CN111304178B

CN111304178B - Herbicide tolerance protein, coding gene and application thereof

Info

Publication number: CN111304178B
Application number: CN202010194585.0A
Authority: CN
Inventors: 贺芹; 刘斌; 彭乾; 肖翔; 何健; 陶青
Original assignee: Nanjing Agricultural University; Beijing Dabeinong Biotechnology Co Ltd
Current assignee: Nanjing Agricultural University; Beijing Dabeinong Biotechnology Co Ltd
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2022-06-03
Anticipated expiration: 2040-03-19
Also published as: CN111304178A; CN114854702A; CN114854702B

Abstract

The invention relates to a herbicide tolerance protein, a coding gene and application thereof, wherein the protein comprises: (a) has an amino acid sequence shown as SEQ ID NO. 6 or SEQ ID NO. 9; (b) a protein derived from (a) having a hydroxyphenylpyruvate dioxygenase, wherein the protein is obtained by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence of (a). The herbicide tolerance protein can endow plants with tolerance to HPPD inhibitor herbicides, and has wide application prospect in plants.

Description

Herbicide tolerance protein, coding gene and application thereof

Technical Field

The invention relates to a herbicide tolerance protein, a coding gene and application thereof, in particular to a protein with tolerance to an HPPD inhibitor herbicide, a coding gene and application thereof.

Background

Hydroxyphenylpyruvate dioxygenase (HPPD) is an enzyme that is found in iron ions (Fe)²⁺) And in the presence of oxygen, catalyzing the conversion of a tyrosine degradation product, namely, hydroxyphenylpyruvic acid (HPP) into plantsReaction of tocopherol with homogentisone (PQ) precursor-homogentisate/Homogentisate (HG). Tocopherol has the effect of a membrane-associated antioxidant; PQ is not only an electron carrier between PS II and the cytochrome b6/f complex, but also an essential cofactor for phytoene desaturase in carotenoid biosynthesis.

Herbicides that act by inhibiting HPPD are mainly three chemical families of triketones, isoxazoles and pyrazolones. In plants, they block the biosynthesis of PQ from tyrosine by inhibiting HPPD, leading to PQ depletion, carotenoid deficiency. The above mentioned HPPD inhibiting herbicides are plant phloem mobile bleaches which cause the new meristems and leaves exposed to light to appear white, whereas carotenoids are essential for photoprotection, in the absence of carotenoids, uv radiation and reactive oxygen intermediates disrupt chlorophyll synthesis and function, leading to plant growth inhibition, even death.

The method for providing a plant tolerant to HPPD inhibitor herbicides essentially comprises: 1) HPPDs are overexpressed to produce high quantities of HPPD in plants, and despite the presence of the HPPD inhibitor herbicide, the HPPDs are sufficiently reactive with the HPPD inhibitor herbicide to have sufficient functional enzyme available for use. 2) The target HPPD is mutated to a functional HPPD that is less sensitive to herbicides or active metabolites thereof, but which retains the property of being converted to HG. With respect to mutant HPPD classes, while a given mutant HPPD may provide a useful level of tolerance to some HPPD inhibitor herbicides, the same mutant HPPD may not be sufficient to provide a commercial level of tolerance to a different, more desirable HPPD inhibitor herbicide; for example, HPPD inhibitor herbicides can differ in the range of weeds they control, their cost of manufacture, and their environmental friendliness. Thus, there is a need for new methods and/or compositions for conferring HPPD inhibitor herbicide tolerance to different crops and crop varieties.

Disclosure of Invention

The invention aims to provide a novel protein, a coding gene thereof and application thereof, wherein the protein not only has HPPD enzyme activity, but also enables plants transferred with the coding gene of the protein to have tolerance to HPPD inhibitor herbicides.

To achieve the above object, the present invention provides a protein comprising:

(a) has an amino acid sequence shown as SEQ ID NO. 6 or SEQ ID NO. 9;

(b) and (b) a protein derived from (a) and having hydroxyphenylpyruvate dioxygenase activity, wherein the amino acid sequence in (a) is substituted and/or deleted and/or one or more amino acids are added.

To achieve the above object, the present invention provides a gene comprising:

(a) a nucleotide sequence encoding the protein of claim 1; or

(b) A nucleotide sequence which hybridizes with the nucleotide sequence defined in (a) under strict conditions and codes a protein with hydroxyphenylpyruvate dioxygenase activity; or

(c) Has the nucleotide sequence shown in SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10 or SEQ ID NO 11.

The stringent conditions may be hybridization in a 6 XSSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution at 65 ℃ and then washing the membrane 1 time each with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

To achieve the above object, the present invention also provides an expression cassette comprising the gene under the control of an operably linked regulatory sequence.

In order to achieve the above object, the present invention also provides a recombinant vector comprising the gene or the expression cassette

To achieve the above object, the present invention also provides a method for extending the herbicide tolerance of a plant, comprising: expressing said protein or the protein encoded by said expression cassette in a plant together with at least one second herbicide tolerance protein different from said protein or the protein encoded by said expression cassette.

Further, the second herbicide tolerance protein is 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha ketoglutarate-dependent dioxygenase, dicamba monooxygenase, acetolactate synthase, a cytochrome-like protein and/or protoporphyrinogen oxidase.

To achieve the above object, the present invention also provides a method for selecting a transformed plant cell, comprising: transforming a plurality of plant cells with said gene or said expression cassette and culturing said cells at a concentration of HPPD inhibitor herbicide that allows growth of transformed cells expressing said gene or said expression cassette, while killing or inhibiting growth of untransformed cells;

preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, rape, potato, tomato or arabidopsis;

preferably, the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide; more preferably, the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.

To achieve the above object, the present invention also provides a method for controlling weeds, comprising: applying an effective dose of an HPPD inhibitor herbicide to a field growing a plant of interest, said plant of interest comprising said gene or said expression cassette or said recombinant vector;

preferably, the plant of interest includes a monocotyledon and a dicotyledon; more preferably, the plant of interest is oat, wheat, barley, millet, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, rape, potato, tomato or arabidopsis; further preferably, the plant of interest is a glyphosate tolerant plant and the weeds are glyphosate resistant weeds;

To achieve the above objects, the present invention also provides a method for protecting a plant from damage caused by or conferring tolerance to a HPPD inhibitor herbicide to a plant, comprising: introducing the gene or the expression cassette or the recombinant vector into a plant such that the introduced plant produces an amount of herbicide tolerance protein sufficient to protect it from damage by HPPD inhibitor herbicides;

preferably, the plant includes monocotyledons and dicotyledons; more preferably, the plant is oat, wheat, barley, millet, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, rape, potato, tomato, or arabidopsis;

To achieve the above object, the present invention also provides a method for producing a plant tolerant to an HPPD inhibitor herbicide, comprising introducing the gene into the genome of the plant;

preferably, the method of introduction includes a genetic transformation method, a genome editing method, or a gene mutation method;

specifically, the method of producing a plant tolerant to an HPPD inhibitor herbicide comprises: producing an HPPD inhibitor herbicide tolerant plant by selfing a parent plant or crossing with a second plant, said parent plant and/or second plant comprising said gene or said expression cassette, said HPPD inhibitor herbicide tolerant plant inheriting said gene or said expression cassette from said parent plant and/or second plant;

preferably, the plant includes a monocotyledon and a dicotyledon; more preferably, the plant is oat, wheat, barley, millet, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, rape, potato, tomato or arabidopsis;

To achieve the above objects, the present invention also provides a method of growing a plant tolerant to an HPPD inhibitor herbicide, comprising:

growing at least one plant propagule comprising in its genome the gene or the expression cassette;

growing the plant propagule into a plant;

applying an effective dose of an HPPD inhibitor herbicide to a plant growing environment comprising at least said plant, harvesting plants having reduced plant damage and/or increased plant yield as compared to other plants not having said gene or said expression cassette;

The present invention also provides a method for obtaining a processed agricultural product comprising treating a harvest of HPPD inhibitor herbicide tolerant plants obtained by said method to obtain a processed agricultural product.

To achieve the above object, the present invention also provides a planting system for controlling weed growth, comprising a HPPD inhibitor herbicide and a plant growing environment in which at least one plant of interest is present, said plant of interest comprising said gene or said expression cassette;

To achieve the above objects, the present invention also provides the use of the protein for conferring a HPPD inhibitor herbicide on a plant;

The articles "a" and "an" as used herein mean one or more than one (i.e., at least one). For example, "an element" means one or more elements (components). Furthermore, the terms "comprises" or variations such as "comprising" or "comprising" are to be understood as meaning the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

In the present invention, the term "hydroxyphenylpyruvate dioxygenase (HPPD)" is synonymous with "4-hydroxyphenylpyruvate dioxygenase (4-HPPD)" and "p-hydroxyphenylpyruvate dioxygenase (p-HPPD)".

The term "HPPD-inhibiting herbicide" is synonymous with "HPPD herbicide" and refers to herbicides that directly or indirectly inhibit HPPD, which herbicides are bleaches, and whose primary site of action is HPPD. The most commercially available HPPD inhibiting herbicides belong to one of three chemical families: (1) triketones, for example, sulcotrione (i.e., 2- [ 2-chloro-4- (methylsulfonyl) benzoyl ] -1, 3-cyclohexanedione), mesotrione (mesotrione) (i.e., 2- [4- (methylsulfonyl) -2-nitrobenzoyl ] -1, 3-cyclohexanedione), tembotrione (i.e., 2- [ 2-chloro-4- (methylsulfonyl) -3- [ (2,2, 2-trifluoroethoxy) methyl ] benzoyl ] -1, 3-cyclohexanedione); (2) isoxazoles (in plants, isoxazole HPPD herbicides are rapidly converted to biologically active Diketonitriles (DKN) to exert an inhibitory effect on HPPD, and thus diketonitriles are the active form of isoxazole HPPD inhibiting herbicides), for example, isoxaflutole (i.e. 5-cyclopropyl-4-isoxazolyl [2- (methylsulfonyl) -4- (trifluoromethyl) phenyl ] methanone); (3) pyrazolones (pyrazolonates), for example topramezone (i.e. [3- (4, 5-dihydro-3-isoxazolyl) -2-methyl-4- (methylsulfonyl) phenyl ] (5-hydroxy-1-methylpyrazol-4-yl) methanone), pyrasulfopyra-zole (5-hydroxy-1, 3-dimethylpyrazol-4-yl (2-methanesulfonyl-4-trifluoromethylphenyl) methanone).

The topramezone, also called topramezone, in the present invention refers to [3- (4, 5-dihydro-3-isoxazolyl) -2-methyl-4- (methylsulfonyl) phenyl ] (5-hydroxy-1-methylpyrazol-4-yl) methanone as a white crystalline solid. Belongs to a pyrazolone (pyrazolonate) type internal absorption conduction type HPPD herbicide in postemergence treatment, and a common preparation formulation is a 30% suspending agent. Commercial preparations of topramezone, such as fennel, can control grassy and broadleaf weeds, 5.6-6.7g of the preparation per mu can effectively control weeds, including, but not limited to, large crabgrass (cocksfoot grass), barnyard grass, goosegrass, wild millet, green bristlegrass (nigella sativa), chenopodium quinoa, polygonum, ramie, abutilon, amaranthus, purslane, xanthium, and nightshade. The bracteal defense has obvious synergistic effect after adding atrazine, has excellent control effect on the weeds, and also has good control effect on malignant broadleaf weeds such as cephalanoplos segetum (herba Cirsii), endive, acalypha australis and dayflower (herba brassicae), and particularly can effectively control setaria viridis, digitaria sanguinalis, eleusine indica and broomcorn millet with poor control effect on mesotrione.

The effective dose of topramezone in the invention is 25-100g ai/ha, including 50-100g ai/ha, 60-90g ai/ha or 75-85g ai/ha.

In the present invention, the term "resistant" is heritable and allows plants to grow and reproduce under conditions where herbicides are effective for a given plant with typical herbicides. As recognized by those skilled in the art, even if a given plant is subjected to some degree of herbicide treatment, such as little necrosis, lysis, chlorosis, or other damage, but at least not significantly affected in yield, the plant can still be considered "resistant", i.e., the given plant has an increased ability to withstand herbicide-induced damage of various degrees, while typically causing damage to wild-type plants of the same genotype at the same herbicide dose. The term "resistance" or "tolerance" in the present invention is broader than the term "resistance" and includes "resistance".

