Methods and compositions for conferring and / or enhancing herbicide tolerance using various prochlorophyllide IX oxidase enzymes of cyanobacteria or variants thereof

Abstract

Protoporphyrinogen IX oxidases derived from various organisms or their variants are provided, along with their uses for conferring and / or enhancing herbicide tolerance in plants and / or algae.

Description

Technical Field

[0001] Protoporphyrinogen IX oxidases derived from various organisms or their variants are provided, along with their uses for conferring and / or enhancing herbicide tolerance in plants and / or algae. Background Technology

[0002] The porphyrin biosynthesis pathway is used to synthesize chlorophyll and heme, which play crucial roles in plant metabolism, and this process occurs within 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 oxidation to protoporphyrin IX, protoporphyrin IX binds to magnesium via magnesium chelate to synthesize chlorophyll, or binds to iron via iron chelate to synthesize heme.

[0003] Therefore, when PPO activity is inhibited, chlorophyll and heme synthesis is suppressed, and the substrate protoporphyrinogen IX exits the normal porphyrin biosynthesis pathway. This leads to the rapid export of protoporphyrinogen IX from the chloroplast to the cytoplasm, where it accumulates through non-specific peroxidases and auto-oxidation. The accumulated protoporphyrinogen IX, in the presence of light and oxygen molecules, produces highly reactive singlet oxygen (…). 1 O2 (oxygen 2) can damage cell membranes and rapidly lead to plant cell death. Based on this principle, herbicides that inhibit PPO activity have been developed. To date, classified by chemical structure, there are 10 families of PPO-inhibiting herbicides, including pyrimidine diones, diphenyl ethers, phenylpyrazoles, N-phenylphthalimides, thiadiazoles, oxadiazoles, triazinones, triazolinones, oxazolidinones, and other herbicides.

[0004] Furthermore, in order to prevent the impact of herbicides on crop growth when they are used, it is necessary to provide crops with herbicide tolerance.

[0005] At the same time, algae are photosynthetic organisms that 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 materials.

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

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

[0008] (Patent Document 1) US 6,308,458 (October 30, 2001) Summary of the Invention

[0009] Technical issues

[0010] In this disclosure, it was found that the hemY type PPO gene derived from prokaryotes and their variants exhibits broad herbicide tolerance to protoporphyrinogen IX oxidase (PPO)-inhibiting herbicides, thus indicating that the hemY type PPO gene can confer and / or enhance herbicide tolerance when introduced into the substrate and / or algae.

[0011] One embodiment provides a polypeptide comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0012] Another implementation provides a peptide variant comprising:

[0013] (1) A modified amino acid sequence of SEQ ID NO:1, wherein the modification comprises the deletion and / or substitution with an amino acid different from the original amino acid, of one or more amino acids selected from those involved in the interaction between the polypeptide of SEQ ID NO:1 and the PPO inhibitory herbicide (e.g., at least one amino acid selected from those located at the polypeptide binding site of SEQ ID NO:1 that interacts with the PPO inhibitory herbicide), or

[0014] (2) An amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence (1).

[0015] For example, at least one amino acid selected from the group consisting of amino acids of the polypeptide of SEQ ID NO:1 that participates in the interaction with PPO inhibitory herbicides can be at least one amino acid selected from the group consisting of N59, S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360 and I408 of the amino acid sequence of SEQ ID NO:1.

[0016] Another implementation provides a peptide variant comprising:

[0017] (1) A modified amino acid sequence of SEQ ID NO:2, wherein the modification comprises the deletion and / or substitution with an amino acid different from the original amino acid, selected from the amino acids involved in the interaction between the polypeptide of SEQ ID NO:2 and the PPO inhibitory herbicide (e.g., at least one amino acid selected from the amino acids located at the binding site of the polypeptide of SEQ ID NO:2 that interacts with the PPO inhibitory herbicide), or

[0018] (2) An amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence (1).

[0019] For example, at least one amino acid selected from the group consisting of amino acids of the polypeptide of SEQ ID NO:2 that participates in the interaction with PPO inhibitory herbicides can be at least one amino acid selected from the group consisting of N59, S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360 and I408 of the amino acid sequence of SEQ ID NO:2.

[0020] Another implementation provides a peptide variant comprising:

[0021] (1) A modified amino acid sequence of SEQ ID NO:3, wherein the modification comprises the deletion and / or substitution with an amino acid different from the original amino acid, of one or more amino acids selected from those involved in the interaction between the polypeptide of SEQ ID NO:3 and the PPO inhibitory herbicide (e.g., at least one amino acid selected from those located at the polypeptide binding site of SEQ ID NO:3 that interacts with the PPO inhibitory herbicide), or

[0022] (2) An amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence (1).

[0023] For example, at least one amino acid selected from the group consisting of amino acids of the polypeptide of SEQ ID NO:3 that participates in the interaction with PPO inhibitory herbicides can be at least one amino acid selected from the group consisting of N59, S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360 and I408 of the amino acid sequence of SEQ ID NO:3.

[0024] Another implementation provides a polynucleotide encoding the polypeptide or a variant of the polypeptide.

[0025] Another embodiment provides a recombinant vector containing the polynucleotide. The recombinant vector can be used as an expression vector for expressing the polynucleotide in a suitable host cell.

[0026] Another embodiment provides recombinant cells comprising the recombinant vector.

[0027] Another embodiment provides a composition for conferring and / or enhancing herbicide tolerance in plants and / or algae, comprising at least one selected from the group consisting of:

[0028] (1) Selected from the group consisting of at least one polypeptide of SEQ ID NO:1, polypeptide of SEQ ID NO:2, polypeptide of SEQ ID NO:3, polypeptide variant having modification of SEQ ID NO:1, polypeptide variant having modification of SEQ ID NO:2, polypeptide variant having modification of SEQ ID NO:3, and polypeptide comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with said polypeptide or said polypeptide variant;

[0029] (2) A polynucleotide encoding a polypeptide or polypeptide variant of (1);

[0030] (3) A recombinant vector containing the polynucleotides of (2); and

[0031] (4) Recombinant cells containing the recombinant vector of (3).

[0032] In a specific embodiment, the polynucleotide encoding the polypeptide of SEQ ID NO:1 may contain the nucleic acid sequence of SEQ ID NO:80, the polynucleotide encoding the polypeptide of SEQ ID NO:2 may contain the nucleic acid sequence of SEQ ID NO:81, and the polynucleotide encoding the polypeptide of SEQ ID NO:3 may contain the nucleic acid sequence of SEQ ID NO:82; however, the polynucleotide may not be limited to these. The polynucleotide may contain various nucleic acid sequences capable of encoding the amino acid sequence according to codon degeneracy.

[0033] Herbicides can be herbicides that inhibit the activity of protoporphyrinogen IX oxidase.

[0034] For example, the herbicide may be at least one selected from the group consisting of pyrimidine dione, diphenyl ether, phenylpyrazole, N-phenylphthalimide, phenyl ester, thiadiazole, oxadiazole, triazine one, triazolone, oxazolidinone, and other herbicides, but is not limited thereto.

[0035] In specific embodiments, the herbicide may be at least one selected from the group consisting of: tiafenacil, butafenacil, saflufenacil, benzfendizone, fomesafen, oxyfluorfen, aclofen, acifluorfen, bifenox, ethoxyfen, lactofen, chlomethoxyfen, chlornitrofen, fluoroglycofen-ethyl, halosafen, pyraflufen-ethyl, fluazolate, and flumiflufen-methyl. oxazin), indole-ethyl, flumiclorac-pentyl, fluthiacet, thidiazimin, oxadiargyl, oxadiazon, carfentrazone, sulfentrazone, trifludimoxazin, azafenidin, pentoxazone, pyraclonil, flufenpyr-ethyl, profluazol, phenopylate (2,4-dichlorophenyl-1-pyrrolidinecarboxylate), and carbamate analogs of phenopylate (e.g., O-phenylpyrrololine carbamate and piperidine carbamate analogs (see "Ujjana")). B. Nandihalli, Mary V. Duke, Stephen O. Duke, Relationships between molecular properties and biological activities of O-phenyl pyrrolidino- and piperidinocarbamate herbicides., J. Agric. Food Chem., 40(10) 1993-2000, 1992”), their agriculturally acceptable salts and combinations thereof, but not limited to these.

[0036] Plants can refer to multicellular eukaryotic organisms capable of photosynthesis, and can be monocotyledonous or dicotyledonous, or herbaceous or woody. Algae can refer to organisms capable of photosynthesis, and can be eukaryotic algae.

[0037] In one embodiment, plants or algae may be genetically manipulated to further include a second herbicide tolerance polypeptide or a gene encoding a second herbicide tolerance polypeptide, thereby conferring and / or enhancing herbicide tolerance to a second herbicide. Plants or algae genetically manipulated to include a second herbicide tolerance polypeptide or a gene encoding a second herbicide tolerance polypeptide can be prepared using the second herbicide tolerance polypeptide or a gene encoding a second herbicide tolerance polypeptide, in addition to the compositions described above for conferring and / or enhancing herbicide tolerance. Therefore, compositions for conferring and / or enhancing herbicide tolerance may further include a second herbicide tolerance polypeptide or a gene encoding a second herbicide tolerance polypeptide.

[0038] Examples of second herbicides may include, but are not limited to, cell division inhibitory herbicides, photosynthesis inhibitory herbicides, amino acid synthesis inhibitory herbicides, plastid inhibitory herbicides, cell membrane inhibitory herbicides, etc.

[0039] In specific embodiments, the second herbicide may be exemplified as glyphosate, glufosinate, dicamba, 2,4-D (2,4-dichlorophenoxyacetic acid), isoxaflutole, ALS (acetyllactate synthase) inhibitory herbicide, photosystem II inhibitory herbicide, phenylurea herbicide, bromobenzonitrile herbicide, or combinations thereof, but is not limited thereto.

[0040] For example, a second herbicide-resistant polypeptide can be exemplified as at least one 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 or AAD (aryloxyalkylene ester dioxygenase); ALS-inhibiting sulfonylurea herbicide-resistant ALS (acetolactate synthase), AHAS (acetylhydroxy acid synthase), or AtAHASL (Arabidopsis thaliana). (thaliana) acetylhydroxy acid synthase large subunit; photosystem II inhibitory herbicide-resistant photosystem II protein D1; phenylurea herbicide-resistant cytochrome P450; plastid-inhibiting herbicide-resistant HPPD (hydroxyphenylpyruvate dioxygenase); bromobenzonitrile herbicide-resistant nitrile hydrolase; and combinations thereof, but not limited thereto.

[0041] Additionally, the gene encoding the second herbicide tolerance polypeptide can be exemplified as being selected from at least one of the following groups: glyphosate herbicide tolerance genes cp4 epsps, mepsps, 2 mepsps, 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 isoxaflutole herbicide tolerance genes HPPDPF. The W336 gene and the bxn gene for herbicide tolerance to bromobenzonitrile; and combinations thereof, but not limited thereto.

[0042] Another implementation provides herbicide-tolerant plant and / or algae transformed with polynucleotides or their clones or progeny.

[0043] Another embodiment provides a method for preparing transgenic plants or transgenic algae with herbicide tolerance or enhanced herbicide tolerance, including the step of transforming plants and / or algae with polynucleotides.

[0044] Another implementation provides a method for conferring or enhancing herbicide tolerance in plants and / or algae, including the step of converting plants and / or algae with polynucleotides.

[0045] It can transform algae, and / or plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, buds, or the whole plant.

[0046] Transformers can be algae, and / or plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, buds, or whole organisms. Transformers may include offspring obtained from the first transformant (e.g., T1 to T8 generations).

[0047] Another implementation provides a method for controlling weeds in farmland, comprising:

[0048] Providing plants to farmland, wherein the plants comprise at least one selected from the group consisting of the polypeptide, a variant of the polypeptide, a polynucleotide encoding the polypeptide, a polynucleotide encoding the polypeptide variant, a recombinant vector comprising the polynucleotide, and recombinant cells comprising the recombinant vector; and

[0049] Apply an effective amount of protoporphyrinogen IX oxidase-inhibiting herbicide to farmland (or to plants).

[0050] In a specific implementation, the step of applying an effective amount of protoporphyrinogen IX oxidase inhibitory herbicide to farmland (or to plants) can be carried out by sequentially or simultaneously applying effective amounts of at least two protoporphyrinogen IX oxidase inhibitory herbicides.

[0051] In another embodiment, the plant may be genetically manipulated to further include a second herbicide-tolerant polypeptide or a gene encoding a second herbicide-tolerant polypeptide, and effective amounts of protoporphyrinogen IX oxidase-inhibiting herbicide and the second herbicide may be applied sequentially or simultaneously.

[0052] Another embodiment provides a method for removing unwanted organisms from a culture medium, comprising providing algae to the culture medium, wherein the algae comprises at least one selected from the group consisting of the polypeptide, a variant of the polypeptide, a polynucleotide encoding the polypeptide, a polynucleotide encoding the variant, a recombinant vector containing the polynucleotide, and recombinant cells containing the recombinant vector; and applying an effective amount of a protoporphyrinogen IX 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] As used herein, “conferring and / or enhancing herbicide tolerance in plants or algae” or “enhancing herbicide tolerance in plants or algae” can be interpreted as conferring herbicide tolerance in plants or algae that do not have herbicide tolerance and / or further enhancing the herbicide tolerance in plants or algae that do have herbicide tolerance.

[0056] As used herein, the terms "consisting of a sequence," "consisting substantially of a sequence," or "including a sequence" may be used to cover both cases where the described sequence is included and / or must be included, but are not intended to exclude the inclusion of other sequences besides the described sequence.

[0057] As used herein, the terms "a protein or polypeptide comprising or consisting of an amino acid sequence identified by SEQ ID NO" and "a gene or polynucleotide comprising or consisting of a nucleic acid sequence identified by SEQ ID NO" can refer to a protein (or polypeptide) or gene (or polynucleotide) substantially composed of or consisting of the amino acid sequence or nucleic acid sequence, or having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence or nucleic acid sequence while retaining its inherent activity and / or function.

[0058] One embodiment provides a polypeptide comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0059] Another embodiment provides a peptide variant, said peptide variant being at least one selected from the group consisting of:

[0060] A polypeptide variant comprising: an amino acid sequence modified with respect to SEQ ID NO:1, wherein the modification comprises the deletion and / or substitution with an amino acid different from the original amino acid, of one or more amino acids selected from those involved in the interaction of the polypeptide of SEQ ID NO:1 with PPO inhibitory herbicides (e.g., at least one amino acid selected from those located at the polypeptide binding site of SEQ ID NO:1 that interacts with PPO inhibitory herbicides), or an amino acid sequence having more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity with the amino acid sequence;

[0061] A peptide variant comprising: an amino acid sequence modified with respect to SEQ ID NO:2, wherein the modification comprises the deletion and / or substitution with an amino acid different from the original amino acid, or an amino acid sequence having 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence; and

[0062] A polypeptide variant comprising: an amino acid sequence modified with respect to SEQ ID NO:3, wherein the modification comprises the deletion and / or substitution with an amino acid different from the original amino acid, of one or more amino acids selected from those involved in the interaction of SEQ ID NO:3 with PPO inhibitory herbicides (e.g., at least one amino acid selected from those located at the polypeptide binding site of SEQ ID NO:3 interacting with PPO inhibitory herbicides), or an amino acid sequence having more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity with the amino acid sequence.

[0063] In other embodiments, a polynucleotide encoding SEQ ID NO: 1, 2, or 3, or a polypeptide variant, is provided; a recombinant vector comprising said polynucleotide; and a recombinant cell comprising said recombinant vector. The polynucleotide can be engineered to contain codons optimized for the cells to be transformed. Optimized codons are readily known to those skilled in the art (e.g., see "http: / / sg.idtdna.com / CodonOpt", etc.).

[0064] Another embodiment provides a composition for conferring and / or enhancing herbicide tolerance in plants and / or algae, said composition comprising at least one selected from the group consisting of:

[0065] (1) Selected from the polypeptide of SEQ ID NO:1, the polypeptide of SEQ ID NO:2, the polypeptide of SEQ ID NO:3, a polypeptide variant having a modification of SEQ ID NO:1, a polypeptide variant having a modification of SEQ ID NO:2, a polypeptide variant having a modification of SEQ ID NO:3, and a polypeptide comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with said polypeptide or said polypeptide variant;

[0066] (2) A polynucleotide encoding a polypeptide or polypeptide variant of (1);

[0067] (3) A recombinant vector containing the polynucleotides of (2); and

[0068] (4) Recombinant cells containing the recombinant vector of (3).

