CN114854702A - Herbicide tolerance protein, coding gene and application thereof - Google Patents

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 proteins, their encoding genes and uses

This application is a divisional application of Chinese Patent Application No. 202010194585.0. The name of the invention is: Herbicide Tolerance Protein, Its Encoding Gene and Use. The application date is March 19, 2020.

technical field

The present invention relates to a herbicide tolerance protein, its encoding gene and use, in particular to a protein with tolerance to HPPD inhibitor herbicide, its encoding gene and use.

Background technique

Hydroxyphenylpyruvate dioxygenase (HPPD) is an enzyme that catalyzes the degradation product of tyrosine-p-hydroxyphenylpyruvate (hydroxyphenylpyruvate) in the presence of iron ions (Fe ²⁺ ) and oxygen. acid (HPP for short) is converted into the precursor of tocopherol and plastoquinone (PQ) in plants - the reaction of homogentisate/homogentisate (HG). Tocopherols act as membrane-associated antioxidants; PQ is not only an electron carrier between PSII and the cytochrome b6/f complex, but also an essential cofactor for phytoene desaturase in carotenoid biosynthesis.

The herbicides that act by inhibiting HPPD mainly include three chemical families: triketones, isoxazoles and pyrazolones. In plants, they block PQ biosynthesis from tyrosine by inhibiting HPPD, resulting in PQ depletion and carotenoid deficiency. The above HPPD-inhibiting herbicides are phloem mobile bleaching agents that can cause new meristems and leaves exposed to light to appear white, and carotenoids are necessary for photoprotection, and in the absence of carotenoids Under certain circumstances, ultraviolet radiation and reactive oxygen intermediates can disrupt the synthesis and function of chlorophyll, resulting in the inhibition of plant growth and even death.

Methods for providing plants tolerant to HPPD inhibitor herbicides mainly include: 1) HPPD is overexpressed to produce large amounts of HPPD in plants that, despite the presence of HPPD inhibitor herbicides, are not related to the HPPD inhibitor herbicide. Sufficiently functioning, so that there is sufficient functional enzyme available. 2) Mutation of the target HPPD into a functional HPPD that is less sensitive to the herbicide or its active metabolite, but which retains the property of being converted to HG. With regard to the class of mutant HPPDs, although 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 different, more desirable HPPD inhibitor herbicides; for example, HPPD inhibitor herbicides may vary in the range of weeds they control, their cost of manufacture, and their environmental friendliness. Therefore, there is a need for new methods and/or compositions for conferring HPPD inhibitor herbicide tolerance to different crops and crop varieties.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a novel protein, its encoding gene and use, which not only has HPPD enzyme activity, but also makes plants into which the protein encoding gene is transformed have tolerance to HPPD inhibitor herbicides.

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

(a) has the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:9;

(b) A protein derived from (a) having the amino acid sequence in (a) substituted and/or deleted and/or added with one or several amino acids and having hydroxyphenylpyruvate dioxygenase activity.

To achieve the above object, the invention provides a kind of gene, including:

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

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

(c) having 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 can be hybridization in 6×SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution at 65°C, followed by 2×SSC, 0.1% SDS and 1×SSC, The membranes were washed once with 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.

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

In order to achieve the above object, the present invention also provides a method for extending the range of herbicides tolerated by plants, comprising: combining the protein or the protein encoded by the expression cassette with at least one protein different from the protein or the protein in the plant. The protein encoded by the expression cassette is expressed together with a second herbicide tolerance protein.

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.

In order to achieve the above object, the present invention also provides a method for selecting transformed plant cells, comprising: transforming a plurality of plant cells with the gene or the expression cassette, and in a condition that allows the gene or the expression cassette to be expressed Transformed cells are grown while culturing the cells at a concentration of an HPPD inhibitor herbicide that kills untransformed cells or inhibits the growth of untransformed cells;

Preferably, the plants include monocotyledonous and dicotyledonous plants; more preferably, the plants are oat, wheat, barley, millet, corn, sorghum, Brassica, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis;

Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile.

In order 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 planting a target plant, the target plant comprising the gene or the expression cassette or the recombinant vector;

Preferably, the target plants include monocotyledonous plants and dicotyledonous plants; more preferably, the target plants are oat, wheat, barley, millet, corn, sorghum, Brassica oleracea, rice, tobacco, sunflower, alfalfa , soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or Arabidopsis; further preferably, the target plant is a glyphosate-tolerant plant, and the weed is glyphosate resistant weeds;

Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile.

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

Preferably, the plants include monocotyledonous and dicotyledonous plants; more preferably, the plants are oat, wheat, barley, millet, corn, sorghum, Brassica, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis;

Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile.

In order 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 introduced method includes genetic transformation method, genome editing method or 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 or crossing a parent plant with a second plant, the parent plant and/or or a second plant comprising said gene or said expression cassette, said HPPD inhibitor herbicide tolerant plant having inherited said gene or said expression cassette from said parental plant and/or said second plant;

Preferably, the plants include monocotyledonous and dicotyledonous plants; more preferably, the plants are oat, wheat, barley, millet, corn, sorghum, Brassica, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis;

Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile.

To achieve the above object, the present invention also provides a method for culturing plants resistant to HPPD inhibitor herbicides, 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;

An effective dose of an HPPD inhibitor herbicide is applied to a plant growth environment comprising at least the plant, and the harvest is harvested with reduced plant damage and/or with increased plant damage compared to other plants not having the gene or the expression cassette. Plant yielding plants;

Preferably, the plants include monocotyledonous and dicotyledonous plants; more preferably, the plants are oat, wheat, barley, millet, corn, sorghum, Brassica, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis;

Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile.

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

In order to achieve the above object, the present invention also provides a planting system for controlling the growth of weeds, comprising adding an HPPD inhibitor herbicide and a plant growth environment in which at least one target plant contains the gene or the target plant. expression cassette;

Preferably, the target plants include monocotyledonous plants and dicotyledonous plants; more preferably, the target plants are oat, wheat, barley, millet, corn, sorghum, Brassica oleracea, rice, tobacco, sunflower, alfalfa , soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or Arabidopsis; further preferably, the target plant is a glyphosate-tolerant plant, and the weed is glyphosate resistant weeds;

Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile.

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

Preferably, the plants include monocotyledonous and dicotyledonous plants; more preferably, the plants are oat, wheat, barley, millet, corn, sorghum, Brassica, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis;

Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile.

The articles "a" and "an" as used herein refer to one (species) or more than one (species) (ie, at least one). For example, "an element" means one or more elements (elements). Furthermore, the term "comprising" or variations thereof such as "comprising" or "comprising" should be understood to mean the inclusion of one stated element, integer or step, or a group of elements, integers or steps, but not the exclusion of any other Elements, integers or steps, or groups of elements, integers or steps.

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

The term "HPPD-inhibiting herbicide" is synonymous with "HPPD herbicide" and refers to herbicides that directly or indirectly inhibit HPPD, these herbicides are bleaches, and their 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 (ie, 2-[2-chloro-4-(methylsulfonyl) ) benzoyl]-1,3-cyclohexanedione), mesotrione (i.e. 2-[4-(methylsulfonyl)-2-nitrobenzoyl]-1,3-ring Hexanedione), tembotrione (ie 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 into bioactive diketonitrile (DKN) to HPPD exerts an inhibitory effect, and thus diketonitriles are the active forms of isoxazole-based HPPD-inhibiting herbicides), for example, isoxaflutole (ie, 5-cyclopropyl-4-isoxazolyl[2- (methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone); (3) pyrazolinates, eg, topramezone (ie [3-(4) ,5-dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5-hydroxy-1-methylpyrazol-4-yl)methanone), sulfonic acid Pyrasulfotole (5-hydroxy-1,3-dimethylpyrazol-4-yl(2-methanesulfonyl-4-trifluoromethylphenyl)methanone).

