CN115340987A - Herbicide tolerance protein, coding gene and application thereof - Google Patents

Herbicide tolerance protein, coding gene and application thereof Download PDF

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CN115340987A
CN115340987A CN202110514777.XA CN202110514777A CN115340987A CN 115340987 A CN115340987 A CN 115340987A CN 202110514777 A CN202110514777 A CN 202110514777A CN 115340987 A CN115340987 A CN 115340987A
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inhibitor herbicide
ppo inhibitor
plant
ppo
herbicide
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CN115340987B (en
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肖翔
宋庆芳
陶青
于彩虹
鲍晓明
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Beijing Dabeinong Biotechnology Co Ltd
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    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
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    • C12Y103/03004—Protoporphyrinogen oxidase (1.3.3.4)

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Abstract

The invention relates to a herbicide tolerance protein, a coding gene and application thereof, wherein the protein comprises: (a) has an amino acid sequence shown in SEQ ID NO: 1; (b) And (b) a protein derived from (a) and having protoporphyrinogen oxidase activity, wherein the amino acid sequence in (a) is substituted and/or deleted and/or added with one or more amino acids. The herbicide tolerance protein has higher tolerance to a PPO inhibitor herbicide, and plants containing the nucleotide sequence for encoding the herbicide tolerance protein have strong tolerance to the PPO inhibitor herbicide, and all show high resistance to oxyfluorfen, saflufenacil and flumioxazin with the concentration 4 times that of a field and sulfentrazone with the concentration 2 times that of the field. Therefore, the application prospect on plants is wide.

Description

Herbicide tolerance protein, coding gene and application thereof
Technical Field
The invention relates to a herbicide tolerance protein, a coding gene and application thereof, in particular to a protein with tolerance to a PPO inhibitor herbicide, a coding gene and application thereof.
Background
The porphyrin biosynthesis pathway is used to synthesize chlorophyll and heme, which play important roles in plant metabolism, and occurs in chloroplasts. In this pathway, protoporphyrinogen oxidase (PPO for short) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. After protoporphyrin IX is produced, protoporphyrin IX synthesizes chlorophyll by binding magnesium to magnesium by magnesium chelatase, or heme by binding iron to iron by iron chelatase.
Herbicides that act by inhibiting PPO include diphenyl ether PPO inhibitor herbicides, oxadiazolone PPO inhibitor herbicides, N-phenylphthalimide PPO inhibitor herbicides, oxazolinone PPO inhibitor herbicides, phenylpyrazole PPO inhibitor herbicides, uracil PPO inhibitor herbicides, thiadiazole PPO inhibitor herbicides, triazolinone PPO inhibitor herbicides, triazinone PPO inhibitor herbicides and other types of PPO inhibitor herbicides. In plants, PPO inhibitors inhibit the enzymatic activity of PPO, resulting in the inhibition of chlorophyll and heme synthesis, and resulting in the accumulation of the substrate protoporphyrinogen IX, which is rapidly exported from chloroplasts to cytoplasm, the conversion of protoporphyrinogen IX in cytoplasm to protoporphyrin IX in a non-enzymatic reaction and further the generation of highly reactive singlet oxygen in the presence of light and oxygen molecules: (a) 1 O 2 ) They can disrupt the cell membrane and rapidly lead to the death of plant cells.
The method for providing a PPO inhibitor herbicide tolerant plant essentially comprises: 1) Herbicides are detoxified using enzymes that convert the herbicide or an active metabolite thereof to a non-toxic product. 2) The sensitive PPO is overexpressed so that, in view of this kinetic constant of the enzyme, a sufficient amount of target enzyme relative to the herbicide is produced in the plant so that, despite the PPO-inhibitor herbicide, the sensitive PPO and the PPO-inhibitor herbicide are sufficiently effective to have enough functional enzyme available for use. 3) A mutant PPO is provided which is less sensitive to herbicides or active metabolites thereof, but which retains the property of catalyzing the oxidation of protoporphyrinogen IX to protoporphyrin IX. With respect to the class of mutant PPOs, while a given mutant PPO may provide a useful level of tolerance to some PPO inhibitor herbicides, the same mutant PPO may not be sufficient to provide a commercial level of tolerance to a different, more desirable PPO inhibitor herbicide; for example, PPO inhibitor herbicides can vary in the range of weeds they control, the cost of their manufacture, and their environmental friendliness. Thus, there is a need for new methods for conferring PPO inhibitor herbicide tolerance to different crops and crop varieties.
Disclosure of Invention
The invention aims to provide a novel protein, a coding gene and application thereof, wherein the protein not only has protoporphyrinogen oxidase activity, but also has better tolerance to PPO inhibitor herbicides by plants transferred with a nucleotide sequence for coding the protein.
To achieve the above object, the present invention provides a protein comprising:
(a) Has an amino acid sequence shown as SEQ ID NO. 1;
(b) And (b) a protein derived from (a) and having protoporphyrinogen oxidase activity, wherein the amino acid sequence in (a) is substituted and/or deleted and/or added with one or more amino acids.
To achieve the above object, the present invention provides a gene comprising:
(a) A nucleotide sequence encoding the protein; or
(b) A nucleotide sequence which hybridizes with the nucleotide sequence defined in (a) under strict conditions and codes for a protein with protoporphyrinogen oxidase activity; or
(c) Has a nucleotide sequence shown in SEQ ID NO. 2.
The stringent conditions can be hybridization at 65 ℃ in a 6 XSSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution, followed by washing the membrane 1 times each with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
To achieve the above object, the present invention also provides an expression cassette comprising the gene under the control of an operably linked regulatory sequence.
In order to achieve the above object, the present invention also provides a recombinant vector comprising the gene or the expression cassette.
To achieve the above objects, the present invention also provides a method for extending the herbicide tolerance of a plant, comprising: expressing said protein or the protein encoded by said expression cassette in a plant together with at least one second herbicide tolerance protein different from said protein or the protein encoded by said expression cassette.
Further, the second herbicide tolerance protein is 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha ketoglutarate-dependent dioxygenase, dicamba monooxygenase, acetolactate synthase, a cytochrome-like protein and/or hydroxyphenylpyruvate dioxygenase.
To achieve the above objects, the present invention also provides a method for selecting a transformed plant cell, comprising: transforming a plurality of plant cells with said gene or said expression cassette and culturing said cells at a concentration of a PPO inhibitor herbicide that allows growth of transformed cells expressing said gene or said expression cassette, while killing or inhibiting growth of untransformed cells;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugarbeet, cucumber, cotton, rape, potato, tomato, or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
To achieve the above object, the present invention also provides a method for controlling weeds, comprising: applying an effective dose of a PPO inhibitor herbicide to a field planted with a plant of interest comprising said gene or said expression cassette or said recombinant vector;
preferably, the plant of interest includes a monocotyledon and a dicotyledon; more preferably, the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis; further preferably, the plant of interest is a glyphosate tolerant plant and the weeds are glyphosate resistant weeds;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
To achieve the above objects, the present invention also provides a method for protecting or conferring tolerance to a PPO inhibitor herbicide on a plant, comprising: introducing said gene or said expression cassette or said recombinant vector into a plant such that the introduced plant produces an amount of herbicide tolerance protein sufficient to protect it from the PPO inhibitor herbicide;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
To achieve the above object, the present invention also provides a method for producing a plant tolerant to a PPO inhibitor herbicide, comprising introducing the gene into the genome of the plant;
preferably, the method of introduction includes a genetic transformation method, a genome editing method, or a gene mutation method;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugarbeet, cucumber, cotton, rape, potato, tomato, or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
To achieve the above objects, the present invention also provides a method of growing a PPO inhibitor herbicide tolerant plant comprising:
growing at least one plant propagule comprising in its genome the gene or the expression cassette;
growing the plant propagule into a plant;
applying an effective dose of a PPO inhibitor herbicide to a plant growing environment comprising at least said plant, harvesting plants having reduced plant damage and/or increased plant yield as compared to other plants not having said gene or said expression cassette;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
The present invention also provides a method for obtaining a processed agricultural product comprising treating a harvest of PPO inhibitor herbicide tolerant plants obtained by the above method to obtain a processed agricultural product.
To achieve the above object, the present invention also provides a planting system for controlling weed growth, comprising a PPO inhibitor herbicide and a plant growing environment in which at least one target plant is present, said target plant comprising said gene or said expression cassette;
preferably, the plant of interest includes a monocotyledon and a dicotyledon; more preferably, the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugarbeet, cucumber, cotton, rape, potato, tomato, or arabidopsis; further preferably, the plant of interest is a glyphosate tolerant plant and the weeds are glyphosate resistant weeds;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
To achieve the above object, the present invention also provides the use of the protein for conferring herbicide tolerance to a PPO inhibitor in a plant;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugarbeet, cucumber, cotton, rape, potato, tomato, or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
As specific embodiments, the PPO inhibitor herbicide may be one or more selected from the group consisting of, but not limited to: diphenyl ethers (cumyl ether, metoxyfen (haloxyfen), bifenox (Bifenox), oxyfluorfen (oxyfluorfen), acifluorfen and its salts and esters, fomesafen (Fomesafen), lactofen (lactofen), fluorofen-ethyl, fluoroglycofen-ethyl, clofenapyr, aclonifen (aclonifen), bifenox (Bifenox), chlorolactofen (ethloxifen), clofenpyr-methyl (chlorintrofen), nitrofen (halosafen)); oxadiazolones (oxadiazon (oxadiarzonan), oxadiargyl (oxadiargyl)); n-phenylphthalamide imines (flumioxazin, fluoroeneoxalic acid, cinidon-ethyl); oxazolinones (cyclopentoxazone); phenylpyrazoles (fluazolate, pyraflufen-ethyl); uracils (bensulfuron-methyl, butafenacil, saflufenacil)); thiadiazoles (thiazachlor (thidiazimin), fluthiacet (fluthiacet)); triazolinones (azafenidin), sulfentrazone (carfentrazone), carfentrazone-ethyl); triazinones (triflumimoxazin); others (flufenpyr-ethyl), pyraclonil (pyraclonil).
The articles "a" and "an" as used herein mean one or more than one (i.e., at least one). For example, "an element" means one or more elements (components). Furthermore, the terms "comprises" or variations such as "comprising" or "comprising" are to be understood as meaning the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The term "herbicide insensitive" in the present invention refers to the ability of protoporphyrinogen oxidase to maintain at least a portion of its enzymatic activity in the presence of one or more PPO inhibitor herbicides. The enzymatic activity of protoporphyrinogen oxidase can be measured by any means known in the art, for example by measuring the amount of protoporphyrinogen oxidase product produced or the amount of protoporphyrinogen oxidase substrate consumed by fluorescence, high Performance Liquid Chromatography (HPLC) or Mass Spectrometry (MS) in the presence of one or more PPO inhibitor herbicides. An "herbicide insensitive" may be a complete or partial insensitivity to a particular herbicide and may be expressed as a tolerance or percent insensitivity to a particular PPO inhibitor herbicide.
The term "herbicide tolerance of a plant, seed, plant tissue or cell" or "herbicide tolerant plant, seed, plant tissue or cell" in the present invention refers to the ability of a plant, seed, plant tissue or cell to withstand the action of a herbicide when the herbicide is applied. For example, herbicide tolerant plants may survive or continue to grow in the presence of the herbicide. Herbicide tolerance of a plant, seed, plant tissue or cell can be measured by comparing the plant, seed, plant tissue or cell to a suitable control. For example, herbicide tolerance can be measured or assessed by applying the herbicide to plants that contain a DNA molecule encoding a protein capable of conferring herbicide tolerance (test plants) and plants that do not contain a DNA molecule encoding a protein capable of conferring herbicide tolerance (control plants), and then comparing plant damage of the two plants, wherein herbicide tolerance of the test plants is indicated by a decrease in the rate of damage compared to the rate of damage of the control plants. The herbicide-tolerant plant, seed, plant tissue or cell exhibits a reduced response to the toxic effect of the herbicide as compared to a control plant, seed, plant tissue or cell. The term "herbicide tolerance trait" refers to a transgenic trait that confers improved herbicide tolerance to a plant as compared to a wild-type plant. Plants that can be produced having the herbicide tolerance trait of the present invention include, for example, any plant, including crop plants such as oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugarbeet, cucumber, cotton, oilseed rape, potato, tomato, and arabidopsis thaliana.
The DNA molecules of the invention may be synthesized and modified, in whole or in part, by methods known in the art, particularly where it is desired to provide sequences for DNA manipulation (such as restriction enzyme recognition sites or recombination-based cloning sites), plant-preferred sequences (such as plant codon usage or Kozak consensus sequences), or sequences for DNA construct design (such as spacer or linker sequences).
