CN115340987B - Herbicide tolerance protein, coding gene and application thereof - Google Patents

Herbicide tolerance protein, coding gene and application thereof Download PDF

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CN115340987B
CN115340987B CN202110514777.XA CN202110514777A CN115340987B CN 115340987 B CN115340987 B CN 115340987B CN 202110514777 A CN202110514777 A CN 202110514777A CN 115340987 B CN115340987 B CN 115340987B
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ppo inhibitor
herbicide
inhibitor herbicide
plants
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CN115340987A (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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    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
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    • C12Y103/03—Oxidoreductases acting on the CH-CH group of donors (1.3) with oxygen as acceptor (1.3.3)
    • 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 the following components: (a) has the amino acid sequence shown in SEQ ID NO. 1; (b) A protein derived from (a) wherein the amino acid sequence in (a) is substituted and/or deleted and/or added with one or more amino acids and has protoporphyrinogen oxidase activity. The herbicide tolerance protein of the invention has higher tolerance to PPO inhibitor herbicide, and plants containing a nucleotide sequence encoding the herbicide tolerance protein of the invention have strong tolerance to PPO inhibitor herbicide, and all of them show high resistance to oxyfluorfen, saflufenacil and flumioxazin at 4 times field concentration and mesotrione at 2 times field concentration. Therefore, the application prospect on plants is wide.

Description

Herbicide tolerance protein, coding gene and application thereof
Technical Field
The invention relates to herbicide tolerance protein, a coding gene and application thereof, in particular to protein with tolerance to PPO inhibitor herbicide, a coding gene and application thereof.
Background
The porphyrin biosynthetic pathway is used to synthesize chlorophyll and heme, which play an important role in plant metabolism, and this pathway occurs in chloroplasts. In this pathway, protoporphyrinogen oxidase (PPO) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. After protoporphyrin IX is produced, protoporphyrin IX is synthesized to chlorophyll by binding magnesium with magnesium chelating enzyme, or heme by binding iron with iron chelating enzyme.
Herbicides that act by inhibiting PPO include diphenyl ether type PPO inhibitor herbicides, oxadiazolone type PPO inhibitor herbicides, N-phenylphthalamide imide type PPO inhibitor herbicides, oxazolinone type PPO inhibitor herbicides, phenylpyrazole type PPO inhibitor herbicides, uracil type PPO inhibitor herbicides, thiadiazole type PPO inhibitor herbicides, triazolone type PPO inhibitor herbicides, triazinone type PPO inhibitor herbicides, and other types of PPO inhibitor herbicides. In plants, PPO inhibitors inhibit the enzymatic activity of PPO, leading to inhibition of chlorophyll and heme synthesis and accumulation of the substrate protoporphyrinogen IX, which is rapidly exported from chloroplasts to the cytoplasm where it is converted to protoporphyrinogen IX under non-enzymatic reactions and further generates highly reactive singlet oxygen in the presence of light and oxygen molecules 1 O 2 ) They damage the cell membrane and rapidly lead to death of plant cells.
The method for providing PPO inhibitor herbicide tolerant plants consists essentially of: 1) Herbicides are detoxified using enzymes that convert the herbicide or its active metabolite into non-toxic products. 2) The sensitive PPO is overexpressed such that, in view of the kinetic constant of this enzyme, a sufficient amount of target enzyme is produced in the plant relative to the herbicide, such that despite the presence of the PPO inhibitor herbicide, these sensitive PPO are sufficiently functional with the PPO inhibitor herbicide to have sufficient functional enzyme available for use. 3) A mutant PPO is provided that is less susceptible to herbicides or active metabolites thereof, but which retains the property of catalyzing the oxidation of protoporphyrinogen IX to protoporphyrin IX. Regarding 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, their cost of manufacture, and their environmental friendliness. Thus, new methods for conferring PPO inhibitor herbicide tolerance to different crops and crop varieties are needed.
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 herbicide when being transferred into a plant 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 in SEQ ID NO. 1;
(b) A protein derived from (a) wherein the amino acid sequence in (a) is substituted and/or deleted and/or added with one or more amino acids and has protoporphyrinogen oxidase activity.
To achieve the above object, the present invention provides a gene comprising:
(a) A nucleotide sequence encoding the protein; or (b)
(b) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence defined in (a) and which encodes a protein having protoporphyrinogen oxidase activity; or (b)
(c) Has the nucleotide sequence shown in SEQ ID NO. 2.
The stringent conditions may be hybridization in 6 XSSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution at 65℃and then washing the membrane 1 time with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS, respectively.
To achieve the above object, the present invention also provides an expression cassette comprising the gene under the control of operably linked regulatory sequences.
To achieve the above object, the present invention also provides a recombinant vector comprising the gene or the expression cassette.
To achieve the above object, the present invention also provides a method for expanding the range of herbicide tolerance of plants, comprising: the protein or the protein encoded by the expression cassette is expressed in a plant together with at least one second herbicide tolerance protein different from the protein or the protein encoded by the 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 protein, and/or hydroxyphenylpyruvate dioxygenase.
To achieve the above object, the present invention also provides a method of 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 PPO inhibitor herbicide concentration that allows growth of transformed cells expressing said gene or said expression cassette while killing or inhibiting growth of untransformed cells;
Preferably, the plant comprises a monocot and a dicot; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether type PPO inhibitor herbicide, an oxadiazolone type PPO inhibitor herbicide, an N-phenylphthalamide imide type PPO inhibitor herbicide, an oxazolinone type PPO inhibitor herbicide, a phenylpyrazole type PPO inhibitor herbicide, a uracil type PPO inhibitor herbicide, a thiadiazolone type PPO inhibitor herbicide, a triazolone type PPO inhibitor herbicide, and/or a triazinone type PPO inhibitor herbicide; more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfenacil and/or flumioxazin.
To achieve the above object, the present invention also provides a method of controlling weeds, comprising: applying an effective dose of a PPO inhibitor herbicide to a field in which a plant of interest is grown, the plant of interest comprising the gene or the expression cassette or the recombinant vector;
Preferably, the plant of interest includes monocotyledonous plants and dicotyledonous plants; more preferably, the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis; further preferably, the plant of interest is a glyphosate tolerant plant and the weed is a glyphosate resistant weed;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether type PPO inhibitor herbicide, an oxadiazolone type PPO inhibitor herbicide, an N-phenylphthalamide imide type PPO inhibitor herbicide, an oxazolinone type PPO inhibitor herbicide, a phenylpyrazole type PPO inhibitor herbicide, a uracil type PPO inhibitor herbicide, a thiadiazolone type PPO inhibitor herbicide, a triazolone type PPO inhibitor herbicide, and/or a triazinone type PPO inhibitor herbicide; more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfenacil and/or flumioxazin.
To achieve the above object, the present invention also provides a method for protecting plants from injury caused by PPO inhibitor herbicides or for conferring PPO inhibitor herbicide tolerance to plants, 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 PPO inhibitor herbicides;
Preferably, the plant comprises a monocot and a dicot; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether type PPO inhibitor herbicide, an oxadiazolone type PPO inhibitor herbicide, an N-phenylphthalamide imide type PPO inhibitor herbicide, an oxazolinone type PPO inhibitor herbicide, a phenylpyrazole type PPO inhibitor herbicide, a uracil type PPO inhibitor herbicide, a thiadiazolone type PPO inhibitor herbicide, a triazolone type PPO inhibitor herbicide, and/or a triazinone type PPO inhibitor herbicide; more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfenacil 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 introducing comprises a genetic transformation method, a genome editing method or a gene mutation method;
Preferably, the plant comprises a monocot and a dicot; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether type PPO inhibitor herbicide, an oxadiazolone type PPO inhibitor herbicide, an N-phenylphthalamide imide type PPO inhibitor herbicide, an oxazolinone type PPO inhibitor herbicide, a phenylpyrazole type PPO inhibitor herbicide, a uracil type PPO inhibitor herbicide, a thiadiazolone type PPO inhibitor herbicide, a triazolone type PPO inhibitor herbicide, and/or a triazinone type PPO inhibitor herbicide; more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfenacil and/or flumioxazin.
