CN114854702B

CN114854702B - Herbicide tolerance protein, coding gene and application thereof

Info

Publication number: CN114854702B
Application number: CN202210330228.1A
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: 2024-04-19
Anticipated expiration: 2040-03-19
Also published as: CN111304178B; CN111304178A; CN114854702A

Abstract

The invention relates to a herbicide tolerance protein, a coding gene and application thereof, wherein the protein comprises the following components: (a) Has an amino acid sequence shown as SEQ ID NO. 6 or SEQ ID NO. 9; (b) The protein derived from (a) wherein the amino acid sequence in (a) is substituted and/or deleted and/or added with one or more amino acids and has hydroxyphenylpyruvate dioxygenase. The herbicide tolerance protein can endow plants with tolerance to HPPD inhibitor herbicides, and has wide application prospects in plants.

Description

Herbicide tolerance protein, coding gene and application thereof

The application is a divisional application of Chinese patent application 202010194585.0, the application name is: herbicide tolerance protein, coding gene and application thereof, and application date: 3 months and 19 days 2020.

Technical Field

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

Background

Hydroxyphenylpyruvate dioxygenase (hydroxyphenylpyruvate dioxygenase, abbreviated as HPPD) is an enzyme which catalyzes the reaction of the degradation product of tyrosine, p-hydroxyphenylpyruvate (hydroxyphenylpyruvic acid, abbreviated as HPP), to homogentisate/homogentisate (homogentisate, abbreviated as HG), a precursor of tocopherols and plastoquinone (plastoquinone, abbreviated as PQ) in plants, in the presence of iron ions (Fe ²⁺) and oxygen. Tocopherol has the function of a membrane-associated antioxidant; PQ is not only an electron carrier between PS II and the cytochrome b6/f complex, but is also an essential cofactor for phytoene desaturase in carotenoid biosynthesis.

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

The method for providing a HPPD inhibitor herbicide tolerant plant consists essentially of: 1) HPPD is overexpressed to produce large amounts of HPPD in plants, which, despite the presence of the HPPD inhibitor herbicide, are sufficiently active 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. Regarding the class of mutant HPPDs, 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 vary 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 object of the present invention is to provide a novel protein which not only has HPPD enzyme activity but also makes plants transformed with the protein-encoding gene tolerant to HPPD inhibitor herbicides, a gene encoding the same and uses thereof.

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 added with one or more amino acids.

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

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

(B) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence defined in (a) and which encodes a protein having hydroxyphenylpyruvate dioxygenase activity; or (b)

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

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

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

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

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

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

To achieve the above object, the present invention also provides a method of selecting a transformed plant cell, comprising: transforming a plurality of plant cells with said gene or said expression cassette and culturing said cells at a 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 comprises a monocot and a dicot; more preferably, the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis;

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

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

Preferably, the plant of interest includes monocotyledonous plants and dicotyledonous plants; more preferably, the plant of interest is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis; further preferably, the plant of interest is a glyphosate tolerant plant and the weed is a glyphosate resistant weed;

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

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 introducing comprises a genetic transformation method, a genome editing method or a gene mutation method;

In particular, the method of producing a HPPD inhibitor herbicide tolerant plant comprises: generating an HPPD inhibitor herbicide tolerant plant by selfing or crossing a parent plant and/or a second plant, said parent plant and/or second plant comprising said gene or said expression cassette, said HPPD inhibitor herbicide tolerant plant inherits said gene or said expression cassette from said parent plant and/or second plant;

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

planting at least one plant propagule comprising the gene or the expression cassette in the genome of the plant propagule;

growing the plant propagules into plants;

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

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

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

To achieve the above object, the present invention also provides the use of the protein in conferring HPPD inhibitor herbicides on plants;

The articles "a" and "an" as used herein mean one or more than one (i.e., at least one). For example, "an element" means one or more elements. Furthermore, the terms "comprises," "comprising," or any other variation thereof, such as "includes" or "including," are to be understood to mean including one of the elements, integers or steps, or a group of elements, integers or steps, but not excluding any other elements, integers or steps, or groups of elements, integers or steps.

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 inhibit HPPD, either directly or indirectly, which 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) Triones, for example, sulcotrione (i.e. 2- [ 2-chloro-4- (methylsulfonyl) benzoyl ] -1, 3-cyclohexanedione), mesotrione (i.e. 2- [4- (methylsulfonyl) -2-nitrobenzoyl ] -1, 3-cyclohexanedione), tembotrione (i.e. 2- [ 2-chloro-4- (methylsulfonyl) -3- [ (2, 2-trifluoroethoxy) methyl ] benzoyl ] -1, 3-cyclohexanedione); (2) Isoxazoles (in plants, isoxazoles HPPD herbicides are rapidly converted to biologically active diketopyrrolopyrroles (diketonitrile, DKN) to exert an inhibitory effect on HPPD, so that the diketopyrrolopyrroles are active forms of isoxazoles HPPD-inhibiting herbicides), for example isoxaflutole (isoxaflutole) (i.e. 5-cyclopropyl-4-isoxazolyl [2- (methylsulfonyl) -4- (trifluoromethyl) phenyl ] methanone); (3) Pyrazolones (pyrazolinate) such as topramezone (i.e., [3- (4, 5-dihydro-3-isoxazolyl) -2-methyl-4- (methylsulfonyl) phenyl ] (5-hydroxy-1-methylpyrazol-4-yl) methanone), sulfonyloxazomet (pyrasulfotole) (5-hydroxy-1, 3-dimethylpyrazol-4-yl (2-methylsulfonyl-4-trifluoromethylphenyl) methanone).

The topramezone, also called topramezone, refers to [3- (4, 5-dihydro-3-isoxazolyl) -2-methyl-4- (methylsulfonyl) phenyl ] (5-hydroxy-1-methylpyrazol-4-yl) methanone, which is a white crystalline solid. The systemic conduction HPPD herbicide belongs to pyrazolone (pyrazolinate) post-emergence stem and leaf treatment, and the common dosage form is 30% suspending agent. Topramezone commercial preparations such as bract guard can control grassy and broadleaf weeds, and 5.6-6.7g of the preparation is used for each mu, and weeds which can be effectively controlled, including but not limited to crabgrass (herba aristolochiae), barnyard grass, goosegrass, wild millet, green bristlegrass (Guzi), chenopodium, polygonum, green hemp, abutilon, wild amaranth, purslane, siberian cocklebur and black nightshade. The atrazine-containing herbicide has remarkable synergistic effect after the atrazine is added, has excellent control effect on the weeds, can also have good control effect on malignant broadleaf weeds such as spiny grass (herba Cephalanoploris), endive, acalypha australis and dayflower (orchid), and can particularly effectively control bristlegrass, crabgrass, goosefoot and wild millet with poor control effect on mesotrione.