In the present invention, topramezone, also known as topramezone, 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. It belongs to pyrazolinate class post-emergence stem and leaf treatment systemic HPPD herbicide, and the commonly used formulation is 30% suspending agent. The commercial preparations of fenpyrazone, such as Baowei, can control grass and broadleaf weeds, and use 5.6-6.7g of the preparation per mu. Weeds that can be effectively controlled include, but are not limited to, crabgrass (chicken's claw), Barnyardgrass, beef tendon grass, wild millet, foxtail (grass), quinoa, polygonum, green hemp, amaranth, wild amaranth, purslane, cocklebur, nightshade. Baowei has a significant synergistic effect after adding atrazine. In addition to having excellent control effects on the above weeds, it can also treat malignant broad-leaved weeds such as spinach (little thistle), endive radish, iron amaranth, Commelina officinalis (orchid) has a good control effect, especially it can effectively control foxtail, crabgrass, beef tendon, and wild millet which have poor control effect of mesotrione.

In the present invention, the effective dose of fenflufen refers to use at 25-100 g ai/ha, including 50-100 g ai/ha, 60-90 g ai/ha or 75-85 g ai/ha.

In the present invention, the term "resistance" is heritable and allows a plant to grow and reproduce with the herbicide under normal herbicide-effective treatment of a given plant. As recognized by those skilled in the art, even if a given plant suffers some degree of damage from herbicide treatment, such as little necrosis, lysis, chlorosis, or other damage, but at least no significant effect on yield, the plant can still be It is considered "resistance", that is, the increased ability of a given plant to resist various degrees of herbicide-induced damage, while the same herbicide dosage generally results in damage to wild-type plants of the same genotype. The term "tolerance" or "tolerance" in the present invention is broader than the term "resistance" and includes "resistance".

The term "conferring" as used herein refers to providing a plant with a characteristic or trait, such as herbicide tolerance and/or other desirable traits.

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 "heterologous" DNA, can be expressed in soybean plants using transgenic methods.

The term "nucleic acid" in the present invention includes reference to polymers of deoxyribonucleotides or ribonucleotides in single- or double-stranded form, and unless otherwise limited, includes known analogs having the essential properties of natural nucleotides (eg peptide nucleic acids) because they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

In the present invention, when the terms "encode" or "encoded" are used in the context of a specific nucleic acid, it means that the nucleic acid contains the necessary information to direct the translation of a nucleotide sequence into a specific protein. The information used to encode the protein is specified through the use of codons. A nucleic acid encoding a protein may contain untranslated sequences within the translated region of the nucleic acid (eg, introns), or may lack such intervening untranslated sequences (eg, in cDNA).

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

Genes encoding the herbicide tolerance proteins of the present invention are useful for producing plants tolerant to HPPD-inhibiting herbicides. The herbicide tolerance gene is particularly suitable for expression in plants in order to impart 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 analog of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers thing. The proteins of the present invention can be produced from a nucleic acid disclosed herein or by using standard molecular biology techniques. For example, a truncated protein of the present invention can be produced by expressing a recombinant nucleic acid of the present invention in a suitable host cell, or alternatively, in combination with ex vivo methods such as protease digestion and purification.

The present invention also provides nucleic acid molecules comprising polynucleotide sequences encoding herbicide tolerance proteins having HPPD enzymatic activity and conferring tolerance in plants to certain classes of HPPD-inhibiting herbicides acceptability. In general, the present invention includes any polynucleotide sequence encoding any of the herbicide tolerance proteins described herein, as well as encoding one or more conservative amino acid substitutions relative to the herbicide tolerance proteins described herein Any polynucleotide sequence of a herbicide tolerance protein. Conservative substitutions of amino acids to provide functionally similar amino acids are well known to those skilled in the art, and the following five groups each contain amino acids that are conservatively substituted for each other: 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), Glutamine Acid (E), Asparagine (N), Glutamine (Q).

Therefore, sequences that have HPPD-inhibiting herbicide tolerance activity and hybridize under stringent conditions to the genes encoding the herbicide tolerance proteins of the present invention are included in the present invention. Illustratively, these sequences are at least about 75% different, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homology.

Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the herbicide tolerance genes of the present invention. Nucleic acid molecules or fragments thereof are capable of specific hybridization with other nucleic acid molecules under certain circumstances. In the present invention, if two nucleic acid molecules can form an anti-parallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules can specifically hybridize with each other. Two nucleic acid molecules are said to be the "complement" of the other if they exhibit complete complementarity. In the present invention, when each nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of another nucleic acid molecule, the two nucleic acid molecules are said to show "complete complementarity". Two nucleic acid molecules are said to be "minimally complementary" if they can hybridize 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 be "complementary" if they can hybridize to each other with sufficient stability so that they anneal and bind to each other under conventional "high stringency" conditions. Deviations from perfect complementarity are permissible as long as such deviations do not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to function as a primer or probe, it only needs to be sufficiently complementary in sequence to allow for the formation of a stable double-stranded structure under the particular solvent and salt concentration employed.

In the present invention, a substantially homologous sequence is a nucleic acid molecule that can specifically hybridize with the complementary strand of another matched nucleic acid molecule under highly stringent conditions. Suitable stringent conditions to promote DNA hybridization, for example, treatment with 6.0x sodium chloride/sodium citrate (SSC) at approximately 45°C, followed by a wash with 2.0x SSC at 50°C, these conditions are very useful to those skilled in the art. is known. For example, the salt concentration in the wash step can be selected from about 2.0×SSC, 50°C under low stringency conditions to about 0.2×SSC, 50°C under highly stringent conditions. In addition, the temperature conditions in the wash steps can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions. Both the temperature conditions and the salt concentration can be changed, or one can be kept constant while the other variable. Preferably, the stringent conditions of the present invention may be specific hybridization with the herbicide tolerance gene of the present invention in 6×SSC, 0.5% SDS solution at 65°C, and then using 2×SSC, 0.1% SDS and The membrane was washed once with 1×SSC and 0.1% SDS each.

In the present invention, the term "hybridizes" or "specifically hybridizes" means that a molecule can only bind, double-stranded or hybridize to a specific nucleotide sequence under stringent conditions, which is when the sequence exists in a complex in a mixture (eg, total cellular) DNA or RNA.

Due to the redundancy of the genetic code, many different DNA sequences can encode the same amino acid sequence. The generation of these alternative DNA sequences encoding the same or substantially the same proteins is within the skill of those skilled in the art. These various DNA sequences are included within the scope of the present invention. The "substantially identical" sequences refer to sequences with amino acid substitutions, deletions, additions or insertions that do not substantially affect herbicide tolerance activity, and also include fragments that retain herbicide tolerance activity.

The term "functional activity" or "activity" in the present invention refers to the ability of the protein/enzyme for use in the present invention (alone or in combination with other proteins) to degrade or attenuate herbicidal activity. Plants producing the proteins of the present invention preferably produce an "effective amount" of the protein such that upon treatment of the plant with the herbicide, the protein is expressed at a level sufficient to confer complete or partial tolerance to the herbicide (or general amount unless otherwise specified) to the plant sex. The herbicides can be used at rates that would normally kill the target plant, normal field rates and concentrations. Preferably, the plant cells and plants of the present invention are protected from growth inhibition or damage caused by herbicide treatment. The transformed plants and plant cells of the present invention preferably are HPPD-inhibiting herbicide tolerant, ie, the transformed plants and plant cells are capable of growing in the presence of an effective amount of the HPPD-inhibiting herbicide.

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

The term "variant" of the present invention means a substantially similar sequence. For polynucleotides, a variant includes deletions and/or additions of one or more nucleotides at one or more internal sites within the reference polynucleotide and/or in herbicide tolerance Substitution of one or more nucleotides at one or more sites in a gene. The term "reference polynucleotide or polypeptide" in the present invention includes a herbicide tolerance nucleotide sequence or amino acid sequence, respectively. The term "native polynucleotide or polypeptide" in the present invention includes correspondingly naturally-occurring nucleotide sequences or amino acid sequences. For polynucleotides, conservative variants include the nucleotide sequence encoding one of these herbicide tolerance proteins of the invention (due to the degeneracy of the genetic code). Naturally occurring allelic variants such as these can be identified using well-known molecular biology techniques, eg, using the polymerase chain reaction (PCR) and hybridization techniques outlined below. Variant polynucleotides also include synthetically derived polynucleotides, eg, sequences generated by using site-directed mutagenesis but which still encode a herbicide tolerance protein of the invention. Typically, a variant 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, as determined by sequence alignment procedures and parameters.

