Protox variants

Abstract

The present invention provides protoporphyrinogen oxidase amino acid variants derived from prokaryotes. The use of the protoporphyrinogen oxidase amino acid variants can confer enhanced herbicide tolerance and / or enhanced herbicide resistance to plants and / or algae.

Description

[0001] This application is a divisional application of a Chinese patent application filed on June 15, 2017, with application number 201780037777.7 and invention name “Methods and compositions for conferring and / or enhancing herbicide tolerance using protoporphyrinogen oxidase or its variants”. Technical Field

[0002] Provided are prokaryotically derived protoporphyrinogen oxidase (PPO) or a variant thereof, and a technology for conferring and / or enhancing herbicide tolerance on plants and / or algae using the PPO or a variant thereof. Background Art

[0003] The porphyrin biosynthesis pathway is used to synthesize chlorophyll and heme, which play an important role in plant metabolism, and occurs in chloroplasts. In this pathway, protoporphyrinogen IX oxidase (hereinafter referred to as PPO; EC: 1.3.3.4) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. After protoporphyrinogen IX is oxidized to protoporphyrin IX, protoporphyrin IX is bound to magnesium by magnesium chelatase to synthesize chlorophyll, or to iron by ferrochelatase to synthesize heme.

[0004] Therefore, when PPO activity is inhibited, the synthesis of chlorophyll and heme is inhibited, and the substrate protoporphyrinogen IX leaves the normal porphyrin biosynthesis pathway, resulting in rapid export of protoporphyrinogen IX from the chloroplast to the cytoplasm and accumulation of cytoplasmic protoporphyrin IX due to oxidation. The accumulated protoporphyrin IX generates highly reactive singlet oxygen ( 1 O2), which destroys the cell membrane and quickly leads to plant cell death. Based on this principle, herbicides that inhibit PPO activity have been developed. To date, there are 9 families of PPO-inhibiting herbicides classified according to their chemical structure, including pyrimidinedione, diphenyl ether, phenylpyrazole, N-phenylphthalimide, thiadiazole, Oxadiazole, triazolinone, Oxazolidinediones and other herbicides.

[0005] Furthermore, in order to prevent the effects of herbicides on crop growth when using them, it is necessary to provide crops with herbicide tolerance.

[0006] Algae are photosynthetic organisms that convert light energy into chemical energy that can be used to synthesize a variety of 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. Furthermore, large-scale cultivation of algae can produce a variety of substances, such as industrial enzymes, therapeutic compounds and proteins, nutrients, commercial substances, and fuels.

[0007] However, in the case of large-scale cultivation of algae in bioreactors or open or closed ponds, contamination by undesirable competing organisms, such as undesirable algae, fungi, rotifers or zooplankton, may occur.

[0008] Therefore, there is a need for a technology to harvest desired plants and / or algae on a large scale after conferring herbicide tolerance to the desired plants and / or algae by treating the herbicide at a concentration that inhibits the growth of competing organisms without herbicide tolerance.

[0009] References

[0010] (Patent Document 1) U.S. Patent Application Registration Publication No. 6,308,458 (October 30, 2001)

[0011] (Patent Document 2) U.S. Patent Application Registration Publication No. 6,808,904 (October 26, 2004)

[0012] (Patent Document 3) U.S. Patent Application Registration Publication No. 7,563,950 (July 21, 2009)

[0013] (Patent Document 4) International Patent Application Publication No. WO2011 / 085221 (July 14, 2011)

[0014] (Non-Patent Document 1) Li X, Volrath SL, Chilcott CE, Johnson MA, Ward ER, Law MD. Development of protoporphyrinogen oxidase as an efficient selection marker for agrobacterium tumefaciens-mediated transformation of maize. Plant Physiology, 133: 736-747, 2003. Summary of the Invention

[0015] Technical issues

[0016] In this specification, it was found that the hemY-type PPO gene and its mutants derived from prokaryotes showed a wide range of herbicide tolerance to protoporphyrinogen oxidase (PPO)-inhibiting herbicides, thereby proposing that if the hemY-type PPO gene and its mutants are provided to plants and / or algae, herbicide tolerance can be conferred and / or enhanced.

[0017] One embodiment provides a polypeptide variant comprising, consisting essentially of, or consisting of:

[0018] (1) an amino acid sequence in which one or more selected from the group consisting of amino acids that affect the interaction between a PPO-inhibiting herbicide and the PPO polypeptide of SEQ ID NO: 2 (e.g., amino acids located at the binding site of SEQ ID NO: 2 that interacts with the PPO-inhibiting herbicide) are respectively and independently deleted or substituted with an amino acid different from the original amino acid at the corresponding position, or

[0019] (2) An amino acid sequence having a sequence identity of 95% or greater, 98% or greater, or 99% or greater to the amino acid sequence of (1).

[0020] One or more selected from the group consisting of amino acids affecting the interaction between the PPO-inhibiting herbicide and the PPO polypeptide SEQ ID NO: 2 can be one or more selected from the group consisting of N59, S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360 and I408 in the amino acid sequence of SEQ ID NO: 2.

[0021] Another embodiment provides a polypeptide variant comprising, consisting essentially of, or consisting of:

[0022] (1) an amino acid sequence in which one or more selected from the group consisting of amino acids that affect the interaction between a PPO-inhibiting herbicide and the PPO polypeptide of SEQ ID NO: 4 (e.g., amino acids located at the binding site of SEQ ID NO: 4 that interacts with the PPO-inhibiting herbicide) are respectively and independently deleted or substituted with an amino acid different from the original amino acid at the corresponding position, or

[0023] (2) An amino acid sequence having 95% or more, 98% or more, or 99% or more homology to the amino acid sequence (1).

[0024] One or more selected from the group consisting of amino acids affecting the interaction between the PPO-inhibiting herbicide and the PPO polypeptide SEQ ID NO: 4 can be one or more selected from the group consisting of R101, F171, V175, A177, G194, P316, V318, F337, L340, I353 and F373 in the amino acid sequence of SEQ ID NO: 4.

[0025] Other embodiments provide polynucleotides encoding polypeptides or polypeptide variants.

[0026] Other embodiments provide a recombinant vector comprising the polynucleotide.

[0027] Other embodiments provide a recombinant cell comprising the recombinant vector.