The effective dose of the topramezone disclosed by 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 "resistance" is heritable and allows plants to grow and reproduce with herbicide treatment of a given plant in general herbicide-effective manner. As recognized by those skilled in the art, even if a given plant is subjected to some degree of injury, such as little necrosis, solubilization, chlorosis, or other injury, but at least not significantly affected in yield, the plant may still be considered "resistant", i.e., the given plant has an increased ability to resist various degrees of injury induced by the herbicide, while generally resulting in injury to wild type plants of the same genotype at the same herbicide dose. The term "resistance" or "tolerance" is used herein more broadly than the term "resistance" and includes "resistance".

The term "conferring" in the present invention refers to providing a plant with a characteristic or trait, such as herbicide tolerance and/or other desired trait.

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

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

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

The herbicide tolerance proteins of the invention have HPPD enzyme activity and confer tolerance in plants to certain classes of HPPD-inhibiting herbicides. The DNA sequences encoding the herbicide tolerance proteins of the invention are useful for providing plants, crops, plant cells and seeds of the invention that provide enhanced tolerance to one or more HPPD herbicides.

Genes encoding the herbicide tolerance proteins of the 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 plants.

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

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

Thus, sequences that have HPPD inhibiting herbicide tolerance activity and hybridize under stringent conditions to genes encoding herbicide tolerance proteins of the invention are included in the 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, SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 11.

Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the herbicide tolerance genes of the present invention. The nucleic acid molecule or fragment thereof is capable of specifically hybridizing to other nucleic acid molecules under certain conditions. In the present invention, two nucleic acid molecules can be said to specifically hybridize to each other if they form an antiparallel double-stranded nucleic acid structure. Two nucleic acid molecules are said to be "complements" of one nucleic acid molecule if they exhibit complete complementarity. In the present invention, a nucleic acid molecule is said to exhibit "complete complementarity" when each nucleotide of the two molecules is complementary to a corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to have "complementarity" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under conventional "highly stringent" conditions. Deviations from complete complementarity are permissible provided that such deviations do not completely prevent the formation of double-stranded structures by the two molecules. In order to enable a nucleic acid molecule to act as a primer or probe, it is only necessary to ensure sufficient complementarity in sequence to allow the formation of a stable double-stranded structure at the particular solvent and salt concentration employed.

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

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

Due to the redundancy of the genetic code, a variety of different DNA sequences may encode the same amino acid sequence. The generation of these alternative DNA sequences encoding the same or substantially the same protein is within the skill level of those skilled in the art. These different DNA sequences are included within the scope of the present invention. The term "substantially identical" sequence refers to a sequence having amino acid substitutions, deletions, additions or insertions without substantially affecting the herbicide tolerance activity, and also includes fragments that retain the herbicide tolerance activity.

The term "functional activity" or "activity" in the present invention refers to the ability of the protein/enzyme of use of the invention (alone or in combination with other proteins) to degrade or attenuate the activity of a herbicide. Plants producing a protein of the invention preferably produce an "effective amount" of the protein such that when the plant is treated with the herbicide, the protein is expressed at a level sufficient to render the plant wholly or partially tolerant to the herbicide (typically in amounts unless specifically indicated). The herbicide may be used in amounts, in normal field amounts and concentrations that would normally kill the target plant. Preferably, the plant cells and plants of the invention are protected from growth inhibition or damage caused by herbicide treatment. The transformed plants and plant cells of the invention 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 an HPPD inhibiting herbicide.

Genes and proteins described herein include not only the specific exemplified sequences, but also portions and/or fragments (including internal and/or terminal deletions as compared to the full-length protein), variants, mutants, variant proteins, substitutions (proteins with alternative amino acids), chimeras and fusion proteins that preserve the HPPD-inhibiting herbicide tolerance activity characteristics of the specific exemplified proteins.

The term "variant" according to the invention means a substantially similar sequence. For polynucleotides, a variant includes a deletion and/or addition of one or more nucleotides at one or more internal sites within the reference polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the herbicide tolerance gene. The term "reference polynucleotide or polypeptide" in the present invention correspondingly includes herbicide tolerance nucleotide sequences or amino acid sequences. The term "native polynucleotide or polypeptide" in the present invention correspondingly includes naturally occurring nucleotide sequences or amino acid sequences. For polynucleotides, conservative variants include nucleotide sequences encoding one of these herbicide tolerance proteins of the invention (due to the degeneracy of the genetic code). Such naturally occurring allelic variants can be identified using well known molecular biological techniques, for example using the Polymerase Chain Reaction (PCR) and hybridization techniques outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as sequences produced by using site-directed mutagenesis but which nevertheless encode a 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, the homology being determined by sequence alignment procedures and parameters.

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

Methods for alignment are well known in the art and can be accomplished using mathematical algorithms such as the MYERS AND MILLER (1988) CABIOS 4:11-17 algorithm; a local alignment algorithm of Smith et al (1981) adv.appl.math.2:482; NEED EMAN AND Wunsch (1970) J.mol.biol.48:443-453 global alignment algorithm; and KARLIN AND Altschul (1990) Proc.Natl. Acad.Sci.USA 872264, as modified in KARLIN AND Altschul (1993) Proc.Natl. Acad.Sci.USA 90:5873-5877. Computer implementations of these mathematical algorithms may be utilized for sequence comparison to determine sequence homology, such implementations including, but not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, mountain View, california); ALLGN programs (version 2.0) and GAP, BESTFIT, BLAST, FASTA and TFASTA (available from accelrys inc.,9685Scranton Road,San Diego,California,USA) in GCG Wisconsin Genetics Software Package version 10.