[0069] In a specific embodiment, the polynucleotide encoding the polypeptide of SEQ ID NO:1 may contain the nucleic acid sequence of SEQ ID NO:80, the polynucleotide encoding the polypeptide of SEQ ID NO:2 may contain the nucleic acid sequence of SEQ ID NO:81, and the polynucleotide encoding the polypeptide of SEQ ID NO:3 may contain the nucleic acid sequence of SEQ ID NO:82; however, the polynucleotide may not be limited to these. The polynucleotide may contain various nucleic acid sequences capable of encoding the amino acid sequence according to codon degeneracy.

[0070] In other embodiments, a transformant of a herbicide-resistant plant and / or algae is provided, which is transformed with a polynucleotide encoding the polypeptide or a variant of the polypeptide. The polynucleotide can be engineered to contain codons optimized for the cells to be transformed. Optimized codons are readily known to those skilled in the art (e.g., see "http: / / sg.idtdna.com / CodonOpt", etc.).

[0071] Another embodiment provides a method for preparing transgenic plants or transgenic algae with herbicide tolerance or enhanced herbicide tolerance, including the step of transforming algae, or plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, buds, or the whole plant, with polynucleotides.

[0072] Another implementation provides a method for conferring or enhancing herbicide tolerance to plants and / or algae, including the step of converting algae, or plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, buds, or the whole plant, with polynucleotides.

[0073] The invention will now be described in more detail.

[0074] The polypeptides described in this article, SEQ ID NO:1, 2 and 3, are PPO proteins derived from cyanobacteria that are resistant to PPO-inhibiting herbicides.

[0075] Specifically, a PPO protein (containing GenBank accession number CP032152.1) derived from the Thermosynechococcus elongatus strain PKUAC-SCTE542 is provided. This protein is named CyPPO19 in this paper. The amino acid sequence of the protein is represented by SEQ ID NO:1, and the nucleotide sequence of the gene encoding the protein is represented by SEQ ID NO:80.

[0076] In addition, a PPO protein (GenBank accession number RMH63851.1) derived from the cyanobacterium strain J003 is provided. This protein is named CyPPO20, and its amino acid sequence is represented by SEQ ID NO:2. The nucleotide sequence of the gene encoding this protein is represented by SEQ ID NO:81.

[0077] In addition, a PPO protein (GenBank accession number BAY51976.1) derived from the Thermosynechococcus vulcanus strain NIES-2134 is provided. This protein is named CyPPO18, and its amino acid sequence is represented by SEQ ID NO:3. The nucleotide sequence of the gene encoding this protein is represented by SEQ ID NO:82.

[0078] In this document, peptides and peptide variants may be expressed as herbicide-resistant PPO proteins or herbicide-resistant PPO protein variants that are resistant to PPO-inhibiting herbicides. Additionally, as used herein, the term "herbicide-resistant PPO or its variants" may be used to refer to the above-mentioned herbicide-resistant PPO proteins or herbicide-resistant PPO protein variants, herbicide-resistant PPO protein encoding genes or herbicide-resistant PPO protein variant encoding genes, or combinations thereof.

[0079] Compared to plant-derived PPO proteins, cyanobacterial PPO proteins may exhibit superior enzymatic activity and confer tolerance to PPO-inhibiting herbicides. Furthermore, when cyanobacterial PPO proteins are modified through amino acid mutations (variations) within a range that maintains their overall enzymatic activity, their tolerance to PPO-inhibiting herbicides can be further enhanced compared to wild-type PPO proteins. Such amino acid mutations may include substitutions, deletions, additions, and / or additions of one or more amino acid residues selected from the interaction sites between the PPO protein and the herbicide.

[0080] The PPO protein variants will be described in more detail below.

[0081] One embodiment provides a polypeptide variant that is a variant of the polypeptide of SEQ ID NO:1 (CyPPO19), the variant comprising or consisting of the following amino acid sequences: an amino acid sequence modified with respect to SEQ ID NO:1, wherein the modification comprises the deletion and / or substitution with an amino acid different from the original amino acid (i.e., the amino acid corresponding to the wild type) of one or more amino acids of SEQ ID NO:1 involved in the interaction with PPO inhibitory herbicides (e.g., at least one amino acid selected from the amino acids located at the binding site of the polypeptide of SEQ ID NO:1 interacting with PPO inhibitory herbicides); or an amino acid sequence having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher sequence identity with the amino acid sequence.

[0082] Another embodiment provides a polypeptide variant, which is a variant of the polypeptide of SEQ ID NO:2 (CyPPO20), the variant comprising or consisting of the following amino acid sequences: an amino acid sequence modified with respect to SEQ ID NO:2, wherein the modification comprises the deletion and / or substitution with an amino acid different from the original amino acid (i.e., the amino acid corresponding to the wild type) of one or more amino acids of SEQ ID NO:2 involved in the interaction with PPO inhibitory herbicides (e.g., at least one amino acid selected from the amino acids located at the binding site of the polypeptide of SEQ ID NO:2 interacting with PPO inhibitory herbicides); or an amino acid sequence having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher identity with the amino acid sequence.

[0083] Another embodiment provides a polypeptide variant that is a variant of the polypeptide of SEQ ID NO:3 (CyPPO18), the variant comprising or consisting of the following amino acid sequences: an amino acid sequence modified with respect to SEQ ID NO:3, wherein the modification comprises the deletion and / or substitution with an amino acid different from the original amino acid (i.e., the amino acid corresponding to the wild type) of one or more amino acids of SEQ ID NO:3 involved in the interaction with PPO inhibitory herbicides; or an amino acid sequence having 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher identity with the amino acid sequence.

[0084] The amino acid to be missing or replaced with another amino acid different from the original amino acid (e.g., at least one residue selected from the group consisting of amino acids located at the binding site of the polypeptide to the PPO inhibitory herbicide in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3) can be at least one of the group consisting of N59 (referring to N(Asn) at position 59; the expression of the following amino acid residues is interpreted in this way), S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360, and I408 in the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, for example, SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. The amino acid sequence of IDNO:3 may contain at least one, at least two, at least three, at least four, at least five, at least six, or all of the amino acid sequences R89, V165, A167, V305, L327, F360, and I408.

[0085] In one specific embodiment, the polypeptide variant may include: an amino acid sequence modified for SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, wherein one or more amino acid residues selected from the group consisting of N59, S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360, and I408 of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 (e.g., at least one, at least two, at least three, at least four, at least five, at least six, or all of R89, V165, A167, V305, L327, F360, and I408) are 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 (Tr Amino acid substitutions that are different from the corresponding amino acids in the wild type, such as those consisting of the groups of M (Met), V (Val), I (Ile), T (Thr), L (Leu), C (Cys), A (Ala), S (Ser), R (Arg), and W (Trp); or amino acid sequences that have at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence.

[0086] For example, peptide variants may include:

[0087] (a) An amino acid sequence having a modification of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, wherein the modification comprises at least one, at least two, at least three, at least four, at least five, at least six, or all of the following amino acid mutations selected from the group consisting of amino acids in the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3:

[0088] (i) F360M (referring to a variant or mutation in which the amino acid residue at position 360 is replaced by M (Met) instead of F (Phe); the expression of the following amino acid mutations is interpreted in this way), F360V, F360I, F360T, or F360L,

[0089] (ii) A167C, A167L, or A167I,

[0090] (iii) V305M or V305L,

[0091] (iv)R89A,

[0092] (v)V165S or V165C,

[0093] (vi)L327T, and

[0094] (vii) I408R or I408W; or

[0095] (b) An amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence.