The oxyfluorfen (oxyfluorfen) in the invention is 2-chloro-1- (3-ethoxy-4-nitrophenoxy) -4-trifluoromethyl benzene, and is colorless crystalline solid. Belongs to a PPO inhibitor herbicide with ultralow dosage selectivity of diphenyl ether and pre-bud and post-bud contact killing, and can be prepared into missible oil for use. Weeds are killed mainly by absorbing the medicament through coleoptiles and mesocotyl. Oxyfluorfen can effectively control weeds in the fields of crops such as rice, soybean, corn, cotton, vegetables, grapes, fruit trees and the like, and the preventable weeds include, but are not limited to, barnyard grass, sesbania, sparrow, green bristlegrass, stramonium, creeping wheatgrass, ragweed, yellowweed, abutilon, mustard monocotyledonous and broadleaf weeds.
The effective dose of oxyfluorfen in the invention is 180-720g ai/ha, including 190-700g ai/ha, 250-650g ai/ha, 300-600g ai/ha or 400-500g ai/ha.
In the invention, the saflufenacil is N' - [ 2-chloro-4-fluoro-5- (3-methyl-2, 6-dioxo-4- (trifluoromethyl) -3, 6-dihydro-1 (2H) -pyrimidine) benzoyl ] -N-isopropyl-N-methyl sulfamide, and is a light brown strip-extruding granular solid. Belongs to a uracil biocidal PPO inhibitor herbicide, and can be prepared into 70 percent water dispersible granule formulation. Saflufenacil is effective in controlling a variety of broadleaf weeds, including weeds that are resistant to glyphosate, ALS, and triazines, have a fast kill action and soil residues degrade rapidly.
The effective dose of saflufenacil in the invention is 25-100g ai/ha, including 30-95g ai/ha, 40-90g ai/ha, 50-85g ai/ha or 60-80g ai/ha.
In the present invention, flumioxazin (Flumioxazin) refers to 2- [ 7-fluoro-3, 4-dihydro-3-oxo-4- (2-propynyl) -2H-1, 4-benzoxazin-6-yl ] -4,5,6, 7-tetrahydro-1H-isoindole-1, 3 (2H) -dione. Belongs to N-phenylphthalimide budlet and leaf absorption type PPO inhibitor herbicide, and the common formulation is 50 percent of wettable powder and 48 percent of suspending agent. Flumioxazin is effective in controlling 1-year-old broadleaf weeds and part of grassy weeds. It is easy to degrade in the environment and is safe to the succeeding crops.
In the present invention, the effective dose of flumioxazin is 60-240g ai/ha, including 70-220g ai/ha, 85-200g ai/ha, 90-185g ai/ha or 100-150g ai/ha.
In the invention, sulfentrazone (Sulfenpyrazone) refers to N- (2, 4-dichloro-5- (4-difluoromethyl-4, 5-dihydro-3-methyl-5-oxo-1H-1, 2, 4-triazol-1-yl) phenyl) methanesulfonamide and is a brown yellow solid. Belongs to a triazolinone PPO inhibitor herbicide, and the common formulation is suspending agent with the concentration of 38.9 percent and 44.5 percent. The sulfentrazone can be used for preventing and treating 1-year-old broadleaf weeds, gramineous weeds, nutgrass flatsedge and the like in fields such as corn, sorghum, soybean, peanut and the like, morning glory, amaranthus retroflexus, quinoa, stramonium, digitaria sanguinalis, green bristlegrass, xanthium, eleusine indica, cyperus rotundus and the like.
The effective dose of the sulfentrazone is 450-900g ai/ha, and comprises 500-850g ai/ha, 550-700g ai/ha, 500-685g ai/ha or 550-650g ai/ha.
In the present invention, the term "resistant" is heritable and allows plants to grow and reproduce under conditions where herbicides are effective for a given plant with typical herbicides. As recognized by those skilled in the art, even if a given plant is subjected to some degree of herbicide treatment, such as little necrosis, lysis, chlorosis, or other damage, but at least not significantly affected in yield, the plant can still be considered "resistant", i.e., the given plant has an increased ability to resist herbicide-induced damage of various degrees, while typically causing damage to wild-type plants of the same genotype at the same herbicide dose. The term "resistance" or "tolerance" in the present invention is broader than the term "resistance" and includes "resistance".
As used herein, "glyphosate" refers to N-phosphonomethylglycine and its salts and treatment with "glyphosate herbicide" refers to the use of any glyphosate containing herbicideThe preparation is processed. Commercial formulations of glyphosate include, but are not limited to,
Figure BDA0003061498190000081
(Glyphosate as an isopropylamine salt),
Figure BDA0003061498190000083
WEATHERMAX (glyphosate as potassium salt),
Figure BDA0003061498190000082
DRY and
Figure BDA0003061498190000084
(Glyphosate as an amine salt),
Figure BDA0003061498190000086
GEOFORCE (Glyphosate as the sodium salt) and
Figure BDA0003061498190000085
(Glyphosate as the trimethylsulfonium salt).
The effective dose of the glyphosate is 200-1600g ae/ha, and comprises 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.
As used herein, "glufosinate," also known as glufosinate, refers to ammonium 2-amino-4- [ hydroxy (methyl) phosphono ] butanoate, and treatment with "glufosinate herbicide" refers to treatment with any herbicide formulation containing glufosinate.
The effective dose of glufosinate-ammonium in the invention is 200-800g ae/ha, including 200-750g ae/ha, 250-700g ae/ha, 300-700g ae/ha, 350-650g ae/ha or 400-600g ae/ha.
The application rate of the herbicide in the present invention varies with soil structure, pH, organic matter content, farming system and weed size and is determined by looking at the appropriate herbicide application rate on the herbicide label.
The term "conferring" in the context of the present invention means providing a characteristic or trait, such as herbicide tolerance and/or other desirable traits, to a plant.
The term "heterologous" in the context of the present invention means from another source. In the context of DNA, "heterologous" refers to any foreign "non-self DNA, including DNA from another plant of the same species. For example, in the present invention, transgenic methods may be used to express the soybean PPO gene in soybean plants, which are still considered "heterologous" DNA.
The term "nucleic acid" in the present invention encompasses deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, encompasses known analogs (e.g., peptide nucleic acids) that have the basic properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.
In the present invention, when the term "encoding" or "encoded" is used in the context of a particular nucleic acid, it means that the nucleic acid contains the necessary information to direct the translation of the nucleotide sequence into a particular protein. The information used to encode the protein is specified by the use of codons. A nucleic acid encoding a protein may comprise untranslated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening untranslated sequences (e.g., in cDNA).
The gene encoding the herbicide tolerance protein of the present invention is used to provide plants, plant cells, and seeds of the present invention that provide better tolerance to multiple PPO inhibitor herbicides as compared to the same plant (control plant) that does not comprise the gene encoding the herbicide tolerance protein of the present invention.
The genes encoding the herbicide tolerance proteins of the present invention are useful for producing plants that are tolerant to PPO inhibitor herbicides. The genes encoding the herbicide tolerance proteins of the present invention are particularly suitable for expression in plants in order to confer herbicide tolerance to the plants.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to polymers of amino acid residues in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The polypeptides of the invention can be produced from a nucleic acid disclosed herein or by using standard molecular biology techniques. For example, a truncated protein of the invention may be produced by expressing a recombinant nucleic acid of the invention in a suitable host cell, or alternatively by a combination of ex vivo methods such as protease digestion and purification.
The invention also provides nucleic acid molecules comprising proteins encoding said herbicide tolerance proteins. In general, the invention includes any nucleotide sequence encoding any of the herbicide tolerance proteins described herein, as well as any nucleotide sequence encoding an herbicide tolerance protein having one or more conservative amino acid substitutions relative to the herbicide tolerance protein described herein. Conservative substitutions providing functionally similar amino acids are well known to those skilled in the art, and the following five groups each contain amino acids that are conservative substitutions for one another: aliphatic: glycine (G), alanine (a), valine (V), leucine (L), isoleucine (I); aromatic compounds: phenylalanine (F), tyrosine (Y), tryptophan (W); sulfur-containing: methionine (M), cysteine (C); basic: arginine (I), lysine (K), histidine (H); acidic: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q).
Thus, sequences having PPO inhibitor herbicide tolerance activity and which hybridize under stringent conditions to the nucleotides of the invention encoding the herbicide tolerance proteins are included in the invention. Illustratively, these sequences have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology to the sequence of SEQ ID NO 2 of the present invention.
Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the herbicide tolerance gene of the present invention. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. In the present invention, two nucleic acid molecules can be said to be capable of specifically hybridizing to each other if they can form an antiparallel double-stranded nucleic acid structure. Two nucleic acid molecules are said to be "complements" of one another if they exhibit complete complementarity. In the present invention, two nucleic acid molecules are said to exhibit "perfect complementarity" when each nucleotide of the nucleic acid molecule is complementary to the corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability to allow them to anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to have "complementarity" if they are capable of hybridizing to each other with sufficient stability to allow them to anneal and bind to each other under conventional "highly stringent" conditions. Deviations from perfect complementarity may be tolerated as long as such deviations do not completely prevent the formation of a double-stranded structure by the two molecules. In order for a nucleic acid molecule to be able to act as a primer or probe, it is only necessary to ensure sufficient complementarity in the sequence to allow formation of a stable double-stranded structure at the particular solvent and salt concentrations employed.
In the present invention, a substantially homologous sequence is a nucleic acid molecule that specifically hybridizes under highly stringent conditions to the complementary strand of a compatible nucleic acid molecule. Suitable stringency conditions for promoting DNA hybridization include, for example, treatment with 6.0 XSSC/sodium citrate (SSC) at about 45 ℃ followed by a wash with 2.0 XSSC at 50 ℃, as is well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from about 2.0 XSSC at low stringency conditions, about 50 ℃ to about 0.2 XSSC, about 50 ℃ at high stringency conditions. In addition, the temperature conditions in the washing step can be raised from about 22 ℃ at room temperature for low stringency conditions to about 65 ℃ for high stringency conditions. Both the temperature conditions and the salt concentration may be varied, or one may be held constant while the other is varied. Preferably, the stringent conditions according to the present invention may be such that specific hybridization with the gene encoding the protoporphyrinogen oxidase of the present invention occurs at 65 ℃ in 6 XSSC, 0.5% SDS solution, and then each washing of the membrane 1 time with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
In the present invention, the term "hybridize" or "specifically hybridize" refers to a molecule that can only bind, double-stranded, or hybridize to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
Due to the redundancy of the genetic code, a plurality of different DNA sequences may encode the same amino acid sequence. It is well within the skill of the art to generate such alternative DNA sequences encoding the same or substantially the same protein. These different DNA sequences are included in the scope of the present invention. The "substantially identical" sequences refer to sequences having amino acid substitutions, deletions, additions or insertions which do not substantially affect the herbicide tolerance activity, and also include fragments which retain the herbicide tolerance activity.
The term "functional activity" or "activity" as used herein means that the protein/enzyme (alone or in combination with other proteins) for use according to the invention has the ability to degrade or attenuate the activity of a herbicide. The plants producing the proteins of the invention preferably produce an "effective amount" of the protein such that when the plants are treated with the herbicide, the level of protein expression is sufficient to confer full or partial tolerance to the herbicide (if not specifically stated, the amount normally used) to the plant. The herbicide may be used in an amount that normally kills the target plant, in a normal field amount, and at a concentration. Preferably, the plant cells and plants of the invention are protected from growth inhibition or damage caused by herbicide treatment. The transformed plants and plant cells of the present invention preferably have tolerance to a PPO inhibitor herbicide, i.e., the transformed plants and plant cells are capable of growing in the presence of an effective amount of a PPO inhibitor herbicide.
Genes and proteins described herein include not only the specific exemplary sequences, but also portions and/or fragments (including deletions at and/or termini compared to the full-length protein), variants, mutants, variant proteins, substitutions (proteins with alternative amino acids), chimeras and fusion proteins that preserve the active characteristics of the specific exemplary proteins.
The term "variant" of the invention means substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within a reference polynucleotide and/or a substitution of one or more nucleotides at one or more sites in an herbicide tolerance gene. The term "reference polynucleotide or polypeptide" in the present invention encompasses a herbicide-tolerant nucleotide sequence or amino acid sequence, respectively. The term "native polynucleotide or polypeptide" in the present invention correspondingly includes naturally occurring nucleotide or amino acid sequences. For nucleic acid molecules, conservative variants include a nucleotide sequence encoding one of the herbicide tolerance proteins of the present invention (due to the degeneracy of the genetic code). Such naturally occurring allelic variants can be identified using well-known molecular biology techniques, for example, using the Polymerase Chain Reaction (PCR) and hybridization techniques outlined below. Variant nucleic acid molecules also include synthetically derived nucleic acid molecules, such as nucleic acid sequences generated by using site-directed mutagenesis but still encoding the herbicide tolerance proteins of the present invention. Typically, variants of a particular nucleic acid molecule of the invention will have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology to the particular nucleic acid molecule as determined by sequence alignment programs and parameters.