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

The benefits of combinations of two or more modes of action in improving the controlled weed spectrum and/or naturally more tolerant or resistant weed species can also be extended to the creation of chemicals in crops that are resistant to herbicides other than HPPD tolerant crops by artificial (transgenic or non-transgenic) action, as is well known to those skilled in the art. In fact, the following resistant traits may be stacked singly or in multiple combinations to provide effective control or prevent weed succession from developing resistance to herbicides: glyphosate resistance (e.g., resistant plants or bacteria EPSPS, GOX, GAT), glufosinate resistance (e.g., PAT, bar), acetolactate synthase (ALS) inhibitory herbicide resistance (e.g., imidazolinone, sulfonylurea, triazolopyrimidine, sulfoaniline, pyrimidothiobenzoic acid and other chemical resistance genes such as AHAS, csrl, surA, etc.), phenoxy auxin herbicide resistance (e.g., aryloxyalkanoate dioxygenase-AAD), dicamba herbicide resistance (e.g., dicamba monooxygenase-DMO), bromoxynil resistance (e.g., bxn), resistance to Phytoene Desaturase (PDS) inhibitors, resistance to system ii inhibitory herbicides (e.g., psba), resistance to system i inhibitory herbicides, resistance to protoporphyrinogen oxidase ix (PPO) inhibitory herbicides (e.g., PPO-1), resistance to phenylurea herbicides (e.g., CYP76B 1), dimethoate degrading enzymes, etc.

Glyphosate is widely used because it controls a very broad spectrum of broadleaf and grass weed species. However, repeated use of glyphosate in glyphosate tolerant crops and non-crop applications has (and will continue to) select for species or glyphosate resistant biotypes that will shift weeds to naturally more tolerant. Most herbicide resistance management strategies suggest the use of an effective amount of a tank-mixed herbicide partner as a method of delaying the emergence of resistant weeds, which provides control of the same species, but with a different mode of action. Superimposing the herbicide tolerance genes of the 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 (e.g., topramezone) on the same crop. The application of these herbicides can be simultaneous in a tank mix containing two or more herbicides of different modes of action, separate use of a single herbicide composition in successive uses (e.g. pre-planting, pre-emergence or post-emergence) with intervals ranging from 2 hours to 3 months, or alternatively any combination of numbers of herbicides representing applicable each class of compound can be used at any time (from 7 months in the crop to harvest (or pre-harvest interval for a single herbicide, shortest).

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

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

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

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

The gene encoding the herbicide tolerance protein has higher tolerance to pyrazolone herbicides, and is an important basis for herbicide tolerance crops and the possibility of selecting marker characteristics.

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

The present invention encompasses the transformation of plants with an expression cassette capable of expressing a polynucleotide of interest (i.e., a polynucleotide encoding a herbicide tolerance protein or variant thereof that retains HPPD enzymatic 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 transcription and translation initiation region (i.e., promoter) and a polynucleotide open reading frame in the 5'-3' direction of transcription. The expression cassette may optionally include transcriptional and translational termination regions (i.e., termination regions) that function in the plant. In some embodiments, the expression cassette includes a selectable marker gene to allow selection of stable transformants. The expression constructs of the invention may also include a leader sequence and/or a sequence that allows for inducible expression of the polynucleotide of interest.

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

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

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

Such leader sequences include, but are not limited to, picornaviral leader sequences, such as EMCV leader sequences (encephalomyocarditis virus 5' non-coding region); potyvirus leader sequences, such as MDMV (maize dwarf mosaic virus) leader sequences; human immunoglobulin heavy chain binding proteins (bips); a non-translated leader sequence of alfalfa mosaic virus coat protein mRNA (AMV RNA 4); tobacco Mosaic Virus (TMV) leader sequence.

Such enhancers include, but are not limited to, the cauliflower mosaic virus (CaMV) enhancer, the Figwort Mosaic Virus (FMV) enhancer, the carnation weathered ring virus (CERV) enhancer, the cassava vein mosaic virus (CsVMV) enhancer, the Mirabilis jalapa mosaic virus (MMV) enhancer, the night yellow leaf curl virus (CmYLCV) enhancer, the Multan cotton leaf curl virus (CLCuMV), the dayflower maculopathy virus (CoYMV), and the peanut chlorosis line mosaic virus (PCLSV) enhancer.

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

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

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

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

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

The herbicide tolerance proteins of the present invention can be used in a variety of plants, including but not limited to alfalfa, beans, broccoli, cabbage, carrot, celery, cotton, cucumber, eggplant, lettuce, melon, pea, pepper, zucchini, radish, canola, spinach, soybean, pumpkin, tomato, arabidopsis, peanut, or watermelon; preferably, the dicotyledonous plant is cucumber, soybean, arabidopsis, tobacco, cotton, peanut or canola. Such monocotyledonous plants include, but are not limited to, maize, rice, sorghum, wheat, barley, rye, millet, sugarcane, oat, or turf grass; preferably, the monocotyledonous plant is maize, rice, sorghum, wheat, barley, millet, sugarcane or oat.

In the present invention, the term "plant transformation" refers to cloning a herbicide resistant or tolerant polynucleotide into an expression system, either alone or in combination with one or more additional nucleic acid molecules encoding polypeptides conferring a desired trait, that is, into a plant cell. The receptor and expression cassette of the invention may be introduced into plant cells in a variety of well known ways. In the context of polynucleotides, the term "introducing" (e.g., a nucleotide construct of interest) is intended to mean providing a polynucleotide to the plant in such a way that the polynucleotide gains access to or is achieved within a plant cell. Where more than one polynucleotide is to be introduced, these polynucleotides may be assembled as part of a single nucleotide construct, or as separate nucleotide constructs, and may be located on the same or different transformation vectors. Thus, or as part of a breeding program, such as in a plant, the polynucleotides may be introduced into a host cell of interest in a single transformation event, in separate transformation events. The methods of the invention do not depend on a particular method for introducing one or more polynucleotides into a plant, but merely obtaining access or implementation of the polynucleotide or polynucleotides to the interior of at least one cell of the plant. Methods known in the art for introducing one or more polynucleotides into a plant include, but are not limited to, transient transformation methods, stable transformation methods, virus-mediated methods, or genome editing techniques.