[0096] More specifically, variants of the polypeptide may include: an amino acid sequence having a modification of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with said amino acid sequence, wherein the modification includes SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. The following amino acid mutations in the amino acid sequence of NO:3: R89A, V165C, V165S, A167C, A167I, A167L, V305L, V305M, L327T, F360M, F360I, F360L, F360V, F360T, I408R, I408W, R89A+V165C (referring to all variants or mutations containing the substitution of R for A at residue 89 and V for C at residue 165; the following expressions of two or more amino acid mutations are interpreted in this manner), R89A+V165S, R89A+A1 67C, R89A+A167I, R89A+A167L, R89A+V305L, R89A+V305M, R89A+L327T, R89A+F360M, R89A+F360I, R89A+F360L, R89A+F360V, R8 9A+F360T, R89A+I408R, V165C+A167C, V165C+A167I, V165C+A167L, V165C+V305L, V165C+V305M, V165C+L327T, V165C+F360M, V 165C+F360I, V165C+F360L, V165C+F360V, V165C+F360T, V165C+I408R, V165S+A167C, V165S+A167I, V165S+A167L, V165S+V30 5L, V165S+V305M, V165S+L327T, V165S+F360M, V165S+F360I, V165S+F360L, V165S+F360V, V165S+F360T, V165S+I408W, A167C+ V305L, A167C+V305M, A167C+L327T, A167C+F360M, A167C+F360I, A167C+F360L, A167C+F360V, A167C+F360T, A167C+I408R, A16 7I+V305L, A167I+V305M, A167I+L327T, A167I+F360M, A167I+F360I, A167I+F360L, A167I+F360V, A167I+F360T, A167I+I408W,A167L+V305L, A167L+V305M, A167L+L327T, A167L+F360M, A167L+F360I, A167L+F360L, A167L+F360V, A167L+F360T, A167L+I408R, V305L+L327T, V30 5L+F360M, V305L+F360I, V305L+F360L, V305L+F360V, V305L+F360T, V305L+I408W, V305M+L327T, V305M+F360M, V305M+F360I, V305M+F360L, V305M+ F360V、V305M+F360T、V305M+I408R、L327T+F360M、L327T+F360I、L327T+F 360L、L327T+F360V、L327T+F360T、L327T+I408R、F360M+I408R、F360I+I40 8R、F360L+I408W、F360V+I408W、F360T+I408W、R89A+V165C+A167C、R89A+V 165C+A167I、R89A+V165C+A167L、R89A+V165C+V305L、R89A+V165C+V305M、 R89A+V165C+L327T, R89A+V165C+F360M, R89A+V165C+F360I, R89A+V165C+F360L, R89A+V165C+F360V, R89A+V165C+F360T, R89A+V165C+I408R, R89A +V165S+A167C、R89A+V165S+A167I、R89A+V165S+A167L、R89A+V165S+V30 5L、R89A+V165S+V305M、R89A+V165S+L327T、R89A+V165S+F360M、R89A+V16 5S+F360I、R89A+V165S+F360L、R89A+V165S+F360V、R89A+V165S+F360T、R 89A+V165S+I408W、R89A+A167C+V305L、R89A+A167C+V305M、R89A+A167C+L 327T, R89A+A167C+F360M, R89A+A167C+F360I, R89A+A167C+F360L, R89A+A167C+F360V, R89A+A167C+F360T, R89A+A167C+I408R, R89A+A167I+V305LR89A+A167I+V305M, R89A+A167I+L327T, R89A+A167I+F360M, R89A+A167I+F360I, R89A+A167I+F360L, R89A+A167I+F360V, R89A+A167I+F360T, R89A +A167I+I408W, R89A+A167L+V305L, R89A+A167L+V305M, R89A+A167L+L327T, R89A+A167L+F360M, R89A+A167L+F360I, R89A+A167L+F360L, R89A+A16 7L+F360V, R89A+A167L+F360T, R89A+A167L+I408R, R89A+V305L+L327T, R89A+V305L+F360M, R89A+V305L+F360I, R89A+V305L+F360L, R89A+V305L+F 360V, R89A+V305L+F360T, R89A+V305L+I408W, R89A+V305M+L327T, R89A+V305M+F360M, R89A+V305M+F360I, R89A+V305M+F360L, R89A+V305M+F360V R89A+V305M+F360T, R89A+V305M+I408R, R89A+L327T+F360M, R89A+L327T+F360I, R89A+L327T+F360L, R89A+L327T+F360V, R89A+L327T+F360T, R89 A+L327T+I408R、R89A+F360M+I408R、R89A+F360I+I408R、R89A+F360L+I4 08W、R89A+F360V+I408W、R89A+F360T+I408W、V165C+A167C+V305L、V165C+ A167C+V305M、V165C+A167C+L327T、V165C+A167C+F360M、V165C+A167C+F 360I、V165C+A167C+F360L、V165C+A167C+F360V、V165C+A167C+F360T、V16 5C+A167C+I408R、V165C+A167I+V305L、V165C+A167I+V305M、V165C+A167 I+L327T、V165C+A167I+F360M、V165C+A167I+F360I、V165C+A167I+F360L、V165C+A167I+F360V、V165C+A167I+F360T、V165C+A167I+I408W、V165C+A167L+V305L、V165C+A167L+V305M、V165C+A167L+L327T、V165C+A167L+F360M、V165C+A167L+F360I、V165C+A167L+F360L、V165C+A167L+F360V、V165C+A167L+F360T、V165C+A167L+I408R、V165C+V305L+L327T、V165C+V305L+F360M、V165C+V305L+F360I、V165C+V305L+F360L、V165C+V305L+F360V、V165C+V305L+F360T、V165C+V305L+F360T、V165C+V305L+I408W、V165C+V305M+L327T、V165C+V305M+F360M、V165C+V305M+F360I、V165C+V305M+F360L、V165C+V305M+F360V、V165C+V305M+F360T、V165C+V305M+I408R、V165C+L327T+F360M、V165C+L327T+F360I、V165C+L327T+F360L、V165C+L327T+F360V、V165C+L327T+F360T、V165C+L327T+I408R、V165C+F360M+I408R、V165C+F360I+I408R、V165C+F360L+I408W、V165C+F360V+I408W、V165C+F360T+I408W、V165S+A167C+V305L、V165S+A167C+V305M、V165S+A167C+L327T、V165S+A167C+F360M、V165S+A167C+F360I、V165S+A167C+F360L、V165S+A167C+F360V、V165S+A167C+F360T、V165S+A167C+I408R、V165S+A167I+V305L、V165S+A167I+V305M、V165S+A167I+L327T、V165S+A167I+F360M、V165S+A167I+F360I、V165S+A167I+F360L、V165S+A167I+F360V、V165S+A167I+F360T、V165S+A167I+I408W、V165S+A167L+V305L、V165S+A167L+V305M、V165S+A167L+L327T、V165S+A167L+F360M、V165S+A167L+F360I、V165S+A167L+F360L、V165S+A167L+F360V、V165S+A167L+F360T、V165S+A167L+I408R、V165S+V305L+L327T、V165S+V305L+F360M、V165S+V305L+F360I、V165S+V305L+F360L、V165S+V305L+F360V、V165S+V305L+F360T、V165S+V305L+I408W、V165S+V305M+L327T、V165S+V305M+F360M、V165S+V305M+F360I、V165S+V305M+F360L、V165S+V305M+F360V、V165S+V305M+F360T、V165S+V305M+I408R、V165S+L327T+F360M、V165S+L327T+F360I、V165S+L327T+F360L、V165S+L327T+F360V、V165S+L327T+F360T、V165S+L327T+I408R、V165S+F360M+I408R、V165S+F360I+I408R、V165S+F360L+I408W、V165S+F360V+I408W、V165S+F360T+I408W、A167C+V305L+L327T、A167C+V305L+F360M、A167C+V305L+F360I、A167C+V305L+F360L、A167C+V305L+F360V、A167C+V305L+F360T、A167C+V305L+I408W、A167C+V305M+L327T、A167C+V305M+F360M、A167C+V305M+F360I、A167C+V305M+F360L、A167C+V305M+F360V、A167C+V305M+F360T、A167C+V305M+I408R、A167C+L327T+F360M、A167C+L327T+F360I、A167C+L327T+F360L、A167C+L327T+F360V、A167C+L327T+F360T、A167C+L327T+I408R、A167C+F360M+I408R、A167C+F360I+I408R、A167C+F360L+I408W、A167C+F360V+I408W、A167C+F360T+I408W、A167I+V305L+L327T、A167I+V305L+F360M、A167I+V305L+F360I、A167I+V305L+F360L、A167I+V305L+F360V、A167I+V305L+F360T、A167I+V305L+I408W、A167I+V305M+L327T、A167I+V305M+F360M、A167I+V305M+F360I、A167I+V305M+F360L、A167I+V305M+F360V、A167I+V305M+F360T、A167I+V305M+I408R、A167I+L327T+F360M、A167I+L327T+F360I、A167I+L327T+F360L、A167I+L327T+F360V、A167I+L327T+F360T、A167I+L327T+I408R、A167I+F360M+I408R、A167I+F360I+I408R、A167I+F360L+I408W、A167I+F360V+I408W、A167I+F360T+I408W、A167L+V305L+L327T、A167L+V305L+F360M、A167L+V305L+F360I、A167L+V305L+F360L、A167L+V305L+F360V、A167L+V305L+F360T、A167L+V305L+I408W、A167L+V305M+L327T、A167L+V305M+F360M、A167L+V305M+F360I、A167L+V305M+F360L、A167L+V305M+F360V、A167L+V305M+F360T、A167L+V305M+I408R、A167L+L327T+F360M、A167L+L327T+F360I、A167L+L327T+F360L、A167L+L327T+F360V、A167L+L327T+F360T、A167L+L327T+I408R、A167L+F360M+I408R、A167L+F360I+I408R、A167L+F360L+I408W、A167L+F360V+I408W、A167L+F360T+I408W、V305L+L327T+F360M、V305L+L327T+F360I、V305L+L327T+F360L、V305L+L327T+F360V、V305L+L327T+F360T、V305L+L327T+I408R、V305L+F360M+I408R、V305L+F360I+I408R、V305L+F360L+I408W、V305L+F360V+I408W、V305L+F360T+I408W、V305M+L327T+F360M、V305M+L327T+F360I、V305M+L327T+F360L、V305M+L327T+F360V、V305M+L327T+F360T、V305M+L327T+I408R、V305M+F360M+I408R、V305M+F360I+I408R、V305M+F360L+I408W、V305M+F360V+I408W、V305M+F360T+I408W、L327T+F360M+I408R、L327T+F360I+I408R、L327T+F360T+I408R、L327T+F360L+I408W、L327T+F360V+I408W、L327T+F360T+I408W、R89A+V165C+A167C+V305L、R89A+V165C+A167C+V305M、R89A+V165C+A167C+L327T、R89A+V165C+A167C+F360M、R89A+V165C+A167C+F360I、R89A+V165C+A167C+F360L、R89A+V165C+A167C+F360V、R89A+V165C+A167C+F360T、R89A+V165C+A167C+I408R、R89A+V165C+A167I+V305L、R89A+V165C+A167I+V305M、R89A+V165C+A167I+L327T、R89A+V165C+A167I+F360M、R89A+V165C+A167I+F360I、R89A+V165C+A167I+F360L、R89A+V165C+A167I+F360V、R89A+V165C+A167I+I408W、R89A+V165C+A167L+V305L、R89A+V165C+A167L+V305M、R89A+V165C+A167L+L327T、R89A+V165C+A167L+F360M、R89A+V165C+A167L+F360I、R89A+V165C+A167L+F360L、R89A+V165C+A167L+F360V、R89A+V165C+A167L+I408R、R89A+V165C+V305L+L327T、R89A+V165C+V305L+F360M、R89A+V165C+V305L+F360I、R89A+V165C+V305L+F360L、R89A+V165C+V305L+F360V、R89A+V165C+V305L+I408W、R89A+V165C+V305M+L327T、R89A+V165C+V305M+F360M、R89A+V165C+V305M+F360I、R89A+V165C+V305M+F360L、R89A+V165C+V305M+F360V、R89A+V165C+V305M+I408R、R89A+V165C+L327T+F360M、R89A+V165C+L327T+F360I、R89A+V165C+L327T+F360L、R89A+V165C+L327T+F360V、R89A+V165C+L327T+I408R、R89A+V165C+F360M+I408R、R89A+V165C+F360I+I408R、R89A+V165C+F360L+I408W、R89A+V165C+F360V+I408W、R89A+V165S+A167C+V305L、R89A+V165S+A167C+V305M、R89A+V165S+A167C+L327T、R89A+V165S+A167C+F360M、R89A+V165S+A167C+F360I、R89A+V165S+A167C+F360L、R89A+V165S+A167C+F360V、R89A+V165S+A167C+I408R、R89A+V165S+A167I+V305L、R89A+V165S+A167I+V305M、R89A+V165S+A167I+L327T、R89A+V165S+A167I+F360M、R89A+V165S+A167I+F360I、R89A+V165S+A167I+F360L、R89A+V165S+A167I+F360V、R89A+V165S+A167I+I408W、R89A+V165S+A167L+V305L、R89A+V165S+A167L+V305M、R89A+V165S+A167L+L327T, R89A+V165S+A167L+F360M, R89A+V165S+A167L+F360I, R89A+V165S+A167L+F360L, R89A+V165S+A167L+F360V, R89A+V16 5S+A167L+I408R、R89A+V165S+V305L+L327T、R89A+V165S+V305L+F360M、R 89A+V165S+V305L+F360I、R89A+V165S+V305L+F360L、R89A+V165S+V305L+ F360V、R89A+V165S+V305L+I408W、R89A+V165S+V305M+L327T、R89A+V165 S+V305M+F360M、R89A+V165S+V305M+F360I、R89A+V165S+V305M+F360L、R8 9A+V165S+V305M+F360V, R89A+V165S+V305M+I408R, R89A+V165S+L327T+F360M, R89A+V165S+L327T+F360I, R89A+V165S+L327T+F360L, R89A+V165S+ L327T+F360V、R89A+V165S+L327T+I408R、R89A+V165S+F360M+I408R、R89 A+V165S+F360I+I408R、R89A+V165S+F360L+I408W、R89A+V165S+F360V+I4 08W, R89A+A167C+V305L+L327T, R89A+A167C+V305L+F360M, R89A+A167C+V305L+F360I, R89A+A167C+V305L+F360L, R89A+A167C+V305L+F360V, R89A+ A167C+V305L+I408W, R89A+A167C+V305M+L327T, R89A+A167C+V305M+F360M, R89A+A167C+V305M+F360I, R89A+A167C+V305M+F360L, R89A+A167C+V30 5M+F360V, R89A+A167C+V305M+I408R, R89A+A167C+L327T+F360M, R89A+A167C+L327T+F360I, R89A+A167C+L327T+F360L, R89A+A167C+L327T+F360VR89A+A167C+L327T+I408R, R89A+A167C+F360M+I408R, R89A+A167C+F360I+I408R, R89A+A167C+F360L+I408W, R89A+A167C+F360V+I408W, R89A+A16 7I+V305L+L327T, R89A+A167I+V305L+F360M, R89A+A167I+V305L+F360I, R89A+A167I+V305L+F360L, R89A+A167I+V305L+F360V, R89A+A167I+V305L+ I408W, R89A+A167I+V305M+L327T, R89A+A167I+V305M+F360M, R89A+A167I+V305M+F360I, R89A+A167I+V305M+F360L, R89A+A167I+V305M+F360V, R8 9A+A167I+V305M+I408R, R89A+A167I+L327T+F360M, R89A+A167I+L327T+F360I, R89A+A167I+L327T+F360L, R89A+A167I+L327T+F360V, R89A+A167I+ L327T+I408R, R89A+A167I+F360M+I408R, R89A+A167I+F360I+I408R, R89A+A167I+F360L+I408W, R89A+A167I+F360V+I408W, R89A+A167L+V305L+L3 27T, R89A+A167L+V305L+F360M, R89A+A167L+V305L+F360I, R89A+A167L+V305L+F360L, R89A+A167L+V305L+F360V, R89A+A167L+V305L+I408W, R89A+ A167L+V305M+L327T, R89A+A167L+V305M+F360M, R89A+A167L+V305M+F360I, R89A+A167L+V305M+F360L, R89A+A167L+V305M+F360V, R89A+A167L+V30 5M+I408R, R89A+A167L+L327T+F360M, R89A+A167L+L327T+F360I, R89A+A167L+L327T+F360L, R89A+A167L+L327T+F360V, 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T+F360M、R89A+V165C+A167L+L327T+F360I、R89A+V165C+A167L+L327T+F360L、R89A+V165C+A167L+L327T+F360V、R89A+V165C+A167L+L327T+I408R、R89A+V165C+A167L+F360M+I408R、R89A+V165C+A167L+F360I+I408R、R89A+V165C+A167L+F360L+I408W、R89A+V165C+A167L+F360V+I408W、R89A+V165C+V305L+L327T+F360M、R89A+V165C+V305L+L327T+F360I、R89A+V165C+V305L+L327T+F360L、R89A+V165C+V305L+L327T+F360V、R89A+V165C+V305L+L327T+I408R、R89A+V165C+V305L+F360M+I408R、R89A+V165C+V305L+F360I+I408R、R89A+V165C+V305L+F360L+I408W、R89A+V165C+V305L+F360V+I408W、R89A+V165C+V305M+L327T+F360M、R89A+V165C+V305M+L327T+F360I、R89A+V165C+V305M+L327T+F360L、R89A+V165C+V305M+L327T+F360V、R89A+V165C+V305M+L327T+I408R、R89A+V165C+V305M+F360M+I408R、R89A+V165C+V305M+F360I+I408R、R89A+V165C+V305M+F360L+I408W、R89A+V165C+V305M+F360V+I408W、R89A+V165C+L327T+F360M+I408R、R89A+V165C+L327T+F360I+I408R、R89A+V165C+L327T+F360L+I408W、R89A+V165C+L327T+F360V+I408W、R89A+V165S+A167C+V305L+L327T、R89A+V165S+A167C+V305L+F360M、R89A+V165S+A167C+V305L+F360I、R89A+V165S+A167C+V305L+F360L、R89A+V165S+A167C+V305L+F360V、R89A+V165S+A167C+V305L+I408W、R89A+V165S+A167C+V305M+L327T、R89A+V165S+A167C+V305M+F360M、R89A+V165S+A167C+V305M+F360I、R89A+V165S+A167C+V305M+F360L、R89A+V165S+A167C+V305M+F360V、R89A+V165S+A167C+V305M+I408R、R89A+V165S+A167C+L327T+F360M、R89A+V165S+A167C+L327T+F360I、R89A+V165S+A167C+L327T+F360L、R89A+V165S+A167C+L327T+F360V、R89A+V165S+A167C+L327T+I408R、R89A+V165S+A167C+F360M+I408R、R89A+V165S+A167C+F360I+I408R、R89A+V165S+A167C+F360L+I408W、R89A+V165S+A167C+F360V+I408W、R89A+V165S+A167I+V305L+L327T、R89A+V165S+A167I+V305L+F360M、R89A+V165S+A167I+V305L+F360I、R89A+V165S+A167I+V305L+F360L、R89A+V165S+A167I+V305L+F360V、R89A+V165S+A167I+V305L+I408W、R89A+V165S+A167I+V305M+L327T、R89A+V165S+A167I+V305M+F360M、R89A+V165S+A167I+V305M+F360I、R89A+V165S+A167I+V305M+F360L、R89A+V165S+A167I+V305M+F360V、R89A+V165S+A167I+V305M+I408R、R89A+V165S+A167I+L327T+F360M、R89A+V165S+A167I+L327T+F360I、R89A+V165S+A167I+L327T+F360L、R89A+V165S+A167I+L327T+F360V、R89A+V165S+A167I+L327T+I408R、R89A+V165S+A167I+F360M+I408R、R89A+V165S+A167I+F360I+I408R、R89A+V165S+A167I+F360L+I408W、R89A+V165S+A167I+F360V+I408W、R89A+V165S+A167L+V305L+L327T、R89A+V165S+A167L+V305L+F360M、R89A+V165S+A167L+V305L+F360I、R89A+V165S+A167L+V305L+F360L、R89A+V165S+A167L+V305L+F360V、R89A+V165S+A167L+V305L+I408W、R89A+V165S+A167L+V305M+L327T、R89A+V165S+A167L+V305M+F360M、R89A+V165S+A167L+V305M+F360I、R89A+V165S+A167L+V305M+F360L、R89A+V165S+A167L+V305M+F360V、R89A+V165S+A167L+V305M+I408R、R89A+V165S+A167L+L327T+F360M、R89A+V165S+A167L+L327T+F360I、R89A+V165S+A167L+L327T+F360L、R89A+V165S+A167L+L327T+F360V、R89A+V165S+A167L+L327T+I408R、R89A+V165S+A167L+F360M+I408R、R89A+V165S+A167L+F360I+I408R、R89A+V165S+A167L+F360L+I408W、R89A+V165S+A167L+F360V+I408W、R89A+V165S+V305L+L327T+F360M、R89A+V165S+V305L+L327T+F360I、R89A+V165S+V305L+L327T+F360L、R89A+V165S+V305L+L327T+F360V、R89A+V165S+V305L+L327T+I408R、R89A+V165S+V305L+F360M+I408R、R89A+V165S+V305L+F360I+I408R、R89A+V165S+V305L+F360L+I408W、R89A+V165S+V305L+F360V+I408W、R89A+V165S+V305M+L327T+F360M、R89A+V165S+V305M+L327T+F360I、R89A+V165S+V305M+L327T+F360L、R89A+V165S+V305M+L327T+F360V、R89A+V165S+V305M+L327T+I408R、R89A+V165S+V305M+F360M+I408R、R89A+V165S+V305M+F360I+I408R、R89A+V165S+V305M+F360L+I408W、R89A+V165S+V305M+F360V+I408W、R89A+V165S+L327T+F360M+I408R、R89A+V165S+L327T+F360I+I408R、R89A+V165S+L327T+F360L+I408W、R89A+V165S+L327T+F360V+I408W、R89A+A167C+V305L+L327T+F360M、R89A+A167C+V305L+L327T+F360I、R89A+A167C+V305L+L327T+F360L、R89A+A167C+V305L+L327T+F360V、R89A+A167C+V305L+L327T+I408R、R89A+A167C+V305L+F360M+I408R、R89A+A167C+V305L+F360I+I408R、R89A+A167C+V305L+F360L+I408W、R89A+A167C+V305L+F360V+I408W、R89A+A167C+V305M+L327T+F360M、R89A+A167C+V305M+L327T+F360I、R89A+A167C+V305M+L327T+F360L、R89A+A167C+V305M+L327T+F360V、R89A+A167C+V305M+L327T+I408R、R89A+A167C+V305M+F360M+I408R、R89A+A167C+V305M+F360I+I408R、R89A+A167C+V305M+F360L+I408W、R89A+A167C+V305M+F360V+I408W, R89A+A167C+L327T+F360M+I408R, R89A+A167C+L327T+F360I+I408R, R89A+A167C+L327T+F360L+I408W, R89A+A1 67C+L327T+F360V+I408W, R89A+A167I+V305L+L327T+F360M, R89A+A167I+V305L+L327T+F360I, R89A+A167I+V305L+L327T+F360L, R89A+A167I+V30 5L+L327T+F360V, R89A+A167I+V305L+L327T+I408R, R89A+A167I+V305L+F360M+I408R, R89A+A167I+V305L+F360I+I408R, R89A+A167I+V305L+F360 L+I408W, R89A+A167I+V305L+F360V+I408W, R89A+A167I+V305M+L327T+F360M, R89A+A167I+V305M+L327T+F360I, R89A+A167I+V305M+L327T+F360L R89A+A167I+V305M+L327T+F360V, R89A+A167I+V305M+L327T+I408R, R89A+A167I+V305M+F360M+I408R, R89A+A167I+V305M+F360I+I408R, R89A+A1 