The term "conferring" in the context of the present invention means providing a characteristic or trait, such as herbicide tolerance and/or other desirable traits, to a plant.

The term "heterologous" in the context of the present invention means from another source. In the context of DNA, "heterologous" refers to any foreign "non-self DNA, including DNA from another plant of the same species. For example, in the present invention, transgenic methods can be used to express the soybean HPPD gene in soybean plants, which is still considered "heterologous" DNA.

The term "nucleic acid" in the present invention encompasses deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, encompasses known analogs (e.g., peptide nucleic acids) that have the basic properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

In the present invention, when the term "encoding" or "encoded" is used in the context of a particular nucleic acid, it means that the nucleic acid contains the necessary information to direct the translation of the nucleotide sequence into a particular protein. The information used to encode the protein is specified by the use of codons. A nucleic acid encoding a protein may comprise untranslated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening untranslated sequences (e.g., in cDNA).

The herbicide tolerance protein of the present invention has HPPD enzymatic activity and confers tolerance in plants to certain classes of HPPD inhibiting herbicides. DNA sequences encoding herbicide tolerance proteins of the invention are useful in providing plants, crops, plant cells and seeds of the invention which provide enhanced tolerance to one or more HPPD herbicides.

The genes encoding the herbicide tolerance proteins of the present invention are useful for producing plants that are tolerant to HPPD inhibiting herbicides. The herbicide tolerance gene is particularly suitable for expression in plants in order to confer herbicide tolerance to the plant.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to polymers of amino acid residues in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The protein of the invention can be produced from a nucleic acid disclosed herein or by using standard molecular biology techniques. For example, a truncated protein of the invention may be produced by expressing a recombinant nucleic acid of the invention in a suitable host cell, or alternatively by a combination of ex vivo methods such as protease digestion and purification.

The invention also provides nucleic acid molecules comprising a polynucleotide sequence encoding an herbicide tolerance protein that has HPPD enzyme activity and confers tolerance in plants to certain classes of HPPD inhibiting herbicides. In general, the invention includes any polynucleotide sequence encoding any of the herbicide tolerance proteins described herein, as well as any polynucleotide sequence encoding an herbicide tolerance protein having one or more conservative amino acid substitutions relative to the herbicide tolerance protein described herein. Conservative substitutions providing functionally similar amino acids are well known to those skilled in the art, and the following five groups each contain amino acids that are conservative substitutions for one another: aliphatic: glycine (G), alanine (a), valine (V), leucine (L), isoleucine (I); aromatic: phenylalanine (F), tyrosine (Y), tryptophan (W); sulfur-containing: methionine (M), cysteine (C); basic: arginine (I), lysine (K), histidine (H); acidic: aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q).

Therefore, sequences having HPPD activity to inhibit herbicide tolerance and hybridizing under stringent conditions to the gene encoding the herbicide tolerance protein of the present invention are included in the present invention. Illustratively, these sequences have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology to the sequences of the invention SEQ ID NO 2, 3, 7, 8, 10, 11.

Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the herbicide tolerance gene of the present invention. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. In the present invention, two nucleic acid molecules can be said to be capable of specifically hybridizing to each other if they can form an antiparallel double-stranded nucleic acid structure. Two nucleic acid molecules are said to be "complements" of one another if they exhibit complete complementarity. In the present invention, two nucleic acid molecules are said to exhibit "perfect complementarity" when each nucleotide of the two nucleic acid molecules is complementary to the corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability to allow them to anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to have "complementarity" if they are capable of hybridizing to each other with sufficient stability to allow them to anneal and bind to each other under conventional "highly stringent" conditions. Deviations from perfect complementarity may be tolerated as long as such deviations do not completely prevent the formation of a double-stranded structure by the two molecules. In order to allow 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 in the particular solvent and salt concentrations employed.

In the present invention, a substantially homologous sequence is a nucleic acid molecule that specifically hybridizes under highly stringent conditions to the complementary strand of a compatible nucleic acid molecule. Suitable stringency conditions for facilitating DNA hybridization, for example, 6.0 XSSC/sodium citrate (SSC) at about 45 ℃ followed by a 2.0 XSSC wash at 50 ℃, are well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from the group consisting of about 2.0 XSSC for low stringency conditions, 50 ℃ to about 0.2 XSSC for high stringency conditions, 50 ℃. In addition, the temperature conditions in the washing step can be raised from about 22 ℃ at room temperature for low stringency conditions to about 65 ℃ for high stringency conditions. Both the temperature conditions and the salt concentration may be varied, or one may be held constant while the other is varied. Preferably, the stringent conditions of the present invention may be those in which specific hybridization with the herbicide tolerance gene of the present invention occurs in a 6 XSSC solution of 0.5% SDS at 65 ℃ and then the membranes are washed 1 time each with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In the present invention, the term "hybridize" or "specifically hybridize" refers to a molecule that can only bind, double-stranded, or hybridize to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

Due to the redundancy of the genetic code, a plurality of different DNA sequences may encode the same amino acid sequence. It is well within the skill of the art to generate such alternative DNA sequences encoding the same or substantially the same protein. These different DNA sequences are included within the scope of the present invention. The "substantially identical" sequences refer to sequences having amino acid substitutions, deletions, additions or insertions which do not substantially affect the herbicide tolerance activity, and also include fragments which retain the herbicide tolerance activity.

The term "functional activity" or "activity" as used herein means that the protein/enzyme (alone or in combination with other proteins) for use according to the invention has the ability to degrade or attenuate the activity of a herbicide. The plants producing the proteins of the invention preferably produce an "effective amount" of the protein such that when the plants are treated with the herbicide, the level of protein expression is sufficient to confer full or partial tolerance to the herbicide (if not specifically stated, the amount normally used) to the plant. The herbicide may be used in an amount that normally kills the target plant, in a normal field amount, and at a concentration. Preferably, the plant cells and plants of the invention are protected from growth inhibition or damage caused by herbicide treatment. The transformed plants and plant cells of the invention preferably have tolerance to HPPD inhibiting herbicides, i.e., the transformed plants and plant cells are capable of growing in the presence of an effective amount of a HPPD inhibiting herbicide.

The genes and proteins described in the present invention include not only the specific exemplary sequences, but also portions and/or fragments (including internal and/or terminal deletions compared to the full-length protein), variants, mutants, variant proteins, substitutions (proteins with substituted amino acids), chimeras and fusion proteins that preserve the HPPD herbicide tolerance-inhibiting activity characteristics of the specific exemplary proteins.

The term "variant" of the invention means substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within a reference polynucleotide and/or a substitution of one or more nucleotides at one or more sites in an herbicide tolerance gene. The term "reference polynucleotide or polypeptide" in the present invention encompasses a herbicide-tolerant nucleotide sequence or amino acid sequence, respectively. The term "native polynucleotide or polypeptide" in the present invention correspondingly includes naturally occurring nucleotide sequences or amino acid sequences. For polynucleotides, conservative variants include a nucleotide sequence encoding one of these herbicide tolerance proteins of the present invention (due to the degeneracy of the genetic code). Such naturally occurring allelic variants can be identified using well-known molecular biology techniques, for example, using the Polymerase Chain Reaction (PCR) and hybridization techniques outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as sequences produced by use of site-directed mutagenesis, but which still encode an herbicide tolerance protein of the invention. Typically, variants of a particular polynucleotide of the invention will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology to the particular polynucleotide as determined by sequence alignment programs and parameters.

By "variant protein" is meant a protein derived from a reference protein by deletion or addition of one or more amino acids at one or more internal sites in said herbicide tolerance protein and/or substitution of one or more amino acids at one or more sites in the herbicide tolerance protein. Variant proteins encompassed by the present invention are biologically active, i.e. they continue to have the desired activity of the herbicide tolerance protein, i.e. still have said HPPD enzyme activity and/or herbicide tolerance. Such variants may arise, for example, from genetic polymorphisms or from artificial manipulation. A biologically active variant of an herbicide tolerance protein of the invention will have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology to the entire amino acid sequence of the herbicide tolerance protein as determined by sequence alignment programs and parameters. A biologically active variant of a protein of the invention may differ from a protein in as few as 1-15 amino acid residues, as few as 1-10 (e.g., 6-10), as few as 5 (e.g., 4, 3, 2, or even 1) amino acid residues.

Methods for sequence 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; smith et al (1981) Adv.appl.Math.2: 482; need the global alignment algorithm of Need eman and Wunsch (1970) J.Mol.biol.48: 443-; and the algorithm of Karlin and Al tschul (1990) Proc.Natl.Acad.Sci.USA 872264, as modified in Karlin and Al tschul (1993) Proc.Natl.Acad.Sci.USA 90: 5873-. Computer implementations of these mathematical algorithms can be utilized for sequence comparison to determine sequence homology, such implementations including, but not limited to: CLUSTAL (available from Intelligenetics, mountain n View, California) in the PC/Gene program; ALLGN program (version 2.0) and GAP, BESTFIT, BLAST, FASTA and TFASTA in GCG Wiscons in Genetics Software Package version 10 (available from Accelrys Inc.,9685 Scanton Road, San Diego, California, USA).

In certain embodiments, amino acids encoding the herbicide tolerance proteins of the present invention or variants thereof that retain HPPD enzymatic activity can be stacked with any combination of polynucleotide sequences of interest to produce plants with a desired trait. The term "trait" refers to a phenotype derived from a particular sequence or group of sequences. For example, a polynucleotide encoding an amino acid of the herbicide tolerance protein or variant retaining HPPD enzyme activity may be stacked with any other polynucleotide encoding a polypeptide conferring a desired trait including, but not limited to: resistance to diseases, insects, and herbicides, tolerance to heat and drought, reduced crop maturation time, improved industrial processing (e.g., for converting starch or biomass into fermentable sugars), and improved agronomic quality (e.g., high oil content and high protein content).

The benefits of the combination of two or more modes of action in improving the controlled weed spectrum and/or the naturally more tolerant or resistant weed species can also be extended to the creation of herbicide tolerant chemicals in crops other than HPPD tolerant crops by man (transgenic or non-transgenic), as is well known to those skilled in the art. Indeed, the traits encoding the following resistances may be stacked, alone or in multiple combinations, to provide effective control or prevent the ability of a weed succession to develop resistance to herbicides: glyphosate resistance (e.g., resistant plant or bacteria EPSPS, GOX, GAT), glufosinate resistance (e.g., PAT, Bar), acetolactate synthase (ALS) inhibitory herbicide resistance (e.g., imidazolinone, sulfonylurea, triazolopyrimidine, sulfonanilide, pyrimidine thiobenzoate, 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 (PDS) inhibitors, resistance to systemic ii inhibitory herbicides (e.g., psbA), resistance to systemic i inhibitory herbicides, resistance to protoporphyrinogen oxidase ix (PPO) inhibitory herbicide desaturase (e.g., PPO-1), resistance to phenylurea (e.g., CYP76B1), Dichloromethoxybenzoic acid degrading enzymes, and the like.

Glyphosate is widely used because it controls a very broad spectrum of broadleaf and grass weed species. However, repeated use of glyphosate in glyphosate-tolerant crop and non-crop applications has (and will continue to) choose to allow weeds to evolve into a more naturally-tolerant species or glyphosate-resistant biotype. Most herbicide resistance management strategies suggest the use of an effective amount of a tank-mix herbicide partner that provides control of the same species but has a different mode of action as a means of delaying the emergence of resistant weeds. The superposition of the herbicide tolerance genes of the present invention with glyphosate tolerance traits (and/or other herbicide tolerance traits) can achieve control of glyphosate resistant weed species (broadleaf weed species controlled by one or more pyrazolone herbicides) in glyphosate tolerant crops by allowing selective use of glyphosate and pyrazolone herbicides (such as topramezone) on the same crop. The application of these herbicides can be simultaneous use in tank mixes containing two or more herbicides of different modes of action, separate use of individual herbicide compositions (with time intervals ranging from 2 hours to 3 months between uses) in successive uses (e.g., pre-planting, pre-emergence, or post-emergence), or alternatively, a combination of any number of herbicides representing the applicable classes of each compound can be used at any time from within 7 months of planting the crop to when the crop is harvested (or for a single herbicide, at the shortest interval).