[0028] Other embodiments provide a composition for conferring or enhancing herbicide tolerance in a plant or alga, comprising one or more selected from the group consisting of:

[0029] a polypeptide of SEQ ID NO: 2, a polypeptide of SEQ ID NO: 4, a polypeptide variant as described above, and a polypeptide having an amino acid sequence that has 95% or more, 98% or more, or 99% or more sequence homology to the polypeptide or polypeptide variant;

[0030] a polynucleotide encoding a polypeptide, a polypeptide variant, and a polypeptide having an amino acid sequence that has 95% or more, 98% or more, or 99% or more sequence homology to the polypeptide or variant;

[0031] a recombinant vector comprising the polynucleotide; and

[0032] a recombinant cell comprising the recombinant vector.

[0033] For example, the polynucleotide encoding the polypeptide of SEQ ID NO: 2 can comprise the polynucleotide sequence of SEQ ID NO: 1, and the polynucleotide encoding the polypeptide of SEQ ID NO: 4 can comprise the polynucleotide sequence of SEQ ID NO: 3, but is not limited thereto.

[0034] The herbicide can be a protoporphyrinogen oxidase inhibitor herbicide.

[0035] As a specific embodiment, the herbicide can be one or more selected from the group consisting of a pyrimidinedione, a diphenyl ether, a phenylpyrazole, an N-phenylphthalimide, a phenyl ester, a thiadiazole, a triazolinone, an oxazolidinedione, and other herbicides, but is not limited thereto.

[0036] As a specific embodiment, the herbicide can be one or more selected from the group consisting of fluazifop-butyl, fluazifop-butyl, fluazifop-butyl, tiafenacil, fomesafen, oxyfluorfen, benzopyrene, acifluorfen, bifenox, ethoxyfen, lactofen, chlomethoxyfen, chlorintrofen, fluoroglycofen-ethyl, halosafen, pyraflufen-ethyl, fluazolate, flumioxazin, cinidon-ethyl, flumiclorac-pentyl, fluthiacet, thidiazimin, propyne Oxadiargyl, Oxadiazon, carfentrazone, sulfentrazone, azafenidin, cyclopentane The present invention also includes pentoxazone, pyraclonil, flufenpyr-ethyl, profluazol, phenopylate (2,4-dichlorophenyl-1-pyrrolidinecarboxylate), carbamate analogs of phenopylate (for example, O-phenyl pyrrolidino- and piperidinocarbamate 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., 1992, 40 (10) 1993-2000"), agriculturally acceptable salts thereof, and combinations thereof, but are not limited thereto.

[0037] Plants refer to multicellular eukaryotic organisms with photosynthetic ability, which can be monocots or dicots and can be herbaceous or woody. Algae refer to single-celled organisms with photosynthetic ability, which can be prokaryotic or eukaryotic.

[0038] In one embodiment, plants and algae are genetically manipulated to further include a second herbicide tolerance polypeptide or a gene encoding it, and can confer and / or enhance herbicide tolerance to a wider range of second herbicides. Plants and algae genetically manipulated to include a greater number of second herbicide tolerance polypeptides or genes encoding them can be prepared using a composition for conferring and / or enhancing herbicide tolerance, which further includes a second herbicide tolerance polypeptide or a gene encoding it. Therefore, a composition for conferring and / or enhancing herbicide tolerance can further include a second herbicide tolerance polypeptide or a gene encoding it.

[0039] As specific embodiments, the second herbicide may include cell division inhibiting herbicides, photosynthesis inhibiting herbicides, amino acid synthesis inhibiting herbicides, plastid inhibiting herbicides, and cell membrane inhibiting herbicides, but is not limited thereto.

[0040] As a specific embodiment, examples of the second herbicide can be glyphosate, glufosinate, dicamba, 2,4-D (2,4-dichlorophenoxyacetic acid), isothiophene, Mesotrione, ALS (acetolactate synthase)-inhibiting herbicides, photosystem II-inhibiting herbicides, phenylurea-based herbicides, bromoxynil-based herbicides, and combinations thereof, but are not limited thereto.

[0041] As a specific embodiment, an example of the second herbicide can be one or more selected from the group consisting of: glyphosate herbicide-tolerant EPSPS (glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase), GOX (glyphosate oxidase), GAT (glyphosate-N-acetyltransferase) or glyphosate decarboxylase; glufosinate herbicide-tolerant PAT (phosphinothricin-N-acetyltransferase); dicamba herbicide-tolerant DMO (dicamba monooxygenase); 2,4-D herbicide-tolerant 2,4-D monooxygenase or AAD (aryloxyalkanoate dioxygenase); Dioxygenase); ALS-inhibiting sulfonylurea herbicide-tolerant ALS (acetolactate synthase), AHAS (acetohydroxyacid synthase) or Athahasl (acetohydroxyacid synthase large subunit); photosystem II-inhibiting herbicide-tolerant photosystem II protein D1; phenylurea-based herbicide-tolerant cytochrome P450; plastid-inhibiting herbicide-tolerant HPPD (hydroxyphenylpyruvate dioxygenase); bromoxynil herbicide-tolerant nitrilase; and combinations thereof, but are not limited thereto.

[0042] Further, examples of genes encoding the second herbicide tolerance polypeptide can be one or more selected from the group consisting of: glyphosate herbicide tolerance cp4 epsps, epsps (AG), mepsps, 2mepsps, goxv247, gat4601 or gat4621 genes; glufosinate herbicide tolerance bar, pat or pat (SYN) genes; dicamba herbicide tolerance dmo genes; 2,4-D herbicide tolerance AAD-1, AAD-12 genes; ALS-inhibiting sulfonylurea herbicide tolerance ALS, GM-HRA, S4-HRA, ZM-HRA, Csr1, Csr1-1, Csr1-2, SurA or SurB; photosystem II inhibitory herbicide tolerance psbA gene; phenylurea herbicide tolerance CYP76B1 gene; isoenzyme ALS, ... isoenzyme ALS, sulfonylurea herbicide tolerance psbA gene; isoenzyme ALS, The herbicide-tolerant HPPDPF W336 gene of foramsulfuron and the herbicide-tolerant bxn gene of bromoxynil; and combinations thereof, but not limited thereto.

