Methods and compositions for conferring and / or enhancing herbicide tolerance using protoporphyrinogen oxidase or its variants.

Abstract

A technique is provided that uses protoporphyrinogen oxidase or its amino acid variants derived from prokaryotes to confer enhanced herbicide tolerance and / or improve herbicide tolerance in plants and / or algae.

Description

Technical Field

[0001] Protoporphyrinogen oxidase or variants thereof derived from prokaryotes are provided, along with techniques for conferring and / or enhancing herbicide tolerance in plants and / or algae using the protoporphyrinogen oxidase or variants thereof. Background Technology

[0002] The porphyrin biosynthesis pathway is used to synthesize chlorophyll and heme, which play important roles in plant metabolism, and it occurs in chloroplasts. In this pathway, protoporphyrinogen IX oxidase (hereinafter referred to as PPO; EC: 1.3.3.4) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. After the oxidation of protoporphyrinogen IX to protoporphyrin IX, protoporphyrin IX is used to synthesize chlorophyll by binding with magnesium via magnesium chelase, or to synthesize heme by binding with iron via iron chelase.

[0003] Therefore, when PPO activity is inhibited, chlorophyll and heme synthesis is suppressed, and the substrate protoporphyrinogen IX leaves the normal porphyrin biosynthesis pathway, leading to the rapid export of protoporphyrinogen IX from the chloroplast to the cytoplasm, and the accumulation of cytoplasmic protoporphyrin IX due to oxidation. The accumulated protoporphyrin IX produces highly reactive singlet oxygen in the presence of light and oxygen molecules. 1 O2 (oxygen 2) damages cell membranes and rapidly leads to plant cell death. Based on this principle, herbicides that inhibit PPO activity have been developed. To date, there are nine families of PPO-inhibiting herbicides classified according to their chemical structures, including pyrimidine diones, diphenyl ethers, phenylpyrazoles, N-phenylphthalimides, thiadiazoles, etc. diazole, triazolinone, Alzolidinedione and other herbicides.

[0004] In addition, to prevent the effects of herbicides on crop growth when using them, it is necessary to provide crops with herbicide tolerance.

[0005] At the same time, algae are photosynthetic organisms, which can convert light energy into chemical energy that can be used to synthesize various useful compounds. For example, algae can fix carbon through photosynthesis and convert carbon dioxide into sugars, starches, lipids, fats, or other biomolecules, thereby removing greenhouse gases from the atmosphere. In addition, large-scale cultivation of algae can produce a variety of substances, such as industrial enzymes, therapeutic compounds and proteins, nutrients, commercial substances, and fuel substances.

[0006] However, in the case of large-scale cultivation of algae in bioreactors or open or closed ponds, contamination may occur due to undesirable competing organisms, such as undesirable algae, fungi, rotifers, or zooplankton.

[0007] Therefore, there is a need for a technology to harvest desired plants and / or algae on a large scale by treating them with herbicides at concentrations that inhibit the growth of competing organisms without herbicide tolerance, after conferring herbicide tolerance to the desired plants and / or algae.

[0008] References

[0009] (Patent Document 1) U.S. Patent Application Registration Publication US 6,308,458 (October 30, 2001)

[0010] (Patent Document 2) U.S. Patent Application Registration Publication US 6,808,904 (October 26, 2004)

[0011] (Patent Document 3) US Patent Application Registration Publication US 7,563,950 (July 21, 2009)

[0012] (Patent Document 4) International Patent Application Publication WO2011 / 085221 (July 14, 2011)

[0013] (Non-Patent Literature 1) Li X, Volrath SL, Chilcott CE, Johnson MA, Ward ER, Law MD, Development of protoporphyrinogen oxidase as an efficient selection marker for agrobacterium tumefaciens-mediated transformation of maize, Plant Physiology 133: 736-747, 2003 Summary of the Invention

[0014] Technical issues

[0015] In this specification, it was found that the hemY type PPO gene and its mutants derived from prokaryotes exhibit broad herbicide tolerance to protoporphyrinogen oxidase (PPO)-inhibiting herbicides, thus suggesting that providing the hemY type PPO gene and its mutants to plants and / or algae could confer and / or enhance herbicide tolerance.

[0016] One implementation provides a polypeptide variant that comprises, substantially consists of, or consists of the following:

[0017] (1) The following amino acid sequence, wherein one or more amino acids selected from the group consisting of amino acids that affect the interaction between the PPO inhibitory herbicide and the PPO polypeptide SEQ ID NO: 2 (e.g., amino acids located at the binding site of SEQ ID NO: 2 that interacts with the PPO inhibitory herbicide) are respectively and independently deleted or substituted by amino acids different from the original amino acid at the corresponding position, or

[0018] (2) An amino acid sequence that has 95% or higher, 98% or higher, or 99% or higher sequence homology with the amino acid sequence (1).

[0019] The amino acid sequence selected from the group consisting of amino acids that affect the interaction between PPO inhibitory herbicides and PPO polypeptide SEQ ID NO: 2 may be one or more selected from the group consisting of N59, S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360 and I408 in the amino acid sequence of SEQ ID NO: 2.

[0020] Another implementation provides a polypeptide variant that comprises, is substantially composed of, or is composed of:

[0021] (1) The following amino acid sequence, wherein one or more amino acids selected from the group consisting of amino acids that affect the interaction between PPO inhibitory herbicides and PPO polypeptide SEQ ID NO: 4 (e.g., amino acids located at the binding site of SEQ ID NO: 4 that interacts with PPO inhibitory herbicides) are respectively and independently deleted or substituted by amino acids different from the original amino acids at the corresponding positions, or

[0022] (2) An amino acid sequence that has 95% or more, 98% or more, or 99% or more homology with the amino acid sequence (1).

[0023] The amino acids selected from the group consisting of amino acids that affect the interaction between PPO inhibitory herbicides and PPO polypeptide SEQ ID NO: 4 may be one or more selected from the group consisting of R101, F171, V175, A177, G194, P316, V318, F337, L340, I353 and F373 in the amino acid sequence of SEQ ID NO: 4.

[0024] Other implementations provide polynucleotides encoding polypeptides or polypeptide variants.

[0025] Other embodiments provide recombinant vectors containing the polynucleotide.

[0026] Other embodiments provide recombinant cells containing the recombinant vector.

[0027] Other embodiments provide compositions for imparting or enhancing herbicide tolerance to plants or algae, comprising one or more of the following:

[0028] The polypeptide of SEQ ID NO: 2, the polypeptide of SEQ ID NO: 4, the polypeptide variant as described above, and the polypeptide whose amino acid sequence has 95% or more, 98% or more, or 99% or more sequence homology with the polypeptide or polypeptide variant.

[0029] The polynucleotide encoding the polypeptide, the polypeptide variant, and the amino acid sequence are polypeptides having 95% or more, 98% or more, or 99% or more amino acid sequence homology with said polypeptide or variant;

[0030] A recombinant vector containing the polynucleotide; and

[0031] Recombinant cells containing the recombinant vector.

[0032] For example, the polynucleotide encoding the polypeptide of SEQ ID NO:2 may contain the polynucleotide sequence of SEQ ID NO:1, and the polynucleotide encoding the polypeptide of SEQ ID NO:4 may contain the polynucleotide sequence of SEQ ID NO:3, but is not limited thereto.

[0033] Herbicides can be protoporphyrinogen oxidase inhibitory herbicides.

[0034] As a specific implementation method, the herbicide can be selected from pyrimidine dione, diphenyl ether, phenylpyrazole, N-phenylphthalimide, phenyl ester, thiadiazole, etc. diazole, triazolinone, It is one or more of the group consisting of aziridine dione and other herbicides, but is not limited thereto.

[0035] In a specific embodiment, the herbicide may be one or more selected from the group consisting of: flufenoxuron, bensulfuron-methyl, bispyribac-methyl, tiafenacil, flufenoxuron, ethoxyfen, bensulfuron-methyl, bifenoxuron, ethoxyfen, lactoferrin, chlomethoxyfen, chlorintrofen, fluoroglycofen-ethyl, halosafen, pyraflufen-ethyl, fluazolate, flumioxazin, cinidon-ethyl, flumiclorac-pentyl, fluthiacet, thidiazimin, and propyzine. Oxadiargyl Oxadiazon, carfentrazone, sulfentrazone, azafenidin, cyclopentadiazon Pentoxazone, pyraclonil, flufenpyr-ethyl, profluazol, phenopylate (2,4-dichlorophenyl-1-pyrrolidinocarbamate), carbamate analogs of phenopylate (e.g., O-phenylpyrrolidino- and piperidinocarbamate analogs (see “Ujjana B. Nandihalli, Mary V. Duke, Stephen O. Duke, Relationships between molecular properties and biological activities of O-phenylpyrrolidino- and piperidinocarbamate herbicides. J. Agric. Food Chem., 1992, 40(10) 1993-2000”), their agriculturally acceptable salts and combinations thereof, but not limited thereto.

[0036] Plants are multicellular eukaryotic organisms capable of photosynthesis; they can be monocotyledonous or dicotyledonous, and can be herbaceous or woody. Algae are single-celled organisms capable of photosynthesis; they can be prokaryotic or eukaryotic algae.

[0037] In one embodiment, plants and algae are genetically manipulated to further include a second herbicide tolerance peptide or its encoding gene, and this can confer and / or enhance broader herbicide tolerance to the second herbicide. Plants and algae genetically manipulated to further include the second herbicide tolerance peptide or its encoding gene can be prepared using compositions for conferring and / or enhancing herbicide tolerance, said compositions further including the second herbicide tolerance peptide or its encoding gene. Therefore, compositions for conferring and / or enhancing herbicide tolerance can further include the second herbicide tolerance peptide or its encoding gene.

[0038] As a specific implementation, the second herbicide may include, but is not limited to, cell division inhibitory herbicides, photosynthesis inhibitory herbicides, amino acid synthesis inhibitory herbicides, plastid inhibitory herbicides, and cell membrane inhibitory herbicides.

[0039] As a specific implementation, examples of the second herbicide may be glyphosate, glufosinate, dicamba, 2,4-D (2,4-dichlorophenoxyacetic acid), isopropanol, etc. Herbicides including but not limited to acetochlor, ALS (acetyllactate synthase) inhibitors, photosystem II inhibitors, phenylurea-based herbicides, bromobenzonitrile-based herbicides, and combinations thereof.

[0040] As a specific embodiment, an example of the second herbicide may be one or more selected from the group consisting of: glyphosate herbicide-tolerant EPSPS (glyphosate-tolerant 5-enolpyruvate-shikimate-3-phosphate synthase), GOX (glyphosate oxidase), GAT (glyphosate-N-acetyltransferase), or glyphosate decarboxylase; glufosinate herbicide-tolerant PAT (glufosinate-N-acetyltransferase); dicamba herbicide-tolerant DMO (dicamba monooxygenase); 2,4-D herbicide-tolerant 2,4-D monooxygenase or AAD (aryloxyalkanoate dioxygenase). Dioxygenase; ALS-inhibiting sulfonylurea herbicide-resistant ALS (acetyllactate synthase), AHAS (acetylhydroxylate synthase), or Athahasl (acetylhydroxylate synthase large subunit); photosystem II-inhibiting herbicide-resistant photosystem II protein D1; phenylurea-based herbicide-resistant cytochrome P450; plastid-inhibiting herbicide-resistant HPPD (hydroxyphenylpyruvate dioxygenase); bromobenzonitrile herbicide-resistant nitrile hydrolase; and combinations thereof, but not limited thereto.

[0041] Further, examples of genes encoding second herbicide tolerance peptides may be selected from one or more of the following groups: glyphosate herbicide tolerance genes cp4 epsps, epsps(AG), mepsps, 2mepsps, goxv247, gat4601, or gat4621; glufosinate herbicide tolerance genes bar, pat, or pat(SYN); dicamba herbicide tolerance genes dmo; 2,4-D herbicide tolerance genes AAD-1 or AAD-12; ALS-inhibiting sulfonylurea herbicide tolerance genes ALS, GM-HRA, S4-HRA, ZM-HRA, Csr1, Csr1-1, Csr1-2, SurA, or SurB; photosystem II-inhibiting herbicide tolerance genes psbA; phenylurea herbicide tolerance genes CYP76B1; and isocyanate herbicide tolerance genes. The HPPDPF W336 gene for tolerance to cyclohexane herbicides and the bxn gene for tolerance to bromobenzonitrile herbicides; and combinations thereof, but not limited thereto.

[0042] Other embodiments provide herbicide-tolerant plant and / or algal transformants, which are transformed with polynucleotides or their clones or progeny.

[0043] Other embodiments provide methods for preparing herbicide-resistant plants or algae, which include the step of transforming the plants and / or algae with the polynucleotide.

[0044] Other embodiments provide methods for conferring or enhancing herbicide tolerance in plants and / or algae, which include the step of converting plants and / or algae with the polynucleotide.

[0045] It can transform algal and / or plant cells, protoplasts, callus tissue, hypocotyls, seeds, cotyledons, branches, or whole plants.

[0046] Transformers can be algal and / or plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, branches, or whole plants.

[0047] Other embodiments provide a method for controlling weeds in farmland, including:

[0048] The step of providing plants to farmland, said plants comprising one or more of a polypeptide selected from SEQ ID NO: 2 or 4, a polypeptide variant, a polynucleotide encoding the polypeptide, a recombinant vector containing the polynucleotide, and a recombinant cell containing the recombinant vector; and

[0049] The steps of applying an effective dose of protoporphyrinogen oxidase-inhibiting herbicide to farmland (or plants).

[0050] In one specific implementation, the step of applying an effective dose of protoporphyrinogen oxidase-inhibiting herbicide to farmland can be carried out by sequentially or simultaneously applying effective doses of two or more protoporphyrinogen oxidase-inhibiting herbicides.

[0051] As an alternative implementation, the plant may be genetically manipulated to further include a second herbicide-tolerant polypeptide or its encoding gene, and an effective dose of a protoporphyrinogen oxidase-inhibiting herbicide and a second herbicide may be applied sequentially or simultaneously.

[0052] Other embodiments provide methods for removing unwanted aquatic organisms from a culture medium, including: the step of providing algae to the culture medium, said algae comprising one or more of the group consisting of a polypeptide, a polypeptide variant, a polynucleotide encoding a polypeptide or a polypeptide variant, a recombinant vector comprising the polynucleotide, and recombinant cells comprising the recombinant vector; and the step of applying an effective dose of a protoporphyrinogen oxidase-inhibiting herbicide to the culture medium.

[0053] Technical solution

[0054] Technologies that impart and / or enhance herbicide tolerance to plants or algae are provided.

[0055] In this article, “conferring and / or enhancing herbicide tolerance in plants or algae” or “enhancing herbicide tolerance in plants or algae” is interpreted as conferring tolerance to plants or algae that do not have herbicide tolerance, or enhancing the tolerance of plants or algae that do have herbicide tolerance, or encompassing the broader meaning of both.

[0056] As used herein, the terms “composed of a sequence,” “essentially composed of a sequence,” or “includes a sequence” are used to refer to either the inclusion of the described sequence or the necessary inclusion of the sequence, and can be interpreted as including sequences other than the described sequence and / or containing mutations (additions, deletions, and / or substitutions of amino acids or nucleic acids), provided that the protein, polypeptide, or nucleic acid molecule retains its intrinsic activity and exhibits the intended function.

