquisition
1, recombinant expression carrier converts Agrobacterium
To oneself constructed correct recombinant expression carrier DBN100632, DBN100631, DBN100634, DBN100633, DBN100636 and DBN100635 is transformed into Agrobacterium GV3101 with liquid nitrogen method, conversion condition are as follows: 100 μ L Agrobacteriums GV3101,3 μ L Plasmid DNA (recombinant expression carrier)；Be placed in liquid nitrogen 10 minutes, 37 DEG C tepidarium 10 minutes；After conversion Agrobacterium GV3101 is inoculated in LB test tube in 28 DEG C of temperature, revolving speed to cultivate 2 hours under the conditions of 200rpm, is applied to containing 50mg/L Rifampin (Rifampicin) and 50mg/L spectinomycin LB plate on until grow positive monoclonal, picking monoclonal Its plasmid is cultivated and extracted, carries out digestion verification with restriction enzyme, the results showed that recombinant expression carrier DBN100632, DBN100631, DBN100634, DBN100633, DBN100636 and DBN100635 structure are completely correct.
2, transgenic Arabidopsis plants are obtained
By wildtype Arabidopsis thaliana seed suspension in 0.1% (w/v) agarose solution.The seed of suspension is protected at 4 DEG C It deposits 2 days and guarantees that seed is synchronous to complete the needs to suspend mode and sprout.With vermiculite mixing horsehit soil and with water sub-irrigation to wet Profit drains soil mixture 24 hours.It is covered 7 days by pretreated seed kind on soil mixture and with moisture preserving cover. Sprout seed and in 120-150 μm of ol/m of constant temperature (22 DEG C) constant humidity (40-50%) luminous intensity2The long-day conditions (16 of second Hour illumination/8 hour dark) under cultivate plant in the greenhouse.Start to use Huo Gelan nutrition liquid irrigation plant, then uses deionization Water is irrigated, and is kept soil moisture but is not drenched.
Use flower infusion method arabidopsis thaliana transformation.With the Agrobacterium colony inoculation one or more parts 15-30mL of selection containing grand The pre-culture of the YEP culture solution of mycin (50mg/L) and rifampin (10mg/L).With 220rpm by culture in 28 DEG C of constant speed It shakes and is incubated overnight.Each pre-culture contains spectinomycin (50mg/L) and rifampin (10mg/L) for being inoculated with two parts of 500mL YEP culture solution culture and culture is incubated overnight in 28 DEG C of lasting shake.Room temperature was with about 8700 × g centrifugation 10 minutes Sedimentation cell, the supernatant discarded.Cell precipitation is softly resuspended in 500mL osmotic medium, the infiltration culture Base contains that 1/2 × MS salt/B5 vitamin, 10% (w/v) sucrose, (10 μ L/L are (in 1mg/mL DMSO for 0.044 μM of benayl aminopurine Stoste)) and 300 μ L/L Silvet L-77.The plant at about 1 monthly age is impregnated 15 seconds in the medium, it is ensured that submergence is newest Inflorescence.Then plant side was fallen and covered (transparent or opaque) 24 hours, and be then washed with water and place vertically.? 22 DEG C are cultivated plant with 16 hours illumination/8 hour dark photoperiod.Seed is harvested after impregnating about 4 weeks.
(ALT nucleotide sequence) T that will newly harvest1Seed was at drying at room temperature 7 days.Seed kind is sprouted in 26.5 × 51cm In disk, every disk receives 200mgT1Seed (about 10000 seeds), the seed has been suspended in 40mL 0.1% (w/v) fine jade in advance Lipolysaccharide solution simultaneously saves 2 days needs with completion to suspend mode at 4 DEG C to guarantee the synchronous sprouting of seed.
With vermiculite mixing horsehit soil and with water sub-irrigation to moistening, gravity drainage is utilized.After being pre-processed with pipette Seed (each 40mL) equably plant on soil mixture, and with moisture preserving cover cover 4-5 days.Grass is sprayed after using emergence Ammonium phosphine (pat gene of selection cotransformation) carries out initial transformant and selects first 1 day to remove cover.
After 7 plantation number of days (DAP) and DeVilbiss compressed-air atomizer is reused with 10mL/ disk in 11DAP 0.2% spray solution T of the sprinkling volume of (703L/ha) Liberty herbicide (glufosinate-ammonium of 200g ai/L)1Plant (point Wei cotyledon period and 2-4 leaf phase), a effective amount of glufosinate-ammonium of 280g ai/ha is applied every time to provide.4-7 days after last sprinkling Identification survival strain (plant of active growth), and be transplanted to respectively (every in the square basin of the 7cmx7cm with horsehit soil and vermiculite preparation 3-5, disk).With plant 3-4 days of moisture preserving cover covering transplanting, and such as preposition in 22 DEG C of culturing room or directly immigration greenhouse.It connects Remove cover and before the ability that test ALT gene provides pyrazosulfuron Herbicid resistant at least 1 day by plant cultivating to warm Room (22 ± 5 DEG C, 50 ± 30%RH, 14 hours illumination: 10 hours dark, 500 μ E/m of minimum2s1Naturally+supplement light).
Fourth embodiment, the herbicide tolerant effect detection of transgenic Arabidopsis plants
It uses in the unconverted seed background of glufosinate-ammonium selection scheme first and selects T1Transformant.About 40000 T are screened1 In seed and identify 380 plants of T1For positive transformant (pat gene), about 0.95% transformation efficiency.Conversion recombinant expression carries Body DBN100632 is Arabidopsis plant (the At cytoplasm ALT-1- for being transferred to ALT-1-01 nucleotide sequence for being positioned at cytoplasm 01), conversion recombinant expression carrier DBN100631 is the arabidopsis for being transferred to ALT-1-01 nucleotide sequence for being positioned at chloroplaset Plant (At chloroplaset ALT-1-01)；Conversion recombinant expression carrier DBN100634 be positioned at cytoplasm be transferred to ALT-2-01 The Arabidopsis plant (At cytoplasm ALT-2-01) of nucleotide sequence, conversion recombinant expression carrier DBN100633 is to be positioned at leaf The Arabidopsis plant (At chloroplaset ALT-2-01) for being transferred to ALT-2-01 nucleotide sequence of green body；Convert recombinant expression carrier DBN100636 is the Arabidopsis plant (At cytoplasm ALT-3-01) for being transferred to ALT-3-01 nucleotide sequence for being positioned at cytoplasm, Converting recombinant expression carrier DBN100635 is the Arabidopsis plant for being transferred to ALT-3-01 nucleotide sequence for being positioned at chloroplaset (At chloroplaset ALT-3-01).By the T of At cytoplasm ALT-1-011Plant, At chloroplaset ALT-1-01 T1Plant, At cytoplasm The T of ALT-2-011Plant, At chloroplaset ALT-2-01 T1Plant, At cytoplasm ALT-3-01 T1Plant, At chloroplaset ALT- The T of 3-011Plant and wild-type Arabidopsis plants (after planting 14 days) carry out herbicide tolerant effect to pyrazosulfuron respectively Detection.
Respectively by the T of At cytoplasm ALT-1-011Plant, At chloroplaset ALT-1-01 T1Plant, At cytoplasm ALT-2-01 T1Plant, At chloroplaset ALT-2-01 T1Plant, At cytoplasm ALT-3-01 T1Plant, At chloroplaset ALT-3-01 T1It plants Strain and wild-type Arabidopsis plants pyrazosulfuron (25g ai/ha, 1 times of crop field concentration) and blank solvent (water) are sprayed.It sprays Plant resistance situation is counted after 14 days: upgrowth situation and blank solvent (water) is consistent divides highly resistance plant into after 14 days, after 14 days Bolting height is that it is low that dividing into for bolting is still unable to after 14 days lower than anti-plant in the dividing into of 1/2 blank solvent (water) bolting height Anti- plant, it is dead after 14 days to divide not anti-plant into.Due to every plant of arabidopsis T1Plant is independent transformation event, it is contemplated that The individual T in given dose1The significant difference of response.As a result as shown in table 1 and Fig. 4.
Table 1, transgenic arabidopsis T1Plant pair pyrazosulfuron herbicide tolerant experimental result
For arabidopsis, 25g ai/ha pyrazosulfuron herbicide is by sensitive plant and the plant with average resistance level The effective dose that object distinguishes.Table 1 and Fig. 4's the result shows that: thifensulfuronmethyl hydrolase (ALT-1, ALT-2 and ALT-3) assign Giving individual arabidopsis thaliana pyrazosulfuron herbicide tolerant, (individual plant are due to T without the reason of tolerance1Dai Zhi Object insertion point is random, thus the expression of genes conferring resistance is variant, shows the difference of durability level)；Phase Than in the T of At cytoplasm ALT-1-011Plant, At cytoplasm ALT-2-01 T1The T of plant and At cytoplasm ALT-3-011Plant, At leaf The T of green body ALT-1-011Plant, At chloroplaset ALT-2-01 T1The T of plant and At chloroplaset ALT-3-011Plant can produce Raw higher pyrazosulfuron herbicide tolerant, shows that thifensulfuronmethyl hydrolase (ALT-1, the ALT-2 and ALT-3) gene is fixed Arabidopsis thaliana can be enhanced to the tolerance of pyrazosulfuron herbicide in expression in chloroplaset；And wild-type Arabidopsis plants Do not have the tolerance to pyrazosulfuron herbicide then.
5th embodiment has unexpected technical effect for different sulfonylurea herbicides
The thifensulfuronmethyl hydrolase is also referred to as sulfonylurea herbicide and removes esterase, makes to have by hydrolysis of ester bonds with The sulfonylurea herbicide (such as thifensulfuronmethyl) of ester bond is degraded to female acid of no herbicidal activity, thus it cannot be dropped Sulfonylurea herbicide (such as nicosulfuron, chlorsulfuron) of the solution without ester bond.Have similar in ester bond and structure in the prior art There are many sulfonylurea herbicide, such as tribenuron-methyl, iodine metsulfuron-methyl, oxasulfuron, mesosulfuron (mesosulfuronmethyl), the phonetic sulphur of pyrrole Grand, sulfometuronmethyl, halosulfuronmethyl etc..