"Variant protein" in the present invention means 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 in A protein derived from the 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, ie they continue to possess the desired activity of a herbicide tolerance protein, ie, still possess the described HPPD enzyme activity and/or herbicide tolerance. Such variants can arise, for example, from genetic polymorphisms or from human manipulation. A biologically active variant of a herbicide tolerance protein of the invention will have at least about 85%, 90%, 91%, 92%, 93%, 94% of the total amino acid sequence of the herbicide tolerance protein %, 95%, 96%, 97%, 98%, 99% or more sequence homology as determined by sequence alignment programs and parameters. A biologically active variant of a protein of the invention may differ from as few as 1-15 amino acid residues, as few as 1-10 (eg, 6-10), as few as 5 (eg, 4, 3) , 2, or even 1) amino acid residues of the protein.

Methods for alignment of sequences are well known in the art and can be accomplished using mathematical algorithms such as those of Myers and Miller (1988) CABIOS 4: 11-17; Smith et al. (1981) Adv. Appl .Math. 2:482 Local Alignment Algorithm; Need eman and Wunsch (1970) J.Mol.Biol.48:443-453 Global Alignment Algorithm; and Karlin and Altschul (1990) Proc.Natl.Acad.Sci . The algorithm of USA872264, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Computer implementations of these mathematical algorithms can be used for sequence comparison to determine sequence homology, such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, MountainView, California); the ALLGN program ( Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in GCG Wisconsin Genetics Software Package Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA).

In certain embodiments, the amino acids encoding the herbicide tolerance proteins of the invention or their variants that retain HPPD enzymatic activity can be stacked with any combination of polynucleotide sequences of interest to produce a desired traits of plants. 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 can be stacked with any other polynucleotide encoding a polypeptide conferring a desired trait, the Traits include, but are not limited to: resistance to disease, insects, and herbicides, tolerance to heat and drought, shortened crop maturation time, improved industrial processing (for example, for converting starch or biomass to fermentable sugars) species) and improved agronomic qualities (eg, high oil content and high protein content).

As is well known to those skilled in the art, the benefits of a combination of two or more modes of action in increasing the controlled weed spectrum and/or in naturally more tolerant or resistant weed species may also extend to artificial ( Transgenic or non-transgenic) chemicals that confer herbicide tolerance in crops other than HPPD-tolerant crops. Indeed, traits encoding the following resistances can be stacked individually or in multiple combinations to provide effective control or prevent weed succession from developing resistance to herbicides: glyphosate resistance (eg resistant plants or bacteria EPSPS, GOX, GAT), glufosinate resistance (e.g. PAT, Bar), acetolactate synthase (ALS) inhibitory herbicide resistance (e.g. imidazolidinone, sulfonylurea, triazolopyrimidine, sulfaniline, pyrimidine thiobenzoic acid) and other chemical resistance genes such as AHAS, Csrl, SurA, etc.), phenoxy auxin herbicide resistance (such as aryloxyalkanoate dioxygenase-AAD), dicamba herbicide resistance ( Such as dicamba monooxygenase-DMO), bromoxynil resistance (such as Bxn), resistance to phytoene desaturase (PDS) inhibitors, resistance to photosystem II-inhibiting herbicides (such as psbA), resistance to photosystem I-inhibiting herbicides, resistance to protoporphyrinogen oxidase IX (PPO)-inhibiting herbicides (such as PPO-1), resistance to phenylurea herbicides ( Such as CYP76B1), dichloromethoxybenzoic acid degrading enzyme and so on.

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 be) selected for the succession of weeds to naturally more tolerant species or glyphosate resistant biotypes. Most herbicide resistance management strategies recommend the use of effective amounts of tank-mixed herbicide companions that provide control of the same species but with different modes of action as a means of delaying the emergence of resistant weeds. Stacking the herbicide tolerance genes of the present invention with glyphosate tolerance traits (and/or other herbicide tolerance traits) may allow selective use of glyphosate and pyrazolone herbicides (eg, fenpyridine) on the same crop. control of glyphosate-resistant weed species (broadleaf weed species controlled by one or more pyrazolone herbicides) in glyphosate-tolerant crops. The applications of these herbicides can be simultaneous use in tank mixtures containing two or more herbicides with different modes of action, individual herbicide compositions in sequential use (eg pre-plant, pre-emergence or post-emergence) (Intervals used ranged from 2 hours to 3 months), or alternatively, at any time (within 7 months from planting the crop to when the crop was harvested (or the pre-harvest interval for individual herbicides, whichever is the shortest) )) use combinations representing any number of herbicides to which each compound class can be applied.

It is important to have flexibility in controlling broadleaf weeds, namely timing of application, individual herbicide rates and the ability to control stubborn or resistant weeds. The application range of glyphosate superimposed with the glyphosate resistance gene/herbicide tolerance gene of the present invention in crops can be from 250 to 2500 g ae/ha; From 25-500gai/ha. The optimal combination of times for these applications depends on the specific conditions, species and environment.

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

In addition, the genes encoding the herbicide tolerance proteins of the invention can be combined with one or more other inputs such as insect resistance, fungal resistance or stress tolerance, alone or stacked with other herbicide tolerant crop traits. etc.) or output (eg increased yield, improved oil yield, improved fiber quality, etc.) trait stacking. 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 improve crop quality.

These stacked combinations can be produced by any method including, but not limited to, cross-breeding plants or genetic transformation by conventional or topcrossing methods. If the sequences are stacked by genetically transforming the 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 can be used as targets for the introduction of additional traits by subsequent transformation. These traits can be introduced simultaneously with the polynucleotides 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 can be contained in separate expression cassettes (trans) or in the same expression cassette (cis). Expression of these sequences can 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 suppressor expression cassettes or overexpression cassettes to produce the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system.

The gene encoding the herbicide tolerance protein of the present invention has high tolerance to pyrazolone herbicides, and is the basis for the feature possibility of important herbicide tolerance crops and selection 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 a suitable host cell, comprising operably linked to a nucleotide sequence of interest (ie, a single or a polynucleotide encoding a herbicide tolerance protein or a variant that retains HPPD enzymatic activity in combination with one or more additional nucleic acid molecules encoding a polypeptide conferring the desired trait), The nucleotide sequence of interest is operably linked to a termination signal. The coding region typically encodes a protein of interest, but can also encode a functional RNA of interest, such as antisense RNA or an untranslated RNA in the sense or antisense orientation. An 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 can 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, ie, the particular DNA sequence of the expression cassette does not naturally occur in the host cell and must have been introduced into the new host cell by a transformation event. Expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some specific external stimulus. Additionally, the promoter is specific for a particular tissue or organ or stage of development.

The present invention contemplates encoding a herbicide-resistant polynucleotide capable of expressing a polynucleotide of interest (ie, a single or in combination with one or more additional nucleic acid molecules encoding a polypeptide conferring a desired trait). Plants are transformed with an expression cassette of a receptor protein or a polynucleotide of its variant that retains HPPD enzymatic activity. The expression cassette includes transcription and translation initiation regions (i.e., promoters) and a polynucleotide open reading frame in the 5'-3' transcriptional direction. The expression cassette may optionally include transcriptional and translational termination regions (ie, termination regions) that function in plants. In some embodiments, the expression cassette includes a selectable marker gene to allow selection of stable transformants. Expression constructs of the present invention may also include leader sequences and/or sequences that allow 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 described in the present invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leader sequences, introns, and other operably linked to the herbicide tolerance protein encoding the herbicide tolerance. gene regulatory sequences.

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

The transit peptide (also known as a secretion signal sequence or targeting sequence) directs the transgene product to a specific organelle or cellular compartment, the transit peptide may be heterologous to the receptor protein, for example, by encoding chloroplast transport The peptide sequences were targeted to the chloroplast, either to the endoplasmic reticulum using the 'KDEL' retention sequence, or to the vacuole using the CTPP of the barley lectin gene.

The leader sequence includes, but is not limited to, picornavirus leader sequence, such as EMCV leader sequence (encephalomyocarditis virus 5' non-coding region); Potato gamma virus group leader sequence, such as MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin heavy chain binding protein (BiP); untranslated leader sequence of coat protein mRNA of alfalfa mosaic virus (AMVRNA4); tobacco mosaic virus (TMV) leader sequence.