[0057] In one implementation, one or more polypeptide variants selected from the group consisting of:

[0058] A polypeptide variant comprising, substantially consisting of, or consisting of the following amino acid sequences, wherein one or more amino acids selected from the group consisting of those affecting the interaction between the PPO inhibitory herbicide and the PPO polypeptide SEQ ID NO: 2 (e.g., amino acids located at the binding site of SEQ ID NO: 2 interacting with the PPO inhibitory herbicide) are respectively and independently deleted or substituted by other amino acids different from the original amino acid; or an amino acid sequence having 95% or higher, 98% or higher, or 99% or higher homology to the amino acid sequence of the polypeptide; and

[0059] A polypeptide variant comprising, substantially consisting of, or consisting of the following amino acid sequences, amino acids that affect the interaction between the PPO inhibitory herbicide and the PPO polypeptide SEQ ID NO: 4 (e.g., amino acids located at the binding site of SEQ ID NO: 4 that interacts with the PPO inhibitory herbicide), wherein the amino acids are individually and independently missing or substituted with other amino acids different from the original amino acids at the corresponding positions; or an amino acid sequence having 95% or more, 98% or more, or 99% or more homology with the amino acid sequence of the polypeptide.

[0060] In other embodiments, a polynucleotide encoding a polypeptide or a polypeptide variant is provided, a recombinant vector containing the polynucleotide, and a recombinant cell containing the recombinant vector. The polynucleotide can be engineered such that optimized codons in the codons encoding each amino acid are contained in the cell to be transformed. Optimized codons are readily known to those skilled in the art (e.g., see "http: / / www.genscript.com / codon-opt.html", "http: / / sg.idtdna.com / CodonOpt", etc.).

[0061] In other embodiments, compositions are provided for conferring or enhancing herbicide tolerance to plants or algae, comprising one or more of the following:

[0062] The polypeptide of SEQ ID NO: 2, the polypeptide of SEQ ID NO: 4, its polypeptide variants, and the amino acid sequence of which are 95% or higher, 98% or higher, or 99% or higher homology with the amino acid sequence of the polypeptide or polypeptide variant.

[0063] The polynucleotide encoding the polypeptide or a polypeptide variant;

[0064] A recombinant vector containing the polynucleotide; and

[0065] Recombinant cells containing the recombinant vector.

[0066] For example, the polynucleotide encoding the polypeptide of SEQ ID NO:2 may contain the polynucleotide sequence of SEQ ID NO:1, and the polynucleotide encoding the polypeptide of SEQ ID NO:4 may contain the polynucleotide sequence of SEQ ID NO:3, but is not limited thereto.

[0067] Another implementation provides a transformant of a herbicide-resistant plant or algae, which is transformed with a polynucleotide encoding a polypeptide or a polypeptide variant. The polynucleotide can be engineered such that an optimized codon is included in the codon encoding each amino acid in the cell to be transformed. The optimized codon is readily known to those skilled in the art (e.g., see "http: / / www.genscript.com / codon-opt.html", "http: / / sg.idtdna.com / CodonOpt", etc.).

[0068] In other embodiments, a method for preparing herbicide-resistant plants or algae is provided, comprising the step of transforming algae, or plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, branches, or whole plants with the polynucleotide.

[0069] In other embodiments, methods are provided for conferring or enhancing herbicide tolerance to plants or algae, comprising the step of converting algae, or the plant’s cells, protoplasts, callus, hypocotyls, seeds, cotyledons, branches, or the whole plant, with the polynucleotide.

[0070] The invention will be described in more detail below.

[0071] A polypeptide having the amino acid sequence of SEQ ID NO: 2 or 4, or an amino acid sequence having 95% or higher, 98% or higher, or 99% or higher homology with it, or a variant thereof provided herein, is a PPO protein derived from a prokaryote (e.g., cyanobacteria) and is a herbicide-resistant PPO protein resistant to PPO-inhibiting herbicides. Specifically, a PPO protein derived from *Thermosynechococcus elongatus* BP-1 is provided, named CyPPO10, whose amino acid sequence is represented by SEQ ID NO: 2, and whose encoding gene has a nucleotide sequence represented by SEQ ID NO: 1. Additionally, a PPO protein derived from *Thermosynechococcus sp.* JA-3-3Ab strain is provided, named CyPPO13, whose amino acid sequence is represented by SEQ ID NO: 4, and whose encoding gene has a nucleotide sequence represented by SEQ ID NO: 3.

[0072] In this document, the aforementioned polypeptides and polypeptide variants may refer to herbicide-resistant PPO proteins or herbicide-resistant PPO protein variants that are resistant to PPO-inhibiting herbicides. Furthermore, as used herein, "herbicide-resistant PPO or its variants" may refer to the aforementioned herbicide-resistant PPO protein or herbicide-resistant PPO protein variants, the gene encoding the herbicide-resistant PPO protein or the gene encoding the herbicide-resistant PPO protein variant, or all of them.

[0073] Cyanobacterial PPO proteins themselves possess superior enzymatic activity compared to plant PPOs, and these PPO proteins can confer tolerance to PPO-inhibiting herbicides by incorporating amino acid mutations relative to wild-type PPO proteins within a range that maintains overall enzyme activity. Such amino acid mutations can include substitution, deletion, addition, and / or introduction of one or more amino acids selected from the interaction sites between the PPO protein and the herbicide.

[0074] The PPO protein variants will be described in more detail below.

[0075] One embodiment provides a polypeptide variant comprising, substantially consisting of, or consisting of the following amino acid sequences:

[0076] The amino acid sequence, wherein one or more amino acids selected from the group consisting of those that affect the interaction between the PPO inhibitory herbicide and the PPO polypeptide SEQ ID NO: 2 (CyPPO10) (e.g., amino acids located at the binding site of SEQ ID NO: 2 that interacts with the PPO inhibitory herbicide) are respectively and independently deleted or substituted by other amino acids different from the original amino acid (i.e., the corresponding amino acid in the wild type), or

[0077] An amino acid sequence having 95% or higher, 98% or higher, or 99% or higher homology with the amino acid sequence.

[0078] The deletion or substitution of amino acid residues of the polypeptide of SEQ ID NO: 2 with other amino acids different from the original amino acid (i.e., one or more amino acids selected from the group consisting of the amino acids located at the binding site of the polypeptide of SEQ ID NO: 2 with the PPO inhibitory herbicide) can be one or more amino acid residues selected from the group consisting of N59 (meaning "N(Asn) at position 59"; the representation of the following amino acid residues is interpreted in the same way), S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360 and I408 of the amino acid sequence of SEQ ID NO: 2.

[0079] In one specific embodiment, the polypeptide variant may comprise, consist substantially of, or consist of the following amino acid sequences:

[0080] Amino acid sequences, wherein one or more of the amino acid sequences selected from the group consisting of N59, S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360, and I408 of SEQ ID NO: 2 are respectively and independently deleted or selected from M (Met), V (Val), I (Ile), T (Thr), L (Leu), C (Cys), A (Ala), S (Ser), F (Phe), P (Pro), W (Trp), N (Asn), and Q (Gln). Amino acid substitutions that are different from the original amino acid at the corresponding position in the group consisting of G (Gly), Y (Tyr), D (Asp), E (Glu), R (Arg), H (His), K (Lys), etc. (e.g., amino acid substitutions selected from the group consisting of M (Met), V (Val), I (Ile), T (Thr), L (Leu), C (Cys), A (Ala), S (Ser), R (Arg), W (Trp), G (Gly), etc., and different from the original amino acid at the corresponding position in the wild type), or

[0081] An amino acid sequence having 95% or higher, 98% or higher, or 99% or higher homology with the amino acid sequence.

[0082] For example, peptide variants may contain the following amino acid sequences, consist essentially of the following amino acid sequences, or consist of the following amino acid sequences:

[0083] An amino acid sequence comprising one or more amino acid mutations selected from the amino acid sequence of SEQ ID NO: 2, specifically F360M (meaning "the amino acid residue at position 360 is replaced by M (Met) instead of F (Phe)"; the following amino acid mutations are interpreted in the same manner), F360V, F360I, F360T, F360L, F360C, A167C, A167L, A167I, P303L, V305L, V305M, V305T, N59T, S60T, R89A, R89L, R89V, F161A, V165S, V165C, Q184G, F324V, L327T, I340T, I408R, and I408W, or

[0084] An amino acid sequence having 95% or higher, 98% or higher, or 99% or higher homology with the amino acid sequence.

[0085] More specifically, peptide variants may contain the following amino acid sequences, consist essentially of the following amino acid sequences, or consist of the following amino acid sequences:

[0086] The amino acid sequence comprising the amino acids selected from SEQ ID NO: 2: F360M, F360V, F360I, F360T, F360L, F360C, A167C, A167L, A167I, P303L, N59T, S60T, R89A, R89L, R89V, F161A, V165S, V165C, Q184G, V305L, V305M, V305T, F324V, L327T, I340T, I408R. I408W, P303L+V305L (meaning a mutant or mutation including substitutions of all residues at position 303 from P to L and substitutions of residues at position 305 from V to L; representations of two or more mutations below are interpreted in the same manner), N59T+F360V, S60T+V165S+F360M, S60T+V165S+F360I, S60T+I340T+F360I, R89A+F360M, R89A+F36 0I, R89A+F360L, R89L+F360I, R89V+F360I, R89A+A167L+F360M, R89A+V305T+F360M, V165S+F360M, V165S+F360I, V165S+F360L, V165S+F360V, V165C+F360M, V165C+A167C+F360M, V165C+A167I+F360 One or more amino acid mutations in the group consisting of M, V165C+A167L+F360M, A167L+F360M, A167L+F360I, A167C+F360M, A167C+F360I, A167I+F360M, V305M+F360M, V305T+F360I, V305L+F360M, I408R+F360M, or I408W+F360M, or

[0087] An amino acid sequence having 95% or higher, 98% or higher, or 99% or higher homology with the amino acid sequence.

[0088] Other embodiments provide polypeptide variants that comprise, consist substantially of, or consist of the following amino acid sequences:

[0089] The amino acid sequence, wherein one or more amino acids selected from the group consisting of those that affect the interaction between the PPO inhibitory herbicide and the PPO polypeptide SEQ ID NO: 4 (CyPPO13) (e.g., amino acids located at the binding site of the polypeptide of SEQ ID NO: 4 that interacts with the PPO inhibitory herbicide) are respectively and independently deleted or substituted by other amino acids different from the original amino acid at the corresponding position (i.e., the corresponding amino acid in the wild type), or

[0090] An amino acid sequence having 95% or higher, 98% or higher, or 99% or higher homology with the amino acid sequence.

[0091] The amino acid residues in the deletion of the polypeptide of SEQ ID NO: 4 or the substitution of other amino acids that are different from the original amino acid at the corresponding position (e.g., one or more selected from the group consisting of amino acids located at the binding site of the polypeptide of SEQ ID NO: 3 and the PPO inhibitory herbicide) can be one or more selected from the group consisting of R101, F171, V175, A177, G194, P316, V318, F337, L340, I353 and F373 in the amino acid sequence of SEQ ID NO: 4.

[0092] In one specific embodiment, the polypeptide variant may comprise, consist substantially of, or consist of the following amino acid sequences:

[0093] Amino acid sequences, wherein one or more of the following amino acid sequences selected from the group consisting of R101, F171, V175, A177, G194, P316, V318, F337, L340, I353, and F373 in the amino acid sequence of SEQ ID NO: 4 are respectively and independently deleted or selected from M (Met), V (Val), I (Ile), T (Thr), L (Leu), C (Cys), A (Ala), S (Ser), F (Phe), P (Pro), W (Trp), N (Asn), Q (Gln), G (Gly), Y (Tyr). Amino acid substitutions that are different from the original amino acid at the corresponding position in the wild type, such as those consisting of groups like D(Asp), E(Glu), R(Arg), H(His), and K(Lys), or those selected from groups like M(Met), V(Val), I(Ile), T(Thr), L(Leu), C(Cys), A(Ala), E(Glu), Q(Gln), K(Lys), R(Arg), H(His), and N(Asn), or those selected from groups like M(Met), V(Val), I(Ile), T(Thr), L(Leu), C(Cys), A(Ala), E(Glu), Q(Gln), K(Lys), R(Arg), H(His), and N(Asn), or

[0094] An amino acid sequence having 95% or higher, 98% or higher, or 99% or higher homology with the amino acid sequence.

[0095] For example, peptide variants may contain the following amino acid sequences, consist essentially of the following amino acid sequences, or consist of the following amino acid sequences:

[0096] An amino acid sequence comprising one or more amino acid mutations selected from the group consisting of F373M, F373V, F373I, F373T, F373L, F373C, F373N, F373H, A177C, A177L, A177I, P316A, P316L, V318L, V318M, R101A, F171A, V175C, V175L, G194E, G194Q, G194M, G194K, G194R, F337V, L340T, and I353T, or an amino acid sequence having 95% or higher, 98% or higher, or 99% or higher homology with said amino acid sequence. More specifically, the polypeptide variant may comprise: an ... The amino acid sequence of NO:4 includes F373M, F373V, F373I, F373T, F373L, F373C, F373N, F373H, A177C, A177L, A177I, P316A, P316L, V318L, V318M, R101A, F171A, V175C, V175L, G194E, G194Q, G194M, G194K, G194R, F337V, L340T, I353T, P316L+V318L, P316A+V318L, R101A+F373M, A1 One or more amino acid mutations in the group consisting of amino acid mutations of 77C+F373M, A177I+F373M, A177L+F373M, A177L+F373I, A177L+F373L, A177L+F373T, A177L+F373V, A177C+F373T, A177C+F373V, V175L+F373M, G194E+F373M, G194Q+F373M, G194M+F373M, G194K+F373M, G194R+F373M, or V318M+F373M, or

[0097] An amino acid sequence having 95% or higher, 98% or higher, or 99% or higher homology with the amino acid sequence.

[0098] Peptide variants containing amino acid sequences having the sequence homology described herein (e.g., 95% or higher, 98% or higher, or 99% or higher sequence homology) can maintain enzymatic activity equivalent to peptides having amino acid sequences as a standard for sequence homology identification (e.g., PPO proteins with the aforementioned amino acid mutations). For example, compared to peptides having standard amino acid sequences in plants (whole plants, plant cells or cell cultures, plant tissues, etc.), algae, and / or in vitro, the peptide variants have 5% or higher, 10% or higher, 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, or 95% or higher enzymatic activity and possess the function of conferring herbicide tolerance. The description of sequence homology is used to clarify that the herbicide-tolerant PPO protein variants or peptide variants described herein can contain all sequence mutations within the range of the above conditions (maintaining enzyme activity and possessing the function of conferring herbicide tolerance).

[0099] The names of the amino acids used in the description are listed below:

[0100]

[0101]

[0102] Herbicide-tolerant PPO protein variants can maintain the enzymatic activity of PPO protein and exhibit enhanced herbicide tolerance compared to the wild type.

[0103] Furthermore, herbicide-tolerant PPO protein variants may contain further mutations that exhibit the same biological activity in a polypeptide consisting of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or having the aforementioned amino acid mutations. For example, additional mutations may be amino acid substitutions that do not alter the overall molecular activity, and such amino acid substitutions are well known in the art. In one instance, additional substitutions may be, but are not limited to, substitutions of amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, or Asp / Gly. In some cases, herbicide-tolerant PPO protein variants may have modifications made by one or more of the group consisting of phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, etc. In addition, herbicide-tolerant PPO protein variants may include protein variants in which the structural stability of the protein to heat, pH, etc., or the protein activity is increased through amino acid mutations and / or modifications.