[0043] Other embodiments provide herbicide-tolerant plant and / or algal transformants transformed with the polynucleotide or a clone or progeny thereof.

[0044] Other embodiments provide methods for producing plants or algae with herbicide tolerance, comprising the step of transforming the plants and / or algae with the polynucleotide.

[0045] Other embodiments provide methods of conferring or enhancing herbicide tolerance in plants and / or algae, comprising the step of transforming the plants and / or algae with the polynucleotides described.

[0046] Transformation can be performed on algal and / or plant cells, protoplasts, callus tissue, hypocotyls, seeds, cotyledons, shoots or whole plants.

[0047] The transformant can be a cell, protoplast, callus, hypocotyl, seed, cotyledon, shoot or whole plant of algae and / or plants.

[0048] Other embodiments provide a method for controlling weeds in a farmland, comprising:

[0049] The step of providing a plant to a farmland, wherein the plant comprises one or more selected from the group consisting of a polypeptide of SEQ ID NO: 2 or 4, a polypeptide variant, a polynucleotide encoding the same, a recombinant vector comprising the polynucleotide, and a recombinant cell comprising the recombinant vector; and

[0050] The step of applying an effective dose of a protoporphyrinogen oxidase-inhibiting herbicide to agricultural land (or plants).

[0051] As a specific embodiment, the step of applying an effective dose of a protox-inhibiting herbicide to a farmland can be performed by sequentially or simultaneously applying effective doses of two or more protox-inhibiting herbicides.

[0052] As another embodiment, the plant can be genetically manipulated to further contain a second herbicide tolerance polypeptide or a gene encoding it, and an effective dose of a protoporphyrinogen oxidase-inhibiting herbicide and the second herbicide can be applied sequentially or simultaneously.

[0053] Other embodiments provide methods for removing unwanted aquatic organisms from a culture medium, comprising: providing algae to the culture medium, wherein the algae comprises one or more selected from the group consisting of a polypeptide, a polypeptide variant, a polynucleotide encoding the polypeptide or polypeptide variant, a recombinant vector comprising the polynucleotide, and a recombinant cell comprising the recombinant vector; and applying an effective amount of a protoporphyrinogen oxidase-inhibiting herbicide to the culture medium.

[0054] Technical Solution

[0055] Technologies are provided for conferring and / or enhancing herbicide tolerance in plants or algae.

[0056] Herein, "conferring and / or enhancing herbicide tolerance to plants or algae" or "enhancing herbicide tolerance of plants or algae" is interpreted as conferring tolerance to plants or algae that do not have herbicide tolerance, or enhancing the tolerance of plants or algae that have herbicide tolerance, or covering a broad meaning of both.

[0057] As used herein, the use of "consisting of a sequence", "consisting essentially of a sequence" or "comprising a sequence" is intended to refer to both cases of including the described sequence or necessarily including the sequence, and can be interpreted as including sequences other than the described sequence and / or containing mutations (additions, deletions and / or substitutions of amino acids or nucleic acids), as long as it maintains the intrinsic activity of the protein, polypeptide or nucleic acid molecule and exhibits the intended function.

[0058] In one embodiment, one or more polypeptide variants are provided selected from the group consisting of:

[0059] A polypeptide variant comprising, consisting essentially of, or consisting of an amino acid sequence in which one or more selected from the group consisting of amino acids that affect the interaction between a PPO-inhibiting herbicide and the PPO polypeptide SEQ ID NO: 2 (e.g., amino acids located at the binding site of SEQ ID NO: 2 that interacts with a PPO-inhibiting herbicide) is respectively and independently deleted or substituted with other amino acids different from the original amino acids; or an amino acid sequence having 95% or more, 98% or more, or 99% or more homology to the amino acid sequence of the polypeptide; and

[0060] A polypeptide variant comprising, consisting essentially of, or consisting of the following amino acid sequence, wherein the amino acids that affect the interaction between a PPO-inhibiting herbicide and the PPO polypeptide of SEQ ID NO: 4 (e.g., amino acids located at the binding site of SEQ ID NO: 4 that interacts with the PPO-inhibiting herbicide) are individually and independently deleted or substituted with other amino acids that are different from the original amino acids at the corresponding positions; or an amino acid sequence having 95% or more, 98% or more, or 99% or more homology to the amino acid sequence of the polypeptide.

[0061] In other embodiments, polynucleotides encoding polypeptides or polypeptide variants, recombinant vectors comprising the polynucleotides, and recombinant cells comprising the recombinant vectors are provided. The polynucleotides can be designed so that optimized codons encoding each amino acid are included in the cell to be transformed. Optimized codons are readily known to those skilled in the art (e.g., see "http: / / www.genscript.com / codon-opt.html," "http: / / sg.idtdna.com / CodonOpt," etc.).

[0062] In other embodiments, a composition for conferring or enhancing herbicide tolerance to plants or algae is provided, comprising one or more selected from the group consisting of:

[0063] The polypeptide of SEQ ID NO: 2, the polypeptide of SEQ ID NO: 4, polypeptide variants thereof, and polypeptides having an amino acid sequence having 95% or greater, 98% or greater, or 99% or greater homology to the amino acid sequence of the polypeptide or polypeptide variant;

[0064] a polynucleotide encoding the polypeptide or polypeptide variant;

[0065] a recombinant vector comprising the polynucleotide; and

[0066] A recombinant cell comprising the recombinant vector.

[0067] For example, a polynucleotide encoding the polypeptide of SEQ ID NO: 2 may include the polynucleotide sequence of SEQ ID NO: 1, and a polynucleotide encoding the polypeptide of SEQ ID NO: 4 may include the polynucleotide sequence of SEQ ID NO: 3, but are not limited thereto.

[0068] Another embodiment provides a transformant of a plant or algae having herbicide tolerance, which is transformed with a polynucleotide encoding a polypeptide or polypeptide variant. The polynucleotide can be designed so that an optimized codon encoding each amino acid is included in the cell to be transformed. Optimized codons are readily known to those skilled in the art (e.g., see "http: / / www.genscript.com / codon-opt.html," "http: / / sg.idtdna.com / CodonOpt," etc.).

[0069] In other embodiments, a method for preparing a plant or algae with herbicide tolerance is provided, comprising the step of transforming algae, or plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, shoots or whole plants with the polynucleotide.