The term "stable transformation" refers to the introduction of a foreign gene into the genome of a plant and the stable integration into the genome of the plant and any successive generations thereof, resulting in stable inheritance of the foreign gene.

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

The term "genome editing technology" refers to a genome modification technology capable of performing accurate manipulation of a genome sequence, realizing manipulation of site-directed mutation, insertion, deletion, and the like of genes. Currently, genome editing techniques mainly include HE (homing endonuclease ), ZFN (zinc-finger nuclease), TALEN (transcription activator-like effector nuclease ), CRISPR (Clustered regulatory interspaced short palindromic repeat, clustered regularly interspaced short palindromic repeats).

Numerous transformation vectors available for plant transformation are known to those of skill in the art, and genes relevant to the present invention may be used in combination with any of the above-described vectors. The choice of vector will depend on the preferred transformation technique and the target species used for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers conventionally used in transformation include the nptl gene conferring resistance to kanamycin and related antibiotics or related herbicides (this gene was published by bevan et al, nature science, volume 304, pages 184-187, 1983), the herbicide glufosinate (also known as glufosinate; see the pat and bar genes of white et al published in 1990 on page 18 of Nucl. AcidsRes, volume 18 of 1062, spencer et al published in 1990 on pages 79-631 of Theor. Appl. Genet, and U.S. Pat. Nos. 5561236 and 5276268), the hpn gene conferring resistance to the antibiotic hygromycin (Blochinger & DIGGELMANN, mol. Cell biol.4: 2929-2931) and the dnfr gene conferring resistance to methotrexate (Bourouis et al, 1983 on EMBO J. Volume 2 of 1099-1104), the EPSPS gene conferring resistance to glyphosate (U.S. Pat. No. 4940935 and 5188642), the N-acetyltransferase gene of Glyphosate (GAT) also conferring resistance to glyphosate (Castle et al on page 304, manna number 1-1154 of science, and the mannase patent applications No. 3 of mannase, and the mannase genes described in U.S. Pat. No. 5,518 and the mannase patent publication No. 5767378-4225 are provided.

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

The planting system described herein refers to a combination of plants, any herbicide tolerance that it displays, and/or herbicide treatments that are available at different stages of plant development, resulting in high yielding and/or reduced wounding plants.

In the present invention, the weeds refer to plants which compete with cultivated plants of interest in a plant growing environment.

The terms "control" and/or "control" according to the present invention mean that at least an effective dose of the pyrazolone herbicide is applied directly (e.g., by spraying) into the plant growing environment to minimize weed development and/or stop growth. At the same time, the cultivated plants of interest should be morphologically normal and can be cultivated under conventional methods for consumption and/or production of the product; preferably, there is reduced plant damage and/or increased plant yield compared to a non-transgenic wild type plant. Such plants with reduced damage include, but are not limited to, improved stalk resistance, and/or increased grain weight, among others. The "control" and/or "control" effect of the herbicide tolerance protein on weeds can be independent and is not attenuated and/or lost by the presence of other "control" and/or "control" weed species. In particular, any tissue of a transgenic plant (containing a gene encoding a herbicide tolerance protein) is present and/or produced simultaneously and/or asynchronously, said herbicide tolerance protein and/or another substance that can control weeds, the presence of which does not affect nor cause the "control" and/or "control" effect of said herbicide tolerance protein on weeds to be achieved entirely and/or partially by said another substance, independently of said herbicide tolerance protein.

"Plant propagules" as used herein include, but are not limited to, plant sexual propagules and plant asexual propagules. Such plant propagules include, but are not limited to, plant seeds; the plant vegetative propagation body refers to a vegetative organ or a special tissue of a plant body, which can produce a new plant under ex vivo conditions; the vegetative organ or a particular tissue includes, but is not limited to, roots, stems and leaves, such as: plants using roots as vegetative propagation bodies include strawberries, sweet potatoes and the like; plants with stems as vegetative propagation material include sugarcane, potato (tuber) and the like; plants with leaves as vegetative propagation material include aloe and begonia etc.

The present invention can confer new herbicide resistance traits to plants and no adverse effect on phenotype, including yield, is observed. Plants of the invention are tolerant to levels as generally found in 2×,3×, or 4× of at least one herbicide tested. These improvements in tolerance levels are within the scope of the present invention. For example, a number of techniques known in the art may be predictably optimized and further developed to increase expression of a given gene.

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

1. the herbicide tolerance proteins of the invention can confer tolerance to HPPD inhibitor herbicides on plants, at least 1-fold field concentration of topramezone.

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

The technical scheme of the invention is further described in detail through the drawings and the embodiments.

Drawings

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

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

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

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

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

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

Detailed Description

The herbicide tolerance protein, the coding gene and the technical scheme of the application are further described by specific examples.

First example, mutation and screening of HP1 Gene

1. Synthesis of HP1 Gene

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

2. Construction of HP1 Gene mutation library

After amplifying the above synthesized HP1 gene by PCR, cloning the amplified product onto the vector pUC118 according to the procedure described in the Takara product pUC118 vector (Takara, CAT: 3318), and introducing the ligated product into E.coli DH 5. Alpha. As a template, and error-prone PCR was performed using forward and reverse primers to mutate the HP1 gene due to random base mismatches.

The primer and error-prone PCR reaction system is as follows:

forward primer: 5'-cccaagcttgatggccgccgattccgaaaatc-3' as shown in SEQ ID NO. 4 of the sequence Listing (HindIII cleavage site underlined);

reverse primer: 5'-cgcggatcctcaggcatcgaccgtcacaacg-3' as shown in SEQ ID NO:5 of the sequence Listing (underlined as BamHI cleavage 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 (3) carrying out agarose gel electrophoresis on the error-prone PCR product by 7g/L, cutting and recovering the error-prone PCR product, carrying out double enzyme digestion on the error-prone PCR product after the cutting and recovering and a pUC118 vector by using HindIII and BamHI, purifying the enzyme-digested product, carrying out enzyme linkage by using T4-DNA ligase, and then converting the enzyme-digested product into E.coli DH5 alpha sensitive to topramezone to construct an HP1 gene random mutation library.