[0104] The term "sequence homology" refers to the degree of similarity to a wild-type or reference amino acid or nucleotide sequence, and any protein may be included within the scope of this invention as long as it contains an amino acid sequence with 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% or higher, or 99% or higher identity with the amino acid sequence of a herbicide-resistant PPO protein and maintains equivalent biological activity to the herbicide-resistant PPO protein variant. These protein homologs may contain active sites equivalent to the target protein. Such homology comparisons can be performed or by means of readily available comparison procedures. Homology between two or more sequences can be calculated as a percentage (%) using online available analytical procedures. Sequence alignment for sequence comparison can be performed by any conventional method known in the relevant art, for example, conventional methods may include, but are not limited to, GAP, BESTFIT, BLAST, and Clustal Omega.

[0105] Herbicide-resistant PPO proteins or variants thereof can be obtained from nature by methods well known in the art. Alternatively, they can be obtained as recombinant proteins using gene recombination technology. When using gene recombination technology, recombinant proteins can be obtained by introducing nucleic acids encoding herbicide-resistant PPO proteins or variants into a suitable expression vector, transforming host cells with this vector to express the target protein, and then collecting the herbicide-resistant PPO proteins or variants from the host cells. After expression in selected host cells, the protein can be separated and purified using general biochemical separation techniques, such as treatment with a protein precipitant (salting out), centrifugation, ultrasonic disruption, ultrafiltration, dialysis, chromatography (e.g., molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography), etc., and these methods can be used in combination to separate the protein with high purity.

[0106] Herbicide-resistant PPO nucleic acid molecules (polynucleotides encoding PPO proteins or variants thereof) can be isolated or prepared using standard molecular biology techniques (such as chemical synthesis or recombination methods), or commercially available techniques can be used.

[0107] In one specific embodiment, using a herbicide tolerance testing system employing PPO-deficient *E. coli* BT3 (ΔPPO), PPO proteins were found to exhibit broad herbicide tolerance to nine representative families of PPO-inhibiting herbicides classified according to their chemical structures. They were also found to be expressible in plant chloroplasts via transport peptides (TPs). Furthermore, PPO proteins were found to be expressed in the *Arabidopsis thaliana* ecotype Columbia via plant expression vectors. Germination and growth were observed even in plants treated with PPO-inhibiting herbicides. Moreover, genetic studies confirmed the inheritance of these herbicide tolerance traits in the next generation.

[0108] Therefore, the PPO protein and its variants provided in this article can be introduced into plants or algae to enhance their herbicide tolerance.

[0109] In this article, herbicides refer to active ingredients that kill, control, or otherwise adversely alter the growth of plants or algae. Furthermore, herbicide tolerance or herbicide tolerance refers to the reduction or elimination of growth inhibition in plants compared to normal or wild-type plants, even after treatment with herbicides that typically kill or inhibit their growth, thus allowing the plant to continue growing. Herbicides include those that inhibit protoporphyrinogen oxidase (PPO) in plants or algae. Based on their chemical structure, these PPO-inhibiting herbicides can be classified into pyrimidine diones, diphenyl ethers, phenylpyrazoles, N-phenylphthalimides, thiadiazoles, etc. diazoles, triazolones, Alzolidinediones and other herbicides.

[0110] As a specific implementation, pyrimidine dione herbicides include, but are not limited to, flupropargyl, pyrimisulfuron, bispyribac-sodium, and tefenazate.

[0111] Diphenyl ether herbicides include, but are not limited to, flufenoxuron, ethoxyflufenoxuron, bensulfuron, trifluralin, chlorpyrifos, clethodim, methoxyflufenoxuron, chlorintrofen, ethoxyflufenoxuron, and flunisulfuron.

[0112] Phenylepiazole herbicides include, but are not limited to, flupyrazole and isopyrazosulfuron.

[0113] Phenyl phthalimide herbicides include, but are not limited to, propyzoxystrobin, indole-3-propargyl, and fluroxypyr.

[0114] Phenyl ester herbicides include, but are not limited to, pyrrolidino (2,4-dichlorophenyl-1-pyrrolidinecarboxylate) and pyrrolidino carbamate analogs (e.g., O-phenylpyrrolidino- and piperidinocarbamate analogs (see “Ujjana B. Nandihalli, Mary V. Duke, Stephen O. Duke, Relationships between molecular properties and biological activities of O-phenylpyrrolidino- and piperidinocarbamate herbicides., J. Agric. Food Chem., 1992, 40(10) 1993-2000”). In one embodiment, the carbamate analogue of *Pyrrolizinus oleracea* may be one or more selected from the group consisting of: phenyl pyrrolidine-1-carboxylate (CAS No. 55379-71-0), 1-pyrrolidinecarboxylic acid, 2-chlorophenyl ester (CAS No. 143121-06-6), 4-chlorophenylpyrrolidine-1-carboxylate (CAS No. 1759-02-0), carbamic acid, diethyl-, 2,4-dichloro-5-(2-propynoxy)phenyl ester (9CI) (CAS No. 143121-07-7), 1-pyrrolidinecarboxylic acid, 2,4-dichloro-5-hydroxyphenyl ester (CAS No. 143121-08-8), 2,4-dichloro-5-(methoxycarbonyl)phenylpyrrolidine-1-carboxylate (CAS No. 133636-94-9), 2,4-dichloro-5-[(prop-2-oxy)carbonyl] Phenylated pyrrolidine-1-carboxylic acid ester (CAS No. 133636-96-1), 1-piperidinic acid, 2,4-dichloro-5-(2-propynoxy)phenyl ester (CAS No. 87374-78-5), 2,4-dichloro-5-(prop-2-yn-1-yloxy)phenylpyrrolidine-1-carboxylic acid ester (CAS No. 87365-63-7), 2,4-dichloro-5-(prop-2-yn-1-yloxy) Phenyl 4,4-difluoropiperidine-1-carboxylic acid ester (CAS No. 138926-22-4), 1-pyrrolidinecarboxylic acid, 3,3-difluoro-,2,4-dichloro-5-(2-propyn-1-yloxy)phenyl ester (CAS No. 143121-10-2), 4-chloro-2-fluoro-5-[(propyn-2-yloxy)carbonyl]phenylpyrrolidine-1-carboxylic acid ester (CAS No. 133636-98-3), etc.

[0115] Thiadiazole herbicides include, but are not limited to, methamidophos and thiamethoxam.

[0116] Diazole herbicides include propyne. Herbicides and Herbicides, but not limited to these.

[0117] Triazoline herbicides include, but are not limited to, oxychlorpyrifos, mesotrione, and acetamiprid.

[0118] Alzolidinedione herbicides include cyclopentadione. Herbicides, but not limited to these.

[0119] Other herbicides include, but are not limited to, bispyribac-sodium, flupyridaben, and flupyrazosulfuron.

[0120] The herbicide tolerance PPO gene provided herein can be introduced into plants or algae using various methods known in the art, preferably by using an expression vector for plant or algal transformation.

[0121] In the case of plant transformation, a suitable promoter that can be included in the vector can be any promoter commonly used in the art for introducing genes into plants. For example, promoters may include, but are not limited to, the SP6 promoter, T7 promoter, T3 promoter, PM promoter, maize ubiquitin promoter, cauliflower mosaic virus (CaMV) 35S promoter, cauliflower base synthase (nos) promoter, Scrophularia mosaic virus 35S promoter, sugarcane baculovirus promoter, Commelina communis yellow mottle virus promoter, light-inducible promoters from the small subunit of ribulose-1,5-bisphosphate carboxylase (ssRuBisCO), rice cytosol triose phosphate isomerase (TPI) promoter, Arabidopsis adenine phosphoribosyltransferase (APRT) promoter, octopus base synthase promoter, and BCB (copper-binding protein) promoter.

[0122] Furthermore, the vector may include a poly A signal sequence that induces 3′-terminal polyadenylation, for example, it may include the NOS 3′-terminus of the alkaloid synthase gene from Agrobacterium tumefaciens, the octopaloid synthase terminator from Agrobacterium tumefaciens, the 3′-terminus of the protease inhibitor I or II gene from tomato or potato, the CaMV 35S terminator, the rice α-amylase terminator RAmy1A, and the betaine terminator, but is not limited thereto.

[0123] In addition, chloroplast-specific promoters, nuclear promoters, constitutive promoters, or inducible promoters can be used as promoters to introduce genes into algae. The herbicide-tolerant PPO gene or its variants provided herein can be designed to be operatively linked to the 5′UTR or 3′UTR, thereby expressing function in the algal nucleus. Furthermore, the vector can further contain transcriptional regulatory sequences suitable for algal transformation. Recombinant genes conferring herbicide tolerance can be integrated into the genome of the host algal chloroplast or the genome of the cell nucleus, but are not limited thereto.

[0124] In addition, in the vector, the transport peptide required to target chloroplasts can be linked to the 5′ end of the PPO gene to express the herbicide-tolerant PPO gene in the chloroplasts.

[0125] In addition, optionally, the vector may further include a gene encoding a selection marker as a reporter molecule, examples of which may include, but are not limited to, antibiotic (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, bleomycin, chloramphenicol, etc.) or herbicide (glyphosate, glufosinate, glufosinate, etc.) tolerance genes.

[0126] Furthermore, recombinant vectors for plant expression can include Agrobacterium binary vectors, co-integration vectors, or general vectors that do not have a T-DNA region but are designed for expression in plants. A binary vector refers to a vector containing two independent vector systems: one containing a plasmid responsible for migration, consisting of the left (LB) and right (RB) boundaries of a Ti (tumor-inducible) plasmid; and the other containing a target gene transfer. This vector may include a promoter region and a polyadenylation signal sequence for expression in plants.

[0127] When using binary or co-integrative vectors, the strain used to transform the recombinant vector into plants is preferably Agrobacterium (Agrobacterium-mediated transformation). Agrobacterium tumefaciens or Agrobacterium rhizogenes can be used in this regard. Alternatively, when using vectors without a T-DNA region, recombinant plasmids can be introduced into plants using electroporation, particle bombardment, polyethylene glycol-mediated uptake, etc.

[0128] Plants genetically transformed using the above methods can be redifferentiated into plants using standard techniques known in the art through callus induction, rhizome growth, and soil adaptation.

[0129] In this article, the plants that undergo transformation are understood through the meanings of plant cells (including cells in suspension culture), protoplasts, callus tissue, hypocotyls, seeds, cotyledons, branches, and mature plants.

[0130] Furthermore, the scope of transformants includes transformants of which the introduced gene has been introduced, as well as their clones or progeny (T1, T2, T3, T4, T5, or any progeny). For example, transformed plants also include plants with heritable herbicide tolerance traits as sexual and asexual progeny of plants transformed with the genes provided herein. The scope of the invention also includes all mutants and variants exhibiting characteristics of the initially transformed plant, and all hybridization and fusion products of plants transformed with the genes provided herein. Furthermore, the scope of the invention also includes portions of plants, such as seeds, flowers, stems, fruits, leaves, roots, tubers, and / or rhizomes, derived from a transformed plant or its progeny pre-transformed by the methods of the invention, and portions composed of at least a portion of transformed cells.

[0131] The plants to which this invention is applied are not particularly limited, but include monocotyledonous or dicotyledonous plants. Furthermore, the plants include herbaceous or woody plants. Monocotyledonous plants may include plants belonging to the following families: Alismataceae, Hydrocharitaceae, Juncaginaceae, Scheuchzeriaceae, Potamogetonaceae, Najadaceae, Zosteraceae, Liliaceae, Haemodoraceae, Agavaceae, Amaryllidaceae, Dioscoreaceae, and Pontederiaceae. The families include, but are not limited to, Iridaceae, Burmannaceae, Juncaceae, Commelinaceae, Eriocaulaceae, Gramineae / Poaceae, Araceae, Lemnaceae, Sparganiaceae, Typhaceae, Cyperaceae, Musaceae, Zingiberaceae, Cannaceae, and Orchidaceae.

[0132] Dicotyledonous plants can include those belonging to the following families: Diapensiaceae, Clethraceae, Pyrolaceae, Ericaceae, Myrsinaceae, Primulaceae, Plumbaginaceae, Ebenaceae, Styracaceae, Symplococeae, Oleaceae, Loganiaceae, Gentianaceae, and Menthaaceae. Menyanthaceae, Apocynaceae, Asclepiadaceae, Rubiaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Lamiaceae, Solanaceae, Scrophulariaceae, Bignoniaceae, Acanthaceae, Pedaliaceae, etc. Orboranchaceae, Gesneriaceae, Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, Adoxaceae, Valerianaceae, Dipsacaceae, Campanulaceae, Compositae, Myricaceae, Juglandaceae, Salicaceae The family names of the Chinese family (including Betulaceae, Fagaceae, Ulmaceae, Moraceae, Urticaceae, Santalaceae, Loranthaceae, Polygonaceae, Phytolaccaceae, Nyctaginaceae, Aizoaceae, Portulacaceae, Caryophyllaceae, and Chenopodiaceae) are listed below.Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cercidiphyllaceae, Ranunculaceae, Berberidaceae, Lardizabalaceae, Menispermaceae, Nymphaeaceae, Ceratophyllaceae, Cabombaceae, and others. Saururaceae, Piperaceae, Chloranthaceae, Aristolochiaceae, Actinidiaceae, Theaceae, Guttiferae, Droseraceae, Papaveraceae, Capparidaceae, Cruciferae, Platanaceae, Hamamelidaceae, Crassulaceae Eucommiaceae, Saxifragaceae, Pittosporaceae, Rosaceae, Leguminosae, Oxalidaceae, Geraniaceae, Tropaeolaceae, Zygophyllaceae, Linaceae, Euphorbiaceae, Callitrichaceae, Rutaceae, Simaroubaceae (Simaroubaceae), Meliaceae, Polygalaceae, Anacardiaceae, Aceraceae, Sapindaceae, Hippocastanaceae, Sabiaceae, Balsaminaceae, Aquifoliaceae, Celastraceae, Staphyleaceae, Buxaceae, EmpetraceaeRhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvaceae, Sterculiaceae, Thymelaeaceae, Elaeagnaceae, Flacourtiaceae, Violaceae, Passifloraceae, Tamaricaceae The families listed include, but are not limited to, Elatinaceae, Begoniaceae, Cucurbitaceae, Lythraceae, Punicaceae, Onaraceae, Haloragaceae, Alangiaceae, Cornaceae, Araliaceae, and Umbelliferae / Apiaceae.

[0133] In one specific embodiment, the plant may be one or more selected from the group consisting of: food crops, such as rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; vegetable crops, such as Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, thin-skinned melon, pumpkin, welshanion, anion, and carrot; and special-purpose crops, such as ginseng, tobacco, cotton, soilage, forage, sesame, sugarcane, sugar beets, and perilla. (sp.), peanut, rapeseed, grass and castor oil plants; fruit trees, such as apple, pear, jujube, peach, kiwi, grape, citrus, persimmon, plum, apricot and banana trees; woody plants, such as pine, palm and eucalyptus; flowering crops, such as rose, gladiolus, gerbera, carnation, chrysanthemum, lily and tulip; forage crops, such as ryegrass, red clover, fruit tree grass, alfalfa, tall fescue and perennial ryegrass, but not limited to these. In a specific implementation, the plant may be one or more selected from the group consisting of: dicotyledonous plants, such as Arabidopsis thaliana, potato, eggplant, tobacco, red pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, beet, sweet potato, celery, carrot, water celery, parsley, Chinese cabbage, cabbage, radish, watermelon, thin-skinned melon, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean and pea; and monocotyledonous plants, such as rice, wheat, barley, corn, sorghum, etc., but not limited thereto.