It is important to have flexibility in controlling broadleaf weeds, i.e., time of use, individual herbicide dosage, and the ability to control persistent or resistant weeds. The application range of glyphosate superposed with glyphosate resistance gene/herbicide tolerance gene in crops can be from 250 to 2500g ae/ha; the pyrazolone herbicide(s) may be in the range of from 25 to 500g ai/ha. The optimal combination of times for these applications depends on the specific conditions, species and environment.

Herbicide formulations (such as ester, acid or salt formulations or soluble concentrates, emulsified concentrates or soluble liquids) and tank-mix additives (such as adjuvants or compatibilizers) can significantly affect the weed control of a given herbicide or combination of one or more herbicides. Any chemical combination of any of the foregoing herbicides is within the scope of the invention.

In addition, the gene encoding the herbicide tolerance protein of the present invention can be added to one or more other input (e.g., insect resistance, fungal resistance, or stress tolerance, etc.) or output (e.g., increased yield, improved oil, increased fiber quality, etc.) traits, either alone or in combination with other herbicide tolerance crop traits. Thus, the present invention can be used to provide a complete agronomic solution with the ability to flexibly and economically control any number of agronomic pests and to improve crop quality.

The combination of these overlays can be produced by any method, including but not limited to: cross-breeding plants or genetic transformation by conventional or top-crossing methods. If these sequences are stacked by genetically transforming these plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, transgenic plants comprising one or more desired traits may be used as targets for the introduction of additional traits by subsequent transformation. These traits may be introduced in a co-transformation protocol simultaneously with the polynucleotides of interest provided by any combination of expression cassettes. For example, if two sequences are to be introduced, the two sequences may be contained in separate expression cassettes (trans) or in the same expression cassette (cis). Expression of these sequences may be driven by the same promoter or by different promoters. In some cases, it may be desirable to introduce an expression cassette that inhibits expression of the polynucleotide of interest. This can be combined with any combination of other suppression or overexpression cassettes to produce the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system.

The gene for coding the herbicide tolerance protein has higher tolerance to pyrazolone herbicides, and is the basis of characteristic possibility of important herbicide tolerant crops and selective markers.

The term "expression cassette" in the present invention refers to a nucleic acid molecule capable of directing the 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 an herbicide tolerance protein or variant retaining HPPD enzyme activity, alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring a desired trait), which 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 an antisense RNA or an untranslated RNA in the sense or antisense orientation. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be a naturally occurring expression cassette, but must be obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous to the host, i.e., the particular DNA sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into a new host cell by a transformation event. Expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus. In addition, the promoter is specific for a particular tissue or organ or developmental stage.

The present invention encompasses transforming plants with expression cassettes capable of expressing a polynucleotide of interest (i.e., a polynucleotide encoding an herbicide tolerance protein or a variant thereof that retains HPPD enzyme activity, either alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring a desired trait). The expression cassette includes a transcriptional and translational initiation region (i.e., promoter) and a polynucleotide open reading frame in the 5 '-3' direction of transcription. The expression cassette may optionally include transcriptional and translational termination regions (i.e., termination regions) that are functional in plants. In some embodiments, the expression cassette includes a selectable marker gene to allow for selection of stable transformants. The expression constructs of the invention may also comprise leader sequences and/or sequences which allow inducible expression of the polynucleotide of interest.

The regulatory sequences of the expression cassette are operably linked to the polynucleotide of interest. The regulatory sequences of the present invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the herbicide tolerance gene encoding the herbicide tolerance protein.

The promoter is a promoter capable of being expressed in a plant, and the promoter capable of being expressed in the plant is a promoter which ensures that a coding sequence connected with the promoter is expressed in a plant cell. The promoter expressible in plants may be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, 35S promoter derived from cauliflower mosaic virus, maize Ubi promoter, promoter of rice GOS2 gene, and the like. Alternatively, the plant expressible promoter may be a tissue specific promoter, i.e. a promoter that directs expression of the coding sequence at a higher level in some tissues of the plant, e.g. in green tissues, than in other tissues of the plant (as can be determined by conventional RNA assays), e.g. the PEP carboxylase promoter. Alternatively, the promoter expressible in a plant may be a wound-inducible promoter. A wound-inducible promoter or a promoter that directs a wound-induced expression pattern means that when a plant is subjected to mechanical or insect feeding induced wounds, the expression of the coding sequence under the control of the promoter is significantly increased compared to under normal growth conditions. Examples of wound-inducible promoters include, but are not limited to, promoters of potato and tomato protease-inhibitory genes (pin I and pin II) and maize protease-inhibitory gene (MPI).

The transit peptide (also known as a secretion signal sequence or targeting sequence) is intended to direct the transgene product to a specific organelle or cellular compartment, and for the receptor protein, the transit peptide may be heterologous, e.g., targeting the chloroplast using a chloroplast transit peptide sequence, or targeting the endoplasmic reticulum using a 'KDEL' retention sequence, or targeting the vacuole using the CTPP of the barley lectin gene.

The leader sequence includes, but is not limited to, a small RNA virus leader sequence, such as an EMCV leader sequence (encephalomyocarditis virus 5' non-coding region); potyvirus leaders, such as the MDMV (maize dwarf mosaic virus) leader; human immunoglobulin heavy chain binding protein (BiP); untranslated leader sequences of envelope protein mRNA of alfalfa mosaic virus (AMV RNA 4); tobacco Mosaic Virus (TMV) leader sequence.

Such enhancers include, but are not limited to, cauliflower mosaic virus (CaMV) enhancer, Figwort Mosaic Virus (FMV) enhancer, carnation weathering Circovirus (CERV) enhancer, cassava vein mosaic virus (CsVMV) enhancer, Mirabilis Mosaic Virus (MMV) enhancer, midnight fragrant tree yellowing leaf curl virus (CmYLCV) enhancer, multan cotton leaf curl virus (CLCuMV), dayflower yellow mottle virus (CoYMV), and peanut chlorosis streak mosaic virus (PCLSV) enhancer.

For monocot applications, the intron includes, but is not limited to, the maize hsp70 intron, the maize ubiquitin intron, Adh intron 1, the sucrose synthase intron, or the rice Act1 intron. For dicot applications, the introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "superubiquitin" intron.

The terminator may be a suitable polyadenylation signal sequence that functions in plants, including, but not limited to, polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene, polyadenylation signal sequence derived from the protease inhibitor II (PIN II) gene, polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and polyadenylation signal sequence derived from the alpha-tubulin (alpha-tubulin) gene.

As used herein, "operably linked" refers to the linkage of nucleic acid sequences such that one provides the functionality required of the linked sequence. In the present invention, the "operative linkage" may be a linkage of a promoter to a sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter. "operably linked" when the sequence of interest encodes a protein and expression of the protein is desired indicates that: the promoter is linked to the sequence in such a way that the resulting transcript is translated efficiently. If the linkage of the promoter to the coding sequence is a transcript fusion and expression of the encoded protein is desired, such a linkage is made such that the first translation initiation codon in the resulting transcript is the initiation codon of the coding sequence. Alternatively, if the linkage of the promoter to the coding sequence is a translational fusion and expression of the encoded protein is desired, the linkage is made such that the first translation initiation codon contained in the 5' untranslated sequence is linked to the promoter and is linked in such a way that the resulting translation product is in frame with the translational open reading frame encoding the desired protein. Nucleic acid sequences that may be "operably linked" include, but are not limited to: sequences that provide gene expression functions (i.e., gene expression elements such as promoters, 5 'untranslated regions, introns, protein coding regions, 3' untranslated regions, polyadenylation sites, and/or transcription terminators), sequences that provide DNA transfer and/or integration functions (i.e., T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), sequences that provide selective functions (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scorable marker functions, sequences that facilitate sequence manipulation in vitro or in vivo (i.e., polylinker sequences, site-specific recombination sequences), and sequences that provide replication functions (i.e., bacterial origins of replication, autonomously replicating sequences, centromeric sequences).

The genome of a plant, plant tissue or plant cell as defined in the present invention refers to any genetic material within a plant, plant tissue or plant cell and includes the nuclear and plastid and mitochondrial genomes.

In the present invention, the term "plant part" or "plant tissue" includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, nuclei, ears, cobs, husks, stems, roots, root tips, anthers, and the like.

The herbicide tolerance proteins of the present invention can be applied to a variety of plants, including but not limited to alfalfa, beans, cauliflower, cabbage, carrot, celery, cotton, cucumber, eggplant, lettuce, melon, pea, pepper, pumpkin, radish, rape, spinach, soybean, pumpkin, tomato, arabidopsis, peanut, or watermelon; preferably, the dicotyledonous plant is cucumber, soybean, arabidopsis thaliana, tobacco, cotton, peanut or rape. The monocotyledonous plant includes, but is not limited to, rice, sorghum, wheat, barley, rye, millet, sugarcane, oat or turf grass; preferably, the monocotyledonous plant is rice, sorghum, wheat, barley, millet, sugar cane or oat.

The term "plant transformation" in the context of the present invention refers to the cloning of a herbicide resistant or tolerant polynucleotide into an expression system, alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring a desired trait, into a plant cell. The receptor and target expression cassette of the present invention can be introduced into a plant cell in a variety of well-known methods. In the context of a polynucleotide, the term "introduced" (e.g., a nucleotide construct of interest) is intended to mean that the polynucleotide is provided to the plant in such a way that the polynucleotide gains access to or is achieved by the interior of a plant cell. Where more than one polynucleotide is to be introduced, these polynucleotides may be assembled as part of a single nucleotide construct, or as separate nucleotide constructs, and may be located on the same or different transformation vectors. Thus, or as part of a breeding scheme, the polynucleotides may be introduced into a host cell of interest, e.g., in a single transformation event, in separate transformation events in a plant. The methods of the invention do not depend on a particular method for introducing one or more polynucleotides into a plant, but merely obtain access or achievement of the polynucleotide(s) to the interior of at least one cell of 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 an exogenous gene into the genome of a plant and stable integration into the genome of the plant and any successive generation thereof, resulting in stable inheritance of the exogenous gene.

The term "transient transformation" refers to the introduction of a nucleic acid molecule or protein into a plant cell that performs a function but does not integrate into the plant genome, resulting in the inability of a foreign gene to be stably inherited.

The term "genome editing technology" refers to a genome modification technology that can perform precise operations on genome sequences to achieve operations such as site-directed mutagenesis, insertion, deletion, and the like of genes. Currently, the genome editing technologies mainly include HE (home endonucleases), ZFN (Zinc finger nucleases), TALEN (transcription activator-like effector nucleases), CRISPR (Clustered regularly interspaced short palindromic repeats).

Numerous transformation vectors available for plant transformation are known to those skilled in the art, and the genes associated with the present invention can be used in combination with any of the above vectors. The choice of vector will depend on the preferred transformation technique and the species of interest for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers conventionally used in transformation include the nptll gene conferring resistance to kanamycin and related antibiotics or related herbicides (this gene was published by Bevan et al in 1983 at page 184-187 of Nature Science 304), the pat and bar genes conferring resistance to the herbicide glufosinate (also known as glufosinate; see White et al in 1990 at page 1062 of Nucl. AcidsRes 18, Spencer et al in 1990 at page 79 of the door. Appl. Genet. 631 and US patents 5561236 and 5276268), the hpn gene conferring resistance to the antibiotic hygromycin (Blochinger & Diggelmann, mol. biol.4:2929-2931) and the Boufr gene conferring resistance to methotrexate (Glyphosate et al in 1983 at page 2 of EMBO J9), the Boufr gene conferring resistance to glyphosate (see also WO 1104-519) and the glyphosate gene conferring resistance to methotrexate (Glyphosate gene) and the EP 881 gene conferring resistance to glyphosate (see WO 3, WO 2-WO 2), the glyphosate gene conferring resistance to glyphosate and the SPS-2004 at page 8811) 1154 pages; us published patents 20070004912, 20050246798 and 20050060767) and the 6-phosphomannose isomerase gene that provides for the metabolism of mannose (us patents 5767378 and 5994629 are described).

Methods for regenerating plants are also well known in the art. For example, Ti plasmid vectors have been utilized for delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, microinjection, and microprojectiles.

The planting system in the present invention refers to a combination of plants, any one of which exhibits herbicide tolerance and/or herbicide treatments available at different stages of plant development, resulting in plants with high yield and/or reduced damage.