By the T of At cytoplasm ALT-1-01 in fourth embodiment1Plant, At chloroplaset ALT-1-01 T1Plant, At cytoplasm The T of ALT-2-011Plant, At chloroplaset ALT-2-01 T1Plant, At cytoplasm ALT-3-01 T1Plant, At chloroplaset ALT- The T of 3-011Plant and wild-type Arabidopsis plants are in addition to pyrazosulfuron (25g ai/ha, 1 times of crop field concentration) and blank solvent (water) sprinkling is outer, and also with iodine metsulfuron-methyl (10g ai/ha, 1 times of crop field concentration), mesosulfuron, (14g ai/ha, 1 times greatly respectively Field concentration) and oxasulfuron (60g ai/ha, 1 times of crop field concentration) sprinkling.Statistics plant resistance situation after spraying 14 days: 14 Upgrowth situation and blank solvent (water) is consistent divides highly resistance plant into after it, bolting height is lower than 1/2 blank solvent after 14 days Anti- plant in the dividing into of (water) bolting height, bolting is still unable to after 14 days divides low anti-plant into, dead after 14 days to divide into not Anti- plant.Due to every plant of arabidopsis T1Plant is independent transformation event, it is contemplated that the individual T in given dose1Response is shown Write difference.As a result as shown in table 2 and Fig. 4.
Table 2, transgenic arabidopsis T1Plant pair sulfonylurea herbicide tolerance test result
Table 2 compares ALT-1, ALT-2 and ALT-3 and inputs thifensulfuronmethyl hydrolytic enzyme activities to arabidopsis T1Plant answers It answers.Although the arabidopsis T of all conversions1Plant has been assigned thifensulfuronmethyl hydrolytic enzyme activities, but in given processing (iodine first Sulphur is grand, mesosulfuron and oxasulfuron) in, the arabidopsis T of all conversions1Plant does not show have above-mentioned sulphur of degrading The ability of sulfonylurea herbicide, the arabidopsis T of all conversions1The degree of injury of plant (ALT-1, ALT-2 and ALT-3) and wild Without any difference between type Arabidopsis plant.
Table 2 absolutely prove table 1 the result is that unexpected.Although pyrazosulfuron and iodine metsulfuron-methyl, oxasulfuron and Mesosulfuron be with sulfonylurea herbicide similar in ester bond and chemical structure, and given processing be also have it is comparable (the 1 times of crop field concentration) of property, at the same thifensulfuronmethyl hydrolase (ALT-1, ALT-2 and ALT-3) in plant individual with expection Level is inputted and is expressed, however the plant for expressing thifensulfuronmethyl hydrolase does not have degradation iodine metsulfuron-methyl, mesosulfuron and ring The ability of oxygen Sulfometuron Methyl can not protect itself from the damage of above-mentioned sulfonylurea herbicide, the table with WT lines Now without any difference, these data are enough to confirm: the thifensulfuronmethyl hydrolase (ALT-1, ALT-2 and ALT-3) assigns plant It is to be difficult to expect to pyrazosulfuron herbicide tolerant.
Sixth embodiment, the building of soybean recombinant expression carrier and recombinant expression carrier convert Agrobacterium
1, the soybean recombinant expression carrier containing ALT nucleotide sequence is constructed
Digestion recombinant cloning vector DBN01-T, DBN04- are distinguished with restriction enzyme SpeI and KasI, NcoI and FspI T and expression vector DBNBC-02 (carrier framework: pCAMBIA2301 (CAMBIA mechanism can provide)), the ALT-1- that will be cut 01 nucleotide sequence and EPSPS nucleotide sequence fragment be inserted into respectively the SpeI and KasI of expression vector DBNBC-02, NcoI and Between the site FspI, be using conventional enzymatic cleavage methods carrier construction it is well-known to those skilled in the art, be built into recombination table Up to carrier DBN100828 (being positioned at cytoplasm), process (Spec: spectinomycin gene as shown in Figure 5 is constructed；RB: right margin； PrAtUbi10: arabidopsis Ubiquitin (ubiquitin) 10 gene promoter (SEQ ID NO:12)；ALT-1-01:ALT-1-01 core Nucleotide sequence (SEQ ID NO:2)；TNos: the terminator (SEQ ID NO:13) of rouge alkali synthetase gene；PrBrCBP: oil Dish eukaryon elongation factor gene 1 α (Tsf1) promoter (SEQ ID NO:18)；SpAtCTP2: arabidopsis chloroplast transit peptides (SEQ ID NO:17)；EPSPS:5- enolpyruvylshikimate -3- phosphate synthase gene (SEQ ID NO:11)；TPsE9: pea The terminator (SEQ ID NO:19) of beans RbcS gene；LB: left margin).
Recombinant expression carrier DBN100828 heat shock method is converted into Escherichia coli according in second embodiment 2 method T1 competent cell, and alkalinity extraction its plasmid.It will be identified after restriction enzyme SpeI and the KasI digestion of the plasmid of extraction, And positive colony is subjected to sequencing identification, the results showed that core of the recombinant expression carrier DBN100828 between the site SpeI and KasI Nucleotide sequence is nucleotide sequence, i.e. ALT-1-01 nucleotide sequence shown in SEQ ID NO:2 in sequence table.
According to the method for above-mentioned building recombinant expression carrier DBN100828, building contains ALT-1-01 nucleotide sequence Recombinant expression carrier DBN100827 (is positioned at chloroplaset), carrier structure (carrier framework: pCAMBIA2301 as shown in Figure 6 (CAMBIA mechanism can provide)；Spec: spectinomycin gene；RB: right margin；PrAtUbi10: arabidopsis Ubiquitin is (general Element) 10 gene promoters (SEQ ID NO:12)；SpAtCTP2: arabidopsis chloroplast transit peptides (SEQ ID NO:17)；ALT- 1-01:ALT-1-01 nucleotide sequence (SEQ ID NO:2)；TNos: rouge alkali synthetase gene terminator (SEQ ID NO: 13)；PrBrCBP: rape eukaryon elongation factor gene 1 α (Tsf1) promoter (SEQ ID NO:18)；SpAtCTP2: arabidopsis Chloroplast transit peptides (SEQ ID NO:17)；EPSPS:5- enolpyruvylshikimate -3- phosphate synthase gene (SEQ ID NO: 11)；TPsE9: the terminator (SEQ ID NO:19) of pea RbcS gene；LB: left margin).Sequencing is carried out to positive colony to test Card, the results showed that the ALT-1-01 nucleotides sequence being inserted into recombinant expression carrier DBN100827 is classified as SEQ ID in sequence table Nucleotide sequence shown in NO:2, i.e. ALT-1-01 nucleotide sequence are correctly inserted into.
According to the method for above-mentioned building recombinant expression carrier DBN100828, by SpeI and KasI, NcoI and FspI digestion weight The ALT-2-01 nucleotide sequence and EPSPS nucleotide sequence insertion table that group cloning vector DBN02-T and DBN04-T are cut Up to carrier DBNBC-02, recombinant expression carrier DBN100826 is obtained.Digestion and sequence verification recombinant expression carrier DBN100826 In nucleotide sequence contain for nucleotide sequence, i.e. ALT-2- shown in SEQ ID NO:5 in sequence table and SEQ ID NO:11 01 nucleotide sequence and EPSPS nucleotide sequence are correctly inserted into.
According to the method for above-mentioned building recombinant expression carrier DBN100827, by SpeI and KasI, NcoI and FspI digestion weight The ALT-2-01 nucleotide sequence and EPSPS nucleotide sequence insertion table that group cloning vector DBN02-T and DBN04-T are cut Up to carrier DBNBC-02, recombinant expression carrier DBN100825 (containing spAtCTP2, being positioned at chloroplaset) is obtained.Digestion and survey Nucleotide sequence in sequence verifying recombinant expression carrier DBN100825 contains for SEQ ID NO:5 in sequence table and SEQ ID Nucleotide sequence shown in NO:11, i.e. ALT-2-01 nucleotide sequence and EPSPS nucleotide sequence are correctly inserted into.
According to the method for above-mentioned building recombinant expression carrier DBN100828, by SpeI and KasI, NcoI and FspI digestion weight The ALT-3-01 nucleotide sequence and EPSPS nucleotide sequence insertion table that group cloning vector DBN03-T and DBN04-T are cut Up to carrier DBNBC-02, recombinant expression carrier DBN100824 is obtained.Digestion and sequence verification recombinant expression carrier DBN100824 In nucleotide sequence contain for nucleotide sequence, i.e. ALT-3- shown in SEQ ID NO:8 in sequence table and SEQ ID NO:11 01 nucleotide sequence and EPSPS nucleotide sequence are correctly inserted into.
According to the method for above-mentioned building recombinant expression carrier DBN100827, by SpeI and KasI, NcoI and FspI digestion weight The ALT-3-01 nucleotide sequence and EPSPS nucleotide sequence insertion table that group cloning vector DBN03-T and DBN04-T are cut Up to carrier DBNBC-02, recombinant expression carrier DBN100823 (containing spAtCTP2, being positioned at chloroplaset) is obtained.Digestion and survey Nucleotide sequence in sequence verifying recombinant expression carrier DBN100823 contains for SEQ ID NO:8 in sequence table and SEQ ID Nucleotide sequence shown in NO:11, i.e. ALT-3-01 nucleotide sequence and EPSPS nucleotide sequence are correctly inserted into.
2, recombinant expression carrier converts Agrobacterium
To oneself constructed correct recombinant expression carrier DBN100828, DBN100827, DBN100826, DBN100825, DBN100824 and DBN100823 with liquid nitrogen method be transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: In 18313-015), conversion condition are as follows: 100 μ L Agrobacterium LBA4404s, 3 μ L Plasmid DNA (recombinant expression carrier)；It is placed in liquid 10 minutes in nitrogen, 37 DEG C tepidarium 10 minutes；By the Agrobacterium LBA4404 after conversion be inoculated in LB test tube in 28 DEG C of temperature, Revolving speed is cultivated 2 hours under the conditions of being 200rpm, is applied to the grand mould of rifampin (Rifampicin) containing 50mg/L and 50mg/L Until growing positive monoclonal on the LB plate of element, picking Colony Culture simultaneously extracts its plasmid, is carried out with restriction enzyme Digestion verification, the results showed that recombinant expression carrier DBN100828, DBN100827, DBN100826, DBN100825, DBN100824 and DBN100823 structure is completely correct.