[0134] The algae used in applying this invention are not particularly limited to, but include, prokaryotic or eukaryotic algae. For example, the algae can be cyanobacteria, green algae, red algae, brown algae, macroalgae, or microalgae.

[0135] Cyanobacteria include: Chroococcales phylum (e.g., genera *Aphanocapsa*, *Aphanothece*, *Chamaesiphon*, *Chondrocystis*, *Chroococcus*, *Chroogloeocystis*, *Crocosphaera*, *Cyanobacterium*, *Cyanobium*, *Cyanodictyon*, *Cyanosarcina*, *Cyanothece*, *Dactylococcopsis*, and *Croococcales*). Genus (Gloeocapsa), Gloeothece, Halothece, Johannesbaptistia, Merismopedia, Microcystis, Radiocystis, Rhabdoderma, Snowella, Synechococcus, Synechocystis, Thermosynechococcus, Woronichinia), Gloeobacteria phylum, Nostocalesphylum (e.g., Microchaetaceae, Nostocaceae, Rivulariaceae, Scytonemataceae), Oscillatoriales phylum (e.g.,*Arthronema*, *Arthrospira*, *Blennothrix*, *Crinalium*, *Geitlerinema*, *Halomicronema*, *Halospirulina*, *Hydrocoleum*, *Jaaginema*, *Katagnymene*, *Komvophoron*, *Leptolyngbya*, *Limnothrix*, *Lyngbya*, *Microcole* us), Oscillatoria, Phormidium, Planktothricoides, Planktothrix, Plectonema, Pseudanabaena, Pseudophormidium, Schizothrix, Spirulina, Starria, Symplocosmium, Trichodesmium, Tychonema, Pleurocapsales Phyllium (e.g., *Chroococcidiopsis*, *Dermocarpa*, *Dermocarpella*, *Myxosarcina*, *Pleurocapsa*, *Solentia*, *Stanieria*, *Xenococcus*), Prochlorales phylum, or Stigonematales Phylum (e.g., *Capsosira*, *Chlorogloeopsis*, *Fischerella*, *Hapalosiphon*, *Mastigocladopsis*, *Mastigocladus*, *Nostochopsis*, *Stigonema*, *Symphyonema*, *Symphonemopsis*, *Umezakia*, *Westiellopsis*), etc.

[0136] As another example of algae, examples could be the phylum Chlorophyta, the genus Chlamydomonas, the order Volvacales, the genus Dunaliella, the genus Scenedesmus, the genus Chlorella, or the genus Hematococcm.

[0137] Other examples of algae include *Phaeodactylum tricornutum*, *Amphiprora hyaline*, *Amphora* spp., *Chaetocerosmuelleri*, *Navicula saprophila*, *Nitzschia communis*, *Scenedesmus dimorphus*, *Scenedesmus obliquus*, *Tetraselmis suecica*, *Chlamydomonas reinhardtii*, *Chlorella vulgaris*, *Haematococcus pluvialis*, *Neochloris oleoabundans*, *Synechococcus elongatus*, *Botryococcus braunii*, and *Gloeobacter*. violaceus), Synechocystis, Thermosynechococcus elongatus, Nannochloropsis oculata, Nannochloropsis salina, Nannochloropsis gaditana, Isochrysis galbana, Botryococcus sudeticus, Euglena gracilis, Neochlorisoleoabundans, Nitzschia palea, Pleurochrysis carterae, Tetraselmis chuii, Pavlova spp., Aphanococcus aspp., Synechosystis Examples include *Nannochloris* spp. and others. However, the scope is not limited to the species listed above and can include algae belonging to other genera and families.

[0138] Plants or algae that have been introduced with the herbicide-tolerant PPOs or variants thereof described herein may exhibit tolerance to two or more PPO-inhibiting herbicides.

[0139] Therefore, by using two or more herbicides that inhibit PPO sequentially or simultaneously, the techniques presented herein can be used to control weeds or remove unwanted aquatic organisms.

[0140] One embodiment provides a method for controlling weeds in farmland, comprising the steps of providing the farmland with plants containing the herbicide-tolerant PPO protein, its variants, or their encoding genes, and applying an effective dose of a protoporphyrinogen oxidase-inhibiting herbicide to the farmland.

[0141] Another embodiment provides a method for removing unwanted aquatic organisms from a culture medium, comprising the steps of providing the culture medium with algae containing the aforementioned herbicide-resistant PPO protein, its variants, or its encoding gene, and applying an effective dose of a protoporphyrinogen oxidase-inhibiting herbicide to the culture medium.

[0142] Furthermore, the herbicide-resistant PPO protein, its variants, or their encoding genes provided in this article can be used in combination with a second herbicide-resistant polypeptide or its encoding gene.

[0143] Therefore, plants or algae incorporating the herbicide-tolerant PPOs provided herein can exhibit tolerance to two or more herbicides with different mechanisms of action. In this invention, two or more different herbicides (including PPO-inhibiting herbicides) with different mechanisms of action can be used sequentially or simultaneously to control weeds and / or remove unwanted aquatic organisms. Hereinafter, herbicides with mechanisms of action different from PPO-inhibiting herbicides are referred to as "second herbicides".

[0144] One embodiment provides a composition for conferring or enhancing herbicide tolerance to plants or algae, comprising the aforementioned herbicide-tolerant PPO protein, a variant thereof, or its encoding gene; and a second herbicide-tolerant polypeptide or its encoding gene.

[0145] Another embodiment provides a herbicide-tolerant transformant of a plant or algae, a clone thereof or its progeny, said transformant, clone thereof or its progeny comprising the herbicide-tolerant PPO protein described above, its variants or its encoding gene; and a second herbicide-tolerant polypeptide or its encoding gene.

[0146] Other embodiments provide a method for preparing herbicide-tolerant plants or algae, the method comprising the steps of transforming algae, or plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, branches, or whole plants, with the above-mentioned herbicide-tolerant PPO protein, its variants, or its encoding gene; and a second herbicide-tolerant polypeptide or its encoding gene.

[0147] Other embodiments provide a method for controlling weeds in farmland, comprising: providing plants to the farmland, said plants comprising the above-mentioned herbicide-resistant PPO protein, its variants or its encoding gene; a second herbicide-resistant polypeptide or its encoding gene; and applying an effective dose of a protoporphyrinogen oxidase-inhibiting herbicide to the farmland.

[0148] Other embodiments provide a method for removing unwanted aquatic organisms from a culture medium, comprising: providing algae to the culture medium containing a herbicide-resistant PPO protein, a variant thereof, or a gene encoding therefor; a second herbicide-resistant polypeptide or a gene encoding therefor; and applying an effective dose of a protoporphyrinogen oxidase-inhibiting herbicide to the culture medium.

[0149] For example, plants or algae also include second herbicide tolerance peptides or their encoding genes, thereby giving them new and / or enhanced tolerance to the second herbicide.

[0150] For example, a second herbicide may include, but is not limited to, cell division inhibitory herbicides, photosynthesis inhibitory herbicides, amino acid synthesis inhibitory herbicides, plastid inhibitory herbicides, cell membrane inhibitory herbicides, and / or any combination thereof. Examples of a second herbicide include glyphosate, glufosinate, dicamba, 2,4-D (2,4-dichlorophenoxyacetic acid), ALS (acetyllactone synthase) inhibitory herbicides (e.g., imidazolinone, sulfonylurea, triazolidine, sulfaniline, pyrimidinethiobenzoic acid, etc.), photosystem II inhibitory herbicides, phenylurea-based herbicides, plastid inhibitory herbicides, bromobenzonitrile-based herbicides, and / or any combination thereof.

[0151] For example, a second herbicide-resistant polypeptide can be exemplified as one or more selected from the group consisting of: glyphosate-resistant EPSPS (glyphosate-resistant 5-enolpyruvate-shikimate-3-phosphate synthase), GOX (glyphosate oxidase), GAT (glyphosate-N-acetyltransferase), or glyphosate decarboxylase; glufosinate-resistant PAT (glufosinate-N-acetyltransferase); dicamba-resistant DMO (dicamba monooxygenase); 2,4-D-resistant 2,4-D monooxygenase. Oxygenases or AAD (aryloxyalkyl ester dioxygenase); ALS-inhibiting sulfonylurea herbicide-resistant ALS (acetyllactate synthase), AHAS (acetylhydroxylate synthase), or AtAHASL (acetylhydroxylate synthase large subunit); photosystem II-inhibiting herbicide-resistant photosystem II protein D1; phenylurea-based herbicide-resistant cytochrome P450; plastid-inhibiting herbicide-resistant HPPD (hydroxyphenylpyruvate dioxygenase); bromobenzonitrile herbicide-resistant nitrile hydrolases; and combinations thereof, but not limited to these.

[0152] Further, the gene encoding the second herbicide tolerance polypeptide can be exemplified as one or more selected from the group consisting of: glyphosate herbicide tolerance genes cp4 epsps, epsps(AG), mepsps, 2mepsps, goxv247, gat4601, or gat4621; glufosinate herbicide tolerance genes bar, pat, or pat(SYN); dicamba herbicide tolerance genes dmo; 2,4-D herbicide tolerance genes AAD-1 or AAD-12; ALS-inhibiting sulfonylurea herbicide tolerance genes ALS, GM-HRA, S4-HRA, ZM-HRA, Csr1, Csr1-1, Csr1-2, SurA, or SurB; photosystem II-inhibiting herbicide tolerance genes psbA; phenylurea herbicide tolerance genes CYP76B1; and isocyanate herbicide tolerance genes. The HPPDPF W336 gene for tolerance to cyclohexane herbicides; the bxn gene for tolerance to bromobenzonitrile herbicides; and combinations thereof, but not limited thereto.

[0153] Beneficial effects

[0154] Variants of the herbicide-tolerant PPO protein provided herein, or their encoding genes, can be applied to plants or algae to confer and / or enhance superior herbicide tolerance traits, and to use herbicides for selective control, thereby economically controlling weeds or removing aquatic organisms. Attached Figure Description

[0155] Figure 1 This is a spectrum of the pACBB vector.

[0156] Figure 2The cell growth levels of PPO-deficient BT3 Escherichia coli transformed with pACBB-eGFP vector control (V), PPO-susceptible Arabidopsis thaliana, PPO1 gene (AtPPO1 WT), PPO-tolerant Arabidopsis thaliana PPO1 mutant gene (AtPPO1 SLYM), CyPPO10 gene (Cy10 WT), and CyPPO13 gene (Cy13 WT) are shown after treatment with 0 μM, 100 μM, or 400 μM tafenamic acid, respectively.

[0157] Figure 3 It is a map of the pET303-CT-His vector.

[0158] Figure 4 A schematic diagram of a recombinant vector for a fusion protein is shown, in which MBP (maltose-binding protein) and PPO protein are fused.

[0159] Figure 5 This is a spectrum of the pMAL-c2X vector.

[0160] Figure 6 This is an exemplary schematic diagram of the structure of a binary vector used for plant transformation of the CyPPO gene.

[0161] Figure 7 The results are from Western blot analysis, showing the expression levels of CyPPO variant proteins in T2 Arabidopsis thaliana transformed with either the CyPPO10 variant (F360I or F360M) or the CyPPO13 variant (F373M).

[0162] Figure 8 The damage levels of Arabidopsis transformants (T3) transformed with the wild-type CyPPO10 or CyPPO13 gene are shown when treated with 1 μM tefena. Col-O indicates non-transgenic Arabidopsis.

[0163] Figure 9 The damage levels of Arabidopsis transformants (T2) transformed with genes encoding CyPPO10 variants (F360C, F360I, F360L, F360M, F360V, F360T, A167C, A167L, A167L+F360M or A167C+F360I) are shown when treated with tefena at concentrations of 1 μM, 5 μM or 25 μM.

[0164] Figure 10 The damage levels of Arabidopsis transformants (T2) transformed with genes encoding CyPPO13 variants (A177C, F373C, F373I, F373M, A177L+F373L, or A177L+F373I) are shown when treated with tefena at concentrations of 1 μM or 10 μM.

[0165] Figure 11 The cell growth levels of PPO-deficient BT3 Escherichia coli (ΔPPO) transformants, converted with wild-type CyPPO10 gene (denoted as CY10 WT) or various CyPPO10 mutant genes, are shown when treated with tefena at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM.

[0166] Figure 12 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from CY10 WT or various CyPPO10 mutant genes are shown when treated with pyrimisulfuron at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM.

[0167] Figure 13 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from CY10 WT or various CyPPO10 mutant genes are shown when treated with flufenoxuron at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM.

[0168] Figure 14 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from CY10 WT or various CyPPO10 mutant genes are shown when treated with trifluralin at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM.

[0169] Figure 15 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from CY10 WT or various CyPPO10 mutant genes are shown when treated with propyzamide at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM.

[0170] Figure 16 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from CY10 WT or various CyPPO10 mutant genes are shown when treated with mesotrione at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM.

[0171] Figure 17 The results show the effects of using cyclopentyl alcohol at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM. Cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted with CY10 WT or various CyPPO10 mutant genes during cyclophosphamide treatment.

[0172] Figure 18 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from CY10 WT or various CyPPO10 mutant genes are shown when treated with fluoxetine at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM.

[0173] Figure 19 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from CY10 WT or various CyPPO10 mutant genes are shown when treated with 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM of bispyribac-sodium.

[0174] Figure 20 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from CyPPO13 wild-type gene (denoted as Cy13 WT) or various CyPPO13 mutant genes are shown when treated with tefena at concentrations of 0 μM, 5 μM, 25 μM and 50 μM.

[0175] Figure 21 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from Cy13WT or various CyPPO13 mutant genes are shown when treated with 0 μM, 5 μM, 25 μM and 50 μM bensulfuron-methyl.

[0176] Figure 22 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from Cy13 WT or various CyPPO13 mutant genes are shown when treated with flufenoxuron at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM.

[0177] Figure 23 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from Cy13 WT or various CyPPO13 mutant genes are shown when treated with trifluralin at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM.

[0178] Figure 24 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from Cy13WT or various CyPPO13 mutant genes are shown when treated with propyzamide at concentrations of 0 μM, 5 μM, 25 μM and 50 μM.

[0179] Figure 25The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from Cy13 WT or various CyPPO13 mutant genes are shown when treated with mesotrione at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM.

[0180] Figure 26 The results show the effects of using cyclopentyl alcohol at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM. Cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from Cy13 WT or various CyPPO13 mutant genes during cyclophosphamide treatment.

[0181] Figure 27 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from Cy13 WT or various CyPPO13 mutant genes are shown when treated with fluoxetine at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM.

[0182] Figure 28 The cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from Cy13 WT or various CyPPO13 mutant genes are shown when treated with 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM of bispyribac-sodium.

[0183] Figure 29 The results show the effects of using concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM. Cell growth levels of PPO-deficient BT3 (ΔPPO) transformants converted from Cy13 WT or various CyPPO13 mutant genes during cyclophosphamide treatment.

[0184] Figure 30 This is a spectrum of the pET29b vector.

[0185] Figures 31a to 31c Seed germination results of Arabidopsis thaliana transformants, converted with the wild-type CyPPO10 or CyPPO13 gene or their mutant genes, are shown on 1 / 2 MS medium containing various herbicides on day 7 post-sowing. Col-0 indicates non-transgenic Arabidopsis thaliana.