In the present invention, the weeds mean plants that compete with cultivated target plants in the environment where the plants grow.

The terms "control" and/or "control" herein refer to the direct application (e.g., by spraying) of at least an effective dose of a pyrazolone herbicide to a plant growing environment to minimize weed development and/or stop growth. At the same time, the cultivated plant of interest should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product; preferably, there is reduced plant damage and/or increased plant yield as compared to a non-transgenic wild type plant. The plant with reduced damage is characterized by including but not limited to improved stalk resistance, and/or increased kernel weight. The "controlling" and/or "controlling" action of the herbicide tolerance protein on weeds can be present independently and not diminished and/or eliminated by the presence of other materials that can "control" and/or "control" weeds. In particular, any tissue of the transgenic plant (containing the gene encoding the herbicide tolerance protein) which is present and/or produced simultaneously and/or asynchronously, said herbicide tolerance protein and/or another substance which controls weeds, the presence of said another substance neither affecting the "controlling" and/or "controlling" action of said herbicide tolerance protein on weeds nor causing said "controlling" and/or "controlling" action to be effected wholly and/or partially by said another substance, independently of said herbicide tolerance protein.

"plant propagules" as used herein include, but are not limited to, vegetative propagules and vegetative propagules. The plant sexual propagules include, but are not limited to, plant seeds; the vegetative propagation body of the plant refers to a vegetative organ or a special tissue of the plant body, and can generate a new plant under the condition of in vitro; the vegetative organ or a specific tissue includes, but is not limited to, roots, stems and leaves, such as: plants with roots as vegetative propagules include strawberry, sweet potato, and the like; plants with stems as vegetative propagules include sugarcane and potato (tubers); the plant with leaves as asexual propagules includes aloe, begonia, etc.

The present invention can impart new herbicide resistance traits to plants, and no adverse effect on phenotype including yield was observed. The plants of the invention are tolerant to, for example, at least one herbicide subject to 2 x, 3 x, or 4 x normal use levels. These increased levels of resistance are within the scope of the present invention. For example, various techniques known in the art can be predictably optimized and further developed to increase expression of a given gene.

The invention provides a herbicide tolerance protein, a coding gene and application thereof, and has the following advantages:

1. the herbicide tolerance protein can endow plants with tolerance to HPPD inhibitor herbicide, and can tolerate the topramezone with field concentration of at least 1 time.

2. The herbicide tolerance protein has wide application prospect in plants.

The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.

Drawings

FIG. 1 is a schematic structural diagram of a prokaryotic recombinant expression vector DBN11765 containing the HP1 nucleotide sequence;

FIG. 2 is a graph showing the effect of enzyme activity of the herbicide tolerance protein HP1 under conditions of different concentration gradients of HPPD inhibitor herbicides (topramezone, mesotrione or diketonitrile);

FIG. 3 is a graph showing the effect of enzyme activity of the herbicide tolerance protein HP1-1 under conditions of different concentration gradients of HPPD inhibitor herbicides (topramezone, mesotrione or diketonitrile);

FIG. 4 is a graph showing the effect of enzyme activity of the herbicide tolerance protein HP1-2 under conditions of different concentration gradients of HPPD inhibitor herbicides (topramezone, mesotrione or diketonitrile);

FIG. 5 is a schematic structural diagram of an Arabidopsis thaliana recombinant expression vector DBN11770 containing the HP1M nucleotide sequence;

FIG. 6 is a schematic structural diagram of a control recombinant expression vector DBN11770N of the present invention.

Detailed Description

The technical schemes of the herbicide tolerance protein, the coding gene and the application thereof are further illustrated by the following specific examples.

First example, mutation and screening of HP1 Gene

1. Synthesis of HP1 Gene

The nucleotide sequence of the HP1 gene is synthesized and is shown as SEQ ID NO. 2 in the sequence table, and the HP1 protein is coded and is shown as SEQ ID NO. 1 in the sequence table. The nucleotide sequence of HP1M which codes the amino acid sequence corresponding to the HP1 is obtained according to the preference codon of Arabidopsis thaliana, and is shown as SEQ ID NO. 3 in the sequence table.

2. Construction of HP1 Gene mutation library

The synthesized HP1 gene was PCR amplified, cloned into pUC118 vector according to the procedure described in pUC118 vector (Takara, CAT: 3318) manufactured by Takara, and the ligated product was introduced into E.coli DH 5. alpha. as a template for error-prone PCR using a forward primer and a reverse primer, resulting in mutation of HP1 gene due to random base mismatches.

The primers and error-prone PCR reaction system are as follows:

a forward primer: 5' -cccaagcttgatggccgccgattccgaaaatc-3' as shown in SEQ ID NO:4 in the sequence Listing (HindIII restriction sites are underlined);

reverse primer: 5' -cgcggatcctcaggcatcgaccgtcacaacg-3', as shown in SEQ ID NO:5 in the sequence Listing (underlined is BamHI enzyme cutting site);

the error-prone PCR reaction system (total volume 50. mu.L) was:

the concentration of the plasmid DNA template is 1-10 ng/. mu.L, the concentration of the forward primer is 10. mu.M, and the concentration of the reverse primer is 10. mu.M.

The error-prone PCR reaction conditions were:

and (2) carrying out agarose gel electrophoresis on the error-prone PCR product by 7g/L, cutting the gel and recovering, carrying out double enzyme digestion on the error-prone PCR product subjected to gel cutting recovery and a pUC118 vector by utilizing HindIII and BamHI, purifying the enzyme digestion product, carrying out enzyme ligation by T4-DNA ligase, and then transforming the enzyme digestion product into escherichia coli DH5 alpha sensitive to topramezone to construct an HP1 gene random mutation library.

3. Screening of HP1 Gene mutation library

The transformants in the library were inoculated into 48-well plates containing 300. mu.M of topramezone in 2mL of LB liquid medium (ampicillin 100mg/L, tyrosine 800mg/L) and shake-cultured in a shaker at a constant temperature of 180rpm and 37 ℃. Observing the growth condition of the strain, adding 1mM IPTG into the LB liquid culture medium when the strain grows to OD 0.4-0.6, continuing to culture, and observing the color change of the culture medium.

As tyrosine aminotransferase exists in Escherichia coli, tyrosine can be catalyzed to generate p-hydroxyphenyl pyruvic acid (HPP), the HPP can be used as a substrate of the HPPD, a product HG after the catalytic reaction of the HPPD can be spontaneously oxidized and polymerized to generate brownish red puama, but the HPPD inhibitor can inhibit the activity of the HPPD enzyme and further inhibit the color generation. Thus, the mutant library can be screened at high throughput based on whether it produces a red-brown color in LB liquid medium, and E.coli DH 5. alpha. that still produces a red-brown color on the medium containing 300. mu.M topramezone can be isolated to obtain a resistance gene.

4. Obtaining a mutated resistance gene

Sequencing results show that two HP1 mutant resistance genes are obtained and are respectively named as HP1-1 and HP1-2, the 475 th site of the HP1-1 nucleotide sequence is mutated from the original A to G, and the 159 th site of the amino acid sequence is mutated from the original threonine to alanine; the 772 th and 773 th positions of the HP1-2 nucleotide sequence are mutated from original CA to AT, so that the 258 th position of the amino acid sequence is mutated from original glutamine to methionine.

The amino acid sequence of the herbicide tolerance protein HP1-1 is shown as SEQ ID NO. 6 in the sequence table, and the nucleotide sequence of HP1-1 which codes the amino acid sequence corresponding to the herbicide tolerance protein HP1-1 is shown as SEQ ID NO. 7 in the sequence table; an HP1-1M nucleotide sequence encoding an amino acid sequence corresponding to the herbicide tolerance protein HP1-1 is obtained according to the Arabidopsis thaliana preference codon, and is shown as SEQ ID NO:8 in the sequence table.

The amino acid sequence of the herbicide tolerance protein HP1-2 is shown as SEQ ID NO. 9 in the sequence table, and the nucleotide sequence of HP1-2 which codes the amino acid sequence corresponding to the herbicide tolerance protein HP1-2 is shown as SEQ ID NO. 10 in the sequence table; an HP1-2M nucleotide sequence encoding an amino acid sequence corresponding to the herbicide tolerance protein HP1-2 was obtained according to Arabidopsis thaliana bias codons, as shown in SEQ ID NO:11 of the sequence Listing.

Second example, testing of the tolerance Effect of the herbicide tolerance proteins HP1-1 and HP1-2 on HPPD inhibitor herbicides

1. Synthesis of nucleotide sequences for HP1, HP1-1, and HP1-2

The 5 'and 3' ends of the HP1 nucleotide sequence (SEQ ID NO:2), the HP-1 nucleotide sequence (SEQ ID NO:7) and the HP1-2 nucleotide sequence (SEQ ID NO:10) were respectively connected to a universal adapter primer 1:

5' end universal adaptor primer 1: 5'-taagaaggagatatacatatg-3', as shown in SEQ ID NO:12 of the sequence Listing;

3' end universal adaptor primer 1: 5'-gtggtggtggtggtgctcgag-3', as shown in SEQ ID NO 13 of the sequence list.

2. Construction of prokaryotic recombinant expression vector and obtaining of recombinant strain

The prokaryotic expression vector DBNBC-01 is linearized by utilizing restriction enzymes Nde I and Xho I, the digestion product is purified to obtain a linearized DBNBC-01 expression vector skeleton (vector skeleton: pET-29a (+)), the HP1 nucleotide sequence connected with the universal joint primer 1 and the linearized DBNBC-01 expression vector skeleton are subjected to recombination reaction, the operation steps are carried out according to the instruction of an In-Fusion seamless connection product kit (Clontech, CA, USA, CAT: 121416) of Takara company, a recombinant expression vector DBN11765 is constructed, the vector structure is shown as figure 1 (F1 Ori: the replication origin of bacteriophage F1; Kan: kanamycin resistance gene; Ori: the replication origin; Rop: Rop gene, encoding ROP protein; T7 promoter: T7 RNA promoter; HP 1: HP1 nucleotide sequence (SEQ ID NO: 2); affinity tag 6xHis, encoding 6 consecutive His as a tag for purification of the target protein; t7 terminator: a terminator of T7 RNA polymerase; LacI operator: a manipulation region of the lactose operon; lacI promoter: a lacI promoter; lacI: lactose operon regulatory gene).

The recombinant expression vector DBN11765 is used for transforming escherichia coli BL21(DE3) competent cells by a heat shock method, wherein the heat shock conditions are as follows: 50 μ L of E.coli BL21(DE3) competent cells, 10 μ L of plasmid DNA (recombinant expression vector DBN11765), water bath at 42 ℃ for 30 s; shaking at 37 deg.C for 1h (shaking table at 100 rpm); then, the mixture was cultured on the LB solid plate containing 50mg/L of Kanamycin (Kanamycin) at 37 ℃ for 12 hours, and white colonies were picked up and cultured overnight at 37 ℃ in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, Kanamycin 50mg/L, pH adjusted to 7.5 with NaOH). The plasmid is extracted by an alkaline method. The extracted plasmid is subjected to sequencing identification, the result shows that the nucleotide sequences of the recombinant expression vector DBN11765 between Nde I and Xho I sites are respectively the nucleotide sequences shown by SEQ ID NO. 2 in the sequence table, and the recombinant strain BL21(HP1) is preserved for later use.

According to the method for constructing the recombinant expression vector DBN11765, the HP1-1 nucleotide sequence and the HP1-2 nucleotide sequence connected with the universal joint primer 1 are respectively subjected to recombination reaction with the linearized DBNBC-01 expression vector skeleton, and the recombinant expression vectors DBN11774 and DBN11775 are sequentially obtained. The recombinant expression vectors DBN11774 and DBN11775 are respectively transformed into competent cells of escherichia coli BL21(DE3) by a heat shock method, plasmids of the competent cells are extracted by an alkaline method, and sequencing verification is carried out on the extracted plasmids, so that the nucleotide sequences in the recombinant expression vectors DBN11774 and DBN11775 respectively contain the nucleotide sequence shown by SEQ ID NO. 7 and the nucleotide sequence shown by SEQ ID NO. 10 in a sequence table, namely the nucleotide sequence of HP1-1 and the nucleotide sequence of HP1-2 are correctly inserted. The resulting recombinant strains BL21(HP1-1) and BL21(HP1-2) were stored for future use.