The acquisition and verifying of 7th embodiment, Transgenic soybean plants
1, Transgenic soybean plants are obtained
According to the Agrobacterium infestation method routinely used, by the cotyledonary node tissue of Huang 13 in the soybean varieties of sterile culture and the The co-cultivation of Agrobacterium described in 2 in six embodiments, the recombinant expression carrier DBN100828 that in sixth embodiment 1 is constructed, T-DNA (including arabidopsis in DBN100827, DBN100826, DBN100825, DBN100824 and DBN100823 Promoter sequence, ALT-1-01 nucleotide sequence, ALT-2-01 nucleotide sequence, the ALT-3-01 core of Ubiquitin10 gene Nucleotide sequence, tNos terminator, 1 α promoter of rape eukaryon elongation factor gene, arabidopsis chloroplast transit peptides, 5- enol third The terminator of ketone acid shikimic acid -3- phosphate synthase gene, pea RbcS gene) it is transferred in soybean genome, turned Changing recombinant expression carrier DBN100828 is soybean plant strain (the Gm born of the same parents for being transferred to ALT-1-01 nucleotide sequence for being positioned at cytoplasm Matter ALT-1-01), conversion recombinant expression carrier DBN100827 be positioned at chloroplaset be transferred to ALT-1-01 nucleotide sequence Soybean plant strain (Gm chloroplaset ALT-1-01)；Converting recombinant expression carrier DBN100826 is to be positioned at being transferred to for cytoplasm The soybean plant strain (Gm cytoplasm ALT-2-01) of ALT-2-01 nucleotide sequence, conversion recombinant expression carrier DBN100825 are fixed Positioned at the soybean plant strain (Gm chloroplaset ALT-2-01) for being transferred to ALT-2-01 nucleotide sequence of chloroplaset；Conversion recombinant expression carries Body DBN100824 is the soybean plant strain (Gm cytoplasm ALT-3-01) for being transferred to ALT-3-01 nucleotide sequence for being positioned at cytoplasm, Converting recombinant expression carrier DBN100823 is the soybean plant strain for being transferred to ALT-3-01 nucleotide sequence for being positioned at chloroplaset (Gm chloroplaset ALT-3-01)；Simultaneously using Wild-type soy plant as control.
For the transformation of soybean of mediated by agriculture bacillus, briefly, by mature soya seeds in soybean germination culture medium (B5 salt 3.1g/L, B5 vitamin, sucrose 20g/L, agar 8g/L, pH5.6) in sprouted, seed is inoculated on germination medium, By the following conditions culture: 25 ± 1 DEG C of temperature；Photoperiod (light dark) is 16/8h.It is taken after sprouting 4-6 days swollen at bud green cotyledonary node Big soybean aseptic seedling, cuts hypocotyl under cotyledonary node at 3-4 millimeter, longitudinally slit cotyledon removes terminal bud, lateral bud and seed Root.Wound is carried out at cotyledonary node with the knife spine of scalpel, the cotyledonary node tissue crossed with agrobacterium suspension contact wound, wherein Agrobacterium can transmit the ALT-1-01 nucleotide sequence, ALT-2-01 nucleotide sequence, ALT-3-01 nucleotide sequence In this step, cotyledonary node tissue preferably immerses Agrobacterium suspension to the cotyledonary node tissue (step 1: infecting step) crossed to wound Liquid (OD660=0.5-0.8 infects culture medium (MS salt 2.15g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, acetyl fourth Ketone musk (AS) 40mg/L, 2-morpholine ethane sulfonic acid (MES) 4g/L, zeatin (ZT) 2mg/L, pH5.3) in start inoculation.Cotyledon It saves tissue and Agrobacterium co-cultures one period (3 days) (step 2: co-culturing step).Preferably, cotyledonary node group, which is woven in, infects step In solid medium (MS salt 4.3g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, 2-morpholine ethane sulfonic acid (MES) after rapid 4g/L, zeatin 2mg/L, agar 8g/L, pH5.6) on cultivate.After the stage of co-cultivation herein, there can be the " extensive of a selectivity It is multiple " step.In " recovery " step, recovery media (B5 salt 3.1g/L, B5 vitamin, 2-morpholine ethane sulfonic acid (MES) 1g/L, Sucrose 30g/L, zeatin (ZT) 2mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, pH5.6) at least in the presence of it is a kind of oneself know and inhibit the antibiotic (cephalosporin) of Agrobacterium growth, do not add plant and turn Change the selective agent (step 3: recovering step) of body.Preferably, the tissue block of cotyledon node regeneration is having antibiotic but no selective agent Solid medium on cultivate, to eliminate Agrobacterium and provide convalescence for infected cell.Then, the tissue block of cotyledon node regeneration It is cultivated on the culture medium containing selective agent (glyphosate) and selects the transformed calli (step 4: selection step) grown.It is excellent Selection of land, the tissue block of cotyledon node regeneration is in screening solid medium (B5 salt 3.1g/L, B5 vitamin, 2- morpholine for having selective agent Ethanesulfonic acid (MES) 1g/L, sucrose 30g/L, 6-benzyladenine (6-BAP) 1mg/L, agar 8g/L, cephalosporin 150mg/L, Glutamic acid 100mg/L, aspartic acid 100mg/L, N- (phosphine carboxymerhyl) glycine 0.25mol/L, pH5.6) on cultivate, cause turn The cell selective of change is grown.Then, the cytothesis of conversion is at plant (step 5: regeneration step), it is preferable that containing selection The tissue block of the cotyledon node regeneration grown on the culture medium of agent is at solid medium (B5 differential medium and B5 root media) Upper culture is with aftergrowth.
It screens obtained resistant tissues block and is transferred to the B5 differential medium (B5 salt 3.1g/L, B5 vitamin, 2- morpholine Ethanesulfonic acid (MES) 1g/L, sucrose 30g/L, zeatin (ZT) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L, N- (phosphine carboxymerhyl) glycine 0.25mol/L, PH5.6 on), differentiation is cultivated at 25 DEG C.It differentiates the seedling come and is transferred to the B5 root media (B5 salt 3.1g/L, B5 dimension His life, 2-morpholine ethane sulfonic acid (MES) 1g/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/L, indole -3-butyric acid (IBA) 1mg/L), in culture of rootage, culture moves to hot-house culture to solid to about 10cm high at 25 DEG C.In the greenhouse, often It is cultivated 16 hours at 26 DEG C, is cultivated 8 hours at 20 DEG C.
2, Transgenic soybean plants are verified with TaqMan
The soybean plant strain of Gm cytoplasm ALT-1-01, the soybean plant strain of Gm chloroplaset ALT-1-01, Gm cytoplasm ALT- are taken respectively The soybean plant strain of 2-01, the soybean plant strain of Gm chloroplaset ALT-2-01, Gm cytoplasm ALT-3-01 soybean plant strain and Gm chloroplaset The blade of the soybean plant strain of ALT-3-01 about 100mg extracts it as sample, with the DNeasy Plant Maxi Kit of Qiagen Genomic DNA detects EPSPS gene copy number by Taqman fluorescence probe quantitative PCR method to determine the copy of ALT gene Number.Simultaneously using Wild-type soy plant as control, tested and analyzed according to the method described above.Experiment sets 3 repetitions, is averaged Value.
Detecting EPSPS gene copy number, the specific method is as follows:
Step 11, the soybean plant strain for taking Gm cytoplasm ALT-1-01 respectively, the soybean plant strain of Gm chloroplaset ALT-1-01, Gm born of the same parents The soybean plant strain of matter ALT-2-01, the soybean plant strain of Gm chloroplaset ALT-2-01, Gm cytoplasm ALT-3-01 soybean plant strain, Gm leaf Each 100mg of blade of the soybean plant strain and Wild-type soy plant of green body ALT-3-01 is ground into mortar with liquid nitrogen even respectively Slurry, each sample take 3 repetitions；
Step 12, the genomic DNA that above-mentioned sample is extracted using the DNeasy Plant Mini Kit of Qiagen, specifically Method refers to its product description；
Step 13, the genomic DNA concentration that above-mentioned sample is measured with NanoDrop 2000 (Thermo Scientific)；
Step 14, the genomic DNA concentration of the above-mentioned sample of adjustment to same concentration value, the range of the concentration value is 80- 100ng/μL；
Step 15, the copy number that sample is identified using Taqman fluorescence probe quantitative PCR method, by being copied known to identification The sample of shellfish number is as standard items, and using the sample of Wild-type soy plant as control, 3 repetitions of each sample take it average Value；Fluorescence quantification PCR primer and probe sequence are respectively:
Following primer and probe is used to detect EPSPS gene order:
Primer 1:CTGGAAGGCGAGGACGTCATCAATA is as shown in SEQ ID NO:20 in sequence table；
Primer 2: TGGCGGCATTGCCGAAATCGAG is as shown in SEQ ID NO:21 in sequence table；
Probe 1:ATGCAGGCGATGGGCGCCCGCATCCGTA is as shown in SEQ ID NO:22 in sequence table；
PCR reaction system are as follows:
50 × the primer/probe mixture includes each 45 μ L of every kind of primer, 50 μ of probe of 100 μM of concentration of 1mM concentration L and 860 μ L 1 × TE buffers, and at 4 DEG C, it is housed in amber tube.
PCR reaction condition are as follows:
Data are analyzed using SDS2.3 software (Applied Biosystems).
By analyzing the experimental result of EPSPS gene copy number, and then confirm ALT-1-01 nucleotide sequence, ALT-2-01 Oneself is integrated into the genome of soybean plant strain detected for nucleotide sequence and ALT-3-01 nucleotide sequence, and Gm born of the same parents The soybean plant strain of matter ALT-1-01, the soybean plant strain of Gm chloroplaset ALT-1-01, Gm cytoplasm ALT-2-01 soybean plant strain, Gm leaf The soybean plant strain of the soybean plant strain of green body ALT-2-01, the soybean plant strain of Gm cytoplasm ALT-3-01 and Gm chloroplaset ALT-3-01 is equal The Transgenic soybean plants singly copied.