[0070] In other embodiments, a method for conferring or enhancing herbicide tolerance to plants or algae is provided, comprising the step of transforming algae, or plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, shoots or whole plants with the polynucleotide.

[0071] Hereinafter, the present invention will be described in more detail.

[0072] Polypeptides having the amino acid sequence of SEQ ID NO: 2 or 4, or amino acid sequences having 95% or greater, 98% or greater, or 99% or greater homology thereto, and variants thereof provided herein are PPO proteins derived from prokaryotes (e.g., cyanobacteria) and are herbicide-tolerant PPO proteins tolerant to PPO-inhibiting herbicides. Specifically, a PPO protein derived from Thermosynechococcus elongatus BP-1 is provided, designated CyPPO10, the amino acid sequence of which is represented by SEQ ID NO: 2, and the nucleotide sequence of the gene encoding it is represented by SEQ ID NO: 1. Furthermore, a PPO protein derived from Thermosynechococcus sp. strain JA-3-3Ab is provided, designated CyPPO13, the amino acid sequence of which is represented by SEQ ID NO: 4, and the nucleotide sequence of the gene encoding it is represented by SEQ ID NO: 3.

[0073] Herein, the above-mentioned polypeptides and polypeptide variants can be respectively expressed as herbicide-tolerant PPO proteins or herbicide-tolerant PPO protein variants that are tolerant to PPO-inhibiting herbicides. In addition, as used herein, "herbicide-tolerant PPO or its variant" can be used to refer to the above-mentioned herbicide-tolerant PPO protein or herbicide-tolerant PPO protein variant, the herbicide-tolerant PPO protein encoding gene or the herbicide-tolerant PPO protein variant encoding gene, or all of them.

[0074] Cyanobacterial PPO proteins inherently possess superior enzymatic activity compared to plant PPOs, and these PPO proteins can confer tolerance to PPO-inhibiting herbicides. Herbicide tolerance can be enhanced by including amino acid mutations relative to wild-type PPO proteins while maintaining overall enzymatic activity. Such amino acid mutations can include substitutions, deletions, additions, and / or introductions of one or more amino acids selected from amino acid residues at sites of interaction between the PPO protein and the herbicide.

[0075] The PPO protein variants will be described in more detail as follows.

[0076] One embodiment provides a polypeptide variant comprising, consisting essentially of, or consisting of the following amino acid sequence:

[0077] an amino acid sequence, wherein one or more selected from the group consisting of amino acids that affect the interaction between a PPO-inhibiting herbicide and the PPO polypeptide SEQ ID NO: 2 (CyPPO10) (e.g., amino acids located at the binding site of SEQ ID NO: 2 that interacts with the PPO-inhibiting herbicide) are respectively and independently deleted or substituted with other amino acids that are different from the original amino acids (i.e., the amino acids at the corresponding positions of the wild type), or

[0078] An amino acid sequence having 95% or greater, 98% or greater, or 99% or greater homology to the amino acid sequence.

[0079] The amino acid residues deleted or substituted with amino acids different from the original amino acids in the polypeptide of SEQ ID NO: 2 (i.e., one or more selected from the group consisting of amino acids located at the binding site of the polypeptide of SEQ ID NO: 2 and the PPO-inhibiting herbicide) can be one or more selected from the group consisting of N59 (meaning "N (Asn) at position 59"; the representation of amino acid residues below is interpreted in the same manner), S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360 and I408 of the amino acid sequence of SEQ ID NO: 2.

[0080] In one embodiment, the polypeptide variant may comprise, consist essentially of, or consist of the following amino acid sequence:

[0081] An amino acid sequence, wherein one or more selected from the group consisting of N59, S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360 and I408 in the amino acid sequence of SEQ ID NO: 2 are respectively and independently deleted or selected from M (Met), V (Val), I (Ile), T (Thr), L (Leu), C (Cys), A (Ala), S (Ser), F (Phe), P (Pro), W (Trp), N (Asn), Q (Gln) , G (Gly), Y (Tyr), D (Asp), E (Glu), R (Arg), H (His), K (Lys), etc., and are substituted with an amino acid different from the original amino acid at the corresponding position (for example, substituted with an amino acid selected from the group consisting of M (Met), V (Val), I (Ile), T (Thr), L (Leu), C (Cys), A (Ala), S (Ser), R (Arg), W (Trp), G (Gly), etc., and are different from the original amino acid at the corresponding position in the wild type), or

[0082] An amino acid sequence having 95% or greater, 98% or greater, or 99% or greater homology to the amino acid sequence.

[0083] For example, a polypeptide variant may comprise, consist essentially of, or consist of the following amino acid sequence:

[0084] An amino acid sequence comprising one or more amino acid mutations selected from the group consisting of F360M (meaning "the amino acid residue at position 360 is substituted from F (Phe) to M (Met)"; the following expressions of amino acid mutations are interpreted in the same manner), F360V, F360I, F360T, F360L, F360C, A167C, A167L, A167I, P303L, V305L, V305M, V305T, N59T, S60T, R89A, R89L, R89V, F161A, V165S, V165C, Q184G, F324V, L327T, I340T, I408R and I408W in the amino acid sequence of SEQ ID NO: 2, or

[0085] An amino acid sequence having 95% or greater, 98% or greater, or 99% or greater homology to the amino acid sequence.

[0086] More specifically, the polypeptide variant may comprise, consist essentially of, or consist of the following amino acid sequence:

[0087] An amino acid sequence comprising F360M, F360V, F360I, F360T, F360L, F360C, A167C, A167L, A167I, P303L, N59T, S60T, R89A, R89L, R89V, F161A, V165S, V165C, Q184G, V305L, V305M, V305T, F324V, L327T, I340T, I408R, I408W, P303L+V305L (meaning all mutants or mutations including substitution of residue 303 from P to L and substitution of residue 305 from V to L; the following representation of two or more mutations is interpreted in the same way), N59T+F360V, S60T+V165S+F360M, S60T+V165S+F360I, S60T+I340T+F360I, R89A+F360M, R89A+F360 0I, R89A+F360L, R89L+F360I, R89V+F360I, R89A+A167L+F360M, R89A+V305T+F360M, V165S+F360M, V165S+F360I, V165S+F360L, V165S+F360V, V165C+F360M, V165C+A167C+F360M, V165C+A167I+F360 one or more amino acid mutations selected from the group consisting of: V165M+F360M, V165C+A167L+F360M, A167L+F360M, A167L+F360I, A167C+F360M, A167C+F360I, A167I+F360M, V305M+F360M, V305T+F360I, V305L+F360M, I408R+F360M, or I408W+F360M, or

[0088] An amino acid sequence having 95% or greater, 98% or greater, or 99% or greater homology to the amino acid sequence.