3. Screening of HP1 Gene mutation library

The transformation products in the mutation library were inoculated into a 48-well plate containing 300. Mu.M of topramezone in 2mL LB liquid medium (ampicillin 100mg/L, tyrosine 800 mg/L) and shake-cultured in a shaking table at a constant temperature of 37℃and 180 rpm. And (3) observing the growth condition of the strain, and when the strain grows to OD 0.4-0.6, adding 1mM IPTG into the LB liquid medium, continuing culturing, and observing the color change of the medium.

Because of the presence of tyrosine aminotransferase in E.coli, which catalyzes the production of p-Hydroxyphenylpyruvate (HPP), HPP can be utilized as a substrate for HPPD, and the product HG after HPPD catalytic reaction can spontaneously oxidize and polymerize to produce purplish red purplish melanin, but HPPD inhibitors can inhibit HPPD enzyme activity and thus inhibit color production. Thus, the above mutant library can be subjected to high throughput screening according to whether or not reddish brown color is produced in LB liquid medium, and E.coli DH 5. Alpha. Still producing reddish brown color on the above medium containing 300. Mu.M topramezone can be isolated to obtain a resistance gene.

4. Obtaining mutated resistance genes

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

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

The amino acid sequence of the herbicide tolerance protein HP1-2 is shown as SEQ ID NO 9 in a sequence table, and the nucleotide sequence of the HP1-2, which codes for the amino acid sequence corresponding to the herbicide tolerance protein HP1-2, is shown as SEQ ID NO 10 in the sequence table; the HP1-2M nucleotide sequence encoding the amino acid sequence corresponding to the herbicide tolerance protein HP1-2 is obtained according to the Arabidopsis preference codon, and is shown as SEQ ID NO. 11 in the sequence table.

Second example, tolerance Effect detection of herbicide tolerance proteins HP1-1 and HP1-2 against HPPD inhibitor herbicides

1. Synthesis of nucleotide sequences of 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) are respectively connected with a universal joint primer 1:

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

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

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

The prokaryotic expression vector DBNBC-01 is subjected to double digestion reaction by using restriction endonucleases Nde I and Xho I, so that the prokaryotic expression vector is linearized, 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 Takara company In-Fusion seamless ligation product kit (Clontech, CA, USA, CAT 121416) to construct a recombinant expression vector DBN11765, the vector structure of which is shown In figure 1 (F1 Ori: replication origin of F1; kan: kanamycin resistance gene; ori: replication origin; rop: rop gene; encoding ROP protein; T7 protter: T7 RNA polymerase promoter; HP1: HP1 nucleotide sequence (SEQ ID NO: 2)), 6xHis: affinity tag, 6 continuous His, and a lacmap 3R 7 RNA polymerase promoter; lacmap operator I, lacter lacmap 3, lacmap operator I, lacter lacmap 7 RNA polymerase promoter, lacmap gene, lacr gene 3. Lacc 7 RNA polymerase promoter.

The recombinant expression vector DBN11765 is used for transforming competent cells of the escherichia coli BL21 (DE 3) by a heat shock method, and the heat shock conditions are as follows: 50. Mu.L of E.coli BL21 (DE 3) competent cells, 10. Mu.L of plasmid DNA (recombinant expression vector DBN 11765), 42℃in a water bath for 30s; shake culturing at 37deg.C for 1 hr (shaking table at 100 rpm); then, the white colonies were picked up and cultured overnight at 37℃in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, naCl g/L, kanamycin 50mg/L, pH adjusted to 7.5 with NaOH) on the LB solid plate containing 50mg/L Kanamycin (KANAMYCIN) at 37℃for 12 hours. Extracting the plasmid by an alkaline method. Sequencing and identifying the extracted plasmid, and the result shows that the nucleotide sequence of the recombinant expression vector DBN11765 between Nde I and Xho I sites is the nucleotide sequence shown as SEQ ID NO. 2 in the sequence table, and the recombinant strain BL21 (HP 1) is preserved for standby.

According to the method for constructing the recombinant expression vector DBN11765, the HP1-1 nucleotide sequence and the HP1-2 nucleotide sequence which are connected with the universal joint primer 1 are respectively subjected to recombination reaction with the linearized DBNBC-01 expression vector skeleton, so that 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 (DE 3) by a heat shock method, plasmids are extracted by an alkaline method, sequencing verification is carried out on the extracted plasmids, and the result shows that the nucleotide sequences in the recombinant expression vectors DBN11774 and DBN11775 respectively contain a nucleotide sequence shown as SEQ ID NO. 7 and a nucleotide sequence shown as SEQ ID NO.10 in a sequence table, namely, the HP1-1 nucleotide sequence and the HP1-2 nucleotide sequence are correctly inserted. The resulting recombinant strains BL21 (HP 1-1) and BL21 (HP 1-2) were stored for use.

3. Expression and purification of herbicide tolerance proteins in E.coli

The recombinant strains BL21 (HP 1), BL21 (HP 1-1) and BL21 (HP 1-2) were individually inoculated in 100mL of LB medium (tryptone 10g/L, yeast extract 5g/L, naCl g/L, ampicillin 100mg/L, pH was adjusted to 7.5 with NaOH) and cultured to a concentration of OD _600nm =0.6-0.8, IPTG at a concentration of 0.4mM was added, and induction was performed at 16℃for 12 hours. The cells were collected, resuspended in 15mL of PBS buffer (50 mM, pH 7.4), sonicated (X0-900D ultrasonic processor ultrasonic processor,30%intensity) for 10min, centrifuged, the supernatant collected, and the obtained herbicide-resistant proteins were purified by nickel ion affinity chromatography, and the purified results were detected by SDS-PAGE protein electrophoresis, with a band size consistent with that predicted by theory.

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

Enzymatic reaction system (1 mL): comprising 25. Mu.g of the reaction enzyme (the herbicide tolerance protein HP1, HP1-1 or HP1-2 obtained by the above purification), 0.2mM HPP substrate, 1mM Fe ²⁺, 1mM ascorbic acid, different concentration gradients (concentrations of 0. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M and 50. Mu.M respectively) and the buffer system was 50mM PBS buffer (pH 7.4) and reacted at 30℃for 20min in a water bath, each reaction starting with the addition of the reaction enzyme, and the boiling water bath was terminated for 5 min.