8th embodiment, the herbicide tolerant effect detection of Transgenic soybean plants
1, pyrazosulfuron tolerance
By the soybean plant strain of Gm cytoplasm ALT-1-01, the soybean plant strain of Gm chloroplaset ALT-1-01, Gm cytoplasm ALT-2-01 Soybean plant strain, the soybean plant strain of Gm chloroplaset ALT-2-01, the soybean plant strain of Gm cytoplasm ALT-3-01, Gm chloroplaset ALT-3- 01 soybean plant strain and Wild-type soy plant (Seedling Stage) carry out herbicide tolerant effect detection to pyrazosulfuron respectively.
The soybean plant strain of Gm cytoplasm ALT-1-01, the soybean plant strain of Gm chloroplaset ALT-1-01, Gm cytoplasm ALT- are taken respectively The soybean plant strain of 2-01, the soybean plant strain of Gm chloroplaset ALT-2-01, Gm cytoplasm ALT-3-01 soybean plant strain, Gm chloroplaset The soybean plant strain of ALT-3-01 and Wild-type soy plant, it is molten with pyrazosulfuron (25g ai/ha, 1 times of crop field concentration) and blank Agent (water) sprinkling.Respectively 3 days (3DAT) after spraying, 7 days (7DAT), 14 days (14DAT) and after 21 days (21DAT), according to leaf Piece amount of crimp and growing point degree of injury count every plant of plant by the degree of injury of herbicide: for example untreated with leveling blade Plant, growing point are intactly 0%；Vein part browning and young leaves deformity, plant strain growth more slowly be 50%；Vein is blue extremely It is 100% that whole strain death and growing point browning, which dry up,.The soybean plant strain of Gm cytoplasm ALT-1-01 totally 2 strains (S1 and S2), Gm The soybean plant strain of chloroplaset ALT-1-01 totally 2 strains (S3 and S4), the soybean plant strain of Gm cytoplasm ALT-2-01 totally 2 strains (S5 and S6), the soybean plant strain of Gm chloroplaset ALT-2-01 totally 2 strains (S7 and S8), the soybean plant strain of Gm cytoplasm ALT-3-01 Totally 2 strains (S9 and S10), plant by totally 2 strains (S11 and S12), Wild-type soy for the soybean plant strain of Gm chloroplaset ALT-3-01 Strain (CK1) totally 1 strain；10-15 plants are selected to be tested from each strain.The results are shown in Table 3.
Table 3, genetically engineered soybean T1Plant herbicide tolerant experimental result
For soybean, 25g ai/ha pyrazosulfuron herbicide is by sensitive plant and the plant with average resistance level The effective dose distinguished.Table 3 the result shows that: thifensulfuronmethyl hydrolase (ALT-1, ALT-2 and ALT-3) assign transgenosis Bean plant high level pyrazosulfuron herbicide tolerant；Soybean plant strain, Gm cytoplasm ALT- compared to Gm cytoplasm ALT-1-01 The soybean plant strain of 2-01 and the soybean plant strain of Gm cytoplasm ALT-3-01, soybean plant strain, the Gm chloroplaset of Gm chloroplaset ALT-1-01 It is resistance to that the soybean plant strain of ALT-2-01 and the soybean plant strain of Gm chloroplaset ALT-3-01 can generate higher pyrazosulfuron herbicide By property, showing that thifensulfuronmethyl hydrolase (ALT-1, the ALT-2 and ALT-3) assignment of genes gene mapping is expressed in chloroplaset be can be enhanced Tolerance of the bean plant to pyrazosulfuron herbicide；And Wild-type soy plant does not have then to the resistance to of pyrazosulfuron herbicide By property.
2, glyphosate tolerant
By the soybean plant strain of Gm cytoplasm ALT-1-01, the soybean plant strain of Gm chloroplaset ALT-1-01, Gm cytoplasm ALT-2-01 Soybean plant strain, the soybean plant strain of Gm chloroplaset ALT-2-01, the soybean plant strain of Gm cytoplasm ALT-3-01, Gm chloroplaset ALT-3- 01 soybean plant strain and Wild-type soy plant (Seedling Stage) carry out herbicide tolerant effect detection to glyphosate respectively.
The soybean plant strain of Gm cytoplasm ALT-1-01, the soybean plant strain of Gm chloroplaset ALT-1-01, Gm cytoplasm ALT- are taken respectively The soybean plant strain of 2-01, the soybean plant strain of Gm chloroplaset ALT-2-01, Gm cytoplasm ALT-3-01 soybean plant strain, Gm chloroplaset The soybean plant strain of ALT-3-01 and each 2 strains of Wild-type soy plant, select 10-15 plants to be tested from each strain.With grass Sweet phosphine (840g ae/ha, 1 times of crop field concentration) and blank solvent (water) sprinkling.After spraying 14 days (14DAT), according to phytotoxicity disease Shape is come the aggrieved rate of herbicide that counts every plant of plant: the aggrieved rate of herbicide (%)=∑ (aggrieved strain number × number of levels at the same level)/(total Strain number × highest level).Symptom of chemical damage classification is as shown in table 5.
Table 5, glyphosate herbicidal are to the grade scale of soybean phytotoxicity degree
Phytotoxicity rank Symptom description
1 Growth is normal, without any damage symptoms
2 Slight phytotoxicity, phytotoxicity are less than 10%
3 Medium phytotoxicity can restore later
4 Phytotoxicity is heavier, it is difficult to restore
5 Phytotoxicity is serious, cannot restore
The result shows that: the soybean plant strain of Gm cytoplasm ALT-1-01, the soybean plant strain of Gm chloroplaset ALT-1-01, Gm cytoplasm The soybean plant strain of ALT-2-01, the soybean plant strain of Gm chloroplaset ALT-2-01, Gm cytoplasm ALT-3-01 soybean plant strain and Gm leaf The aggrieved rate of glyphosate herbicidal of the soybean plant strain of green body ALT-3-01 is essentially 0%, and the grass of Wild-type soy plant (CK1) The sweet aggrieved rate of phosphine herbicide is up to 90% or more；The soybean plant strain of Gm cytoplasm ALT-1-01, Gm chloroplaset ALT-1-01 as a result, Soybean plant strain, the soybean plant strain of Gm cytoplasm ALT-2-01, the soybean plant strain of Gm chloroplaset ALT-2-01, Gm cytoplasm ALT-3-01 The soybean plant strain of soybean plant strain and Gm chloroplaset ALT-3-01 have good glyphosate herbicide tolerance.
The building of 9th embodiment, corn recombinant expression carrier
1, the corn recombinant cloning vector containing ALT nucleotide sequence is constructed
By the ALT-1-02 nucleotide sequence of synthesis be connected into cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600 on), operating procedure is carried out by Promega Products pGEM-T carrier specification, obtains recombinant cloning vector DBN05- T, (wherein, Amp indicates ampicillin resistance gene to building process as shown in Figure 7；F1 indicates that the duplication of bacteriophage f1 rises Point；LacZ is LacZ initiation codon；SP6 is SP6 RNA polymerase promoter；T7 is T7 RNA polymerase promoter；ALT- 1-02 is ALT-1-02 nucleotide sequence (SEQ ID NO:3)；MCS is multiple cloning sites).
Recombinant cloning vector DBN05-T heat shock method is converted into Escherichia coli T1 according in second embodiment 1 method Competent cell, and with its plasmid of alkalinity extraction, the plasmid of extraction carries out positive colony after SpeI and KasI digestion identification Sequence verification, the results showed that the ALT-1-02 nucleotides sequence being inserted into recombinant cloning vector DBN05-T is classified as in sequence table Nucleotide sequence shown in SEQ ID NO:3, i.e. ALT-1-02 nucleotide sequence are correctly inserted into.
According to the method for above-mentioned building recombinant cloning vector DBN05-T, by the ALT-2-02 nucleotide sequence of synthesis It is connected on cloning vector pGEM-T, obtains recombinant cloning vector DBN06-T, wherein ALT-2-02 is ALT-2-02 nucleotides sequence It arranges (SEQ ID NO:6).ALT-2-02 nucleotide sequence described in digestion and sequence verification recombinant cloning vector DBN06-T is correct Insertion.
According to the method for above-mentioned building recombinant cloning vector DBN05-T, by the ALT-3-02 nucleotide sequence of synthesis It is connected on cloning vector pGEM-T, obtains recombinant cloning vector DBN07-T, wherein ALT-3-02 is ALT-3-02 nucleotides sequence It arranges (SEQ ID NO:9).ALT-3-02 nucleotide sequence described in digestion and sequence verification recombinant cloning vector DBN07-T is correct Insertion.
2, the corn recombinant expression carrier containing ALT nucleotide sequence is constructed
Digestion recombinant cloning vector DBN05-T and expression vector DBNBC-03 is distinguished with restriction enzyme SpeI and KasI (carrier framework: pCAMBIA2301 (CAMBIA mechanism can provide)), the ALT-1-02 nucleotide sequence fragment cut is inserted into It is those skilled in the art using conventional enzymatic cleavage methods carrier construction between the site SpeI and KasI of expression vector DBNBC-03 Member known to, be built into recombinant expression carrier DBN100830 (being positioned at cytoplasm), building process as shown in Figure 8 (Spec: Spectinomycin gene；RB: right margin；PrUbi: corn Ubiquitin (ubiquitin) 1 gene promoter (SEQ ID NO:23)； ALT-1-02:ALT-1-02 nucleotide sequence (SEQ ID NO:3)；TNos: terminator (the SEQ ID of rouge alkali synthetase gene NO:13)；PMI: Phophomannose isomerase gene (SEQ ID NO:24)；LB: left margin).
Recombinant expression carrier DBN100830 heat shock method is converted into Escherichia coli according in second embodiment 2 method T1 competent cell, and alkalinity extraction its plasmid.It will be identified after restriction enzyme SpeI and the KasI digestion of the plasmid of extraction, And positive colony is subjected to sequencing identification, the results showed that core of the recombinant expression carrier DBN100830 between the site SpeI and KasI Nucleotide sequence is nucleotide sequence, i.e. ALT-1-02 nucleotide sequence shown in SEQ ID NO:3 in sequence table.