[0186] Figure 32The damage levels of Arabidopsis transformants (T3) transformed with genes encoding CyPPO10 variants (F360I, F360L, F360M, A167C+F360I, A167C+F360M, or V305M+F360M) are shown when treated with 25 μM tebufenozide or 100 μM pyrimethanil.

[0187] Figure 33a The damage levels of Arabidopsis transformants (T3) transformed with the gene encoding the CyPPO10 variant (F360I or A167L+F360M) were shown when treated with 50 μM of tefenazate, pyrimisulfuron, propyzoxystrobin, or mesotrione.

[0188] Figure 33b The damage levels of Arabidopsis transformants (T3) transformed with the gene encoding the CyPPO13 variant (A177L+F373L or A177L+F373I) are shown when treated with 50 μM of pyrimethanil, tefenazate, propyzoxystrobin, mesotrione, ethoxyfluazuron, or bispyribac-sodium.

[0189] Figure 34 The damage levels of Arabidopsis thaliana transformants (T4) transformed with CyPPO10 F360I are shown when treated with 15 μM tebufenozide and 150 μM fenbufenozide.

[0190] Figure 35 The damage levels of Arabidopsis thaliana transformants (T5) transformed with CyPPO10 F360I are shown when treated with 15 μM tebufenozide or 150 μM fenbufenozide. Col-0 indicates non-transgenic Arabidopsis thaliana.

[0191] Figure 36 The results of Western blot analysis show the expression of CyPPO10 F360I protein in Arabidopsis transformants (T4 or T5) transformed with CyPPO10 F360I.

[0192] Figure 37 This is a map of the pB2GW7.0 binary carrier.

[0193] Figure 38 The damage levels in leaves of T0 soybean transformed with the CyPPO10 A167L+F360M mutant gene are shown when treated with 5 μM or 15 μM tefena. Kwangan soybean refers to non-GMO soybean (cultivar).

[0194] Figure 39DNA (Southern) blot results were provided, showing the presence of transgenes in soybeans transformed with CyPPO10 A167L+F360M.

[0195] Figure 40 The herbicide tolerance of T1 transgenic soybean (CyPPO10 A167L+F360M) was shown 5 days after spraying with 25 μM tebufenozide or 150 μM fenproxil fumarate. Guang'an soybean refers to non-transgenic soybean (cultivar).

[0196] Figure 41 The cell growth levels of BT3(ΔPPO) Escherichia coli transformed with the mutant gene CyPPO10 when cultured in a medium containing herbicides are shown. Detailed Implementation

[0197] The present invention will be described in detail below by way of examples. However, the following examples are for illustrative purposes only, and the present invention is not limited to the following examples.

[0198] Example 1. Isolation of the PPO gene from prokaryotes

[0199] PPO genes were collected from the Genbank database of *Synechococcus BP-1* and *Synechococcus* JA-3-3Ab, and synthesized based on codon-optimized information for efficient herbicide resistance screening in *E. coli* BT3. The synthesized PPO genes were amplified and cloned into the pACBB vector using primers from Table 1 under the following conditions.

[0200] A 50 μL (50 μl) PCR reaction mixture was prepared by mixing 1 μl template (synthetic DNA for each gene), 5 μl 10X buffer, 1 μl dNTP mixture (10 mM each), 1 μl forward primer (see Table 1; 10 μM), 1 μl reverse primer (see Table 1; 10 μM), 40 μl DDW, and 1 μl Pfu-X (Solgent, 2.5 units / μl), and amplified under the following conditions: 94 °C for 4 min, 1 cycle; 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1.5 min, 25 cycles; 72 °C for 5 min, 1 cycle.

[0201] Specifically, the PPO isolated from *Synechococcus slenderus* BP-1 was named CyPPO10, and the PPO isolated from *Synechococcus* JA-3-3Ab strain was named CyPPO13.

[0202] Table 1

[0203]

[0204] Example 2. Herbicide tolerance of CyPPO10 and CyPPO13

[0205] The tolerance of CyPPO10 and CyPPO13 to herbicides was tested using PPO-deficient Escherichia coli.

[0206] After transforming PPO-deficient BT3 Escherichia coli (ΔPPO) with CyPPO10 or CyPPO13, the transformed BT3 (ΔPPO) was cultured on LB agar plates containing a PPO-inhibiting herbicide to detect the growth level of the transformed BT3 (ΔPPO). The BT3 (ΔPPO) strain was obtained from Hokkaido University (Japan). The BT3(ΔPPO) strain lacks hemG-type PPO and is kanamycin resistant (see Watanabe et al., Dual targeting of spinach protoporphyrinogen oxidase II to mitochondria and chloroplasts by alternative use of two in-frame inhibition codons, JBC 2001 276(23): 20474-20481; Che et al., Molecular Characterization and Subcellular Localization of Protoporphyrinogen Oxidase in Spinach Chloroplasts, Plant Physiol. 2000 Sep; 124(1): 59-70″).

[0207] The specific testing process is as follows:

[0208] The CyPPO10 and CyPPO13 genes were cloned into the pACBB vector (plasmid #32551; Addgene; see also). Figure 1 )middle.

[0209] Specifically, the PCR products amplified in Example 1 were treated with BamHI and XhoI restriction enzymes (New England Biolabs) and ligated with the pACBB-eGFP vector treated with the same restriction enzymes.

[0210] Treatment with restriction enzymes was carried out under the following conditions:

[0211] 30 μl of PCR product, 0.5 μl each of BamHI and XhoI (New England Biolabs), 4 μl of 10X buffer, and 5.5 μl of water; the restriction enzyme reaction was carried out at 37°C for 1 hour.

[0212] The ligation reaction is carried out under the following conditions:

[0213] 0.5 μl T4 DNA ligase (RBC), 1 μl A buffer, 1 μl B buffer, PCR product and vector treated with restriction enzyme, total 10 μl; run at 22°C for 30 minutes.

[0214] The cloned plasmids were added to 100 μl of BT3 competent cells (Hokkaido University; Japan) for transformation via heat shock. E. coli transformed with each PPO gene were cultured in LB (Luria-Bertani) agar medium containing chloramphenicol (Duchefa).

[0215] For seed culture of *E. coli* transformed with each gene, incubate each individual *E. coli* transformant colony as provided above in 3 ml LB broth containing chloramphenicol overnight (220 rpm, 37°C), and subculture 50 to 100 μl in fresh 3 ml LB broth until the absorbance (OD) reaches a certain level. 600 The absorbance (OD) was 0.5 to 1, diluted with LB broth to a value of 0.5 to 1. 600 The concentration was 0.5. The diluted solution was then diluted five times consecutively with LB broth at a factor of one-tenth. Subsequently, 10 μl of each diluted solution was added dropwise to LB agar medium (petition dish) containing 0 μM, 100 μM, and 400 μM teifenafil. The LB agar medium was incubated at 37°C under light conditions, and the level of growth inhibition was observed after 16 to 20 hours of incubation.

[0216] For comparison, the pACBB-eGFP vector (plasmid #32551; Addgene; see below) was used. Figure 1BT3 E. coli transformants transformed with the wild-type Arabidopsis PPO1 gene (AtPPO1 WT, wild-type AtPPO1; PPO susceptibility) (SEQ ID NO: 6) were tested in the same way as BT3 E. coli transformants transformed with the Arabidopsis mutant PPO1 gene encoding a mutant AtPPO1 (AtPPO1SLYM, SEQ ID NO: 7), which has amino acid substitutions based on the amino acid sequence of wild-type AtPPO1 (SEQ ID NO: 5) of Y426M (tyrosine substituted for methionine at amino acid residue 426) and S305L (serine substituted for leucine at amino acid residue 305). (Li et al. Development of protoporphyrinogen oxidase as an efficient selection marker for agrobacterium tumefaciens-mediated maize transformation) transformation of maize).Plantphysiol.2003 133:736-747).

[0217] Figure 2 The results obtained are shown in the figure. Figure 2 As shown, on a medium free of herbicide (Tifena 0 μM), the growth of BT3 transformants (V) transformed with pACBB-eGFP without the PPO gene did not recover, while the growth of BT3 transformants transformed with the PPO-susceptible Arabidopsis PPO1 wild-type gene (AtPPO1 WT), the PPO-tolerant Arabidopsis PPO1 mutant gene (AtPPO1 SLYM), the CyPPO10 gene (Cy10 WT), or the CyPPO13 gene (Cy13 WT) recovered, because each introduced gene acts as a PPO enzyme in BT3. These results indicate that both CyPPO10 and CyPPO13 perform normal PPO function.

[0218] BT3 transformants (AtPPO1 WT) transformed with the wild-type PPO1 gene of Arabidopsis thaliana, which is susceptible to tafenac, grew normally in herbicide-free media, but did not grow in media containing 100 μM tafenac. BT3 transformants (AtPPO1 SLYM) transformed with the tafenac-resistant PPO1 mutant gene of Arabidopsis thaliana showed a gradual increase in growth starting with 100 μM tafenac, and almost no growth at 400 μM. BT3 transformants transformed with the CyPPO10 or CyPPO13 genes grew at similar growth levels in media containing 100 μM tafenac as in media without tafenac, and also grew well even in media containing 400 μM tafenac. These results demonstrate that the CyPPO10 and CyPPO13 genes can exhibit significantly higher tolerance to tefena compared to wild-type Arabidopsis PPO1, which is susceptible to tefena, and similar or higher tolerance compared to tefena-tolerant Arabidopsis PPO1 mutants.

[0219] Example 3. Identification of interactions with PPO inhibitors from PPO and PPO inhibitory herbicide complexes. PPO amino acid residues

[0220] To investigate the binding structure of PPO proteins with herbicides, tebufenozide, pyrimethanil, propyzoxystrobin, or metsulfuron-methyl were used as representative examples of PPO-inhibiting herbicides. The gene encoding the CyPPO10 protein was cloned into the pET29b vector (catalog number: 69872-3; EMD Biosciences; see [link]). Figure 30 The CyPPO10 protein was expressed in *E. coli* using an *E. coli* system. The expressed CyPPO10 protein was purified by nickel affinity chromatography and crystallized with PPO-inhibiting herbicides. Then, using a synchrotron radiation accelerator, complexes of CyPPO10 with tebufenozide, pyrimisulfuron, propyzamide, or mesotrione were obtained. High-resolution X-ray diffraction data were used to identify the three-dimensional structure of the complex. This process allowed for the collection of information on the amino acid mutation locations in the CyPPO10 protein, which confers herbicide tolerance.

[0221] Analysis of the structure of the CyPPO10-Tefena complex led to the following conclusion: amino acids N59, S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360, and I408 of the CyPPO10 protein (SEQ ID NO: 2) interact with Tefena.

[0222] Using binding information derived from the structure of the CyPPO10-Tefena complex, sequence homology analysis between the amino acids of CyPPO10 (SEQ ID NO: 2) and CyPPO13 was used to identify the amino acid residues in the CyPPO13 (SEQ ID NO: 4) protein that interact with Tefena (NCBI BLAST, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome).

[0223] As a result, it can be understood that the amino acids at positions R101, F171, V175, A177, G194, P316, V318, F337, L340, I353 and F373 of the CyPPO13 protein (SEQ ID NO: 4) interact with tefena.

[0224] Example 4. Preparation of PPO variants

[0225] To enhance tolerance to PPO-inhibiting herbicides such as CyPPO10 and CyPPO13, mutant genes that increase tolerance to PPO-inhibiting herbicides were prepared by mutating the amino acid positions of both genes at the herbicide-interacting sites as identified in Example 3.

[0226] Using the primers in Table 3, the mutated PPO gene was isolated and amplified by PCR under the following conditions:

[0227] Material

[0228] Template (CyPPO10 or CyPPO13 synthetic DNA) 1 μl

[0229] 10X buffer 5μl

[0230] dNTP mixture (10mM each) 1μl

[0231] Forward primer (10 μM) 1 μl

[0232] Reverse primer (10 μM) 1 μl

[0233] DDW 40μl

[0234] Pfu-X (Solgent, 2.5 units / μl) 1μl

[0235] 50 μl in total

[0236] Table 2

[0237] PCR conditions

[0238]

[0239] Table 3

[0240]

[0241] The amplified gene products were cut with XbaI and XhoI and the pET303-CT His vector (VT0163; Novagen; see below). Figure 3 Then, pET303-CyPPO10 and pET303-CyPPO13 plasmids were prepared using T4 DNA ligase (RBC, 3 units / μl).

[0242] The mutant genes of CyPPO10 and CyPPO13 were prepared by performing PCR using the primers in Table 5 and 6 below under the following conditions, and using CyPPO10 and CyPPO13 cloned into the pET303-CT His vector as templates.

[0243] Material

[0244] Template 1μl

[0245] 10X buffer 5μl

[0246] dNTP mixture (10mM each) 1μl

[0247] Forward primer (10 μM) 1 μl

[0248] Reverse primer (10 μM) 1 μl

[0249] DDW 40μl

[0250] Pfu-X (Solgent, 2.5 units / μl) 1μl

[0251] 50 μl in total

[0252] Table 4

[0253] PCR conditions

[0254]

[0255] Table 5

[0256] List of primers used to construct the CyPPO13 mutant gene

[0257]

[0258]

[0259]

[0260]

[0261] Table 6

[0262] List of primers used to construct the CyPPO13 mutant gene

[0263]

[0264]

[0265]

[0266] Example 5. PPO-inhibiting herbicide tolerance of PPO and its variants

[0267] To enhance tolerance to PPO-inhibiting herbicides CyPPO10 and CyPPO13, the amino acids that interact with the herbicides, as identified in Example 3, were mutated. PPO-deficient BT3 *E. coli* (ΔPPO) was transformed with the mutated PPO gene and then cultured with a PPO-inhibiting herbicide to observe the growth of the transformed *E. coli*, as follows:

[0268] The pET303-CyPPO10 or pET303-CyPPO13 plasmids prepared in Example 4 and plasmids containing each mutant gene were transformed into BT3 competent cells by heat shock method and cultured in LB agar medium containing ampicillin (100 μg / ml).

[0269] For seed culture of BT3 transformants, single colonies were cultured in 3 ml of LB broth (LPSS) containing ampicillin for 12 hours or longer, and then 50-100 μl of culture was taken and cultured until the absorbance (OD) reached a certain level. 600 The absorbance (OD) was then increased to 0.5 to 1. The resulting culture medium was then diluted with LB broth to adjust the absorbance (OD). 600 Adjust to 0.5, then dilute 5 times with LB broth at a factor of one-tenth.

[0270] Prepare a herbicide-containing culture medium by mixing LB (25 g / L), Bacto agar (12 g / L), ampicillin (100 μg / ml) and various herbicides (0 to 200 μM).

[0271] 10 μL of diluted solution was dropped onto a medium containing the herbicide, and the medium was incubated at 37°C under light for 16 to 20 hours. The growth level and tolerance to PPO-inhibiting herbicides of BT3 transformed with each gene were assessed.

[0272] The herbicides used in the experiment are listed in Table 7 below:

[0273] Table 7

[0274]

[0275] Herbicide tolerance was evaluated relatively compared to CyPPO wild type, and is shown in Tables 8 to 11 below. Figures 11 to 29 middle.