By "variant protein" is meant a protein derived from a reference protein by deletion or addition of one or more amino acids at one or more internal sites in the herbicide tolerance protein and/or substitution of one or more amino acids at one or more sites in the herbicide tolerance protein. Variant proteins encompassed by the present invention are biologically active, i.e. they continue to have the desired activity of the protoporphyrinogen oxidase of the present invention, i.e. still have the recited protoporphyrinogen oxidase activity and/or herbicide tolerance. Such variants may arise, for example, from genetic polymorphisms or from artificial manipulation. A biologically active variant of an herbicide tolerance protein of the invention will have at least about 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology to the entire amino acid sequence of the herbicide tolerance protein, as determined by sequence alignment programs and parameters. A biologically active variant of a protein of the invention may differ from a protein in as few as 1-15 amino acid residues, as few as 1-10 (e.g., 6-10), as few as 5 (e.g., 4, 3, 2, or even 1) amino acid residues.
Methods for alignment are well known in the art and can be accomplished using mathematical algorithms such as those of Myers and Miller (1988) cabaos 4; smith et al (1981) Adv.appl.Math.2: 482; need email and Wunsch (1970) J.mol.biol.48: 443-453; and the algorithm of Karlin and Al tschul (1990) proc.natl.acad.sci.usa 872264, as modified in Karlin and Al tschul (1993) proc.natl.acad.sci.usa 90. Computer implementations of these mathematical algorithms can be utilized for sequence comparison to determine sequence homology, such implementations including, but not limited to: CLUSTAL (available from Intelligenetics, mountain n View, california) in the PC/Gene program; ALLGN program (version 2.0) and GAP, BESTFIT, BLAST, FASTA and TFASTA in GCG Wiscons in Genetics Software Package version 10 (available from Accelrys Inc., 9685Scanton road, san Diego, calif. USA).
In certain embodiments, the nucleic acid sequences encoding the herbicide-tolerant proteins of the invention, or their variants that retain protoporphyrinogen oxidase activity, can be stacked in combination with any nucleic acid sequence of interest to produce plants with a desired trait. The term "trait" refers to a phenotype derived from a particular sequence or group of sequences. For example, the nucleic acid sequence encoding a herbicide tolerance protein of the present invention or a variant that retains protoporphyrinogen oxidase activity can be stacked with any other nucleic acid encoding a polypeptide that confers a desired trait including, but not limited to: resistance to diseases, insects, and herbicides, tolerance to heat and drought, reduced crop maturation time, improved industrial processing (e.g., for converting starch or biomass into fermentable sugars), and improved agronomic quality (e.g., high oil content and high protein content).
As is well known to those skilled in the art, the benefits of the combination of two or more modes of action in improving the controlled weed spectrum and/or naturally more tolerant or resistant weed species may also be extended to the production of herbicide tolerant chemicals in crops other than PPO tolerant crops by man (transgenic or non-transgenic). Indeed, the traits encoding for resistance to the following can be stacked individually or in multiple combinations to provide effective control or prevent the ability of weed succession to develop resistance to herbicides: glyphosate resistance (e.g., resistant plant or bacteria EPSPS, GOX, GAT), glufosinate resistance (e.g., PAT, bar), acetolactate synthase (ALS) inhibitory herbicide resistance (e.g., imidazolinone, sulfonylurea, triazolopyrimidine, sulfonanilide, pyrimidthiobenzoate, and other chemical resistance genes such as AHAS, csrl, surA, etc.), phenoxy auxin herbicide resistance (e.g., aryloxyalkanoate dioxygenase-AAD), dicamba herbicide resistance (e.g., dicamba monooxygenase-DMO), bromoxynil resistance (e.g., bxn), resistance to Phytoene Desaturase (PDS) inhibitors, resistance to photosystem ii inhibitory herbicides (e.g., psbA), pyruvate i inhibitory herbicides, 4-hydroxyphenyl dioxygenase inhibitory herbicides (e.g., HPPD), resistance to phenylurea herbicides (e.g., CYP76B 1), dicamba degrading enzymes, and the like.
Glyphosate is widely used because it controls a very broad spectrum of broadleaf and grass weed species. However, repeated use of glyphosate in glyphosate-tolerant crop and non-crop applications has (and will continue to) choose to allow weeds to evolve into a more naturally-tolerant species or glyphosate-resistant biotype. Most herbicide resistance management strategies suggest the use of an effective amount of a tank-mix herbicide partner that provides control of the same species but has a different mode of action as a means of delaying the emergence of resistant weeds. Superimposing the gene encoding the herbicide tolerance protein of the present invention with a glyphosate tolerance trait (and/or other herbicide tolerance traits) can enable control of glyphosate resistant weed species (broadleaf weed species controlled by one or more PPO inhibitor herbicides) in glyphosate tolerant crops by allowing selective use of glyphosate and PPO inhibitor herbicides (such as oxyfluorfen, saflufenacil and flumioxazin) on the same crop. The application of these herbicides can be simultaneous use in tank mixes containing two or more herbicides of different modes of action, separate use of individual herbicide compositions (with time intervals ranging from 2 hours to 3 months between uses) in successive uses (such as pre-planting, pre-emergence or post-emergence), or alternatively, any number of combinations of herbicides representing each applicable chemical class can be used at any time from the time of planting the crop within 7 months to the time of harvesting the crop (or for a single herbicide, the shortest time).
It is important to have flexibility in controlling broadleaf weeds, i.e., time of use, individual herbicide dosage, and the ability to control persistent or resistant weeds. The application range of glyphosate superposed with glyphosate resistance gene/gene for encoding herbicide tolerance protein in crops can be from 250 to 2500g ae/ha; the PPO inhibitor herbicide(s) may be present in an amount of from 10 to 1000g ai/ha. The optimal combination of times for these applications depends on the specific conditions, species and environment.
Herbicide formulations (such as ester, acid or salt formulations or soluble concentrates, emulsified concentrates or soluble liquids) and tank mix additives (such as adjuvants or compatibilizers) can significantly affect weed control for a given herbicide or combination of one or more herbicides. Any chemical combination of any of the foregoing herbicides is within the scope of the invention.
In addition, the gene encoding the herbicide tolerance protein of the present invention can be stacked with one or more other input (e.g., insect resistance, fungal resistance, or stress tolerance, etc.) or output (e.g., increased yield, improved oil, increased fiber quality, etc.) traits, either alone or in combination with other herbicide tolerant crop traits. Thus, the present invention can be used to provide a complete agronomic solution with the ability to flexibly and economically control any number of agronomic pests and to improve crop quality.
The combination of these overlays can be produced by any method, including but not limited to: cross-breeding plants or genetic transformation by conventional or top-crossing methods. If these sequences are stacked by genetically transforming these plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, transgenic plants comprising one or more desired traits may be used as targets for the introduction of additional traits by subsequent transformation. These traits may be introduced in a co-transformation protocol simultaneously with the polynucleotides of interest provided by any combination of expression cassettes. For example, if two sequences are to be introduced, the two sequences may be contained in separate expression cassettes (trans) or in the same expression cassette (cis). Expression of these sequences may be driven by the same promoter or by different promoters. In some cases, it may be desirable to introduce an expression cassette that inhibits expression of the polynucleotide of interest. This can be combined with any combination of other suppression or overexpression cassettes to produce the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system.
The gene encoding the herbicide tolerance protein has higher tolerance to PPO inhibitor herbicides, and is the basis of characteristic possibilities of important herbicide tolerant crops and selection markers.
The term "expression cassette" as used herein refers to a nucleic acid molecule capable of directing the expression of a particular nucleotide sequence in a suitable host cell, comprising a promoter operably linked to a nucleotide sequence of interest (i.e., a polynucleotide encoding a protoporphyrinogen oxidase or a variant protein retaining protoporphyrinogen oxidase activity as described herein, either alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring a desired trait) operably linked to a termination signal. The coding region typically encodes a protein of interest, but may also encode a functional RNA of interest, such as an antisense RNA or an untranslated RNA in the sense or antisense orientation. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be a naturally occurring expression cassette, but must be obtained in recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into a new host cell by a transformation event. Expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In addition, the promoter is specific for a particular tissue or organ or developmental stage.
The present invention encompasses transforming plants with expression cassettes capable of expressing a polynucleotide of interest (i.e., a polynucleotide encoding a protoporphyrinogen oxidase of the present invention, or its variant protein retaining protoporphyrinogen oxidase activity, either alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring desired traits). The expression cassette includes a transcriptional and translational initiation region (i.e., promoter) and a polynucleotide open reading frame in the 5'-3' direction of transcription. The expression cassette may optionally include transcriptional and translational termination regions (i.e., termination regions) that are functional in plants. In some embodiments, the expression cassette includes a selectable marker gene to allow for selection of stable transformants. The expression constructs of the invention may also comprise leader sequences and/or sequences which allow inducible expression of the polynucleotide of interest.
The regulatory sequences of the expression cassette are operably linked to the polynucleotide of interest. The regulatory sequences of the present invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the gene encoding the protoporphyrinogen oxidase.
The promoter is a promoter capable of being expressed in a plant, and the promoter capable of being expressed in the plant is a promoter which ensures that a coding sequence connected with the promoter is expressed in a plant cell. The promoter expressible in plants may be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, 35S promoter derived from cauliflower mosaic virus, maize Ubi promoter, promoter of rice GOS2 gene, and the like. Alternatively, the plant expressible promoter may be a tissue specific promoter, i.e. a promoter that directs expression of the coding sequence at a higher level in some tissues of the plant, e.g. in green tissues, than in other tissues of the plant (as can be determined by conventional RNA assays), e.g. the PEP carboxylase promoter. Alternatively, the promoter expressible in plants may be a wound-inducible promoter. A wound-inducible promoter or a promoter that directs a wound-induced expression pattern means that when a plant is subjected to mechanical or insect feeding induced wounds, the expression of the coding sequence under the control of the promoter is significantly increased compared to under normal growth conditions. Examples of wound-inducible promoters include, but are not limited to, promoters of potato and tomato protease-inhibitory genes (pin I and pin II) and maize protease-inhibitory gene (MPI).
The transit peptide (also known as a secretion signal sequence or targeting sequence) is intended to direct the transgene product to a specific organelle or cellular compartment, and for the receptor protein, the transit peptide may be heterologous, e.g., targeting the chloroplast using a chloroplast transit peptide sequence, or targeting the endoplasmic reticulum using a 'KDEL' retention sequence, or targeting the vacuole using the CTPP of the barley lectin gene.
The leader sequence includes, but is not limited to, a small RNA virus leader sequence, such as an EMCV leader sequence (encephalomyocarditis virus 5' non-coding region); potyvirus leaders, such as the MDMV (maize dwarf mosaic virus) leader; human immunoglobulin heavy chain binding protein (BiP); untranslated leader sequences of envelope protein mRNA of alfalfa mosaic virus (AMV RNA 4); tobacco Mosaic Virus (TMV) leader sequence.
Such enhancers include, but are not limited to, cauliflower mosaic virus (CaMV) enhancer, figwort Mosaic Virus (FMV) enhancer, carnation weathering Circovirus (CERV) enhancer, cassava vein mosaic virus (CsVMV) enhancer, mirabilis jalapa mosaic virus (MMV) enhancer, night fragrant tree yellowing leaf curl virus (CmYLCV) enhancer, multan cotton leaf curl virus (CLCuMV), dayflower yellow mottle virus (CoYMV), and peanut chlorosis streak mosaic virus (PCLSV) enhancer.
For monocot applications, the intron includes, but is not limited to, the maize hsp70 intron, the maize ubiquitin intron, adh intron 1, the sucrose synthase intron, or the rice Act1 intron. For dicot applications, the introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "superubiquitin" intron.
The terminator may be a suitable polyadenylation signal sequence that functions in plants, including, but not limited to, polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene, polyadenylation signal sequence derived from the protease inhibitor II (PIN II) gene, polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and polyadenylation signal sequence derived from the alpha-tubulin (alpha-tubulin) gene.
As used herein, "operably linked" refers to the linkage of nucleic acid sequences such that one provides the functionality required of the linked sequence. In the present invention, the "operative linkage" may be a linkage of a promoter to a sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter. "operably linked" when the sequence of interest encodes a protein and expression of the protein is desired indicates that: the promoter is linked to the sequence in such a way that the resulting transcript is translated efficiently. If the linkage of the promoter to the coding sequence is a transcript fusion and expression of the encoded protein is desired, such a linkage is made such that the first translation initiation codon in the resulting transcript is the initiation codon of the coding sequence. Alternatively, if the linkage of the promoter to the coding sequence is a translational fusion and expression of the encoded protein is desired, the linkage is made such that the first translation initiation codon contained in the 5' untranslated sequence is linked to the promoter and is linked in such a way that the resulting translation product is in frame with the translational open reading frame encoding the desired protein. Nucleic acid sequences that may be "operably linked" include, but are not limited to: sequences that provide gene expression functions (i.e., gene expression elements such as promoters, 5 'untranslated regions, introns, protein coding regions, 3' untranslated regions, polyadenylation sites, and/or transcription terminators), sequences that provide DNA transfer and/or integration functions (i.e., T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), sequences that provide selective functions (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scorable marker functions, sequences that facilitate sequence manipulation in vitro or in vivo (i.e., polylinker sequences, site-specific recombination sequences), and sequences that provide replication functions (i.e., bacterial origins of replication, autonomously replicating sequences, centromeric sequences).