Centrifugally filtering the terminated enzyme reaction system, and taking 20 mu L of filtrate for High Performance Liquid Chromatography (HPLC) analysis, wherein the HPLC conditions are as follows: the mobile phase was acetonitrile in water (30:70, V/V) to which was added 0.1% trifluoroacetic acid. C18 reversed phase chromatographic column (5 μm,250 mm. Times.4.6 mm), column temperature of 40deg.C, VWD-3100 single wavelength ultraviolet detector, detection wavelength of 292nm, sample injection amount of 20 μl, and flow rate of 1.0mL/min. According to HPLC analysis, the peak time of the enzymatic reaction product is consistent with that of a homogentisate standard sample, and the reaction system without adding enzyme does not generate homogentisate. The resistance of HP1, HP1-1 and HP1-2 was tested according to the different concentrations of HPPD inhibitor herbicide (topramezone, mesotrione or diketophenolide) added to the system, specifically: the IC ₅₀ of the HPPD inhibitor herbicide on HP1, HP1-1 and HP1-2 (concentration of HPPD inhibitor herbicide when the enzymatic activity catalytic reaction was inhibited by 50%) was calculated by measuring the amount of product homogentisate by HPLC, based on the relative enzymatic activity when the HPPD inhibitor herbicide was not added as 100%. One enzyme activity unit is defined as: the amount of enzyme required to produce 1. Mu. Mol of homogentisate in 1min at pH 7.4 and 30 ℃. The results of the experiments are shown in FIGS. 2-4 (graphs of the enzyme activity of the herbicide tolerance proteins HP1, HP1-1 and HP1-2 under different concentration gradients of HPPD inhibitor herbicides (topramezone, mesotrione or diketopyronitrile)) and the IC ₅₀ values of topramezone, mesotrione and diketopyronitrile for HP1, HP1-1 and HP1-2 were calculated by nonlinear fitting using Prism 6.0 software and the results are shown in Table 1.

TABLE 1 half inhibitory concentration of HPPD inhibitor herbicides topramezone, mesotrione and diketopyronitrile on HP1, HP1-1 and HP1-2

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

Third example, acquisition and verification of transgenic Arabidopsis plants

1. Construction of recombinant expression vectors of Arabidopsis thaliana containing HP1, HP1-1 or HP1-2 genes, respectively

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) and the HP1-2M nucleotide sequence (SEQ ID NO: 11) described in the above first example 4 were ligated to the universal adaptor primer 2, respectively:

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

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

Carrying out double digestion reaction on a plant expression vector DBNBC-02 by utilizing restriction endonucleases SpeI and Asc I, thereby linearizing the plant expression vector, purifying digestion products to obtain a linearized DBNBC-02 expression vector skeleton (vector skeleton: pCAMBIA2301 (available from CAMBIA mechanism)), 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 specification of an In-Fusion seamless connection product kit (Clontech, CA, USA, CAT: 121416) of Takara company, wherein the structure schematic diagram of the recombinant expression vector DBN11770 is shown In FIG. 5 (Spec: spectinomycin gene; RB right border, eFMV 34S enhancer of figwort mosaic virus (SEQ ID NO: 16), prBrCBP promoter of rape eukaryotic elongation factor gene 1 alpha (Tsf 1) (SEQ ID NO: 17), spAtCTP 2A Arabidopsis chloroplast transit peptide (SEQ ID NO: 18), EPSPS 5-enolpyruvshikimate-3-phosphate synthase gene (SEQ ID NO: 19), tPsE9 terminator of pea RbcS gene (SEQ ID NO: 20), prAtUbi10 promoter of Arabidopsis Ubiquitin (Ubiquinin) 10 gene (SEQ ID NO: 21), spAtCTP 2A Arabidopsis chloroplast transit peptide (SEQ ID NO: 18), HP1M nucleotide sequence (SEQ ID NO: 3), tNos A terminator of nopaline synthase gene (SEQ ID NO: 22), pr35S cauliflower mosaic virus 35S promoter (SEQ ID NO: 23), phosphinothricin N17) An acetyltransferase gene (SEQ ID NO: 24); t35S: the cauliflower mosaic virus 35S terminator (SEQ ID NO: 25); LB: left boundary).

The recombinant expression vector DBN11770 is used for transforming competent cells of the escherichia coli T ₁ by a heat shock method, and the heat shock conditions are as follows: 50. Mu.L of E.coli T ₁ competent cells, 10. Mu.L of plasmid DNA (recombinant expression vector DBN 11770), 42℃in a water bath for 30s; shake culturing at 37deg.C for 1 hr (shaking table at 100 rpm); then cultured on the LB solid plate (tryptone 10g/L, yeast extract 5g/L, naCl g/L, agar 15g/L, pH adjusted to 7.5 with NaOH) containing 50mg/L of spectinomycin (Spectinomycin) at 37℃for 12 hours, white colonies were picked up, and cultured in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, naCl g/L, spectinomycin 50mg/L, pH adjusted to 7.5 with NaOH) at 37℃for overnight. Extracting the plasmid by an alkaline method: centrifuging the bacterial solution at 12000rpm for 1min, removing supernatant, and suspending the precipitated bacterial cells with 100 μl ice-precooled solution I (25 mM Tris-HCl, 10mM EDTA (ethylenediamine tetraacetic acid), 50mM glucose, pH 8.0); 200. Mu.L of freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)) was added, the tube was inverted 4 times, mixed, and placed on ice for 3-5min; adding 150 μl ice-cold solution III (3M potassium acetate, 5M acetic acid), immediately mixing, and standing on ice for 5-10min; centrifuging at 4deg.C and 12000rpm for 5min, adding 2 times volume of absolute ethanol into the supernatant, mixing, and standing at room temperature for 5min; centrifuging at 4deg.C and 12000rpm for 5min, removing supernatant, washing precipitate with 70% ethanol (V/V), and air drying; adding 30. Mu.L of TE (10 mM Tris-HCl, 1mM EDTA, pH 8.0) containing RNase (20. Mu.g/mL) to dissolve the precipitate; digesting RNA in a water bath at 37 ℃ for 30 min; preserving at-20deg.C for use. Sequencing and identifying the extracted plasmid, and the result shows that the nucleotide sequence of the recombinant expression vector DBN11770 between the Spe I and Asc I sites is the nucleotide sequence shown in 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, so that the recombinant expression vectors DBN11777 and DBN11778 are sequentially obtained. Sequencing verifies that the nucleotide sequences in the recombinant expression vectors DBN11777 and DBN11778 respectively contain the nucleotide sequence shown in SEQ ID NO. 8 and the nucleotide sequence shown in SEQ ID NO. 11 in the sequence table, namely, the HP1-1M nucleotide sequence and the HP1-2M nucleotide sequence are correctly inserted.

