WO2022127894A1

WO2022127894A1 - Herbicide-resistant plant

Info

Publication number: WO2022127894A1
Application number: PCT/CN2021/139052
Authority: WO
Inventors: 高彩霞; 陈宇航; 张瑞; 王梅花; 陈沙; 张纯瑞
Original assignee: 上海蓝十字医学科学研究所
Priority date: 2020-12-17
Filing date: 2021-12-17
Publication date: 2022-06-23
Also published as: CN116724119A; AR124409A1

Abstract

Disclosed is a p-hydroxyphenylpyruvate dioxygenase (HPPD) mutant that can confer herbicide resistance in a plant, and a method for producing a plant comprising the p-hydroxyphenylpyruvate dioxygenase (HPPD) mutant.

Description

herbicide resistant plants

technical field

The present invention relates to the field of plant genetic engineering. In particular, the present invention relates to mutants of p-hydroxyphenylpyruvate dioxidase (HPPD) capable of conferring herbicide resistance in plants, and production of mutants comprising said p-hydroxyphenylpyruvate dioxidase (HPPD) Methods of herbicide-resistant plants in vivo.

Background of the Invention

A major threat to the production of crops such as rice is weed competition, which can lead to lower grain yields and poor quality. Although tillage can be used to eliminate weeds, the soil of cultivated land is susceptible to erosion by wind and water. Due to ease of application and effectiveness, herbicide treatments are the preferred method of weed control. Herbicides can also provide weed control in direct cropping systems that reduce arable land or are designed to leave high levels of residue on the soil surface to prevent erosion.

The development of herbicide resistance in plants provides important production and economic advantages; therefore, the use of herbicides to control weeds or undesired plants in crops has become almost universal practice. However, the use of such herbicides can also result in the death or reduced growth of the desired crop plants, making the timing and method of herbicide application critical or in some cases not feasible at all. One way to address this is to develop herbicide-resistant varieties. In this method, the herbicide is applied to the crop to control weeds without causing damage to the herbicide-resistant crop.

Of particular interest to farmers is the use of herbicides with greater potency, broad weed spectrum effectiveness and rapid soil degradation. Plants, plant tissues and seeds that are resistant to these compounds offer an attractive solution by allowing the use of herbicides to control weed growth without the risk of crop damage. One class of broad-spectrum herbicides are those compounds that inhibit the activity of p-hydroxyphenylpyruvate dioxidase (HPPD) in plants. However, many crops, such as rice, are susceptible to many HPPD-inhibiting herbicides targeting monocots, making the use of these herbicides to control grass weeds nearly impossible.

Therefore, there remains a need in the art to develop crop plants, such as rice, that are resistant to HPPD-inhibiting herbicides.

Brief description of the invention

This application includes at least the following embodiments:

Embodiment 1. An HPPD mutant or functional fragment thereof having an amino acid mutation such as an amino acid substitution at one or more positions selected from positions 365, 378, 414, 415, 417 or 419 relative to wild-type HPPD , the amino acid position is with reference to SEQ ID NO: 1.

Embodiment 2. The HPPD mutant according to embodiment 1, or a functional fragment thereof, having an amino acid mutation, such as an amino acid substitution, relative to wild-type HPPD, at a position selected from the group consisting of amino acid positions with reference to SEQ ID NO: 1:

i) positions 378 and 415;

ii) positions 378 and 417;

iii) positions 414 and 417; or

iv) Positions 378, 415 and 417.

Embodiment 3. The HPPD mutant or functional fragment thereof according to

embodiment

1 or 2, said wild-type HPPD comprising the amino acid sequence of one of SEQ ID NOs: 1-13.

Embodiment 4. The HPPD mutant or functional fragment thereof according to any one of embodiments 1 to 3, which, when expressed in a plant, is capable of conferring resistance to a herbicide to the plant sex.

Embodiment 5. The HPPD mutant or functional fragment thereof according to Embodiment 4, wherein the herbicide is selected from the group consisting of pyrazole compounds, such as fenflufen, sulfapyr, and fenazopyramide; three Ketones such as sulcotrione, mesotrione, terbotrione, furfuryl ketone, bicyclopyrone, benzobicyclone; isoxazoles such as isoxaflutole; diketonitrile compounds such as 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-4-trifluoromethylphenyl)-propane-1,3-dione and 2-cyano-1 -[4-(Methylsulfonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-dione; and benzophenone compounds, or them any combination of .

Embodiment 6. The HPPD mutant or functional fragment thereof according to any one of embodiments 1-5, wherein relative to wild-type HPPD, the HPPD mutant or functional fragment thereof comprises the group selected from L365K, F378A, G414A, One or more amino acid substitutions of said amino acids G414V, G415A, G415V, G417S, G417A, G417K, G419W, G417V, G417R, G417N, G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T, The position is referenced to SEQ ID NO:1.

Embodiment 7. The HPPD mutant or functional fragment thereof according to embodiment 6, wherein relative to wild-type HPPD, the HPPD mutant or functional fragment thereof comprises an amino acid substitution selected from the group consisting of

i) F378A and G415A;

ii) F378A and G415V;

iii) F378A and G417K;

iv) F378A and G417R;

v) F378A and G417V;

vi) F378A and G417A;

vii) G414V and G417K;

viii) F378A, G415A and G417A; or

ix) F378A, G415V and G417K;

wherein the amino acid positions are referenced to SEQ ID NO: 1.

Embodiment 8. The HPPD mutant or functional fragment thereof according to any one of embodiments 1-7, said HPPD mutant comprising the amino acid sequence shown in any one of SEQ ID NOs: 14-55.

Embodiment 9. A nucleic acid comprising a nucleotide sequence encoding the HPPD mutant of any one of embodiments 1-8, or a functional fragment thereof.

Embodiment 10. An expression cassette comprising a nucleotide sequence encoding the HPPD mutant of any one of embodiments 1-8, or a functional fragment thereof, operably linked to a regulatory sequence.

Embodiment 11. An expression construct comprising the expression cassette of embodiment 10.

Embodiment 12. A method of transgenic production of a herbicide-resistant plant comprising introducing the nucleic acid of embodiment 9, the expression cassette of embodiment 10, and/or the expression construct of embodiment 11 into said plant.

Embodiment 13. A method of producing a herbicide-resistant plant, the method comprising targeted modification of an endogenous HPPD coding sequence of a plant, thereby causing the endogenous HPPD to be selected from the group consisting of: 365, 378, 414, 415, Amino acid mutations at one or more of positions 417 or 419 referenced to SEQ ID NO: 1.

Embodiment 14. The method according to embodiment 13, wherein the targeted modification results in the endogenous HPPD comprising the group consisting of L365K, F378A, G414A, G414V, G415A, G415V, G417S, G417A, G417K, G419W, F378A/G417A, One or more amino acid substitutions of G417V, G417R, G417N, G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T, the amino acid positions referenced to SEQ ID NO:11.

Embodiment 15. The method according to embodiment 13 or 14, wherein the targeted modification results in the HPPD mutant of any one of embodiments 1-8.

Embodiment 16. The method according to any one of embodiments 13-15, wherein the coding sequence of the endogenous HPPD is targetedly modified by gene editing or homologous recombination.

Embodiment 17. The method according to embodiment 16, wherein the gene editing is base editing or prime editing.

Embodiment 18. A method of producing herbicide-resistant plants, comprising subjecting a population of said plants to physical or chemical mutagenesis, and screening for endogenous HPPD at least selected from the group consisting of 365, 378, 414, 415, 417 Or a plant comprising amino acid mutations at one or more positions, such as 1, 2, 3, 4, 5 or 6 positions at position 419, wherein the amino acid positions are referenced to SEQ ID NO: 1.

Embodiment 19. The method according to embodiment 18, wherein screening for endogenous HPPD comprises at least selected from L365K, F378A, G414A, G414V, G415A, G415V, G417S, G417A, G417K, G419W, F378A/G417A, G417V, G417R, G417N, Plants with one or more amino acid substitutions of G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T, the amino acid positions referenced to SEQ ID NO:1.

Embodiment 20. The method according to embodiment 18 or 19, wherein a plant is screened for comprising or expressing the HPPD mutant of any one of embodiments 1-8.

Embodiment 21. The method according to any one of embodiments 18-20, wherein the physical mutagenesis comprises by irradiation of the plant population and the chemical mutagenesis comprises by treatment with ethyl methanesulfonate (EMS) the plant population.

Embodiment 22. The method according to any one of embodiments 12-21, wherein the plant comprises a monocotyledonous or dicotyledonous plant, preferably the plant is a crop plant, such as a monocotyledonous crop plant.

Embodiment 23. The method according to embodiment 22, wherein the plant is selected from the group consisting of rice, wheat, barley, sorghum, maize, oat, Arabidopsis, ryegrass, brome, wild soybean, soybean, and tobacco.

Embodiment 24. A herbicide-resistant plant or progeny thereof comprising or expressing the HPPD mutant or functional fragment thereof of any one of embodiments 1-8, or a passage thereof by any of embodiments 12-21 A method is generated.

Embodiment 25. The herbicide resistant plant or progeny thereof according to embodiment 24, wherein the plant comprises a monocotyledonous or dicotyledonous plant, preferably the plant is a crop plant, eg a monocotyledonous crop plant.

Embodiment 26. The herbicide-resistant plant or progeny thereof according to embodiment 25, wherein the plant is selected from the group consisting of rice, wheat, barley, sorghum, maize, oat, Arabidopsis, ryegrass, land brome, wild soybean , soy and tobacco.

Description of drawings

Figures 1-6 show mesotrione herbicide resistance mutants tested in in vitro biochemical experiments. Compared with the wild type, the herbicide-resistant mutants in the figure are L365K, F378A, G419W, G417A, G417A/F378A, G417S, G417K, G415A, G415V, G414A, G414V, G417V, G417R, G417N, G417D, G417C , G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T. The first picture 1 shows that these mutants are all enzymatically active, which means that they can catalyze the substrate (HPPA) to generate products, and have the most basic enzymatic activities for plant growth. Figures 2 to 6 show that each mutant has herbicide resistance activity compared with the wild type, the herbicide used is mesotrione, the abscissa is the concentration gradient of mesotrione, and the ordinate is the measured light absorption value calculation The velocity (V) of the resulting product.

Figure 1. Kinetic parameters of enzyme reactions for each mutant.

Figure 2. Mutants L365K and F378A are more herbicide resistant than wild type.