[0276] Table 8

[0277]

[0278] NT (Untested)

[0279] Table 9

[0280]

[0281]

[0282] NT (Untested)

[0283] Table 10

[0284]

[0285] NT (Untested)

[0286] Table 11

[0287]

[0288]

[0289] NT (Untested)

[0290] In Tables 8 to 11, the herbicide tolerance level of the wild type is represented by "-". The herbicide tolerance level is gradually represented by "-" to indicate the same tolerance level, and "+" is added if it is higher, up to the maximum "+++++".

[0291] Figures 11 to 19 (Wild type and variants of CyPPO10) and Figures 20 to 29 (Wild-type and variant of CyPPO13) shows the culture results of E. coli transformed with the CyPPO gene (wild-type and variant), and the concentrations described above are the concentrations of the herbicides treated. Six columns of each concentration were successively diluted 5-fold to the right with E. coli culture at a factor of one-tenth. The leftmost column shows the result of the E. coli culture with OD600 = 0.5.

[0292] As shown in Tables 8 to 11 and Figures 11 to 29As shown, compared with transformants of the wild-type gene, all transformants transformed with the mutant genes of CyPPO10 and CyPPO13 demonstrated the same or increased levels of herbicide tolerance to various herbicides.

[0293] Example 6: Measurement of enzyme activity and IC50 using PPO as a herbicide 50 value

[0294] Enzymatic activities of PPO protein and its variants were detected, and inhibition assays against PPO-inhibiting herbicides were performed. PPO protein has been shown to have low water solubility, but when expressed as a fusion protein with MBP (maltose-binding protein) (MBP-PPO), it can be stably expressed in a water-soluble form. Therefore, wild-type and variant proteins expressed as fusion proteins with MBP (see [link to study]) were used in this study. Figure 4 ).

[0295] To express the wild-type and mutant genes of CyPPO10 and CyPPO13 (see Examples 1 and 4), these genes were introduced into the pMAL-c2X vector (see Example 4). Figure 5 Then cloned into BL21(DE3) Escherichia coli (CodonPlus).

[0296] Transformed E. coli were cultured under the following conditions to express the introduced PPO gene:

[0297] Induction: OD 600 =0.2, add IPTG to a final concentration of 0.3 mM;

[0298] Expression temperature: 23℃, incubated with shaking at 200 rpm;

[0299] Presentation time: 16 hours;

[0300] Culture scale: 200ml / 1000ml flask.

[0301] The following methods were used to perform cell lysis and protein extraction on cultured E. coli cells:

[0302] Extraction buffer: Column buffer (50mM Tris-Cl, pH 8.0, 200mM NaCl) 5ml buffer / g cells;

[0303] Ultrasonic treatment: SONICS & MATERIALS VCX130 (130 watts);

[0304] 15 seconds to turn on, 10 seconds to turn off, 5 minutes on the ice;

[0305] Centrifuge at 4°C for 20 minutes (20,000×g); dilute the supernatant obtained by centrifugation with column buffer at a ratio of 1:6.

[0306] The following method for purifying PPO protein was performed in a 4°C cold chamber. A 1.5 × 15 cm column (Bio-Rad Econo 1.5 × 10 cm glass column, maximum volume) was packed with linear starch resin (New England Biolabs), and the obtained protein extract was loaded into the column at a flow rate of 0.2 mL / min. The column was washed with 3 column volumes of buffer, and the amount of protein in the wash solution was determined. Washing was stopped when no more protein was detected. Then, MBP-PPO protein was eluted with approximately 2 column volumes of buffer containing 20 mM maltose. The protein concentration of each eluent was determined, and elution was stopped when no more protein was detected. Ten μL fractions of each fraction were studied for protein quantification and SDS-PAGE analysis. The high-purity fractions containing PPO protein were used for enzyme activity assays.

[0307] The enzyme activities of purified wild-type and variant CyPPO10 and CyPPO13 proteins were measured using the following methods.

[0308] First, the substrate for PPO protein protoporphyrinogen IX was synthesized. The process was carried out in a nitrogen-fluidized space. 6 mg of protoporphyrinogen IX was dissolved in 20 mL of 20% (v / v) EtOH and stirred for 30 min in the dark. The resulting protoporphyrinogen IX solution was transferred in 800 μl to a 15 mL spiral tube and rinsed with nitrogen for 5 min. 1 g of sodium amalgam was added, and the tube was vigorously shaken for 2 min. The cap was opened to purge hydrogen gas from the tube. The cap was then closed and incubated for 3 min. The protoporphyrinogen IX solution was filtered using a syringe and a cellulose membrane filter. Approximately 300 μl of 2 MMOPS [3-(N-morpholino)propanesulfonic acid] was added to 600 μl of the obtained protoporphyrinogen IX solution to adjust the pH to 8.0. To determine the enzymatic activity of PPO protein, a reaction mixture was prepared with the following composition (based on 10 ml): 50 mM Tris-Cl (pH 8.0); 50 mM NaCl; 0.04% (v / v) Tween 20; 40 mM glucose (0.072 g); 5 units of glucose oxidase (16.6 mg); and 10 units of catalase (1 μl).

[0309] A 200 μL (200 μl) reaction mixture containing purified PPO protein was placed in a 96-well plate and preheated at room temperature for 30 minutes to reduce oxygen concentration via a glucose oxidase-catalase reaction. The mineral oil was separated into layers, and the reaction was initiated by adding substrate protoporphyrin IX solution to a final concentration of 50 μM. The reaction was allowed to proceed at room temperature for 30 minutes, and the fluorescence of protoporphyrin IX was measured using a microplate reader (Sense, Hidex) (excitation: 405 nm; emission: 633 nm). To calculate PPO enzyme activity, the protoporphyrin IX solution was kept open in air to oxidize the solution (overnight). 2.7 N HCl was added, and the absorbance was measured at 408 nm. A standard curve was generated using standard protoporphyrin IX, and PPO activity was measured by calibrating protoporphyrin IX using the standard curve.

[0310] The enzyme activities of the obtained PPO wild-type and variants are shown in Table 12.

[0311] Simultaneously, the Michaelis constant (Km) and maximum rate (Vmax) values ​​for each enzyme were calculated to evaluate the kinetic parameters of the PPO proteins (CyPPO10 and CyPPO13). Initial reaction rates were measured, with the reaction rate proportional to the substrate concentration, and the amount of protoporphyrin IX produced as a product of the enzymatic reaction was measured over 20 minutes at room temperature according to the time history. Km and Vmax values ​​were calculated using the Michaelis equation via an enzyme kinetic analysis program, with plant PPO serving as a control group. The results are shown in Table 12.

[0312] Table 12

[0313]

[0314] As shown in Table 12, CyPPO10 and CyPPO13 have superior abilities as PPO enzymes compared to Arabidopsis PPO1 (AtPPO1) and Amaranth PPO1.

[0315] The concentration (IC50) of the PPO inhibitory herbicide at which each herbicide inhibited PPO enzyme activity to 50% was measured. 50 The final concentrations of each herbicide are as follows:

[0316] 0, 10, 50, 100, 250, 500, 1,000, 2,500, 5,000nM

[0317] IC 50 The value is calculated as the herbicide concentration that inhibits PPO enzyme activity to 50%, and then the herbicide at the above concentration is added to perform the above enzyme activity measurement process.

[0318] IC50 of different herbicides 50 The values ​​are shown in Table 13 below.

[0319] Table 13

[0320]

[0321]

[0322]

[0323]

[0324] NT (Untested)

[0325] As shown in Table 13, compared with wild-type CyPPO protein, CyPPO protein variants exhibited significantly increased IC50. 50 These results indicate that amino acid mutations at certain positions in the PPO protein can lead to increased herbicide tolerance. Although this data shows that the CyPPO protein variant has reduced enzyme activity compared to the wild type, this may be due to different protein folding conditions and / or the hydrophobicity of the recombinant PPO compared to the natural PPO. While the natural PPO is hydrophobic and localized in the chloroplast membrane of plants, the recombinant PPO produced in *E. coli* containing MBP as a fusion partner is hydrophilic. Therefore, when the PPO variant is properly assembled and localized in the chloroplast membrane of plants, the enzyme activity is not significantly affected.

[0326] Example 7. Production of Arabidopsis thaliana transformants using CyPPO and its variants, and tolerance test to PPO-inhibiting herbicides.

[0327] 7-1. Construction of Arabidopsis transformation vectors and transformation of Arabidopsis

[0328] Arabidopsis thaliana was transformed using a binary vector containing selectively marked ORFs, the bar gene (for glufosinate tolerance), and ORFs for each coding gene of either the CyPPO10 or CyPPO13 variant. Cross-tolerance to glufosinate and PPO-inhibiting herbicides was examined in the transgenic plants. The bar gene was also used to examine whether the transgene was stably inherited across generations. The NOS promoter and E9 terminator were used for bar gene expression.

[0329] To express CyPPO10, CyPPO10 variants, CyPPO13, and CyPPO13 variants in plants, respectively, the CaMV35S promoter and NOS terminator were used. The coding genes for CyPPO10, CyPPO10 variants, CyPPO13, and CyPPO13 variants were cloned using XhoI and BamHI restriction enzymes. To identify the expressed protein, a hemagglutinin (HA) tag was fused to the 3′ terminal region using BamHI and SacI restriction enzymes. A NOS terminator was inserted after the HA tag to terminate the transcription of the PPO genes. Additionally, to transport the protein to chloroplasts, the transport peptide (TP) (SEQ ID NO: 10) of the AtPPO1 gene was inserted 5′ anterior to the inserted gene using XbaI and XhoI restriction enzymes. The transport peptide region in the inserted vector is represented by SEQ ID NO: 27, and the inserted HA tag sequence is represented by SEQ ID NO: 28. A schematic diagram of the plant transformation binary vector is shown in [image / description missing]. Figure 6 As shown in the image.

[0330] The vectors constructed above were introduced into *Agrobacterium tumefaciens* GV3101 competent cells using a freeze-thaw method. To prepare *Agrobacterium tumefaciens* GV3101 competent cells, *Agrobacterium tumefaciens* GV3101 seed cells were cultured in 5 ml LB medium at 30°C and 200 rpm for 12 hours. The medium was then inoculated into 200 ml LB medium and cultured at 30°C and 200 rpm for 3 to 4 hours, followed by centrifugation at 3000 × g for 20 minutes at 4°C. The precipitate was washed with sterile distilled water and resuspended in 20 ml LB medium. 200 μl aliquots of the samples were flash-frozen in liquid nitrogen and stored in a deep freezer.

[0331] Each transformed Agrobacterium was cultured and screened on antibiotic medium (LB agar containing spectinomycin). Screened colonies were then liquid cultured in LB broth. After harvesting the Agrobacterium from the medium, it was measured at an absorbance of 0.8 (OD). 600 Resuspended in 5% (w / v) sucrose and 0.05% (v / v) Silwet L-77 solution (Momentive Performance Materials). Col-0 ecotype Arabidopsis wild-type was transformed by floral dipping, and seeds were harvested 1 to 2 months later (T1).

[0332] The Bar gene in the binary vector was used to screen for individual transformants. The obtained T1 seeds were sown in 1 / 2 MS medium (2.25 g / L MS salt, 10 g / L sucrose, 7 g / L agar) supplemented with 25 μM glufosinate, and surviving plants were selected 7 days after sowing and transplanted into soil.

[0333] To test the tolerance of transgenic plants to PPO-inhibiting herbicides, each 40×60cm area (0.24m²) was surveyed. 2 Four-week-old plants were uniformly sprayed with 100 ml of 1 μM Tefena solution (0.05% Silwet L-77). Although wild-type Arabidopsis thaliana (Col-0 ecotype; Columbia-0 ecotype) died completely within 7 days after treatment, none of the transformants showed damage to the PPO-inhibiting herbicide treatment.

[0334] T2 seeds harvested from surviving plants were sown in 1 / 2 MS medium supplemented with 25 μM glufosinate (2.25 g / L MS salt, 10 g / L sucrose, 7 g / L agar), and surviving plants were transplanted into soil one week later.

[0335] To determine the copy number of each strain, the segregation rate was studied using T2 seeds.

[0336] By analyzing each 40×60cm area (0.24m) 2 Spray 100 ml of tafina solution (1 μM, 5 μM, 10 μM or 25 μM tafina + 0.05% Silwet L-77) to confirm tafina tolerance in 4-week-old transformants. Harvest T3 seeds from tafina-tolerant T2 plants.

[0337] Seeds were selected from 1 / 2 MS medium containing 25 μM glufosinate, and lines in which all individuals were tolerant to glufosinate were identified as homolines.

[0338] 7-2. Seed germination

[0339] Herbicide tolerance of Arabidopsis thaliana transformants incorporating wild-type or variant genes CyPPO10 and CyPPO13 was confirmed.

[0340] T3 generation seeds of each transformant were sown in 1 / 2 MS medium containing a herbicide. Col-0 ecotype (wild-type Arabidopsis thaliana) seeds were used as a control. The types and concentrations of herbicides are as follows:

[0341] Figure 31a : 25μM glufosinate (PPT), 70nM tefenazate, 100nM bensulfuron-methyl, 25μM glufosinate + 70nM tefenazate, or 25μM glufosinate + 30nM tefenazate + 40nM bensulfuron-methyl;

[0342] Figure 31b and 31c: 25 μM glufosinate (PPT), 0.1 μM or 1 μM tebufenozide, 0.3 μM or 3 μM benzosulfuron, 0.1 μM or 1 μM propyzoxystrobin, 0.5 μM or 5 μM cyprodinil, or 1 μM or 10 μM mesotrione.

[0343] The result of seed germination 7 days after sowing Figure 31a , 31b As shown in 31c. Figures 31a to 31c In this context, 10-3 refers to the wild-type CyPPO10, 10FM-4-7 refers to the CyPPO10 F360M transgenic line, 10FL-1-9 refers to the CyPPO10 F360L transgenic line, 10FC-3-5 refers to the CyPPO10 F360C transgenic line, 10AC-5-4 refers to the CyPPO10 A167C transgenic line, 13-1 refers to the wild-type CyPPO13, 13FM-3-1 refers to the CyPPO13 F373M transgenic line, 13FC-1-1 refers to the CyPPO13 F373C transgenic line, 13FI-2-1 refers to the CyPPO13 F373I transgenic line, 13AC-1-3 refers to the CyPPO13 A177C transgenic line, and CyPPO13_ALFL refers to CyPPO13... A177L+F373L transgenic line and CyPPO13_ALFI refer to CyPPO13 A177L+F373I transgenic line.

[0344] like Figures 31a to 31c As shown, while wild-type Arabidopsis thaliana (Col-0 ecotype) germinated in herbicide-free 1 / 2 MS medium, it did not germinate in herbicide-containing 1 / 2 MS medium. Therefore, germination tests on herbicide-containing media can be used to assess herbicide tolerance.

[0345] Simultaneously, the Arabidopsis T3 line was transformed, and CyPPO10 wild-type, CyPPO10 mutant genes (F360M, F360I, F360L, F360C, A167C), CyPPO13 wild-type, or CyPPO13 mutant genes (F373M, F373C, F373I, A177C, A177L+F373L, A177L+F373I) germinated in media containing herbicides (25 μM glufosinate, 25 μM glufosinate + 70 nM tebufenozide, or 25 μM glufosinate + 30 nM tebufenozide + 40 nM pyrimethanil). These results indicate that the bar gene (glufosinate tolerance gene) and the CyPPO gene (PPO inhibitory herbicide tolerance gene) simultaneously and independently function as herbicide tolerance traits in transgenic plants.