The genome of a plant, plant tissue or plant cell as defined in the present invention refers to any genetic material within a plant, plant tissue or plant cell and includes the nuclear and plastid and mitochondrial genomes.
In the present invention, the term "plant part" or "plant tissue" includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, nuclei, ears, cobs, husks, stems, roots, root tips, anthers, and the like.
The herbicide tolerant proteins of the present invention may be applied in a variety of plants, including but not limited to alfalfa, kidney bean, cauliflower, cabbage, carrot, celery, cotton, cucumber, eggplant, lettuce, melon, pea, pepper, pumpkin, radish, rape, spinach, soybean, squash, tomato, arabidopsis, peanut, or watermelon; preferably, the dicotyledonous plant is cucumber, soybean, arabidopsis thaliana, tobacco, cotton, peanut or rape. The monocotyledonous plant includes, but is not limited to, maize, rice, sorghum, wheat, barley, rye, millet, sugarcane, oats or turf grass; preferably, the monocotyledonous plant is maize, rice, sorghum, wheat, barley, millet, sugar cane or oats.
The term "plant transformation" in the context of the present invention means that a herbicide-resistant or tolerant nucleic acid molecule encoding a protoporphyrinogen oxidase of the present invention, alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring desired traits, is cloned into an expression system, which is transformed into a plant cell. The receptor and target expression cassette of the present invention can be introduced into a plant cell in a variety of well-known methods. In the context of a polynucleotide, the term "introduced" (e.g., a nucleotide construct of interest) is intended to mean that the polynucleotide is provided to the plant in such a way that the polynucleotide gains access to or is achieved by the interior of a plant cell. Where more than one polynucleotide is to be introduced, these polynucleotides may be assembled as part of a single nucleotide construct, or as separate nucleotide constructs, and may be located on the same or different transformation vectors. Thus, or as part of a breeding scheme, the polynucleotides may be introduced into a host cell of interest, e.g., in a single transformation event, in separate transformation events in a plant. The methods of the invention do not depend on a particular method for introducing one or more polynucleotides into a plant, but merely obtain access or achievement of the polynucleotide(s) to the interior of at least one cell of a plant. Methods known in the art for introducing one or more polynucleotides into a plant include, but are not limited to, transient transformation methods, stable transformation methods, virus-mediated methods, or genome editing techniques.
The term "stable transformation" refers to the introduction of a foreign gene into the genome of a plant and stable integration into the genome of the plant and any successive generation thereof, resulting in stable inheritance of the foreign gene.
The term "transient transformation" refers to the introduction of a nucleic acid molecule or protein into a plant cell that performs a function but does not integrate into the plant genome, resulting in the inability of a foreign gene to be stably inherited.
The term "genome editing technology" refers to a genome modification technology that can perform precise operations on genome sequences to achieve operations such as site-directed mutagenesis, insertion, deletion, and the like of genes. Currently, the genome editing technologies mainly include HE (home endonucleases), ZFN (Zinc finger nucleases), TALEN (transcription activator-like effector nucleases), CRISPR (Clustered regularly interspaced short palindromic repeats).
Numerous transformation vectors available for plant transformation are known to those skilled in the art, and the genes associated with the present invention can be used in combination with any of the above vectors. The choice of vector will depend on the preferred transformation technique and the species of interest for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers routinely used in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics or related herbicides (this gene was published by Bevan et al in 1983 at pages 184-187 of the "nature Science" volume 304), the gene conferring resistance to the herbicide glufosinate (also known as glufosinate; see White et al, nucl. Acids Rs.18, 1062, 1990, spencer et al, theor. Appl. Genet, 79, 625-631, and U.S. Patents 5561236 and 5276268, hpn genes conferring resistance to the antibiotic hygromycin (Blochinger & Diggelmann, mol. Cell biol.4: 2929-2931), and dnfr genes conferring resistance to methotrexate (Bourouis et al, 1983, EMBO J. 2, 1099-1104), EPSPS genes conferring resistance to glyphosate (U.S. Patents Nos. 4940935 and 5188642), glyphosate N-acetyltransferase (GAT) genes also conferring resistance to glyphosate (mannose N-acetyltransferase (GAT) genes (mannose isomerase genes described in Cassia, 2004, science 304, 1151-0044, published U.S. Pat. Nos. 1154940935 and 20011588467) and mannose isomerase genes 200677657767, and mannose isomerase genes described in U.S. 2009459767, 1990, 2009460579 and 67596).
Methods for regenerating plants are also well known in the art. For example, ti plasmid vectors have been utilized for delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles.
The planting system in the present invention refers to a combination of plants, any one of which exhibits herbicide tolerance and/or herbicide treatments available at different stages of plant development, resulting in plants with high yield and/or reduced damage.
In the present invention, the weeds mean plants that compete with cultivated transgenic plants in the plant growing environment.
The term "control" and/or "control" as used herein means that at least an effective dose of a PPO inhibitor herbicide is applied directly (e.g., by spraying) to the environment in which the plant is growing to minimize weed development and/or stop growth. At the same time, the cultivated transgenic plants should be morphologically normal and can be cultured under conventional methods for the consumption and/or production of the product; preferably, there is reduced plant damage and/or increased plant yield as compared to a non-transgenic wild type plant. The plant with reduced damage, including but not limited to improved stalk resistance, and/or increased kernel weight, etc. The "controlling" and/or "controlling" action of the protoporphyrinogen oxidase on weeds can be independently present and not attenuated and/or eliminated by the presence of other weed-controlling and/or "controlling" substances. In particular, any tissue of a transgenic plant (containing a gene encoding a protoporphyrinogen oxidase of the present invention), which is present and/or produced simultaneously and/or asynchronously, will be such that the protoporphyrinogen oxidase and/or another weed-controlling substance is present, and the presence of such another substance will neither affect the "controlling" and/or "controlling" action of the protoporphyrinogen oxidase on weeds, nor will it result in such a "controlling" and/or "controlling" action being achieved, in whole and/or in part, by such another substance, regardless of the protoporphyrinogen oxidase.
"plant propagules" as used herein include, but are not limited to, vegetative propagules and vegetative propagules. The plant sexual propagules include, but are not limited to, plant seeds; the vegetative propagation body of the plant refers to a vegetative organ or a special tissue of the plant body, and can generate a new plant under the condition of in vitro; the vegetative organ or a specific tissue includes, but is not limited to, roots, stems and leaves, such as: plants with roots as vegetative propagules include strawberry, sweet potato, and the like; plants with stems as vegetative propagules include sugarcane and potato (tubers); plants with leaves as the vegetative propagules include aloe, begonia, and the like.
The present invention can impart new herbicide resistance traits to plants, and no adverse effect on phenotype including yield was observed. The plants of the invention are tolerant to, for example, at least one herbicide subject to 2X, 3X or 4X common application levels. These increased levels of resistance are within the scope of the present invention. For example, a variety of techniques known in the art can be predictably optimized and further developed to increase expression of a given gene.
The invention provides a herbicide tolerance protein, a coding gene and application thereof, and has the following advantages:
1. has wide tolerance to herbicide. The herbicide tolerance protein can show higher tolerance to the PPO inhibitor herbicide, so that the application prospect on plants is wide.
2. Has strong tolerance to herbicide. The herbicide tolerance protein has strong tolerance to a PPO inhibitor herbicide, and all the herbicide tolerance protein has high resistance to oxyfluorfen, saflufenacil and flumioxazin with field concentration of 4 times and sulfentrazone with field concentration of 2 times.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
FIG. 1 is a schematic structural diagram of an Arabidopsis thaliana recombinant expression vector DBN12353 containing the PPO15A nucleotide sequence;
FIG. 2 is a schematic structural diagram of a control recombinant expression vector DBN12353N of the present invention.
Detailed Description
The technical scheme of the use of protoporphyrinogen oxidase of the present invention is further illustrated by the following specific examples.
First example, acquisition and validation of transgenic Arabidopsis plants
1. Obtaining PPO15 gene
The amino acid sequence of the herbicide tolerance protein PPO15 is shown as SEQ ID NO. 1 in a sequence table, and a PPO15A nucleotide sequence which codes the herbicide tolerance protein PPO15 is obtained according to common preference codons of arabidopsis thaliana and soybean, and is shown as SEQ ID NO. 2 in the sequence table.
The amino acid sequence of Escherichia coli (Escherichia coli) protoporphyrinogen oxidase PPO-EC is shown as SEQ ID NO. 3 in the sequence table; a PPO-EC nucleotide sequence corresponding to the Escherichia coli protoporphyrinogen oxidase PPO-EC is coded and is shown as SEQ ID NO. 4 in a sequence table; the PPO-ECA nucleotide sequence of the Escherichia coli protoporphyrinogen oxidase PPO-EC is obtained according to the common preference codon of Arabidopsis thaliana and soybean, and is shown as SEQ ID NO:5 in the sequence table.
The amino acid sequence of Arabidopsis protoporphyrinogen oxidase PPO-AT is shown as SEQ ID NO 6 in the sequence table; encoding a PPO-AT nucleotide sequence corresponding to the Arabidopsis thaliana protoporphyrinogen oxidase PPO-AT, as shown by SEQ ID NO. 7 in the sequence table; and obtaining a PPO-ATA nucleotide sequence which codes the PPO-ATA corresponding to the Arabidopsis protoporphyrinogen oxidase PPO-AT according to the common preference codon of Arabidopsis thaliana and soybean, and the sequence is shown as SEQ ID NO. 8 in the sequence table.
2. Synthesis of the above nucleotide sequence
Respectively connecting the 5 'end and the 3' end of the PPO15A nucleotide sequence, the PPO-ECA nucleotide sequence and the PPO-ATA nucleotide sequence (SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO: 8) with a universal joint primer 1:
5' end universal adaptor primer 1:5 'taagagagatatacatatacatatg-3' is shown as SEQ ID NO:9 in the sequence table;
3' end universal adaptor primer 1:5 'gtggtggtggtggtgctcgag-3' is shown as SEQ ID NO:10 in the sequence table.
3. Respectively constructing arabidopsis thaliana recombinant expression vectors containing PPO15A nucleotide sequence, PPO-ECA nucleotide sequence and PPO-ATA nucleotide sequence
Carrying out double enzyme digestion reaction on a plant expression vector DBNBC-01 by utilizing restriction enzymes Spe I and Asc I, thereby linearizing the plant expression vector, purifying enzyme digestion products to obtain a linearized DBNBC-01 expression vector skeleton (the vector skeleton: pCAMBIA2301 (can be provided by CAMBIA organization)), carrying out recombination reaction on the PPO15A nucleotide sequence (SEQ ID NO: 2) connected with the universal joint primer 1 and the linearized DBNBC-01 expression vector skeleton, and constructing a recombinant expression vector DBN12353 according to the instruction of an In-Fusion seamless connection product kit (Clontech, CA, USA, CAT: 121416) of Takara company, wherein the structural schematic diagram is shown In figure 1 (Spec: spectinomycin gene; RB (Right Border), eFMV (Figwort mosaic Virus) 34S enhancer (SEQ ID NO: 11), prBrCBP (rapeseed eukaryotic elongation factor gene 1 alpha (Tsf 1) promoter (SEQ ID NO: 12), spAtCTP2 (Arabidopsis chloroplast transit peptide) (SEQ ID NO: 13), EPSPS 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 14), tPES 9 (SEQ ID NO: 15), prAtubi10 (Ubiquitin) 10 gene promoter (SEQ ID NO: 16), spAtCLP1 (albino or aqua) transit peptide (SEQ ID NO: 17), PPO15A (PPO 15A) nucleotide sequence (SEQ ID NO: 2), tNos: nopaline synthase gene terminator (SEQ ID NO: 18), prCLP 35S (Cauliflower mosaic Virus) 35S promoter (SEQ ID NO: 19), PAT (Phosphomycin N: 19), and pTAT (SEQ ID NO: 11) -acetyltransferase gene (SEQ ID NO: 20); t35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 21); LB: left border).