67I+V305M+F360L+I408W、R89A+A167I+V305M+F360V+I408W、R89A+A167I+L327T+F360M+I408R、R89A+A167I+L327T+F360I+I408R、R89A+A167I+L32 7T+F360L+I408W, R89A+A167I+L327T+F360V+I408W, R89A+A167L+V305L+L327T+F360M, R89A+A167L+V305L+L327T+F360I, R89A+A167L+V305L+L327 T+F360L、R89A+A167L+V305L+L327T+F360V、R89A+A167L+V305L+L327T+I4 08R、R89A+A167L+V305L+F360M+I408R、R89A+A167L+V305L+F360I+I408R、R89A+A167L+V305L+F360L+I408W、R89A+A167L+V305L+F360V+I408W、R89A+A167L+V305M+L327T+F360M、R89A+A167L+V305M+L327T+F360I、R89A+A167L+V305M+L327T+F360L、R89A+A167L+V305M+L327T+F360V、R89A+A167L+V305M+L327T+I408R、R89A+A167L+V305M+F360M+I408R、R89A+A167L+V305M+F360I+I408R、R89A+A167L+V305M+F360L+I408W、R89A+A167L+V305M+F360V+I408W、R89A+A167L+L327T+F360M+I408R、R89A+A167L+L327T+F360I+I408R、R89A+A167L+L327T+F360L+I408W、R89A+A167L+L327T+F360V+I408W、R89A+V305L+L327T+F360M+I408R、R89A+V305L+L327T+F360I+I408R、R89A+V305L+L327T+F360L+I408W、R89A+V305L+L327T+F360V+I408W、R89A+V305M+L327T+F360M+I408R、R89A+V305M+L327T+F360I+I408R、R89A+V305M+L327T+F360L+I408W、R89A+V305M+L327T+F360V+I408W、V165C+A167C+V305L+L327T+F360M、V165C+A167C+V305L+L327T+F360I、V165C+A167C+V305L+L327T+F360L、V165C+A167C+V305L+L327T+F360V、V165C+A167C+V305L+L327T+I408R、V165C+A167C+V305L+F360M+I408R、V165C+A167C+V305L+F360I+I408R、V165C+A167C+V305L+F360L+I408W、V165C+A167C+V305L+F360V+I408W、V165C+A167C+V305M+L327T+F360M、V165C+A167C+V305M+L327T+F360I、V165C+A167C+V305M+L327T+F360L、V165C+A167C+V305M+L327T+F360V、V165C+A167C+V305M+L327T+I408R、V165C+A167C+V305M+F360M+I408R、V165C+A167C+V305M+F360I+I408R、V165C+A167C+V305M+F360L+I408W、V165C+A167C+V305M+F360V+I408W、V165C+A167C+L327T+F360M+I408R、V165C+A167C+L327T+F360I+I408R、V165C+A167C+L327T+F360L+I408W、V165C+A167C+L327T+F360V+I408W、V165C+A167I+V305L+L327T+F360M、V165C+A167I+V305L+L327T+F360I、V165C+A167I+V305L+L327T+F360L、V165C+A167I+V305L+L327T+F360V、V165C+A167I+V305L+L327T+I408R、V165C+A167I+V305L+F360M+I408R、V165C+A167I+V305L+F360I+I408R、V165C+A167I+V305L+F360L+I408W、V165C+A167I+V305L+F360V+I408W、V165C+A167I+V305M+L327T+F360M、V165C+A167I+V305M+L327T+F360I、V165C+A167I+V305M+L327T+F360L、V165C+A167I+V305M+L327T+F360V、V165C+A167I+V305M+L327T+I408R、V165C+A167I+V305M+F360M+I408R、V165C+A167I+V305M+F360I+I408R、V165C+A167I+V305M+F360L+I408W、V165C+A167I+V305M+F360V+I408W、V165C+A167I+L327T+F360M+I408R、V165C+A167I+L327T+F360I+I408R、V165C+A167I+L327T+F360L+I408W、V165C+A167I+L327T+F360V+I408W、V165C+A167L+V305L+L327T+F360M、V165C+A167L+V305L+L327T+F360I、V165C+A167L+V305L+L327T+F360L、V165C+A167L+V305L+L327T+F360V、V165C+A167L+V305L+L327T+I408R、V165C+A167L+V305L+F360M+I408R、V165C+A167L+V305L+F360I+I408R、V165C+A167L+V305L+F360L+I408W、V165C+A167L+V305L+F360V+I408W、V165C+A167L+V305M+L327T+F360M、V165C+A167L+V305M+L327T+F360I、V165C+A167L+V305M+L327T+F360L、V165C+A167L+V305M+L327T+F360V、V165C+A167L+V305M+L327T+I408R、V165C+A167L+V305M+F360M+I408R、V165C+A167L+V305M+F360I+I408R、V165C+A167L+V305M+F360L+I408W、V165C+A167L+V305M+F360V+I408W、V165C+A167L+L327T+F360M+I408R、V165C+A167L+L327T+F360I+I408R、V165C+A167L+L327T+F360L+I408W、V165C+A167L+L327T+F360V+I408W、V165C+V305L+L327T+F360M+I408R、V165C+V305L+L327T+F360I+I408R、V165C+V305L+L327T+F360L+I408W、V165C+V305L+L327T+F360V+I408W、V165C+V305M+L327T+F360M+I408R、V165C+V305M+L327T+F360I+I408R、V165C+V305M+L327T+F360L+I408W、V165C+V305M+L327T+F360V+I408W、V165S+A167C+V305L+L327T+F360M、V165S+A167C+V305L+L327T+F360I、V165S+A167C+V305L+L327T+F360L、V165S+A167C+V305L+L327T+F360V、V165S+A167C+V305L+L327T+I408R、V165S+A167C+V305L+F360M+I408R、V165S+A167C+V305L+F360I+I408R、V165S+A167C+V305L+F360L+I408W、V165S+A167C+V305L+F360V+I408W、V165S+A167C+V305M+L327T+F360M、V165S+A167C+V305M+L327T+F360I、V165S+A167C+V305M+L327T+F360L、V165S+A167C+V305M+L327T+F360V、V165S+A167C+V305M+L327T+I408R、V165S+A167C+V305M+F360M+I408R、V165S+A167C+V305M+F360I+I408R、V165S+A167C+V305M+F360L+I408W、V165S+A167C+V305M+F360V+I408W、V165S+A167C+L327T+F360M+I408R、V165S+A167C+L327T+F360I+I408R、V165S+A167C+L327T+F360L+I408W、V165S+A167C+L327T+F360V+I408W、V165S+A167I+V305L+L327T+F360M、V165S+A167I+V305L+L327T+F360I、V165S+A167I+V305L+L327T+F360L、V165S+A167I+V305L+L327T+F360V、V165S+A167I+V305L+L327T+I408R、V165S+A167I+V305L+F360M+I408R、V165S+A167I+V305L+F360I+I408R、V165S+A167I+V305L+F360L+I408W、V165S+A167I+V305L+F360V+I408W、V165S+A167I+V305M+L327T+F360M、V165S+A167I+V305M+L327T+F360I、V165S+A167I+V305M+L327T+F360L、V165S+A167I+V305M+L327T+F360V、V165S+A167I+V305M+L327T+I408R、V165S+A167I+V305M+F360M+I408R、V165S+A167I+V305M+F360I+I408R、V165S+A167I+V305M+F360L+I408W、V165S+A167I+V305M+F360V+I408W、V165S+A167I+L327T+F360M+I408R、V165S+A167I+L327T+F360I+I408R、V165S+A167I+L327T+F360L+I408W、V165S+A167I+L327T+F360V+I408W、V165S+A167L+V305L+L327T+F360M、V165S+A167L+V305L+L327T+F360I、V165S+A167L+V305L+L327T+F360L、V165S+A167L+V305L+L327T+F360V、V165S+A167L+V305L+L327T+I408R、V165S+A167L+V305L+F360M+I408R、V165S+A167L+V305L+F360I+I408R、V165S+A167L+V305L+F360L+I408W、V165S+A167L+V305L+F360V+I408W、V165S+A167L+V305M+L327T+F360M、V165S+A167L+V305M+L327T+F360I、V165S+A167L+V305M+L327T+F360L、V165S+A167L+V305M+L327T+F360V、V165S+A167L+V305M+L327T+I408R、V165S+A167L+V305M+F360M+I408R、V165S+A167L+V305M+F360I+I408R、V165S+A167L+V305M+F360L+I408W、V165S+A167L+V305M+F360V+I408W、V165S+A167L+L327T+F360M+I408R、V165S+A167L+L327T+F360I+I408R、V165S+A167L+L327T+F360L+I408W、V165S+A167L+L327T+F360V+I408W、V165S+V305L+L327T+F360M+I408R、V165S+V305L+L327T+F360I+I408R、V165S+V305L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R89A+V165C+A167I+V305M+F360T, R89A+V165C+A167I+L327T+F360T, R89A+V165C+A167I+F360T+I408W, R89A+V1 65C+A167L+V305L+F360T、R89A+V165C+A167L+V305M+F360T、R89A+V165C +A167L+L327T+F360T、R89A+V165C+A167L+F360T+I408R、R89A+V165C+V30 5L+L327T+F360T, R89A+V165C+V305L+F360T+I408W, R89A+V165C+V305M+L327T+F360T, R89A+V165C+V305M+F360T+I408R, R89A+V165C+L327T+F360 T+I408W、R89A+V165S+A167C+V305L+F360T、R89A+V165S+A167C+V305M+F3 60T、R89A+V165S+A167C+L327T+F360T、R89A+V165S+A167C+F360T+I408R、 R89A+V165S+A167I+V305L+F360T, R89A+V165S+A167I+V305M+F360T, R89A+V165S+A167I+L327T+F360T, R89A+V165S+A167I+F360T+I408W, R89A+V1 65S+A167L+V305L+F360T, R89A+V165S+A167L+V305M+F360T, R89A+V165S+A167L+L327T+F360T, R89A+V165S+A167L+F360T+I408R, R89A+V165S+V30 5L+L327T+F360T, R89A+V165S+V305L+F360T+I408W, R89A+V165S+V305M+L327T+F360T, R89A+V165S+V305M+F360T+I408R, R89A+V165S+L327T+F360 T+I408W、R89A+A167C+V305L+L327T+F360T、R89A+A167C+V305L+F360T+I4 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89A+V165S+A167I+V305L+L327T+F360M、R89A+V165S+A167I+V305L+L327T+F360I、R89A+V165S+A167I+V305L+L327T+F360L、R89A+V165S+A167I+V305L+L327T+F360V、R89A+V165S+A167I+V305L+L327T+I408R、R89A+V165S+A167I+V305L+F360M+I408R、R89A+V165S+A167I+V305L+F360I+I408R、R89A+V165S+A167I+V305L+F360L+I408W、R89A+V165S+A167I+V305L+F360V+I408W、R89A+V165S+A167I+V305M+L327T+F360M、R89A+V165S+A167I+V305M+L327T+F360I、R89A+V165S+A167I+V305M+L327T+F360L、R89A+V165S+A167I+V305M+L327T+F360V、R89A+V165S+A167I+V305M+L327T+I408R、R89A+V165S+A167I+V305M+F360M+I408R、R89A+V165S+A167I+V305M+F360I+I408R、R89A+V165S+A167I+V305M+F360L+I408W、R89A+V165S+A167I+V305M+F360V+I408W、R89A+V165S+A167I+L327T+F360M+I408R、R89A+V165S+A167I+L327T+F360I+I408R、R89A+V165S+A167I+L327T+F360L+I408W、R89A+V165S+A167I+L327T+F360V+I408W、R89A+V165S+A167L+V305L+L327T+F360M、R89A+V165S+A167L+V305L+L327T+F360I、R89A+V165S+A167L+V305L+L327T+F360L、R89A+V165S+A167L+V305L+L327T+F360V、R89A+V165S+A167L+V305L+L327T+I408R、R89A+V165S+A167L+V305L+F360M+I408R、R89A+V165S+A167L+V305L+F360I+I408R、R89A+V165S+A167L+V305L+F360L+I408W、R89A+V165S+A167L+V305L+F360V+I408W、R89A+V165S+A167L+V305M+L327T+F360M、R89A+V165S+A167L+V305M+L327T+F360I、R89A+V165S+A167L+V305M+L327T+F360L、R89A+V165S+A167L+V305M+L327T+F360V、R89A+V165S+A167L+V305M+L327T+I408R、R89A+V165S+A167L+V305M+F360M+I408R、R89A+V165S+A167L+V305M+F360I+I408R、R89A+V165S+A167L+V305M+F360L+I408W、R89A+V165S+A167L+V305M+F360V+I408W、R89A+V165S+A167L+L327T+F360M+I408R、R89A+V165S+A167L+L327T+F360I+I408R、R89A+V165S+A167L+L327T+F360L+I408W、R89A+V165S+A167L+L327T+F360V+I408W、R89A+V165S+V305L+L327T+F360M+I408R、R89A+V165S+V305L+L327T+F360I+I408R、R89A+V165S+V305L+L327T+F360L+I408W、R89A+V165S+V305L+L327T+F360V+I408W、R89A+V165S+V305M+L327T+F360M+I408R、R89A+V165S+V305M+L327T+F360I+I408R、R89A+V165S+V305M+L327T+F360L+I408W、R89A+V165S+V305M+L327T+F360V+I408W、R89A+A167C+V305L+L327T+F360M+I408R、R89A+A167C+V305L+L327T+F360I+I408R、R89A+A167C+V305L+L327T+F360L+I408W、R89A+A167C+V305L+L327T+F360V+I408W、R89A+A167C+V305M+L327T+F360M+I408R、R89A+A167C+V305M+L327T+F360I+I408R、R89A+A167C+V305M+L327T+F360L+I408W、R89A+A167C+V305M+L327T+F360V+I408W、R89A+A167I+V305L+L327T+F360M+I408R、R89A+A167I+V305L+L327T+F360I+I408R、R89A+A167I+V305L+L327T+F360L+I408W、R89A+A167I+V305L+L327T+F360V+I408W、R89A+A167I+V305M+L327T+F360M+I408R、R89A+A167I+V305M+L327T+F360I+I408R、R89A+A167I+V305M+L327T+F360L+I408W、R89A+A167I+V305M+L327T+F360V+I408W、R89A+A167L+V305L+L327T+F360M+I408R、R89A+A167L+V305L+L327T+F360I+I408R、R89A+A167L+V305L+L327T+F360L+I408W、R89A+A167L+V305L+L327T+F360V+I408W、R89A+A167L+V305M+L327T+F360M+I408R、R89A+A167L+V305M+L327T+F360I+I408R、R89A+A167L+V305M+L327T+F360L+I408W、R89A+A167L+V305M+L327T+F360V+I408W、V165C+A167C+V305L+L327T+F360M+I408R、V165C+A167C+V305L+L327T+F360I+I408R、V165C+A167C+V305L+L327T+F360L+I408W、V165C+A167C+V305L+L327T+F360V+I408W、V165C+A167C+V305M+L327T+F360M+I408R、V165C+A167C+V305M+L327T+F360I+I408R、V165C+A167C+V305M+L327T+F360L+I408W、V165C+A167C+V305M+L327T+F360V+I408W、V165C+A167I+V305L+L327T+F360M+I408R、V165C+A167I+V305L+L327T+F360I+I408R、V165C+A167I+V305L+L327T+F360L+I408W、V165C+A167I+V305L+L327T+F360V+I408W、V165C+A167I+V305M+L327T+F360M+I408R、V165C+A167I+V305M+L327T+F360I+I408R、V165C+A167I+V305M+L327T+F360L+I408W、V165C+A167I+V305M+L327T+F360V+I408W、V165C+A167L+V305L+L327T+F360M+I408R、V165C+A167L+V305L+L327T+F360I+I408R、V165C+A167L+V305L+L327T+F360L+I408W、V165C+A167L+V305L+L327T+F360V+I408W、V165C+A167L+V305M+L327T+F360M+I408R、V165C+A167L+V305M+L327T+F360I+I408R、V165C+A167L+V305M+L327T+F360L+I408W、V165C+A167L+V305M+L327T+F360V+I408W、V165S+A167C+V305L+L327T+F360M+I408R、V165S+A167C+V305L+L327T+F360I+I408R、V165S+A167C+V305L+L327T+F360L+I408W、V165S+A167C+V305L+L327T+F360V+I408W、V165S+A167C+V305M+L327T+F360M+I408R、V165S+A167C+V305M+L327T+F360I+I408R、V165S+A167C+V305M+L327T+F360L+I408W、V165S+A167C+V305M+L327T+F360V+I408W、V165S+A167I+V305L+L327T+F360M+I408R、V165S+A167I+V305L+L327T+F360I+I408R、V165S+A167I+V305L+L327T+F360L+I408W、V165S+A167I+V305L+L327T+F360V+I408W、V165S+A167I+V305M+L327T+F360M+I408R、V165S+A167I+V305M+L327T+F360I+I408R、V165S+A167I+V305M+L327T+F360L+I408W、V165S+A167I+V305M+L327T+F360V+I408W、V165S+A167L+V305L+L327T+F360M+I408R、V165S+A167L+V305L+L327T+F360I+I408R、V165S+A167L+V305L+L327T+F360L+I408W、V165S+A167L+V305L+L327T+F360V+I408W、V165S+A167L+V305M+L327T+F360M+I408R、V165S+A167L+V305M+L327T+F360I+I408R、V165S+A167L+V305M+L327T+F360L+I408W、V165S+A167L+V305M+L327T+F360V+I408W、R89A+V165C+A167C+V305L+L327T+F360T、R89A+V165C+A167C+V305L+F360T+I408W、R89A+V165C+A167C+V305M+L327T+F360T、R89A+V165C+A167C+V305M+F360T+I408R、R89A+V165C+A167C+L327T+F360T+I408W、R89A+V165C+A167I+V305L+L327T+F360T、R89A+V165C+A167I+V305L+F360T+I408R、R89A+V165C+A167I+V305M+L327T+F360T、R89A+V165C+A167I+V305M+F360T+I408W、R89A+V165C+A167I+L327T+F360T+I408R、R89A+V165C+A167L+V305L+L327T+F360T、R89A+V165C+A167L+V305L+F360T+I408W、R89A+V165C+A167L+V305M+L327T+F360T、R89A+V165C+A167L+V305M+F360T+I408R、R89A+V165C+A167L+L327T+F360T+I408W、R89A+V165C+V305L+L327T+F360T+I408R、R89A+V165C+V305M+L327T+F360T+I408W、R89A+V165S+A167C+V305L+L327T+F360T、R89A+V165S+A167C+V305L+F360T+I408R、R89A+V165S+A167C+V305M+L327T+F360T、R89A+V165S+A167C+V305M+F360T+I408W、R89A+V165S+A167C+L327T+F360T+I408R、R89A+V165S+A167I+V305L+L327T+F360T、R89A+V165S+A167I+V305L+F360T+I408W、R89A+V165S+A167I+V305M+L327T+F360T、R89A+V165S+A167I+V305M+F360T+I408R、R89A+V165S+A167I+L327T+F360T+I408W、R89A+V165S+A167L+V305L+L327T+F360T、R89A+V165S+A167L+V305L+F360T+I408R、R89A+V165S+A167L+V305M+L327T+F360T、R89A+V165S+A167L+V305M+F360T+I408W、R89A+V165S+A167L+L327T+F360T+I408R、R89A+V165S+V305L+L327T+F360T+I408W、R89A+V165S+V305M+L327T+F360T+I408R、R89A+A167C+V305L+L327T+F360T+I408W、R89A+A167C+V305M+L327T+F360T+I408R、R89A+A167I+V305L+L327T+F360T+I408W、R89A+A167I+V305M+L327T+F360T+I408R、R89A+A167L+V305L+L327T+F360T+I408W、R89A+A167L+V305M+L327T+F360T+I408R、V165C+A167C+V305L+L327T+F360T+I408W、V165C+A167C+V305M+L327T+F360T+I408R、V165C+A167I+V305L+L327T+F360T+I408W、V165C+A167I+V305M+L327T+F360T+I408R、V165C+A167L+V305L+L327T+F360T+I408W、V165C+A167L+V305M+L327T+F360T+I408R、V165S+A167C+V305L+L327T+F360T+I408W、V165S+A167C+V305M+L327T+F360T+I408R、V165S+A167I+V305L+L327T+F360T+I408W、V165S+A167I+V305M+L327T+F360T+I408R、V165S+A167L+V305L+L327T+F360T+I408W、V165S+A167L+V305M+L327T+F360T+I408R、R89A+V165C+A167C+V305L+L327T+F360M+I408R、R89A+V165C+A167C+V305L+L327T+F360I+I408R、R89A+V165C+A167C+V305L+L327T+F360L+I408W、R89A+V165C+A167C+V305L+L327T+F360V+I408W、R89A+V165C+A167C+V305M+L327T+F360M+I408R、R89A+V165C+A167C+V305M+L327T+F360I+I408R、R89A+V165C+A167C+V305M+L327T+F360L+I408W、R89A+V165C+A167C+V305M+L327T+F360V+I408W、R89A+V165C+A167I+V305L+L327T+F360M+I408R、R89A+V165C+A167I+V305L+L327T+F360I+I408R、R89A+V165C+A167I+V305L+L327T+F360L+I408W、R89A+V165C+A167I+V305L+L327T+F360V+I408W、R89A+V165C+A167I+V305M+L327T+F360M+I408R、R89A+V165C+A167I+V305M+L327T+F360I+I408R、R89A+V165C+A167I+V305M+L327T+F360L+I408W、R89A+V165C+A167I+V305M+L327T+F360V+I408W、R89A+V165C+A167L+V305L+L327T+F360M+I408R、R89A+V165C+A167L+V305L+L327T+F360I+I408R、R89A+V165C+A167L+V305L+L327T+F360L+I408W、R89A+V165C+A167L+V305L+L327T+F360V+I408W、R89A+V165C+A167L+V305M+L327T+F360M+I408R、R89A+V165C+A167L+V305M+L327T+F360I+I408R、R89A+V165C+A167L+V305M+L327T+F360L+I408W、R89A+V165C+A167L+V305M+L327T+F360V+I408W、R89A+V165S+A167C+V305L+L327T+F360M+I408R、R89A+V165S+A167C+V305L+L327T+F360I+I408R、R89A+V165S+A167C+V305L+L327T+F360L+I408W、R89A+V165S+A167C+V305L+L327T+F360V+I408W、R89A+V165S+A167C+V305M+L327T+F360M+I408R、R89A+V165S+A167C+V305M+L327T+F360I+I408R、R89A+V165S+A167C+V305M+L327T+F360L+I408W、R89A+V165S+A167C+V305M+L327T+F360V+I408W、R89A+V165S+A167I+V305L+L327T+F360M+I408R、R89A+V165S+A167I+V305L+L327T+F360I+I408R、R89A+V165S+A167I+V305L+L327T+F360L+I408W、R89A+V165S+A167I+V305L+L327T+F360V+I408W、R89A+V165S+A167I+V305M+L327T+F360M+I408R、R89A+V165S+A167I+V305M+L327T+F360I+I408R、R89A+V165S+A167I+V305M+L327T+F360L+I408W、R89A+V165S+A167I+V305M+L327T+F360V+I408W, R89A+V165S+A167L+V305L+L327T+F360M+I408R, R89A+V165S+A167L+V305L+L3 27T+F360I+I408R, R89A+V165S+A167L+V305L+L327T+F360L+I408W, R89A+V165S+A167L+V305L+L327T+F360V+I408W, R89A+V165S +A167L+V305M+L327T+F360M+I408R, R89A+V165S+A167L+V305M+L327T+F360I+I408R, R89A+V165S+A167L+V305M+L327T+F360L+ I408W, R89A+V165S+A167L+V305M+L327T+F360V+I408W, R89A+V165C+A167C+V305L+L327T+F360T+I408W, R89A+V165C+A167C+V30 5M+L327T+F360T+I408R, R89A+V165C+A167I+V305L+L327T+F360T+I408W, R89A+V165C+A167I+V305M+L327T+F360T+I408R, R89A +V165C+A167L+V305L+L327T+F360T+I408W、R89A+V165C+A167L+V305M+L327T+F360T+I408R、R89A+V165S+A167C+V305L+L327T+F 360T+I408W, R89A+V165S+A167C+V305M+L327T+F360T+I408R, R89A+V165S+A167I+V305L+L327T+F360T+I408W, R89A+V165S+A167I+V305M+L327T+F360T+I408R, R89A+V165S+A167L+V305M+L327T+F360T+I408W or R89A+V165S+A167L+V305M+L327T+F360T+I408R.