Figure 3. Mutants G419W and G417A are more herbicide resistant than wild type.

Figure 4. Mutants G417A/F378A and G417S are more herbicide resistant than wild type.

Figure 5. Mutants G417K, G415A, G415V, G414A, G414V are more herbicide resistant than wild type.

Figure 6 Mutants G417V, G417R, G417N, G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T are more herbicide resistant than wild type.

Figure 7 shows double and triple resistance of rice HPPD high resistance loci. The Y-axis on the left represents the enzymatic activity, and the Y-axis on the right represents the inhibition constant, and the higher the inhibition constant Ki, the stronger the resistance.

Figure 8 shows the kinetic constants and inhibition constants (resistance) of the enzymatic reaction of HPPD mutants derived from corn and soybean. The corresponding rice-derived HPPD loci are: F378A/G417A, G414A, G417K, G417Q, G417R.

Figure 9 shows a schematic diagram of the overexpression vector of rice HPPD protein.

Figure 10 shows the screened phenotypes of rice expressing different resistance mutations when the relative expression levels of HPPD-mRNA are known.

Figure 11 shows that tobacco expressing the G417K mutation has stronger drug resistance when the relative mRNA expression levels are similar.

SUMMARY OF THE INVENTION

1. Definition

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory procedures used herein are the terms and routine procedures widely used in the corresponding fields. For example, standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those of skill in the art and are more fully described in Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook"). Meanwhile, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "and/or" covers all combinations of the items linked by the term, as if each combination had been individually listed herein. For example, "A and/or B" covers "A", "A and B", and "B". For example, "A, B and/or C" encompasses "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

When the word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence or may have additional amino acids or nuclei at one or both ends of the protein or nucleic acid Glycosides, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the initiation codon at the N-terminus of the polypeptide is retained in some practical situations (eg, when expressed in a specific expression system), but does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain the methionine encoded by the initiation codon at the N-terminus, the methionine containing the methionine is also covered at this time. The sequence, correspondingly, its encoding nucleotide sequence may also contain an initiation codon; and vice versa.

"Foreign" with respect to a sequence means a sequence from a foreign species, or, if from the same species, a sequence that has been significantly altered in composition and/or locus from its native form by deliberate human intervention.

"Polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid" are used interchangeably and are polymers of single- or double-stranded RNA or DNA, optionally containing synthetic, non-natural or Altered nucleotide bases. Nucleotides are referred to by their single-letter names as follows: "A" for adenosine or deoxyadenosine (for RNA or DNA, respectively), "C" for cytidine or deoxycytidine, "G" for guanosine or Deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, " H" means A or C or T, "D" means A, T or G, "I" means inosine, and "N" means any nucleotide.

Codon optimization refers to replacing at least one codon of a native sequence (eg, about or more than about 1, 2, 3, 4, 5, 10) with a codon that is more or most frequently used in a gene in a host cell. , 15, 20, 25, 50 or more codons while maintaining the native amino acid sequence and modifying the nucleic acid sequence to enhance expression in a host cell of interest. Different species display certain codons for specific amino acids Codon bias (differences in codon usage between organisms) is often related to the translation efficiency of messenger RNA (mRNA), which is thought to depend on the nature and nature of the codons being translated. Availability of a specific transfer RNA (tRNA) molecule. The predominance of a selected tRNA within a cell generally reflects the codons most frequently used for peptide synthesis. Thus, genes can be tailored based on codon optimization for the most efficient use in a given organism Optimal gene expression. Codon utilization tables are readily available, for example in the codon usage database ("Codon Usage Database") available at www.kazusa.orjp/codon/, and these tables can be adjusted in different ways Applicable. See, Nakamura Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

"Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modified forms including, but not limited to, glycosylation, lipid linkage, sulfation, gamma carboxylation of glutamic acid residues, hydroxyl sylation and ADP-ribosylation.

Sequence "identity" has an art-recognized meaning, and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along regions of the molecule. (See, e.g.: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While many methods exist for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled artisan (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).

In peptides or proteins, suitable conservative amino acid substitutions are known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. In general, those skilled in the art recognize that single amino acid substitutions in non-essential regions of polypeptides do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub .co., p.224).

As used in the present invention, "expression construct" refers to a vector such as a recombinant vector suitable for expression of a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, expression of a nucleotide sequence can refer to transcription of the nucleotide sequence (eg, transcription to produce mRNA or functional RNA) and/or translation of RNA into a precursor or mature protein.

The "expression construct" of the present invention may be a linear nucleic acid fragment, circular plasmid, viral vector, or, in some embodiments, may be an RNA (eg, mRNA) capable of translation, eg, RNA produced by in vitro transcription.

An "expression construct" of the present invention may comprise regulatory sequences and nucleotide sequences of interest from different sources, or regulatory sequences and nucleotide sequences of interest from the same source but arranged in a manner different from that normally found in nature.

"Regulatory sequence" and "regulatory element" are used interchangeably and refer to a coding sequence upstream (5' non-coding sequence), intermediate or downstream (3' non-coding sequence) and affecting transcription, RNA processing or Stability or translated nucleotide sequence. Regulatory sequences can include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

"Promoter" refers to a nucleic acid segment capable of controlling the transcription of another nucleic acid segment. In some embodiments of the invention, a promoter is a promoter capable of controlling the transcription of a gene in a cell, whether or not it is derived from the cell. The promoter may be a constitutive promoter or a tissue-specific promoter or a developmentally regulated promoter or an inducible promoter.

A "constitutive promoter" refers to a promoter that will generally cause a gene to be expressed in most cell types under most circumstances. "Tissue-specific promoter" and "tissue-preferred promoter" are used interchangeably and refer to expression primarily, but not necessarily exclusively, in a tissue or organ, but also in a particular cell or cell type promoter. "Developmentally regulated promoter" refers to a promoter whose activity is determined by developmental events. An "inducible promoter" selectively expresses an operably linked DNA sequence in response to endogenous or exogenous stimuli (environmental, hormonal, chemical signals, etc.).

Examples of promoters include, but are not limited to, polymerase (pol) I, pol II, or pol III promoters. When used in plants, the promoter may be the cauliflower mosaic virus 35S promoter, the maize Ubi-1 promoter, the wheat U6 promoter, the rice U3 promoter, the maize U3 promoter, the rice actin promoter.

As used herein, the term "operably linked" refers to regulatory elements (eg, but not limited to, promoter sequences, transcription termination sequences, etc.) are linked to a nucleic acid sequence (eg, a coding sequence or open reading frame) such that nucleotides Transcription of the sequence is controlled and regulated by the transcriptional regulatory elements. Techniques for operably linking regions of regulatory elements to nucleic acid molecules are known in the art.

"Introducing" a nucleic acid molecule (eg, plasmid, linear nucleic acid fragment, RNA, etc.) or protein into an organism refers to transforming the cells of the organism with the nucleic acid or protein so that the nucleic acid or protein can function in the cell. "Transformation" as used in the present invention includes stable transformation and transient transformation. "Stable transformation" refers to the introduction of a foreign nucleotide sequence into the genome, resulting in the stable inheritance of the foreign gene. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof. "Transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell to perform a function without the stable inheritance of an exogenous gene. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.

As used herein, the term "plant" includes whole plants and any progeny, cells, tissues, or parts of plants. The term "plant part" includes any part of a plant, including, for example, but not limited to: seeds (including mature seeds, immature embryos without seed coats, and immature seeds); plant cuttings; plant cells; Plant cell cultures; plant organs (eg, pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and related explants). A plant tissue or plant organ can be a seed, callus, or any other population of plant cells organized into structural or functional units. A plant cell or tissue culture is capable of regenerating plants having the physiological and morphological characteristics of the plant from which the cell or tissue is derived, and capable of regenerating plants having substantially the same genotype as the plant. In contrast, some plant cells are unable to regenerate to produce plants. The regenerable cells in plant cells or tissue culture can be embryos, protoplasts, meristems, callus, pollen, leaves, anthers, roots, root tips, silks, flowers, nuts, ears, cobs, shells , or stem.

Plant "progeny" includes any subsequent generation of the plant.

2. Mutants of p-hydroxyphenylpyruvate dioxidase (HPPD) conferring herbicide resistance

P-hydroxyphenylpyruvate dioxidase (HPPD) is involved in two metabolic pathways, tyrosine catabolism and plastoquinone and tocopherol biosynthesis. p-Hydroxyphenylpyruvate is converted to homogentisate under the catalysis of HPPD enzyme. Urogentine is further decarboxylated and alkylated to generate plastoquinone and tocopherol. Plastoquinone acts as the final electron acceptor and electron transporter in the photosynthetic chain in carotenoid biosynthesis, and the lack of plastoquinone in thylakoid will lead to reduced carotenoid biosynthesis. p-Hydroxyphenylpyruvate dioxidase (HPPD) inhibitor has broad-spectrum herbicidal activity and can control broad-leaved weeds in broad-leaved crops. It can be used before or after emergence. It has high activity, Low residue, good environmental compatibility and safe use.

HPPD-inhibiting herbicides include, but are not limited to, pyrazoles, triketones, isoxazoles, diketonitriles, and benzophenones, or any combination thereof. Suitable pyrazole compounds include, but are not limited to, fenflufenazone (Baowei), sulfofenapyr, acid fenazopyramide, and the like. Suitable triketone compounds include, but are not limited to, sulcotrione, mesotrione, terbotrione, tefurfuryl ketone, bicyclopyrone, benzobicyclone, and the like. Suitable isoxazoles include, but are not limited to, isoxaflutole. Suitable diketonitriles include, but are not limited to, 2-cyano-3-cyclopropyl-1-(2-methylsulfonyl-4-trifluoromethylphenyl)-propane-1,3-dione and 2-cyano-1-[4-(methylsulfonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-dione.

In the present invention, the inventors created and identified novel HPPD mutants resistant to HPPD-inhibiting herbicides by prokaryotic expression and enzyme activity analysis techniques.

Therefore, in one aspect, the present invention provides a p-hydroxyphenylpyruvate dioxidase (HPPD) mutant or a functional fragment thereof, which is at least selected from the group consisting of 365, 378, 414, 415 relative to the wild-type HPPD. There are amino acid mutations at one or more of positions , 417 or 419 with reference to SEQ ID NO: 1.

In some embodiments, the HPPD mutant or functional fragment thereof, relative to wild-type HPPD, has at least 1, 2, 3, There were amino acid mutations at

positions

5 or 6, referenced to SEQ ID NO: 1.