3. Expression and purification of herbicide tolerance protein in escherichia coli

The recombinant strains BL21(HP1), BL21(HP1-1) and BL21(HP1-2) were respectively and monoclonally inoculated in 100mL of LB medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, ampicillin 100mg/L, pH adjusted to 7.5 with NaOH) and cultured to OD concentration_600nm0.6-0.8, IPTG was added at a concentration of 0.4mM and induced at 16 ℃ for 12 h. The cells were collected, resuspended in 15mL of PBS buffer (50mM, pH 7.4), sonicated (X0-900D ultrasonic processor ultrasonic processor, 30% intensity) for 10min, centrifuged, and the supernatant collected, and the obtained herbicide-resistant proteins were purified by a nickel ion affinity column, and the size of the band was determined to be identical to the theoretically predicted band size by SDS-PAGE protein electrophoresis.

4. Determination of the tolerance of HP1, HP1-1 and HP1-2 to HPPD inhibitor herbicides

Enzyme activity reaction system (1 mL): contains 25. mu.g of a reaction enzyme (the herbicide-tolerant protein HP1, HP1-1 or HP1-2 obtained by the above purification), 0.2mM HPP substrate, 1mM Fe²⁺1mM ascorbic acid, a gradient of topramezone (mesotrione or diketonitrile) at different concentrations (0. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 40. mu.M and 50. mu.M, respectively), in a buffer system of 50mM PBS buffer (pH 7.4), and reacted in a water bath at 30 ℃ for 20min, each reaction being started with the addition of the reaction enzyme and stopped in a boiling water bath for 5 min.

Separating the enzyme reaction system from the reaction systemFiltering, and analyzing 20 μ L of filtrate by High Performance Liquid Chromatography (HPLC) under the following conditions: the mobile phase was acetonitrile/water (30:70, V/V), to which was added 0.1% trifluoroacetic acid. C18 reversed phase chromatographic column (5 μm, 250mm × 4.6mm), column temperature 40 deg.C, VWD-3100 single wavelength ultraviolet detector, detection wavelength 292nm, sample injection amount 20 μ L, and flow rate 1.0 mL/min. By HPLC analysis, the peak time of the enzymatic reaction product is consistent with that of the homogentisate standard sample, and no homogentisate is generated in the reaction system without adding enzyme. The resistance conditions of HP1, HP1-1 and HP1-2 are detected according to different concentrations of HPPD inhibitor herbicides (topramezone, mesotrione or diketonitrile) added into the system, and specifically are as follows: calculating relative enzyme activity when HPPD inhibitor herbicide is added by taking the enzyme activity when the HPPD inhibitor herbicide is not added as 100 percent, and calculating the IC of the HPPD inhibitor herbicide to HP1, HP1-1 and HP1-2 by measuring the generation amount of a product, namely homogentisate by HPLC₅₀(concentration of HPPD inhibitor herbicide at which the enzyme activity catalyzes 50% inhibition). One unit of enzyme activity is defined as: the amount of enzyme required to produce 1. mu. mol of homogentisate within 1min at 30 ℃ and pH 7.4. The results of the experiments are shown in FIGS. 2-4 (graphs of the enzyme activity effects of the herbicide tolerance proteins HP1, HP1-1 and HP1-2 under different concentration gradients of HPPD inhibitor herbicides (topramezone, mesotrione or diketonitrile)), and IC of topramezone, mesotrione and diketonitrile for HP1, HP1-1 and HP1-2 were calculated by non-linear fitting using Prism 6.0 software₅₀The results are shown in Table 1.

TABLE 1 half inhibitory concentrations of HPPD inhibitor herbicides topramezone, mesotrione and diketonitrile for HP1, HP1-1 and HP1-2

The results in table 1 show that: the herbicide tolerance proteins HP1, HP1-1 and HP1-2 all have varying degrees of tolerance to HPPD inhibitor herbicides (topramezone, mesotrione and diketonitrile); the purified herbicide tolerance proteins HP1-1 and HP1-2 were more resistant to HPPD inhibitor herbicides than the herbicide tolerance protein HP 1.

Third example, acquisition and validation of transgenic Arabidopsis plants

1. Respectively constructing arabidopsis thaliana recombinant expression vectors containing HP1, HP1-1 or HP1-2 genes

The 5 'and 3' ends of the HP1M nucleotide sequence (SEQ ID NO:3) described in the above first example 1, the HP1-1M nucleotide sequence (SEQ ID NO:8) described in the above first example 4, and the HP1-2M nucleotide sequence (SEQ ID NO:11) were ligated to universal adapter primer 2:

5' end universal adaptor primer 2: 5'-agtttttctgattaacagactagt-3', as shown in SEQ ID NO:14 of the sequence Listing;

3' end universal adaptor primer 2: 5'-caaatgtttgaacgatcggcgcgcc-3', as shown in SEQ ID NO. 15 of the sequence list.

Carrying out double enzyme digestion reaction on a plant expression vector DBNBC-02 by using restriction enzymes Spe I and Asc I, thereby linearizing the plant expression vector, purifying enzyme digestion products to obtain a linearized DBNBC-02 expression vector skeleton (the vector skeleton: pCAMBIA2301 (can be provided by CAMBIA organization)), carrying out recombination reaction on the HP1M nucleotide sequence connected with the universal joint primer 2 and the linearized DBNBC-02 expression vector skeleton, and constructing a recombinant expression vector DBN11770 according to the instruction of Takara In-Fusion seamless connection product kit (Clontech, CA, USA, CAT: 121416), wherein the structural schematic diagram of the recombinant expression vector DBN11770 is shown In figure 5 (Spec: spectinomycin gene; right border; 34S enhancer of the mosaic figwort virus (TsCBV: 16); prCBP: promoter of rape eukaryotic elongation factor gene 1 alpha (chloroplast f1) (AtCTP: 17), sp 2: Arabidopsis peptide (SEQ ID NO: 18); EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 19); tPSE 9: the terminator of the pea RbcS gene (SEQ ID NO: 20); prAtUbi 10: the promoter of the Arabidopsis Ubiquitin (Ubiquitin)10 gene (SEQ ID NO: 21); spAtCTP 2: arabidopsis chloroplast transit peptide (SEQ ID NO: 18); HP 1M: HP1M nucleotide sequence (SEQ ID NO: 3); tNos: a terminator of the nopaline synthase gene (SEQ ID NO: 22); pr 35S: the cauliflower mosaic virus 35S promoter (SEQ ID NO: 23); PAT: phosphinothricin N-acetyltransferase gene (SEQ ID NO: 24); t 35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 25); LB: left border).

Transforming the recombinant expression vector DBN11770 into Escherichia coli T by a heat shock method₁Competent cells under heat shock conditions: 50 μ L of E.coli T₁Competent cells, 10. mu.L of plasmid DNA (recombinant expression vector DBN11770), water bath at 42 ℃ for 30 s; shaking at 37 deg.C for 1h (shaking table at 100 rpm); then, the mixture was cultured on the LB solid plate (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L, pH adjusted to 7.5 with NaOH) containing 50mg/L Spectinomycin (Spectinomycin) at 37 ℃ for 12 hours, and white colonies were picked up and cultured overnight at 37 ℃ in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, Spectinomycin 50mg/L, pH adjusted to 7.5 with NaOH). Extracting the plasmid by an alkaline method: centrifuging the bacterial solution at 12000rpm for 1min, removing supernatant, and suspending the precipitated bacterial solution with 100 μ l ice-precooled solution I (25mM Tris-HCl, 10mM EDTA (ethylene diamine tetraacetic acid), 50mM glucose, pH 8.0); add 200. mu.L of freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)), invert the tube 4 times, mix, and place on ice for 3-5 min; adding 150 μ L ice-cold solution III (3M potassium acetate, 5M acetic acid), mixing well immediately, and standing on ice for 5-10 min; centrifuging at 4 deg.C and 12000rpm for 5min, adding 2 times volume of anhydrous ethanol into the supernatant, mixing, and standing at room temperature for 5 min; centrifuging at 4 deg.C and 12000rpm for 5min, removing supernatant, washing precipitate with 70% ethanol (V/V), and air drying; adding 30. mu.L of TE (10mM Tris-HCl, 1mM EDTA, pH8.0) containing RNase (20. mu.g/mL) to dissolve the precipitate; bathing in water at 37 deg.C for 30min to digest RNA; storing at-20 deg.C for use. The extracted plasmid is subjected to sequencing identification, and the result shows that the nucleotide sequence of the recombinant expression vector DBN11770 between Spe I and Asc I sites is the nucleotide sequence shown by SEQ ID NO. 3 in the sequence table, namely the HP1M nucleotide sequence.

According to the method for constructing the recombinant expression vector DBN11770, the HP1-1M nucleotide sequence and the HP1-2M nucleotide sequence which are respectively connected with the universal joint primer 2 are respectively subjected to recombination reaction with the linearized DBNBC-02 expression vector skeleton, and the recombinant expression vectors DBN11777 and DBN11778 are sequentially obtained. The nucleotide sequences in the recombinant expression vectors DBN11777 and DBN11778 respectively contain the nucleotide sequence shown by SEQ ID NO. 8 and the nucleotide sequence shown by SEQ ID NO. 11 in the sequence table through sequencing verification, namely the HP1-1M nucleotide sequence and the HP1-2M nucleotide sequence are correctly inserted.

According to the above-mentioned method for constructing recombinant expression vector DBN11770, a control recombinant expression vector DBN11770N was constructed, and its vector structure was shown in FIG. 6 (Spec: spectinomycin gene; RB: right border; eFMV: enhancer of 34S of figwort mosaic virus (SEQ ID NO:16), prBrCBP: promoter of rape eukaryotic elongation factor gene 1 α (Tsf1) (SEQ ID NO:17), spAtCTP 2: Arabidopsis chloroplast transit peptide (SEQ ID NO:18), EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO:19), tPSE 9: terminator of pea RbcS gene (SEQ ID NO:20), pr 35S: cauliflower mosaic virus 35S promoter (SEQ ID NO:23), PAT: phosphinothricin N-acetyltransferase gene (SEQ ID NO:24), t 35S: cauliflower mosaic virus terminator (SEQ ID NO:25), LB: left border).

2. Agrobacterium transformed by arabidopsis recombinant expression vector

The correctly constructed recombinant expression vectors DBN11770, DBN11777, DBN11778 and the control recombinant expression vector DBN11770N are transformed into agrobacterium GV3101 by a liquid nitrogen method respectively, wherein the transformation conditions are as follows: 100. mu.L of Agrobacterium GV3101, 3. mu.L of plasmid DNA (recombinant expression vector); placing in liquid nitrogen for 10min, and heating in 37 deg.C water bath for 10 min; inoculating the transformed agrobacterium GV3101 into an LB test tube, culturing for 2h at the temperature of 28 ℃ and the rotation speed of 200rpm, smearing on an LB solid plate containing 50mg/L Rifampicin (Rifampicin) and 50mg/L spectinomycin until a positive monoclonal grows out, selecting the monoclonal for culturing, extracting a plasmid, and sequencing and identifying the extracted plasmid to show that the structures of the recombinant expression vectors DBN11770, DBN11777, DBN11778 and DBN11770N are completely correct.

3. Obtaining transgenic Arabidopsis plants

Wild type Arabidopsis seeds were suspended in 0.1% (w/v) agarose solution. The suspended seeds were stored at 4 ℃ for 2 days toThe need for dormancy is accomplished to ensure synchronous germination of the seeds. Horse dung soil was mixed with vermiculite and irrigated with water underground until moist, and the soil mixture was drained for 24 h. The pretreated seeds were planted on the soil mixture and covered with a moisture-retaining cover for 7 days. Germinating the seeds and keeping the light intensity at a constant temperature (22 ℃) and a constant humidity (40-50%) of 120-150 mu mol/m²s^-1Under long-day conditions (16h light/8 h dark) plants were grown in a greenhouse. Plants were initially irrigated with Hoagland nutrient solution followed by deionized water to keep the soil moist but not drenched.