The enhancers include, but are not limited to, cauliflower mosaic virus (CaMV) enhancers, scrophularia mosaic virus (FMV) enhancers, carnation weathering ring virus (CERV) enhancers, cassava vein mosaic virus (CsVMV) enhancers , Purple jasmine mosaic virus (MMV) enhancer, Nougat yellow leaf curl virus (CmYLCV) enhancer, Multan cotton leaf curl virus (CLCuMV), Commelina yellow mottle virus (CoYMV) and Peanut chlorotic streak Leaf virus (PCLSV) enhancer.

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

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

"Operably linked" in the context of the present invention refers to the association of nucleic acid sequences such that one sequence can provide the desired function for the linked sequences. In the present invention, the "operably linked" can be to link a promoter with a sequence of interest, so that the 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 to mean: a 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 are linked, and are linked in such a way that the resulting translation product is in-frame with respect to 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 function (i.e., gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, poly adenylation sites and/or transcription terminators), sequences providing DNA transfer and/or integration functions (i.e. T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), providing selection Sequences for sexual function (i.e. antibiotic resistance markers, biosynthetic genes), sequences providing scoreable marker function, sequences assisting in sequence manipulation in vitro or in vivo (i.e. polylinker sequences, site-specific recombination sequences) and providing Sequences of replication function (ie bacterial origins of replication, autonomously replicating sequences, centromeric sequences).

The genome of a plant, plant tissue or plant cell in the present invention refers to any genetic material in the plant, plant tissue or plant cell, and includes the nucleus and plastid and mitochondrial genome.

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 parts of plants on or below plants In complete plant cells, these plant parts are embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, cores, ears, cobs, husks, stems, roots, root tips, anthers, and the like.

The herbicide-tolerant protein of the present invention can be applied to a variety of plants, including but not limited to alfalfa, kidney bean, cauliflower, cabbage, carrot, celery, cotton, cucumber, eggplant, lettuce, melon, pea , pepper, zucchini, radish, rape, spinach, soybean, pumpkin, tomato, Arabidopsis, peanut or watermelon; preferably, the dicotyledonous plant refers to cucumber, soybean, Arabidopsis, tobacco, cotton, peanut or rapeseed . The monocots include but are not limited to corn, rice, sorghum, wheat, barley, rye, millet, sugar cane, oats or turfgrass; preferably, the monocots refer to corn, rice, sorghum, wheat, barley , millet, sugar cane or oats.

The term "plant transformation" in the present invention refers to the cloning of a herbicide-resistant or tolerant polynucleotide, alone or in combination with one or more additional nucleic acid molecules encoding a polypeptide conferring a desired trait, into In an expression system, it is transformed into a plant cell. The receptors and target expression cassettes of the present invention can be introduced into plant cells in a variety of well-known ways. In the context of a polynucleotide, the term "introduced" (eg, a nucleotide construct of interest) is intended to mean that the polynucleotide is provided to the plant in such a way that the polynucleotide acquires support for a The access or realization of 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 regimen, eg, in plants, the polynucleotides can be introduced into host cells of interest in a single transformation event, in separate transformation events. The methods of the present invention do not depend on a particular method for introducing one or more polynucleotides into a plant, merely obtaining access or realization 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 plants 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 generations thereof, resulting in the 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 the foreign gene to be stably inherited.

The term "genome editing technology" refers to a genome modification technology that enables precise manipulation of genome sequences to achieve gene site-directed mutation, insertion, deletion and other operations. At present, genome editing technologies mainly include HE (homingendonuclease, homing endonuclease), ZFN technology (Zinc-finger nuclease, zinc finger nuclease), TALEN technology (transcription activator-like effector nuclease, transcription activator-like effector nuclease) , CRISPR technology (Clustered regulatory interspaced short palindromic repeat, clustered regularly interspaced short palindromic repeat).

Numerous transformation vectors available for plant transformation are known to those skilled in the art, and the 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. Selectable markers routinely used in transformation include the nptll gene that confers resistance to kanamycin and related antibiotics or related herbicides (this gene was published by Bevan et al. page), conferring resistance to the herbicide glufosinate (also known as glufosinate-ammonium; see White et al., Nucl. AcidsRes, Vol. 18, p. 1062, 1990, Spencer et al., 1990, Theor. Appl. Genet, Vol. 79, pp. 625-631 and U.S. Pat. 2931) and the dnfr gene conferring resistance to methotrexate (Bourouis et al. 1983 in EMBO J. Vol. 2 pp. 1099-1104), the EPSPS gene conferring resistance to glyphosate (U.S. Pat. 4940935 and 5188642), the glyphosate N-acetyltransferase (GAT) gene that also confers resistance to glyphosate (Castle et al., Science vol. 304, pp. 1151-1154, 2004; US application published Patents 20070004912, 20050246798 and 20050060767) and the 6-phosphate mannose isomerase gene that provides the metabolism of mannose (described in US Patents 5,767,378 and 5,994,629).

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

Planting systems in the context of the present invention refer to plants, which exhibit any combination of herbicide tolerance and/or herbicide treatments available at different stages of plant development, resulting in high yielding and/or reduced damage plants.

In the present invention, the weeds refer to plants that compete with the target plant to be cultivated in the plant growth environment.

The terms "control" and/or "control" herein refer to the direct application (eg, by spraying) of at least an effective dose of a pyrazolone herbicide to the plant growth environment to minimize and/or stop weed development . At the same time, the plant of interest to be cultivated should be morphologically normal and can be cultivated under conventional methods for product consumption and/or production; preferably, with reduced plant damage and /or have increased plant yield. Specific manifestations of the reduced plant damage include, but are not limited to, improved stalk resistance, and/or increased grain weight, and the like. The "control" and/or "control" effect of the herbicide tolerance protein on weeds can exist independently, and is not weakened by the presence of other substances that can "control" and/or "control" weeds. / or disappear. Specifically, any tissue of a transgenic plant (containing a gene encoding a herbicide tolerance protein) exists and/or produces simultaneously and/or asynchronously, the herbicide tolerance protein and/or the weed-controlling another substance, the presence of the other substance neither affects nor results in the "control" and/or "control" of the herbicide tolerance protein on weeds or "control" effect is achieved entirely and/or in part by the other substance independent of the herbicide tolerance protein.

The "plant propagules" described in the present invention include, but are not limited to, plant sexual propagules and plant vegetative propagules. The plant sexual propagules include, but are not limited to, plant seeds; the plant vegetative propagules refer to the vegetative organs or certain special tissues of the plant body, which can produce new plants under in vitro conditions; the vegetative organs or certain Special tissues include but are not limited to roots, stems and leaves, for example: plants with roots as vegetative bodies include strawberries and sweet potatoes, etc.; plants with stems as vegetative bodies include sugarcane and potato (tuber), etc.; leaves as vegetative bodies Plants that produce propagules include aloe vera and begonia.

The present invention can confer novel herbicide resistance traits to plants, and no adverse effects on phenotype, including yield, are observed. Plants of the present invention can tolerate typical application levels such as 2x, 3x, or 4x of at least one of the herbicides tested. Improvements in these tolerance levels are within the scope of the present invention. For example, various techniques known in the art can be foreseen for optimization and further development to increase the expression of a given gene.

The present invention provides a herbicide tolerance protein, its encoding gene and application, and has the following advantages:

1. The herbicide tolerance protein of the present invention can endow plants with tolerance to HPPD inhibitor herbicides, and can tolerate at least 1-fold field concentration of fenflufenac.

2. The herbicide tolerance protein of the present invention has broad application prospects in plants.

The technical solutions of the present invention will be further described in detail below through the accompanying drawings and embodiments.

Description of drawings

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

Figure 2 is a graph showing the effect of the enzyme activity of the herbicide-tolerant protein HP1 under the conditions of different concentration gradients of HPPD inhibitor herbicides (fenflumetrizone, mesotrione or diketonitrile);

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

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

Figure 5 is a schematic structural diagram of the Arabidopsis thaliana recombinant expression vector DBN11770 containing the HP1M nucleotide sequence of the present invention;

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

Detailed ways

The technical solutions of the herbicide tolerance protein of the present invention, its encoding gene and its use are further described below through 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 listing, which encodes the HP1 protein, as shown in SEQ ID NO: 1 in the sequence listing. The HP1M nucleotide sequence encoding the amino acid sequence corresponding to the HP1 was obtained according to Arabidopsis thaliana-preferred codons, as shown in SEQ ID NO: 3 in the sequence listing.