In some embodiments, the HPPD mutant or functional fragment thereof has amino acid mutations at least at positions 378 and 415 relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant, or a functional fragment thereof, has amino acid mutations at least at positions 378 and 417 relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant, or a functional fragment thereof, has amino acid mutations at least at positions 414 and 417 relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof has amino acid mutations at least at positions 378, 415 and 417 relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO: 1.

As used herein, "an amino acid position with reference to SEQ ID NO:x" (SEQ ID NO:x being a specific sequence listed herein) refers to the position number of the specific amino acid being described that is the amino acid in SEQ ID NO:x The position number of the corresponding amino acid above. The correspondence of amino acids in different sequences can be determined according to sequence alignment methods well known in the art. For example amino acid correspondences can be determined by the online alignment tool of EMBL-EBI (https://www.ebi.ac.uk/Tools/psa/), where two sequences can be determined using the Needleman-Wunsch algorithm using default parameters to Align. For example, the alanine at position 360 of a polypeptide from its N-terminus is aligned with the amino acid at position 365 of SEQ ID NO:x in a sequence alignment, then the alanine in the polypeptide may also be used herein. Described as "Alanine at position 365 of the polypeptide, the amino acid position referenced to SEQ ID NO:x". For another example, the glycine at position 412 in the real position of the amino acid sequence SEQ ID NO: 9 of the present invention is aligned with the glycine at position 417 in SEQ ID NO: 1 in the sequence alignment, then the glycine in SEQ ID NO: 9 is aligned. The glycine may also be described herein as "the glycine at position 417 of SEQ ID NO:9, the amino acid position referenced to SEQ ID NO:1".

In some embodiments, the HPPD mutant or functional fragment thereof, when expressed in a plant, is capable of conferring resistance to a herbicide, eg, an HPPD-inhibiting herbicide, to the plant. "Conferring resistance to a herbicide, such as an HPPD-inhibiting herbicide, to the plant" refers to the resistance of a plant comprising or expressing the HPPD mutant or functional fragment thereof to a herbicide, such as an HPPD-inhibiting herbicide, relative to Plants that do not contain or express the HPPD mutant or a functional fragment thereof or only contain or express (a comparable amount) wild-type HPPD are enhanced, e.g. by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or higher. Methods for determining resistance to HPPD-inhibiting herbicides are known in the art. Resistance can be readily determined by one skilled in the art for a specific plant and specific herbicide. In some embodiments, the herbicide-resistant plants of the present invention are capable of at least 1 μmol/L, at least 1.5 μmol/L, at least 1.8 μmol/L, at least 2 μmol/L, at least 3 μmol/L, at least 5 μmol/L, at least 10 mol Normal growth was exhibited in the presence of herbicides (eg, HPPD-inhibiting herbicides such as mesotrione) at concentrations of /L or higher.

Resistance of HPPD mutants or functional fragments thereof of the present invention to HPPD-inhibiting herbicides can be determined in vitro by the enzymatic reaction kinetics method described herein, eg, see the method described in Example 4. The resistance of HPPD mutants or functional fragments thereof of the present invention to HPPD-inhibiting herbicides can be determined in vivo by detecting the growth status of plants comprising said mutants or functional fragments thereof in the presence of HPPD-inhibiting herbicides, See, for example, the method described in Example 5.

In some embodiments, the wild-type HPPD comprises the amino acid sequence of one of SEQ ID NOs: 1-13. In some embodiments, the amino acid sequence of the wild-type HPPD is, for example, one of SEQ ID NOs: 1-13. In some embodiments, the wild-type HPPD is, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% of the amino acid sequence of one of SEQ ID NOs: 1-13 , a naturally occurring variant of one of SEQ ID NOs: 1-13 having at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity. In some embodiments, the HPPD mutant is derived from rice wild-type HPPD. An exemplary rice wild-type HPPD comprises the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the HPPD mutant is derived from a wheat (Triticum aestivum) wild-type HPPD. Exemplary wheat wild-type HPPDs comprise the amino acid sequences set forth in SEQ ID NO: 2, 3 or 4. In some embodiments, the HPPD mutant is derived from Avena sativa wild-type HPPD. An exemplary oat wild-type HPPD comprises the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the HPPD mutant is derived from Pseudomonas fluorescens wild-type HPPD. An exemplary Pseudomonas fluorescens wild-type HPPD comprises the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the HPPD mutant is derived from Lolium rigidum wild-type HPPD. An exemplary ryegrass wild-type HPPD comprises the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the HPPD mutant is derived from Bromus tectorum wild-type HPPD. An exemplary land brome wild-type HPPD comprises the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the HPPD mutant is derived from Zea mays wild-type HPPD. An exemplary corn wild-type HPPD comprises the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the HPPD mutant is derived from a barley (Hordeum vulgare) wild-type HPPD. An exemplary barley wild-type HPPD comprises the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the HPPD mutant is derived from Glycine soja wild-type HPPD. An exemplary wild soybean wild-type HPPD comprises the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the HPPD mutant is derived from Arabidopsis thaliana wild-type HPPD. An exemplary Arabidopsis wild-type HPPD comprises the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the HPPD mutant is derived from soybean (Glycine max) wild-type HPPD. An exemplary soybean wild-type HPPD comprises the amino acid sequence set forth in SEQ ID NO:13.

In some embodiments, the amino acid mutation is an amino acid substitution.

In some embodiments, the HPPD mutant or functional fragment thereof is substituted at the leucine (L) at position 365 relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof is substituted at phenylalanine (F) at position 378 relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) substitution at position 414, relative to wild-type HPPD, referenced to SEQ ID NO: 1 for the amino acid position. In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) substitution at position 415, relative to wild-type HPPD, referenced to SEQ ID NO: 1 for the amino acid position. In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) substitution at position 417 relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof is substituted at glycine (G) at position 419, relative to wild-type HPPD, at the amino acid position referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof is substituted at phenylalanine (F) at position 378 and glycine (G) at position 417 relative to wild-type HPPD, the amino acid positions See SEQ ID NO:1. In some embodiments, the HPPD mutant or functional fragment thereof is substituted at phenylalanine (F) at position 378 and glycine (G) at position 415 relative to wild-type HPPD, the amino acid positions See SEQ ID NO:1. In some embodiments, the HPPD mutant or functional fragment thereof is substituted at glycine (G) at position 414 and glycine (G) at position 417 relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof has a phenylalanine (F) at position 378, a glycine (G) at position 415, and a glycine at position 417 relative to wild-type HPPD (G) is substituted, and the amino acid positions are referenced to SEQ ID NO: 1.

In some embodiments, the HPPD mutant or functional fragment thereof has a leucine (L) at position 365 replaced by a lysine (K) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO :1. In some embodiments, the HPPD mutant or functional fragment thereof is substituted with alanine (A) at position 378 of phenylalanine (F) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof is substituted with an alanine (A) at position 414 of glycine (G) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 3 . In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) at position 414 replaced by a valine (V) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 . In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) at position 415 replaced by an alanine (A) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 . In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) at position 415 replaced by a valine (V) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 . In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) at position 417 replaced by a serine (S) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) at position 417 replaced by an alanine (A) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 . In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) at position 417 replaced by a lysine (K) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 . In some embodiments, the HPPD mutant or functional fragment thereof is substituted with tryptophan (W) at glycine (G) at position 419, relative to wild-type HPPD, at the amino acid position referenced in SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof is replaced by an alanine (A) at a phenylalanine (F) at position 378 and a glycine (G) at position 417, respectively, relative to wild-type HPPD ) substitution, the amino acid positions are referred to SEQ ID NO: 13. In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) at position 417 replaced by a valine (V) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 . In some embodiments, the HPPD mutant or functional fragment thereof is substituted with an arginine (R) at position 417 of glycine (G) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 . In some embodiments, the HPPD mutant or functional fragment thereof is substituted with an asparagine (N) at position 417 of glycine (G) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 . In some embodiments, the HPPD mutant or functional fragment thereof is substituted with an aspartic acid (D) at position 417 of glycine (G) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) at position 417 replaced by a cysteine (C) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof is substituted with glutamine (Q) at position 417 of glycine (G) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 . In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) at position 417 replaced by a glutamic acid (E) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 . In some embodiments, the HPPD mutant or functional fragment thereof is substituted with a histidine (H) at position 417 of glycine (G) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 . In some embodiments, the HPPD mutant or functional fragment thereof is substituted with isoleucine (I) at position 417 of glycine (G) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) at position 417 replaced with a methionine (M) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) at position 417 replaced by a phenylalanine (F) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof has a glycine (G) at position 417 replaced by a proline (P) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 . In some embodiments, the HPPD mutant or functional fragment thereof is substituted with a glycine (G) at position 417 by a threonine (T) relative to wild-type HPPD, the amino acid position being referenced to SEQ ID NO: 1 .

In some embodiments, relative to wild-type HPPD, the HPPD mutant or functional fragment thereof comprises at least one selected from the group consisting of L365K, F378A, G414A, G414V, G415A, G415V, G417S, G417A, G417K, G419W, F378A/G417A, One or more amino acid substitutions of G417V, G417R, G417N, G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T, the amino acid positions referenced to SEQ ID NO:1.

In some embodiments, relative to wild-type HPPD, the HPPD mutant or functional fragment thereof comprises at least one selected from the group consisting of L365K, F378A, G414A, G414V, G415A, G415V, G417S, G417A, G417K, G419W, F378A/G417A, One amino acid substitution of G417V, G417R, G417N, G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T, the amino acid positions referenced to SEQ ID NO:1.

In some embodiments, the HPPD mutant or functional fragment thereof comprises at least the amino acid substitutions F378A and G415A relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof comprises at least the amino acid substitutions F378A and G415V relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof comprises at least the amino acid substitutions F378A and G417K relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO:1. In some embodiments, the HPPD mutant or functional fragment thereof comprises at least the amino acid substitutions F378A and G417R relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof comprises at least the amino acid substitutions F378A and G417V relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO: 1. In some embodiments, the HPPD mutant or functional fragment thereof comprises at least the amino acid substitutions F378A and G417A relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO:1. In some embodiments, the HPPD mutant or functional fragment thereof comprises at least the amino acid substitutions G414V and G417K relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO:1. In some embodiments, the HPPD mutant or functional fragment thereof comprises at least the amino acid substitutions F378A, G415A and G417A relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO:1. In some embodiments, the HPPD mutant or functional fragment thereof comprises at least the amino acid substitutions F378A, G415V and G417K relative to wild-type HPPD, the amino acid positions being referenced to SEQ ID NO:1.