[0097] For example, peptide variants may include:

[0098] The modified amino acid sequence of SEQ ID NO:1, wherein the modification may include the amino acid sequence of SEQ ID NO:1. The following amino acid mutations in the amino acid sequence of NO:1: R89A, V165C, V165S, A167C, A167I, A167L, V305M, V305L, L327T, F360M, F360I, F360L, F360V, F360T, I408R, I408W, R89A+F360M, R89A+F360V, R89A+F360I, R89A+F360L, R89A+F360T, V165C+F360I, V165C+F360M, V165C+F360V, V165C+F360L, V165S+F360V, V165S+F360V, V165S+F360T. 0L, V165S+F360T, A167C+F360I, A167L+F360M, A167I+F360L, A167L+F36 0T, A167C+F360M, A167C+F360L, A167C+F360V, V305M+F360I, V305L+F36 0M, V305M+F360M, V305M+F360V, V305M+F360L, V305L+F360L, V305M+F36 0T, L327T+F360I, L327T+F360M, L327T+F360V, L327T+F360L, L327T+F36 0T, I408W+F360I, I408R+F360M, I408W+F360V, I408R+F360L, I408W+F36 0T, R89A+V165C+F360I, R89A+V165S+F360M, R89A+V165C+F360V, R89A+A 167I+F360V, R89A+A167C+F360L, R89A+A167L+F360T, R89A+V305M+F360 I. R89A+V305M+F360V, R89A+V305M+F360T, R89A+L327T+F360I, R89A+L3 27T+F360M, R89A+L327T+F360T, R89A+I408R+F360M, R89A+I408W+F360V , R89A+I408R+F360L, V165S+A167I+F360M, V165S+A167C+F360L, V165C+ A167L+F360T, V165C+V305M+F360I, V165C+V305M+F360V, V165S+V305L+ F360L, V165C+L327T+F360I, V165C+L327T+F360V, V165S+L327T+F360T,V165C+I408W+F360V, V165C+I408W+F360T, A167I+V305M+F360L, A167C+V305L+F360V, A167L+V30 5M+F360T, A167C+L327T+F360I, A167L+L327T+F360M, A167I+L327T+F360V, A167C+I408W+F360I, A167I+I408W+F360V, A167L+I408R+F360L, V305M+L327T+F360I, V305L+L327T+F360V, V305M+L32 7T+F360T, V305M+I408W+F360I, V305M+I408W+F360V, V305L+I408R+F360L, L327T+I408R+F360M, L327T+I408W+F360V, R89A+V165C+A167C+F360I, R89A+V165S+V305M+F360M, R89A+V165C+L327T+ F360V, R89A+V165S+I408R+F360L, R89A+A167L+V305L+F360T, R89A+A167L+L327T+F360I, R89A+A 167C+I408R+F360M, V165C+A167I+V305M+F360V, V165S+A167C+L327T+F360M, V165C+A167C+I408W+F360T, A167I+V305L+L327T+F360V, A167L+V305M+I408R+F360M or V305L+L327T+I408W+F360V, or...

[0099] An amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence.

[0100] For example, peptide variants may include:

[0101] The modified amino acid sequence of SEQ ID NO:3 may include the following amino acid mutations in the amino acid sequence of SEQ ID NO:3: F360M, F360V, F360I, F360L, A167C, A167L, A167I, V305M, R89A, V165S, V165C, L327T, R89A+F360M, A167L+F360M, L327T+F360M, R89A+F360V, A167L+F360I, V305M+F360I, R89A+V 165C+F360I, V165C+A167L+F360I, V165C+A167L+F360M, A167L+V305M+F360M, V165S+A167L+V305M+ F360I, R89A+V165S+V305M+F360I, V165S+A167C+L327T+F360M, R89A+V165S+A167C+L327T+F360M, or R89A+V165C+A167L+V305M+F360I, or

[0102] An amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence.

[0103] The polypeptide variants described herein, comprising amino acid sequences having sequence identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity), can maintain enzymatic activity equivalent to that of polypeptides having amino acid sequences used as standards for identifying sequence identity (e.g., PPO proteins with the aforementioned amino acid mutations). For example, they maintain 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the enzymatic activity of polypeptides having amino acid sequences used as standards in plants (whole plants, plant cells or cell cultures, or plant tissues), algae, and / or in vitro, and possess the function of conferring herbicide tolerance. The use of sequence identity description is to clarify that the herbicide-tolerant PPO protein (polypeptide) or its variants described herein can contain any sequence mutation within the range capable of satisfying the above conditions (maintaining enzymatic activity and possessing the function of conferring herbicide tolerance).

[0104] The amino acids used in this description are summarized below:

[0105]

[0106]

[0107] The peptide variant (herbicide-tolerant PPO protein variant) can retain its enzymatic activity as a PPO protein and exhibits increased herbicide tolerance compared to the wild type.

[0108] Additionally, the polypeptide (herbicide-resistant PPO protein) and its variants (herbicide-resistant PPO protein variants) may contain other mutations exhibiting biological activity equivalent to that of the polypeptide composed of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or having the aforementioned amino acid mutations. For example, the additional mutation may be an amino acid substitution that does not completely alter the molecular activity, and such substitution may be suitably selected by those skilled in the art. In one example, the additional substitution may be a substitution between, but not limited to, the 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 undergo at least one modification selected from the group consisting of phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, etc. Additionally, herbicide-tolerant PPO protein variants may be variants that exhibit increased structural stability to heat, pH, etc., or variants that exhibit increased protein activity through amino acid variations (mutations) and / or modifications.

[0109] The term "sequence identity" refers to the degree of similarity to a wild-type or reference amino acid or nucleotide sequence, and any protein can be included within the scope of this invention as long as it comprises amino acid residues having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity with the above-described herbicide-resistant PPO protein variant, and maintains equivalent biological activity to the herbicide-resistant PPO protein variant. Such protein homologs may contain active sites equivalent to the active sites of the target protein. This identity comparison can be performed visually or by means of readily available comparison procedures. The identity 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.

[0110] Herbicide-resistant PPO proteins or their variants can be obtained from nature by methods well-known in the relevant field. Alternatively, herbicide-resistant PPO proteins or their variants can be obtained as recombinant proteins using gene recombination technology. In the case of gene recombination technology, herbicide-resistant PPO proteins or their variants are harvested by introducing nucleic acids encoding herbicide-resistant PPO proteins or their variants into a suitable expression vector, introducing the expression vector into host cells to express the herbicide-resistant PPO protein or its variants, and then collecting the expressed herbicide-resistant PPO protein or its variants from the host cells. After expression of the protein in selected host cells, the protein can be separated and / or purified using conventional biochemical separation techniques, such as treatment with a protein precipitant (salting out), centrifugation, ultrasonic disruption, ultrafiltration, dialysis, and chromatography (e.g., molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.). These methods can be combined to separate proteins of high purity.

[0111] Herbicide-resistant PPO nucleic acid molecules (polynucleotides encoding PPO proteins or their variants) can be isolated or prepared using standard molecular biology techniques, such as chemical synthesis or recombination methods, or commercially available herbicide-resistant PPO nucleic acid molecules can be used.

[0112] In this disclosure, using a herbicide tolerance testing system employing PPO-deficient *Escherichia coli* BT3 (ΔPPO), PPO proteins / nucleic acids or their variants were found to exhibit broad herbicide tolerance to 10 representative families of PPO-inhibiting herbicides classified according to their chemical structures. It was also found that the protein could be expressed in the chloroplasts of plants using transport peptides (TPs). Furthermore, it was discovered that PPO proteins / nucleic acids or their variants could also be expressed in monocotyledonous plants (e.g., rice (*Oryza sativa*)) or dicotyledonous plants (e.g., *Arabidopsis thaliana* ecotype Columbia-0). Germination and growth were observed even when transformed plants were treated with PPO-inhibiting herbicides. Moreover, genetic studies confirmed that the aforementioned herbicide tolerance traits can be successfully inherited by the next generation.

[0113] Therefore, the PPO proteins and their variants provided in this article can be introduced into plants or algae to confer herbicide tolerance and / or enhance herbicide tolerance in plants or algae.

[0114] One embodiment provides a composition for conferring and / or enhancing herbicide tolerance in plants and / or algae, the composition comprising at least one selected from the group consisting of:

[0115] (1) Selected from at least one of the following groups: the polypeptide of SEQ ID NO:1, the polypeptide of SEQ ID NO:2, the polypeptide of SEQ ID NO:3, the polypeptide variant thereof as described above, and a polypeptide comprising an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with said polypeptide or said polypeptide variant;

[0116] (2) A polynucleotide encoding a polypeptide or polypeptide variant of (1);

[0117] (3) A recombinant vector containing the polynucleotides of (2); and

[0118] (4) Recombinant cells containing the recombinant vector of (3).