2. Construction of HP1 gene mutation library

After the above-mentioned synthetic HP1 gene is amplified by PCR, according to the operation steps of the Takara company product pUC118 carrier (Takara, CAT: 3318) instruction manual, clone on the carrier pUC118, then import the product after the above-mentioned connection into Escherichia coli DH5α as a template, Error-prone PCR with forward and reverse primers allowed the HP1 gene to mutate due to random base mismatches.

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

Forward primer: 5'-ccc aagctt gatggccgccgattccgaaaatc-3', as shown in SEQ ID NO: 4 in the sequence listing (the underline is the HindIII restriction site);

Reverse primer: 5'-cgc ggatcc tcaggcatcgaccgtcacaacg-3', as shown in SEQ ID NO: 5 in the sequence listing (the underline is the BamHI restriction site);

The error-prone PCR reaction system (total volume 50 μL) was:

The concentration of the plasmid DNA template is 1-10 ng/μL, the concentration of the forward primer is 10 μM, and the concentration of the reverse primer is 10 μM.

Error-prone PCR reaction conditions are:

The above-mentioned error-prone PCR product was subjected to 7g/L agarose gel electrophoresis and recovered by cutting the gel. The error-prone PCR product and the pUC118 vector after the recovery of the cutting gel were double digested by HindIII and BamHI, and the digested product was purified. T4-DNA ligase was used for enzymatic ligation, and then it was transformed into E. coli DH5α, which is sensitive to fenflufen, to construct a random mutation library of HP1 gene.

3. Screening the HP1 gene mutation library

The transformation product in the above mutant library was inoculated into a 48-well plate containing 2mL LB liquid medium (100mg/L of ampicillin, 800mg/L of tyrosine) containing 300 μM of fenflumezone, and the temperature was kept at 37°C and 180rpm. Shake the culture in a shaker. The growth status of the strain was observed. When the strain grew to an OD of 0.4-0.6, 1 mM IPTG was added to the above-mentioned LB liquid medium and the culture was continued, and the color change of the medium was observed.

Due to the presence of tyrosine aminotransferase in Escherichia coli, it can catalyze the formation of p-hydroxyphenylpyruvate (HPP) from tyrosine. HPP can be used as a substrate for HPPD, and the product HG catalyzed by HPPD can be oxidized spontaneously. Polymerization produces brown-red pyomelanin, but HPPD inhibitors can inhibit HPPD enzyme activity and thus inhibit color production. Therefore, high-throughput screening of the above-mentioned mutant library can be performed according to whether brown-red color is produced in the LB liquid medium. sex genes.

4. Acquire a mutated resistance gene

The sequencing results showed that two HP1 mutant resistance genes were obtained, named HP1-1 and HP1-2 respectively. The 475th position of the HP1-1 nucleotide sequence was mutated from the original A to G, resulting in the 159th amino acid sequence. The 772nd and 773rd positions of the HP1-2 nucleotide sequence are mutated from the original CA to AT, resulting in the mutation of the 258th position of the amino acid sequence from the original glutamine for methionine.

The amino acid sequence of the herbicide tolerance protein HP1-1, as shown in SEQ ID NO: 6 in the sequence listing, encodes the HP1-1 nucleotide sequence corresponding to the amino acid sequence of the herbicide tolerance protein HP1-1 , as shown in SEQ ID NO: 7 in the sequence listing; obtain the HP1-1M nucleotide sequence encoding the amino acid sequence corresponding to the herbicide tolerance protein HP1-1 according to the Arabidopsis preference codons, as shown in the sequence listing shown in SEQ ID NO:8.

The amino acid sequence of the herbicide tolerance protein HP1-2, as shown in SEQ ID NO: 9 in the sequence listing, encodes the HP1-2 nucleotide sequence corresponding to the amino acid sequence of the herbicide tolerance protein HP1-2 , as shown in SEQ ID NO: 10 in the sequence listing; 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 codons, as shown in the sequence listing shown in SEQ ID NO: 11.

The second example, the detection of the tolerance effect of herbicide tolerance proteins HP1-1 and HP1-2 on HPPD inhibitor herbicides

1. Nucleotide sequences for the synthesis of HP1, HP1-1 and HP1-2

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

5'-end universal adapter primer 1: 5'-taagaaggatatacatatg-3', as shown in SEQ ID NO: 12 in the sequence listing;

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

2. Construction of prokaryotic recombinant expression vector and acquisition of recombinant strains

The prokaryotic expression vector DBNBC-01 was subjected to double digestion reaction with restriction enzymes Nde I and Xho I to linearize the prokaryotic expression vector, and the digestion product was purified to obtain the linearized DBNBC-01 expression vector backbone (vector backbone: pET-29a(+)), the HP1 nucleotide sequence connected with the universal linker primer 1 is subjected to a recombination reaction with the linearized DBNBC-01 expression vector backbone, and the operation steps follow the Takara company In-Fusion seamless The ligation product kit (Clontech, CA, USA, CAT: 121416) was used to construct a recombinant expression vector DBN11765, the structure of which is shown in Figure 1 (F1 ori: origin of replication of phage F1; Kan: kanamycin resistance Sex gene; Ori: origin of replication; Rop: Rop gene, encoding ROP protein; T7 promoter: T7 RNA polymerase promoter; HP1: HP1 nucleotide sequence (SEQ ID NO: 2); 6xHis: affinity tag, encoding 6 A continuous His is used as a tag to purify the target protein; T7terminator: the terminator of T7 RNA polymerase; LacI operator: the operator region of the lactose operon; lacIpromoter: the lacI promoter; lacI: the lacI operon regulatory gene).

The recombinant expression vector DBN11765 was transformed into E. coli BL21(DE3) competent cells by heat shock method, and the heat shock conditions were: 50 μL E. coli BL21 (DE3) competent cells, 10 μL plasmid DNA (recombinant expression vector DBN11765), 42 ℃ water bath 30s; 37°C shaking culture for 1h (shaking at 100rpm); then cultured on the LB solid plate containing 50mg/L kanamycin (Kanamycin) for 12h at 37°C, pick white colonies, In LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, kanamycin 50 mg/L, adjusted to pH 7.5 with NaOH) overnight at 37°C. Its plasmid was extracted by alkaline method. The extracted plasmid is sequenced and identified, and the results show that the nucleotide sequence of the recombinant expression vector DBN11765 between the Nde I and Xho I sites is the nucleotide sequence shown in SEQ ID NO: 2 in the sequence table, respectively, and the recombinant strain is preserved. BL21(HP1) is spare.

According to the above-mentioned method for constructing the recombinant expression vector DBN11765, the HP1-1 nucleotide sequence and HP1-2 nucleotide sequence of the universal linker primer 1 were respectively connected with the linearized DBNBC-01 expression vector backbone. After the recombination reaction, the recombinant expression vectors DBN11774 and DBN11775 were obtained in turn. The recombinant expression vectors DBN11774 and DBN11775 were transformed into Escherichia coli BL21(DE3) competent cells by heat shock method and their plasmids were extracted by alkaline method. The extracted plasmids were sequenced and verified. The results showed that the nucleotides in the recombinant expression vectors DBN11774 and DBN11775 The sequences respectively contain the nucleotide sequence shown in SEQ ID NO: 7 and the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing, that is, the HP1-1 nucleotide sequence and the HP1-2 nucleotide sequence are correctly inserted . The obtained recombinant strains BL21 (HP1-1) and BL21 (HP1-2) were saved 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 monoclonally inoculated in 100 mL of LB medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L). L. Ampicillin 100 mg/L, adjusted to pH 7.5 with NaOH), cultured to a concentration of OD _600nm = 0.6-0.8, added IPTG with a concentration of 0.4 mM, and induced at a temperature of 16 °C for 12 h. Collect the bacteria, resuspend the bacteria with 15mL PBS buffer (50mM, pH 7.4), ultrasonically disrupt (X0-900Dultrasonic processor ultrasonic processor, 30% intensity) for 10min, then centrifuge, collect the supernatant, and use nickel ion affinity chromatography Columns were used to purify the above herbicide-tolerant proteins obtained, and the purification results were detected by SDS-PAGE protein electrophoresis. The band size was consistent with the theoretically predicted band size.