As used herein, "functional fragment" refers to a fragment that retains at least partially or fully the function of the full-length HPPD mutant from which it is derived.

In some embodiments, the HPPD mutant or functional fragment thereof further comprises one or more additional amino acid mutations, eg, conservative amino acid substitutions, relative to wild-type HPPD. In addition, those skilled in the art will appreciate that small amino acid insertions, deletions or additions at the termini (C-terminus and/or N-terminus) of a protein generally do not significantly alter the function of the protein. For example, a tag, such as a histidine tag, can be added to the end of the protein to facilitate protein purification and/or detection.

In some embodiments, the HPPD mutant comprises at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of any of SEQ ID NOs: 14-55 %, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% amino acid sequences of sequence identity. In some embodiments, the HPPD mutant comprises the amino acid sequence set forth in any of SEQ ID NOs: 14-55.

In another aspect, the present invention also provides a nucleic acid comprising a nucleotide sequence encoding the HPPD mutant of the present invention or a functional fragment thereof. In some embodiments, the nucleic acid is an isolated nucleic acid or a recombinant nucleic acid.

In some embodiments, the nucleotide sequences encoding the HPPD mutants of the invention or functional fragments thereof may be codon-optimized for the plant of interest.

In another aspect, the present invention also provides an expression cassette comprising a nucleotide sequence encoding an HPPD mutant or a functional fragment thereof operably linked to a regulatory sequence.

In another aspect, the present invention also provides an expression construct comprising the expression cassette of the present invention.

In another aspect, the present invention also provides the use of an HPPD mutant of the present invention or a functional fragment thereof, an isolated nucleic acid of the present invention, an expression cassette of the present invention, or an expression construct of the present invention in the production of herbicide-resistant plants .

In another aspect, the present invention also provides a herbicide-resistant plant comprising or expressing the HPPD mutant of the present invention or a functional fragment thereof.

The plants described in various aspects of the invention may be plants susceptible to HPPD inhibitors, including monocotyledonous or dicotyledonous plants. Preferably, the plant is a crop plant, such as a monocotyledonous crop plant. Examples of suitable plants include, but are not limited to, rice, wheat, barley, sorghum, maize, oats, Arabidopsis, ryegrass, brome, wild soybean, soybean, or tobacco, and the like. In some preferred embodiments, the plant is rice. In other preferred embodiments, the plant is maize. In other preferred embodiments, the plant is soybean. In other preferred embodiments, the plant is tobacco.

3. Methods of producing herbicide-resistant plants by transgenic

In another aspect, the present invention also provides a method of transgenic production of herbicide-resistant plants, comprising introducing a nucleic acid of the present invention, an expression cassette of the present invention and/or an expression construct of the present invention into a plant. In some embodiments, introduction of the nucleic acid of the invention, the expression cassette of the invention and/or the expression construct of the invention results in the plant comprising or expressing the HPPD mutant of the invention or a functional fragment thereof.

The nucleic acid of the invention, the expression cassette of the invention and/or the expression construct of the invention can be introduced into the plant using various methods known in the art. Suitable methods of introduction include, but are not limited to, biolistic, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube passage, and ovary injection.

In some embodiments, the nucleic acid of the invention, the expression cassette of the invention and/or the expression construct of the invention are integrated into the genome of the plant. The isolated nucleic acids of the invention, the expression cassettes of the invention and/or the expression constructs of the invention will confer resistance to said plants to herbicides capable of inhibiting HPPD activity.

In another aspect, the present invention also provides a herbicide-resistant plant comprising, or consisting of, an expression cassette of the present invention, a nucleic acid or an expression construct of the present invention, or a nucleic acid of the present invention, an expression cassette of the present invention and/or The expression constructs of the present invention are transformed. The present invention also encompasses the progeny of said herbicide-resistant plants.

The plants described in various aspects of the invention may be plants susceptible to HPPD inhibitors, including monocotyledonous or dicotyledonous plants. Preferably, the plant is a crop plant, such as a monocotyledonous crop plant. Examples of suitable plants include, but are not limited to, rice, wheat, barley, sorghum, corn, oats, Arabidopsis, ryegrass, ryegrass, wild soybean, soybean, and tobacco, among others. In some preferred embodiments, the plant is rice. In other preferred embodiments, the plant is maize. In other preferred embodiments, the plant is soybean. In other preferred embodiments, the plant is tobacco.

4. Methods for generating herbicide-resistant plants by targeted modification of plant endogenous HPPD

Based on the HPPD mutants and corresponding mutation sites identified in the present invention that confer herbicide resistance, the plant endogenous HPPD can be engineered through targeted mutation, thereby generating herbicide-resistant plants.

Accordingly, in one aspect, the present invention also provides a method of producing a herbicide-resistant plant, the method comprising modifying, eg, targeted modification of an endogenous HPPD coding sequence of the plant, thereby resulting in the endogenous HPPD (eg, an expressed endogenous HPPD) amino acid mutation at one or more positions selected from the group consisting of positions 365, 378, 414, 415, 417 or 419, for example at

positions

1, 2, 3, 4, 5 or 6, the amino acid The position is referenced to SEQ ID NO:1.

The plant's endogenous HPPD, for example, comprises the amino acid sequence of one of SEQ ID NOs: 1-13 or is at least 80%, at least 85%, at least 90%, at least 80%, at least 85%, at least 90% identical to the amino acid sequence of one of SEQ ID NO: 1-13. A naturally occurring variant of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% sequence identity.

In some embodiments, the modification results in the substitution of a leucine (L) at position 365 and/or a phenylalanine at position 378 ( F) is substituted, and/or glycine (G) at position 414 is substituted, and/or glycine (G) at position 415 is substituted, and/or glycine (G) at position 417 is substituted, and/or Glycine (G) at position 419 is substituted, the amino acid position is referenced to SEQ ID NO: 1.

In some embodiments, the modification results in the replacement of a leucine (L) with a lysine (K) at position 365 of the endogenous HPPD (eg, expressed endogenous HPPD), and/or at position 378 The phenylalanine (F) is replaced by alanine (A), and/or the glycine (G) at position 414 is replaced by alanine (A) or valine (V), and/or at position 414 Glycine (G) at position 415 is replaced by alanine (A) or valine (V), and/or glycine (G) at position 417 is replaced by serine (S) or alanine (A) or lysine acid (K) or valine (V) or arginine (R) or asparagine (N) or aspartic acid (D) or cysteine (C) or glutamine (Q) or glutamine Amino acid (E) or histidine (H) or isoleucine (I) or methionine (M) or phenylalanine (F) or proline (P) or threonine (T) Substitution, and/or substitution of glycine (G) with tryptophan (W) at position 419, the amino acid position is referenced to SEQ ID NO: 1.

In some embodiments, the modification results in that the endogenous HPPD (eg, expressed endogenous HPPD) comprises a group selected from the group consisting of L365K, F378A, G414A, G414V, G415A, G415V, G417S, G417A, G417K, G419W, F378A/G417A, One or more amino acid substitutions of G417V, G417R, G417N, G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T, the amino acid positions referenced to SEQ ID NO:1.

In some embodiments, the modification results in the HPPD mutant of the invention described above, eg, causes the plant to express the HPPD mutant of the invention described above.

Accordingly, the present invention also provides a method of producing a herbicide-resistant plant, the method comprising modifying, for example, the targeted modification of an endogenous HPPD coding sequence of the plant, thereby resulting in the HPPD mutant of the invention hereinabove, for example, resulting in the The plants express the HPPD mutants of the present invention described above.

A "herbicide-resistant plant" may refer to a plant in which the endogenous HPPD coding sequence of the invention has been targetedly modified, relative to a plant without said targeted modification, having increased resistance to HPPD-inhibiting herbicides, eg, enhanced 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or higher. Methods for determining resistance to HPPD-inhibiting herbicides are known in the art. Resistance can be readily determined by one skilled in the art for a specific plant and specific herbicide. In some embodiments, the herbicide-resistant plants of the present invention are capable of at least 1 μmol/L, at least 1.5 μmol/L, at least 1.8 μmol/L, at least 2 μmol/L, at least 3 μmol/L, at least 5 μmol/L, at least 10 mol Normal growth was exhibited in the presence of herbicides (eg, HPPD-inhibiting herbicides such as mesotrione) at concentrations of /L or higher.

In some embodiments, the endogenous HPPD coding sequence of the plant is targeted for modification by homologous recombination. Methods of modifying endogenous genes in plants by homologous recombination are well known to those skilled in the art.

In some embodiments, the endogenous HPPD coding sequence of the plant is targeted for modification by gene editing. In some embodiments, the plant's endogenous HPPD coding sequence is targeted for modification by introducing into the plant a gene editing system that targets the endogenous HPPD coding region in the plant genome.

In some embodiments, introduction of the gene editing system results in an amino acid mutation of the endogenous HPPD at one or more positions selected from positions 365, 378, 414, 415, 417, or 419, the amino acid positions See SEQ ID NO:1.

In some embodiments, introduction of the gene editing system results in the substitution of the endogenous HPPD for a leucine (L) at position 365, and/or a substitution for a phenylalanine (F) at position 378 , and/or substituted glycine (G) at position 414, and/or substituted glycine (G) at position 415, and/or substituted glycine (G) at position 417, and/or substituted at position 417 Glycine (G) at position 419 is substituted, and the amino acid position is referenced to SEQ ID NO: 1.

In some embodiments, introduction of the gene editing system results in the substitution of leucine (L) with lysine (K) at position 365 of the endogenous HPPD, and/or phenylalanine at position 378 Acid (F) is replaced by alanine (A), and/or glycine (G) at position 414 is replaced by alanine (A) or valine (V), and/or glycine at position 415 (G) is replaced by alanine (A) or valine (V), and/or glycine (G) at position 417 is replaced by serine (S) or alanine (A) or lysine (K) or valine (V) or arginine (R) or asparagine (N) or aspartic acid (D) or cysteine (C) or glutamine (Q) or glutamic acid (E ) or histidine (H) or isoleucine (I) or methionine (M) or phenylalanine (F) or proline (P) or threonine (T) substitution, and/ Or glycine (G) at position 419 is substituted with tryptophan (W), the amino acid position is referenced to SEQ ID NO: 1.