To achieve the above object, the present invention also provides a method of culturing a plant tolerant to a PPO inhibitor herbicide, comprising:
planting at least one plant propagule comprising the gene or the expression cassette in the genome of the plant propagule;
Growing the plant propagules into plants;
applying an effective dose of a PPO inhibitor herbicide to a plant growth environment comprising at least said plant, harvesting a plant 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 comprises a monocot and a dicot; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether type PPO inhibitor herbicide, an oxadiazolone type PPO inhibitor herbicide, an N-phenylphthalamide imide type PPO inhibitor herbicide, an oxazolinone type PPO inhibitor herbicide, a phenylpyrazole type PPO inhibitor herbicide, a uracil type PPO inhibitor herbicide, a thiadiazolone type PPO inhibitor herbicide, a triazolone type PPO inhibitor herbicide, and/or a triazinone type PPO inhibitor herbicide; more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfenacil and/or flumioxazin.
The present invention also provides a method of 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 applying a PPO inhibitor herbicide and a plant growth environment in which at least one plant of interest is present, said plant of interest comprising said gene or said expression cassette;
preferably, the plant of interest includes monocotyledonous plants and dicotyledonous plants; more preferably, the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis; further preferably, the plant of interest is a glyphosate tolerant plant and the weed is a glyphosate resistant weed;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether type PPO inhibitor herbicide, an oxadiazolone type PPO inhibitor herbicide, an N-phenylphthalamide imide type PPO inhibitor herbicide, an oxazolinone type PPO inhibitor herbicide, a phenylpyrazole type PPO inhibitor herbicide, a uracil type PPO inhibitor herbicide, a thiadiazolone type PPO inhibitor herbicide, a triazolone type PPO inhibitor herbicide, and/or a triazinone type PPO inhibitor herbicide; more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfenacil and/or flumioxazin.
To achieve the above object, the present invention also provides the use of the protein for conferring herbicide tolerance to PPO inhibitors in plants;
preferably, the plant comprises a monocot and a dicot; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis;
preferably, the PPO inhibitor herbicide comprises a diphenyl ether type PPO inhibitor herbicide, an oxadiazolone type PPO inhibitor herbicide, an N-phenylphthalamide imide type PPO inhibitor herbicide, an oxazolinone type PPO inhibitor herbicide, a phenylpyrazole type PPO inhibitor herbicide, a uracil type PPO inhibitor herbicide, a thiadiazolone type PPO inhibitor herbicide, a triazolone type PPO inhibitor herbicide, and/or a triazinone type PPO inhibitor herbicide; more preferably, the PPO inhibitor herbicide comprises oxyfluorfen, saflufenacil, sulfenacil and/or flumioxazin.
As a specific embodiment, the PPO inhibitor herbicide may be one or more selected from the group consisting of, but not limited to: diphenyl ethers (cumyl ether, methoxyl herbicidal ether (chloromethoxfen), carboxin herbicidal ether (Bifenox), oxyfluorfen (oxyfluorfen), acifluorfen, salts and esters thereof, fomesafen (Fomesafen), lactofen (lactofen), fluoroglycofen (fluoroglyfen-ethyl), chlorofluorophenyl ether, aclonifen (aclonifen), bifenox (Bifenox), chlorolactofen (ethoxyfen), chloroherbicidal ether (chlorotrofen), fluoronitro sulfonamide (halosaffen)); oxadiazolones (oxadiazon, oxadiargyl); n-phenylphthalamide imines (flumioxazin), flumetofen (flumium-penyl), indoxacarb (cinidon-ethyl)); oxazolinones (cyclopentaoxazone); phenylpyrazoles (pyraclostrobin), pyriproxyfen; uracil (bupirimate, flumetsulam, saflufenacil); thiadiazoles (thiabendazole, oxazin methyl ester (fluthiacet)); triazolinones (carfentrazone), sulfentrazone, carfentrazone-ethyl); triazinones (trifludioxazin); other classes (fluorofenpyr-ethyl, 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. Furthermore, the terms "comprises," "comprising," or any other variation thereof, such as "includes" or "including," are to be understood to mean including one of the elements, integers or steps, or a group of elements, integers or steps, but not excluding any other elements, integers or steps, or groups of elements, integers or steps.
The term "herbicide insensitive" in the present invention refers to the ability of a 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 the protox enzyme may be measured by any means known in the art, for example, measuring the amount of protox product produced or the amount of protox substrate consumed by fluorescence, high Performance Liquid Chromatography (HPLC) or Mass Spectrometry (MS) in the presence of one or more PPO inhibitor herbicides. "herbicide insensitive" may be completely or partially insensitive to a particular herbicide and may be expressed as a percentage of tolerance or 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 resist 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 an appropriate control. For example, herbicide tolerance can be measured or assessed by applying the herbicide to a plant comprising a DNA molecule encoding a protein capable of conferring herbicide tolerance (test plant) and a plant not comprising a DNA molecule encoding a protein capable of conferring herbicide tolerance (control plant), and then comparing the plant lesions of the two plants, wherein herbicide tolerance of the test plant is indicated by a reduced rate of lesion compared to the rate of lesion of the control plant. Herbicide-tolerant plants, seeds, plant tissues or cells exhibit reduced response to herbicide toxic effects compared to control plants, seeds, plant tissues or cells. The term "herbicide tolerance trait" refers to a transgenic trait that imparts improved herbicide tolerance to a plant as compared to a wild-type plant. Plants that may be produced having the herbicide tolerance traits of the 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, beet, cucumber, cotton, canola, 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) refers to 2-chloro-1- (3-ethoxy-4-nitrophenoxy) -4-trifluoromethyl benzene, and is a colorless crystalline solid. Belongs to a diphenyl ether type selective pre-bud and post-bud contact-killing type PPO inhibitor herbicide with ultralow dosage, and can be prepared into emulsifiable concentrates for use. Weeds are mainly killed by absorption of the medicament by coleoptile and mesocotyl. The oxyfluorfen can effectively control weeds in rice, soybean, corn, cotton, vegetables, grapes, fruit trees and other crop fields, and the weeds which can be prevented include, but are not limited to, barnyard grass, sesbania, eclosion, green bristlegrass, stramonium, creeping wheatgrass, ragweed, daylily, abutilon, tian Gai menu cotyledon and broadleaf weeds.
The effective dose of oxyfluorfen in the invention refers to 180-720g ai/ha, and comprises 190-700g ai/ha, 250-650g ai/ha, 300-600g ai/ha or 400-500g ai/ha.
The saflufenacil (saflufenacil) refers to 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 extruded granular solid. Belongs to uracil biocidal PPO inhibitor herbicide, and can be prepared into 70% water dispersible granule formulation. Saflufenacil is effective against a wide variety of broadleaf weeds, including weeds that are resistant to glyphosate, ALS and triazines, and has a rapid biocidal effect and the soil residue degrades rapidly.
The effective dose of saflufenacil in the present invention is 25-100g ai/ha, including 30-95g ai/ha, 40-90g ai/ha, 50-85g ai/ha or 60-80g ai/ha.
The 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. The herbicide belongs to N-phenyl phthalimide type bud and leaf absorption PPO inhibitor herbicide, and the common dosage forms are 50% wettable powder and 48% suspending agent. The flumioxazin can effectively prevent and treat 1-year-old broadleaf weeds and part of gramineous weeds. It is easy to degrade in the environment and is safe for the succeeding crop.
The effective dose of flumioxazin in the invention is 60-240g ai/ha, including 70-220g ai/ha, 85-200g ai/ha, 90-185g ai/ha or 100-150g ai/ha.
The sulfenamide (sulfenazone) 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 triazolinone PPO inhibitor herbicides, and the common dosage forms are 38.9% and 44.5% suspending agents. The sulfenamide can be used for preventing and controlling 1 year old broadleaf weeds such as semen Maydis, jowar, semen glycines, semen Arachidis Hypogaeae, herba Amaranthi Tricoloris, fructus Chenopodii, herba Daturae, crabgrass, herba Setariae viridis, herba Xanthii, herba Eleusines Indicae, rhizoma Cyperi, etc., grassy weeds, and Cyperus rotundus.
The effective dose of the sulfenamide is 450-900g ai/ha, including 500-850g ai/ha, 550-700g ai/ha, 500-685g ai/ha or 550-650g ai/ha.