According to the method for above-mentioned building recombinant expression carrier DBN100830, building contains ALT-1-02 nucleotide sequence Recombinant expression carrier DBN100829 (is positioned at chloroplaset), carrier structure (carrier framework: pCAMBIA2301 as shown in Figure 9 (CAMBIA mechanism can provide)；Spec: spectinomycin gene；RB: right margin；PrUbi: corn Ubiquitin (ubiquitin) 1 base Because of promoter (SEQ ID NO:23)；SpAtCTP2: arabidopsis chloroplast transit peptides (SEQ ID NO:17)；ALT-1-02: ALT-1-02 nucleotide sequence (SEQ ID NO:3)；TNos: the terminator (SEQ ID NO:13) of rouge alkali synthetase gene； PMI: Phophomannose isomerase gene (SEQ ID NO:24)；LB: left margin).Sequence verification is carried out to positive colony, as a result Show that the ALT-1-02 nucleotides sequence being inserted into recombinant expression carrier DBN100829 is classified as in sequence table shown in SEQ ID NO:3 Nucleotide sequence, i.e. ALT-1-02 nucleotide sequence is correctly inserted into.
According to the method for above-mentioned building recombinant expression carrier DBN100830, by SpeI and KasI digestion recombinant cloning vector The ALT-2-02 nucleotide sequence that DBN06-T is cut is inserted into expression vector DBNBC-03, obtains recombinant expression carrier DBN100832.Nucleotide sequence in digestion and sequence verification recombinant expression carrier DBN100832 contains for SEQ in sequence table Nucleotide sequence shown in ID NO:6, i.e. ALT-2-02 nucleotide sequence are correctly inserted into.
According to the method for above-mentioned building recombinant expression carrier DBN100829, by SpeI and KasI digestion recombinant cloning vector The ALT-2-02 nucleotide sequence that DBN06-T is cut is inserted into expression vector DBNBC-03, obtains recombinant expression carrier DBN100831 (containing spAtCTP2, is positioned at chloroplaset).In digestion and sequence verification recombinant expression carrier DBN100831 Nucleotide sequence contains correctly to be inserted for nucleotide sequence shown in SEQ ID NO:6, i.e. ALT-2-02 nucleotide sequence in sequence table Enter.
According to the method for above-mentioned building recombinant expression carrier DBN100830, by SpeI and KasI digestion recombinant cloning vector The ALT-3-02 nucleotide sequence that DBN07-T is cut is inserted into expression vector DBNBC-03, obtains recombinant expression carrier DBN100834.Nucleotide sequence in digestion and sequence verification recombinant expression carrier DBN100834 contains for SEQ in sequence table Nucleotide sequence shown in ID NO:9, i.e. ALT-3-02 nucleotide sequence are correctly inserted into.
According to the method for above-mentioned building recombinant expression carrier DBN100829, by SpeI and KasI digestion recombinant cloning vector The ALT-3-02 nucleotide sequence that DBN07-T is cut is inserted into expression vector DBNBC-03, obtains recombinant expression carrier DBN100833 (containing spAtCTP2, is positioned at chloroplaset).In digestion and sequence verification recombinant expression carrier DBN100833 Nucleotide sequence contains correctly to be inserted for nucleotide sequence shown in SEQ ID NO:9, i.e. ALT-3-02 nucleotide sequence in sequence table Enter.
3, corn recombinant expression carrier converts Agrobacterium
To oneself constructed correct recombinant expression carrier DBN100830, DBN100829, DBN100832, DBN100831, DBN100834 and DBN100833 with liquid nitrogen method be transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: In 18313-015), conversion condition are as follows: 100 μ L Agrobacterium LBA4404s, 3 μ L Plasmid DNA (recombinant expression carrier)；It is placed in liquid 10 minutes in nitrogen, 37 DEG C tepidarium 10 minutes；By the Agrobacterium LBA4404 after conversion be inoculated in LB test tube in 28 DEG C of temperature, Revolving speed is cultivated 2 hours under the conditions of being 200rpm, is applied to the grand mould of rifampin (Rifampicin) containing 50mg/L and 50mg/L Until growing positive monoclonal on the LB plate of element, picking Colony Culture simultaneously extracts its plasmid, is carried out with restriction enzyme Digestion verification, the results showed that recombinant expression carrier DBN100830, DBN100829, DBN100832, DBN100831, DBN100834 and DBN100833 structure is completely correct.
The acquisition and verifying of tenth embodiment, transgenic corn plant
According to the Agrobacterium infestation method routinely used, by the rataria and the 9th of the corn variety of sterile culture comprehensive 31 (Z31) The co-cultivation of Agrobacterium described in 3 in embodiment, the recombinant expression carrier DBN100830 that in the 9th embodiment 2 are constructed, T-DNA (including corn in DBN100829, DBN100832, DBN100831, DBN100834 and DBN100833 Promoter sequence, ALT-1-02 nucleotide sequence, ALT-2-02 nucleotide sequence, the ALT-3-02 nucleosides of Ubiquitin1 gene Acid sequence, arabidopsis chloroplast transit peptides, PMI gene and tNos terminator sequence) it is transferred in maize chromosome group, it obtains Converting recombinant expression carrier DBN100830 is the plant (Zm for being transferred to ALT-1-02 nucleotide sequence for being positioned at cytoplasm Cytoplasm ALT-1-02), conversion recombinant expression carrier DBN100829 be positioned at chloroplaset be transferred to ALT-1-02 nucleotides sequence The plant (Zm chloroplaset ALT-1-02) of column；Converting recombinant expression carrier DBN100832 is to be positioned at being transferred to for cytoplasm The plant (Zm cytoplasm ALT-2-02) of ALT-2-02 nucleotide sequence, conversion recombinant expression carrier DBN100831 are fixed Positioned at the plant (Zm chloroplaset ALT-2-02) for being transferred to ALT-2-02 nucleotide sequence of chloroplaset；Conversion recombinant expression carries Body DBN100834 is the plant (Zm cytoplasm ALT-3-02) for being transferred to ALT-3-02 nucleotide sequence for being positioned at cytoplasm, Converting recombinant expression carrier DBN100833 is the plant for being transferred to ALT-3-02 nucleotide sequence for being positioned at chloroplaset (Zm chloroplaset ALT-3-02)；Simultaneously using wild-type corn plant as control.
For the corn transformation of mediated by agriculture bacillus, briefly, immature rataria is separated from corn, is suspended with Agrobacterium Liquid contacts rataria, and wherein Agrobacterium can be by ALT-1-02 nucleotide sequence, ALT-2-02 nucleotide sequence, ALT-3-02 nucleosides Acid sequence is transferred at least one cell (step 1: infecting step) of one of rataria.In this step, rataria preferably immerses Agrobacterium suspension (OD660=0.4-0.6 infects culture medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, glucose 36g/L, acetosyringone (AS) 40mg/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, pH5.3)) In with start inoculation.Rataria and Agrobacterium co-culture one period (3 days) (step 2: co-culturing step).Preferably, rataria exists It infects after step in solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose 10g/ L, acetosyringone (AS) 100mg/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, agar 8g/L, pH5.8) on cultivate.? After this co-cultivation stage, there can be " recovery " step of a selectivity.In " recovery " step, recovery media (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 2,4 dichlorophenoxyacetic acid (2,4-D) 1mg/L, plant gel 3g/L, pH5.8) at least in the presence of it is a kind of oneself know and inhibit the antibiotic (cephalosporin) of Agrobacterium growth, do not add Plant Transformation The selective agent (step 3: recovering step) of body.Preferably, rataria is trained on having antibiotic but the not solid medium of selective agent It supports, to eliminate Agrobacterium and provide convalescence for infected cell.Then, the rataria of inoculation is in the culture for containing selective agent (mannose) It is cultivated on base and selects the transformed calli (step 4: selection step) grown.Preferably, rataria is in the sieve for having selective agent Select solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, mannose 12.5g/L, 2,4- bis- Chlorophenoxyacetic acid (2,4-D) 1mg/L, plant gel 3g/L, pH5.8) on cultivate, cause conversion cell selective growth.So Afterwards, callus regeneration is at plant (step 5: regeneration step), it is preferable that the callus group grown on the culture medium containing selective agent It is woven on solid medium (MS differential medium and MS root media) and cultivates with aftergrowth.
It screens obtained resistant calli and is transferred to the MS differential medium (MS salt 4.3g/L, MS vitamin, cheese Plain 300mg/L, sucrose 30g/L, 6-benzyladenine 2mg/L, mannose 5g/L, plant gel 3g/L, pH5.8) on, at 25 DEG C Culture differentiation.It differentiates the seedling come and is transferred to the MS root media (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) on, it cultivates at 25 DEG C to about 10cm Height moves to hot-house culture to solid.In the greenhouse, it cultivates 16 hours at 28 DEG C, is cultivated 8 hours at 20 DEG C daily.
2, transgenic corn plant is verified with TaqMan
The method for verifying Transgenic soybean plants with TaqMan according in the 7th embodiment 2, to Zm cytoplasm ALT-1-02's Plant, the plant of Zm chloroplaset ALT-1-02, the plant of Zm cytoplasm ALT-2-02, Zm chloroplaset ALT-2-02 Plant, the plant of Zm cytoplasm ALT-3-02 and the plant of Zm chloroplaset ALT-3-02 tested and analyzed. PMI gene copy number is detected by Taqman fluorescence probe quantitative PCR method to determine the copy number of ALT gene.Simultaneously with open country Raw type plant is tested and analyzed according to the method described above as control.Experiment sets 3 repetitions, is averaged.
Following primer and probe is used to detect PMI gene order:
Primer 3:GCTGTAAGAGCTTACTGAAAAAATTAACA is as shown in SEQ ID NO:25 in sequence table；
Primer 4:CGATCTGCAGGTCGACGG is as shown in SEQ ID NO:26 in sequence table；
Probe 2:TCTCTTGCTAAGCTGGGAGCTCGATCC is as shown in SEQ ID NO:27 in sequence table.