[0346] like Figures 31a to 31cAs shown, in culture media containing various types and concentrations of PPO-inhibiting herbicides, transformed Arabidopsis thaliana typically germinated and survived, while Col-0 typically failed to germinate. These results suggest that transformed Arabidopsis thaliana is conferred tolerance to various PPO-inhibiting herbicides or maintains enhanced tolerance to them through gene insertion in the transformant.

[0347] 7-3. Study on CyPPO protein expression in Arabidopsis thaliana (T2) with CyPPO gene introduced.

[0348] The expression of each protein was investigated in Arabidopsis transformants (T2) that had genes encoding CyPPO10, CyPPO10 variants (F360I or F360M), CyPPO13, or CyPPO13 variants (F373M) inserted, respectively.

[0349] Leaves of 4-week-old Arabidopsis transformants were ground with liquid nitrogen, and protein extraction buffer (0.05M Tris-Cl pH 7.5, 0.1M NaCl, 0.01M EDTA, 1% Triton X-100, 1mM DTT) was added to extract proteins. Western blotting was then performed using an anti-HA antibody (Santa Cruz). HA tagging was used to detect protein expression in the transformants. To compare the amount of loaded protein, the amount of the RuBisCO large subunit was confirmed by Coomassie blue staining. Two independent lines of each variant were tested, with Col-0 used as a control.

[0350] The result is Figure 7 As shown in the figure, all Arabidopsis transformants infused with the CyPPO10 variant (F360I or F360M) or CyPPO13 variant (F373M) genes exhibited successful expression of the PPO protein.

[0351] 7-4. Validation of herbicide tolerance in transformed Arabidopsis thaliana (T2 or T3)

[0352] Herbicide tolerance was tested using Arabidopsis transformants (T2 or T3) with genes encoding CyPPO10, CyPPO10 variants (F360C, F360I, F360L, F360M, F360V, F360T, A167C, A167L, A167L+F360M, A167C+F360M, A167C+F360I or V305M+F360M), CyPPO13, or CyPPO13 variants (A177C, F373C, F373I, F373M, A177L+F373I or A177L+F373L).

[0353] Use tefena solution (1 μM tefena + 0.05% (v / v) Silwet L-77) in each 40 × 60 cm area (0.24 m²). 2After treating CyPPO10 or CyPPO13 transformants (T3) with 100 ml of solution, the level of damage to the plants was determined on day 7. For comparison, the same test was performed using wild-type Arabidopsis thaliana (Col-0 ecotype).

[0354] The result is Figure 8 As shown in the image.

[0355] In addition, each 40×60cm area (0.24m) 2 Plant damage levels were determined on day 7 after treatment with 100 ml of tefena solution (1 μM, 5 μM, 10 μM or 25 μM tefena + 0.05% (v / v) Silwet L-77) encoding genes of CyPPO10 variants (F360C, F360I, F360L, F360M, F360V, F360T, A167C, A167L, A167L+F360M or A167C+F360I) or CyPPO13 variants (A177C, F373C, F373I, F373M, A177L+F373I or A177L+F373L).

[0356] The result is Figure 9 (T2 transformants with CyPPO10 variant gene introduced) and Figure 10 (The T2 transformant with CyPPO13 variant gene introduced) is shown in the figure.

[0357] in addition, Figures 8 to 10 The damage levels (damage index) of each strain after tefena treatment are shown as numerical indices in Table 14 below.

[0358] Table 14

[0359] T2 Injury Index (Injury Level)

[0360]

[0361]

[0362] Each 40×60cm area (0.24m) 2 Transformers (T3) containing the encoding genes of CyPPO10 variants (F360I, F360L, F360M, A167C+F360I, A167C+F360M or V305M+F360M) were treated with 100 ml of teifenna solution (25 μM teifenna + 0.05% (v / v) Silwet L-77) or bensulfuron-methyl solution (100 μM bensulfuron-methyl + 0.05% (v / v) Silwet L-77), and the damage level of the plants was determined on day 7.

[0363] The results of introducing T3 transformants encoding the CyPPO10 variant gene were in Figure 32 As shown in the image.

[0364] In addition, the damage levels (damage index) of Arabidopsis transformants introduced with the CyPPO10 mutant gene after treatment with tefenazate or fensulfuron-methyl are shown as numerical indices in Table 15 below.

[0365] Table 15

[0366] T3 Damage Index (Damage Level)

[0367]

[0368]

[0369] Tables 14 and 15 show the average damage levels of individuals (10 to 20 individuals) tested according to the standards in Table 16 below.

[0370] Table 16

[0371] Definition of damage level

[0372]

[0373] Following treatment with tebufenozide, fensulfuron-methyl, propyzoxystrobin, or metsulfuron-methyl (50 μM each), the tolerance levels of Arabidopsis thaliana transformants (T3) infused with the CyPPO10 mutant gene (F360I or A167L+F360M) or the CyPPO13 mutant gene (A177L+F373L or A177L+F373I) were confirmed. For comparison, the wild-type Arabidopsis thaliana or the Arabidopsis thaliana PPO1 SLYM (AtPPO1 SLYM, S305L+Y426M) transformants (T3) were tested under the same conditions.

[0374] In tolerance tests using various herbicides, each 40×60cm area (0.24m) was tested. 2 100 ml of each herbicide at a concentration of 50 μM was sprayed evenly. The molecular weights (MW) of teifen, bensulfuron-methyl, propyzoxystrobin, and mesotrione were 511.87, 500.85, 354.34, and 387.18, respectively. The treatment doses corresponding to switching were 106.7 g ai / ha for teifen, 104.4 g ai / ha for bensulfuron-methyl, 73.8 g ai / ha for propyzoxystrobin, and 80.7 g ai / ha for mesotrione.

[0375] The results are as follows Figure 33a and 33b As shown.

[0376] In addition, the damage level (damage index) of the transformant is shown as a numerical index in Figure 33 and Table 17.

[0377] Table 17

[0378] T3 Damage Index (Damage Level)

[0379]

[0380] exist Figure 33a In Table 17, Cy10FI, AtPPO1 SLYM, and Cy10 ALFM represent CyPPO10 F360I transformant, AtPPO1 S305L+Y426M transformant (control), and CyPPO10 A167L+F360M transformant, respectively.

[0381] exist Figure 33b In the diagram, Col-0, Cy13 ALFL, and Cy13 ALFI represent the wild type, CyPPO13 A177L+F373L transformant, and CyPPO13 A177L+F373I transformant, respectively.

[0382] like Figure 33a As shown, the transformants of the mutant gene exhibited the same or higher tolerance than AtPPO1 SLYM. This confirmed that all CyPPO10 FI and CyPPO10 ALFM conferred higher tolerance to various herbicides compared to AtPPO1 SLYM.

[0383] As shown in Table 14 and Figure 8 As shown, after treatment with 1 μM tafenamic acid, almost all transformants with wild-type CyPPO10, its variant gene, wild-type CyPPO13, or its variant gene grew, while wild-type Arabidopsis (Col-0) died.

[0384] Additionally, as shown in Tables 15 and 17, Figures 9 to 10 and Figures 32 to 3 As shown in Figure 3, Arabidopsis transformants infused with CyPPO10 or CyPPO13 variant genes showed no or weak damage after treatment with 5 μM tefena. These results indicate that the introduction of CyPPO10, CyPPO13, or their mutant genes confers and / or enhances herbicide tolerance in Arabidopsis.

[0385] It was confirmed that herbicide tolerance was maintained from generation T2 to T3, indicating that herbicide tolerance can be stably transferred even with generational evolution.

[0386] Based on these results, CyPPO variants are expected to provide tolerance to a variety of PPO-inhibiting herbicides in other plants as well as Arabidopsis thaliana.

[0387] 7-5. Confirm transgenic stability during passage.

[0388] In this embodiment, it was confirmed whether the gene introduced into Arabidopsis thaliana was stably inherited across generations.

[0389] Transformers of the T3 lines 7-2, 10-2, and 10-5, transformed with CyPPO10 F360I, were further developed into the T4 and T5 generations, thereby confirming the tolerance of tefenazate or bensulfuron-methyl in each line and the expression of the introduced gene in the T4 and T5 generations.

[0390] protein extraction

[0391] Proteins were extracted from each generation of plants. After grinding seedlings with liquid nitrogen, protein extraction buffer (0.05M Tris-Cl pH 7.5, 0.1M NaCl, 0.01M EDTA, 1% Triton X-100, 1mM DTT) was added, and total protein was extracted. After electrophoresis, the extracted proteins were transferred to a PVDF membrane and then Western blotted using an anti-HA antibody (Santacruz).

[0392] Confirm herbicide tolerance

[0393] Four weeks after transplanting, spray 100 ml of a herbicide solution containing 15 μM tebufenozide or 150 μM fenproxilsulfuron-methyl evenly over a 40 × 60 cm area (0.24 m²). 2 In Arabidopsis thaliana, herbicide damage levels were observed on day 7 post-treatment.

[0394] The results of herbicide tolerance in Figure 34 (T4) and Figure 35 As shown in (T5), the level of damage to the transformant by the herbicide (damage index) is shown in Table 18.

[0395] Table 18

[0396] T4 and T5 damage indices (damage levels)

[0397]

[0398] Although the negative control (Col-0; Arabidopsis wild-type) was sensitive to herbicide treatment, the T4 and T5 Arabidopsis transformants of CyPPO10 F360I were tolerant.

[0399] In addition, Western blot analysis of transgenic expression in Figure 36 As shown in the figure, CyPPO10 F360I protein was detected only in all T4 and T5 generation transformants.

[0400] Therefore, it is demonstrated that the herbicide tolerance obtained by introducing the CyPPO10 variant is stably inherited and maintained through the T4 and T5 generations.

[0401] Example 8. Construction of soybean transformants and PPO-inhibiting herbicide tolerance test using CyPPO and its variants.

[0402] 8-1. Recombinant vectors for soybean conversion and construction of soybean transformants using these recombinant vectors

[0403] A vector was constructed for soybean plant transformation to confer tefena tolerance by expressing the CyPPO10 A167L+F360M gene.

[0404] Specifically, a vector for Arabidopsis transformation is used (see [link to Arabidopsis thaliana transformation]). Figure 6 Using a template, the CyPPO10 A167L+F360M gene, which combines with the transport peptide of the Arabidopsis PPO1 gene, was amplified by PCR. The amplified product was cloned using the pENTR Directional TOPO Cloning Kit (Invitrogen) and transformed into DH5α competent cells (Invitrogen). Then, the cloned gene was transferred into the pB2GW7.0 binary vector for plant transformation using the Gateway LR Clonase II enzyme mixture kit (Invitrogen). Figure 37 After mixing the pENTR / D-TOPO vector containing the CyPPO10A167L+F360M gene, TE buffer, and LR Clonase II enzyme, the mixture was incubated at 25°C for 1 hour. Proteinase K solution (Invitrogen) was added to the reaction mixture, which was then incubated at 37°C for 10 minutes and transformed into DH5α competent cells.

[0405] The binary vector constructed above was used to electroconvert Agrobacterium EHA105.

[0406] Guang'an soybean plants are used to construct soybean transformants.

[0407] After removing the seed coat from soybean seeds, the hypocotyl was cut and damaged 7-8 times using a surgical scalpel (#11 cutter). Approximately 50 explants were mixed with transformed Agrobacterium tumefaciens EHA105 (Hood et al., New Agrobacterium helper plasmid for gene transfer to plants (EHA105)). Trans Res. 1993 2: 208-218, and the mixture was sonicated for 20 seconds and then incubated for 30 minutes before inoculation. The explants were placed on CCM (co-medium; 0.32 g / L Gamborg B5, 4.26 g / L MES, 30 g / L sucrose, 0.7% agar). They were then co-cultured in a growth chamber (25°C, 18 h light / 6 h dark) for 5 days.

[0408] Afterward, it was washed for 10 minutes in liquid 1 / 2 SIM (twig induction medium; 3.2 g / L Gamborg B5, 1.67 mg / L BA, 3 mM MES, 0.8% (w / v) agar, 3% (w / v) sucrose, 250 mg / L cefotaxime, 50 mg / L vancomycin, 100 mg / L ticarcillin, pH 5.6) and placed on antibiotic-free SIM and cultured in a growth chamber (25°C, 18 hours light / 6 hours dark) for 2 weeks.

[0409] The branch-induced explants were transplanted into SIM-1 medium (SIM medium supplemented with 10 mg / L DL-glufosinate, pH 5.6).

[0410] Brown branches were transplanted onto a SEM (branch elongation medium; 4.4 g / L MS salt, 3 mM MES, 0.5 mg / L GA3, 50 mg / L asparagine, 100 mg / L pyroglutamic acid, 0.1 mg / L IAA, 1 mg / L zeatin, 3% (w / v) sucrose, 0.8% (w / v) agar, 250 mg / L cefotaxime, 50 mg / L vancomycin, 100 mg / L ticarcillin, 5 mg / L DL-glufosinate, pH 5.6). Elongated branches with a height of 4 cm were transferred to RIM (root induction medium; 4.4 g / L MS salt, 3 mM MES, 3% sucrose, 0.8% agar, 50 mg / L cefotaxime, 50 mg / L vancomycin, 50 mg / L ticarcillin, 25 mg / L asparagine, 25 mg / L pyroglutamic acid, pH 5.6).

[0411] Once the roots have fully developed, transplant the plant to a bed of soil (Bioplug No. 2, Farmhannong) mixed with vermiculite in a 2:1 (v / v) ratio. Ten days later, apply 100 mg / L DL-phosphidin to the leaves.

[0412] 8-2. Verify the herbicide tolerance of transformed soybeans

[0413] Apply 5 μmol or 15 μM Tefena solution 2 to 3 times by brush to the leaves of CyPPO10 A167L+F360M transformed soybean line 2 (T0 generation) and untransformed soybean (Guang'an; wild-type soybean, control). The Tefena solution contains 0.05% (v / v) Silwet L-77 as a surfactant.

[0414] like Figure 38 As shown, Guang'an (non-converted soybeans) showed severe damage 7 days after treatment with 5 μM Tefena, but soybeans converted by CyPPO10 A167L+F360M did not show damage even after treatment with 15 μM Tefena.

[0415] Simultaneously, in stages V2 and V3, the T1 generation of CyPPO10 A167L+F360M transformant line 2 was treated with either tefenazate or bensulfuron-methyl. 100 ml of 25 μM tefenazate or 150 μM bensulfuron-methyl was evenly sprayed over a 40 × 60 cm area (0.24 m²). 2 On the surface, assess the level of damage 5 days after spraying.

[0416] exist Figure 40 In this study, Guang'an soybean was used as a control. Compared with the control, the CyPPO10 A167L+F360M(10ALFM) transformant soybean did not show any damage even after treatment with relatively high concentrations of tebufenozide or pyrimethanil.

[0417] 8-3. Confirm the number of inserted genes in the transformed soybeans.

[0418] Genomic DNA was extracted from 250 mg of leaf tissue from strains 2 or 23 transformed with CyPPO10 A167L+F360M to analyze the copy number of the transgene.