In some embodiments, introduction of the gene editing system results in the endogenous HPPD comprising L365K, F378A, G414A, G414V, G415A, G415V, G417S, G417A, G417K, G419W, F378A/G417A, G417V, G417R, One or more amino acid substitutions of G417N, G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T, the amino acid positions referenced to SEQ ID NO:1.

In some embodiments, introduction of the gene editing system results in the HPPD mutant of the invention above, eg, causes the plant to express the HPPD mutant of the invention above.

The gene editing system usable in the present invention may be various gene editing systems known in the art as long as it enables targeted genome editing in plants. The gene editing system may be a CRISPR, ZFN or TALEN based gene editing system. Preferably, the gene editing system is a CRISPR-based gene editing system.

In some preferred embodiments, the gene editing system is a base editing system. The base editing system usable in the present invention may be various base editing systems known in the art as long as it can perform targeted genome base editing in plants. For example, such base editing systems include, but are not limited to, those described in WO 2018/056623, WO 2019/120283, WO 2019/120310.

In some embodiments, the base editing system comprises a base editing fusion protein or an expression construct comprising a nucleotide sequence encoding the same, and at least one guide RNA or an expression construct comprising a nucleotide sequence encoding the same body, such as the system comprising at least one of the following i) to v):

i) a base editing fusion protein, and at least one guide RNA;

ii) an expression construct comprising a nucleotide sequence encoding a base-edited fusion protein, and at least one guide RNA;

iii) base editing fusion proteins, and expression constructs comprising nucleotide sequences encoding at least one guide RNA;

iv) an expression construct comprising a nucleotide sequence encoding a base-edited fusion protein, and an expression construct comprising a nucleotide sequence encoding at least one guide RNA;

v) an expression construct comprising a nucleotide sequence encoding a base-edited fusion protein and a nucleotide sequence encoding at least one guide RNA;

Wherein the base editing fusion protein comprises a CRISPR effector protein and a deaminase domain, and the at least one guide RNA is capable of targeting the base editing fusion protein to an endogenous HPPD coding sequence in the plant genome.

In the embodiments herein, "base editing fusion protein" and "base editor" are used interchangeably and refer to a protein that can mediate one or more nucleotide substitutions of a target sequence in a genome in a sequence-specific manner protein.

As used herein, the term "CRISPR effector protein" generally refers to nucleases (CRISPR nucleases) or functional variants thereof that occur in naturally occurring CRISPR systems. The term encompasses any effector protein based on the CRISPR system capable of sequence-specific targeting within a cell.

As used herein, a "functional variant" with respect to a CRISPR nuclease means that it retains at least the guide RNA-mediated sequence-specific targeting ability. Preferably, the functional variant is a nuclease inactive variant, ie it lacks double-stranded nucleic acid cleavage activity. However, CRISPR nucleases lacking double-stranded nucleic acid cleavage activity also encompass nickases that form a nick in a double-stranded nucleic acid molecule, but do not completely cleave the double-stranded nucleic acid. In some preferred embodiments of the present invention, the CRISPR effector protein of the present invention has nickase activity. In some embodiments, the functional variant recognizes a different PAM (Prospacer Adjacent Motif) sequence relative to the wild-type nuclease.

"CRISPR effector proteins" can be derived from Cas9 nucleases, including Cas9 nucleases or functional variants thereof. The Cas9 nuclease may be a Cas9 nuclease from a different species, eg spCas9 from S. pyogenes or SaCas9 from S. aureus. "Cas9 nuclease" and "Cas9" are used interchangeably herein to refer to an RNA comprising a Cas9 protein or fragment thereof (eg, a protein comprising the active DNA cleavage domain of Cas9 and/or the gRNA binding domain of Cas9) directed nucleases. Cas9 is a component of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats and related systems) genome editing system, which can target and cleave DNA target sequences under the guidance of guide RNA to form DNA double-strand breaks (DSBs). ).

"CRISPR effector proteins" can also be derived from Cpf1 nucleases, including Cpf1 nucleases or functional variants thereof. The Cpf1 nuclease may be a Cpf1 nuclease from a different species, such as Cpf1 nucleases from Francisella novicida U112, Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006.

Useful "CRISPR effector proteins" can also be derived from Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Csn2 , Cas4, C2c1, C2c3 or C2c2 nucleases, for example including these nucleases or functional variants thereof.

In some embodiments, the CRISPR effector protein is nuclease-inactive Cas9. The DNA cleavage domain of the Cas9 nuclease is known to contain two subdomains: the HNH nuclease subdomain and the RuvC subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC subdomain cleaves the non-complementary strand. Mutations in these subdomains can inactivate the nuclease activity of Cas9, forming "nuclease-inactive Cas9". The nuclease-inactivated Cas9 still retains the DNA binding ability directed by the gRNA.

The nuclease-inactive Cas9 of the present invention can be derived from different species of Cas9, for example, from S. pyogenes Cas9 (SpCas9), or from S. aureus Cas9 (SaCas9) ). Simultaneous mutation of the HNH nuclease subdomain and RuvC subdomain of Cas9 (eg, comprising mutations D10A and H840A) inactivates the nuclease of Cas9, becoming nuclease-dead Cas9 (dCas9). Mutational inactivation of one of the subdomains allows Cas9 to have nickase activity, ie to obtain a Cas9 nickase (nCas9), eg, nCas9 with only mutation D10A.

When Cas9 nuclease is used for gene editing, it is usually required that the target sequence has a PAM (prespacer adjacent motif) sequence of 5'-NGG-3' at the 3' end. However, this PAM sequence appears infrequently in some species such as rice, greatly limiting gene editing in these species such as rice. For this reason, CRISPR effector proteins that recognize different PAM sequences, such as Cas9 nuclease functional variants with different PAM sequences, are preferably used in the present invention.

In some preferred embodiments, the CRISPR effector protein is a Cas9 variant that recognizes the PAM sequence 5'-NG-3'. In some preferred embodiments, the CRISPR effector protein is a nuclease-inactive Cas9 variant that recognizes the PAM sequence 5'-NG-3'.

The deaminase domains described herein can be cytosine deamination domains or adenine deamination domains.

As used herein, "cytosine deamination domain" refers to a domain capable of accepting single-stranded DNA as a substrate to catalyze the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively.

Examples of cytosine deaminase useful in the present invention include, but are not limited to, eg, APOBEC1 deaminase, activation-induced cytidine deaminase (AID), APOBEC3G, CDA1, human APOBEC3A deaminase, or functional variants thereof. body. In some embodiments, the cytosine deaminase is human APOBEC3A deaminase or a functional variant thereof.

As used herein, "adenine deamination domain" refers to a domain capable of accepting single-stranded DNA as a substrate and catalyzing the formation of inosine (I) from adenosine or deoxyadenosine (A). In some embodiments, the adenine deaminase is a variant of the E. coli tRNA adenine deaminase TadA (ecTadA).

As used herein, "guide RNA" and "gRNA" are used interchangeably and refer to an RNA capable of forming a complex with a CRISPR effector protein and capable of targeting the complex to a target sequence due to some identity to the target sequence molecular. The guide RNA targets the target sequence by base pairing with the complementary strand of the target sequence. For example, a gRNA employed by a Cas9 nuclease or a functional variant thereof typically consists of partially complementary crRNA and tracrRNA molecules forming a complex, wherein the crRNA comprises sufficient identity to the target sequence to hybridize to the complementary strand of the target sequence and direct The CRISPR complex (Cas9+crRNA+tracrRNA) is a guide sequence (also called a seed sequence) that specifically binds to the target sequence. However, it is known in the art that it is possible to design single guide RNAs (sgRNAs) that contain features of both crRNA and tracrRNA. The gRNAs employed by Cpf1 nucleases or functional variants thereof usually consist of only mature crRNA molecules, which may also be referred to as sgRNAs. It is within the skill of the artisan to design a suitable gRNA based on the CRISPR nuclease used and the target sequence to be edited.

In some embodiments, the at least one gRNA of the present invention comprises a target sequence in the coding region of the endogenous HPPD, and the amino acid sequence encoded by the target sequence comprises the endogenous HPPD selected from the group consisting of 365, 378, An amino acid at one or more of positions 414, 415, 417, or 419, such as at

positions

1, 2, 3, 4, 5, or 6, with reference to SEQ ID NO: 1.

In some embodiments, the gene editing system is a so-called prime editing system. The system includes a fusion of a Cas nuclease with target strand nicking activity (eg, Cas9-H840A) and a reverse transcriptase (eg, M-MLV reverse transcriptase), and a 3' end with a repair template (RT template) and Free single-stranded binding region (PBS) pegRNA (prime editing gRNA, guide editing gRNA). The system uses PBS to bind the free single strands generated by Cas nickase (such as Cas9-H840A), and make it transcribe single-stranded DNA sequences according to a given RT template. After cell repair, the PAM sequence can be located in the genome. - Any change in DNA sequence downstream of position 3. For example, for a prime editing system, see Anzalone, A. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature https://doi.org/10.1038/s41586-019-1711 -4 (2019).

In the methods of the present invention, the gene editing system can be introduced into plants by various methods well known to those skilled in the art. Methods that can be used to introduce the gene editing system of the present invention into plants include, but are not limited to: biolistic, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube pathway, and ovary injection.

In the method of the present invention, the modification of the target sequence can be achieved only by introducing or producing the gene editing system in a plant cell, and the modification can be stably inherited without the need to stably transform the gene editing system into a plant. This avoids potential off-target effects of stably existing gene editing systems, and also avoids the integration of exogenous nucleotide sequences into the plant genome, resulting in higher biological safety.

In some preferred embodiments, the introduction is performed in the absence of selective pressure, thereby avoiding integration of foreign nucleotide sequences into the plant genome.

In some embodiments, the introducing comprises transforming the gene editing system of the invention into an isolated plant cell or tissue, and then regenerating the transformed plant cell or tissue into a whole plant. Preferably, the regeneration is carried out in the absence of selective pressure, that is, without the use of any selection agent for the selection gene carried on the expression vector during tissue culture. Without the use of a selection agent, the regeneration efficiency of plants can be improved, and herbicide-resistant plants without exogenous nucleotide sequences can be obtained.

In other embodiments, the gene editing systems of the present invention can be transformed into specific sites on intact plants, such as leaves, shoot tips, pollen tubes, young ears, or hypocotyls. This is particularly suitable for the transformation of plants that are difficult to regenerate in tissue culture.