By analyzing the experimental result of PMI gene copy number, and then confirm ALT-1-02 nucleotide sequence, ALT-2-02 core Oneself is integrated into the genome of plant detected for nucleotide sequence and ALT-3-02 nucleotide sequence, and Zm cytoplasm The plant of ALT-1-02, the plant of Zm chloroplaset ALT-1-02, the plant of Zm cytoplasm ALT-2-02, Zm leaf are green The plant of the plant of body ALT-2-02, the plant of Zm cytoplasm ALT-3-02 and Zm chloroplaset ALT-3-02 is obtained Obtained the transgenic corn plant of single copy.
11st embodiment, the herbicide tolerant effect detection of transgenic corn plant
By the plant of Zm cytoplasm ALT-1-02, the plant of Zm chloroplaset ALT-1-02, Zm cytoplasm ALT-2-02 Plant, the plant of Zm chloroplaset ALT-2-02, the plant of Zm cytoplasm ALT-3-02, Zm chloroplaset ALT-3- 02 plant and wild-type corn plant (V3-V4 period) carry out the inspection of herbicide tolerant effect to pyrazosulfuron respectively It surveys.
The plant of Zm cytoplasm ALT-1-02, the plant of Zm chloroplaset ALT-1-02, Zm cytoplasm ALT- are taken respectively The plant of 2-02, the plant of Zm chloroplaset ALT-2-02, the plant of Zm cytoplasm ALT-3-02, Zm chloroplaset The plant of ALT-3-02 and wild-type corn plant, it is molten with pyrazosulfuron (25g ai/ha, 1 times of crop field concentration) and blank Agent (water) sprinkling.Respectively 3 days (3DAT) after spraying, 7 days (7DAT), 14 days (14DAT) and after 21 days (21DAT), according to plant The upgrowth situation of strain counts every plant of plant by the degree of injury of herbicide: with comparable with untreated plant growth condition being 0%；It is 50% that blade part chlorisis, which turns to be yellow but has substantially no effect on normal plants,；Blue be at death's door of whole strain be 100%.The plant of Zm cytoplasm ALT-1-02 totally 2 strains (S13 and S14), the plant of Zm chloroplaset ALT-1-02 Totally 2 strains (S15 and S16), the plant of Zm cytoplasm ALT-2-02 totally 2 strains (S17 and S18), Zm chloroplaset ALT- The plant of 2-02 totally 2 strains (S19 and S20), the plant of Zm cytoplasm ALT-3-02 totally 2 strains (S21 and S22), the plant of Zm chloroplaset ALT-3-02 totally 2 strains (S23 and S24), wild-type corn plant (CK2) totally 1 strain System；10-15 plants are selected to be tested from each strain.The results are shown in Table 4.
Table 4, transgenic corns T1Plant herbicide tolerant experimental result
For corn, 25g ai/ha pyrazosulfuron herbicide is by sensitive plant and the plant with average resistance level The effective dose distinguished.Table 4 the result shows that: thifensulfuronmethyl hydrolase (ALT-1, ALT-2 and ALT-3) assign transgenosis Corn plant high level pyrazosulfuron herbicide tolerant；Plant, Zm cytoplasm ALT- compared to Zm cytoplasm ALT-1-02 The plant of 2-02 and the plant of Zm cytoplasm ALT-3-02, the plant of Zm chloroplaset ALT-1-02, Zm chloroplaset It is resistance to that the plant of ALT-2-02 and the plant of Zm chloroplaset ALT-3-02 can generate higher pyrazosulfuron herbicide By property, showing that thifensulfuronmethyl hydrolase (ALT-1, the ALT-2 and ALT-3) assignment of genes gene mapping is expressed in chloroplaset be can be enhanced Tolerance of the corn plant to pyrazosulfuron herbicide；And wild-type corn plant does not have then to the resistance to of pyrazosulfuron herbicide By property.
It can be phonetic to pyrrole in conclusion present invention firstly discloses thifensulfuronmethyl hydrolases (ALT-1, ALT-2 and ALT-3) The grand herbicide of sulphur shows higher tolerance, and the Arabidopsis plant containing coding thifensulfuronmethyl hydrolase nucleotide sequence, Soybean plant strain and plant are strong to the tolerance of pyrazosulfuron herbicide, can at least be resistant to 1 times of crop field concentration, therefore planting It has a extensive future on object.
It should be noted last that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although ginseng It is described the invention in detail according to preferred embodiment, those skilled in the art should understand that, it can be to the present invention Technical solution be modified or replaced equivalently, without departing from the spirit and scope of the technical solution of the present invention.

Claims (97)

1. a kind of method for controlling weeds, which is characterized in that including the herbicide containing effective dose pyrazosulfuron to be applied to There are in the plant growth environment of at least one genetically modified plants, the genetically modified plants include coding thiophene in its genome The nucleotide sequence of the grand hydrolase of sulphur, the genetically modified plants and other nucleotides sequences without coding thifensulfuronmethyl hydrolase The plant of column is compared with the plant injury weakened and/or has increased plant products；The wherein thifensulfuronmethyl hydrolase For amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7.
2. according to claim 1 control weeds method, which is characterized in that the genetically modified plants be monocotyledon or Dicotyledon.
3. controlling the method for weeds according to claim 1, which is characterized in that the genetically modified plants are corn and soybean, intend Southern mustard, cotton, rape, rice, sorghum, wheat, barley, grain, sugarcane or oat.
4. the method for any one of -3 control weeds according to claim 1, which is characterized in that the effective dose pyrazosulfuron For 9-50 g ai/ha.
5. the method for any one of -3 control weeds according to claim 1, which is characterized in that the thifensulfuronmethyl hydrolase Nucleotide sequence are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
6. controlling the method for weeds according to claim 4, which is characterized in that the nucleotides sequence of the thifensulfuronmethyl hydrolase It is classified as:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
7. controlling the method for weeds according to claim 5, which is characterized in that the genetically modified plants further include at least one Different from encoding second of nucleotide of the nucleotide sequence of the thifensulfuronmethyl hydrolase.
8. controlling the method for weeds according to claim 7, which is characterized in that second of nucleotide coding selected marker Protein, synthesizing activity protein, degrading activity protein, biotic protein, resisting abiotic stress protein, male Sterile protein, the protein for influencing plant products and/or the protein for influencing plant quality.
9. controlling the method for weeds according to claim 8, which is characterized in that second of nucleotide coding 5- enol third Keto acyl shikimic acid -3- phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyl transferase, glyphosate decarboxylase, careless ammonium Phosphinothricin acetyl transferase, alpha Ketoglutarate dependence dioxygenase, dicamba monooxygenase enzyme, 4- hydroxyphenyl pyravate dioxygenase, second Acyl lactic acid synthase, cytochromes proteinoid and/or proporphyrinogen oxidase.
10. according to the method for any one of the claim 7-9 control weeds, which is characterized in that described to contain effective dose pyrrole The herbicide of Sulfometuron Methyl further includes glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins herbicide, grass family weeding Before agent, germination selective herbicide and/or germination after selective herbicide.
11. controlling the method for weeds according to claim 4, which is characterized in that the genetically modified plants further include at least one Kind is different from encoding second of nucleotide of the nucleotide sequence of the thifensulfuronmethyl hydrolase.
12. controlling the method for weeds according to claim 11, which is characterized in that second of nucleotide coding selection mark Remember protein, synthesizing activity protein, degrading activity protein, biotic protein, resisting abiotic stress protein, hero Property infertility protein, influence the protein of plant products and/or influence the protein of plant quality.
13. controlling the method for weeds according to claim 12, which is characterized in that second of nucleotide coding 5- enol Pyruvoyl shikimic acid -3- phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyl transferase, glyphosate decarboxylase, grass Ammonium phosphinothricin acetyl transferase, alpha Ketoglutarate dependence dioxygenase, dicamba monooxygenase enzyme, 4- hydroxyphenyl pyravate dioxygenase, Acetolactate synthase, cytochromes proteinoid and/or proporphyrinogen oxidase.
14. the method for any one of 1-13 control weeds according to claim 1, which is characterized in that described to contain effective dose The herbicide of pyrazosulfuron further includes glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins herbicide, grass family weeding Before agent, germination selective herbicide and/or germination after selective herbicide.
15. the method for any one of -3 or 6 control weeds according to claim 1, which is characterized in that the genetically modified plants are also Second of the nucleotide including at least one nucleotide sequence for being different from encoding the thifensulfuronmethyl hydrolase.
16. controlling the method for weeds according to claim 15, which is characterized in that second of nucleotide coding selection mark Remember protein, synthesizing activity protein, degrading activity protein, biotic protein, resisting abiotic stress protein, hero Property infertility protein, influence the protein of plant products and/or influence the protein of plant quality.
17. the method for the 6 control weeds according to claim 1, which is characterized in that second of nucleotide coding 5- enol Pyruvoyl shikimic acid -3- phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyl transferase, glyphosate decarboxylase, grass Ammonium phosphinothricin acetyl transferase, alpha Ketoglutarate dependence dioxygenase, dicamba monooxygenase enzyme, 4- hydroxyphenyl pyravate dioxygenase, Acetolactate synthase, cytochromes proteinoid and/or proporphyrinogen oxidase.
18. the method for the 6 or 17 control weeds according to claim 1, which is characterized in that described to contain the phonetic sulphur of effective dose pyrrole Grand herbicide further includes glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins herbicide, gramineous herbicide, hair Before bud selective herbicide and/or germination after selective herbicide.
19. controlling the method for weeds according to claim 15, which is characterized in that described containing effective dose pyrazosulfuron Before herbicide further includes glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins herbicide, gramineous herbicide, germination Selective herbicide after selective herbicide and/or germination.
20. controlling the method for weeds according to claim 5, which is characterized in that described containing effective dose pyrazosulfuron Before herbicide further includes glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins herbicide, gramineous herbicide, germination Selective herbicide after selective herbicide and/or germination.
21. controlling the method for weeds according to claim 4, which is characterized in that described containing effective dose pyrazosulfuron Before herbicide further includes glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins herbicide, gramineous herbicide, germination Selective herbicide after selective herbicide and/or germination.