In the present invention, the term "resistance" is heritable and allows plants to grow and reproduce with herbicide treatment of a given plant in general herbicide-effective manner. As recognized by those skilled in the art, even if a given plant is subjected to some degree of injury, such as little necrosis, solubilization, sallow or other injury, but at least not significantly affected in yield, the plant may still be considered "resistant", i.e., the given plant has an increased ability to resist various degrees of injury induced by the herbicide, while generally resulting in injury to wild type plants of the same genotype at the same herbicide dose. The term "resistance" or "tolerance" is used herein more broadly than the term "resistance" and includes "resistance".
As used herein, "glyphosate" refers to N-phosphonomethylglycine and its salts, and treatment with a "glyphosate herbicide" refers to treatment with any herbicide formulation containing glyphosate. Commercial formulations of glyphosate include but are not limited to,(glyphosate as isopropylamine salt),>WEATHERMAX (glyphosate as potassium salt), +.>DRY and->(glyphosate as an amine salt),>GEOFORCE (glyphosate as sodium salt) and +.>(glyphosate as a trimethyl sulphur salt).
The effective dose of glyphosate is 200-1600g ae/ha, including 250-1600g ae/ha, 300-1600g ae/ha, 500-1600g ae/ha, 800-1500g ae/ha, 1000-1500g ae/ha or 1200-1500g ae/ha.
The term "glufosinate" as used herein, also known as glufosinate, refers to 2-amino-4- [ hydroxy (methyl) phosphono ] butanoic acid ammonium, and treatment with a "glufosinate herbicide" refers to treatment with any herbicide formulation containing glufosinate.
The effective dose of glufosinate 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 amount of herbicide applied in the present invention varies with soil structure, pH, organic content, farming system and weed size, and is determined by looking at the appropriate herbicide application on the herbicide label.
The term "conferring" in the present invention refers to providing a plant with a characteristic or trait, such as herbicide tolerance and/or other desired trait.
The term "heterologous" in 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, the soybean PPO gene, which is still considered to be "heterologous" DNA, can be expressed in soybean plants using transgenic methods.
The term "nucleic acid" in the present invention includes reference to deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, includes known analogues having the basic properties of natural nucleotides (e.g., peptide nucleic acids) 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 "encoding" 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. The nucleic acid encoding the protein may comprise an untranslated sequence (e.g., an intron) within the translated region of the nucleic acid, or may lack such an inserted untranslated sequence (e.g., in a cDNA).
Genes encoding the herbicide tolerance proteins of the invention are used to provide plants, plant cells and seeds of the invention that provide better tolerance to a variety of PPO inhibitor herbicides than the same plants (control plants) that do not contain the genes encoding the herbicide tolerance proteins of the invention.
Genes encoding the herbicide tolerance proteins of the invention are useful for producing plants that are tolerant to PPO inhibitor herbicides. Genes encoding the herbicide tolerance proteins of the 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, one or more of which are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. The polypeptides of the invention may be produced from a nucleic acid of the disclosure 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 combining ex vivo methods (e.g., protease digestion and purification).
The invention also provides nucleic acid molecules comprising the proteins encoding the herbicide tolerance proteins. In general, the invention includes any nucleotide sequence encoding any herbicide tolerance protein described herein, as well as any nucleotide sequence encoding a herbicide tolerance protein having one or more conservative amino acid substitutions relative to the herbicide tolerance proteins described herein. It is well known to those skilled in the art to provide functionally similar amino acid conservative substitutions, and the following five groups each comprise amino acids that are conservative substitutions for one another: aliphatic: glycine (G), alanine (a), valine (V), leucine (L), isoleucine (I); aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W); sulfur-containing: methionine (M), cysteine (C); alkaline: arginine (R), lysine (K), histidine (H); acidic: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q).
Thus, sequences that have PPO inhibitor herbicide tolerance activity and hybridize under stringent conditions to the nucleotides encoding the herbicide tolerance proteins of the invention are included in the invention. Illustratively, these sequences are at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology to the sequence of the invention SEQ ID NO. 2.
Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the herbicide tolerance genes of the present invention. The nucleic acid molecule or fragment thereof is capable of specifically hybridizing to other nucleic acid molecules under certain conditions. In the present invention, two nucleic acid molecules can be said to specifically hybridize to each other if they form an antiparallel double-stranded nucleic acid structure. Two nucleic acid molecules are said to be "complements" of one nucleic acid molecule if they exhibit complete complementarity. In the present invention, a nucleic acid molecule is said to exhibit "complete complementarity" when each nucleotide of the two molecules is complementary to a 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 such that they 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 such that they anneal and bind to each other under conventional "highly stringent" conditions. Deviations from complete complementarity are permissible provided that such deviations do not completely prevent the formation of double-stranded structures by the two molecules. In order to enable a nucleic acid molecule to act as a primer or probe, it is only necessary to ensure sufficient complementarity in sequence to allow the formation of a stable double-stranded structure at the particular solvent and salt concentration employed.
In the present invention, a substantially homologous sequence is a nucleic acid molecule that specifically hybridizes to the complementary strand of a matching nucleic acid molecule under highly stringent conditions. Suitable stringent conditions for promoting DNA hybridization, for example, treatment with 6.0 XSSC/sodium citrate (SSC) at about 45℃followed by washing with 2.0 XSSC at 50℃are well known to those skilled in the art. For example, the salt concentration in the washing step may be selected from about 2.0 XSSC at low stringency conditions, about 0.2 XSSC at 50℃to high stringency conditions, about 50 ℃. In addition, the temperature conditions in the washing step may be raised from about 22 ℃ at room temperature under low stringency conditions to about 65 ℃ under high stringency conditions. The temperature conditions and salt concentration may both be varied, or one may remain unchanged while the other variable is varied. Preferably, the stringent conditions of the present invention may be specific hybridization with the gene encoding the protoporphyrinogen oxidase of the present invention in a 6 XSSC, 0.5% SDS solution at 65℃and then washing the membrane 1 time with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
In the present invention, the term "hybridization" or "specific hybridization" refers to a molecule that can bind, double-stranded or hybridize only to a specific nucleotide sequence under stringent conditions when the sequence is present in a complex mixture (e.g., total cell) DNA or RNA.
Due to the redundancy of the genetic code, a variety of different DNA sequences may encode the same amino acid sequence. The generation of these alternative DNA sequences encoding the same or substantially the same protein is within the skill level of those skilled in the art. These different DNA sequences are included within the scope of the present invention. The term "substantially identical" sequence refers to a sequence having amino acid substitutions, deletions, additions or insertions without substantially affecting the herbicide tolerance activity, and also includes fragments that retain the herbicide tolerance activity.
The term "functional activity" or "activity" in the present invention refers to the ability of the protein/enzyme of use of the invention (alone or in combination with other proteins) to degrade or attenuate the activity of a herbicide. Plants producing a protein of the invention preferably produce an "effective amount" of the protein such that when the plant is treated with the herbicide, the protein is expressed at a level sufficient to render the plant wholly or partially tolerant to the herbicide (typically in amounts unless specifically indicated). The herbicide may be used in amounts, in normal field amounts and concentrations that would normally kill the target plant. 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 invention are preferably tolerant to PPO inhibitor herbicides, 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 exemplified sequences, but also portions and/or fragments (including internal and/or terminal deletions as compared to the full-length protein), variants, mutants, variant proteins, substitutions (proteins with substituted amino acids), chimeras, and fusion proteins that preserve the active characteristics of the specific exemplified proteins.
The term "variant" according to the invention means a substantially similar sequence. For polynucleotides, a variant includes a deletion and/or addition of one or more nucleotides at one or more internal sites within the reference polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the herbicide tolerance gene. The term "reference polynucleotide or polypeptide" in the present invention correspondingly includes herbicide tolerance nucleotide sequences or amino acid sequences. The term "native polynucleotide or polypeptide" in the present invention correspondingly includes naturally occurring nucleotide sequences or amino acid sequences. For nucleic acid molecules, conservative variants include nucleotide sequences (due to the degeneracy of the genetic code) that encode one of the herbicide tolerance proteins described in the present invention. Such naturally occurring allelic variants can be identified using well known molecular biological 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 produced by using site-directed mutagenesis but which nevertheless encode the herbicide tolerance proteins of the 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 procedures and parameters.