In some embodiments of the invention, the in vitro expressed protein and/or in vitro transcribed RNA molecule is directly transformed into the plant. The protein and/or RNA molecules enable gene editing in plant cells and are subsequently degraded by the cells, avoiding the integration of exogenous nucleotide sequences into the plant genome.

In another aspect, the present invention also provides a herbicide-resistant plant produced by the method of the present invention for targeted modification of endogenous HPPD in a plant. The present invention also encompasses the progeny of said herbicide-resistant plants.

In some embodiments of the invention, the herbicide-resistant plant is non-transgenic.

The plants described in various aspects of the invention may be plants susceptible to HPPD inhibitors, including monocotyledonous or dicotyledonous plants. Preferably, the plant is a crop plant, such as a monocotyledonous crop plant. Examples of suitable plants include, but are not limited to, rice, wheat, barley, sorghum, corn, oats, Arabidopsis, ryegrass, ryegrass, wild soybean, soybean, and tobacco, and the like. In some preferred embodiments, the plant is rice. In other preferred embodiments, the plant is maize. In other preferred embodiments, the plant is soybean. In other preferred embodiments, the plant is tobacco.

5. Methods of producing herbicide-resistant plants by physical or chemical mutagenesis

In another aspect, the present invention also provides a method of producing herbicide-resistant plants comprising physically or chemically mutagenizing a population of said plants and screening for endogenous HPPD (eg, expressed endogenous HPPD) for at least A plant comprising an amino acid mutation at one or more positions selected from the group consisting of positions 365, 378, 414, 415, 417 or 419, eg, 1, 2, 3, 4, 5 or 6 positions, wherein said amino acid positions refer to SEQ ID NO: 1.

In some embodiments, the screening endogenous HPPD (eg, expressed endogenous HPPD) has at least a leucine (L) substitution at position 365, and/or a phenylalanine (F) substitution at position 378 , and/or substituted glycine (G) at position 414, and/or substituted glycine (G) at position 415, and/or substituted glycine (G) at position 417, and/or substituted at position 417 Plants in which glycine (G) at No. 419 is substituted, the amino acid position is referred to in SEQ ID NO: 1.

In some embodiments, the endogenous HPPD (eg, expressed endogenous HPPD) is screened for at least substitution of leucine (L) with lysine (K) at position 365, and/or phenylalanine at position 378 Acid (F) is replaced by alanine (A), and/or glycine (G) at position 414 is replaced by alanine (A) or valine (V), and/or glycine at position 415 (G) is replaced by alanine (A) or valine (V), and/or glycine (G) at position 417 is replaced by serine (S) or alanine (A) or lysine (K) or valine (V) or arginine (R) or asparagine (N) or aspartic acid (D) or cysteine (C) or glutamine (Q) or glutamic acid (E ) or histidine (H) or isoleucine (I) or methionine (M) or phenylalanine (F) or proline (P) or threonine (T) substitution, and/ Or a plant in which glycine (G) at position 419 is substituted with tryptophan (W), the amino acid position being referenced to SEQ ID NO: 1.

In some embodiments, screening for endogenous HPPD (eg, expressed endogenous HPPD) comprises at least one selected from the group consisting of L365K, F378A, G414A, G414V, G415A, G415V, G417S, G417A, G417K, G419W, F378A/G417A, G417V, G417R, Plants with one or more amino acid substitutions of G417N, G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T, the amino acid positions are referenced to SEQ ID NO:1.

In some embodiments, plants are screened for endogenous HPPD (eg, expressed endogenous HPPD) mutated to the HPPD mutants described above in the invention.

Accordingly, the present invention also provides a method of producing herbicide-resistant plants, comprising subjecting a population of said plants to physical or chemical mutagenesis, and screening for plants comprising or expressing the HPPD mutants of the present invention described above .

A "herbicide-resistant plant" may refer to an endogenous HPPD mutagenized plant of the invention having enhanced resistance to HPPD-inhibiting herbicides, eg, enhanced 10 %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or higher. Methods for determining resistance to HPPD-inhibiting herbicides are known in the art. Resistance can be readily determined by one skilled in the art for a specific plant and specific herbicide. In some embodiments, the herbicide-resistant plants of the present invention are capable of at least 1 μmol/L, at least 1.5 μmol/L, at least 1.8 μmol/L, at least 2 μmol/L, at least 3 μmol/L, at least 5 μmol/L, at least 10 mol Normal growth was exhibited in the presence of herbicides (eg, HPPD-inhibiting herbicides such as mesotrione) at concentrations of /L or higher.

In some embodiments, the physical mutagenesis can be accomplished by radioactively irradiating the plant population (eg, irradiating plant seeds). In some embodiments, the chemical mutagenesis can be accomplished by treating the plant population (eg, treating plant seeds) with ethyl methanesulfonate (EMS).

In some embodiments, the screening can be accomplished by sequencing the coding sequence of the endogenous HPPD.

In another aspect, the present invention also provides a herbicide-resistant plant produced by the physical or chemical mutagenesis method of the present invention. The present invention also encompasses the progeny of said herbicide-resistant plants.

6. Plant Breeding Methods

In another aspect, the present invention provides a method of plant breeding, comprising crossing a first plant having herbicide resistance obtained by the above-described method of the present invention with a second plant not containing said herbicide resistance, thereby bringing The herbicide resistance is introduced into the second plant.

Example

Example 1 Construction of OsHPPD mutant plasmid

Wild-type OsHPPD (amino acid sequence shown in SEQ ID NO: 1) was expressed and purified in pET-smt vector. In order to obtain mutants, circular PCR is used to design forward and reverse primers at the sites that need to be mutated. After PCR, transformation, sequencing and other steps, mutant plasmids with correct point mutations are obtained for the next experiment.

Example 2 Expression of OsHPPD mutant protein

Transform E. coli ^BL21 (DE3) competent cells with the mutant plasmid confirmed by sequencing (12-14h). Observe the turbidity and vigorous growth of the bacterial liquid cultured in a small amount of the test tube. Take 2 mL of bacterial liquid and inoculate it into a small triangular flask containing 120 mL of freshly prepared liquid LB medium, and expand the culture at 37°C and 220 rpm for 12 hours. The bacterial liquid in the small triangular flask was inoculated into the freshly prepared TB medium, and 10 mL of bacterial liquid was added to each 500 mL of TB medium. Cultivate at 37°C at 250rpm until the OD value of the bacterial solution reaches about 0.6, add IPTG with a final concentration of 0.4mM, and induce the expression of the target protein at 16°C. After the induction and expression, use a high-speed centrifuge to collect the bacteria.

Example 3 Purification of OsHPPD mutant protein

3.1 Purification of target protein by nickel affinity chromatography

The expressed HPPD protein is tagged with 10 histidines, so it can be combined with nickel ions on the chromatography medium to achieve the purpose of separation from other impurities. The target protein adsorbed on the chromatographic medium can be eluted by using the free ligand solvent to complete the preliminary purification of the target protein.

3.2 Purification of target protein by gel filtration chromatography

In this experiment, GE's model Superdex 200 increase molecular sieve was used for gel filtration chromatography purification. The protein sample after passing through the nickel column was concentrated with a Millipore tube, concentrated to a volume of 1ml, and transferred to a new 1.5mL EP tube. The pellet was removed by centrifugation at 13,000 rpm for 10 minutes at 4°C. The centrifuged protein was injected into the molecular sieve by the high-efficiency protein liquid-phase purification system AKTA purifier 10 for further purification and separation of the target HPPD protein, and then SDS-PAGE gel electrophoresis was performed to observe the size and purity of the HPPD protein.

Example 4 Activity determination of enzyme reaction kinetics of OsHPPD, zmHPPD and gmHPPD proteins and identification of resistant mutants based on inhibition kinetics

4.1 Activity assay for enzymatic reaction kinetics: This is a coupled method for HPPD activity assay and herbicide screening. This experiment combines the formation of conversion of HPPA to HGA by HPPD with the production of maleylacetoacetate from HGA by HGA dioxygenase. The absorption peak of maleate acetoacetate is about 318 nm, which is used for real-time spectrophotometric monitoring. In vitro activity and coupled enzyme assays for HPPD inhibition were determined by modifying previously reported methods (ref. 1). Monitoring was performed in a 96-well plate at 30°C using a UV/visible plate reader to monitor the formation of acetoacetate maleate at 318 nm. The total assay volume of the reaction mixture was 200 uL, containing appropriate amounts of HPPA, 100 uM FeSO4, 2 mM sodium ascorbate, 20 mM HEPES buffer (pH=7.0), HPPD (125 nM) and HGD (750 nM). All reaction components were pre-equilibrated at 30°C for at least 30 minutes prior to analysis. The amount of HGD activity was predetermined to greatly exceed HPPD activity to ensure tight coupling of the reaction. Each experiment was performed around 30 min and repeated at least 3 times, and the average was taken.

4.2 Identification of resistant mutants based on inhibition kinetics: The HPPD herbicide mesotrione, dissolved in dimethyl sulfoxide (DMSO) as a stock solution, was diluted to various concentrations with reaction buffer prior to use (Reference 2). Inhibition constant (Ki, inhibition constant) is an indicator of inhibitor efficacy, which reflects the inhibitory strength of the inhibitor to the target. The smaller the value, the stronger the inhibitory effect. In other words, the larger the Ki value, the more resistant the mutant is. the stronger. Ki was obtained by inputting 1/v relative to inhibitor concentration (7 concentrations in total, specific concentrations determined by inhibitor potency) at fixed substrate concentrations (final concentrations of HPPA were 40 uM and 80 uM, respectively) into the software GraphPad. Figures 1-5 show the changes in the velocity V0 calculated from the measured absorbance values of the mutants under seven mesotrione concentration gradients. The values of these mutants are higher than those of the wild type, indicating that they have Herbicide-Mesotrione Resistance. In Figure 6, there are 13 mutants whose Ki value is larger than that of the wild type at the amino acid G417 position. The larger the Ki value, the stronger the resistance of the mutants. Taking wild-type HPPD as a control, at different mesotrione concentrations, a total of 24 mutants (containing amino acid substitutions L365K, F378A, G419W, G417A, G417A/F378A, G417S, G417K, G415A, G415V, G414A, G414V, respectively, were found) , G417V, G417R, G417N, G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T, numbering relative to SEQ ID NO: 1; their amino acid sequences are shown in SEQ ID NO: 14-37) more resistant than wild type. Similarly, rice HPPD mutants with double or triple mutations (containing amino acid substitutions F378A/G415A, F378A/G415V, F378A/G417K, F378A/G417R, F378A/G417V, G414V/G417K, F378A/G417A/G415A, F378A, respectively /G417K/G415V, numbered relative to SEQ ID NO: 1; their amino acid sequences are shown in SEQ ID NO: 48-55, respectively) also showed resistance to herbicides (Figure 7). The corn-derived HPPD mutants shown in Figure 8 (containing amino acid substitutions F373A/G412A, G409A, G412K, G412Q, G412R, respectively, numbered relative to SEQ ID NO: 9; or containing amino acid substitutions F378A/G417A, G414A, G417K, respectively, G417Q, G417R, numbered relative to SEQ ID NO: 1; their amino acid sequences are shown in SEQ ID NO: 38-42, respectively) and soybean-derived HPPD mutants (containing amino acid substitutions F386A/G425A, G422A, G425K, G425Q, G425R, respectively) , numbering relative to SEQ ID NO: 13; or respectively comprising amino acid substitutions F378A/G417A, G414A, G417K, G417Q, G417R, numbering relative to SEQ ID NO: 1; their amino acid sequences are shown in SEQ ID NO: 43-47, respectively) It has a corresponding mutation site with the rice-derived HPPD mutant, and also shows resistance to herbicides.