[0089] Other embodiments provide polypeptide variants comprising, consisting essentially of, or consisting of the following amino acid sequence:

[0090] an amino acid sequence, wherein one or more selected from the group consisting of amino acids that affect the interaction between a PPO-inhibiting herbicide and the PPO polypeptide of SEQ ID NO: 4 (CyPPO13) (e.g., amino acids located at the binding site of the polypeptide of SEQ ID NO: 4 that interacts with a PPO-inhibiting herbicide) are respectively and independently deleted or substituted with other amino acids that are different from the original amino acids at the corresponding positions (i.e., the amino acids at the corresponding positions of the wild type), or

[0091] An amino acid sequence having 95% or greater, 98% or greater, or 99% or greater homology to the amino acid sequence.

[0092] The amino acid residues deleted from the polypeptide of SEQ ID NO: 4 or substituted with other amino acids different from the original amino acids at the corresponding positions (for example, one or more selected from the group consisting of amino acids located at the binding site of the polypeptide of SEQ ID NO: 3 and PPO-inhibiting herbicides) can be one or more selected from the group consisting of R101, F171, V175, A177, G194, P316, V318, F337, L340, I353 and F373 in the amino acid sequence of SEQ ID NO: 4.

[0093] In one embodiment, the polypeptide variant may comprise, consist essentially of, or consist of the following amino acid sequence:

[0094] An amino acid sequence, wherein one or more selected from the group consisting of R101, F171, V175, A177, G194, P316, V318, F337, L340, I353 and F373 in the amino acid sequence of SEQ ID NO: 4 are respectively and independently deleted or selected from M (Met), V (Val), I (Ile), T (Thr), L (Leu), C (Cys), A (Ala), S (Ser), F (Phe), P (Pro), W (Trp), N (Asn), Q (Gln), G (Gly), Y (Tyr), D (Asp), E (Glu), R (Arg), H (His), K (Lys), etc., and substituted with an amino acid different from the original amino acid at the corresponding position in the wild type (for example, substituted with an amino acid selected from the group consisting of M (Met), V (Val), I (Ile), T (Thr), L (Leu), C (Cys), A (Ala), E (Glu), Q (Gln), K (Lys), R (Arg), H (His), N (Asn), etc., and substituted with an amino acid different from the amino acid at the corresponding position in the wild type), or

[0095] An amino acid sequence having 95% or greater, 98% or greater, or 99% or greater homology to the amino acid sequence.

[0096] For example, a polypeptide variant may comprise, consist essentially of, or consist of the following amino acid sequence:

[0097] An amino acid sequence comprising one or more amino acid mutations selected from the group consisting of F373M, F373V, F373I, F373T, F373L, F373C, F373N, F373H, A177C, A177L, A177I, P316A, P316L, V318L, V318M, R101A, F171A, V175C, V175L, G194E, G194Q, G194M, G194K, G194R, F337V, L340T and I353T in the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having 95% or more, 98% or more, or 99% or more homology to the amino acid sequence. More specifically, the polypeptide variant may comprise: an amino acid sequence comprising one or more amino acid mutations selected from the group consisting of SEQ ID NO: 4 F373M, F373V, F373I, F373T, F373L, F373C, F373N, F373H, A177C, A177L, A177I, P316A, P316L, V318L, V318M, R101A, F171A, V175C, V175L, G194E, G194Q, G194M, G194K, G194R, F337V, L340T, I353T, P316L+V318L, P316A+V318L, R101A+F373M, A177 one or more amino acid mutations selected from the group consisting of A177C+F373M, A177I+F373M, A177L+F373M, A177L+F373I, A177L+F373L, A177L+F373T, A177L+F373V, A177C+F373T, A177C+F373V, V175L+F373M, G194E+F373M, G194Q+F373M, G194M+F373M, G194K+F373M, G194R+F373M, or V318M+F373M, or

[0098] An amino acid sequence having 95% or greater, 98% or greater, or 99% or greater homology to the amino acid sequence.

[0099] Polypeptide variants comprising an amino acid sequence having sequence homology as described herein (e.g., 95% or greater, 98% or greater, or 99% or greater sequence homology) can maintain an enzyme activity equivalent to that of a polypeptide having an amino acid sequence as a standard for sequence homology identification (e.g., a PPO protein having the above-mentioned amino acid mutations). For example, the polypeptide variants can have 5% or greater, 10% or greater, 20% or greater, 30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, or 95% or greater enzyme activity compared to a polypeptide having the amino acid sequence as a standard in plants (whole plants, plant cells or cell cultures, plant tissues, etc.), in algae, and / or in vitro, and can also confer herbicide tolerance. The description of sequence homology is used to clarify that the herbicide-tolerant PPO protein variants or polypeptide variants described herein can contain all sequence mutations that meet the above conditions (maintaining enzyme activity and conferring herbicide tolerance).

[0100] The names of the amino acids used in the description are arranged as follows:

[0101]

[0102]

[0103] The herbicide-tolerant PPO protein variant can maintain the enzymatic activity of the PPO protein and exhibit enhanced herbicide tolerance compared to the wild type.

[0104] In addition, the herbicide-tolerant PPO protein variant may comprise further mutations that exhibit biologically equivalent activity to a polypeptide consisting of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or a polypeptide having the above amino acid mutations. For example, the additional mutation may be an amino acid substitution that does not generally alter the activity of the molecule, and such amino acid substitutions are well known in the art. In one example, the additional substitution may be a substitution of amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, or Asp / Gly, but is not limited thereto. In some cases, the herbicide-tolerant PPO protein variant may have modifications selected from the group consisting of phosphorylation, sulfation, acylation, glycosylation, methylation, farnesylation, and the like. Furthermore, herbicide-tolerant PPO protein variants may include protein variants in which the structural stability of the protein to heat, pH, etc. or the activity of the protein is increased by amino acid mutation and / or modification.