By "variant protein" in the present invention 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 possess the desired activity of the protoporphyrinogen oxidase of the present invention, i.e. still possess the protoporphyrinogen oxidase activity and/or herbicide tolerance. Such variants may arise, for example, from genetic polymorphisms or from manual manipulation. A biologically active variant of a 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 all of the amino acid sequences of the herbicide tolerance protein as determined by sequence alignment procedures and parameters. A biologically active variant of a protein of the invention may differ from a protein with 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 the algorithm of Myers and Miller (1988) CABIOS 4:11-17; a local alignment algorithm of Smith et al (1981) adv.appl.math.2:482; need eman andWunsch (1970) J.mol.biol.48:443-453 global alignment algorithm; and Karlin and Al tschul (1990) Proc.Natl. Acad.Sci.USA 872264, as modified in Karlin and Al tschul (1993) Proc.Natl. Acad.Sci.USA 90:5873-5877. Computer implementations of these mathematical algorithms may be utilized for sequence comparison to determine sequence homology, such implementations including, but not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, mountain View, california); all program (version 2.0) and GAP, BESTFIT, BLAST, FASTA in version 10 of GCG Wiscons in Genetics Software Package and TFASTA (available from Accelrys inc.,9685Scranton Road,San Diego,Calif ornia,USA).
In certain embodiments, nucleic acid sequences encoding herbicide tolerance proteins of the invention or variants thereof that retain protoporphyrinogen oxidase activity may be superimposed with any combination of nucleic acid sequences of interest to produce plants having 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 the herbicide tolerance protein of the invention or variants that retain protoporphyrinogen oxidase activity may be superimposed 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 to fermentable sugars), and improved agronomic quality (e.g., high oil content and high protein content).
The benefits of combinations of two or more modes of action in improving the controlled weed spectrum and/or naturally more tolerant or resistant weed species can also be extended to the creation of chemicals in crops that are herbicide tolerant in addition to PPO tolerant crops by artificial (transgenic or non-transgenic) as is well known to those skilled in the art. In fact, the following resistant traits may be stacked singly or in multiple combinations to provide effective control or prevent weed succession from developing resistance to herbicides: glyphosate resistance (e.g., resistant plants or bacteria EPSPS, GOX, GAT), glufosinate resistance (e.g., PAT, bar), acetolactate synthase (ALS) inhibitory herbicide resistance (e.g., imidazolinone, sulfonylurea, triazolopyrimidine, sulfoaniline, pyrimidothiobenzoic acid 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 system ii inhibitory herbicides (e.g., psbA), resistance to system i inhibitory herbicides, resistance to 4-hydroxyphenylpyruvate dioxygenase inhibitory herbicides (e.g., HPPD), resistance to phenylurea herbicides (e.g., 76B 1), dichloromethoxybenzoate degrading enzymes, etc.
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 crops and non-crop applications has (and will continue to) select for species or glyphosate resistant biotypes that will shift weeds to naturally more tolerant. Most herbicide resistance management strategies suggest the use of an effective amount of a tank-mixed herbicide partner as a method of delaying the emergence of resistant weeds, which provides control of the same species, but with a different mode of action. Superimposing the gene encoding the herbicide tolerance protein of the invention with a glyphosate tolerance trait (and/or other herbicide tolerance traits) can achieve 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 in a tank mix containing two or more herbicides of different modes of action, separate use of a single herbicide composition in successive uses (e.g. pre-planting, pre-emergence or post-emergence) with intervals ranging from 2 hours to 3 months, or alternatively any combination of numbers of herbicides representing applicable each class of compound can be used at any time (from 7 months in the crop to harvest (or pre-harvest interval for a single herbicide, shortest).
It is important to have flexibility in controlling broadleaf weeds, i.e., time of use, individual herbicide usage, and the ability to control refractory or resistant weeds. The application of glyphosate superimposed with the glyphosate resistance gene/gene encoding the herbicide tolerance protein of the invention in crops can range from 250 to 2500g ae/ha; the PPO inhibitor herbicide(s) may be in the range of from 10 to 1000g ai/ha. The optimal combination of times for these applications depends on the particular conditions, species and circumstances.
Herbicide formulations (e.g., ester, acid or salt formulations or soluble concentrates, emulsifying concentrates or soluble liquids) and tank-mix additives (e.g., 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 present invention.
In addition, genes encoding the herbicide tolerance proteins of the invention can be superimposed with one or more other input (e.g., insect resistance, fungal resistance, stress tolerance, etc.) or output (e.g., increased yield, improved oil mass, increased fiber quality, etc.) traits, alone or in combination with other herbicide tolerance crop characteristics. Thus, the present invention can be used to provide a complete agronomic solution to flexibly and economically control any number of agronomic pests and to improve crop quality.
The combination of these stacks may be produced by any method including, but not limited to: crossbred plants or genetic transformation by conventional or top crossing methods. If these sequences are superimposed by genetic transformation of these plants, the polynucleotide sequences of interest may be combined at any time and in any order. For example, transgenic plants comprising one or more desired traits can be used as targets for introducing additional traits by subsequent transformation. These traits can be introduced simultaneously with the polynucleotide of interest provided by any combination of expression cassettes in a co-transformation scheme. 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 the expression of the polynucleotide of interest. This can be combined with any combination of other suppression or overexpression cassettes to produce the desired trait combination in the plant. It is further recognized that polynucleotide sequences may 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 herbicide, and is an important herbicide tolerance crop and the basis of the characteristic possibility of a selection marker.
The term "expression cassette" in the present invention refers to a nucleic acid molecule capable of directing expression of a particular nucleotide sequence in an appropriate host cell, including a promoter operably linked to the nucleotide sequence of interest (i.e., a polynucleotide encoding a protoporphyrinogen oxidase or variant protein retaining protoporphyrinogen oxidase activity of the present invention, alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring a desired trait), which nucleotide sequence of interest is 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 antisense RNA or an untranslated RNA in 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 naturally occur 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 or inducible promoter that 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 stage of development.
The present invention encompasses the transformation of plants with an expression cassette capable of expressing a polynucleotide of interest (i.e., a polynucleotide encoding a protoporphyrinogen oxidase or a variant protein thereof retaining protoporphyrinogen oxidase activity of the present invention, either alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring a desired trait). The expression cassette includes a transcription and translation 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 function in the plant. In some embodiments, the expression cassette includes a selectable marker gene to allow selection of stable transformants. The expression constructs of the invention may also include a leader sequence and/or a sequence that allows for 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 an expressible promoter in a plant, and the expression promoter in the plant refers to a promoter which ensures that a coding sequence connected with the promoter is expressed in a plant cell. The promoter expressible in the plant may be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, 35S promoters derived from cauliflower mosaic virus, maize Ubi promoter, promoters of rice GOS2 gene, and the like. Alternatively, the promoter that is expressible in a plant may be a tissue-specific promoter, i.e. the promoter directs higher expression of the coding sequence in some tissues of the plant, such as in green tissues, than in other tissues of the plant (as may be determined by conventional RNA assays), such as the PEP carboxylase promoter. Alternatively, the promoter expressible in the plant may be a wound-inducible promoter. A wound-inducible promoter or a promoter that directs the pattern of wound-induced expression refers to a promoter that significantly increases expression of a coding sequence under the control of the promoter when the plant is subjected to a wound caused by mechanical or insect feeding, as compared to normal growth conditions. Examples of wound-inducible promoters include, but are not limited to, promoters of the protease inhibitor genes (pinI and pinII) and the maize protease inhibitor gene (MPI) of potato and tomato.
The transit peptide (also known as a secretion signal sequence or targeting sequence) is directed to direct the transgene product to a specific organelle or cellular compartment, and may be heterologous to the receptor protein, for example, targeting to the chloroplast using a sequence encoding a chloroplast transit peptide, or to the endoplasmic reticulum using a 'KDEL' retention sequence, or to the vacuole using CTPP of the barley plant lectin gene.
Such leader sequences include, but are not limited to, picornaviral leader sequences, such as EMCV leader sequences (encephalomyocarditis virus 5' non-coding region); potyvirus leader sequences, such as MDMV (maize dwarf mosaic virus) leader sequences; human immunoglobulin heavy chain binding proteins (bips); a non-translated leader sequence of alfalfa mosaic virus coat protein mRNA (AMV RNA 4); tobacco Mosaic Virus (TMV) leader sequence.