Arabidopsis thaliana was transformed using the floral dip method. One or more 15-30mL precultures of LB broth containing spectinomycin (50mg/L) and rifampicin (10mg/L) were inoculated with selected Agrobacterium colonies. The preculture was incubated overnight at a temperature of 28 ℃ with constant shaking at 220 rpm. Each preculture was used to inoculate two 500ml cultures of the YEP broth containing spectinomycin (50mg/L) and rifampicin (10mg/L) and the cultures were incubated overnight at a temperature of 28 ℃ with continuous shaking. The cells were pelleted by centrifugation at about 4000rpm for 20min at room temperature and the resulting supernatant was discarded. The cell pellet was gently resuspended in 500mL of osmotic medium containing 1/2 XMS salt/B5 vitamins, 10% (w/v) sucrose, 0.044. mu.M benzylaminopurine (10. mu.L/L (stock in 1mg/mL DMSO)), and 300. mu.L/L Silvet L-77. About 1 month old Arabidopsis plants were soaked for 5min in osmotic medium containing resuspended cells to ensure that the latest inflorescences were submerged. Then, the Arabidopsis thaliana plants were laid down and covered on their side, and after keeping moisture for 24 hours in a dark environment, the Arabidopsis thaliana plants were normally cultured at a temperature of 22 ℃ with 16 hours of light/8 hours of dark photoperiod. The seeds were harvested after about 4 weeks.

Freshly harvested (HP1M nucleotide sequence, HP1-1M nucleotide sequence, HP1-2M nucleotide sequence, and control vector DBN11770N) T₁The seeds were dried at room temperature for 7 days. Seeds were planted in 26.5cm by 51cm germinating discs, each receiving 200mg of T₁Seeds (approximately 10000 seeds) which had been previously suspended in distilled water and stored at a temperature of 4 ℃ for 2 days to complete the need for dormancy to ensure synchronous germination of the seeds.

Mixing the horse dung with vermiculite, irrigating with water until the mixture is wet, and draining water by gravity.The pretreated seeds were evenly seeded on the soil mixture with a pipette and covered with a moisture hood for 4-5 days. The hood was removed 1 day prior to initial transformant selection using post-emergence glufosinate-ammonium spray (selection for co-transformed PAT gene). After 7 planting Days (DAP) and again at 11DAP, using a DeVilbiss compressed air nozzle, T is sprayed with a 0.2% solution of Liberty herbicide (200g ai/L glufosinate) at a spray volume of 10 mL/dish (703L/ha)₁Plants (cotyledonary stage and 2-4 leaf stage, respectively) to provide an effective amount of glufosinate of 280g ai/ha per application. Surviving plants (actively growing plants) were identified 4-7 days after the final spray and transplanted into 7cm x 7cm square pots (3-5 per dish) made with horse manure and vermiculite, respectively. The transplanted plants were covered with a moisture-retaining cover for 3-4 days and placed in a 22 ℃ culture room as before or directly transferred to a greenhouse. The caps were then removed and the plants were planted in a greenhouse (temperature 22 + -5 deg.C, 50 + -30% RH, 14h light: 10h dark, minimum 500. mu.E/M) for at least 1 day before testing the ability of the HP1M gene, HP1-1M gene, and HP1-2M gene to provide tolerance to pyrazoxolone herbicides²s^-1Natural + supplemental light).

4. Detection of herbicide tolerance effects in transgenic Arabidopsis plants

First T was selected from a background of untransformed seeds using a glufosinate selection protocol₁A transformant. The transgenic recombinant expression vector DBN11770 is an Arabidopsis plant (HP1) transferred with an HP1M nucleotide sequence, the transgenic recombinant expression vector DBN11777 is an Arabidopsis plant (HP1-1) transferred with an HP1-1M nucleotide sequence, the transgenic recombinant expression vector DBN11778 is an Arabidopsis plant (HP1-2) transferred with an HP1-2M nucleotide sequence, and the transgenic recombinant expression vector DBN11770N is an Arabidopsis plant (control vector) transferred with a control recombinant expression vector. About 20000T HP1 were screened₁Seeds and 213T strains were identified₁Passage-positive transformants (PAT gene), a transformation efficiency of about 1.0%; about 20000 HP 1-1T were screened₁Seeds and 195T strains were identified₁Generation-positive transformants (PAT gene), transformation efficiency of about 1.0%; about 20000 HP 1-2T were screened₁Seeds and 195T strains were identified₁Passage-positive transformants (PAT gene), a transformation efficiency of about 1.0%; screen outT of about 20000 control vectors₁Seeds, and 172T strains were identified₁Passage-positive transformants (PAT gene), about 0.86% transformation efficiency.

HP1 Arabidopsis thaliana T₁Plant, HP1-1 Arabidopsis thaliana T₁Plant, HP1-2 Arabidopsis thaliana T₁Arabidopsis thaliana T of plant and control vector₁Plants and wild type arabidopsis thaliana plants (CK) (18 days after sowing) were each sprayed with 3 concentrations of topramezone to test arabidopsis thaliana for herbicide tolerance, namely 25g ai/ha (1 times field strength, 1 ×), 100g ai/ha (4 times field strength, 4 ×) and 0g ai/ha (water, 0 ×). After 7 days of spraying (7DAT), the extent of damage to each plant by the herbicide was counted according to the leaf whitening area ratio (leaf whitening area ratio ═ leaf whitening area/total leaf area × 100%): the substantially non-whitening phenotype is grade 0, the leaf whitening area proportion is less than 50% and grade 1, the leaf whitening area proportion is more than 50% and grade 2, and the leaf whitening area proportion is 100% and grade 3. According to the formula X ═ Σ (NxS)/(TxM)]X 100 the resistance performance of the transformation event for each recombinant expression vector was scored. (X-phytotoxicity score, N-same-stage damaged plant number, S-phytotoxicity grade number, T-total plant number and M-highest phytotoxicity grade), and carrying out resistance evaluation according to the score: high resistant plants (0-15 points), medium resistant plants (16-33 points), low resistant plants (34-67 points) and non-resistant plants (68-100 points). The results of the experiment are shown in table 2.

TABLE 2 transgenic Arabidopsis T₁Tolerance experiment result of plant topramezone

The results in table 2 show that: compared with Arabidopsis thaliana T transferred into a control vector DBN11770N₁Plants and wild type Arabidopsis thaliana plants, Arabidopsis thaliana T transformed with HP1M nucleotide sequence₁Plant, Arabidopsis thaliana T transferred with HP1-1M nucleotide sequence₁Plant and Arabidopsis thaliana T transferred with HP1-2M nucleotide sequence₁The plants can generate tolerance to topramezone to different degrees; compared with Arabidopsis thaliana T with HP1M nucleotide sequence₁The plant is cultured in a culture medium,arabidopsis thaliana T transferred with HP1-1M nucleotide sequence₁Plant and Arabidopsis thaliana T transferred with HP1-2M nucleotide sequence₁The plant has better resistance to topramezone.

In conclusion, when the amino acid sequence of the herbicide tolerance protein HP1 is mutated from threonine to alanine at the 159 th position or from glutamine to methionine at the 258 th position (the herbicide tolerance protein HP1-1 or HP1-2) shows high tolerance to HPPD inhibitor herbicides, and the herbicide tolerance protein can tolerate topramezone (middle resistance) with the field concentration being 1 time higher, so that the application prospect on plants is wide.

Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> Beijing Dabei agricultural Biotechnology Co., Ltd

Nanjing Agricultural University

<120> herbicide tolerance protein, encoding gene and use thereof

<130> DBNBC152

<160> 25

<170> SIPOSequenceListing 1.0

<210> 1

<211> 350

<212> PRT

<213> amino acid sequence of HP1 (Sphingobacterium)

<400> 1

Met Ala Ala Asp Ser Glu Asn Pro Leu Gly Leu His Gly Phe Ala Phe

1 5 10 15

Ala Glu Phe Thr Ser Pro Asp Pro Ala Ala Met Ala Arg Gln Phe Glu

20 25 30

Gln Leu Gly Phe Val Pro Ala Ala Arg Arg Lys Asp Arg Gly Leu Thr

35 40 45

Leu Tyr Arg Gln Gly Arg Ile Ala Phe Ile Leu Asn Ala Gly Glu Gly

50 55 60

Glu Gln Ala Ser Ala Phe Arg Ala Ala His Gly Pro Ser Ala Asn Gly

65 70 75 80

Met Ala Phe Asn Val Ala Asp Ala Lys Ala Ala His Arg His Ala Ile

85 90 95

Gln Ser Gly Ala Thr Asp Ala Asp Thr Ala Gln Ser Ala Leu Pro Gly

100 105 110

Thr Tyr Ala Ile Glu Gly Ile Gly Asp Ser Leu Leu Tyr Leu Val Asp

115 120 125

Ser Asp Pro Phe Ala Asp Trp Asp Glu Val Pro Gly Trp Arg Glu Ala

130 135 140

Ser Ala Glu Arg Gly Val Gly Leu Asp Leu Leu Asp His Leu Thr His

145 150 155 160

Asn Val Arg Arg Gly Gln Met Arg Val Trp Ser Glu Phe Tyr Ala Thr

165 170 175

Leu Phe Gly Phe Glu Glu Gln Lys Phe Phe Asp Ile Lys Gly Gln Ala

180 185 190

Thr Gly Leu Phe Ser Gln Ala Met Ile Ala Pro Asp His Glu Ile Arg

195 200 205

Ile Pro Leu Asn Glu Ser Gln Asp Asp Asn Ser Gln Ile Glu Glu Phe

210 215 220

Ile Arg Glu Tyr Asn Gly Glu Gly Ile Gln His Leu Ala Leu Thr Thr

225 230 235 240

Pro Asp Ile Tyr Ser Thr Val Glu Lys Leu Arg Ala Asn Gly Val Arg

245 250 255

Leu Gln Asp Thr Ile Asp Thr Tyr Tyr Asp Leu Val Asp Glu Arg Val

260 265 270

Pro Gly His Gly Glu Asp Leu Ala Arg Leu Arg Lys Asn Arg Ile Leu

275 280 285

Ile Asp Gly Asp Val His Asp Glu Gly Leu Leu Leu Gln Ile Phe Thr

290 295 300

Glu Thr Met Phe Gly Pro Ile Phe Phe Glu Ile Ile Gln Arg Lys Gly

305 310 315 320

Asn Glu Gly Phe Gly Asn Gly Asn Phe Gln Val Leu Tyr Glu Ser Ile

325 330 335

Glu Leu Asp Gln Ile Arg Arg Gly Val Val Thr Val Asp Ala

340 345 350

<210> 2

<211> 1053

<212> DNA

<213> nucleotide sequence of HP1 (Sphingobacterium)

<400> 2

atggccgccg attccgaaaa tccgttgggc ctgcatggct ttgcgtttgc tgaatttacc 60

tcacccgatc cggccgcgat ggcgcggcag ttcgagcagc ttggatttgt tccggcggcg 120

cgccggaagg acagggggct gacgctttac cggcaggggc gcatcgcgtt cattctgaat 180

gccggggagg gcgaacaggc cagcgcgttt cgcgccgccc atggcccatc cgccaacggc 240

atggcgttta acgtcgccga cgcgaaagca gcgcaccggc acgccattca aagtggggcg 300

acagacgctg ataccgcgca gagcgctctg ccggggactt atgccattga gggcataggc 360

gactcgctgc tctatctggt ggacagcgac ccctttgcgg actgggatga ggtgcccggt 420

tggcgagagg cgtcggcgga gcggggcgtc ggcctcgacc tgctcgacca cctgacccat 480

aatgtccgcc gggggcagat gcgcgtatgg tccgaattct acgcgacgct gttcggtttc 540

gaggaacaga agttcttcga catcaaaggg caggcgaccg gtctgttcag ccaggcgatg 600

atcgccccgg atcatgaaat ccgcatccct ctgaatgaaa gccaggatga taacagccag 660

atcgaggagt ttatccgcga atataatggc gagggtatcc agcatctcgc cctgacgacg 720

ccggatatct attccacggt ggagaagcta cgcgccaatg gcgtgcggct gcaggatacg 780

atcgacacct attatgatct ggtcgacgag cgggtgccgg ggcatggcga ggatctggcg 840

cgattgcgca aaaaccgcat cctgatcgat ggcgatgtcc atgatgaggg gctgctgctg 900

cagatattca ccgagactat gttcggcccg atcttcttcg agatcatcca gcgcaagggc 960

aatgagggtt tcgggaacgg caatttccag gtactctacg aatccataga attggaccag 1020

atccggcgcg gcgttgtgac ggtcgatgcc tga 1053

<210> 3

<211> 1053

<212> DNA

<213> nucleotide Sequence of HP1M (Artificial Sequence)