Transforming the recombinant expression vector DBN12353 into the escherichia coli T1 competent cells by a heat shock method, wherein the heat shock condition is as follows: 50 μ L of E.coli T1 competent cells, 10 μ L of plasmid DNA (recombinant expression vector DBN 12353), water bath at 42 ℃ for 30s; shaking at 37 deg.C for 1h (shaking at 100 rpm); then, the mixture was cultured on the LB solid plate (tryptone 10g/L, yeast extract 5g/L, naCl 10g/L, agar 15g/L, pH adjusted to 7.5 with NaOH) containing 50mg/L Spectinomycin (Spectinomycin) at 37 ℃ for 12 hours, and white colonies were picked up and cultured overnight at 37 ℃ in an LB liquid medium (tryptone 10g/L, yeast extract 5g/L, naCl 10g/L, spectinomycin 50mg/L, pH adjusted to 7.5 with NaOH). Extracting the plasmid by an alkaline method: centrifuging the bacterial solution at 12000rpm for 1min, removing supernatant, and suspending the precipitated bacterial solution with 100 μ l ice-precooled solution I (25 mM Tris-HCl, 10mM EDTA (ethylene diamine tetraacetic acid), 50mM glucose, pH 8.0); add 200. Mu.L of freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)), invert the tube 4 times, mix and place on ice for 3-5min; adding 150 μ L ice-cold solution III (3M potassium acetate, 5M acetic acid), mixing well immediately, and standing on ice for 5-10min; centrifuging at 4 deg.C and 12000rpm for 5min, adding 2 times volume of anhydrous ethanol into the supernatant, mixing, and standing at room temperature for 5min; centrifuging at 4 deg.C and 12000rpm for 5min, removing supernatant, washing precipitate with 70% ethanol (V/V), and air drying; the precipitate was dissolved by adding 30. Mu.L of TE (10 mM Tris-HCl, 1mM EDTA, pH 8.0) containing RNase (20. Mu.g/mL); bathing in water at 37 deg.C for 30min to digest RNA; storing at-20 deg.C for use. Sequencing and identifying the extracted plasmid, wherein the result shows that the nucleotide sequence of the recombinant expression vector DBN12353 between Spe I and Asc I sites is the nucleotide sequence shown by SEQ ID NO. 2 in the sequence table, namely the PPO15A nucleotide sequence.
According to the method for constructing the recombinant expression vector DBN12353, the PPO-ECA nucleotide sequence and the PPO-ATA nucleotide sequence which are respectively connected with the universal joint primer 1 are respectively subjected to recombination reaction with the linearized DBNBC-01 expression vector skeleton, and the recombinant expression vectors DBN12354 and DBN12355 are sequentially obtained. The correct insertion of the nucleotide sequences in the recombinant expression vectors DBN12354 and DBN12355 was verified by sequencing.
According to the above method for constructing recombinant expression vector DBN12353, a control recombinant expression vector DBN12353N was constructed, and its vector structure is shown in FIG. 2 (Spec: spectinomycin gene; RB: right border; eFMV: enhancer of 34S of figwort mosaic virus (SEQ ID NO: 11), prBrCBP: promoter of rape eukaryotic elongation factor gene 1 alpha (Tsf 1) (SEQ ID NO: 12), spAtCTP2: arabidopsis chloroplast transit peptide (SEQ ID NO: 513), EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 14), tPSE9: terminator of pea RbcS gene (SEQ ID NO: 15), pr35S: cauliflower mosaic virus 35S promoter (SEQ ID NO: 19), PAT: phosphinothricin N-acetyltransferase gene (SEQ ID NO: 20), t35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 21), LB: left border).
4. Agrobacterium transformed by arabidopsis recombinant expression vector
The correctly constructed recombinant expression vectors DBN12353 to DBN12355 and the control recombinant expression vector DBN12353N are transformed into Agrobacterium GV3101 by liquid nitrogen method respectively, and the transformation conditions are as follows: 100. Mu.L of Agrobacterium GV3101, 3. Mu.L of plasmid DNA (recombinant expression vectors DBN12353 to DBN12355, DBN 12353N); placing in liquid nitrogen for 10min, and heating in 37 deg.C water bath for 10min; inoculating the transformed agrobacterium GV3101 into an LB test tube, culturing for 2h at the temperature of 28 ℃ and the rotation speed of 200rpm, smearing the agrobacterium GV3101 on an LB solid plate containing 50mg/L Rifampicin (Rifampicin) and 50mg/L spectinomycin until positive monoclonals grow out, selecting monoclonals, culturing and extracting plasmids, and sequencing and identifying the extracted plasmids, wherein the results show that the structures of the recombinant expression vectors DBN 12353-DBN 12355 and DBN12353N are completely correct.
5. Obtaining transgenic Arabidopsis plants
Wild type Arabidopsis seeds were suspended in 0.1% (w/v) agarose solution. The suspended seeds were stored at 4 ℃ for 2 days to complete the need for dormancy to ensure synchronous germination of the seeds. Horse dung soil was mixed with vermiculite and irrigated with water underground until moist, allowing the soil mixture to drain for 24h. The pretreated seeds were planted on the soil mixture and covered with a moisture mask for 7 days. Germinating the seeds and maintaining the light intensity at constant temperature (22 deg.C) and constant humidity (40-50%) at 120-150 μmol/m 2 s -1 Under long-day conditions (16 h light/8 h dark) plants were grown in a greenhouse. Plants were initially irrigated with Hoagland nutrient solution followed by deionized water to keep the soil moist but not drenched.
Arabidopsis thaliana was transformed using the floral dip method. One or more 15-30mL precultures of LB broth containing spectinomycin (50 mg/L) and rifampicin (10 mg/L) were inoculated with selected Agrobacterium colonies. The preculture was incubated overnight at a temperature of 28 ℃ with constant shaking at 220 rpm. Each preculture was used to inoculate two 500ml cultures of the YEP broth containing spectinomycin (50 mg/L) and rifampicin (10 mg/L) and the cultures were incubated overnight at a temperature of 28 ℃ with continuous shaking. The cells were pelleted by centrifugation at about 4000rpm for 20min at room temperature and the resulting supernatant was discarded. The cell pellet was gently resuspended in 500mL of osmotic medium containing 1/2 XMS salt/B5 vitamins, 10% (w/v) sucrose, 0.044. Mu.M benzylaminopurine (10. Mu.L/L (stock in 1mg/mL DMSO)), and 300. Mu.L/LSilvet L-77. About 1 month old Arabidopsis plants were soaked for 5min in the osmotic medium containing resuspended cells to ensure that the latest inflorescences were submerged. Then, the Arabidopsis thaliana plants were placed on their sides and covered, and were kept moist for 24 hours in a dark environment, and then were normally cultured at a temperature of 22 ℃ under a 16 hour light/8 hour dark photoperiod. The seeds were harvested after about 4 weeks.
Newly harvested (PPO 15A nucleotide sequence, PPO-ECA nucleotide sequence, PPO-ATA nucleotide sequence and control vector DBN 12353N) T 1 The seeds were dried at room temperature for 7 days. Seeds were planted in 26.5cm by 51cm germinating discs receiving 200mg T per disc 1 Seeds (approximately 10000 seeds) which had been previously suspended in distilled water and stored at a temperature of 4 ℃ for 2 days to fulfill the need for dormancy to ensure synchronous germination of the seeds.
Mixing the horse dung with vermiculite, irrigating with water until the mixture is wet, and draining water by gravity. The pretreated seeds were evenly seeded on the soil mixture with a pipette and covered with a moisture hood for 4-5 days. The hood was removed 1 day prior to initial transformant selection using post-emergence glufosinate-ammonium spray (selection for co-transformed PAT gene).
After 7 planting Days (DAP) and again at 11DAP, using a DeVilbiss compressed air nozzle, T is sprayed with a 0.2% solution of Liberty herbicide (200 g ai/L glufosinate) at a spray volume of 10 mL/dish (703L/ha) 1 Plants (cotyledon stage and 2-4 leaf stage, respectively) to provide an effective amount of glufosinate-ammonium per application of 280 gai/ha. Surviving plants (actively growing plants) were identified 4-7 days after the last spray and individuallyTransplanting into 7cm × 7cm square pots (3-5 per dish) prepared from horse dung and vermiculite. Transplanted plants were covered with a moisture-retaining hood for 3-4 days and either placed in a 22 ℃ culture room as before or directly transferred to a greenhouse. The caps were then removed and plants were planted at least 1 day into a greenhouse (temperature 22 + -5 deg.C, 50 + -30 deg.C RH,14h light: 10h dark, minimum 500. Mu.E/m, light: 10h dark) before testing the ability of PPO15A nucleotide sequences, PPO-ECA nucleotide sequences, PPO-ATA nucleotide sequences and control vectors to provide tolerance to PPO inhibitor herbicides 2 s -1 Natural + supplemental light).
6. Detection of herbicide tolerance effects in transgenic Arabidopsis plants
First, transformed Arabidopsis thaliana T was selected using glufosinate herbicide 1 And (5) plant growing. Respectively transferring the Arabidopsis thaliana T with PPO15A nucleotide sequence 1 Plant (PPO 15A), arabidopsis thaliana T with PPO-ECA nucleotide sequence transferred 1 Plant (PPO-ECA), arabidopsis thaliana T with PPO-ATA nucleotide sequence transferred therein 1 Plant (PPO-ATA), arabidopsis thaliana T transferred into control vector 1 Plants (control vehicle) and wild-type arabidopsis thaliana plants (CK) 24 plants (18 days after sowing) were tested with 3 concentrations of oxyfluorfen (180 g ai/ha (1-fold field strength, 1 ×), 720g ai/ha (4-fold field strength, 4 ×) and 0g ai/ha (water, 0 × 0)), 3 concentrations of saflufenacil (25 g ai/ha (1-fold field strength, 1 × 1), 100g ai/ha (4-fold field strength, 4 × 2) and 0g ai/ha (water, 0 ×), 3 concentrations of flumioxazin (60 g ai/ha (1-fold field strength, 1 ×), 240g ai/ha (4-fold field strength, 4 ×) and 0g ai/ha (water, 0 ×) respectively, and 3 concentrations of alachlor (450 g ai/ha, 1 ×,0 ×) and 3 concentrations of butachlor/ha (water, 0 ×) at 900-fold field strength. After 7 days of spraying (7 DAT), the extent of damage of the herbicide to each plant was evaluated according to the plant average damage percentage scale (plant average damage percentage = leaf damage area/total leaf area x 100%), i.e. the pesticide damage scale: the 0 grade is that the growth condition is basically consistent with the spraying of a blank solvent (water), the 1 grade is that the average damage percentage of the plants is less than 10 percent, the 2 grade is that the average damage percentage of the plants is more than 10 percent, and the 3 grade is that the average damage percentage of the plants is 100 percent. The plant growth status is divided into 0 grade and 1 gradeThe plants with high resistance are classified into 2 grades according to the growth conditions of the plants, the plants with medium and low resistance are classified into 3 grades according to the growth conditions of the plants, and the plants with medium and low resistance are not resistant. The results of the experiments are shown in tables 1-4.
TABLE 1 Arabidopsis thaliana T with PPO15A, PPO-ECA, PPO-ATA and control vectors 1 Experimental result of tolerance of plants to oxyfluorfen
Figure BDA0003061498190000231
For Arabidopsis, 180g of ai/ha oxyfluorfen herbicide is an effective dose to distinguish sensitive plants from those with an average level of resistance. The results in Table 1 show that (1) both PPO15A and PPO-ECA are capable of developing varying degrees of tolerance to oxyfluorfen compared to the control vehicle and CK, while PPO-ATA is essentially intolerant to oxyfluorfen; (2) For oxyfluorfen with 1 time of field concentration, the phytotoxicity grades of PPO15A are all 0 grade, and the phytotoxicity grades of PPO-ECA are all 1 grade; (3) For oxyfluorfen with 4 times field concentration, PPO15A shows high tolerance, while 50% of PPO-ECA has no tolerance, and about 50% of PPO-ECA only has medium and low resistance. This demonstrates that PPO15A has a significantly increased tolerance to oxyfluorfen.
TABLE 2 Arabidopsis thaliana T transferred with PPO15A nucleotide sequence, PPO-ECA nucleotide sequence, PPO-ATA nucleotide sequence and contrast carrier 1 Experimental result of tolerance of plants to saflufenacil
Figure BDA0003061498190000241
For Arabidopsis, 25g ai/ha saflufenacil herbicide is an effective dose to distinguish sensitive plants from plants with an average level of resistance. The results in table 2 show that (1) both PPO15A and PPO-ECA are able to produce varying degrees of tolerance to saflufenacil, whereas PPO-ATA is not tolerant to saflufenacil, compared to the control vehicle and CK; (2) For 1 time of field concentration of saflufenacil, the phytotoxicity grades of PPO15A are all 0 grade, while the phytotoxicity grade of about 33 percent of plants in PPO-ECA is 1 grade; (3) With 4 times field concentration of saflufenacil, PPO15A all showed high resistance tolerance, while about 25% of the plants in PPO-ECA did not have high resistance tolerance. This demonstrates that PPO15A has significantly increased tolerance to saflufenacil.
TABLE 3 Arabidopsis thaliana T with PPO15A, PPO-ECA, PPO-ATA and control vectors 1 Experimental result of tolerance of plants to flumioxazin
Figure BDA0003061498190000251
For Arabidopsis, 60g ai/ha flumioxazin herbicide is an effective dose to distinguish sensitive plants from those with an average level of resistance. The results in table 3 show that both PPO15A and PPO-ECA are able to produce varying degrees of tolerance to flumioxazin compared to the control vector and CK, whereas PPO-ATA is essentially not tolerant to flumioxazin; (2) For 1-fold field concentration of flumioxazin, PPO15A all showed high resistance tolerance, whereas about 66% of the plants in PPO-ECA did not. (3) For 4 times of field concentration of flumioxazin, PPO15A all showed high resistance tolerance, while PPO-ECA did not have high resistance tolerance. This demonstrates that PPO15A has significantly increased tolerance to flumioxazin.