22. the method for any one of -3 or 6 control weeds according to claim 1, which is characterized in that described to contain effective dose The herbicide of pyrazosulfuron further includes glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins herbicide, grass family weeding Before agent, germination selective herbicide and/or germination after selective herbicide.
23. a kind of method for controlling glyphosate tolerant weeds, which is characterized in that including by the pyrazosulfuron weeding of effective dose Agent and glyphosate herbicidal are applied to the big Tanaka for planting at least one genetically modified plants, and the big Tanaka includes glyphosate tolerance Property weeds or its seed, the genetically modified plants include in its genome encode thifensulfuronmethyl hydrolase nucleotide sequence and The nucleotide sequence of glyphosate tolerant protein is encoded, the genetically modified plants do not have coding thifensulfuronmethyl hydrolysis with other The plant of the nucleotide sequence of the nucleotide sequence and/or coding glyphosate tolerant protein of enzyme is compared with the plant weakened Damage and/or have increased plant products；Wherein the thifensulfuronmethyl hydrolase be SEQ ID NO:1, SEQ ID NO:4 or Amino acid sequence shown in SEQ ID NO:7.
24. controlling the method for glyphosate tolerant weeds according to claim 23, which is characterized in that the genetically modified plants For monocotyledon or dicotyledon.
25. controlling the method for glyphosate tolerant weeds according to claim 23, which is characterized in that the genetically modified plants For corn and soybean, arabidopsis, cotton, rape, rice, sorghum, wheat, barley, grain, sugarcane or oat.
26. according to the method for any one of the claim 23-25 control glyphosate tolerant weeds, which is characterized in that described Effective dose pyrazosulfuron is 9-50 g ai/ha.
27. controlling the method for glyphosate tolerant weeds according to claim 26, which is characterized in that the effective dose grass Sweet phosphine is 200-1600 g ae/ha.
28. according to the method for any one of the claim 23-25 control glyphosate tolerant weeds, which is characterized in that described Effective dose glyphosate is 200-1600 g ae/ha.
29. according to the method for any one of the claim 23-25 or 27 control glyphosate tolerant weeds, which is characterized in that The nucleotide sequence of the thifensulfuronmethyl hydrolase are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
30. controlling the method for glyphosate tolerant weeds according to claim 26, which is characterized in that the thifensulfuronmethyl water Solve the nucleotide sequence of enzyme are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
31. controlling the method for glyphosate tolerant weeds according to claim 28, which is characterized in that the thifensulfuronmethyl water Solve the nucleotide sequence of enzyme are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
32. according to the method for any one of claim 23-25,27,30 or 31 the control glyphosate tolerant weeds, feature It is, the glyphosate tolerant protein includes 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate reduction Enzyme, glyphosate-N-acetyl transferase or glyphosate decarboxylase.
33. controlling the method for glyphosate tolerant weeds according to claim 32, which is characterized in that the glyphosate tolerance Property protein be SEQ ID NO:10 shown in amino acid sequence.
34. controlling the method for glyphosate tolerant weeds according to claim 33, which is characterized in that the glyphosate tolerance The nucleotides sequence of property protein is classified as nucleotide sequence shown in SEQ ID NO:11.
35. controlling the method for glyphosate tolerant weeds according to claim 26, which is characterized in that the glyphosate tolerance Property protein include 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyl transfer Enzyme or glyphosate decarboxylase.
36. controlling the method for glyphosate tolerant weeds according to claim 35, which is characterized in that the glyphosate tolerance Property protein be SEQ ID NO:10 shown in amino acid sequence.
37. controlling the method for glyphosate tolerant weeds according to claim 36, which is characterized in that the glyphosate tolerance The nucleotides sequence of property protein is classified as nucleotide sequence shown in SEQ ID NO:11.
38. controlling the method for glyphosate tolerant weeds according to claim 28, which is characterized in that the glyphosate tolerance Property protein include 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyl transfer Enzyme or glyphosate decarboxylase.
39. controlling the method for glyphosate tolerant weeds according to claim 38, which is characterized in that the glyphosate tolerance Property protein be SEQ ID NO:10 shown in amino acid sequence.
40. controlling the method for glyphosate tolerant weeds according to claim 39, which is characterized in that the glyphosate tolerance The nucleotides sequence of property protein is classified as nucleotide sequence shown in SEQ ID NO:11.
41. controlling the method for glyphosate tolerant weeds according to claim 29, which is characterized in that the glyphosate tolerance Property protein include 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyl transfer Enzyme or glyphosate decarboxylase.
42. controlling the method for glyphosate tolerant weeds according to claim 41, which is characterized in that the glyphosate tolerance Property protein be SEQ ID NO:10 shown in amino acid sequence.
43. controlling the method for glyphosate tolerant weeds according to claim 42, which is characterized in that the glyphosate tolerance The nucleotides sequence of property protein is classified as nucleotide sequence shown in SEQ ID NO:11.
44. a kind of implant system for controlling weed growth, which is characterized in that including pyrazosulfuron herbicide and exist at least one The pyrazosulfuron herbicide containing effective dose is applied to described in the presence of at least by the plant growth environment of genetically modified plants In a kind of plant growth environment of genetically modified plants, the genetically modified plants include coding thifensulfuronmethyl hydrolysis in its genome The nucleotide sequence of enzyme, the plant of the genetically modified plants and other nucleotide sequences without coding thifensulfuronmethyl hydrolase Compared to the plant injury weakened and/or with increased plant products；Wherein the thifensulfuronmethyl hydrolase is SEQ ID Amino acid sequence shown in NO:1, SEQ ID NO:4 or SEQ ID NO:7.
45. controlling the implant system of weed growth according to claim 44, which is characterized in that the genetically modified plants are single Cotyledon plant or dicotyledon.
46. controlling the implant system of weed growth according to claim 44, which is characterized in that the genetically modified plants are jade Rice, soybean, arabidopsis, cotton, rape, rice, sorghum, wheat, barley, grain, sugarcane or oat.
47. according to the implant system of any one of the claim 44-46 control weed growth, which is characterized in that described effective Dosage pyrazosulfuron is 9-50 g ai/ha.
48. according to the implant system of any one of the claim 44-46 control weed growth, which is characterized in that the thiophene The nucleotide sequence of the grand hydrolase of sulphur are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
49. controlling the implant system of weed growth according to claim 47, which is characterized in that the thifensulfuronmethyl hydrolase Nucleotide sequence are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
50. controlling the implant system of weed growth according to claim 48, which is characterized in that the genetically modified plants are also wrapped Include second of nucleotide of at least one nucleotide sequence for being different from encoding the thifensulfuronmethyl hydrolase.
51. controlling the implant system of weed growth according to claim 50, which is characterized in that second of the nucleotide is compiled Code selected marker protein, synthesizing activity protein, degrading activity protein, biotic protein, resisting abiotic stress Protein, male sterility protein, the protein for influencing plant products and/or the protein for influencing plant quality.
52. controlling the implant system of weed growth according to claim 51, which is characterized in that second of the nucleotide is compiled Code 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyl transferase, glyphosate are de- Carboxylic acid, glufosinate-ammonium transacetylase, alpha Ketoglutarate dependence dioxygenase, 4- hydroxyphenyl pyravate dioxygenase, acetolactic acid Synthase, cytochromes proteinoid and/or proporphyrinogen oxidase.
53. according to the implant system of any one of the claim 50-52 control weed growth, which is characterized in that described to contain The herbicide of weeding effective dose pyrazosulfuron further includes glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins weeding Before agent, gramineous herbicide, germination selective herbicide and/or germination after selective herbicide.
54. controlling the implant system of weed growth according to claim 47, which is characterized in that the genetically modified plants are also wrapped Include second of nucleotide of at least one nucleotide sequence for being different from encoding the thifensulfuronmethyl hydrolase.
55. controlling the implant system of weed growth according to claim 54, which is characterized in that second of the nucleotide is compiled Code selected marker protein, synthesizing activity protein, degrading activity protein, biotic protein, resisting abiotic stress Protein, male sterility protein, the protein for influencing plant products and/or the protein for influencing plant quality.
56. controlling the implant system of weed growth according to claim 55, which is characterized in that second of the nucleotide is compiled Code 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyl transferase, glyphosate are de- Carboxylic acid, glufosinate-ammonium transacetylase, alpha Ketoglutarate dependence dioxygenase, 4- hydroxyphenyl pyravate dioxygenase, acetolactic acid Synthase, cytochromes proteinoid and/or proporphyrinogen oxidase.
57. according to the implant system of any one of the claim 54-56 control weed growth, which is characterized in that described to contain The herbicide of weeding effective dose pyrazosulfuron further includes glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins weeding Before agent, gramineous herbicide, germination selective herbicide and/or germination after selective herbicide.
58. according to the implant system of any one of the claim 44-46 or 49 control weed growth, which is characterized in that described Genetically modified plants further include second of nucleosides of at least one nucleotide sequence for being different from encoding the thifensulfuronmethyl hydrolase Acid.
59. controlling the implant system of weed growth according to claim 58, which is characterized in that second of the nucleotide is compiled Code selected marker protein, synthesizing activity protein, degrading activity protein, biotic protein, resisting abiotic stress Protein, male sterility protein, the protein for influencing plant products and/or the protein for influencing plant quality.
60. controlling the implant system of weed growth according to claim 59, which is characterized in that second of the nucleotide is compiled Code 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyl transferase, glyphosate are de- Carboxylic acid, glufosinate-ammonium transacetylase, alpha Ketoglutarate dependence dioxygenase, 4- hydroxyphenyl pyravate dioxygenase, acetolactic acid Synthase, cytochromes proteinoid and/or proporphyrinogen oxidase.
61. according to the implant system of the control weed growth of claim 59 or 60, which is characterized in that described to have containing weeding The herbicide for imitating dosage pyrazosulfuron further includes glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins herbicide, grass Before section's herbicide, germination selective herbicide and/or germination after selective herbicide.
62. controlling the implant system of weed growth according to claim 58, which is characterized in that described to contain weeding effective agent The herbicide of amount pyrazosulfuron further includes that glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins herbicide, grass family remove Before careless agent, germination selective herbicide and/or germination after selective herbicide.