<400> 3

atggctgcag attctgagaa cccattgggt cttcatggat ttgctttcgc agagtttact 60

tctccagatc cagctgcaat ggctagacaa tttgagcagt tgggtttcgt tcctgctgca 120

agaaggaagg ataggggttt gactctttat agacaaggaa ggattgcttt tattcttaat 180

gcaggagagg gtgaacaggc ttctgcattc agagctgcac acggtccatc agctaacgga 240

atggctttta acgtggctga tgcaaaggct gcacataggc acgctattca atctggtgct 300

actgatgcag atacagctca gtcagcattg cctggaacat atgctattga gggaattgga 360

gattctcttt tgtaccttgt tgattcagat ccattcgcag attgggatga agtgcctgga 420

tggagagagg cttctgcaga aaggggagtt ggtttggatc ttttggatca tcttacccac 480

aacgttagaa ggggtcaaat gagagtgtgg tcagagttct acgctacttt gtttggattc 540

gaggaacaga agtttttcga tattaagggt caagcaacag gattgttttc tcaggctatg 600

attgcaccag atcatgagat taggattcca cttaacgaat ctcaagatga taactcacag 660

atcgaggagt ttattaggga gtataacgga gaaggtattc aacacttggc tcttactaca 720

ccagatatct attcaacagt tgaaaagttg agagcaaatg gtgtgaggct tcaagataca 780

atcgatacct attacgattt ggttgatgag agagtgcctg gacatggtga agatttggct 840

agacttagga agaacaggat tcttattgat ggagatgttc acgatgaggg acttttgctt 900

caaattttta ccgaaactat gttcggtcca attttctttg agattattca gaggaaggga 960

aacgagggat ttggtaacgg aaacttccaa gttttgtacg agtctatcga acttgatcag 1020

attagaaggg gagttgtgac cgtggatgct tga 1053

<210> 4

<211> 32

<212> DNA

<213> Forward primer (Artificial Sequence)

<400> 4

cccaagcttg atggccgccg attccgaaaa tc 32

<210> 5

<211> 31

<212> DNA

<213> reverse primer (Artificial Sequence)

<400> 5

cgcggatcct caggcatcga ccgtcacaac g 31

<210> 6

<211> 350

<212> PRT

<213> amino acid Sequence of HP1-1 (Artificial Sequence)

<400> 6

Met Ala Ala Asp Ser Glu Asn Pro Leu Gly Leu His Gly Phe Ala Phe

1 5 10 15

Ala Glu Phe Thr Ser Pro Asp Pro Ala Ala Met Ala Arg Gln Phe Glu

20 25 30

Gln Leu Gly Phe Val Pro Ala Ala Arg Arg Lys Asp Arg Gly Leu Thr

35 40 45

Leu Tyr Arg Gln Gly Arg Ile Ala Phe Ile Leu Asn Ala Gly Glu Gly

50 55 60

Glu Gln Ala Ser Ala Phe Arg Ala Ala His Gly Pro Ser Ala Asn Gly

65 70 75 80

Met Ala Phe Asn Val Ala Asp Ala Lys Ala Ala His Arg His Ala Ile

85 90 95

Gln Ser Gly Ala Thr Asp Ala Asp Thr Ala Gln Ser Ala Leu Pro Gly

100 105 110

Thr Tyr Ala Ile Glu Gly Ile Gly Asp Ser Leu Leu Tyr Leu Val Asp

115 120 125

Ser Asp Pro Phe Ala Asp Trp Asp Glu Val Pro Gly Trp Arg Glu Ala

130 135 140

Ser Ala Glu Arg Gly Val Gly Leu Asp Leu Leu Asp His Leu Ala His

145 150 155 160

Asn Val Arg Arg Gly Gln Met Arg Val Trp Ser Glu Phe Tyr Ala Thr

165 170 175

Leu Phe Gly Phe Glu Glu Gln Lys Phe Phe Asp Ile Lys Gly Gln Ala

180 185 190

Thr Gly Leu Phe Ser Gln Ala Met Ile Ala Pro Asp His Glu Ile Arg

195 200 205

Ile Pro Leu Asn Glu Ser Gln Asp Asp Asn Ser Gln Ile Glu Glu Phe

210 215 220

Ile Arg Glu Tyr Asn Gly Glu Gly Ile Gln His Leu Ala Leu Thr Thr

225 230 235 240

Pro Asp Ile Tyr Ser Thr Val Glu Lys Leu Arg Ala Asn Gly Val Arg

245 250 255

Leu Gln Asp Thr Ile Asp Thr Tyr Tyr Asp Leu Val Asp Glu Arg Val

260 265 270

Pro Gly His Gly Glu Asp Leu Ala Arg Leu Arg Lys Asn Arg Ile Leu

275 280 285

Ile Asp Gly Asp Val His Asp Glu Gly Leu Leu Leu Gln Ile Phe Thr

290 295 300

Glu Thr Met Phe Gly Pro Ile Phe Phe Glu Ile Ile Gln Arg Lys Gly

305 310 315 320

Asn Glu Gly Phe Gly Asn Gly Asn Phe Gln Val Leu Tyr Glu Ser Ile

325 330 335

Glu Leu Asp Gln Ile Arg Arg Gly Val Val Thr Val Asp Ala

340 345 350

<210> 7

<211> 1053

<212> DNA

<213> HP1-1 nucleotide Sequence (Artificial Sequence)

<400> 7

atggccgccg attccgaaaa tccgttgggc ctgcatggct ttgcgtttgc tgaatttacc 60

tcacccgatc cggccgcgat ggcgcggcag ttcgagcagc ttggatttgt tccggcggcg 120

cgccggaagg acagggggct gacgctttac cggcaggggc gcatcgcgtt cattctgaat 180

gccggggagg gcgaacaggc cagcgcgttt cgcgccgccc atggcccatc cgccaacggc 240

atggcgttta acgtcgccga cgcgaaagca gcgcaccggc acgccattca aagtggggcg 300

acagacgctg ataccgcgca gagcgctctg ccggggactt atgccattga gggcataggc 360

gactcgctgc tctatctggt ggacagcgac ccctttgcgg actgggatga ggtgcccggt 420

tggcgagagg cgtcggcgga gcggggcgtc ggcctcgacc tgctcgacca cctggcccat 480

aatgtccgcc gggggcagat gcgcgtatgg tccgaattct acgcgacgct gttcggtttc 540

gaggaacaga agttcttcga catcaaaggg caggcgaccg gtctgttcag ccaggcgatg 600

atcgccccgg atcatgaaat ccgcatccct ctgaatgaaa gccaggatga taacagccag 660

atcgaggagt ttatccgcga atataatggc gagggtatcc agcatctcgc cctgacgacg 720

ccggatatct attccacggt ggagaagcta cgcgccaatg gcgtgcggct gcaggatacg 780

atcgacacct attatgatct ggtcgacgag cgggtgccgg ggcatggcga ggatctggcg 840

cgattgcgca aaaaccgcat cctgatcgat ggcgatgtcc atgatgaggg gctgctgctg 900

cagatattca ccgagactat gttcggcccg atcttcttcg agatcatcca gcgcaagggc 960

aatgagggtt tcgggaacgg caatttccag gtactctacg aatccataga attggaccag 1020

atccggcgcg gcgttgtgac ggtcgatgcc tga 1053

<210> 8

<211> 1053

<212> DNA

<213> HP1-1M nucleotide Sequence (Artificial Sequence)

<400> 8

atggctgcag attctgagaa cccattgggt cttcatggat ttgctttcgc agagtttact 60

tctccagatc cagctgcaat ggctagacaa tttgagcagt tgggtttcgt tcctgctgca 120

agaaggaagg ataggggttt gactctttat agacaaggaa ggattgcttt tattcttaat 180

gcaggagagg gtgaacaggc ttctgcattc agagctgcac acggtccatc agctaacgga 240

atggctttta acgtggctga tgcaaaggct gcacataggc acgctattca atctggtgct 300

actgatgcag atacagctca gtcagcattg cctggaacat atgctattga gggaattgga 360

gattctcttt tgtaccttgt tgattcagat ccattcgcag attgggatga agtgcctgga 420

tggagagagg cttctgcaga aaggggagtt ggtttggatc ttttggatca tcttgcccac 480

aacgttagaa ggggtcaaat gagagtgtgg tcagagttct acgctacttt gtttggattc 540

gaggaacaga agtttttcga tattaagggt caagcaacag gattgttttc tcaggctatg 600

attgcaccag atcatgagat taggattcca cttaacgaat ctcaagatga taactcacag 660

atcgaggagt ttattaggga gtataacgga gaaggtattc aacacttggc tcttactaca 720

ccagatatct attcaacagt tgaaaagttg agagcaaatg gtgtgaggct tcaagataca 780

atcgatacct attacgattt ggttgatgag agagtgcctg gacatggtga agatttggct 840

agacttagga agaacaggat tcttattgat ggagatgttc acgatgaggg acttttgctt 900

caaattttta ccgaaactat gttcggtcca attttctttg agattattca gaggaaggga 960

aacgagggat ttggtaacgg aaacttccaa gttttgtacg agtctatcga acttgatcag 1020

attagaaggg gagttgtgac cgtggatgct tga 1053

<210> 9

<211> 350

<212> PRT

<213> amino acid Sequence of HP1-2 (Artificial Sequence)

<400> 9

Met Ala Ala Asp Ser Glu Asn Pro Leu Gly Leu His Gly Phe Ala Phe

1 5 10 15

Ala Glu Phe Thr Ser Pro Asp Pro Ala Ala Met Ala Arg Gln Phe Glu

20 25 30

Gln Leu Gly Phe Val Pro Ala Ala Arg Arg Lys Asp Arg Gly Leu Thr

35 40 45

Leu Tyr Arg Gln Gly Arg Ile Ala Phe Ile Leu Asn Ala Gly Glu Gly

50 55 60

Glu Gln Ala Ser Ala Phe Arg Ala Ala His Gly Pro Ser Ala Asn Gly

65 70 75 80

Met Ala Phe Asn Val Ala Asp Ala Lys Ala Ala His Arg His Ala Ile

85 90 95

Gln Ser Gly Ala Thr Asp Ala Asp Thr Ala Gln Ser Ala Leu Pro Gly

100 105 110

Thr Tyr Ala Ile Glu Gly Ile Gly Asp Ser Leu Leu Tyr Leu Val Asp

115 120 125

Ser Asp Pro Phe Ala Asp Trp Asp Glu Val Pro Gly Trp Arg Glu Ala

130 135 140

Ser Ala Glu Arg Gly Val Gly Leu Asp Leu Leu Asp His Leu Thr His

145 150 155 160

Asn Val Arg Arg Gly Gln Met Arg Val Trp Ser Glu Phe Tyr Ala Thr

165 170 175

Leu Phe Gly Phe Glu Glu Gln Lys Phe Phe Asp Ile Lys Gly Gln Ala

180 185 190

Thr Gly Leu Phe Ser Gln Ala Met Ile Ala Pro Asp His Glu Ile Arg

195 200 205

Ile Pro Leu Asn Glu Ser Gln Asp Asp Asn Ser Gln Ile Glu Glu Phe

210 215 220

Ile Arg Glu Tyr Asn Gly Glu Gly Ile Gln His Leu Ala Leu Thr Thr

225 230 235 240

Pro Asp Ile Tyr Ser Thr Val Glu Lys Leu Arg Ala Asn Gly Val Arg

245 250 255

Leu Met Asp Thr Ile Asp Thr Tyr Tyr Asp Leu Val Asp Glu Arg Val

260 265 270

Pro Gly His Gly Glu Asp Leu Ala Arg Leu Arg Lys Asn Arg Ile Leu

275 280 285

Ile Asp Gly Asp Val His Asp Glu Gly Leu Leu Leu Gln Ile Phe Thr

290 295 300

Glu Thr Met Phe Gly Pro Ile Phe Phe Glu Ile Ile Gln Arg Lys Gly

305 310 315 320

Asn Glu Gly Phe Gly Asn Gly Asn Phe Gln Val Leu Tyr Glu Ser Ile

325 330 335

Glu Leu Asp Gln Ile Arg Arg Gly Val Val Thr Val Asp Ala

340 345 350

<210> 10

<211> 1053

<212> DNA

<213> HP1-2 nucleotide Sequence (Artificial Sequence)