Such enhancers include, but are not limited to, the cauliflower mosaic virus (CaMV) enhancer, the Figwort Mosaic Virus (FMV) enhancer, the carnation weathered ring virus (CERV) enhancer, the cassava vein mosaic virus (CsVMV) enhancer, the Mirabilis jalapa mosaic virus (MMV) enhancer, the night yellow leaf curl virus (CmYLCV) enhancer, the Multan cotton leaf curl virus (CLCuMV), the Commelina maculosa refute virus (CoYMV), and the peanut chlorosis line mosaic virus (PCLSV) enhancer.
For monocot applications, the introns include, but are 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 for functioning in plants, including, but not limited to, a polyadenylation signal sequence derived from the Agrobacterium (Agrobacterium tumefaciens) nopaline synthase (NOS) gene, a polyadenylation signal sequence derived from the protease inhibitor II (pin II) gene, a polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and a polyadenylation signal sequence derived from the alpha-tubulin (alpha-tubulin) gene.
"operably linked" as used herein refers to a linkage of nucleic acid sequences such that one sequence provides the desired function for the linked sequences. In the present invention, the term "operably linked" may be used to link 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 a sequence of interest encodes a protein and it is desired to obtain expression of the protein means: the promoter is linked to the sequence in such a way that the resulting transcript is efficiently translated. If the linkage of the promoter to the coding sequence is a transcript fusion and expression of the encoded protein is desired, the 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 the linkage is such that the resulting translational product is in frame with the translational open reading frame encoding the desired protein. Nucleic acid sequences that can 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 assist 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, autonomous replication sequences, centromere sequences).
The genome of a plant, plant tissue or plant cell as used herein 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 clusters, and plant cells that are intact in parts of plants or plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, cores, ears, cobs, husks, stems, roots, root tips, anthers, and the like.
The herbicide tolerance proteins of the present invention can be used in a variety of plants, including but not limited to alfalfa, kidney bean, broccoli, cabbage, carrot, celery, cotton, cucumber, eggplant, lettuce, melon, pea, pepper, zucchini, radish, canola, spinach, soybean, pumpkin, tomato, arabidopsis thaliana, peanut, or watermelon; preferably, the dicotyledonous plant is cucumber, soybean, arabidopsis, tobacco, cotton, peanut or canola. Such monocotyledonous plants include, but are not limited to, maize, rice, sorghum, wheat, barley, rye, millet, sugarcane, oat, or turf grass; preferably, the monocotyledonous plant is maize, rice, sorghum, wheat, barley, millet, sugarcane or oat.
In the present invention, the term "plant transformation" refers to the cloning of 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 a desired trait, into an expression system, i.e., into a plant cell. The receptor and expression cassette of the invention may be introduced into plant cells in a variety of well known ways. In the context of polynucleotides, the term "introducing" (e.g., a nucleotide construct of interest) is intended to mean providing a polynucleotide to the plant in such a way that the polynucleotide gains access to or is achieved within 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 program, such as in a plant, the polynucleotides may be introduced into a host cell of interest in a single transformation event, in separate transformation events. The methods of the invention do not depend on a particular method for introducing one or more polynucleotides into a plant, but merely obtaining access or implementation of the polynucleotide or polynucleotides to the interior of at least one cell of the 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 the stable integration into the genome of the plant and any successive generations 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 the exogenous gene to be stably inherited.
The term "genome editing technology" refers to a genome modification technology capable of performing accurate manipulation of a genome sequence, realizing manipulation of site-directed mutation, insertion, deletion, and the like of genes. Currently, genome editing techniques mainly include HE (homing endonuclease ), ZFN (Zinc-finger nuclease), TALEN (transcription activator-like effector nuclease ), CRISPR (Clustered regulatory interspaced short palindromic repeat, clustered regularly interspaced short palindromic repeats).
Numerous transformation vectors available for plant transformation are known to those of skill in the art, and genes relevant to the present invention may be used in combination with any of the above-described vectors. The choice of vector will depend on the preferred transformation technique and the target species used for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. The selectable markers conventionally used in transformation include the pat and bar genes conferring resistance to kanamycin and related antibiotics or related herbicides (this gene was published by Bevan et al in 1983 at Nature Science at volume 304, pages 184-187), the glufosinate herbicide (also known as glufosinate; see White et al in 1990 at volume 18, page 1062 of Nucl. AcidsRes; spencer et al in 1990 at volume 625, pages 625-631 and U.S. Pat. No. 5561236 and 5276268), the gene of hpn conferring resistance to the antibiotic hygromycin (Blochinger & Diggelmann, mol. Cellis biol 4:2929-2931) and the dnfr gene conferring resistance to methotrexate (Bourprin et al in 1983 at volume 1092), the gene of glyphosate gene conferring resistance to glyphosate (U.S. Pat. No. 35,642 and 5276268), the gene of mann and the enzyme mannose gene conferring resistance to glyphosate (U.S. Pat. No. 35,642 and 35, and the enzyme of the enzyme mannose (U.S. Pat. No. 35,642 and 35) and the enzyme of the enzyme mannose gene conferring resistance to glyphosate (U.S. Pat. No. 35,35) in U.S. 35,516 and 35,.
Methods for regenerating plants are also well known in the art. For example, ti plasmid vectors have been utilized for delivery of exogenous DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles.
The planting system described herein refers to a combination of plants, any herbicide tolerance that it displays, and/or herbicide treatments that are available at different stages of plant development, resulting in high yielding and/or reduced wounding plants.
In the present invention, the weeds are plants which compete with cultivated transgenic plants in a plant growing environment.
The terms "control" and/or "control" in the present invention mean that at least an effective dose of the PPO inhibitor herbicide is applied (e.g., by spraying) directly into the plant growing environment, minimizing weed development and/or stopping growth. At the same time, the cultivated transgenic plants should be morphologically normal and can be cultivated under conventional methods for consumption and/or production of the product; preferably, there is reduced plant damage and/or increased plant yield compared to a non-transgenic wild type plant. Such plants with reduced damage include, but are not limited to, improved stalk resistance, and/or increased grain weight, among others. The "control" and/or "control" effect of the protoporphyrinogen oxidase on weeds can be independent of the presence of other "control" and/or "control" weed species, and is not attenuated and/or absent. In particular, any tissue of a transgenic plant (containing a gene encoding a protox enzyme according to the invention) is present and/or produced simultaneously and/or asynchronously, said protox enzyme and/or another substance which controls weeds, the presence of which does not affect nor cause a "controlling" and/or "controlling" effect of said protox enzyme on weeds, either entirely and/or partly, being effected by said another substance, independently of said protox enzyme.
"plant propagules" as used herein include, but are not limited to, plant sexual propagules and plant asexual propagules. Such plant propagules include, but are not limited to, plant seeds; the plant vegetative propagation body refers to a vegetative organ or a special tissue of a plant body, which can produce a new plant under ex vivo conditions; the vegetative organ or a particular tissue includes, but is not limited to, roots, stems and leaves, such as: plants using roots as vegetative propagation bodies include strawberries, sweet potatoes and the like; plants with stems as vegetative propagation material include sugarcane, potato (tuber) and the like; plants with leaves as vegetative propagation material include aloe and begonia etc.
The present invention can confer new herbicide resistance traits to plants and no adverse effect on phenotype, including yield, is observed. Plants of the invention are tolerant to levels as generally found in 2×, 3×, or 4× of at least one herbicide tested. These improvements in tolerance levels are within the scope of the present invention. For example, a number of techniques known in the art may be predictably optimized and further developed to increase expression of a given gene.
The invention provides herbicide tolerance protein, a coding gene and application thereof, and the herbicide tolerance protein has the following advantages:
1. Has wide tolerance to herbicide. The herbicide tolerance protein can show higher tolerance to PPO inhibitor herbicide, so the application prospect on plants is wide.
2. Has strong tolerance to herbicide. The herbicide tolerance protein of the invention has strong tolerance to PPO inhibitor herbicides, and has high tolerance to all of oxyfluorfen, saflufenacil and flumioxazin with a field concentration of 4 times and mesotrione with a field concentration of 2 times.
The technical scheme of the invention is further described in detail through the drawings and the embodiments.
Drawings
FIG. 1 is a schematic diagram of the structure of an Arabidopsis recombinant expression vector DBN12353 containing the PPO15A nucleotide sequence;
FIG. 2 is a schematic diagram of the structure of a control recombinant expression vector DBN12353N of the invention.