[0119] Herbicides, as defined in this article, refer to active ingredients that kill, control, or otherwise adversely alter the growth of plants or algae. Additionally, herbicide tolerance is the condition where, even after treatment with a herbicide that typically kills or inhibits the growth of normal or wild-type plants, the inhibitory effect on plant growth is reduced or eliminated compared to normal or wild-type plants, allowing the plant to continue growing. Herbicides include those that inhibit protoporphyrinogen IX oxidase (PPO) in plants or algae. Based on their chemical structures, PPO-inhibiting herbicides can be classified as pyrimidinidones, diphenyl ethers, phenylpyrazoles, N-phenylphthalimides, phenyl esters, thiadiazoles, oxadiazoles, triazinones, triazolinones, oxazolidinones, and other herbicides.

[0120] As a specific implementation method, pyrimidine dione herbicides may include, but are not limited to, flupropargyl, pyrimisulfuron, bispyribac-sodium, and tefena.

[0121] Diphenyl ether herbicides may include, but are not limited to, flusulfanilamide, ethoxyflufenican, bensulfuron-methyl, flusulfanilamide, chlorpyrifos, methylflufenican, methoxyflufenican, glufosinate, ethoxyflufenican, and flunitrazepam.

[0122] Phenylepiazole herbicides may include, but are not limited to, pyrazosulfuron and isopyrazosulfuron.

[0123] Phenyl phthalimide herbicides may include, but are not limited to, propyzoxystrobin, indole-3-propargyl, and fluroxypyr.

[0124] Phenyl ester herbicides may include, but are not limited to, pyrrolizin (2,4-dichlorophenyl-1-pyrrolizin carboxylate) and pyrrolizin carbamate analogs (e.g., O-phenylpyrrolizin carbamate analogs and piperidine carbamate analogs (see “Ujjana B. Nandihalli, Mary V. Duke, Stephen O. Duke, Relationship between molecular properties and bioactivity of O-phenylpyrrolizin carbamate and piperidine carbamate herbicides. J. Agric. Food Chem., 40(10) 1993-2000, 1992”)). In one specific embodiment, the carbamate analogue of *Pyrrolizinus var. praecox* may be one or more selected from the group consisting of: phenyl pyrrolizidine-1-carboxylate (CAS No. 55379-71-0), 1-pyrrolizidinecarboxylate, 2-chlorophenyl ester (CAS No. 143121-06-6), 4-chlorophenylpyrrolizidine-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-pyrrolizidinecarboxylate, 2,4-dichloro-5-hydroxyphenyl ester (CAS No. 143121-08-8), 2,4-dichloro-5-(methoxycarbonyl)phenylpyrrolizidine-1-carboxylate (CAS No. 133636-94-9), 2,4-dichloro-5-[(prop-2-oxy)carbonyl] [2,4-Dichloro-5-(2-propynoxy)phenyl 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)phenyl pyrrolidine-1-carboxylic acid ester (CAS No. 87365-63-7), 2,4-dichloro-5-(prop-2-yn-1-yloxy)phenyl pyrrolidine-1-carboxylic acid ester (CAS No. 87365-63-7), 2,4-dichloro-5-(prop-2-yn-1-yloxy)phenyl Phenylacetic acid 4,4-difluoropiperidine-1-carboxylate (CAS No. 138926-22-4), 1-pyrrolidinecarboxylate, 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-carboxylate (CAS No. 133636-98-3), etc.

[0125] Thiadiazole herbicides may include, but are not limited to, methamidophos and thiamethoxam.

[0126] Oxadiazole herbicides may include, but are not limited to, propyzinoxadiazon and oxadiazon.

[0127] Triazinone herbicides may include, but are not limited to, trifludimoxazin.

[0128] Triazoline herbicides may include, but are not limited to, metsulfuron-methyl, and acetamiprid.

[0129] Oxazolidinone herbicides may include, but are not limited to, cyclooxadione.

[0130] Other herbicides may include, but are not limited to, bispyribac-sodium, flupyridaben, and flupyrazosulfuron.

[0131] The herbicide-tolerant PPO gene or its variants provided herein can be introduced into plants or algae using a variety of methods known in the art, preferably by using an expression vector for plant or algal transformation.

[0132] In the case of introducing a gene into a plant, 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, the promoter may include, but is 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 spot virus promoter, photoinducible promoter from the small subunit of ribose-1,5-bisphosphonate carboxylase (ssRUBISCO), rice cytoplasmic trisaccharide phosphate triose isomerase (TPI) promoter, Arabidopsis thaliana (A. thaliana) adenine phosphoribosyltransferase (APRT) promoter, octopus base synthase promoter, and BCB (blue copper binding protein) promoter.

[0133] Furthermore, the vector may include a poly-A signal sequence that causes 3'-terminal polyadenylation, and for example, it may include, but is not limited to, the NOS 3' end of the alkaloid synthase gene derived from Agrobacterium tumefaciens, the octopaloid synthase terminator derived from the octopaloid synthase gene derived from Agrobacterium tumefaciens, the 3'-end of the protease inhibitor I or II gene of tomato or potato, the CaMV 35S promoter terminator, the rice α-amylase RAmy1 A terminator, and the betaine terminator.

[0134] Furthermore, when introducing genes into algae, chloroplast-specific promoters, nuclear promoters, constitutive promoters, or inducible promoters can be used as promoters. The herbicide-tolerant PPO gene or its variants described herein can be designed to be operatively linked to the 5'UTR or 3'UTR, thereby expressing function in the algal nucleus. Additionally, the vector can further contain transcriptional regulatory sequences suitable for algal transformation. The recombinant gene conferring herbicide tolerance can be integrated into the nuclear genome or chloroplast genome of the host algae, but is not limited to these.

[0135] In addition, in the vector, the transport peptide required to target chloroplasts can be linked to the 5'-terminus of the PPO gene or its variants to express the herbicide-tolerant PPO gene or its variants in the chloroplasts.

[0136] Additionally, optionally, the vector may further include a gene encoding a selection marker as a reporter molecule, and examples of the selection marker may include, but are not limited to, genes that are resistant to antibiotics (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, bleomycin, chloramphenicol, ampicillin, etc.) or herbicides (glyphosate, glufosinate, glufosinate, etc.).

[0137] Furthermore, recombinant vectors for plant expression can include Agrobacterium binary vectors, co-integration vectors, or universal 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 carrying a migration-responsible plasmid composed of the left (LB) and right (RB) boundaries of a Ti (tumor-inducible) plasmid, and the other carrying a target gene transfer-responsible plasmid, which may include a promoter region and a polyadenylation signal sequence for expression in plants.

[0138] When using binary or co-integrative vectors, the strain used to transform the recombinant vector into plants is preferably Agrobacterium (Agrobacterium-mediated transformation). For this transformation, Agrobacterium tumefaciens or Agrobacterium rhizogenes can be used. 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.

[0139] Using standard techniques known in the relevant field, plants genetically transformed through the above methods can be redifferentiated into plants through callus induction, rooting, and soil acclimatization.

[0140] The plants used for transformation in this study can cover not only mature plants, but also plant cells (including cells in suspension culture), protoplasts, callus, hypocotyls, seeds, cotyledons, buds, etc., that can grow into mature plants.

[0141] Furthermore, the scope of transformants can include transformants into which the gene has been introduced, and their clones or progeny (T1, T2, T3, T4, T5, T6, T7, T8, or any subsequent generations). For example, transformed plants also include plants with genetically inherited herbicide tolerance traits, the sexual and asexual progeny of plants transformed with the gene provided herein. The scope of the invention also includes all variants and variants exhibiting characteristics of the initially transformed plant, as well as all hybridization and fusion products of plants transformed with the gene provided herein. Furthermore, the scope of the invention also includes portions of a plant derived from a transformed plant pre-transformed by the method of the invention, or its progeny, and from at least a portion of the transformed cells, such as seeds, flowers, stems, fruits, leaves, roots, tubers, and / or rhizomes.

[0142] The plants used in applying this invention are not particularly limited, but may be at least one selected from the group consisting of monocotyledonous or dicotyledonous plants. Furthermore, the plants may be at least one selected from the group consisting of herbaceous plants and woody plants. Monocotyledonous plants may include plants belonging to the following families: Alismataceae, Hydrocharitaceae, Juncaginaceae, Scheuchzeriaceae, Potamogetonaceae, Najadaceae, Zosteraceae, Liliaceae, Haemodoraceae, Agavaceae, Amaryllidaceae, Dioscoreaceae, Pontederiaceae. The family includes, but is not limited to, Iridaceae, Burmannaceae, Juncaceae, Commelinaceae, Eriocaulaceae, Gramineae / Poaceae, Araceae, Lemnaceae, Sparganiaceae, Typhaceae, Cyperaceae, Musaceae, Zingiberaceae, Cannaceae, and Orchidaceae.

[0143] Dicotyledonous plants can include those belonging to the following families: Diapensiaceae, Clethraceae, Pyrolaceae, Ericaceae, Myrsinaceae, Primulaceae, Plumbaginaceae, Ebenaceae, Styracaceae, Symplocoaceae, Oleaceae, Loganiaceae, and Gentianaceae. (Gentianaceae), Menyanthaceae, Apocynaceae, Asclepiadaceae, Rubiaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Lamiaceae, Solanaceae, Scrophulariaceae, Bignoniaceae, Acanthaceae Pedaliaceae, Orobanchaceae, Gesneriaceae, Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, Adoxaceae, Valerianaceae, Dipsacaceae, Campanulaceae, Compositae, Myricaceae, Juglandaceae andaceae, Salicaceae, Betulaceae, Fagaceae, Ulmaceae, Moraceae, Urticaceae, Santalaceae, Loranthaceae, Polygonaceae, Phytolaccaceae, Nyctaginaceae, Aizoaceae, Portulacaceae, CaryophyllaceaeChenopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cercidiphyllaceae, Ranunculaceae, Berberidaceae, Lardizabalaceae, Menispermaceae, Nymphaeaceae, Ceratophyllaceae ), Cabombaceae, Saururaceae, Piperaceae, Chloranthaceae, Aristolochiaceae, Actinidiaceae, Theaceae, Guttiferae, Droseraceae, Papaveraceae, Capparidaceae, Cruciferae, Platanaceae, Hamamelidaceae The families listed are: lidaceae, Crassulaceae, Saxifragaceae, Eucommiaceae, Pittosporaceae, Rosaceae, Leguminosae, Oxalidaceae, Geraniaceae, Tropaeolaceae, Zygophyllaceae, Linaceae, Euphorbiaceae, and Callitrichaceae. Rutaceae, Simaroubaceae, Meliaceae, Polygalaceae, Anacardiaceae, Aceraceae, Sapindaceae, Hippocastanaceae, Sabiaceae, Balsaminaceae, Aquifoliaceae, Celastraceae, Staphyleaceae, BuxaceaeFamily Empetraceae, Family Rhamnaceae, Family Vitaceae, Family Elaeocarpaceae, Family Tiliaceae, Family Malvaceae, Family Sterculiaceae, Family Thymelaeaceae, Family Elaeagnaceae, Family Flacotyleaceae, Family Violaceae, Family Passifloraceae, Family Tamarixaceae The family names include, but are not limited to, the following families: * Arricaceae*, *Elatiaceae*, *Begoniaceae*, *Cucurbitaceae*, *Lythraceae*, *Punicaceae*, *Onaraceae*, *Haloragaceae*, *Alangiaceae*, *Cornaceae*, *Araliaceae*, and *Umbelliferae / Apiaceae*.

[0144] In specific implementations, the plants can 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, scallion, onion, and carrot; and special-purpose crops, such as ginseng, tobacco, cotton, soilage, forage grass, sesame, sugarcane, sugar beets, and perilla. (Perillasp.), peanuts, rapeseed, grasses and castor oil plants; fruit trees, such as apple trees, pear trees, jujube trees, peach trees, kiwifruit trees, grapevines, citrus trees, persimmon trees, plum trees, apricot trees and banana trees; woody plants, such as pine trees, palm oil trees and eucalyptus trees; flowering crops, such as roses, gladioli, gerberas, carnations, chrysanthemums, lilies and tulips; 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.

[0145] The algae used in applying this invention are not particularly limited, but may be at least one prokaryotic algae and / or eukaryotic algae. For example, the algae may be at least one selected from the group consisting of cyanobacteria, green algae, red algae, brown algae, macroalgae, microalgae, etc.

[0146] Cyanobacteria include: the order Chroococcales (e.g., genera *Aphanocapsa*, *Aphanothece*, *Chamaesiphon*, *Chondrocystis*, *Chroococcus*, *Chroogloeocystis*, *Crocosphaera*, *Cyanobacterium*, *Cyanobium*, *Cyanodictyon*, *Cyanosarcina*, *Cyanothece*, *Dactylococcopsis*, *Gloeocapsa*, *Gloeothece*, *Halothece*, *Jo...*). *Hannasbaptistia*, *Merismopedia*, *Microcystis*, *Radiocystis*, *Rhabdoderma*, *Snowella*, *Synechococcus*, *Synechocystis*, *Thermosynechococcus*, *Woronichinia*), *Gloeobacteria*, *Nostocales* (e.g., Microchaetaceae, Nostocaceae, Rivulariaceae, Scytonemataceae), *Oscillatoriales* (e.g.)*Arthronema*, *Arthrospira*, *Blennothrix*, *Crinalium*, *Geitlerinema*, *Halomicronema*, *Halospirulina*, *Hydrocoleum*, *Jaaginema*, *Katagnymene*, *Komvophoron*, *Leptolyngbya*, *Limnothrix*, *Leptolyngbya* Lyngbya), Microcoleus, Oscillatoria, Phormidium, Planktothricoides, Planktothrix, Plectonema, Pseudanabaena, Pseudophormidium, Schizothrix, Spirulina, Starria, Symplocos, Tricholoma odesmium), Tychonema, Pleurocapsales (e.g., Chroococcidiopsis, Dermocarpa, Dermocarpella, Myxosarcina, Pleurocapsa, Solentia, Stanieria, Xenococcus), Prochlorales, or Stigonematales. (For example, genera such as *Capsosira*, *Chlorogloeopsis*, *Fischerella*, *Hapalosiphon*, *Mastigocladopsis*, *Mastigocladus*, *Nostochopsis*, *Stigonema*, *Symphyonema*, *Symphonemopsis*, *Umezakia*, and *Westiellopsis*)

[0147] 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.

[0148] 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 may include algae belonging to other genera and families.

[0149] In one embodiment, plants or algae having the herbicide-tolerant PPO or variants thereof provided herein may exhibit tolerance to two or more PPO-inhibiting herbicides.

[0150] Therefore, by using at least two PPO inhibitory herbicides sequentially or simultaneously, the technology provided in this disclosure can be used to control weeds or remove unwanted aquatic organisms.

[0151] One embodiment provides a method for controlling weeds in farmland, comprising:

[0152] Providing plants to farmland, said plants containing the herbicide-resistant PPO protein, its variants, or its encoding gene as described above, and

[0153] Apply an effective dose of a protoporphyrinogen IX oxidase-inhibiting herbicide to farmland and / or plants.

[0154] Another implementation provides a method for removing unwanted aquatic organisms from a culture medium, comprising:

[0155] Algae are provided to the culture medium, the algae containing the herbicide-resistant PPO protein, its variants, or its encoding gene as described above, and

[0156] Apply an effective dose of a protoporphyrinogen IX oxidase-inhibiting herbicide to the culture medium.

[0157] In addition, 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.

[0158] 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".

[0159] 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.

[0160] Another embodiment provides a herbicide-tolerant plant or algae transformant or its clone or progeny, the transformant or its clone or progeny comprising the above-described herbicide-tolerant PPO protein, its variants or its encoding gene; and a second herbicide-tolerant polypeptide or its encoding gene.

[0161] Another embodiment provides a method for preparing herbicide-tolerant plants or algae, the method comprising the steps of introducing the following into the cells, protoplasts, callus, hypocotyls, seeds, cotyledons, buds, or whole plants of algae or plants: the herbicide-tolerant PPO protein, its variants or their encoding genes, and a second herbicide-tolerant polypeptide or its encoding gene.

[0162] Another implementation provides a method for controlling weeds in farmland, including

[0163] Providing plants to farmland, said plants comprising the aforementioned herbicide-resistant PPO protein, its variants, or their encoding gene, and a second herbicide-resistant polypeptide or its encoding gene, and

[0164] Apply an effective dose of a protoporphyrinogen IX oxidase-inhibiting herbicide and a second herbicide to farmland simultaneously or in any order.