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

Enzyme activity reaction system (1 mL): containing 25 μg reaction enzyme (the herbicide-tolerant protein HP1, HP1-1 or HP1-2 obtained by the above purification), 0.2 mM HPP substrate, 1 mM Fe ²⁺ , 1 mM ascorbic acid, Different concentration gradients (concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM and 50 μM) of fenflumetrizone (mesotrione or diketonitrile), the buffer system is 50mM PBS buffer (pH 7.4 ), reacted in a water bath for 20 min at a temperature of 30 °C, each reaction was timed by adding the reaction enzyme, and the enzyme reaction system was terminated in a boiling water bath for 5 min.

The above-terminated enzyme reaction system was centrifugally filtered, and 20 μL of the filtrate was taken for high-performance liquid chromatography (HPLC) analysis. The HPLC conditions were: the mobile phase was acetonitrile: water (30:70, V/V), and 0.1% trifluoride was added to the water. Acetic acid. C18 reversed-phase chromatographic column (5μm, 250mm×4.6mm), the column temperature is 40°C, the detection wavelength is 292nm with a VWD-3100 single-wavelength UV detector, the injection volume is 20μL, and the flow rate is 1.0mL/min. The HPLC analysis showed that the peak time of the enzymatic reaction product was consistent with that of the standard sample of homogentisic acid, and no homogentisic acid was generated in the reaction system without enzyme. The resistance of HP1, HP1-1 and HP1-2 was detected according to different concentrations of HPPD inhibitor herbicides (fenflumetrizone, mesotrione or diketonitrile) added to the system, specifically: The enzyme activity when adding HPPD inhibitor herbicide is 100%, calculate the relative enzyme activity when adding HPPD inhibitor herbicide, and use HPLC to measure the production of homogentisic acid to calculate the effect of HPPD inhibitor herbicide on HP1, HP1-1 and the IC ₅₀ of HP1-2 (the concentration of HPPD inhibitor herbicide at which the enzymatic catalytic reaction is inhibited by 50%). One unit of enzyme activity is defined as the amount of enzyme required to generate 1 μmol of homogentisate within 1 min at pH 7.4 and temperature of 30 °C. The experimental results are shown in Figure 2-4 (herbicide tolerance proteins HP1, HP1-1 and HP1-2 in different concentration gradients of HPPD inhibitor herbicides (fenflumetrizone, mesotrione or diketonitrile) ) conditions), using Prism 6.0 software, the IC ₅₀ values of fenflumetrizone, mesotrione and diketonitrile on HP1, HP1-1 and HP1-2 were calculated by nonlinear fitting, The results are shown in Table 1.

Table 1. Half-inhibitory concentrations of HPPD inhibitor herbicides mesotrione, mesotrione and diketonitrile on HP1, HP1-1 and HP1-2

The results in Table 1 show that the herbicide tolerance proteins HP1, HP1-1 and HP1-2 have different degrees of tolerance to HPPD inhibitor herbicides (fenflumetrizone, mesotrione and diketonitrile) Compared with the herbicide tolerance protein HP1, the purified herbicide tolerance proteins HP1-1 and HP1-2 have better resistance to HPPD inhibitor herbicides.

The third example, the acquisition and verification of transgenic Arabidopsis plants

1. Construct Arabidopsis recombinant expression vectors containing HP1, HP1-1 or HP1-2 genes respectively

The HP1M nucleotide sequence (SEQ ID NO: 3) described in the above first embodiment 1, the HP1-1M nucleotide sequence (SEQ ID NO: 8) described in the above first embodiment 4, and HP1-2M The 5' and 3' ends of the nucleotide sequence (SEQ ID NO: 11) were respectively connected with universal linker primer 2:

5'-end universal adapter primer 2: 5'-agttttttctgattaacagactagt-3', as shown in SEQ ID NO: 14 in the sequence listing;

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

The plant expression vector DBNBC-02 was subjected to double digestion reaction with restriction endonucleases Spe I and Asc I to linearize the plant expression vector, and the digestion product was purified to obtain the linearized DBNBC-02 expression vector backbone (vector backbone: pCAMBIA2301 (can be provided by CAMBIA agency)), the HP1M nucleotide sequence connected with the universal linker primer 2 is subjected to a recombination reaction with the linearized DBNBC-02 expression vector backbone, and the operation steps are according to Takara In-Fusion No. The suture ligation product kit (Clontech, CA, USA, CAT: 121416) was carried out according to the instructions, and the recombinant expression vector DBN11770 was constructed, the schematic diagram of which is shown in Figure 5 (Spec: spectinomycin gene; RB: right border; eFMV: mysterious 34S enhancer of ginseng mosaic virus (SEQ ID NO: 16); prBrCBP: promoter of rape eukaryotic elongation factor gene 1α (Tsf1) (SEQ ID NO: 17); spAtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO: 18); EPSPS: 5-enolpyruvate shikimate-3-phosphate synthase gene (SEQ ID NO: 19); tPsE9: terminator of the pea RbcS gene (SEQ ID NO: 20); prAtUbi10: Southern Promoter of Ubiquitin 10 gene (SEQ ID NO:21); spAtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO:18); HP1M: HP1M nucleotide sequence (SEQ ID NO:3); tNos: terminator of nopaline synthase gene (SEQ ID NO: 22); pr35S: cauliflower mosaic virus 35S promoter (SEQ ID NO: 23); PAT: phosphinothricin N-acetyltransferase gene (SEQ ID NO: 23) ID NO: 24); t35S: Cauliflower mosaic virus 35S terminator (SEQ ID NO: 25); LB: left border).

The recombinant expression vector DBN11770 was transformed into E. coli T ₁ competent cells by heat shock method, and the heat shock conditions were: 50 μL E. coli T ₁ competent cells, 10 μL plasmid DNA (recombinant expression vector DBN11770), 42 ℃ water bath for 30 s; 37 ℃ Shaking culture for 1 h (shaking at 100 rpm); then on the LB solid plate (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar containing 50 mg/L Spectinomycin) 15g/L, adjust the pH to 7.5 with NaOH) and cultivate it for 12h at a temperature of 37°C, pick white colonies, and put them in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, Spectinomycin 50mg/L, adjust pH to 7.5 with NaOH) and cultivate overnight at 37°C. Extract its plasmid by alkaline method: Centrifuge the bacterial liquid at 12000rpm for 1min, remove the supernatant, and precipitate the bacterial cells with 100μl ice-precooled solution I (25mM Tris-HCl, 10mM EDTA (ethylenediaminetetraacetic acid), 50mM glucose , pH8.0) suspension; add 200 μL of freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)), invert the tube 4 times, mix, put on ice for 3-5min; add 150 μL ice-cold solution III (3M potassium acetate, 5M acetic acid), immediately mixed thoroughly, placed on ice for 5-10 min; centrifuged for 5 min at a temperature of 4 °C and a rotating speed of 12000 rpm, added 2 times the volume of anhydrous ethanol to the supernatant, mixed After homogenization, place at room temperature for 5 min; centrifuge at 4°C and 12000 rpm for 5 min, discard the supernatant, wash the precipitate with 70% ethanol and dry it; add 30 μL containing RNase (20 μg/mL) TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to dissolve the precipitate; water bath at 37 °C for 30 min to digest RNA; store at -20 °C for later use. The extracted plasmid is sequenced and identified, 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 listing, that is, the HP1M core. nucleotide sequence.

According to the above-mentioned method for constructing the recombinant expression vector DBN11770, the HP1-1M nucleotide sequence and HP1-2M nucleotide sequence respectively connected with the universal linker primer 2 were respectively connected with the linearized DBNBC-02 expression vector backbone. Carry out the recombination reaction to obtain the recombinant expression vectors DBN11777 and DBN11778 in turn. Sequencing to verify 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 listing, that is, the HP1-1M core The nucleotide sequence and the HP1-2M nucleotide sequence were inserted correctly.

According to the above-mentioned method of constructing the recombinant expression vector DBN11770, the control recombinant expression vector DBN11770N was constructed, and its vector structure is shown in Figure 6 (Spec: spectinomycin gene; RB: right border; eFMV: 34S enhancer of Scrophulariaceae mosaic virus ( SEQ ID NO: 16); prBrCBP: promoter of rape eukaryotic elongation factor gene 1α (Tsf1) (SEQ ID NO: 17); spAtCTP2: Arabidopsis chloroplast transit peptide (SEQ ID NO: 18); EPSPS: 5- Enolpyruvate shikimate-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: 20) : 23); PAT: phosphinothricin N-acetyltransferase gene (SEQ ID NO: 24); t35S: cauliflower mosaic virus 35S terminator (SEQ ID NO: 25); LB: left border).