[0105] The term "sequence homology" refers to the degree of similarity to a wild-type or reference amino acid sequence or nucleotide sequence, and any protein is included within the scope of the present invention as long as it comprises an amino acid sequence that is 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, or 99% or greater identical to the amino acid sequence of a herbicide-tolerant PPO protein and retains biological activity equivalent to that of a herbicide-tolerant PPO protein variant. These protein homologs may contain active sites equivalent to those of the target protein. Such homology comparisons can be performed or with the aid of readily available comparison programs. Homology between two or more sequences can be calculated as a percentage (%) using online analysis programs. 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.

[0106] The herbicide-tolerant PPO protein or its variant can be obtained by extraction and purification from nature by methods well known in the art. In addition, it can be obtained as a recombinant protein using genetic recombination technology. When using genetic recombination technology, the recombinant protein can be obtained by the following method: a nucleic acid encoding the herbicide-tolerant PPO protein or its variant is introduced into an appropriate expression vector, and the host cell is transformed with the vector to express the target protein, and then the herbicide-tolerant PPO protein or its variant is collected from the host cell. After the protein is expressed in the selected host cell, general biochemical separation techniques can be used to separate and purify it, such as treatment with a protein precipitant (salting out), centrifugation, ultrasonication, ultrafiltration, dialysis, chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography), etc., and in order to isolate the protein with high purity, these methods can be used in combination.

[0107] Herbicide-tolerant PPO nucleic acid molecules (polynucleotides encoding a PPO protein or variants thereof) can be isolated or prepared using standard molecular biology techniques (eg, chemical synthesis or recombinant methods), or commercially available techniques can be used.

[0108] In one embodiment, in a herbicide tolerance test system using PPO-deficient Escherichia coli (E. coli) BT3 (ΔPPO), PPO proteins were found to exhibit broad herbicide tolerance to nine representative families of PPO-inhibiting herbicides classified according to their chemical structure. They were also found to be expressed in plant chloroplasts using transit peptides (TPs). Furthermore, PPO proteins were found to be expressed in Arabidopsis thaliana ecotype Columbia using plant expression vectors. Even when transformed plants were treated with PPO-inhibiting herbicides, plant germination and growth were observed. Furthermore, genetic studies confirmed the inheritance of the above-mentioned herbicide tolerance trait to the next generation.

[0109] Therefore, the PPO proteins and variants thereof provided herein can be introduced into plants or algae to enhance the herbicide tolerance of the plants or algae.

[0110] As used herein, herbicides refer to active ingredients that kill, control, or otherwise adversely alter the growth of plants or algae. In addition, herbicide tolerance or herbicide tolerance means that, compared to normal plants or wild-type plants, even after treatment with a herbicide that normally kills normal or wild-type plants or normally inhibits their growth, the inhibition of plant growth is reduced or eliminated, so that the plant continues to grow. Herbicides include herbicides that inhibit the protoporphyrinogen oxidase (PPO) enzyme of plants or algae. Based on their chemical structure, such PPO-inhibiting herbicides can be divided into pyrimidinediones, diphenyl ethers, phenylpyrazoles, N-phenylphthalimides, thiadiazoles, Oxadiazoles, triazolinones, Oxazolidinediones and other herbicides.

[0111] As a specific embodiment, the pyrimidinedione herbicide includes foramsulfuron, saflufenacil, bispyribac and tifenad, but is not limited thereto.

[0112] Diphenyl ether herbicides include fomesafen, oxyfluorfen, aclonifen-butyl, acifluorfen-butyl, cyfluthrin, chloranil, lactofen, methoxyfenoxam, chlorintrofen, fluazifop-butyl, and flunixin, but are not limited thereto.

[0113] Phenylpyrazole herbicides include, but are not limited to, fluazifop-butyl and isopyraclostrobin.

[0114] Phenylphthalimide herbicides include fluazifop-butyl, fenoxaline, and flufenoxaline, but are not limited thereto.

[0115] Phenyl ester herbicides include pyrimidine (2,4-dichlorophenyl 1-pyrrolidine carboxylate) and carbamate analogs of pyrimidine (e.g., O-phenyl pyrrolidino- and piperidinocarbamate 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., 1992, 40 (10) 1993-2000"), but are not limited thereto. In one embodiment, the carbamate analog of pyrrolidinecarboxylic acid can be one or more selected from the group consisting of phenyl pyrrolidine-1-carboxylate (CAS No. 55379-71-0), 2-chlorophenyl 1-pyrrolidinecarboxylate (CAS No. 143121-06-6), 4-chlorophenyl pyrrolidine-1-carboxylate (CAS No. 1759-02-0), diethyl 2,4-dichloro-5-(2-propynyloxy)phenyl carbamate (9CI) (CAS No. 143121-07-7), 2,4-dichloro-5-hydroxyphenyl 1-pyrrolidinecarboxylate (CAS No. 143121-08-8), 2,4-dichloro-5-(methoxycarbonyl)phenyl pyrrolidine-1-carboxylate (CAS No. 133636-94-9), 2,4-dichloro-5-[(prop-2-yloxy)carbonyl] Phenylpyrrolidine-1-carboxylate (CAS No. 133636-96-1), 1-piperidinecarboxylic acid, 2,4-dichloro-5-(2-propynyloxy)phenyl ester (CAS No. 87374-78-5), 2,4-dichloro-5-(prop-2-yn-1-yloxy)phenylpyrrolidine-1-carboxylate (CAS No. 87365-63-7), 2,4-dichloro-5-(prop-2-yn-1-yloxy) Phenyl 4,4-difluoropiperidine-1-carboxylate (CAS No. 138926-22-4), 1-pyrrolidinecarboxylic acid, 3,3-difluoro-, 2,4-dichloro-5-(2-propyn-1-yloxy)phenyl ester (CAS No. 143121-10-2), 4-chloro-2-fluoro-5-[(prop-2-yloxy)carbonyl]phenylpyrrolidine-1-carboxylate (CAS No. 133636-98-3), etc.

[0116] Thiadiazole herbicides include, but are not limited to, metsulfuron and thiamethoxam.