Example 5 In vivo testing of rice herbicide resistance

5.1 Materials Methods:

1). Construction of Agrobacterium transformation vector overexpressing HPPD variant protein: First, the cDNA reverse transcribed from the mRNA of rice Zhonghua 11 was used as the amplification template to obtain the unmutated OsHPPD-CDS-WT sequence. Taking the unmutated OsHPPD-CDS sequence as the amplification template, the target mutation was introduced through two rounds of PCR to obtain the sequence containing amino acid mutations such as G414V, G417A, G417K or (F378A+G417K) screened in the previous experiment. Then, the unmutated OsHPPD-CDS-WT sequence and the OsHPPD-CDS-M sequence after introducing the target mutation were cloned into the vector backbone of pHUE411, which was promoted by the maize Ubiquitin-1 (Ubi-1) promoter of RNA polymerase II expression (Figure 9).

2) Rice transformation material - Kitaake

3) Detection of overexpression lines

The total genomic DNA was extracted from the transformed and regenerated rice leaves, and a small amount was used as a PCR amplification template. The sequences of the Ubi-1 promoter and HPPD on the expression vector were used to set primers before and after respectively to identify whether the genome was integrated with the expression vector.

4). qRT-PCR to measure the relative expression of OsHPPD-mRNA in rice

Extract the RNA of the plant with the integrated expression vector and reverse-transcribe it into cDNA, perform real-time fluorescent quantitative PCR (called quantitative RT-PCR or qRT-PCR) reaction, using ChamQTM Universal

qPCR Master Mix reagent. Using OsActin as the internal reference gene, the primers for the target gene and internal reference gene required for qRT-PCR were synthesized and designed, and qRT-PCR was performed according to the manufacturer's instructions.

According to the obtained Ct value, calculate the relative value of the target gene relative to the internal reference gene X=2 ^-ΔCt , ΔCt=Ct-Ct within (Ct is the detected Ct value of the target gene, and Ct is the internal reference gene Ct value). The target gene expression content of the experimental group relative to the control group was X experimental group/X control group.

5). Construction of rice screening system on HPPD inhibitor-mesotrione medium

An appropriate amount of mesotrione stock solution was added to the rooting medium of rice to configure M6 solid medium containing different mesotrione concentration gradients (Table 1). Then, the rice seedlings overexpressing wild-type HPPD and wild-type rice seedlings were tested, and tissue culture seedlings with similar growth status and a plant height of about 6 cm from the growth point were selected and transferred to the medium with different mesotrione concentration gradients. Subculture, three seedlings per dish, 3 dishes per mesotrione concentration of overexpression and wild-type rice seedlings. After about 10 days of culture, the growth phenotype of the seedlings screened by mesotrione was observed: when the concentration of mesotrione in the medium was lower than OsW3, both wild-type and overexpressing rice seedlings grew normally; mesotrione When the concentration is higher than OsW3 and lower than OsW2, the newly grown young leaves of wild-type rice seedlings turn white, and the overexpression material grows normally; when the mesotrione concentration is higher than OsW2, the newly grown young leaves of the overexpression material turn white , while the leaves of wild-type rice seedlings turned white faster. Therefore, OsW2 was chosen as the screening concentration for the overexpression material.

Table 1: Phenotypic identification of rice under different concentration gradients of mesotrione screening.

5.2 Results

After culturing for about two weeks, the growth state of rice seedlings at the screening concentration of OsW2 mesotrione was observed. At the same screening concentration, rice expressing wild-type OsHPPD (relative OsHPPD-mRNA expression levels of 6.1, 7.5, or 14.3) showed a distinct albino phenotype, while rice expressing G414V, G417A or G417K mutations (OsHPPD-mRNA relative The expression level is shown in the bar graph in Figure 10) to maintain the normal growth state, and the rice seedlings overexpressed (F378A+G417K) mutants grow slightly weaker, but the leaves are not albino. Therefore, the G414, G417 or (F378A+G417K) mutations all increased the resistance of rice compared to the wild type (Fig. 10). The resistance phenotypes of more mutant overexpressing plants are summarized in Table 2 below.

Table 2. Screening phenotype of rice overexpressing HPPD protein mutants

Example 6 In vivo testing of tobacco herbicide resistance

6.1 Materials Methods:

1).Construct the expression plasmids of wild type and mutant OsHPPD protein

The wild-type OsHPPD-CDS-WT sequence was cloned into the vector backbone of pBSE401 and expressed by the 35S promoter. Use circular PCR to design forward and reverse primers at the sites that need to be mutated. After PCR, transformation, sequencing and other steps, the point-mutated OsHPPD-CDS-G417K sequence was obtained. Initiate expression.

2). Transformation of ordinary tobacco - variety: cloud 37

3). Detection of overexpression lines

Total genomic DNA was extracted from tobacco leaves, and a small amount was used as a template for PCR amplification. The sequences of the 35S promoter and HPPD on the expression vector were used to set up primers before and after, to identify whether the genome was integrated with the expression vector.

4). qRT-PCR to determine the relative expression of OsHPPD-mRNA in tobacco

qPCR Master Mix reagent. Using NtActin as the internal reference gene, synthesize and design primers for the target gene and internal reference gene required for qRT-PCR, and perform qRT-PCR according to the manufacturer's instructions. .

5). Construction of a screening system for tobacco on HPPD inhibitor-mesotrione medium

First, tobacco rooting media with different mesotrione concentration gradients were prepared (Table 3). Then, the wild-type HPPD-overexpressing tobacco and wild-type tobacco tissue culture seedlings with consistent growth status were selected and transferred to the medium supplemented with mesotrione for subculture. After a period of screening and cultivation, young tobacco leaves with weak resistance gradually turned white. Therefore, the lowest mesotrione concentration that tobacco overexpressing wild-type OsHPPD cannot tolerate was selected as the tobacco screening concentration.

Table 3: Phenotypic identification of tobacco screened at different mesotrione concentrations.

6.2 Results

Cultivate for about 20 days and observe the growth state of tobacco seedlings under mesotrione screening: when the mesotrione concentration is equal to or lower than NbW4, both wild-type and overexpressed tobacco seedlings grow normally; when the screening concentration is NbW3, The newly grown young leaves of the wild-type tobacco seedlings turned white, and the overexpression material grew normally; when the mesotrione concentration was equal to or higher than NbW2, the newly grown young leaves of the overexpression material turned white, while the leaves of the wild-type tobacco seedlings grew Whitens faster. Therefore, mesotrione of NbW2 was selected as the screening concentration for the overexpression material. At the same screening concentration, tobacco expressing wild-type OsHPPD (relative expression of OsHPPD-mRNA, 4.6) showed a pronounced albino phenotype, while tobacco expressing G417K mutation (relative expression of OsHPPD-mRNA, 3.5) maintained normal growth. state, so the G417K mutation increased drug resistance in tobacco compared to wild type (Figure 11).

references

1. Schmidt et al; Murine liver homogentisate 1,2-dioxygenase Purification to homogeneity and novel biochemical properties. Eul: J. Biochem. 228, (1995).

2. Y.L. Xu et al; Pyrazolone–quinazolone hybrids: A novel class of human 4-hydroxyphenylpyruvate dioxygenase inhibitors. Bioorg.Med.Chem.22(2014).

sequence

The following are the specific sequences involved in the present invention, wherein the mutated amino acids are marked in bold and underlined.

>SEQ ID NO: 1 Oryza sativa wild-type HPPD amino acid sequence (446)

>SEQ ID NO:2 Triticum aestivum-A wild-type HPPD amino acid sequence (433)

>SEQ ID NO:3 Triticum aestivum-B wild-type HPPD amino acid sequence (436)

>SEQ ID NO:4 Triticum aestivum-D wild-type HPPD amino acid sequence (436)

>SEQ ID NO:5 Avena sativa wild-type HPPD amino acid sequence (440)

>SEQ ID NO:6 Pseudomonas fluorescens wild-type HPPD amino acid sequence (358)

>SEQ ID NO:7 Lolium rigidum wild-type HPPD amino acid sequence (443)

>SEQ ID NO:8 Bromus tectorum wild-type HPPD amino acid sequence (435)

>SEQ ID NO:9 Zea mays wild-type HPPD amino acid sequence (445)

>SEQ ID NO: 10 Hordeum vulgare wild-type HPPD amino acid sequence (434)

>SEQ ID NO: 11 Glycine soja wild type HPPD amino acid sequence (488)

>SEQ ID NO: 12 Arabidopsis thaliana wild type HPPD amino acid sequence (473)