2. Arabidopsis thaliana recombinant expression vector transformed into Agrobacterium

The correctly constructed recombinant expression vectors DBN11770, DBN11777, DBN11778 and the control recombinant expression vector DBN11770N were transformed into Agrobacterium GV3101 by liquid nitrogen method respectively, and the transformation conditions were: 100 μL Agrobacterium GV3101, 3 μL plasmid DNA (recombinant expression vector) ; placed in liquid nitrogen for 10min, 37 ℃ warm water bath for 10min; inoculated the transformed Agrobacterium GV3101 in LB test tube at a temperature of 28 ℃ and a rotating speed of 200rpm for 2h, coated with rifampicin containing 50mg/L ( Rifampicin) and 50 mg/L spectinomycin on the LB solid plate until a positive single clone grows, pick the single clone and cultivate and extract its plasmid, and the extracted plasmid is sequenced and identified, the results show that the recombinant expression vectors DBN11770, DBN11777 , DBN11778 and DBN11770N structures are completely correct.

3. Obtainment of transgenic Arabidopsis plants

Wild-type Arabidopsis seeds were suspended in 0.1% (w/v) agarose solution. Suspended seeds were stored at 4°C for 2 days to complete the need for dormancy to ensure synchronized seed germination. The horse manure was mixed with vermiculite and sub-irrigated with water until moist, and the soil mixture was allowed to drain for 24 h. The pretreated seeds were sown on the soil mix and covered with a moisturiser for 7 days. Seeds were germinated and plants were grown in a greenhouse under long-day conditions (16h light/8h dark) with a constant temperature (22°C) and constant humidity (40-50%) light intensity of 120-150 μmol/m ² s ⁻¹ . Start by irrigating the plants with Hoagland nutrient solution, followed by deionized water, keeping the soil moist but not soggy.

Arabidopsis thaliana was transformed using the flower soak method. One or more 15-30 mL pre-cultures of LB broth containing spectinomycin (50 mg/L) and rifampicin (10 mg/L) were inoculated with selected Agrobacterium colonies. The preculture was incubated overnight at 28°C with constant shaking at 220 rpm. Each pre-culture was used to inoculate two 500 ml cultures of the YEP broth containing spectinomycin (50 mg/L) and rifampicin (10 mg/L) and the cultures were incubated overnight at 28°C with constant shaking . The cells were pelleted by centrifugation at about 4000 rpm for 20 min at room temperature, and the obtained supernatant was discarded. The cell pellet was gently resuspended in 500 mL of osmotic medium containing 1/2 x MS salts/B5 vitamins, 10% (w/v) sucrose, 0.044 μM benzylaminopurine (10 μL/L (1 mg/ stock solution in mL DMSO)) and 300 μL/L Silvet L-77. About 1 month old Arabidopsis plants were soaked in osmotic medium containing resuspended cells for 5 min, making sure to submerge the newest inflorescences. Next, the Arabidopsis plants were placed sideways and covered, and after moisturizing for 24 hours in a dark environment, the Arabidopsis plants were normally cultured at a temperature of 22°C with a photoperiod of 16h light/8h dark. Seeds are harvested after about 4 weeks.

Freshly harvested (HP1M nucleotide sequence, HP1-1M nucleotide sequence, HP1-2M nucleotide sequence, and control vector _DBN11770N ) Ti seeds were dried at room temperature for 7 days. Seeds were sown in 26.5 cm x 51 cm germination trays, each tray receiving 200 mgT ₁ seeds (approximately 10,000 seeds) that had been previously suspended in distilled water and kept at 4°C for 2 days to complete the need for dormancy to Ensure that the seeds germinate synchronously.

The horse manure was mixed with vermiculite and bottom irrigated with water until moist, draining by gravity. Pipette the pretreated seeds evenly over the soil mix and cover with a moisturiser for 4-5 days. Hoods were removed 1 day prior to initial transformant selection with post-emergence spray glufosinate-ammonium (to select for co-transformed PAT genes). T ₁ was sprayed with a 0.2% solution of Liberty herbicide (200 gai/L of glufosinate) at a spray volume of 10 mL/pan (703 L/ha) after 7 days of planting (DAP) and again at 11 DAP using DeVilbiss compressed air nozzles Plants (cotyledon stage and 2-4 leaf stage, respectively) to provide an effective amount of glufosinate ammonium per application of 280 gai/ha. Survivors (active plants) were identified 4-7 days after the last spray and transplanted into 7 cm x 7 cm square pots (3-5 per tray) prepared with horse manure and vermiculite, respectively. Transplanted plants were covered with a moisturizing hood for 3-4 days and placed in a 22°C culture room as before or moved directly into a greenhouse. The hood was then removed and the plants were planted in a greenhouse (temperature 22±5°C, 50±5°C) at least 1 day prior to testing the HP1M, HP1-1M and HP1-2M genes for their ability to confer tolerance to the herbicide fenflufenac 30% RH, 14h light: 10h dark, min 500μE/m ² s ^-1 natural + supplemental light).

4. Detection of herbicide tolerance of transgenic Arabidopsis plants

T1 transformants were _first selected from an untransformed seed background using the glufosinate selection protocol. The recombinant expression vector DBN11770 was transformed into an Arabidopsis plant (HP1) with the nucleotide sequence of HP1M, and the recombinant expression vector DBN11777 was transformed into an Arabidopsis plant (HP1-1) with the nucleotide sequence of HP1-1M, The Arabidopsis plant (HP1-2) transformed with the HP1-2M nucleotide sequence was transformed into the recombinant expression vector DBN11778, and the Arabidopsis plant transformed into the control recombinant expression vector (control vector) was transformed into the recombinant expression vector DBN11770N. About 20,000 HP1 T1 seeds _were screened, and 213 T1 generation positive transformants (PAT genes) were identified, with _a transformation efficiency of about 1.0%; about 20000 _HP1-1 T1 seeds were screened and identified 195 T ₁ generation positive transformants (PAT gene), about 1.0% transformation efficiency; screened about 20,000 HP1-2 T ₁ seeds, and identified 195 T ₁ generation positive transformants (PAT gene), about 1.0% transformation efficiency; about 20,000 T ₁ seeds of the control vector were screened, and 172 T ₁ generation positive transformants (PAT genes) were identified, with a transformation efficiency of about 0.86%.

The HP1 Arabidopsis T1 plants, _HP1-1 Arabidopsis T1 plants, _HP1-2 Arabidopsis T1 plants, Arabidopsis T1 plants _of the control vector and wild _- type Arabidopsis plants (CK) ( The herbicide tolerance of Arabidopsis thaliana was tested by spraying 18 days after sowing with 3 concentrations of fenflufen, namely 25 gai/ha (1x field concentration, 1×), 100 g ai/ha (4 Double field concentration, 4×) and 0 g ai/ha (water, 0×). After 7 days of spraying (7DAT), the damage degree of each plant by herbicides was calculated according to the proportion of leaf whitening area (the proportion of leaf whitening area = leaf whitening area/total leaf area × 100%): basically no whitening phenotype It is grade 0, the proportion of leaf whitening area is less than 50%, it is grade 1, the proportion of leaf whitening area is more than 50%, it is grade 2, and the proportion of leaf whitening area is 100%, it is grade 3. The 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-the number of injured plants at the same level, S-the number of phytotoxicity grades, T-the total number of plants, M-the highest phytotoxicity grade), resistance evaluation according to the score: highly 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 experimental results are shown in Table 2.

Table 2. Experimental results of the tolerance of transgenic Arabidopsis T ₁ plants to fenflufen

The results in Table 2 show that: compared with the Arabidopsis T1 plants and wild _- type Arabidopsis plants transformed into the control vector DBN11770N, the Arabidopsis T1 plants transformed with the _HP1M nucleotide sequence, transformed into the HP1-1M nucleus Both the Arabidopsis T _{1 plant with the nucleotide sequence and the Arabidopsis T 1 plant} transformed with the HP1-2M nucleotide sequence were able to produce different degrees of tolerance to toflufenazone; Resistance of Arabidopsis T1 plants with acid sequence, Arabidopsis T1 plants transformed with _HP1-1M nucleotide sequence, and Arabidopsis T1 plants transformed with _HP1-2M nucleotide sequence to _oxaflutole better.