[0165] Another implementation provides a method for removing unwanted aquatic organisms from a culture medium, including...

[0166] Algae are provided to the culture medium, said algae comprising a herbicide-resistant PPO protein, a variant thereof or its encoding gene, and a second herbicide-resistant polypeptide or its encoding gene, and

[0167] Apply an effective dose of a protoporphyrinogen IX oxidase inhibitory herbicide and a second herbicide to the culture medium simultaneously or in any order.

[0168] For example, plants or algae may further contain a second herbicide tolerance polypeptide or its encoding gene, thereby acquiring and / or enhancing tolerance to the second herbicide.

[0169] For example, plants or algae may further contain a second herbicide tolerance polypeptide or its encoding gene, thereby giving them new and / or enhanced tolerance to the second herbicide.

[0170] 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 herbicides, plastid inhibitory herbicides, bromobenzonitrile herbicides, and / or any combination thereof.

[0171] 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-3-phosphate synthase), GOX (glyphosate oxidase), GAT (glyphosate-N-acetyltransferase), or glyphosate decarboxylase; glufosinate-resistant PAT (glufosinate-N-acetyltransferase); dicamba-resistant DMO (dicamba monooxygenase); and 2,4-D-resistant 2,4-D monooxygenase. Or AAD (aryloxyalkylene ester dioxygenase); ALS-inhibiting sulfonylurea herbicide-resistant ALS (acetyllactate synthase), AHAS (acetylhydroxy acid synthase), or AtAHASL (Arabidopsis thaliana acetylhydroxy acid synthase large subunit); photosystem II-inhibiting herbicide-resistant photosystem II protein D1; phenylurea herbicide-resistant cytochrome P450; plastid-inhibiting herbicide-resistant HPPD (hydroxyphenylpyruvate dioxygenase); bromobenzonitrile herbicide-resistant nitrile hydrolase; and any combination thereof, but not limited to these.

[0172] 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 isoxaflutole herbicide tolerance genes HPPDPF. The W336 gene; the bxn gene for herbicide tolerance to bromobenzonitrile; and any combination thereof, but not limited to these.

[0173] Beneficial effects of the invention

[0174] Variants of the herbicide-tolerant PPO protein provided herein, or the gene encoding it, can be applied to plants or algae to confer or enhance their herbicide tolerance characteristics. Additionally, herbicides can be used for selective control, thereby economically controlling weeds or removing aquatic organisms. Attached Figure Description

[0175] Figure 1 This is a spectrum of the pMAL-c2X vector.

[0176] Figure 2 It is a map of the pET303-CT-His vector.

[0177] Figures 3 to 30 The cell growth levels of PPO-deficient BT3 Escherichia coli (ΔPPO) transformed with the CyPPO19 wild-type gene (represented by CyPPO19WT) or the CyPPO19 variant gene are shown when treated with various herbicides at various concentrations.

[0178] Figure 31 and Figure 32 The cell growth levels of PPO-deficient BT3 Escherichia coli (ΔPPO) transformed with the CyPPO18 wild-type gene (represented by CyPPO18WT) or the CyPPO18 variant gene are shown when treated with various herbicides at various concentrations.

[0179] Figure 33 The recombinant vector used to prepare the fusion protein is schematically shown, in which maltose-binding protein (MBP) and PPO protein are fused.

[0180] Figure 34 and Figure 35 The results observed on day 7 after spraying transgenic Arabidopsis thaliana (T2) transformed with CyPPO19 WT or its variants (F360M, F360V, F360L, V165C+F360M, V165S+F360V) with 1 μM tefenazate or 1 μM propynol-flufenazate, compared to wild-type Arabidopsis thaliana (Col-0), are shown.

[0181] Figure 36 and Figure 37 The results observed on day 7 after spraying transgenic Arabidopsis thaliana (T2) transformed with CyPPO19 variants (F360M, F360V, F360L, V165C+F360M, V165S+F360V) with 5 μM tefenazate or 5 μM propynol-flufenazate, compared to wild-type Arabidopsis thaliana (Col-0), are shown.

[0182] Figure 38 The results observed on day 7 after spraying transgenic Arabidopsis thaliana (T2) transformed with the CyPPO18 WT gene, compared to wild-type Arabidopsis thaliana (Col-0), are shown.

[0183] Figure 39 The results observed on day 7 after spraying transgenic Arabidopsis thaliana (T2) transformed with the CyPPO18 variant (L327T+F360M) with 1 μM tefena are shown compared to wild-type Arabidopsis thaliana (Col-0). Detailed Implementation

[0184] The present invention will be described in detail below with reference to embodiments. However, these embodiments are for illustrative purposes only, and the present invention is not limited thereto.

[0185] Example 1. Isolation of the PPO gene from a prokaryotic species

[0186] PPO sequence information was obtained from the Genebank database of species including *Thermosynechococcus elongatus* PKUAC-SCTE542, *Cyanobacteria bacterium* J003, and *Thermosynechococcus vulcanus* NIES-2134. The PPO gene was synthesized using integrated DNA technologies. The PPO gene was amplified using the primers listed in Table 1 under the conditions described in Table 2 to clone the PPO gene into the pMAL-c2X vector. Figure 1 ).

[0187] PCR mixture

[0188] Template (synthetic DNA from CyPPO19, CyPPO20, and CyPPO18, respectively) 1 μl

[0189] 10X buffer 5μl

[0190] dNTP mixture (10 mM each) 1 μl

[0191] Forward primer (10 μM, see Table 1) 1 μl

[0192] Reverse primer (10 μM, see Table 1) 1 μl

[0193] DDW 40μl

[0194] Pfu-X (Solgent, 2.5 units / μl) 1μl

[0195] 50 μl in total

[0196] Each PPO gene was named: CyPPO19, isolated from *Synechococcus slenderus* PKUAC-SCTE542; CyPPO20, isolated from cyanobacterial bacterium J003; and CyPPO18, isolated from *Synechococcus vulgaris* NIES-2134.

[0197] [Table 1]

[0198]

[0199] [Table 2] PCR conditions

[0200]

[0201] Example 2. Construction of PPO variants

[0202] To enhance the tolerance of CyPPO19, CyPPO20, and CyPPO18 to PPO-inhibiting herbicides, mutations were introduced at the sites of interaction with the herbicides to prepare variants of CyPPO19, CyPPO20, and CyPPO18.

[0203] The detailed experimental procedure is as follows:

[0204] Using the primers listed in Table 3, perform PCR under the conditions shown in Table 4 to amplify the PPO gene.

[0205] PCR reaction mixture:

[0206] Template (synthetic DNA from CyPPO19, CyPPO20, or CyPPO18) 1 μl

[0207] 10X buffer 5μl

[0208] dNTP mixture (10 mM each) 1 μl

[0209] Forward primer (10 μM) 1 μl

[0210] 1 μl of reverse primer (10 μM)

[0211] DDW 40μl

[0212] Pfu-X (Solgent, 2.5 units / μl) 1μl

[0213] 50 μl in total

[0214] [Table 3] Primer sequence information used for cloning genes into the pET303-CT His vector

[0215] The amplified PCR product and pET303-CT His vector (Invitrogen) were then used. Figure 2 Restriction enzymes XbaI and XhoI were used for digestion, and ligation was performed using T4 DNA ligase (RBC, 3 units / μl) to construct pET303-CyPPO19, pET303-CyPPO20, and pET303-CyPPO18 plasmids. Variants of CyPPO19, CyPPO20, and CyPPO18 were constructed using the CyPPO19, CyPPO20, and CyPPO18 genes cloned in the pET303-CTHis vector, along with the primers listed in Tables 5 to 7, under the following conditions.

[0216] PCR reaction mixture

[0217] 1μl template

[0218] 10X buffer 5μl

[0219] 1 μl of dNTP mixture (10 mM each)

[0220] Forward primer (10 μM) 1 μl

[0221] 1 μl of reverse primer (10 μM)

[0222] DDW 40μl

[0223] Pfu-X (Solgent, 2.5 units / μl) 1μl

[0224] 50μl in total

[0225] [Table 4] PCR reaction conditions

[0226]

[0227] [Table 5] List of primers used for constructing CyPPO19 variants

[0228]

[0229]

[0230] [Table 6] List of primers used for constructing CyPPO20 variants

[0231]

[0232]

[0233] [Table 7] List of primers used for constructing CyPPO18 variants

[0234]

[0235]

[0236] Example 3. Validation of PPO herbicide tolerance in PPO variants (tested in E. coli)

[0237] To enhance the resistance of CyPPO19 and CyPPO18 to PPO-inhibiting herbicides, the PPO variant genes described in Example 2 above were constructed. These were transformed into the PPO-deficient BT3 (ΔPPO) strain and cultured in LB medium with a PPO-inhibiting herbicide to examine whether the growth of the transformed BT3 was not inhibited.

[0238] The detailed experimental procedure is as follows:

[0239] Referring to Example 2, BT3 competent cells were transformed with the constructed pET303-CyPPO19 (wild type) and pET303-CyPPO18 (wild type) plasmids and the mutant plasmids by heat shock, and cultured in LB agar medium containing ampicillin.

[0240] Single colonies transformed with each CyPPO gene were cultured in 3 ml of LB broth (LPSS) containing ampicillin for more than 12 hours, and then subcultured in LB broth until the absorbance (OD) reached a certain level. 600 Reach 0.5 to 1. Then, dilute with LB broth to OD. 600 =0.5. Next, continuously dilute the solution four times to one-tenth.

[0241] LB agar media (LB 25 g / L, bacterial agar 12 g / L) containing ampicillin (100 μg / ml) and various herbicides from 0 to 2,000 μM were prepared. Stock solutions of the herbicides were prepared in DMSO.

[0242] Next, 10 μl of each diluted E. coli solution was dropped onto a plate and incubated at 37°C in the dark for 16 to 20 hours. The growth of E. coli containing each gene was then observed to assess resistance to the PPO-inhibiting herbicide.

[0243] The herbicides used for testing are listed in Table 8.

[0244] [Table 8]

[0245]

[0246] The degree of herbicide resistance was assessed by the relative growth of the variant to the wild type, as listed in Tables 9 and 10. Figures 3 to 32 .

[0247] [Table 9]

[0248]

[0249]

[0250]

[0251]

[0252] [Table 10]

[0253]

[0254] In Tables 9 and 10, the tolerance level of the variant is shown as "-" to be equivalent to the resistance level of the wild type, and tolerance increases by a factor of ten by a factor of "+", up to "+++++" for the maximum tolerance. Figures 3 to 30 as well as Figure 31 and Figure 32 The growth results of *E. coli* transformed with CyPPO19 WT and its variants, and CyPPO18 WT and its variants, are shown respectively. Herbicide concentrations are written on the tolerance test photographs. OD from the leftmost point... 600 =0.5 to the rightmost point of OD 600 =0.00005, a 10-fold dilution series for display points.

[0255] As shown in Tables 9 to 10 and Figures 3 to 32 As shown, all BT3 strains transformed with variants of CyPPO19 or CyPPO18 exhibited significantly higher (at least 10-fold) tolerance levels to various PPO-inhibiting herbicides compared to the wild type.

[0256] Example 4: PPO enzyme activity and IC50 of herbicides 50 Measurement of value

[0257] Enzymatic activity of variants of PPO protein with amino acid mutations at certain positions was measured, and inhibition was determined using a PPO-inhibiting herbicide. Although the solubility of PPO protein under aqueous conditions was significantly low, its solubility was greatly increased when maltose-binding protein (MBP) was fused to it. Therefore, wild-type and variant PPO proteins were expressed as fused with MBP and used in experiments. Figure 33 ).

[0258] To express the wild-type and variant proteins of CyPPO19 and CyPPO18 (see Examples 1 and 2), these genes were introduced into the pMAL-c2X vector (see Example 1). Figure 1 They were then transformed into BL21CodonPlus(DE3) Escherichia coli.

[0259] The transformed E. coli were cultured under the following conditions to express the PPO protein:

[0260] Induction: OD 600 =0.2, add 0.3mM IPTG (final concentration);

[0261] Incubation temperature: 23℃, accompanied by shaking at 200 rpm;

[0262] Incubation time: 16 hours;

[0263] Culture volume: 200ml / 1,000ml flask.

[0264] The following is an example of protein extraction from transformed E. coli cells after lysis culture:

[0265] Extraction buffer: Column buffer (50mM Tris-Cl, pH 8.0, 200mM NaCl) 5ml buffer / g cells;

[0266] Ultrasound: SONICS & MATERIALS VCX130 (130 watts);

[0267] Sonicate on ice for 15 seconds, stop for 10 seconds, and continue for 5 minutes;

[0268] Centrifuge at 4°C for 20 minutes (20,000 × g);

[0269] The supernatant obtained after centrifugation was diluted with column buffer at a ratio of 1:6.

[0270] The following purification procedure for PPO protein was performed in a 4°C cold chamber. A 1.5 x 15 cm column (Bio-Rad, Econo 1.5 x 15 cm glass column, maximum volume) was packed with linear starch resin (New England Biolabs). The obtained protein extract was loaded onto the column at a flow rate of 0.2 mL / min. The column was washed with 3 column volumes of buffer, and the presence of protein in the wash solution was checked. The washing procedure was terminated when no protein was detected. MBP-PPO protein was then 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 protein was detected. Protein quantification and SDS-PAGE analysis of 10 μL fractions of each fraction were investigated. Enzymatic assays were performed using high-purity fractions of the PPO protein variant.

[0271] Enzyme activity was measured using purified proteins from the above-mentioned wild-type and CyPPO19 and CyPPO18 variants as follows:

[0272] First, protoporphyrinogen IX was chemically synthesized in the laboratory. The entire process was carried out under a nitrogen atmosphere. 6 μg of protoporphyrinogen IX was dissolved in 20 mL of 20% (v / v) EtOH and stirred for 30 minutes in the dark. The resulting protoporphyrinogen IX solution was transferred in 1000 μl to a 15 mL spiral tube and rinsed with nitrogen for 5 minutes. 1 g of sodium amalgam was added, and the tube was shaken vigorously for 2 minutes. The cap was opened to purge hydrogen gas from the tube. The tube was then capped and incubated for 3 minutes. The protoporphyrinogen IX solution was filtered using a syringe and a cellulose membrane filter. Approximately 1600 μl of 2M MOPS [3-(N-morpholino)propanesulfonic acid] was added to 800 μl of the obtained protoporphyrinogen IX solution to adjust the pH to 7.5. To determine the enzymatic activity of PPO protein, a reaction mixture (based on 10 ml) was prepared with the following composition: 50 mM Tris-Cl (pH 7.5); 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).

[0273] 180 μl of the reaction mixture was placed in a 96-well plate, and 20 μl of the purified PPO protein (a purified product of the MBP-fused PPO protein) was added. After separation with 50 μl of mineral oil, the reaction was initiated by adding substrate protoporphyrinogen IX solution to a final concentration of 50 μM. The reaction was carried out at room temperature for 30 min, and the fluorescence of protoporphyrinogen IX was measured using a microplate reader (Sense, Hidex) (excitation: 405 nm; emission: 633 nm). To calculate PPO enzyme activity, the protoporphyrinogen IX solution was kept open to air for more than 12 hours to oxidize the solution. 2.7 N HCl was added, and the absorbance was measured at 408 nm. A standard curve was generated using standard protoporphyrinogen IX, and PPO activity was measured by calibration with the standard curve of protoporphyrinogen IX.

[0274] For each herbicide, the concentration (IC50) at which the PPO inhibitory herbicide inhibited PPO enzyme activity by 50% was measured. 50 The final concentrations of each herbicide are as follows:

[0275] - Concentrations of tefenazate, pyrimisulfuron, flumetsulam, propyzoxystrobin, mesotrione, ethoxyflufenazate, and cyclohexane: 0, 10, 50, 100, 250, 500, 1000, 2500, 5000, 10000 nM

[0276] The IC was calculated by adding the herbicide concentration described above to the reaction mixture. 50 Value, which is the concentration of herbicide that inhibits PPO enzyme activity to 50%.

[0277] IC50 of each herbicide 50 The values ​​are shown in Tables 11 and 12 below.