<400> 10

atggccgccg attccgaaaa tccgttgggc ctgcatggct ttgcgtttgc tgaatttacc 60

tcacccgatc cggccgcgat ggcgcggcag ttcgagcagc ttggatttgt tccggcggcg 120

cgccggaagg acagggggct gacgctttac cggcaggggc gcatcgcgtt cattctgaat 180

gccggggagg gcgaacaggc cagcgcgttt cgcgccgccc atggcccatc cgccaacggc 240

atggcgttta acgtcgccga cgcgaaagca gcgcaccggc acgccattca aagtggggcg 300

acagacgctg ataccgcgca gagcgctctg ccggggactt atgccattga gggcataggc 360

gactcgctgc tctatctggt ggacagcgac ccctttgcgg actgggatga ggtgcccggt 420

tggcgagagg cgtcggcgga gcggggcgtc ggcctcgacc tgctcgacca cctgacccat 480

aatgtccgcc gggggcagat gcgcgtatgg tccgaattct acgcgacgct gttcggtttc 540

gaggaacaga agttcttcga catcaaaggg caggcgaccg gtctgttcag ccaggcgatg 600

atcgccccgg atcatgaaat ccgcatccct ctgaatgaaa gccaggatga taacagccag 660

atcgaggagt ttatccgcga atataatggc gagggtatcc agcatctcgc cctgacgacg 720

ccggatatct attccacggt ggagaagcta cgcgccaatg gcgtgcggct gatggatacg 780

atcgacacct attatgatct ggtcgacgag cgggtgccgg ggcatggcga ggatctggcg 840

cgattgcgca aaaaccgcat cctgatcgat ggcgatgtcc atgatgaggg gctgctgctg 900

cagatattca ccgagactat gttcggcccg atcttcttcg agatcatcca gcgcaagggc 960

aatgagggtt tcgggaacgg caatttccag gtactctacg aatccataga attggaccag 1020

atccggcgcg gcgttgtgac ggtcgatgcc tga 1053

<210> 11

<211> 1053

<212> DNA

<213> HP1-2M nucleotide Sequence (Artificial Sequence)

<400> 11

atggctgcag attctgagaa cccattgggt cttcatggat ttgctttcgc agagtttact 60

tctccagatc cagctgcaat ggctagacaa tttgagcagt tgggtttcgt tcctgctgca 120

agaaggaagg ataggggttt gactctttat agacaaggaa ggattgcttt tattcttaat 180

gcaggagagg gtgaacaggc ttctgcattc agagctgcac acggtccatc agctaacgga 240

atggctttta acgtggctga tgcaaaggct gcacataggc acgctattca atctggtgct 300

actgatgcag atacagctca gtcagcattg cctggaacat atgctattga gggaattgga 360

gattctcttt tgtaccttgt tgattcagat ccattcgcag attgggatga agtgcctgga 420

tggagagagg cttctgcaga aaggggagtt ggtttggatc ttttggatca tcttacccac 480

aacgttagaa ggggtcaaat gagagtgtgg tcagagttct acgctacttt gtttggattc 540

gaggaacaga agtttttcga tattaagggt caagcaacag gattgttttc tcaggctatg 600

attgcaccag atcatgagat taggattcca cttaacgaat ctcaagatga taactcacag 660

atcgaggagt ttattaggga gtataacgga gaaggtattc aacacttggc tcttactaca 720

ccagatatct attcaacagt tgaaaagttg agagcaaatg gtgtgaggct tatggataca 780

atcgatacct attacgattt ggttgatgag agagtgcctg gacatggtga agatttggct 840

agacttagga agaacaggat tcttattgat ggagatgttc acgatgaggg acttttgctt 900

caaattttta ccgaaactat gttcggtcca attttctttg agattattca gaggaaggga 960

aacgagggat ttggtaacgg aaacttccaa gttttgtacg agtctatcga acttgatcag 1020

attagaaggg gagttgtgac cgtggatgct tga 1053

<210> 12

<211> 21

<212> DNA

<213> 5' Universal adaptor primer 1(Artificial Sequence)

<400> 12

taagaaggag atatacatat g 21

<210> 13

<211> 21

<212> DNA

<213> 3' terminal Universal adaptor primer 1(Artificial Sequence)

<400> 13

gtggtggtgg tggtgctcga g 21

<210> 14

<211> 24

<212> DNA

<213> 5' Universal adaptor primer 2(Artificial Sequence)

<400> 14

agtttttctg attaacagac tagt 24

<210> 15

<211> 25

<212> DNA

<213> 3' terminal Universal adaptor primer 2(Artificial Sequence)

<400> 15

caaatgtttg aacgatcggc gcgcc 25

<210> 16

<211> 542

<212> DNA

<213> enhancer of 34S of Figwort mosaic virus (Figwort mosaic virus)

<400> 16

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> 17

<211> 1534

<212> DNA

<213> eukaryotic elongation factor Gene 1 α of rapeseed (promoter Brassica napus of Tsf1)

<400> 17

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> 18

<211> 228

<212> DNA

<213> Arabidopsis chloroplast transit peptide (Arabidopsis thaliana)

<400> 18

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> 19

<211> 1368

<212> DNA

<213> 5-enolpyruvylshikimate-3-phosphate synthase gene (Artificial Sequence)

<400> 19

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> 20

<211> 643

<212> DNA

<213> terminator of pea RbcS gene (Pisum sativum)

<400> 20

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> 21

<211> 1322

<212> DNA

<213> Arabidopsis Ubiquitin (Arabidopsis thaliana promoter of Ubiquitin10 Gene)

<400> 21

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> 22

<211> 253

<212> DNA

<213> terminator of nopaline synthase gene (Agrobacterium tumefaciens)

<400> 22

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> 23

<211> 530

<212> DNA

<213> Cauliflower mosaic virus 35S promoter (Cauliflower mosaic virus)

<400> 23

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> 24

<211> 552

<212> DNA

<213> phosphinothricin N-acetyltransferase gene nucleotide sequence (Streptomyces viridochromogenes)

<400> 24

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> 25

<211> 195

<212> DNA

<213> Cauliflower mosaic virus 35S terminator (Cauliflower mosaic virus)

<400> 25

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

1. A protein characterized by having an amino acid sequence represented by SEQ ID NO. 6.

2. A gene, comprising:

(a) a nucleotide sequence encoding the protein of claim 1; or

(b) A nucleotide sequence which is completely complementary to the nucleotide sequence defined in (a); or

(c) The nucleotide sequence shown as SEQ ID NO. 7 or SEQ ID NO. 8.

3. An expression cassette comprising the gene of claim 2 under the control of an operably linked regulatory sequence.

4. A recombinant vector comprising the gene of claim 2 or the expression cassette of claim 3.

5. A method of extending the herbicide tolerance of a plant comprising: expressing in a plant the protein of claim 1 or the protein encoded by the expression cassette of claim 3 together with at least one second herbicide tolerance protein different from the protein of claim 1 or the protein encoded by the expression cassette of claim 3.

6. The method of claim 5, wherein said 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, cytochrome proteins and/or protoporphyrinogen oxidase.

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 HPPD inhibitor herbicide that allows growth of transformed cells expressing the gene or the expression cassette, while killing or inhibiting growth of untransformed cells.

8. The method of claim 7, wherein the plant comprises a monocot and a dicot.

9. The method of claim 8, wherein the plant is oat, wheat, barley, millet, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, canola, potato, tomato, or Arabidopsis thaliana.

10. The method according to any one of claims 7 to 9, characterized in that the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide and/or an isoxazole HPPD inhibitor herbicide.

11. The method of claim 10, wherein the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.

12. A method of controlling weeds, comprising: applying an effective dose of an HPPD inhibitor herbicide to a field planted with a plant of interest comprising the gene of claim 2 or the expression cassette of claim 3 or the recombinant vector of claim 4.

13. The method of claim 12, wherein the plant of interest comprises a monocot and a dicot.

14. The method of claim 13, wherein the plant of interest is selected from the group consisting of oat, wheat, barley, millet, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, canola, potato, tomato, and arabidopsis.

15. The method of claim 14, wherein the plant of interest is a glyphosate tolerant plant and the weed is a glyphosate resistant weed.

16. The method according to any one of claims 12 to 15, characterized in that the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide and/or an isoxazole HPPD inhibitor herbicide.

17. The method of claim 16, wherein the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.

18. A method for protecting or conferring tolerance to a HPPD inhibitor herbicide on a plant from damage caused by the HPPD inhibitor herbicide, comprising: introducing the gene of claim 2 or the expression cassette of claim 3 or the recombinant vector of claim 4 into a plant such that the introduced plant produces an amount of the herbicide tolerance protein sufficient to protect it from damage by HPPD inhibitor herbicides.

19. The method of claim 18, wherein the plant comprises a monocot and a dicot.

20. The method of claim 19, wherein the plant is selected from the group consisting of oat, wheat, barley, millet, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, canola, potato, tomato, and arabidopsis.

21. The method of any one of claims 18 to 20, wherein the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide.

22. The method of claim 21, wherein the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.

23. A method of producing a plant tolerant to a HPPD inhibitor herbicide comprising introducing into the genome of the plant the gene of claim 2.

24. The method of claim 23, wherein the introducing comprises genetic transformation, genome editing, or gene mutation.

25. The method of claim 24, wherein the plant comprises a monocot and a dicot.

26. The method of claim 25, wherein the plant is selected from the group consisting of oat, wheat, barley, millet, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, canola, potato, tomato, and arabidopsis.

27. The method of any one of claims 23 to 26, wherein the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide, and/or an isoxazole HPPD inhibitor herbicide.

28. The method of claim 27, wherein the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.

29. A method of growing a plant tolerant to a HPPD inhibitor herbicide, comprising:

growing at least one plant propagule comprising in its genome the gene of claim 2 or the expression cassette of claim 3;

growing the plant propagule into a plant;

applying an effective dose of an HPPD inhibitor herbicide to a plant growing 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.

30. The method of claim 29, wherein the plant comprises a monocot and a dicot.

31. The method of claim 30, wherein the plant is selected from the group consisting of oat, wheat, barley, millet, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, canola, potato, tomato, and arabidopsis.

32. The method of any one of claims 29 to 31, wherein said HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide and/or an isoxazole HPPD inhibitor herbicide.

33. The method of claim 32, wherein the HPPD inhibitor herbicide is topramezone, mesotrione, and/or a diketonitrile.

34. A method for obtaining a processed agricultural product, comprising treating a harvest of HPPD inhibitor herbicide tolerant plants obtained by the method of any one of claims 29-31 or 33 to obtain a processed agricultural product.

35. A method for obtaining a processed agricultural product, comprising treating a harvest of HPPD inhibitor herbicide tolerant plants obtained by the method of claim 32 to obtain a processed agricultural product.

36. A planting system for controlling weed growth comprising a HPPD inhibitor herbicide and a plant growing environment in which at least one plant of interest is present, said plant of interest comprising a gene of claim 2 or an expression cassette of claim 3.

37. The growing system of claim 36, wherein the plant of interest comprises a monocot and a dicot.

38. The growing system of claim 37, wherein the plant of interest is oat, wheat, barley, millet, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, canola, potato, tomato, or arabidopsis.

39. The growing system of claim 38, wherein the plant of interest is a glyphosate tolerant plant and the weed is a glyphosate resistant weed.

40. The growing system according to any one of claims 36 to 39, wherein said HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide and/or an isoxazole HPPD inhibitor herbicide.

41. The growing system of claim 40, wherein said HPPD inhibitor herbicide is topramezone, mesotrione and/or diketonitrile.

42. Use of a protein according to claim 1 for conferring herbicide tolerance to a plant HPPD inhibitor.

43. The use of claim 42, wherein said plant comprises a monocot and a dicot.

44. The use according to claim 43, wherein the plant is oat, wheat, barley, millet, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or Arabidopsis thaliana.

45. The use according to any one of claims 42 to 44, wherein the HPPD inhibitor herbicide comprises a pyrazolone HPPD inhibitor herbicide, a triketone HPPD inhibitor herbicide and/or an isoxazole HPPD inhibitor herbicide.

46. The use according to claim 45, characterized in that the HPPD inhibitor herbicide is topramezone, mesotrione and/or a diketonitrile.