TABLE 4 Arabidopsis thaliana T with PPO15A, PPO-ECA, PPO-ATA and control vectors 1 Experimental result of tolerance of plants to sulfentrazone
Figure BDA0003061498190000252
Figure BDA0003061498190000261
For Arabidopsis, 450g of ai/ha sulfentrazone herbicide is an effective dose to distinguish sensitive plants from those with an average level of resistance. The results in table 4 show that (1) both PPO15A and PPO-ECA are able to produce varying degrees of tolerance to sulfentrazone compared to the control vehicle and CK, whereas PPO-ATA is not tolerant to sulfentrazone; (2) For 1-fold field concentration of sulfentrazone, PPO15A all shows high-resistance tolerance, while about 50% of plants in PPO-ECA do not have high-resistance tolerance; (3) For 4 times of field concentration of sulfentrazone, PPO15A all shows high-resistance tolerance, while PPO-ECA does not have high-resistance tolerance. This demonstrates that PPO15A has significantly increased tolerance to sulfentrazone.
The applicant needs to demonstrate that the tolerance of plants to herbicides directly corresponds to the yield of plants, that high-resistant plants are not substantially affected by herbicides and thus do not affect the yield of plants, and that the yield of low-resistant plants is much reduced compared to high-resistant plants.
The protoporphyrinogen oxidase PPO15 provided by the invention can not only endow arabidopsis thaliana with better tolerance to PPO inhibitor herbicides, but also endow other dicotyledonous plants (such as soybean, cotton, peanut and the like) and monocotyledonous plants (such as corn, rice, wheat and the like) with better tolerance.
In conclusion, the invention discloses that protoporphyrinogen oxidase PPO15 can endow plants with higher tolerance to PPO inhibitor herbicides, and can tolerate oxyfluorfen, saflufenacil and flumioxazin with the field concentration of at least 4 times and sulfentrazone with the field concentration of 2 times, so that the application prospect on the plants is wide.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
<110> Beijing Dabei agricultural Biotechnology Co., ltd
<120> herbicide tolerance protein, encoding gene and use thereof
<130> DBNBC155
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 174
<212> PRT
<213> Artificial Sequence-amino acid Sequence of PPO15 (Artificial Sequence)
<400> 1
Met Arg Ala Leu Leu Leu Tyr Ser Ser Gln Glu Gly Gln Thr Arg Lys
1 5 10 15
Ile Ile Gln Arg Ile Ala Ala Gln Met Pro Glu Tyr Thr Cys Glu Val
20 25 30
Gln Asp Leu His Gln Ser Ile Asp Ile Asp Trp Ala Glu Tyr Asp Lys
35 40 45
Val Leu Ile Gly Ala Ser Ile Arg Tyr Gly Arg Leu Asn Pro Ala Leu
50 55 60
Tyr Arg Phe Ile Glu His His Leu Val Gly Leu Thr Ser Arg Lys Ala
65 70 75 80
Ala Phe Phe Cys Val Asn Leu Thr Ala Arg Lys Glu Gln Gln Gly Lys
85 90 95
Asp Thr Pro Gln Gly Ser Ala Tyr Ile Gln Thr Phe Leu Lys Lys Ser
100 105 110
Ala Trp Gln Pro Glu Arg Ile Ala Val Phe Ala Gly Ala Leu Arg Tyr
115 120 125
Pro Arg Tyr Arg Trp Ile Asp Lys Val Met Ile Gln Leu Ile Met Arg
130 135 140
Met Thr Gly Gly Glu Thr Asp Thr Ser Gln Glu Val Glu Tyr Thr Asn
145 150 155 160
Trp Asp Lys Val Val Lys Phe Ala Glu Gln Phe Arg Asn Trp
165 170
<210> 2
<211> 525
<212> DNA
<213> Artificial Sequence-PPO 15A nucleotide Sequence (Artificial Sequence)
<400> 2
atgagagctt tactgctcta ctcttctcaa gaaggacaga caaggaagat catccagaga 60
attgcagcac aaatgcctga gtacacttgt gaagtgcaag atcttcacca aagtatagat 120
attgattggg ctgaatatga taaagttttg ataggtgctt ccatccgata tggtcgtctc 180
aaccccgcat tgtatagatt cattgagcat catcttgtag gattgacgag ccgaaaagct 240
gccttctttt gcgtcaattt aactgcaaga aaagaacaac aagggaaaga tacaccacaa 300
ggcagtgctt acatacaaac atttctaaag aaatcagctt ggcagcctga aaggatagct 360
gttttcgccg gagcactgcg ttatccaagg tacagatgga ttgacaaggt gatgattcag 420
cttatcatga ggatgactgg tggagaaact gacacctcac aggaggttga gtatacaaac 480
tgggataagg ttgtgaagtt tgctgagcag tttagaaatt ggtga 525
<210> 3
<211> 181
<212> PRT
<213> amino acid sequence of PPO-EC (Escherichia coli)
<400> 3
Met Lys Thr Leu Ile Leu Phe Ser Thr Arg Asp Gly Gln Thr Arg Glu
1 5 10 15
Ile Ala Ser Tyr Leu Ala Ser Glu Leu Lys Glu Leu Gly Ile Gln Ala
20 25 30
Asp Val Ala Asn Val His Arg Ile Glu Glu Pro Gln Trp Glu Asn Tyr
35 40 45
Asp Arg Val Val Ile Gly Ala Ser Ile Arg Tyr Gly His Tyr His Ser
50 55 60
Ala Phe Gln Glu Phe Val Lys Lys His Ala Thr Arg Leu Asn Ser Met
65 70 75 80
Pro Ser Ala Phe Tyr Ser Val Asn Leu Val Ala Arg Lys Pro Glu Lys
85 90 95
Arg Thr Pro Gln Thr Asn Ser Tyr Ala Arg Lys Phe Leu Met Asn Ser
100 105 110
Gln Trp Arg Pro Asp Arg Cys Ala Val Ile Ala Gly Ala Leu Arg Tyr
115 120 125
Pro Arg Tyr Arg Trp Tyr Asp Arg Phe Met Ile Lys Leu Ile Met Lys
130 135 140
Met Ser Gly Gly Glu Thr Asp Thr Arg Lys Glu Val Val Tyr Thr Asp
145 150 155 160
Trp Glu Gln Val Ala Asn Phe Ala Arg Glu Ile Ala His Leu Thr Asp
165 170 175
Lys Pro Thr Leu Lys
180
<210> 4
<211> 546
<212> DNA
<213> PPO-EC nucleotide sequence (Escherichia coli)
<400> 4
gtgaaaacat taattctttt ctcaacaagg gacggacaaa cgcgcgagat tgcctcctac 60
ctggcttcgg aactgaaaga actggggatc caggcggatg tcgccaatgt gcaccgcatt 120
gaagaaccac agtgggaaaa ctatgaccgt gtggtcattg gtgcttctat tcgctatggt 180
cactaccatt cagcgttcca ggaatttgtc aaaaaacatg cgacgcggct gaattcgatg 240
ccgagcgcct tttactccgt gaatctggtg gcgcgcaaac cggagaagcg tactccacag 300
accaacagct acgcgcgcaa gtttctgatg aactcgcaat ggcgtcccga tcgctgcgcg 360
gtcattgccg gggcgctgcg ttacccacgt tatcgctggt acgaccgttt tatgatcaag 420
ctgattatga agatgtcagg cggtgaaacg gatacgcgca aagaagttgt ctataccgat 480
tgggagcagg tggcgaattt cgcccgagaa atcgcccatt taaccgacaa accgacgctg 540
aaataa 546
<210> 5
<211> 546
<212> DNA
<213> Artificial Sequence-PPO-ECA nucleotide Sequence (Artificial Sequence)
<400> 5
atgaaaacac ttatcttgtt ctcaactaga gatggacaga caagagagat tgcttcttac 60
ttggcttcag aacttaagga gttgggtatt caagctgatg ttgctaatgt tcatagaatt 120
gaagagcctc agtgggaaaa ctatgataga gttgttattg gagcttctat tagatatggt 180
cattaccatt cagcttttca agagttcgtt aagaaacatg ctactagact taactctatg 240
ccatcagctt tttactctgt taacttggtt gctagaaagc ctgagaaaag aactccacaa 300
acaaactctt acgctagaaa gttccttatg aactcacagt ggagacctga tagatgcgct 360
gttattgctg gtgctcttag atatccaaga tacagatggt acgatagatt catgatcaag 420
ttgattatga aaatgtctgg aggtgaaact gatacaagaa aggaggttgt ttacacagat 480
tgggaacagg ttgctaattt cgctagagag attgctcatc ttactgataa gccaacattg 540
aaatga 546
<210> 6
<211> 537
<212> PRT
<213> amino acid sequence of PPO-AT (Arabidopsis thaliana)
<400> 6
Met Glu Leu Ser Leu Leu Arg Pro Thr Thr Gln Ser Leu Leu Pro Ser
1 5 10 15
Phe Ser Lys Pro Asn Leu Arg Leu Asn Val Tyr Lys Pro Leu Arg Leu
20 25 30
Arg Cys Ser Val Ala Gly Gly Pro Thr Val Gly Ser Ser Lys Ile Glu
35 40 45
Gly Gly Gly Gly Thr Thr Ile Thr Thr Asp Cys Val Ile Val Gly Gly
50 55 60
Gly Ile Ser Gly Leu Cys Ile Ala Gln Ala Leu Ala Thr Lys His Pro
65 70 75 80
Asp Ala Ala Pro Asn Leu Ile Val Thr Glu Ala Lys Asp Arg Val Gly
85 90 95
Gly Asn Ile Ile Thr Arg Glu Glu Asn Gly Phe Leu Trp Glu Glu Gly
100 105 110
Pro Asn Ser Phe Gln Pro Ser Asp Pro Met Leu Thr Met Val Val Asp
115 120 125
Ser Gly Leu Lys Asp Asp Leu Val Leu Gly Asp Pro Thr Ala Pro Arg
130 135 140
Phe Val Leu Trp Asn Gly Lys Leu Arg Pro Val Pro Ser Lys Leu Thr
145 150 155 160
Asp Leu Pro Phe Phe Asp Leu Met Ser Ile Gly Gly Lys Ile Arg Ala
165 170 175
Gly Phe Gly Ala Leu Gly Ile Arg Pro Ser Pro Pro Gly Arg Glu Glu
180 185 190
Ser Val Glu Glu Phe Val Arg Arg Asn Leu Gly Asp Glu Val Phe Glu
195 200 205
Arg Leu Ile Glu Pro Phe Cys Ser Gly Val Tyr Ala Gly Asp Pro Ser
210 215 220
Lys Leu Ser Met Lys Ala Ala Phe Gly Lys Val Trp Lys Leu Glu Gln
225 230 235 240
Asn Gly Gly Ser Ile Ile Gly Gly Thr Phe Lys Ala Ile Gln Glu Arg
245 250 255
Lys Asn Ala Pro Lys Ala Glu Arg Asp Pro Arg Leu Pro Lys Pro Gln
260 265 270
Gly Gln Thr Val Gly Ser Phe Arg Lys Gly Leu Arg Met Leu Pro Glu
275 280 285
Ala Ile Ser Ala Arg Leu Gly Ser Lys Val Lys Leu Ser Trp Lys Leu
290 295 300
Ser Gly Ile Thr Lys Leu Glu Ser Gly Gly Tyr Asn Leu Thr Tyr Glu
305 310 315 320
Thr Pro Asp Gly Leu Val Ser Val Gln Ser Lys Ser Val Val Met Thr
325 330 335
Val Pro Ser His Val Ala Ser Gly Leu Leu Arg Pro Leu Ser Glu Ser
340 345 350
Ala Ala Asn Ala Leu Ser Lys Leu Tyr Tyr Pro Pro Val Ala Ala Val
355 360 365
Ser Ile Ser Tyr Pro Lys Glu Ala Ile Arg Thr Glu Cys Leu Ile Asp
370 375 380
Gly Glu Leu Lys Gly Phe Gly Gln Leu His Pro Arg Thr Gln Gly Val
385 390 395 400
Glu Thr Leu Gly Thr Ile Tyr Ser Ser Ser Leu Phe Pro Asn Arg Ala
405 410 415
Pro Pro Gly Arg Ile Leu Leu Leu Asn Tyr Ile Gly Gly Ser Thr Asn
420 425 430
Thr Gly Ile Leu Ser Lys Ser Glu Gly Glu Leu Val Glu Ala Val Asp
435 440 445
Arg Asp Leu Arg Lys Met Leu Ile Lys Pro Asn Ser Thr Asp Pro Leu
450 455 460
Lys Leu Gly Val Arg Val Trp Pro Gln Ala Ile Pro Gln Phe Leu Val
465 470 475 480
Gly His Phe Asp Ile Leu Asp Thr Ala Lys Ser Ser Leu Thr Ser Ser
485 490 495
Gly Tyr Glu Gly Leu Phe Leu Gly Gly Asn Tyr Val Ala Gly Val Ala
500 505 510
Leu Gly Arg Cys Val Glu Gly Ala Tyr Glu Thr Ala Ile Glu Val Asn
515 520 525
Asn Phe Met Ser Arg Tyr Ala Tyr Lys
530 535
<210> 7
<211> 1614
<212> DNA
<213> PPO-AT nucleotide sequence (Arabidopsis thaliana)
<400> 7
atggagttat ctcttctccg tccgacgact caatcgcttc ttccgtcgtt ttcgaagccc 60
aatctccgat taaatgttta taagcctctt agactccgtt gttcagtggc cggtggacca 120
accgtcggat cttcaaaaat cgaaggcgga ggaggcacca ccatcacgac ggattgtgtg 180
attgtcggcg gaggtattag tggtctttgc atcgctcagg cgcttgctac taagcatcct 240
gatgctgctc cgaatttaat tgtgaccgag gctaaggatc gtgttggagg caacattatc 300
actcgtgaag agaatggttt tctctgggaa gaaggtccca atagttttca accgtctgat 360
cctatgctca ctatggtggt agatagtggt ttgaaggatg atttggtgtt gggagatcct 420