63. controlling the implant system of weed growth according to claim 48, which is characterized in that described to contain weeding effective agent The herbicide of amount pyrazosulfuron further includes that glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins herbicide, grass family remove Before careless agent, germination selective herbicide and/or germination after selective herbicide.
64. controlling the implant system of weed growth according to claim 47, which is characterized in that described to contain weeding effective agent The herbicide of amount pyrazosulfuron further includes that glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins herbicide, grass family remove Before careless agent, germination selective herbicide and/or germination after selective herbicide.
65. according to the implant system of any one of the claim 44-46 or 49 control weed growth, which is characterized in that described Herbicide containing weeding effective dose pyrazosulfuron further includes glyphosate herbicidal, glufosinate-ammonium herbicide, plant auxins Before herbicide, gramineous herbicide, germination selective herbicide and/or germination after selective herbicide.
66. a kind of implant system for controlling glyphosate tolerant weeds, which is characterized in that sweet including pyrazosulfuron herbicide, grass Phosphine herbicide and the crop field for planting at least one genetically modified plants, by the pyrazosulfuron herbicide of effective dose and the grass Sweet phosphine herbicide is applied to the big Tanaka of at least one genetically modified plants of plantation, and the big Tanaka includes glyphosate tolerant Weeds or its seed, the genetically modified plants include the nucleotide sequence and volume for encoding thifensulfuronmethyl hydrolase in its genome The nucleotide sequence of code glyphosate tolerant protein, the genetically modified plants do not have coding thifensulfuronmethyl hydrolase with other Nucleotide sequence and/or encode glyphosate tolerant protein nucleotide sequence plant compare with weaken plant damage Hurt and/or have increased plant products；Wherein the thifensulfuronmethyl hydrolase be SEQ ID NO:1, SEQ ID NO:4 or Amino acid sequence shown in SEQ ID NO:7.
67. controlling the implant system of glyphosate tolerant weeds according to claim 66, which is characterized in that the transgenosis Plant is monocotyledon or dicotyledon.
68. controlling the implant system of glyphosate tolerant weeds according to claim 66, which is characterized in that the transgenosis Plant is corn and soybean, arabidopsis, cotton, rape, rice, sorghum, wheat, barley, grain, sugarcane or oat.
69. according to the implant system of any one of the claim 66-68 control glyphosate tolerant weeds, which is characterized in that The effective dose pyrazosulfuron is 9-50 g ai/ha.
70. controlling the implant system of glyphosate tolerant weeds according to claim 69, which is characterized in that the effective agent Amount glyphosate is 200-1600 g ae/ha.
71. according to the implant system of any one of the claim 66-68 control glyphosate tolerant weeds, which is characterized in that The effective dose glyphosate is 200-1600 g ae/ha.
72. according to the implant system of any one of the claim 66-68 or 70 control glyphosate tolerant weeds, feature exists In the nucleotide sequence of the thifensulfuronmethyl hydrolase are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
73. controlling the implant system of glyphosate tolerant weeds according to claim 69, which is characterized in that the thiophene sulphur The nucleotide sequence of grand hydrolase are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
74. controlling the implant system of glyphosate tolerant weeds according to claim 71, which is characterized in that the thiophene sulphur The nucleotide sequence of grand hydrolase are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
75. according to the implant system of any one of claim 66-68,70,73 or 74 the control glyphosate tolerant weeds, Be characterized in that, the glyphosate tolerant protein include 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate also Protoenzyme, glyphosate-N-acetyl transferase or glyphosate decarboxylase.
76. controlling the implant system of glyphosate tolerant weeds according to claim 75, which is characterized in that the glyphosate Tolerance proteins matter is amino acid sequence shown in SEQ ID NO:10.
77. controlling the implant system of glyphosate tolerant weeds according to claim 76, which is characterized in that the glyphosate The nucleotides sequence of tolerance proteins matter is classified as nucleotide sequence shown in SEQ ID NO:11.
78. controlling the implant system of glyphosate tolerant weeds according to claim 69, which is characterized in that the glyphosate Tolerance proteins matter includes 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyl Transferase or glyphosate decarboxylase.
79. controlling the implant system of glyphosate tolerant weeds according to claim 78, which is characterized in that the glyphosate Tolerance proteins matter is amino acid sequence shown in SEQ ID NO:10.
80. controlling the implant system of glyphosate tolerant weeds according to claim 79, which is characterized in that the glyphosate The nucleotides sequence of tolerance proteins matter is classified as nucleotide sequence shown in SEQ ID NO:11.
81. controlling the implant system of glyphosate tolerant weeds according to claim 71, which is characterized in that the glyphosate Tolerance proteins matter includes 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyl Transferase or glyphosate decarboxylase.
82. controlling the implant system of glyphosate tolerant weeds according to claim 81, which is characterized in that the glyphosate Tolerance proteins matter is amino acid sequence shown in SEQ ID NO:10.
83. controlling the implant system of glyphosate tolerant weeds according to claim 82, which is characterized in that the glyphosate The nucleotides sequence of tolerance proteins matter is classified as nucleotide sequence shown in SEQ ID NO:11.
84. controlling the implant system of glyphosate tolerant weeds according to claim 72, which is characterized in that the glyphosate Tolerance proteins matter includes 5- enol pyruvylshikimate -3- phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyl Transferase or glyphosate decarboxylase.
85. controlling the implant system of glyphosate tolerant weeds according to claim 84, which is characterized in that the glyphosate Tolerance proteins matter is amino acid sequence shown in SEQ ID NO:10.
86. controlling the implant system of glyphosate tolerant weeds according to claim 85, which is characterized in that the glyphosate The nucleotides sequence of tolerance proteins matter is classified as nucleotide sequence shown in SEQ ID NO:11.
87. a kind of method for the plant for generating tolerance pyrazosulfuron herbicide, which is characterized in that including in the genome to plant The nucleotide sequence for introducing coding thifensulfuronmethyl hydrolase, is at least deposited when the herbicide containing effective dose pyrazosulfuron is applied to In the big Tanaka of the plant, the plant does not have the plant phase of the nucleotide sequence of coding thifensulfuronmethyl hydrolase with other Than with the plant injury weakened and/or with increased plant products；Wherein the thifensulfuronmethyl hydrolase is SEQ ID Amino acid sequence shown in NO:1, SEQ ID NO:4 or SEQ ID NO:7.
88. the method for the plant of generation tolerance pyrazosulfuron herbicide according to claim 87, which is characterized in that the thiophene The nucleotide sequence of the grand hydrolase of pheno sulphur are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
89. a kind of method of the plant of culture tolerance pyrazosulfuron herbicide characterized by comprising
At least one propagulum is planted, includes the more of coding thifensulfuronmethyl hydrolase in the genome of the propagulum Nucleotide sequence, the thifensulfuronmethyl hydrolase are amino shown in SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7 Acid sequence；
The propagulum is set to grow up to plant；
Herbicide containing effective dose pyrazosulfuron is applied in the plant growth environment including at least the plant, is harvested With other do not have coding thifensulfuronmethyl hydrolase polynucleotide sequences plant compare with decrease plant injury and/or Plant with increased plant products.
90. the method for the plant of culture tolerance pyrazosulfuron herbicide according to claim 89, which is characterized in that the thiophene The nucleotide sequence of the grand hydrolase of pheno sulphur are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
91. a kind of method for protecting the plants from the damage as caused by pyrazosulfuron herbicide, which is characterized in that including that will contain The herbicide of effective dose pyrazosulfuron is applied to there are in the plant growth environment of at least one genetically modified plants, described to turn base Because plant includes the nucleotide sequence for encoding thifensulfuronmethyl hydrolase in its genome, the genetically modified plants do not have with other There is the plant of the nucleotide sequence of coding thifensulfuronmethyl hydrolase to compare with the plant injury weakened and/or there is increased plant Produce amount；Wherein the thifensulfuronmethyl hydrolase is amino shown in SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7 Acid sequence.
92. the method for protecting the plants from the damage as caused by pyrazosulfuron herbicide according to claim 91, feature It is, the nucleotide sequence of the thifensulfuronmethyl hydrolase are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
93. a kind of method of thifensulfuronmethyl hydrolase pyrazosulfuron herbicide, which is characterized in that including effective agent will be contained The herbicide of amount pyrazosulfuron is applied to there are in the plant growth environment of at least one genetically modified plants, the genetically modified plants Nucleotide sequence in its genome comprising coding thifensulfuronmethyl hydrolase, the genetically modified plants do not have coding with other The plant of the nucleotide sequence of thifensulfuronmethyl hydrolase is compared with the plant injury weakened and/or there is increased plant to produce Amount；Wherein the thifensulfuronmethyl hydrolase is amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:7 Column.
94. the method for thifensulfuronmethyl hydrolase pyrazosulfuron herbicide according to claim 93, which is characterized in that institute State the nucleotide sequence of thifensulfuronmethyl hydrolase are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
95. a kind of purposes of thifensulfuronmethyl hydrolase pyrazosulfuron herbicide, wherein the thifensulfuronmethyl hydrolase is SEQ Amino acid sequence shown in ID NO:1, SEQ ID NO:4 or SEQ ID NO:7.
96. the purposes of thifensulfuronmethyl hydrolase pyrazosulfuron herbicide according to claim 95, which is characterized in that packet It includes and the herbicide containing effective dose pyrazosulfuron is applied to there are in the plant growth environment of at least one genetically modified plants, The genetically modified plants in its genome include encode thifensulfuronmethyl hydrolase nucleotide sequence, the genetically modified plants with Other plants for not having the nucleotide sequence of coding thifensulfuronmethyl hydrolase are compared with the plant injury weakened and/or have Increased plant products.
97. according to the purposes of the thifensulfuronmethyl hydrolase pyrazosulfuron herbicide of claim 95 or 96, feature exists In the nucleotide sequence of the thifensulfuronmethyl hydrolase are as follows:
(a) nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:3；Or
(b) nucleotide sequence shown in SEQ ID NO:5 or SEQ ID NO:6；Or
(c) nucleotide sequence shown in SEQ ID NO:8 or SEQ ID NO:9.
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