[0117] oxadiazole herbicides including propyne oxadiazol and Oxychloride, but not limited to it.

[0118] Triazolinone herbicides include, but are not limited to, carfentrazone, sulfentrazone and pyraclostrobin.

[0119] Oxazolidinedione herbicides include cyclopentane Herbicide, but not limited to.

[0120] Other herbicides include, but are not limited to, bispyribac, fluazifop-butyl, and flupyraclostrobin.

[0121] The herbicide-tolerant PPO gene provided herein can be introduced into plants or algae by various methods known in the art, preferably, by using an expression vector for plant or algae transformation.

[0122] In the case of plant transformation, the 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 an SP6 promoter, a T7 promoter, a T3 promoter, a PM promoter, a maize ubiquitin promoter, a cauliflower mosaic virus (CaMV) 35S promoter, a nopaline synthase (nos) promoter, a figwort mosaic virus 35S promoter, a sugarcane bacilliform virus promoter, a Commelina yellow mottle virus promoter, a light-inducible promoter from the small subunit of ribulose-1,5-bisphosphate carboxylase (ssRuBisCO), a rice cytosolic triosephosphate isomerase (TPI) promoter, an adenine phosphoribosyltransferase (APRT) promoter from Arabidopsis, an octopine synthase promoter, and a BCB (blue copper binding protein) promoter, but is not limited thereto.

[0123] Further, the vector may include a poly A signal sequence that causes 3'-terminal polyadenylation, for example, it may include the NOS 3'-end of the nopaline synthase gene from Agrobacterium tumefaciens, the octopine synthase terminator of octopine synthase from Agrobacterium tumefaciens, the 3'-end of the proteinase inhibitor I or II gene from tomato or potato, the CaMV 35S terminator, the rice α-amylase terminator RAmy1 A and the phaseolin terminator, but is not limited thereto.

[0124] In addition, chloroplast-specific promoters, nuclear promoters, constitutive promoters, or inducible promoters can be used as promoters for gene introduction into algae. The herbicide-tolerant PPO genes or variants thereof provided herein can be designed to be operably linked to the 5'UTR or 3'UTR, thereby expressing function in the nucleus of algae. In addition, the vector can further contain transcriptional regulatory sequences suitable for algal transformation. The recombinant gene conferring herbicide tolerance can be integrated into the chloroplast genome or the nuclear genome of the host algae, but is not limited thereto.

[0125] Additionally, a transit peptide required for chloroplast targeting can be linked to the 5'-end of the PPO gene in the vector in order to express the herbicide-tolerant PPO gene in the chloroplast.

[0126] In addition, optionally, the vector may further include a gene encoding a selection marker as a reporter molecule, examples of which may include antibiotic (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, bleomycin, chloramphenicol, etc.) or herbicide (glyphosate, glufosinate, glufosinate, etc.) tolerance genes, but are not limited thereto.

[0127] Furthermore, recombinant vectors for plant expression may include Agrobacterium binary vectors, co-integration vectors, or general vectors that do not have a T-DNA region but are designed for expression in plants. A binary vector refers to a vector containing two independent vector systems, each of which contains a plasmid responsible for migration, the plasmid consisting of the left border (LB) and right border (RB) of a Ti (tumor-inducing) plasmid, and another plasmid for target gene transfer. The vector may include a promoter region and a polyadenylation signal sequence for expression in plants.

[0128] When using binary vectors or co-integration vectors, the bacterial strain for transforming the recombinant vector into the plant is preferably Agrobacterium (Agrobacterium-mediated transformation). In this respect, Agrobacterium tumefaciens or Agrobacterium rhizogenes can be used. In addition, when using a vector without a T-DNA region, electroporation, particle bombardment, polyethylene glycol-mediated uptake, etc. can be used to import the recombinant plasmid into the plant.

[0129] Plants transformed with genes by the methods described above can be redifferentiated into plants through callus induction, rhizome growth, and soil adaptation using standard techniques known in the art.

[0130] Plants that have undergone transformation are understood herein to include plant cells (including cells in suspension culture), protoplasts, callus tissue, hypocotyls, seeds, cotyledons, shoots, as well as mature plants.

[0131] In addition, the scope of transformants includes transformants and clones or offspring (T1 generation, T2 generation, T3 generation, T4 generation, T5 generation or any offspring) of the introduced gene. For example, the transformed plant also includes plants with hereditary herbicide tolerance traits as sexual and asexual offspring of plants transformed with the gene provided herein. The scope of the present invention also includes all mutants and variants showing the characteristics of the plant initially transformed, and all hybridization and fusion products of plants transformed with the gene provided herein. In addition, the scope of the present invention also includes a part of a plant, such as a seed, flower, stem, fruit, leaf, root, tuber and / or root tuber, which is derived from a transformed plant or its offspring transformed in advance by the method of the present invention, and is composed of at least a portion of transformed cells.

[0132] The plant to which the present invention is applied is not particularly limited, but includes monocots or dicots. In addition, the plant includes herbaceous plants or woody plants. Monocots may include plants belonging to the following families: Alismataceae, Hydrocharitaceae, Juncaginaceae, Scheuchzeriaceae, Potamogetonaceae, Najadaceae, Zosteraceae, Liliaceae, Haemodoraceae, Agavaceae, Amaryllidaceae, Dioscoreaceae, Pontederiaceae ), Iridaceae, Burmanniaceae, Juncaceae, Commelinaceae, Eriocaulaceae, Gramineae / Poaceae, Araceae, Lemnaceae, Sparganiaceae, Typhaceae, Cyperaceae, Musaceae, Zingiberaceae, Cannaceae, and Orchidaceae, but are not limited thereto.