Detailed Description
The use of the protoporphyrinogen oxidase of the present invention will be further described by way of specific examples.
First example, acquisition and verification of transgenic Arabidopsis plants
1. Obtaining the PPO15 gene
The amino acid sequence of the herbicide tolerance protein PPO15 is shown as SEQ ID NO. 1 in a sequence table, and the nucleotide sequence of PPO15A which codes for the herbicide tolerance protein PPO15 is obtained according to the common preference codon of arabidopsis thaliana and soybean and is shown as SEQ ID NO. 2 in the sequence table.
The amino acid sequence of the protoporphyrinogen oxidase PPO-EC of Escherichia coli (Escherichia coli) is shown as SEQ ID NO. 3 in a sequence table; the nucleotide sequence of the PPO-EC corresponding to the E.coli protoporphyrinogen oxidase PPO-EC is shown as SEQ ID NO. 4 in a sequence table; obtaining a PPO-ECA nucleotide sequence which codes for PPO-EC corresponding to the E.coli protoporphyrinogen oxidase according to the common preference codon of Arabidopsis thaliana and soybean, and the nucleotide sequence is shown as SEQ ID NO. 5 in a sequence table.
The amino acid sequence of the arabidopsis protoporphyrinogen oxidase PPO-AT is shown as SEQ ID NO. 6 in a sequence table; the nucleotide sequence of the PPO-AT corresponding to the arabidopsis protoporphyrinogen oxidase PPO-AT is encoded and shown as SEQ ID NO 7 in a sequence table; obtaining a PPO-ATA nucleotide sequence which codes for PPO-AT corresponding to the protoporphyrinogen oxidase of Arabidopsis thaliana according to the common preference codon of Arabidopsis thaliana and soybean, and the nucleotide sequence is shown as SEQ ID NO. 8 in a sequence table.
2. Synthesis of the nucleotide sequence
The 5 'and 3' ends 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) are respectively connected with a universal joint primer 1:
5' -terminal universal adaptor primer 1:5'-taagaaggagatatacatatg-3' is shown as SEQ ID NO 9 in the sequence table;
3' -terminal 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 sequences, PPO-ECA nucleotide sequences and PPO-ATA nucleotide sequences
Carrying out double digestion reaction on a plant expression vector DBNBC-01 by using restriction endonucleases SpeI and Asc I, thereby linearizing the plant expression vector, purifying a digestion product to obtain a linearized DBNBC-01 expression vector skeleton (vector skeleton: pCAMBIA2301 (available from CAMBIA mechanism)), 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 specification of an In-Fusion seamless connection product kit (Clontech, CA, USA, CAT 121416) of Takara company, wherein the structure schematic diagram is shown In FIG. 1 (Spec: spectinomycin gene); RB right border, eFMV 34S enhancer of figwort mosaic virus (SEQ ID NO: 11), prBrCBP promoter of rape eukaryotic elongation factor gene 1 alpha (Tsf 1) (SEQ ID NO: 12), sphtCTP 2. Arabidopsis chloroplast transit peptide (SEQ ID NO: 13), EPSPS 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 14), tPsE9 terminator of pea RbcS gene (SEQ ID NO: 15), prAtubi 10. Arabidopsis Ubiquitin (Ubiquinin) 10 gene promoter (SEQ ID NO: 16), sphtCLP 1. Arabidopsis albino or light green body transit peptide (SEQ ID NO: 17), PPO15A: PPO15A nucleotide sequence (SEQ ID NO: 2), tNos terminator of nopaline synthase gene (SEQ ID NO: 18), pr35S promoter of cauliflower mosaic virus (SEQ ID NO: 18) ID NO: 19); PAT: the phosphinothricin N-acetyl transferase gene (SEQ ID NO: 20); t35S: the cauliflower mosaic virus 35S terminator (SEQ ID NO: 21); LB: left boundary).
The recombinant expression vector DBN12353 is used for transforming competent cells of the escherichia coli T1 by a heat shock method, and the heat shock conditions are as follows: 50. Mu.L of E.coli T1 competent cells, 10. Mu.L of plasmid DNA (recombinant expression vector DBN 12353), 42℃in a water bath for 30s; shake culturing at 37deg.C for 1 hr (shaking table at 100 rpm); then cultured on the LB solid plate (tryptone 10g/L, yeast extract 5g/L, naCl g/L, agar 15g/L, pH adjusted to 7.5 with NaOH) containing 50mg/L of Spectinomycin (Spectinomycin) at 37℃for 12 hours, white colonies were picked up, and cultured on LB liquid medium (tryptone 10g/L, yeast extract 5g/L, naCl g/L, spectinomycin 50mg/L, pH adjusted to 7.5 with NaOH) at 37℃for overnight. Extracting the plasmid by an alkaline method: centrifuging the bacterial solution at 12000rpm for 1min, removing supernatant, and suspending the precipitated bacterial cells with 100 μl ice-precooled solution I (25 mM Tris-HCl, 10mM EDTA (ethylenediamine tetraacetic acid), 50mM glucose, pH 8.0); 200. Mu.L of freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)) was added, the tube was inverted 4 times, mixed, and placed on ice for 3-5min; adding 150 μl ice-cold solution III (3M potassium acetate, 5M acetic acid), immediately mixing, and standing on ice for 5-10min; centrifuging at 4deg.C and 12000rpm for 5min, adding 2 times volume of absolute ethanol into the supernatant, mixing, and standing at room temperature for 5min; centrifuging at 4deg.C and 12000rpm for 5min, removing supernatant, washing precipitate with 70% ethanol (V/V), and air drying; adding 30. Mu.L of TE (10 mM Tris-HCl, 1mM EDTA, pH 8.0) containing RNase (20. Mu.g/mL) to dissolve the precipitate; digesting RNA in a water bath at 37 ℃ for 30 min; preserving at-20deg.C for use. Sequencing and identifying the extracted plasmid, and the result shows that the nucleotide sequence of the recombinant expression vector DBN12353 between SpeI and Asc I sites is the nucleotide sequence shown in SEQ ID NO. 2 in the sequence table, namely the nucleotide sequence of the PPO 15A.
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, so as to sequentially obtain the recombinant expression vectors DBN12354 and DBN12355. Sequencing confirmed that the above nucleotide sequences in recombinant expression vectors DBN12354 and DBN12355 were correctly inserted.
According to the method for constructing the recombinant expression vector DBN12353, a control recombinant expression vector DBN12353N is constructed, the vector structure of which is shown in FIG. 2 (Spec: spectinomycin gene; RB: right border; eFMV: figwort mosaic virus 34S enhancer (SEQ ID NO: 11), prBrCBP: promoter of rape eukaryotic elongation factor 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-acetyl transferase gene (SEQ ID NO: 20), t35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 21), and left border.
4. Agrobacterium transformation of arabidopsis recombinant expression vector
The recombinant expression vectors DBN12353 to DBN12355 and the control recombinant expression vector DBN12353N which are constructed correctly are respectively transformed into agrobacterium GV3101 by a liquid nitrogen method, 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 warm water bath at 37deg.C for 10min; the transformed agrobacterium GV3101 is inoculated in an LB test tube and cultured for 2 hours at the temperature of 28 ℃ and the rotating speed of 200rpm, and the agrobacterium is coated on an LB solid plate containing 50mg/L of Rifampicin (Rifampicin) and 50mg/L of spectinomycin until positive monoclonal grows out, the monoclonal culture is selected, plasmids are extracted, and sequencing identification is carried out on the extracted plasmids, so that the result shows that the structures of recombinant expression vectors DBN12353 to DBN12355 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 kept at 4 ℃ for 2 days to fulfill the need for dormancy to ensure synchronized germination of the seeds. The soil mixture was drained with vermiculite mix Ma Fentu and sub-irrigated with water to wetting for 24h. The pretreated seeds were planted on the soil mixture and covered with a moisture-retaining cover for 7 days. Germinating the seeds and providing a constant humidity (40-50%) at a constant temperature (22deg.C) with a light intensity of 120-150 μmol/m 2 s -1 Plants were grown in a greenhouse under long-day conditions (16 h light/8 h dark). Plants were initially irrigated with Hoagland's nutrient solution followed by deionized water to keep the soil moist but not wet.