>SEQ ID NO:13 soybean wild-type HPPD amino acid sequence

>SEQ ID NO:14 Rice HPPD-resistant mutant L365K amino acid sequence

>SEQ ID NO: 15 Amino acid sequence of rice HPPD-resistant mutant F378A

>SEQ ID NO: 16 Amino acid sequence of rice HPPD-resistant mutant G419W

>SEQ ID NO: 17 Amino acid sequence of rice HPPD-resistant mutant G417A

>SEQ ID NO: 18 Amino acid sequence of rice HPPD resistant mutant G417A/F378A

>SEQ ID NO: 19 Amino acid sequence of rice HPPD-resistant mutant G417S

>SEQ ID NO:20 Rice HPPD-resistant mutant G417K amino acid sequence

>SEQ ID NO: 21 Amino acid sequence of rice HPPD-resistant mutant G415A

>SEQ ID NO:22 rice HPPD resistant mutant G415V amino acid sequence

>SEQ ID NO: 23 Amino acid sequence of rice HPPD resistant mutant G414A

>SEQ ID NO:24 rice HPPD resistant mutant G414V amino acid sequence

>SEQ ID NO: 25 Amino acid sequence of rice HPPD-resistant mutant G417V

>SEQ ID NO: 26 Amino acid sequence of rice HPPD-resistant mutant G417R

>SEQ ID NO:27 Rice HPPD-resistant mutant G417N amino acid sequence

>SEQ ID NO: 28 Amino acid sequence of rice HPPD resistant mutant G417D

>SEQ ID NO: 29 Amino acid sequence of rice HPPD-resistant mutant G417C

>SEQ ID NO:30 Rice HPPD-resistant mutant G417Q amino acid sequence

>SEQ ID NO: 31 Amino acid sequence of rice HPPD resistant mutant G417E

>SEQ ID NO:32 Rice HPPD-resistant mutant G417H amino acid sequence

>SEQ ID NO:33 Rice HPPD-resistant mutant G417I amino acid sequence

>SEQ ID NO:34 Rice HPPD-resistant mutant G417M amino acid sequence

>SEQ ID NO:35 Rice HPPD-resistant mutant G417F amino acid sequence

>SEQ ID NO:36 Rice HPPD-resistant mutant G417P amino acid sequence

>SEQ ID NO: 37 Amino acid sequence of rice HPPD resistant mutant G417T

>SEQ ID NO:38 Maize HPPD-resistant mutant F373A/G412A amino acid sequence (position reference SEQ ID NO:9)

>SEQ ID NO:39 Maize HPPD-resistant mutant G409A amino acid sequence (position reference SEQ ID NO:9)

>SEQ ID NO:40 Maize HPPD-resistant mutant G412K amino acid sequence (position reference SEQ ID NO:9)

>SEQ ID NO:41 Maize HPPD-resistant mutant G412Q amino acid sequence (position reference SEQ ID NO:9)

>SEQ ID NO:42 Maize HPPD-resistant mutant G412R amino acid sequence (position reference SEQ ID NO:9)

>SEQ ID NO:43 soybean HPPD resistant mutant F386A/G425A amino acid sequence (position reference SEQ ID NO:13)

>SEQ ID NO:44 soybean HPPD resistant mutant G422A amino acid sequence (position reference SEQ ID NO:13)

>SEQ ID NO:45 soybean HPPD resistant mutant G425K amino acid sequence (position reference SEQ ID NO:13)

>SEQ ID NO:46 soybean HPPD resistant mutant G425Q amino acid sequence (position reference SEQ ID NO:13)

>SEQ ID NO:47 soybean HPPD resistant mutant G425R amino acid sequence (position reference SEQ ID NO:13)

>SEQ ID NO:48 Rice HPPD-resistant mutant F378A/G415A amino acid sequence

>SEQ ID NO:49 Rice HPPD-resistant mutant F378A/G415V amino acid sequence

>SEQ ID NO:50 rice HPPD resistant mutant F378A/G417K amino acid sequence

>SEQ ID NO:51 Rice HPPD-resistant mutant F378A/G417R amino acid sequence

>SEQ ID NO:52 Rice HPPD-resistant mutant F378A/G417V amino acid sequence

>SEQ ID NO:53 Rice HPPD-resistant mutant G414V/G417K amino acid sequence

>SEQ ID NO:54 rice HPPD resistant mutant F378A/G417A/G415A amino acid sequence

>SEQ ID NO:55 Rice HPPD-resistant mutant F378A/G417K/G415V amino acid sequence

Claims

An HPPD mutant or a functional fragment thereof having an amino acid mutation such as an amino acid substitution at one or more positions selected from the group consisting of positions 417, 365, 378, 414, 415, or 419 relative to wild-type HPPD, said Amino acid positions are referenced to SEQ ID NO: 1.
The HPPD mutant of claim 1, or a functional fragment thereof, having an amino acid mutation, such as an amino acid substitution, at a position selected from the group consisting of, with respect to wild-type HPPD, referenced to SEQ ID NO: 1:

i) positions 378 and 415;

ii) positions 378 and 417;

iii) positions 414 and 417; or

iv) Positions 378, 415 and 417.
The HPPD mutant of claim 1 or 2, or a functional fragment thereof, said wild-type HPPD comprising the amino acid sequence of one of SEQ ID NOs: 1-13.
The HPPD mutant or functional fragment thereof according to any one of claims 1 to 3, which, when expressed in a plant, is capable of conferring resistance to a herbicide to the plant.
The HPPD mutant of claim 4 or a functional fragment thereof, wherein the herbicide is selected from the group consisting of pyrazole compounds, such as fenflufen, sulfapyrazole, fenazopyramide; triketone compounds, such as Sulcotrione, mesotrione, terbotrione, furfuryl ketone, bicyclopyrone, benzobicyclone; isoxazoles such as isoxaflutole; diketonitriles such as 2 -Cyano-3-cyclopropyl-1-(2-methylsulfonyl-4-trifluoromethylphenyl)-propane-1,3-dione and 2-cyano-1-[4-( methylsulfonyl)-2-trifluoromethylphenyl]-3-(1-methylcyclopropyl)propane-1,3-dione; and benzophenones, or any combination thereof.
The HPPD mutant or functional fragment thereof of any one of claims 1-5, wherein, relative to wild-type HPPD, the HPPD mutant or functional fragment thereof comprises a mutant selected from the group consisting of G417K, G417S, G417A, G419W, G417V, G417R , G417N, G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T, L365K, F378A, G414A, G414V, G415A, G415V one or more amino acid substitutions, the amino acid positions are referenced to SEQ ID NO :1.
The HPPD mutant or functional fragment thereof of claim 6, wherein relative to wild-type HPPD, the HPPD mutant or functional fragment thereof comprises an amino acid substitution selected from the group consisting of

i) F378A and G415A;

ii) F378A and G415V;

iii) F378A and G417K;

iv) F378A and G417R;

v) F378A and G417V;

vi) F378A and G417A;

vii) G414V and G417K;

viii) F378A, G415A and G417A; or

ix) F378A, G415V and G417K;

wherein the amino acid positions are referenced to SEQ ID NO: 1.
The HPPD mutant of any one of claims 1-7 or a functional fragment thereof, said HPPD mutant comprising the amino acid sequence shown in any one of SEQ ID NOs: 14-55.
A nucleic acid comprising a nucleotide sequence encoding the HPPD mutant of any one of claims 1-8 or a functional fragment thereof.
An expression cassette comprising a nucleotide sequence encoding the HPPD mutant of any one of claims 1-8, or a functional fragment thereof, operably linked to regulatory sequences.
An expression construct comprising the expression cassette of claim 10.
A method of transgenic production of herbicide-resistant plants, comprising introducing the nucleic acid of claim 9, the expression cassette of claim 10 and/or the expression construct of claim 11 into said plants.
A method of producing a herbicide-resistant plant, the method comprising targeted modification of an endogenous HPPD coding sequence of a plant such that the endogenous HPPD is selected from the group consisting of positions 417, 365, 378, 414, 415 or 419 amino acid mutation at one or more positions of SEQ ID NO: 1.
The method of claim 13, wherein said targeted modification results in said endogenous HPPD comprising a group selected from the group consisting of G417K, G417S, G417A, G419W, G417V, G417R, G417N, G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F , G417P, G417T, F378A/G417A, L365K, F378A, G414A, G414V, G415A, G415V one or more amino acid substitutions, the amino acid positions are referenced to SEQ ID NO:11.
14. The method of claim 13 or 14, wherein the targeted modification results in the HPPD mutant of any one of claims 1-8.
15. The method of any one of claims 13-15, wherein the coding sequence of the endogenous HPPD is targeted to be modified by gene editing or homologous recombination.
17. The method of claim 16, wherein the gene editing is base editing or prime editing.
A method of producing herbicide-resistant plants, comprising physical or chemical mutagenesis of a population of said plants, and screening for endogenous HPPD at least at position 417, 365, 378, 414, 415 or 419 Plants comprising amino acid mutations at one or more positions, e.g., 1, 2, 3, 4, 5, or 6 positions, wherein the amino acid positions are referenced to SEQ ID NO: 1.
The method of claim 18, wherein screening for endogenous HPPD comprises at least one selected from the group consisting of G417K, G417S, G417A, G419W, G417V, G417R, G417N, G417D, G417C, G417Q, G417E, G417H, G417I, G417M, G417F, G417P, G417T, F378A / Plants substituted with one or more amino acids of G417A, L365K, F378A, G414A, G414V, G415A, G415V, the amino acid positions are referenced to SEQ ID NO: 1.
19. The method of claim 18 or 19, wherein a plant is screened for comprising or expressing the HPPD mutant of any one of claims 1-8.
20. The method of any one of claims 18-20, wherein the physical mutagenesis comprises irradiating the plant population by radioactivity and the chemical mutagenesis comprises by treating the plant population with ethyl methanesulfonate (EMS).
The method of any one of claims 12-21, wherein the plant comprises a monocotyledonous or dicotyledonous plant, preferably the plant is a crop plant, such as a monocotyledonous crop plant.
23. The method of claim 22, wherein the plant is selected from the group consisting of rice, wheat, barley, sorghum, maize, oat, Arabidopsis, ryegrass, brome, wild soybean, soybean, and tobacco.
A herbicide-resistant plant or progeny thereof comprising or expressing the HPPD mutant or functional fragment thereof of any one of claims 1-8, or by the method of any one of claims 12-21 produce.
24. The herbicide-resistant plant or progeny thereof of claim 24, wherein the plant comprises a monocotyledonous or dicotyledonous plant, preferably the plant is a crop plant, such as a monocotyledonous crop plant.
26. The herbicide-resistant plant or progeny thereof of claim 25, wherein the plant is selected from the group consisting of rice, wheat, barley, sorghum, corn, oat, Arabidopsis, ryegrass, brome, wild soybean, soybean, and tobacco.
The HPPD mutant of any one of claims 1-8 or a functional fragment thereof, the nucleic acid of claim 9, the expression cassette of claim 10 and/or the expression construct of claim 11 in the production of herbicide-resistant plants use in.