[0278] [Table 11] IC50 of CyPPO19 wild-type and variants against various herbicides 50 Value (nM)

[0279]

[0280]

[0281] [Table 12] IC50 of CyPPO18 wild-type and variants against various herbicides 50 Value (nM)

[0282]

[0283] (In Tables 11 and 12 above, IC) 50 Value '5,000' or IC 50 The value '10,000' means equal to or higher than IC. 50 Value 5,000 or IC 50 The value was 10,000 (because the enzyme activity was not inhibited to 50% even at herbicide concentrations of 5,000 nM or 10,000 nM), as shown in Tables 11 and 12, demonstrating that the variant of the CyPPO protein exhibited a significantly increased IC50 against each herbicide compared to the wild type. 50 The results suggest that herbicide tolerance is increased through amino acid substitution at specific sites in the PPO protein. Although the data showed that the CyPPO protein variant had reduced enzyme activity compared to the wild type, this is likely due to differences in protein folding and hydrophobicity between the proteins. Plant-derived PPO proteins are located in the chloroplast membrane and are hydrophobic, but recombinant PPO proteins fused with MBP are hydrophilic. Therefore, when the PPO variant is correctly assembled and expressed in plant chloroplasts, there is no significant difference in enzyme activity between the variant and the wild type.

[0284] Example 5. PPO protein variants derived from various cyanobacteria, algae, or bacteria.

[0285] Based on the mutation sites of CyPPO19 and CyPPO18 that demonstrated increased resistance in Examples 3 and 4 above, their mutation sites were analyzed through 3D protein structure analysis of PPO proteins derived from various cyanobacteria, algae, or bacteria that have similar effects on herbicide resistance in CyPPO19 and CyPPO18 variants, as shown in Table 13:

[0286] [Table 13]

[0287]

[0288]

[0289]

[0290]

[0291] Example 6. Production of Arabidopsis thaliana transformants using CyPPO and its variants and PPO inhibitory herbicide tolerance tests.

[0292] 6-1. Construction of Arabidopsis transformation vectors and generation of Arabidopsis transformants

[0293] Arabidopsis thaliana was transformed with binary vectors containing selectable markers, the Bar gene (the gene for glufosinate resistance), and the ORFs of each variant of CyPPO19 and CyPPO18. Cross-tolerance to glufosinate and PPO-suppressing herbicides was assessed in the transgenic plants. The bar gene was also used to determine whether the transgene was stably inherited across generations. The NOS promoter and E9 terminator were used for bar gene expression.

[0294] To express the proteins of CyPPO19, CyPPO19 variants, CyPPO18, and CyPPO18 variants in plants, the CaMV35S promoter and NOS terminator were used. The encoding genes of CyPPO19, CyPPO19 variants, CyPPO18, and CyPPO18 variants were amplified by PCR using the primer pairs in Table 14, and then introduced into binary vectors using XhoI and BamHI restriction enzymes.

[0295] [Table 14] Primer Sequences

[0296]

[0297] In addition, to confirm protein expression, a hemagglutinin (HA) tag was fused to the 3' terminal region of the PPO protein-coding gene using BamHI and SacI restriction enzymes. A NOS terminator was inserted at the 3' end of the HA tag to induce transcriptional termination of the PPO gene. Furthermore, to facilitate protein transport to chloroplasts, the transport peptide (TP) gene (SEQ ID NO:301) of the AtPPO1 gene (SEQ ID NO:302) was fused to the 5' terminal region of the PPO protein-coding gene using XbaI and XhoI restriction enzymes. Each constructed vector was transformed into *Agrobacterium tumefaciens* GV3101 competent cells using a freeze-thaw method. *Agrobacterium* GV3101 competent cells were prepared by culturing the *Agrobacterium* GV3101 strain in 5 ml LB medium at 30°C and 200 rpm for 12 hours. Cells were passaged in 200 ml LB medium at 30°C and 200 rpm for 3 to 4 hours, and then centrifuged at 3,000 x g at 4°C for 20 minutes. The cell pellet was washed with sterile distilled water and then resuspended in 20 ml LB medium. 200 μl aliquots of the sample were flash-frozen in liquid nitrogen and stored in a cryogenic freezer.

[0298] Each transformed Agrobacterium was screened in LB medium containing spectinomycin. The screened colonies were cultured in LB broth. After harvesting the Agrobacterium cells from the medium, they were analyzed at an absorbance of 0.8 (OD). 600 The sucrose was resuspended in a solution containing 5% sucrose (w / v) and 0.05% Silwet L-77 (v / v) (Momentive Performance Materials Co., Ltd.). Wild-type Arabidopsis thaliana (Col-0) was transformed by the flower immersion method, and seeds were harvested after 1 to 2 months (T1).

[0299] Transgenic plants were screened for glufosinate tolerance conferred by Bar gene expression in a binary vector. 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 50 μM glufosinate. Surviving plants were selected 7 days after sowing. These were then transplanted into soil and grown to obtain T1 plants.

[0300] To test the tolerance of transgenic plants to PPO-inhibiting herbicides, a 40x60 cm area (0.24 m²) was used. 2In this study, 3- to 4-week-old plants were uniformly sprayed with a herbicide (100 ml of 1 μM tafenamic acid and 0.05% Silwet L-77 (v / v)). Wild-type Arabidopsis thaliana (Col-0) died completely within 7 days after treatment with the same concentration of tafenamic acid, while each transgenic plant showed tolerance to PPO-inhibiting herbicide treatment and survived.

[0301] T2 seeds were harvested from tolerant and surviving T1 transgenic plants and sown in 1 / 2 MS medium (2.25 g / L MS salt, 10 g / L sucrose, 7 g / L agar) supplemented with 50 μM glufosinate. One week later, the surviving plants were transplanted into soil.

[0302] 6-2. Validation of herbicide tolerance in transformed Arabidopsis thaliana plants (T2)

[0303] The herbicide tolerance of Arabidopsis thaliana plants (T2) transformed with genes including CyPPO19, CyPPO19 variants (F360M, F360V, F360L, V165C+F360M, V165S+F360V), CyPPO18, or CyPPO18 variant (L327T+F360M) was tested.

[0304] To examine the tolerance of transgenic plants to PPO-inhibiting herbicides, a 40x60cm area (0.24m) was surveyed. 2 CyPPO19 WT and its variants (F360M, F360V, F360L, V165C+F360M, V165S+F360V) transgenic plants were uniformly sprayed with herbicides (100 ml of 1 μM propyzamide and 0.05% Silwet L-77 (v / v)) / (100 ml of 1 μM propyzamide and 0.05% Silwet L-77 (v / v)). Herbicide tolerance was evaluated 7 days after treatment. Wild-type Arabidopsis thaliana plants (Col-0) were used as controls.

[0305] The tolerance assessment of transgenic Arabidopsis (T2) plants to 1 μM tefenazate or 1 μM propyzamide was shown in Figure 34 and 35 middle.

[0306] To examine the tolerance of transgenic plants to PPO-inhibiting herbicides, a 40x60cm area (0.24m²) was surveyed. 2CyPPO19 WT and its variants (F360M, F360V, F360L, V165C+F360M, V165S+F360V) transgenic plants were uniformly sprayed with herbicides (100 ml of 5 μM tebufenozide and 0.05% Silwet L-77 (v / v)) / (100 ml of 5 μM propyzamide and 0.05% Silwet L-77 (v / v)). Herbicide tolerance was evaluated 7 days after treatment. Wild-type Arabidopsis thaliana plants (Col-0) were used as controls.

[0307] The tolerance assessment of transgenic Arabidopsis (T2) plants to treatment with 5 μM tebufenozide or 5 μM propyzamide showed that... Figure 36 and 37 middle.

[0308] To examine the tolerance of transgenic plants to PPO-inhibiting herbicides, a 40x60cm area (0.24m) was surveyed. 2 CyPPO18 wild-type transgenic plants were uniformly sprayed with a herbicide (100 ml of 1 μM tebufenozide and 0.05% Silwet L-77 (v / v)). Herbicide tolerance was evaluated 7 days after treatment. Wild-type Arabidopsis plants (Col-0) were used as a control.

[0309] Tolerance assessment of transgenic Arabidopsis (T2) plants after treatment with 1 μM tafenamic acid was shown in Figure 38 middle.

[0310] To examine the tolerance of transgenic plants to PPO-inhibiting herbicides, a 40x60cm area (0.24m) was surveyed. 2 Transgenic plants of CyPPO18 variant (L327T+F360M) were uniformly sprayed with a herbicide (100 ml of 1 μM tebufenozide and 0.05% Silwet L-77 (v / v)). Herbicide tolerance was evaluated 7 days after treatment. Wild-type Arabidopsis thaliana plants (Col-0) were used as a control.

[0311] Tolerance assessment of transgenic Arabidopsis thaliana (T2) plants after treatment with 1 μM tafenamic acid was shown in Figure 39 middle.

[0312] based on Figures 34 to 39 The results shown assess herbicide tolerance in transgenic plants using the damage index defined in Table 15.

[0313] [Table 15] Definition of Damage Index

[0314]

[0315] The tolerance level of transgenic plants was assessed according to the definition of the damage index, and is shown in Tables 16 to 19.

[0316] [Table 16]

[0317] Damage index of transgenic plants of CyPPO19 WT and its variants (F360M, F360V, F360L, V165C+F360M, V165S+F360V) after treatment with 1 μM tefenazate or 1 μM propyzamide

[0318]

[0319] [Table 17]

[0320] Damage index of transgenic plants of CyPPO19 WT and its variants (F360M, F360V, F360L, V165C+F360M, V165S+F360V) after treatment with 5 μM tefenazate or 5 μM propyzamide

[0321]

[0322] [Table 18]

[0323] Damage index of CyPPO18 WT transgenic plants after treatment with 1 μM tafenamic acid

[0324]

[0325] [Table 19]

[0326] Damage index of transgenic plants of CyPPO18 WT variant (L327T+F360M) after treatment with 1 μM tafenamic acid

[0327]

[0328] As demonstrated by the results above, transgenic plants transformed with CyPPO19 WT or CyPPO18 WT exhibited increased herbicide tolerance compared to non-transgenic plants. Furthermore, transgenic plants transformed with CyPPO19 variants or CyPPO18 variants showed significantly increased herbicide tolerance compared to non-transgenic plants.

Claims

1. A polypeptide comprising the following amino acid sequence, wherein the amino acid sequence is a mutation of one amino acid selected from the group consisting of the amino acid sequences in SEQ ID NO:1: (i) F360M, F360V, F360I, F360T, or F360L, (ii) A167C, A167L, or A167I, (iii) V305M or V305L, (iv)R89A, (v)V165S or V165C, (vi)L327T, and (vii)I408R or I408W.

2. The polypeptide according to claim 1, wherein the polypeptide comprises the following amino acid sequence, wherein the amino acid sequence is formed by the following mutation in the amino acid sequence of SEQ ID NO:1: (i) F360M, F360V, F360I, F360T or F360L.

3. The polypeptide according to claim 2, wherein the polypeptide comprises the following amino acid sequence, wherein the amino acid sequence is further modified by 1-3 amino acid mutations selected from the group consisting of the following amino acid sequences in the amino acid sequence of SEQ ID NO:1: (ii) A167C, A167L, or A167I, (iii) V305M or V305L, (iv)R89A, (v)V165S or V165C, (vi)L327T, and (vii)I408R or I408W.

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. Use of a composition for conferring or enhancing herbicide tolerance to plants or algae, said composition comprising one or more of the following: (1) One or more of the polypeptides selected from any one of claims 1 to 3; (2) A polynucleotide encoding the polypeptide described in (1); (3) A recombinant vector comprising the polynucleotide described in (2); and (4) Recombinant cells containing the recombinant vector described in (3), in, The herbicide is a herbicide that inhibits protoporphyrinogen IX oxidase.

7. The use according to claim 6, wherein, The herbicide is selected from at least one of the following groups: pyrimidine dione, diphenyl ether, phenylpyrazole, N-phenylphthalimide, phenyl ester, thiadiazole, oxadiazole, triazine, triazoline, oxazolidinone, bispyribac-sodium, flupyridamole, and flupyrazosulfuron.

8. The use according to claim 6, wherein, The herbicide is selected from at least one of the following groups: flufenacet, pyrimisulfuron, bispyribac-methyl, tefenazate, flufenacet, oxyfluorfen, phenazate, flufenacet, cyhalofop-butyl, flufenacet, methoxyfenozide, glufosinate, ethoxysulfuron, flufenacet, isopyram, propyzoxystrobin, indole-methyl, flufenacet, cyhalofop-butyl, thiamethoxam, propyzoxystrobin, oxadiazon, pyrazosulfuron, mesotrione, trifluralin, pyrazosulfuron, cyclopyrazosulfuron, bispyrazosulfuron, flupyridazine, flufenacet, pyrazosulfuron, carbamate analogs of pyrazosulfuron and their agriculturally acceptable salts.

9. The use according to claim 6, wherein, The plant or algae further comprises a second herbicide tolerance polypeptide or its encoding gene, and confers or enhances the plant or algae's tolerance to the second herbicide.

10. The use according to claim 9, wherein, The second herbicide is selected from the group consisting of glyphosate, glufosinate, dicamba, 2,4-D (2,4-dichlorophenoxyacetic acid), isoxaflutole, acetolactate synthase inhibitors, photosystem II inhibitors, phenylurea herbicides, bromobenzonitrile herbicides, and combinations thereof.

11. The use according to claim 9, wherein, The second herbicide-resistant polypeptide is selected from one or more of the following groups: 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 acetolactate synthase, acetylhydroxyl synthase, or Arabidopsis thaliana acetylhydroxyl synthase large subunit; Photosystem II inhibitory herbicide tolerance photosystem II protein D1; Cytochrome P450, a phenylurea-based herbicide tolerance level; plastid-inhibiting herbicide-resistant hydroxyphenylpyruvate dioxygenase; Bromobenzonitrile herbicide-resistant nitrile hydrolase; and Their combination.

12. The use according to claim 9, wherein, The gene encoding the second herbicide tolerance polypeptide is selected from one or more of the following groups: Glyphosate herbicide tolerance genes cp4 epsps, mepsps, 2mepsps, goxv247, gat4601, or gat4621; BAR or PAT genes for glufosinate-ammonium herbicide tolerance; DMO gene for tolerance to dicamba herbicide; 2,4-D (2,4-dichlorophenoxyacetic acid) herbicide tolerance AAD-1 or AAD-12 gene; The HPPDPF W336 gene for tolerance to isoxaflutole herbicides; 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; CYP76B1 gene for tolerance to phenylurea herbicides; The bxn gene for herbicide tolerance in bromobenzonitrile; and Their combination.

13. A method for preparing transgenic plants or algae with herbicide tolerance, the method comprising: The polypeptide of any one of claims 1 to 3, or the polynucleotide encoding the polypeptide of claim (1), is introduced into the cells, protoplasts, callus, hypocotyls, seeds, cotyledons, buds, or whole organisms of algae or plants. The herbicide is a herbicide that inhibits protoporphyrinogen IX oxidase.

14. A method for conferring or enhancing herbicide tolerance to plants or algae, the method comprising: (1) the polypeptide of any one of claims 1 to 3, or (2) a polynucleotide encoding the polypeptide of claim (1) introduced into algae, or plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, buds, or the whole plant. in, The herbicide is a herbicide that inhibits protoporphyrinogen IX oxidase.

15. A method for controlling weeds in farmland, the method comprising: Providing farmland with plants comprising (1) a polypeptide according to any one of claims 1 to 3, or (2) a polynucleotide encoding the polypeptide of claim (1), and Apply an effective dose of protoporphyrinogen IX oxidase-inhibiting herbicide to farmland or plants.

16. The method according to claim 15, wherein, The application of an effective dose of protoporphyrinogen IX oxidase inhibitory herbicide to farmland is carried out by sequentially or simultaneously applying effective doses of two or more protoporphyrinogen IX oxidase inhibitory herbicides.

17. The method according to claim 15, wherein, The plant further comprises a second herbicide tolerance polypeptide or its encoding gene, and The process of applying an effective dose of protoporphyrinogen IX oxidase-inhibiting herbicide to farmland is carried out by sequentially or simultaneously applying an effective dose of protoporphyrinogen IX oxidase-inhibiting herbicide and a second herbicide.

18. A method for removing unwanted aquatic organisms from a culture medium, the method comprising: The culture medium is provided with algae, said algae comprising (1) a polypeptide according to any one of claims 1 to 3, or (2) a polynucleotide encoding the polypeptide of (1), and An effective dose of a protoporphyrinogen IX oxidase-inhibiting herbicide was applied to the culture medium.

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