Arabidopsis thaliana was transformed using the floral dip method. One or more 15-30mL precultures of LB medium containing spectinomycin (50 mg/L) and rifampicin (10 mg/L) were inoculated with the selected Agrobacterium colonies. The preculture was incubated overnight at a constant shaking speed at a temperature of 28℃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 at 28℃with continuous shaking overnight. 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 an osmotic medium containing 1/2 XMS salt/B5 vitamin, 10% (w/v) sucrose, 0.044. Mu.M benzylaminopurine (10. Mu.L/L (stock solution in 1mg/mL DMSO)) and 300. Mu.L/LSilvet L-77. Arabidopsis plants of about 1 month of age were soaked in the permeate medium containing the resuspended cells for 5min, ensuring that the latest inflorescences were submerged. Then the sides of the Arabidopsis plants are laid down and covered, after the Arabidopsis plants are moisturized for 24 hours in a dark environment, the Arabidopsis plants are normally cultivated at the temperature of 22 ℃ with the photoperiod of 16h light/8 h darkness. Seeds were harvested after about 4 weeks.
The 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 26.5 cm. Times.51 cm germination trays, each tray receiving 200mg T 1 Seeds (about 10000 seeds) which have been previously suspended in distilled water and stored at a temperature of 4 ℃ for 2 days to fulfill the need for dormancy to ensure synchronized germination of the seeds.
Vermiculite was mixed Ma Fentu and bottom irrigated to wetting with water and gravity drained. The pretreated seeds were planted uniformly on the soil mixture with a pipette and covered with a moisturizing cap for 4-5 days. The cover was removed 1 day prior to the initial transformant selection using post-emergence spray of glufosinate (co-transformed PAT gene selection).
After 7 days of planting (DAP) and again using a DeVilbiss compressed air nozzle at 11DAP, T was sprayed with a 0.2% solution of Liberty herbicide (200 g ai/L glufosinate) at a spray volume of 10 mL/tray (703L/ha) 1 Plants (cotyledonary stage and 2-4 leaf stage, respectively) to provide an effective amount of glufosinate per application of 280 gai/ha. Surviving plants (actively growing plants) were identified 4-7 days after the final spraying and transplanted into 7cm x 7cm square pots (3-5 plants per tray) prepared with struvite and vermiculite, respectively. The transplanted plants were covered with a moisture-retaining cover for 3-4 days and placed in a culture chamber at a temperature of 22℃as before or directly transferred into a greenhouse. The cover is then removed and the plants are planted in a greenhouse (temperature 22.+ -. 5 ℃, 50.+ -.30% RH,14h light: 10h darkness, minimum 500. Mu.E/m) at least 1 day before testing the PPO15A nucleotide sequence, PPO-ECA nucleotide sequence, PPO-ATA nucleotide sequence and control vector for the ability to provide tolerance to PPO inhibitor herbicides 2 s -1 Natural + supplemental light).
6. Herbicide tolerance effect detection of transgenic arabidopsis plants
First, the transformed Arabidopsis thaliana T is selected using a glufosinate herbicide 1 And (5) a plant. Arabidopsis thaliana T into which PPO15A nucleotide sequence is transferred respectively 1 Plants [ (plant ]PPO 15A), arabidopsis T transfected with PPO-ECA nucleotide sequence 1 Plant (PPO-ECA), arabidopsis thaliana T transformed with PPO-ATA nucleotide sequence 1 Plant (PPO-ATA), arabidopsis T transferred into control vector 1 Plants (control vector) and wild-type Arabidopsis plants (CK) were each subjected to herbicide resistance tests with 3 concentrations of oxyfluorfen (180 g ai/ha (1-fold field concentration, 1×), 720g ai/ha (4-fold field concentration, 4×) and 0g ai/ha (water, 0×)), 3 concentrations of saflufenacil (25 g ai/ha (1-fold field concentration, 1×1), 100g ai/ha (4-fold field concentration, 4×2) and 0g ai/ha (water, 0×)), 3 concentrations of flumioxazin (60 g ai/ha (1-fold field concentration, 1×), 240g ai/ha (4-fold field concentration, 4×) and 0g ai/ha (water, 0×)), and 3 concentrations of mesotrione (450 g ai/ha (1-fold field concentration, 4-fold) and 0g ai/ha (water, 0×)), 3 concentrations of flumioxazin (60 g ai/ha (1-fold field concentration, 1-fold, 0-fold field concentration, 0-fold 2). After 7 days of spraying (7 DAT), the extent of injury to each plant by the herbicide was evaluated according to the plant average injury percentage rating (plant average injury percentage = leaf injury area/leaf total area x 100%), i.e. phytotoxicity rating: the growth condition of the level 0 is basically consistent with that of the spraying of a blank solvent (water), the average damage percentage of the level 1 is less than 10 percent, the average damage percentage of the level 2 is more than 10 percent, and the average damage percentage of the level 3 is 100 percent. The high-resistance plants are classified into 0-level and 1-level according to the plant growth condition, the medium-low-resistance plants are classified into 2-level according to the plant growth condition, and the non-resistance plants are classified into 3-level according to the plant growth condition. The experimental results are shown in tables 1-4.
TABLE 1 Arabidopsis thaliana T transformed with PPO15A nucleotide sequence, PPO-ECA nucleotide sequence, PPO-ATA nucleotide sequence and control vector 1 Experimental results of plant tolerance to oxyfluorfen
For Arabidopsis 180g ai/ha oxyfluorfen herbicide is an effective dose to distinguish sensitive plants from plants with average resistance levels. The results in Table 1 show that (1) both PPO15A and PPO-ECA are able to produce a different degree of tolerance to oxyfluorfen than the control vector and CK, while PPO-ATA is essentially intolerant to oxyfluorfen; (2) For oxyfluorfen with 1-time field concentration, the phytotoxicity grade of PPO15A is 0 grade, and the phytotoxicity grade of PPO-ECA is 1 grade; (3) For 4-fold field concentrations of oxyfluorfen, PPO15A all showed high resistance, whereas 50% of the PPO-ECA plants were not resistant, with about 50% of the plants having only medium-low resistance. Thus, it is demonstrated that PPO15A has a significantly increased tolerance to oxyfluorfen.
TABLE 2 Arabidopsis thaliana T transformed with PPO15A nucleotide sequence, PPO-ECA nucleotide sequence, PPO-ATA nucleotide sequence and control vector 1 Experimental results of tolerance of plants to saflufenacil
For Arabidopsis, 25g ai/ha saflufenacil herbicide is an effective dose to distinguish sensitive plants from plants with average resistance levels. The results in Table 2 show that (1) both PPO15A and PPO-ECA are able to produce a different degree of tolerance to saflufenacil than the control vector and CK, whereas PPO-ATA is not tolerant to saflufenacil; (2) For saflufenacil with 1-time field concentration, the phytotoxicity grade of PPO15A is 0 grade, and the phytotoxicity grade of about 33% of plants in PPO-ECA is 1 grade; (3) For 4-fold field concentrations of saflufenacil, PPO15A all showed high resistance tolerance, whereas approximately 25% of plants in PPO-ECA were not. From this, it was demonstrated that PPO15A had significantly increased tolerance to saflufenacil.
TABLE 3 Arabidopsis thaliana T transformed with PPO15A nucleotide sequence, PPO-ECA nucleotide sequence, PPO-ATA nucleotide sequence and control vector 1 Tolerance test results of plants to flumioxazin
For Arabidopsis, 60g ai/ha flumioxazin herbicide is an effective dose to distinguish sensitive plants from plants with average resistance levels. The results in Table 3 show that both PPO15A and PPO-ECA are able to produce a different degree of tolerance to flumioxazin than the control vector and CK, while PPO-ATA is essentially intolerant to flumioxazin; (2) For 1-fold field concentrations of flumioxazin, PPO15A all showed high resistance, whereas about 66% of plants in PPO-ECA were not. (3) For 4-fold field concentrations of flumioxazin, PPO15A all showed high resistance, whereas none of PPO-ECA had high resistance. Thus, PPO15A has a significantly increased tolerance to flumioxazin.