To sum up, the amino acid sequence of the herbicide tolerance protein HP1 of the present invention is mutated from threonine to alanine at position 159 or from glutamine to methionine at position 258 (the herbicide Tolerance proteins HP1-1 or HP1-2) can show higher tolerance to HPPD inhibitor herbicides, and the above herbicide tolerance proteins can tolerate 1 times the field concentration of fenflufenac (medium resistance), so it has broad application prospects in plants.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be The solutions can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

sequence listing

<110> Beijing Dabeinong Biotechnology Co., Ltd.

Nanjing Agricultural College

<120> Herbicide tolerance protein, its encoding gene and use

<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

gccgggggagg 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

gccgggggagg 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

gccgggggagg 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'-end universal adapter primer 1 (Artificial Sequence)

<400> 12

taagaaggag atatacatat g 21

<210> 13

<211> 21

<212> DNA

<213> 3' Universal Adapter Primer 1 (Artificial Sequence)

<400> 13

gtggtggtgg tggtgctcga g 21

<210> 14

<211> 24

<212> DNA

<213> 5'-end universal adapter primer 2 (Artificial Sequence)

<400> 14

agtttttctg attaacagac tagt 24

<210> 15

<211> 25

<212> DNA

<213> 3' Universal Adapter Primer 2 (Artificial Sequence)

<400> 15

caaatgtttg aacgatcggc gcgcc 25

<210> 16

<211> 542

<212> DNA

<213> 34S enhancer of Scrophulariaceae 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> Rape eukaryotic elongation factor gene 1α (Tsf1 promoter Brassica napus)

<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 ttttattgttt 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 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-enolpyruvate shikimate-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 (promoter of Ubiquitin10 gene Arabidopsis thaliana)

<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 ctagttttgtg cgatcgaatt tgtcgattaa tctgagtttt tctgattaac 1320

ag 1322

<210> 22

<211> 253

<212> DNA

<213> Nopaline synthase gene terminator (Agrobacterium tumefaciens)

<400> 22

gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60

atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120

atgacgttat ttatgagatg ggttttttatg 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

<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> Streptomycesviridochromogenes Gene Nucleotide Sequence (Streptomycesviridochromogenes)

<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

<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 (14)

1. a protein, is characterized in that, comprises: (a) has the amino acid sequence shown in SEQ ID NO: 9; (b) A protein derived from (a) having the amino acid sequence in (a) substituted and/or deleted and/or one or several amino acids added and having hydroxyphenylpyruvate dioxygenase activity. 2. A gene, characterized in that, comprising: (a) a nucleotide sequence encoding the protein of claim 1; or (b) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence defined in (a) and encodes a protein having hydroxyphenylpyruvate dioxygenase activity; or (c) having the nucleotide sequence shown in SEQ ID NO:10 or SEQ ID NO:11. 3. An expression cassette, characterized in that it comprises 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 for expanding the range of herbicides tolerated by plants, comprising: changing the protein of claim 1 or the protein encoded by the expression cassette of claim 3 in a plant from at least one different from claim The protein of 1 or the protein encoded by the expression cassette of claim 3 is expressed together with a second herbicide tolerance protein. 6. The method according to claim 5, wherein the second herbicide tolerance protein is 5-enolpyruvylshikimate-3-phosphate synthase, Glyphosate oxidoreductase, glyphosate-N-acetyltransferase, glyphosate decarboxylase, glufosinate acetyltransferase, alpha-ketoglutarate-dependent dioxygenase, dicamba monooxygenase, acetyl Lactate synthase, cytochrome-like proteins and/or protoporphyrinogen oxidase. 7. a method for selecting the plant cell of transformation, is characterized in that, comprises: transform a plurality of plant cells with the described gene of claim 2 or the described expression cassette of claim 3, and allow to express described gene or described expression Transformed cells of the cassette are grown while culturing the cells at a concentration of an HPPD inhibitor herbicide that kills untransformed cells or inhibits the growth of untransformed cells; Preferably, the plants include monocotyledonous and dicotyledonous plants; more preferably, the plants are oat, wheat, barley, millet, corn, sorghum, Brassica, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis; Preferably, the HPPD inhibitor herbicides include pyrazolone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile. 8. A method for controlling weeds, comprising: applying an effective dose of HPPD inhibitor herbicide to a field planting a plant of interest, wherein the plant of interest comprises the gene of claim 2 or the expression of claim 3 Cassette or the described recombinant vector of claim 4; Preferably, the target plants include monocotyledonous plants and dicotyledonous plants; more preferably, the target plants are oat, wheat, barley, millet, corn, sorghum, Brassica oleracea, rice, tobacco, sunflower, alfalfa , soybean, chickpea, peanut, sugar beet, cucumber, cotton, rapeseed, potato, tomato or Arabidopsis; further preferably, the target plant is a glyphosate-tolerant plant, and the weed is glyphosate resistant weeds; Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile. 9. A method for protecting plants from damage caused by HPPD inhibitor herbicides or imparting HPPD inhibitor herbicide tolerance to plants, comprising: adding the gene of claim 2 or claim 3 The expression cassette or the recombinant vector of claim 4 is introduced into a plant, so that the introduced plant produces a herbicide tolerance protein in an amount sufficient to protect it from damage by an HPPD inhibitor herbicide; Preferably, the plants include monocotyledonous and dicotyledonous plants; more preferably, the plants are oat, wheat, barley, millet, corn, sorghum, Brassica, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis; Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile. 10. A method for producing a plant resistant to an HPPD inhibitor herbicide, comprising introducing the gene of claim 2 into the genome of the plant; Preferably, the introduced method includes genetic transformation method, genome editing method or gene mutation method; Preferably, the plants include monocotyledonous and dicotyledonous plants; more preferably, the plants are oat, wheat, barley, millet, corn, sorghum, Brassica, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis; Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile. 11. A method for culturing plants tolerant to HPPD inhibitor herbicides, comprising: Planting at least one plant propagule comprising the gene of claim 2 or the expression cassette of claim 3 in the genome of the plant propagule; growing the plant propagules into plants; Applying an effective dose of the HPPD inhibitor herbicide to a plant growth environment comprising at least the plant, the harvesting has reduced plant damage compared with other plants not having the gene of claim 2 or the expression cassette of claim 3 and/or plants with increased plant yield; Preferably, the plants include monocotyledonous and dicotyledonous plants; more preferably, the plants are oat, wheat, barley, millet, corn, sorghum, Brassica, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis; Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile. 12. A method for obtaining processed agricultural products, comprising treating the harvested plants of HPPD inhibitor herbicide-tolerant plants obtained by the method of claim 11 to obtain processed agricultural products. 13. A planting system for controlling the growth of weeds, characterized in that it comprises HPPD inhibitor herbicide and a plant growth environment in which at least one target plant is present, said target plant comprising the gene of claim 2 or claim 3 the expression cassette; Preferably, the target plants include monocotyledonous plants and dicotyledonous plants; more preferably, the target plants are oat, wheat, barley, millet, corn, sorghum, Brassica oleracea, rice, tobacco, sunflower, alfalfa , soybean, chickpea, peanut, sugar beet, cucumber, cotton, rape, potato, tomato or Arabidopsis; further preferably, the target plant is a glyphosate-tolerant plant, and the weed is glyphosate resistant weeds; Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile. 14. A use of the protein of claim 1 in conferring tolerance to a plant HPPD inhibitor herbicide; Preferably, the plants include monocotyledonous and dicotyledonous plants; more preferably, the plants are oat, wheat, barley, millet, corn, sorghum, Brassica, rice, tobacco, sunflower, alfalfa, soybean , chickpeas, peanuts, beets, cucumbers, cotton, canola, potatoes, tomatoes or Arabidopsis; Preferably, the HPPD inhibitor herbicides include pyrazolinone HPPD inhibitor herbicides, triketone HPPD inhibitor herbicides and/or isoxazole HPPD inhibitor herbicides; more preferably, the HPPD inhibitor herbicides Inhibitor herbicides are fenflufen, mesotrione and/or diketonitrile.

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