According to the method for constructing the recombinant expression vector DBN11770, a control recombinant expression vector DBN11770N is constructed, the vector structure of which is shown in FIG. 6 (Spec: spectinomycin gene; RB: right border; eFMV: figwort mosaic virus 34S enhancer (SEQ ID NO: 16), prBrCBP: promoter of rape eukaryotic elongation factor gene 1α (Tsf 1) (SEQ ID NO: 17), spAtCTP2: arabidopsis chloroplast transit peptide (SEQ ID NO: 18), EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 19), tPsE9: terminator of pea RbcS gene (SEQ ID NO: 20), pr35S: cauliflower mosaic virus 35S promoter (SEQ ID NO: 23), PAT: phosphinothricin N-acetyl transferase gene (SEQ ID NO: 24), t35S: cauliflower mosaic virus 35S terminator (SEQ ID NO:25 LB) and left border.

2. Agrobacterium transformation of arabidopsis recombinant expression vector

The correctly constructed recombinant expression vectors DBN11770, DBN11777, DBN11778 and the control recombinant expression vector DBN11770N are respectively transformed into agrobacterium GV3101 by a liquid nitrogen method, and the transformation conditions are as follows: 100. Mu.L of Agrobacterium GV3101, 3. Mu.L of plasmid DNA (recombinant expression vector); placing in liquid nitrogen for 10min, and warm water bath at 37deg.C for 10min; the transformed agrobacterium GV3101 is inoculated in an LB test tube and cultured for 2 hours at the temperature of 28 ℃ and the rotating speed of 200rpm, and the agrobacterium is coated on an LB solid plate containing 50mg/L of Rifampicin (RIFAMPICIN) and 50mg/L of spectinomycin until positive monoclone grows, the monoclone is selected for culture and plasmid is extracted, and the extracted plasmid is subjected to sequencing identification, so that the result shows 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 kept at 4 ℃ for 2 days to fulfill the need for dormancy to ensure synchronized germination of the seeds. The soil mixture was drained for 24 hours by mixing Ma Fentu with vermiculite and sub-irrigating with water until wet. The pretreated seeds were planted on the soil mixture and covered with a moisture-retaining cover for 7 days. Seeds were germinated and plants were grown in a greenhouse under long-day conditions (16 h light/8 h dark) with a constant temperature (22 ℃) and constant humidity (40-50%) and a light intensity of 120-150. Mu. Mol/m ²s^-1. Plants were initially irrigated with Hoagland's nutrient solution followed by deionized water to keep the soil moist but not wet.

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

The newly harvested (HP 1M nucleotide sequence, HP1-2M nucleotide sequence, and control vector DBN 11770N) T ₁ seeds were dried at room temperature for 7 days. Seeds were planted in 26.5cm x 51cm germination trays, each tray receiving 200mg of T ₁ seeds (about 10000 seeds) which had been previously suspended in distilled water and stored at a temperature of 4 ℃ for 2 days to fulfill the need for dormancy to ensure synchronized germination of the seeds.

Mixing Ma Fentu with vermiculite and irrigating with water bottom until wet, draining with gravity. The pretreated seeds were planted uniformly on the soil mixture with a pipette and covered with a moisturizing cap for 4-5 days. The cover was removed 1 day prior to the initial transformant selection using post-emergence spray of glufosinate (co-transformed PAT gene selection). After 7 days of planting (DAP) and again using a DeVilbiss compressed air nozzle at 11DAP, T ₁ plants (cotyledonary and 2-4 foliar stages, respectively) were sprayed with a 0.2% solution of Liberty herbicide (200 g ai/L glufosinate) at a spray volume of 10 mL/tray (703L/ha) to provide an effective amount of glufosinate per application of 280g ai/ha. Surviving plants (actively growing plants) were identified 4-7 days after the final spraying and transplanted into 7cm x 7cm square pots (3-5 plants per tray) prepared with struvite and vermiculite, respectively. The transplanted plants were covered with a moisture-retaining cover for 3-4 days and placed in a culture chamber at a temperature of 22℃as before or directly transferred into a greenhouse. The cover was then removed and the plants were planted in the greenhouse (22.+ -. 5 ℃ C., 50.+ -. 30% RH,14h light: 10h darkness, minimum 500. Mu.E/M ²s^-1 natural+ supplemental light) at least 1 day before testing the ability of the HP1M gene, HP1-1M gene and HP1-2M gene to provide topramezone herbicide tolerance.

4. Herbicide tolerance effect detection of transgenic arabidopsis plants

The T ₁ transformants were first selected from the untransformed seed background using the glufosinate selection protocol. The transgenic recombinant expression vector DBN11770 is an Arabidopsis plant (HP 1) with the HP1M nucleotide sequence, the transgenic recombinant expression vector DBN11777 is an Arabidopsis plant (HP 1-1) with the HP1-1M nucleotide sequence, the transgenic recombinant expression vector DBN11778 is an Arabidopsis plant (HP 1-2) with the HP1-2M nucleotide sequence, and the transgenic recombinant expression vector DBN11770N is an Arabidopsis plant (control vector) with the control recombinant expression vector. About 20000T ₁ seeds of HP1 were screened and 213T ₁ generation positive transformants (PAT gene) were identified, about 1.0% conversion efficiency; about 20000T ₁ seeds of HP1-1 were screened, and 195T ₁ generation positive transformants (PAT gene) were identified, about 1.0% conversion efficiency; about 20000T ₁ seeds of HP1-2 were screened, and 195T ₁ generation positive transformants (PAT gene) were identified, about 1.0% conversion efficiency; t ₁ seeds of about 20000 control vectors were screened, and 172T ₁ generation positive transformants (PAT gene) were identified with a transformation efficiency of about 0.86%.