[0419] Genomic DNA was extracted using the CTAB buffer method. Leaf tissue was ground in liquid nitrogen using a pestle and mortar, followed by the addition of 1.25 ml of DNA separation buffer (2% (w / v) CTAB, 1.5 M NaCl, 25 mM EDTA, 0.2% (v / v) β-mercaptoethanol, 100 mM Tris-Cl (pH 8.0)) and vortexing. After heating at 60 °C for 1 hour, 1 volume of chloroform:isoamyl alcohol (24:1) was added and mixed by inversion. After centrifugation at 7000 × g for 10 minutes at 4 °C, the supernatant was transferred to a new tube and mixed with 2.5 volumes of ethanol. After centrifugation at 5000 × g for 5 minutes at 4 °C, the supernatant was discarded, and the precipitate was dissolved in TE buffer (LPSS). 20 μg / ml RNase A (Bioneer) was added, and the tube was incubated at 37 °C for 30 minutes. Add 1 volume of phenol:chloroform (1:1), mix, and centrifuge at 10,000×g for 10 minutes at 4°C. Transfer the supernatant to a new tube, then add 1 volume of chloroform:isoamyl alcohol (24:1) and mix. Centrifuge at 10,000×g for 10 minutes at 4°C, transfer the supernatant to a new tube, add 0.1 volume of NaOAc (pH 5.2) and 2 volumes of ethanol, and mix. Centrifuge at 5,000×g for 5 minutes at 4°C, and wash with 70% ethanol. After air drying, dissolve the genomic DNA in an appropriate amount of TE buffer.

[0420] 10 to 40 μg of extracted DNA was digested overnight using EcoRI (Enzynomics).

[0421] Then, after electrophoresis on a 0.8% (w / v) agarose gel (50V), the gel was processed as follows:

[0422] 1) Depurine: 0.25N HCl, shake for 15 minutes

[0423] 2) Denaturation: 0.5M NaOH, 1.5M NaCl, shake for 30 minutes.

[0424] 3) Neutralization: 0.5M Tris (pH 7.5), 1.5M NaCl, shake for 20 minutes.

[0425] Subsequently, the DNA fragments were transferred to a nitrocellulose membrane using capillary transfer and cross-linked using a UV cross-linking agent (UVC-508; ULTRA LUM Inc.).

[0426] Hybridization was performed as follows: A nitrocellulose membrane was immersed in DIG Easy hybridization solution (Roche) and incubated at 42°C for 3 hours. Then, the solution was discarded and replaced with fresh DIG Easy hybridization solution containing a DIG-labeled probe, and incubated at 42°C for 16 to 18 hours.

[0427] The probe (DIG-labeled CyPPO8-M probe) was labeled using the following PCR reaction:

[0428] probe PCR

[0429] The DIG-tagged bar gene was amplified using DIG dUTP (Jena bioscience). The primers used were as follows:

[0430] Forward primer for the bar probe: 5′-TTC CGT ACC GAG CCG CAG GA-3′ (SEQ ID NO: 124)

[0431] Reverse primer for the bar probe: 5′-CGT TGG GCA GCC CGA TGA CA-3′ (SEQ ID NO: 125)

[0432] PCR: using the Solgent e-Taq kit

[0433] Conditions: 5 minutes at 95℃; 30 seconds at 94℃, 30 seconds at 60℃, 30 seconds at 72℃, for a total of 35 cycles; 2 minutes at 72℃.

[0434] After hybridization, the membrane was washed in both low-tightness wash buffer (2×SSC, 0.1% SDS) and high-tightness wash buffer (0.5×SSC, 0.1% SDS). The DNA blot signal was detected as follows:

[0435] 1) Add blocking buffer (Roche) to the membrane and shake for 30 minutes.

[0436] 2) After adding DIG antibody (anti-digoxin-AP Fab fragment, Roche), shake for 30 minutes.

[0437] 3) Shake in the wash buffer for 15 minutes (Roche)

[0438] 4) Add the detection buffer (Roche) and shake for 3 minutes.

[0439] 5) After applying CDP-Star (Roche) to the membrane, develop the imprint on X-ray film.

[0440] For the negative control, genomic DNA from untransformed soybean plants from Guang'an was used for DNA blotting.

[0441] exist Figure 39 In the diagram, the number of bands displayed on the membrane indicates the number of transgenes. Since a single band was observed in CyPPO10 A167L+F360M transformants lines 2 and 23, it was determined that each transgenic plant possessed a single copy of the transgene.

[0442] Example 9: Activity test of mutant genes with sequence homology to PPO variants

[0443] Using CyPPO plasmid (pACBB vector) as a template, error-prone PCR was performed under the following conditions to induce random mutations in CyPPO:

[0444]

[0445] 10× buffer: 100mM Tris-Cl, pH 8.3; 500mM KCl, 70mM MgCl2, 0.1% (w / v) gelatin

[0446] dNTP: 10mM dATP, 10mM dGTP, 100mM dCTP, 100mM dTTP

[0447] 35 cycles of 94℃ for 3 minutes; (94℃ for 30 seconds, 57℃ for 30 seconds, 72℃ for 1.5 minutes, 72℃ for 5 minutes)

[0448] Primer sequences:

[0449] CyPPO10_BamHI F

[0450] ccccggatccATGATTGAAGTGGATGTGGCTA (SEQ ID NO: 126)

[0451] CyPPO10_XhoI R

[0452] ccccctcgagTGATTGTCCACCAGCGAGGTAAG (SEQ ID NO: 127)

[0453] CyPPO13_BamHI F

[0454] ccccggatccATGAACCCTGCTACCCTGAAC(SEQ ID NO: 128)

[0455] CyPPO13_XhoI R

[0456] ccccctcgagCACCTGTGATAACAACTGCTGAG(SEQ ID NO: 129)

[0457] The obtained error-prone PCR products were electrophoresed on an agarose gel, then purified from the gel, and the pACBB vector and PCR products were digested with BamHI and XhoI restriction enzymes. The digested vector and PCR products, after electrophoresis on the agarose gel, were purified and ligated. The ligation products were transformed into BT3 competent cells, and the mutant CyPPO gene from the growing BT3 colonies was sequenced. The presence of mutant CyPPO gene in BT3 cells was confirmed to produce spots on LB agar plates containing different concentrations (0 μM, 50 μM, 100 μM, and 200 μM) of tebufenozide or fenproxilsulfuron-methyl, thereby studying the growth of *E. coli* and testing the level of herbicide tolerance.

[0458] In the mutant clones, clones with the following mutations will be used for the herbicide tolerance test:

[0459] CyPPO10m-6: Contains 9 amino acid mutations (E225G, G258S, Q266L, T336I, V356F, F360M, A364D, R406G, W419R); Nucleic acid sequence - SEQ ID NO: 130, amino acid sequence - SEQ ID NO: 131 (98% sequence homology with wild-type CyPPO10 amino acid sequence)

[0460] BT3 cells transformed with a CyPPO10 mutant gene were cultured in a medium containing herbicides, and cell growth inhibition was measured. Figure 41 In the text, 'AtPPO1WT' refers to the wild-type PPO1 of Arabidopsis thaliana, 'AtPPO1SLYM' refers to the mutant PPO1 (Y426M+S305L) of Arabidopsis thaliana, 'CyPPO10WT' refers to the wild-type CyPPO10, and 'CyPPO10m-6' refers to the mutant CyPPO10 as described above.

[0461] like Figure 41 As shown, cells transformed with CyPPO10 mutants possessing 98% or higher sequence homology to wild-type CyPPO10 exhibited similar cell viability to cells transformed with wild-type CyPPO10, even in media containing high concentrations (up to 200 μM) of tebufenozide or fenproxetine. This result indicates that CyPPO10 mutants with 98% or higher sequence homology can retain the herbicide tolerance (viability in herbicide-containing media) of the wild type.

Claims

1. A polypeptide comprising the following amino acid sequence, wherein the amino acid sequence is an amino acid sequence obtained by substituting the following amino acid sequence into the amino acid sequence of SEQ ID NO:2: (1) F360 is replaced by M (Met), V (Val), I (Ile), C (Cys) or L (Leu), R89 is replaced by A (Ala), V165 is replaced by C (Cys), A167 is replaced by C (Cys) or L (Leu), V305 is replaced by M (Met), or L327 is replaced by T (Thr); (2) The F360 was replaced by the M (Met) and further the following replacements were introduced: V165 was replaced by C (Cys) or S (Ser); or A167 is replaced by C (Cys), L (Leu), or I (Ile); or (3) F360 is replaced by M (Met), V165 is replaced by C (Cys), and the following replacements are further introduced: A167 is replaced by C (Cys), L (Leu), or I (Ile).

2. The polypeptide of claim 1, wherein, The polypeptide is a polypeptide composed of the following amino acid sequence, wherein the amino acid sequence is obtained by substituting the following amino acid sequence into the amino acid sequence of SEQ ID NO:

2. (1) F360 is replaced by M (Met), V (Val), I (Ile), C (Cys) or L (Leu); (2) The F360 was replaced by the M (Met) and further the following replacements were introduced: V165 was replaced by C (Cys) or S (Ser); or A167 is replaced by C (Cys), L (Leu), or I (Ile) or (3) F360 is replaced by M (Met), V165 is replaced by C (Cys), and the following replacements are further introduced: A167 is replaced by C (Cys), L (Leu), or I (Ile).

3. The polypeptide of claim 1, wherein, The polypeptide is a polypeptide composed of the following amino acid sequence, which is an amino acid sequence obtained by performing the following mutations on the amino acid sequence of SEQ ID NO:2: F360M, F360C, F360V, F360I, F360L, R89A, V165C, A167C, A167L, L327T, V305M, F360M+V165C, F360M+V165S, F360M+A167C, F360M+A167L, F360M+A167I, F360M+V165C+A167C, F360M+V165C+A167L, or F360M+V165C+A167I.

4. A polynucleotide encoding a polypeptide according to any one of claims 1 to 3.

5. A recombinant vector comprising the polynucleotide of claim 4.

6. A recombinant cell comprising the recombinant vector of claim 5.

7. A composition for conferring or enhancing herbicide tolerance to plants, comprising at least one selected from the group consisting of: a polypeptide of SEQ ID NO:2; a polypeptide of any one of claims 1 to 3; a polynucleotide encoding said polypeptide; a recombinant vector comprising said polynucleotide; and a recombinant cell comprising said recombinant vector.

8. The composition of claim 7, wherein, The herbicide is a protoporphyrinogen oxidase inhibitory herbicide.

9. The composition of claim 8, wherein, The herbicide is at least one selected from the group consisting of pyrimidinediones, diphenyl ethers, phenylpyrazoles, N-phenylphthalimides, phenyl esters, thiadiazoles, triazolinones, oxazolidinediones, pyroxasulfone, flufenpyr-ethyl, and pyroxasulfone.

10. The composition of claim 9, wherein, The herbicide is selected from at least one of the following groups: flufenacet, sulfadiazine, pyrimethanil, tebufenozide, flufenacet, oxyfluorfen, sulfadiazine, trifluralin, chlorpyrifos, quizalofop-P-ethyl, methoxyfenozide, chlorpyrifos, ethylflufenozide, flufenacetamide, flufenacet, isopyrazosulfuron, propyzoxystrobin, indole-methyl, flufenacet, cyhalofop-P-ethyl, thiamethoxam, propyzoxystrobin ... ketone, Herbicides, cyclohexane, mesotrione, cyclohexane, cyclohexane Herbicides, bispyribac-methyl, flupyridaben, flupyrazosulfuron, pyrrolizum, carbamate analogs of pyrrolizum and their agriculturally acceptable salts.

11. The composition of claim 7, wherein, The plant also contains a second herbicide tolerance polypeptide or its encoding gene, and confers or enhances tolerance to the second herbicide.

12. The composition of claim 11, wherein, The second herbicide is selected from glyphosate, glufosinate, dicamba, 2,4-D (2,4-dichlorophenoxyacetic acid), and isopropylamine. The group consists of herbicides such as acetochlor, acetolactate synthase inhibitors, photosystem II inhibitors, phenylurea-based herbicides, bromobenzonitrile-based herbicides, and combinations thereof.

13. The composition of claim 11, wherein, The second herbicide-resistant polypeptide is selected from at least one of the following: Glyphosate herbicide tolerance; glyphosate tolerance to 5-enolacetone shikimate-3-phosphate synthase, glyphosate oxidase, glyphosate-N-acetyltransferase or glyphosate decarboxylase. glufosinate-N-acetyltransferase, a herbicide resistant to glufosinate-N-acetyltransferase; Dicamba herbicide tolerance dicamba monooxygenase; 2,4-D (2,4-dichlorophenoxyacetic acid) herbicide tolerance to 2,4-D monooxygenase or aryloxyalkyl ester dioxygenase; Acetolactate synthase inhibitory sulfonylurea herbicide-resistant acetylhydroxyl synthase, acetylhydroxyl synthase or acetylhydroxyl synthase large subunit; Photosystem II inhibitory herbicide tolerance photosystem II protein D1; Cytochrome P450, a herbicide-resistant phenylurea compound; plastid-inhibiting herbicide-resistant hydroxyphenylpyruvate dioxygenase; Bromobenzonitrile herbicide-resistant nitrile hydrolase; and their combinations.

14. The composition of claim 11, wherein, The gene encoding the second herbicide tolerance polypeptide is selected from at least one of the following groups: Glyphosate herbicide tolerance genes cp4 epsps, epsps AG, mepsps, 2mepsps, goxv247, gat4601 or gat4621; glufosinate-ammonium herbicide tolerance bar or pat gene; DMO gene for tolerance to dicamba herbicide; 2,4-D (2,4-dichlorophenoxyacetic acid) herbicide tolerance AAD-1 or AAD-12 gene; different HPPDPF W336 gene for tolerance to cyclophosphamide herbicide; Sulfonylurea herbicide tolerance ALS, Csr1, Csr1-1, Csr1-2, GM-HRA, S4-HRA, Zm-HRA, SurA or SurB genes; The psbA gene, representing photosystem II-mediated herbicide tolerance; The CYP76B1 gene for tolerance to phenylurea herbicides; bxn gene for herbicide tolerance in bromobenzonitrile; and their combinations.

15. A method for preparing a herbicide-resistant plant, the method comprising transforming plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, branches, or the whole plant with a polypeptide of SEQ ID NO:2, a polypeptide of any one of claims 1 to 3, or a polynucleotide encoding said polypeptide.

16. A method for conferring or enhancing herbicide tolerance to plants, the method comprising transforming plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, branches, or the whole plant with a polypeptide of SEQ ID NO:2, a polypeptide of any one of claims 1 to 3, or a polynucleotide encoding said polypeptide.

17. A method for controlling weeds in farmland, the method comprising: Plants are provided to the farmland, the plants comprising a polypeptide of SEQ ID NO:2, a polypeptide of any one of claims 1 to 3, or a polynucleotide encoding the polypeptide; and An effective dose of a protoporphyrinogen oxidase-inhibiting herbicide was applied to the farmland.

18. The method of claim 17, wherein, The step of applying an effective dose of protoporphyrinogen oxidase inhibitory herbicide to the farmland is carried out by sequentially or simultaneously applying effective doses of two or more protoporphyrinogen oxidase inhibitory herbicides.

19. The method of claim 17, wherein, The plant also contains a second herbicide tolerance polypeptide or its encoding gene, and The step of applying an effective dose of protoporphyrinogen oxidase-inhibiting herbicide to the farmland is carried out by sequentially or simultaneously applying an effective dose of protoporphyrinogen oxidase-inhibiting herbicide and a second herbicide.

20. A method for removing unwanted aquatic organisms from a culture medium, the method comprising: Provide the culture medium with algae comprising a polypeptide of SEQ ID NO:2, a polypeptide of any one of claims 1 to 3, or a polynucleotide encoding said polypeptide; and An effective dose of a protoporphyrinogen oxidase-inhibiting herbicide was applied to the culture medium.

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