actgcgccaa ggtttgtgtt gtggaatggg aaattgaggc cggttccatc gaagctaaca 480
gacttaccgt tctttgattt gatgagtatt ggtgggaaga ttagagctgg ttttggtgca 540
cttggcattc gaccgtcacc tccaggtcgt gaagaatctg tggaggagtt tgtacggcgt 600
aacctcggtg atgaggtttt tgagcgcctg attgaaccgt tttgttcagg tgtttatgct 660
ggtgatcctt caaaactgag catgaaagca gcgtttggga aggtttggaa actagagcaa 720
aatggtggaa gcataatagg tggtactttt aaggcaattc aggagaggaa aaacgctccc 780
aaggcagaac gagacccgcg cctgccaaaa ccacagggcc aaacagttgg ttctttcagg 840
aagggacttc gaatgttgcc agaagcaata tctgcaagat taggtagcaa agttaagttg 900
tcttggaagc tctcaggtat cactaagctg gagagcggag gatacaactt aacatatgag 960
actccagatg gtttagtttc cgtgcagagc aaaagtgttg taatgacggt gccatctcat 1020
gttgcaagtg gtctcttgcg ccctctttct gaatctgctg caaatgcact ctcaaaacta 1080
tattacccac cagttgcagc agtatctatc tcgtacccga aagaagcaat ccgaacagaa 1140
tgtttgatag atggtgaact aaagggtttt gggcaattgc atccacgcac gcaaggagtt 1200
gaaacattag gaactatcta cagctcctca ctctttccaa atcgcgcacc gcccggaaga 1260
attttgctgt tgaactacat tggcgggtct acaaacaccg gaattctgtc caagtctgaa 1320
ggtgagttag tggaagcagt tgacagagat ttgaggaaaa tgctaattaa gcctaattcg 1380
accgatccac ttaaattagg agttagggta tggcctcaag ccattcctca gtttctagtt 1440
ggtcactttg atatccttga cacggctaaa tcatctctaa cgtcttcggg ctacgaaggg 1500
ctatttttgg gtggcaatta cgtcgctggt gtagccttag gccggtgtgt agaaggcgca 1560
tatgaaaccg cgattgaggt caacaacttc atgtcacggt acgcttacaa gtaa 1614
<210> 8
<211> 1614
<212> DNA
<213> Artificial Sequence-PPO-ATA nucleotide Sequence (Artificial Sequence)
<400> 8
atggagttgt ctcttttaag accaacaaca caatctttgc ttccttcgtt ctctaaaccc 60
aatctcagat tgaatgtcta caaaccacta cgattgcgat gctctgtggc tggtggtcct 120
actgttggaa gttcaaagat agaaggaggc ggtggtacaa ccatcactac agattgtgtg 180
attgtaggtg gtggcatttc gggtctttgc attgctcaag ctctcgctac aaaacatcct 240
gacgctgcgc ccaacttaat agtgacggaa gcaaaagaca gagttggtgg gaatattatc 300
acaagagaag agaatgggtt tttatgggaa gaaggaccga acagctttca accgtcagat 360
ccaatgctta caatggtggt tgattctgga ctgaaagatg atctcgtact tggtgatccg 420
acggccccaa ggtttgtttt gtggaatgga aaattgcgac ctgttccaag caaattaaca 480
gatttacctt tcttcgatct catgagtata ggtgggaaga tcagggccgg ttttggtgca 540
ctgggaatcc gtccttctcc tcctggaaga gaggagagcg tagaagagtt tgtgcgtagg 600
aacctgggag atgaagtttt tgagaggttg attgagccat tttgctcagg cgtttatgca 660
ggtgacccga gcaagctgtc tatgaaggct gctttcggca aagtttggaa gctcgagcag 720
aatggtggat ctattattgg aggaactttc aaggccattc aagaaagaaa gaatgctcct 780
aaagcagaga gggaccctag acttcccaag cctcaaggac aaaccgtcgg ctctttcagg 840
aaaggattaa gaatgttgcc agaggctatc agtgcgcgcc ttggaagcaa ggttaaactt 900
tcatggaaac tatctgggat aaccaaactt gaatcaggag gttacaattt aacttatgaa 960
actccggatg gacttgtttc tgtacagagt aagtcagttg tgatgacggt tccttcacat 1020
gtcgcttctg gtctgttgcg tccattatcc gaatctgccg ctaatgcatt atccaaactc 1080
tactatcctc ccgtggcagc agtcagtatc tcttatccaa aagaagctat tagaacagaa 1140
tgtcttattg atggtgagct taagggattt ggacagcttc accctcgaac tcagggtgtg 1200
gaaacactgg ggactattta tagttcctcc ttgtttccta accgtgcacc acctgggaga 1260
attcttcttc taaactacat tggagggtca acaaacaccg gtatcctatc gaagagtgag 1320
ggagaattag ttgaagctgt cgacagagac ctaaggaaga tgctgataaa gccaaattca 1380
accgaccccc tcaagctcgg agttcgcgtg tggcctcagg caattccgca attcctggta 1440
ggacattttg atatattgga tactgccaaa tcttctctta cttcatcggg ctacgaaggt 1500
ttgtttcttg gcggaaacta tgttgctgga gttgccttgg ggcgctgtgt agagggtgct 1560
tatgagactg caatagaggt gaacaatttc atgtctagat atgcttacaa gtga 1614
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence-5' terminal Universal adaptor primer 1 (Artificial Sequence)
<400> 9
taagaaggag atatacatat g 21
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence-3' terminal Universal adaptor primer 1 (Artificial Sequence)
<400> 10
gtggtggtgg tggtgctcga g 21
<210> 11
<211> 542
<212> DNA
<213> enhancer of 34S of Figwort mosaic virus (Figwort mosaic virus)
<400> 11
aattctcagt ccaaagcctc aacaaggtca gggtacagag tctccaaacc attagccaaa 60
agctacagga gatcaatgaa gaatcttcaa tcaaagtaaa ctactgttcc agcacatgca 120
tcatggtcag taagtttcag aaaaagacat ccaccgaaga cttaaagtta gtgggcatct 180
ttgaaagtaa tcttgtcaac atcgagcagc tggcttgtgg ggaccagaca aaaaaggaat 240
ggtgcagaat tgttaggcgc acctaccaaa agcatctttg cctttattgc aaagataaag 300
cagattcctc tagtacaagt ggggaacaaa ataacgtgga aaagagctgt cctgacagcc 360
cactcactaa tgcgtatgac gaacgcagtg acgaccacaa aagaattagc ttgagctcag 420
gatttagcag cattccagat tgggttcaat caacaaggta cgagccatat cactttattc 480
aaattggtat cgccaaaacc aagaaggaac tcccatcctc aaaggtttgt aaggaagaat 540
tc 542
<210> 12
<211> 1534
<212> DNA
<213> eukaryotic elongation factor gene 1 α of Brassica napus (promoter Brassica napus of Tsf 1)
<400> 12
gattatgaca ttgctcgtgg aatgggacag ttatggtatt tttttgtaat aaattgtttc 60
cattgtcatg agattttgag gttaatctat gagacattga atcacttagc attagggatt 120
aagtagtcac aaatcgcatt caagaagctg aagaacacgt tatggtctaa tggttgtgtc 180
tctttattag aaaatgttgg tcagtagcta tatgcactgt ttctgtaaaa ccatgttggt 240
gttgtgttta tttcaagaca catgttgagt ccgttgattc agagcttttg tcttcgaaca 300
caatctagag agcaaatttg ggttcaattt ggatatcaat atgggttcga ttcagataga 360
acaataccct ttgatgtcgg gtttcgattt ggttgagatt catttttatc gggtttggtt 420
cgattttcga attcggttta ttcgccccct catagcatct acattctgca gattaatgta 480
caagttatgg aaaaaaaaat gtggttttcg aattcggttt agtagctaaa cgttgcttgc 540
agtgtagtta tgggaattat gaaacacgac cgaaggtatc aattagaaga acgggtcaac 600
gggtaagtat tgagaaatta ccggagggta aaaataaaca gtattctttt tttttcttaa 660
cgaccgacca aggttaaaaa aagaaaggag gacgagatac aggggcatga ctgtaattgt 720
acataagatc tgatctttaa accctaggtt tccttcgcat cagcaactat aaataattct 780
gagtgccact cttcttcatt cctagatctt tcgccttatc gctttagctg aggtaagcct 840
ttctatacgc atagacgctc tcttttctct tctctcgatc ttcgttgaaa cggtcctcga 900
tacgcatagg atcggttaga atcgttaatc tatcgtctta gatcttcttg attgttgaat 960
tgagcttcta ggatgtattg tatcatgtga tggatagttg attggatctc tttgagtgaa 1020
ctagctagct ttcgatgcgt gtgatttcag tataacagga tccgatgaat tatagctcgc 1080
ttacaattaa tctctgcaga tttattgttt aatcttggat ttgatgctcg ttgttgatag 1140
aggatcgttt atagaactta ttgattctgg aattgagctt gtgtgatgta ttgtatcatg 1200
tgatcgatag ctgatggatc tatttgagtg aactagcgta cgatcttaag atgagtgtgt 1260
attgtgaact gatgattcga gatcagcaaa acaagatctg atgatatctt cgtcttgtat 1320
gcatcttgaa tttcatgatt ttttattaat tatagctcgc ttagctcaaa ggatagagca 1380
ccacaaaatt ttattgtggt agaaatcggt tcgattccga tagcagctta ctgtgatgaa 1440
tgattttgag atttggtatt tgatatatgt ctactgtgtt gaatgatcgt ttatgcattg 1500
tttaatcgct gcagatttgc attgacaagt agcc 1534
<210> 13
<211> 228
<212> DNA
<213> Arabidopsis chloroplast transit peptide (Arabidopsis thaliana)
<400> 13
atggcgcaag ttagcagaat ctgcaatggt gtgcagaacc catctcttat ctccaatctc 60
tcgaaatcca gtcaacgcaa atctccctta tcggtttctc tgaagacgca gcagcatcca 120
cgagcttatc cgatttcgtc gtcgtgggga ttgaagaaga gtgggatgac gttaattggc 180
tctgagcttc gtcctcttaa ggtcatgtct tctgtttcca cggcgtgc 228
<210> 14
<211> 1368
<212> DNA
<213> 5-enolpyruvylshikimate-3-phosphate synthase gene (Artificial Sequence)
<400> 14
atgcttcacg gtgcaagcag ccgtccagca actgctcgta agtcctctgg tctttctgga 60
accgtccgta ttccaggtga caagtctatc tcccacaggt ccttcatgtt tggaggtctc 120
gctagcggtg aaactcgtat caccggtctt ttggaaggtg aagatgttat caacactggt 180
aaggctatgc aagctatggg tgccagaatc cgtaaggaag gtgatacttg gatcattgat 240
ggtgttggta acggtggact ccttgctcct gaggctcctc tcgatttcgg taacgctgca 300
actggttgcc gtttgactat gggtcttgtt ggtgtttacg atttcgatag cactttcatt 360
ggtgacgctt ctctcactaa gcgtccaatg ggtcgtgtgt tgaacccact tcgcgaaatg 420
ggtgtgcagg tgaagtctga agacggtgat cgtcttccag ttaccttgcg tggaccaaag 480
actccaacgc caatcaccta cagggtacct atggcttccg ctcaagtgaa gtccgctgtt 540
ctgcttgctg gtctcaacac cccaggtatc accactgtta tcgagccaat catgactcgt 600
gaccacactg aaaagatgct tcaaggtttt ggtgctaacc ttaccgttga gactgatgct 660
gacggtgtgc gtaccatccg tcttgaaggt cgtggtaagc tcaccggtca agtgattgat 720
gttccaggtg atccatcctc tactgctttc ccattggttg ctgccttgct tgttccaggt 780
tccgacgtca ccatccttaa cgttttgatg aacccaaccc gtactggtct catcttgact 840
ctgcaggaaa tgggtgccga catcgaagtg atcaacccac gtcttgctgg tggagaagac 900
gtggctgact tgcgtgttcg ttcttctact ttgaagggtg ttactgttcc agaagaccgt 960
gctccttcta tgatcgacga gtatccaatt ctcgctgttg cagctgcatt cgctgaaggt 1020
gctaccgtta tgaacggttt ggaagaactc cgtgttaagg aaagcgaccg tctttctgct 1080
gtcgcaaacg gtctcaagct caacggtgtt gattgcgatg aaggtgagac ttctctcgtc 1140
gtgcgtggtc gtcctgacgg taagggtctc ggtaacgctt ctggagcagc tgtcgctacc 1200
cacctcgatc accgtatcgc tatgagcttc ctcgttatgg gtctcgtttc tgaaaaccct 1260
gttactgttg atgatgctac tatgatcgct actagcttcc cagagttcat ggatttgatg 1320
gctggtcttg gagctaagat cgaactctcc gacactaagg ctgcttga 1368
<210> 15
<211> 643
<212> DNA
<213> terminator of pea RbcS gene (Pisum sativum)
<400> 15
agctttcgtt cgtatcatcg gtttcgacaa cgttcgtcaa gttcaatgca tcagtttcat 60
tgcgcacaca ccagaatcct actgagtttg agtattatgg cattgggaaa actgtttttc 120
ttgtaccatt tgttgtgctt gtaatttact gtgtttttta ttcggttttc gctatcgaac 180
tgtgaaatgg aaatggatgg agaagagtta atgaatgata tggtcctttt gttcattctc 240
aaattaatat tatttgtttt ttctcttatt tgttgtgtgt tgaatttgaa attataagag 300
atatgcaaac attttgtttt gagtaaaaat gtgtcaaatc gtggcctcta atgaccgaag 360
ttaatatgag gagtaaaaca cttgtagttg taccattatg cttattcact aggcaacaaa 420
tatattttca gacctagaaa agctgcaaat gttactgaat acaagtatgt cctcttgtgt 480
tttagacatt tatgaacttt cctttatgta attttccaga atccttgtca gattctaatc 540
attgctttat aattatagtt atactcatgg atttgtagtt gagtatgaaa atatttttta 600
atgcatttta tgacttgcca attgattgac aacatgcatc aat 643
<210> 16
<211> 1322
<212> DNA
<213> Arabidopsis Ubiquitin (Arabidopsis thaliana gene promoter of Ubiquitin 10)
<400> 16
gtcgacctgc aggtcaacgg atcaggatat tcttgtttaa gatgttgaac tctatggagg 60
tttgtatgaa ctgatgatct aggaccggat aagttccctt cttcatagcg aacttattca 120