TABLE 4 Arabidopsis thaliana T transformed with PPO15A nucleotide sequence, PPO-ECA nucleotide sequence, PPO-ATA nucleotide sequence and control vector 1 Tolerance test results of plants to sulfenamide
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For arabidopsis, 450g ai/ha sulfentrazone herbicide is an effective dose to distinguish sensitive plants from plants with average resistance levels. The results in Table 4 show that (1) both PPO15A and PPO-ECA are able to produce a different degree of tolerance to sulfenamide than the control vector and CK, whereas PPO-ATA is not tolerant to sulfenamide; (2) For 1-fold field concentrations of sulfentrazone, PPO15A all showed high resistance, whereas about 50% of plants in PPO-ECA did not have high resistance; (3) For 4-fold field concentrations of sulfenamide, PPO15A all showed high resistance, whereas neither PPO-ECA had high resistance. This demonstrates that PPO15A has significantly increased tolerance to sulfenamide.
The applicant has noted that the tolerance of plants to herbicides has a direct relationship with the yield of plants, and that high-resistance plants are not substantially affected by herbicides and thus do not affect the yield of plants, whereas low-medium-resistance plants yield is much reduced relative to high-resistance plants.
The protoporphyrinogen oxidase PPO15 can not only endow arabidopsis 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 mono and the like) with better tolerance.
In summary, the invention discloses for the first time that protoporphyrinogen oxidase PPO15 can endow plants with higher tolerance to PPO inhibitor herbicides, and can tolerate oxyfluorfen, saflufenacil and flumetsulam with a field concentration of at least 4 times and mesotrione with a field concentration of 2 times, so that the invention has wide application prospects in plants.
Finally, it should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Sequence listing
<110> Beijing Dabei agricultural biotechnology Co., ltd
<120> herbicide tolerance protein, gene encoding the same, 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' Universal Joint primer 1 (Artificial Sequence)
<400> 9
taagaaggag atatacatat g 21
<210> 10
<211> 21
<212> DNA
<213> Artificial sequence-3' Universal Joint primer 1 (Artificial Sequence)
<400> 10
gtggtggtgg tggtgctcga g 21
<210> 11
<211> 542
<212> DNA
<213> 34S enhancer 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> rape eukaryotic elongation factor gene 1α (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 (promoter Arabidopsis thaliana of Ubiquitin10 Gene)
<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 (31)

1. A protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. A gene, comprising:
(a) A nucleotide sequence encoding the protein of claim 1; or (b)
(b) A nucleotide sequence that is fully complementary to the nucleotide sequence defined in (a); or (b)
(c) The nucleotide sequence shown as SEQ ID NO. 2.
3. An expression cassette comprising the gene of claim 2 under the control of operably linked regulatory sequences.
4. A recombinant vector comprising the gene of claim 2 or the expression cassette of claim 3.
5. A method of extending the range of herbicides tolerated by plants comprising: expressing the protein of claim 1 or the protein encoded by the expression cassette of claim 3 in a plant 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 extending the range of herbicide tolerance in plants 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 a PPO inhibitor herbicide that allows growth of transformed cells expressing the gene or the expression cassette, while killing or inhibiting growth of untransformed cells, the PPO inhibitor herbicide being oxyfluorfen, saflufenacil, sulfenacil, and/or flumioxazin.
8. The method of selecting a transformed plant cell according to claim 7, wherein the plant comprises a monocot and a dicot.
9. The method of selecting a transformed plant cell according to claim 8, wherein the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distach, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis thaliana.
10. A method of controlling weeds, comprising: applying an effective dose of a PPO inhibitor herbicide to a field in which a plant of interest is grown, said plant of interest comprising the gene of claim 2 or the expression cassette of claim 3 or the recombinant vector of claim 4, said PPO inhibitor herbicide being oxyfluorfen, saflufenacil, sulfenacil and/or flumioxazin.
11. The method of controlling weeds of claim 10, wherein the plant of interest comprises monocotyledonous plants and dicotyledonous plants.
12. The method of weed control according to claim 11, wherein the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distach, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato or arabidopsis thaliana.
13. A method of controlling weeds according to any one of claims 10 to 12, wherein the plant of interest is a glyphosate tolerant plant and the weeds are glyphosate tolerant weeds.
14. A method for protecting a plant from injury caused by a PPO inhibitor herbicide or for conferring PPO inhibitor herbicide tolerance to a plant, 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 herbicide tolerance protein sufficient to protect it from a PPO inhibitor herbicide, which is oxyfluorfen, saflufenacil, sulfenacil and/or flumioxazin.
15. The method for protecting a plant from injury caused by a PPO inhibitor herbicide or conferring PPO inhibitor herbicide tolerance to a plant according to claim 14, wherein the plant comprises a monocot and a dicot.
16. The method for protecting a plant from injury caused by a PPO inhibitor herbicide or conferring PPO inhibitor herbicide tolerance to a plant according to claim 15, wherein the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyranthes, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis.
17. 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, wherein the PPO inhibitor herbicide is oxyfluorfen, saflufenacil, sulfenacil, and/or flumioxazin.
18. The method of producing a PPO inhibitor herbicide-tolerant plant of claim 17, wherein the method of introducing comprises a genetic transformation method, a genome editing method, or a gene mutation method.
19. The method of producing a PPO inhibitor herbicide-tolerant plant of claim 17, wherein the plant comprises a monocot and a dicot.
20. The method of producing a plant tolerant to a PPO inhibitor herbicide according to claim 19, wherein the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis.
21. 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 propagules into plants;
applying an effective dose of a PPO inhibitor herbicide to a plant growth environment comprising at least said plant, harvesting a plant 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; the PPO inhibitor herbicide is oxyfluorfen, saflufenacil, sulfluramid and/or flumetsulam.
22. The method of culturing a PPO inhibitor herbicide-tolerant plant of claim 21, wherein the plant comprises a monocot and a dicot.
23. The method of claim 22, wherein the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis thaliana.
24. A method of obtaining a processed agricultural product comprising treating a harvest of PPO inhibitor herbicide tolerant plants obtained by the method of any one of claims 21-23 to obtain a processed agricultural product.
25. A planting system for controlling weed growth comprising providing a PPO inhibitor herbicide, which is oxyfluorfen, saflufenacil, sulfenacil and/or flumioxazin, and a plant growth environment in which at least one plant of interest is present, said plant of interest comprising the gene of claim 2 or the expression cassette of claim 3.
26. A planting system for controlling weed growth according to claim 25, wherein the plants of interest comprise monocotyledonous and dicotyledonous plants.
27. The weed growth control planting system according to claim 26, wherein the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distach, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis thaliana.
28. A planting system for controlling the growth of weeds according to any one of claims 25 to 27, wherein the plant of interest is a glyphosate tolerant plant and the weeds are glyphosate tolerant weeds.
29. Use of a protein as defined in claim 1 to confer tolerance to a PPO inhibitor herbicide which is oxyfluorfen, saflufenacil, sulfenacil and/or flumioxazin to a plant.
30. The use according to claim 29, wherein the plants comprise monocotyledonous and dicotyledonous plants.
31. The use according to claim 30, wherein the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distach, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis thaliana.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1212724A (en) * 1996-02-28 1999-03-31 诺瓦提斯公司 DNA molecules encoding plant protoporphyrinogen oxidase and inhibitor-resistant mutant thereof
CN1373811A (en) * 1999-08-13 2002-10-09 辛根塔参与股份公司 Herbicide-tolerant protoporphyrinogen oxidase
CN111465696A (en) * 2017-12-15 2020-07-28 孟山都技术公司 Methods and compositions for tolerance to PPO herbicides

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105802933B (en) * 2016-03-22 2020-05-05 北京大北农科技集团股份有限公司 Herbicide tolerance protein, coding gene and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1212724A (en) * 1996-02-28 1999-03-31 诺瓦提斯公司 DNA molecules encoding plant protoporphyrinogen oxidase and inhibitor-resistant mutant thereof
CN1373811A (en) * 1999-08-13 2002-10-09 辛根塔参与股份公司 Herbicide-tolerant protoporphyrinogen oxidase
CN111465696A (en) * 2017-12-15 2020-07-28 孟山都技术公司 Methods and compositions for tolerance to PPO herbicides

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A0A6G8D3M0_9VIBR;Uniprot;Uniprot;第1-2页 *
WP_166014020.1;NCBI;GenBank;第1页 *
原卟啉原氧化酶抑制性除草剂的发展;苏少泉;;农药研究与应用(第01期);第1-5页 *

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