The HP1 Arabidopsis T ₁ plants, HP1-1 Arabidopsis T ₁ plants, HP1-2 Arabidopsis T ₁ plants, arabidopsis T ₁ plants of the control vector and wild type Arabidopsis plants (CK) (18 days after sowing) were sprayed with 3 concentrations of topramen, respectively, to examine herbicide tolerance of Arabidopsis, namely 25gai/ha (1-fold field concentration, 1×), 100g ai/ha (4-fold field concentration, 4×) and 0g ai/ha (water, 0×). After 7 days of spraying (7 DAT), the extent of injury 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 x 100%). The substantially non-whitening phenotype was rated 0, the proportion of leaf whitening area was rated 1, the proportion of leaf whitening area was rated more than 50% was rated 2, and the proportion of leaf whitening area was rated 100% was rated 3. Transformation event resistance performance of each recombinant expression vector was scored according to the formula x= [ Σ (n×s)/(t×m) ]×100. (X-phytotoxicity score, N-peer victim number, S-phytotoxicity grade number, T-total plant number, M-highest phytotoxicity grade), and resistance evaluation was performed according to the score: high resistant plants (0-15 min), medium resistant plants (16-33 min), low resistant plants (34-67 min), and non-resistant plants (68-100 min). The experimental results are shown in table 2.

TABLE 2 results of experiments on the tolerance of transgenic Arabidopsis T ₁ plants to topramezone

The results in table 2 show that: compared with an arabidopsis T ₁ plant and a wild arabidopsis plant which are transferred into a control vector DBN11770N, the arabidopsis T ₁ plant transferred into an HP1M nucleotide sequence, the arabidopsis T ₁ plant transferred into an HP1-1M nucleotide sequence and the arabidopsis T ₁ plant transferred into an HP1-2M nucleotide sequence can generate tolerance to topramezone to different degrees; compared with an Arabidopsis T ₁ plant with the HP1M nucleotide sequence, an Arabidopsis T ₁ plant with the HP1-1M nucleotide sequence and an Arabidopsis T ₁ plant with the HP1-2M nucleotide sequence have better resistance to topramezone.

In summary, the amino acid sequence 159 of the herbicide tolerance protein HP1 of the invention is mutated from threonine to alanine or 258 from glutamine to methionine (the herbicide tolerance protein HP1-1 or HP 1-2) can show higher tolerance to HPPD inhibitor herbicide, and the herbicide tolerance protein can tolerate topramezone (middle resistance) with 1-time field concentration, so the application prospect on plants is wide.

Finally, it should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A protein characterized by having an amino acid sequence shown in SEQ ID NO. 9.

2. A gene, comprising:

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

(B) The nucleotide sequence shown as SEQ ID NO. 10 or SEQ ID NO. 11.

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

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

5.A method of extending the range of herbicides tolerated by plants comprising: the protein of claim 1 or the protein encoded by the expression cassette of claim 3 is expressed in a plant 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, the second herbicide tolerance protein being 5-enolpyruvylshikimate-3-phosphate synthase, glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha ketoglutarate-dependent dioxygenase, dicamba monooxygenase, acetolactate synthase, cytochrome-like protein and/or protoporphyrinogen oxidase, the plant being oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, potato, tomato or arabidopsis thaliana.

6. 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 an HPPD inhibitor herbicide that kills or inhibits the growth of untransformed cells, said HPPD inhibitor herbicide being topramezone, mesotrione and/or diketo nitrile, said plant being oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato or arabidopsis thaliana.

7. A method of controlling weeds, comprising: applying an effective dose of an HPPD inhibitor herbicide to a field in which a plant of interest is grown, said plant of interest comprising the gene of claim 2 or the expression cassette of claim 3 or the recombinant vector of claim 4, said HPPD inhibitor herbicide being topramezone, mesotrione and/or diketo nitrile, said plant of interest being oat, wheat, barley, millet, corn, sorghum, brachypodium distachyranthes, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato or arabidopsis thaliana.

8. The method of controlling weeds of claim 7, wherein said plant of interest is a glyphosate tolerant plant and said weeds are glyphosate tolerant weeds.

9. A method for protecting a plant from injury caused by or conferring HPPD inhibitor herbicide tolerance to a plant, comprising: introducing the gene of claim 2 or the expression cassette of claim 3 or the recombinant vector of claim 4 into a plant, wherein the introduced plant produces an amount of herbicide tolerance protein sufficient to protect the plant from the HPPD inhibitor herbicide selected from the group consisting of topramezone, mesotrione and/or diketo nitrile, and wherein the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyranthes, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis thaliana.

10. A method of producing a plant resistant to an HPPD inhibitor herbicide, comprising introducing into the genome of a plant the gene of claim 2, wherein the HPPD inhibitor herbicide is topramezone, mesotrione, and/or diketo nitrile, and the plant is oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato, or arabidopsis thaliana.

11. The method of producing a HPPD inhibitor herbicide tolerant plant according to claim 10, wherein the method of introduction comprises a genetic transformation method, a genome editing method or a gene mutation method.

12. 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 of claim 2 or the expression cassette of claim 3;

growing the plant propagules into plants;

Applying an effective dose of an HPPD inhibitor herbicide, which is topramezone, mesotrione and/or diketo nitrile, to a plant growth environment comprising at least said plant, said plant being oat, wheat, barley, millet, corn, sorghum, brachypodium distachyon, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato or arabidopsis thaliana, to a plant growth environment comprising at least said plant, and 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.

13. A method of obtaining a processed agricultural product comprising treating a harvest of HPPD inhibitor herbicide tolerant plants obtained by the method of claim 12 to obtain a processed agricultural product.

14. Use of a protein according to claim 1 for conferring tolerance to HPPD inhibitor herbicides which are topramezone, mesotrione and/or diketene nitrile in plants which are oat, wheat, barley, millet, corn, sorghum, brachypodium distachyranthes, rice, tobacco, sunflower, alfalfa, soybean, chickpea, peanut, beet, cucumber, cotton, canola, potato, tomato or arabidopsis thaliana.