aagaatgttt tgtgtatcat tcttgttaca ttgttattaa tgaaaaaata ttattggtca 180
ttggactgaa cacgagtgtt aaatatggac caggccccaa ataagatcca ttgatatatg 240
aattaaataa caagaataaa tcgagtcacc aaaccacttg ccttttttaa cgagacttgt 300
tcaccaactt gatacaaaag tcattatcct atgcaaatca ataatcatac aaaaatatcc 360
aataacacta aaaaattaaa agaaatggat aatttcacaa tatgttatac gataaagaag 420
ttacttttcc aagaaattca ctgattttat aagcccactt gcattagata aatggcaaaa 480
aaaaacaaaa aggaaaagaa ataaagcacg aagaattcta gaaaatacga aatacgcttc 540
aatgcagtgg gacccacggt tcaattattg ccaattttca gctccaccgt atatttaaaa 600
aataaaacga taatgctaaa aaaatataaa tcgtaacgat cgttaaatct caacggctgg 660
atcttatgac gaccgttaga aattgtggtt gtcgacgagt cagtaataaa cggcgtcaaa 720
gtggttgcag ccggcacaca cgagtcgtgt ttatcaactc aaagcacaaa tacttttcct 780
caacctaaaa ataaggcaat tagccaaaaa caactttgcg tgtaaacaac gctcaataca 840
cgtgtcattt tattattagc tattgcttca ccgccttagc tttctcgtga cctagtcgtc 900
ctcgtctttt cttcttcttc ttctataaaa caatacccaa agcttcttct tcacaattca 960
gatttcaatt tctcaaaatc ttaaaaactt tctctcaatt ctctctaccg tgatcaaggt 1020
aaatttctgt gttccttatt ctctcaaaat cttcgatttt gttttcgttc gatcccaatt 1080
tcgtatatgt tctttggttt agattctgtt aatcttagat cgaagacgat tttctgggtt 1140
tgatcgttag atatcatctt aattctcgat tagggtttca taaatatcat ccgatttgtt 1200
caaataattt gagttttgtc gaataattac tcttcgattt gtgatttcta tctagatctg 1260
gtgttagttt ctagtttgtg cgatcgaatt tgtcgattaa tctgagtttt tctgattaac 1320
ag 1322
<210> 17
<211> 234
<212> DNA
<213> Arabidopsis albino or light green body transit peptide (Arabidopsis thaliana)
<400> 17
atggctacag ctactacaac tgctactgct gctttttcag gagttgtttc tgttggtaca 60
gaaactagaa gaatctattc attctctcat cttcaacctt cagctgcttt tcctgctaag 120
ccatcttcat tcaaatcact taagttgaag cagtctgcta gacttacaag aagattggat 180
catagaccat ttgttgttag atgtgaggct tcttcatcta acggaagact tact 234
<210> 18
<211> 253
<212> DNA
<213> terminator of nopaline synthase gene (Agrobacterium tumefaciens)
<400> 18
gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60
atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120
atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180
gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240
atgttactag atc 253
<210> 19
<211> 530
<212> DNA
<213> Cauliflower mosaic virus 35S promoter (Cauliflower mosaic virus)
<400> 19
ccatggagtc aaagattcaa atagaggacc taacagaact cgccgtaaag actggcgaac 60
agttcataca gagtctctta cgactcaatg acaagaagaa aatcttcgtc aacatggtgg 120
agcacgacac gcttgtctac tccaaaaata tcaaagatac agtctcagaa gaccaaaggg 180
caattgagac ttttcaacaa agggtaatat ccggaaacct cctcggattc cattgcccag 240
ctatctgtca ctttattgtg aagatagtgg aaaaggaagg tggctcctac aaatgccatc 300
attgcgataa aggaaaggcc atcgttgaag atgcctctgc cgacagtggt cccaaagatg 360
gacccccacc cacgaggagc atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc 420
aagtggattg atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt 480
cgcaagaccc ttcctctata taaggaagtt catttcattt ggagaggaca 530
<210> 20
<211> 552
<212> DNA
<213> phosphinothricin N-acetyltransferase gene (Streptomyces viridochromogenes)
<400> 20
atgtctccgg agaggagacc agttgagatt aggccagcta cagcagctga tatggccgcg 60
gtttgtgata tcgttaacca ttacattgag acgtctacag tgaactttag gacagagcca 120
caaacaccac aagagtggat tgatgatcta gagaggttgc aagatagata cccttggttg 180
gttgctgagg ttgagggtgt tgtggctggt attgcttacg ctgggccctg gaaggctagg 240
aacgcttacg attggacagt tgagagtact gtttacgtgt cacataggca tcaaaggttg 300
ggcctaggat ccacattgta cacacatttg cttaagtcta tggaggcgca aggttttaag 360
tctgtggttg ctgttatagg ccttccaaac gatccatctg ttaggttgca tgaggctttg 420
ggatacacag cccggggtac attgcgcgca gctggataca agcatggtgg atggcatgat 480
gttggttttt ggcaaaggga ttttgagttg ccagctcctc caaggccagt taggccagtt 540
acccagatct ga 552
<210> 21
<211> 195
<212> DNA
<213> Cauliflower mosaic virus 35S terminator (Cauliflower mosaic virus)
<400> 21
ctgaaatcac cagtctctct ctacaaatct atctctctct ataataatgt gtgagtagtt 60
cccagataag ggaattaggg ttcttatagg gtttcgctca tgtgttgagc atataagaaa 120
cccttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa 180
accaaaatcc agtgg 195

Claims (14)

1. A protein, comprising:
(a) Has an amino acid sequence shown as SEQ ID NO. 1;
(b) And (b) a protein derived from (a) and having protoporphyrinogen oxidase activity, wherein the amino acid sequence in (a) is substituted and/or deleted and/or added with one or more amino acids.
2. A gene, comprising:
(a) A nucleotide sequence encoding the protein of claim 1; or
(b) A nucleotide sequence which hybridizes with the nucleotide sequence defined in (a) under strict conditions and codes for a protein with protoporphyrinogen oxidase activity; or
(c) Has a nucleotide sequence shown in SEQ ID NO. 2.
3. An expression cassette comprising the gene of claim 2 under the control of an operably linked regulatory sequence.
4. A recombinant vector comprising the gene of claim 2 or the expression cassette of claim 3.
5. A method of extending the herbicide tolerance of a plant comprising: expressing in a plant the protein of claim 1 or the protein encoded by the expression cassette of claim 3 together with at least one second herbicide tolerance protein different from the protein of claim 1 or the protein encoded by the expression cassette of claim 3.
6. The method of claim 5, wherein the second herbicide tolerance protein is 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha ketoglutarate-dependent dioxygenase, dicamba monooxygenase, acetolactate synthase, a cytochrome protein and/or hydroxyphenylpyruvate dioxygenase.
7. A method of selecting a transformed plant cell comprising: transforming a plurality of plant cells with the gene of claim 2 or the expression cassette of claim 3 and culturing the cells at a concentration of the PPO inhibitor herbicide that allows growth of transformed cells expressing the gene or the expression cassette while killing or inhibiting growth of untransformed cells;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
8. A method of controlling weeds, comprising: applying an effective dose of a PPO inhibitor herbicide to a field planted with a plant of interest comprising a gene of claim 2 or an expression cassette of claim 3 or a recombinant vector of claim 4;
preferably, the plant of interest includes a monocotyledon and a dicotyledon; more preferably, the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis; further preferably, the plant of interest is a glyphosate tolerant plant and the weeds are glyphosate resistant weeds;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
9. A method for protecting or conferring tolerance to a PPO inhibitor herbicide on a plant from damage caused by the PPO inhibitor herbicide, comprising: introducing the gene of claim 2 or the expression cassette of claim 3 or the recombinant vector of claim 4 into a plant such that the introduced plant produces an amount of the herbicide tolerance protein sufficient to protect against the PPO inhibitor herbicide;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugarbeet, cucumber, cotton, rape, potato, tomato, or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
10. A method of producing a plant tolerant to a PPO inhibitor herbicide comprising introducing into the genome of the plant the gene of claim 2;
preferably, the method of introduction includes a genetic transformation method, a genome editing method, or a gene mutation method;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
11. A method of growing a PPO inhibitor herbicide tolerant plant comprising:
growing at least one plant propagule comprising in its genome the gene of claim 2 or the expression cassette of claim 3;
growing the plant propagule into a plant;
applying an effective dose of a PPO inhibitor herbicide to a plant growing environment comprising at least said plant, harvesting plants having reduced plant damage and/or increased plant yield as compared to other plants not having the gene of claim 2 or the expression cassette of claim 3;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
12. A method of obtaining a processed agricultural product comprising treating a harvest of PPO inhibitor herbicide tolerant plants obtained by the method of claim 11 to obtain a processed agricultural product.
13. A planting system for controlling the growth of weeds comprising combining a PPO inhibitor herbicide with a plant growing environment in which at least one plant of interest is present, said plant of interest comprising a gene of claim 2 or an expression cassette of claim 3;
preferably, the plant of interest includes a monocotyledon and a dicotyledon; more preferably, the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugarbeet, cucumber, cotton, rape, potato, tomato, or arabidopsis; further preferably, the plant of interest is a glyphosate tolerant plant and the weeds are glyphosate resistant weeds;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
14. Use of a protein according to claim 1 for conferring herbicide tolerance to a PPO inhibitor in a plant;
preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether PPO inhibitor herbicide, an oxadiazolone PPO inhibitor herbicide, an N-phenylphthalamide imine PPO inhibitor herbicide, an oxazolinone PPO inhibitor herbicide, a phenylpyrazole PPO inhibitor herbicide, a uracil PPO inhibitor herbicide, a thiadiazole PPO inhibitor herbicide, a triazolinone PPO inhibitor herbicide and/or a triazinone PPO inhibitor herbicide; more preferably, the PPO-inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfentrazone and/or flumioxazin.
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