[0133] The dicotyledonous plants may include plants belonging to the following families: Diapensiaceae, Clethraceae, Pyrolaceae, Ericaceae, Myrsinaceae, Primulaceae, Plumbaginaceae, Ebenaceae, Styracaceae, Symplococeae, Oleaceae, Loganiaceae, Gentianaceae, Menthaceae, Menyanthaceae), Apocynaceae, Asclepiadaceae, Rubiaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Labiatae, Solanaceae, Scrophulariaceae, Bignoniaceae, Acanthaceae, Pedaliaceae, Orobanchaceae, Gesneriaceae, Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, Adoxaceae, Valerianaceae, Dipsacaceae, Campanulaceae, Compositae, Myricaceae, Juglandaceae, Salicaceae aceae), Betulaceae, Fagaceae, Ulmaceae, Moraceae, Urticaceae, Santalaceae, Loranthaceae, Polygonaceae, Phytolaccaceae, Nyctaginaceae, Aizoaceae, Portulacaceae, Caryophyllaceae, Chenopodiaceae,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, Crassulaceae Eucommia ulceae, Saxifragaceae, Eucommiaceae, Pittosporaceae, Rosaceae, Leguminosae, Oxalidaceae, Geraniaceae, Tropaeolaceae, Zygophyllaceae, Linaceae, Euphorbiaceae, Callitrichaceae, Rutaceae, and Simmonaceae. Simaroubaceae, Meliaceae, Polygalaceae, Anacardiaceae, Aceraceae, Sapindaceae, Hippocastanaceae, Sabiaceae, Balsaminaceae, Aquifoliaceae, Celastraceae, Staphyleaceae, Buxaceae, Empetraceae,Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvaceae, Sterculiaceae, Thymelaeaceae, Elaeagnaceae, Flacourtiaceae, Violaceae, Passifloraceae, Tamaricaceae, Elatinaceae, Begoniaceae, Cucurbitaceae, Lythraceae, Punicaceae, Onaraceae, Haloragaceae, Alangiaceae, Cornaceae, Araliaceae, Umbelliferae / Apiaceae, but are not limited thereto.

[0134] In an embodiment, the plant can be one or more selected from the group consisting of: a food crop such as rice, wheat, barley, corn, soybean, potato, red bean, oat, and sorghum; a vegetable crop such as Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, cantaloupe, pumpkin, welshan ion, anion, and carrot; a special purpose crop such as ginseng, tobacco, cotton, soilage, forage, sesame, sugarcane, sugar beet, Perilla sp., peanut, rape, grass, and castor oil plant; a fruit tree such as an apple tree, a pear tree, a jujube tree, a peach tree, a kiwifruit tree, a grape tree, a citrus tree, a persimmon tree, a plum tree, an apricot tree, and a banana tree; a woody plant such as a pine tree, a palm oil, and an eucalyptus; a flowering crop such as a rose, a gladiolus, a gerbera, a snapdragon, a chrysanthemum, a lily, and a tulip; a forage crop such as ryegrass, red clover, fruit tree grass, alfalfa, tall fescue, and perennial ryegrass, but are not limited thereto. In an embodiment, the plant can be one or more selected from the group consisting of: a dicotyledonous plant such as Arabidopsis thaliana, potato, eggplant, tobacco, red pepper, tomato, burdock, endive, lettuce, gladiolus, spinach, sugar beet, sweet potato, celery, carrot, water dropwort, parsley, Chinese cabbage, cabbage, radish, watermelon, cantaloupe, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, and pea; and a monocotyledonous plant such as rice, wheat, barley, corn, sorghum, etc., but are not limited thereto.

[0135] The algae to which the present invention is applied are not particularly limited, but include prokaryotic algae or eukaryotic algae. For example, the algae may be cyanobacteria, green algae, red algae, brown algae, macroalgae or microalgae.

[0136] Cyanobacteria include: Chroococcales phylum (e.g., Aphanocapsa, Aphanothece, Chamaesiphon, Chondrocystis, Chroococcus, Chroogloeocystis, Crocosphaera, Cyanobacterium, Cyanobium, Cyanodictyon, Cyanosarcina, Cyanothece, Dactylococcopsis, and phylums such as Gloeocapsa, Gloeothece, Halothece, Johannesbaptistia, Merismopedia, Microcystis, Radiocystis, Rhabdoderma, Snowella, Synechococcus, Synechocystis, Thermosynechococcus, Woronichinia), Gloeobacteria phylum, Nostocales phylum (e.g., Microchaetaceae, Nostocaceae, Rivulariaceae, Scytonemataceae), Oscillatoriales phylum (e.g.,Arthronema, Arthrospira, Blennothrix, Crinalium, Geitlerinema, Halomicronema, Halospirulina, Hydrocoleum, Jaaginema, Katagnymene, Komvophoron, Leptolyngbya, Limnothrix, Lyngbya, Microcole us), Oscillatoria, Phormidium, Planktothricoides, Planktothrix, Plectonema, Pseudanabaena, Pseudophormidium, Schizothrix, Spirulina, Starria, Symploca, Trichodesmium, Tychonema), Pleurocapsales phylum) (e.g., Chroococcidiopsis, Dermocarpa, Dermocarpella, Myxosarcina, Pleurocapsa, Solentia, Stanieria, Xenococcus), Prochlorales phylum, or Stigonematales phylum) (e.g., Capsosira, Chlorogloeopsis, Fischerella, Hapalosiphon, Mastigocladopsis, Mastigocladus, Nostochopsis, Stigonema, Symphyonema, Symphonemopsis, Umezakia, Westiellopsis), etc.

[0137] As another example of algae, Chlorophyta, Chlamydomonas, Volvacales, Dunaliella, Scenedesmus, Chlorella, or Hematococcus can be exemplified.

[0138] Other examples of algae include Phaeodactylum tricornutum, Amphiprora hyaline, Amphora spp., Chaetoceros muelleri, Navicula saprophila, Nitzschia communis, Scenedesmus dimorphus, Scenedesmus obliquus, Tetraselmis suecica, Chlamydomonas reinhardtii, Chlorella vulgaris, Haematococcus pluvialis, Neochloris oleoabundans, Synechococcus elongatus, Botryococcus braunii, and Gloeobacter spp. 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., Aphanocapsae spp., Synechosystis spp.), Nannochloris spp., etc. However, it is not limited to the species listed above, and algae belonging to other various genera and families may be included.

[0139] Plants or algae into which the herbicide-tolerant PPOs or variants thereof provided herein are introduced can exhibit tolerance to two or more PPO-inhibiting herbicides.

[0140] Thus, the technology provided herein can be used to control weeds or eliminate undesirable aquatic organisms by using two or more PPO-inhibiting herbicides sequentially or simultaneously.

[0141] One embodiment provides a method for controlling weeds in farmland, comprising the steps of providing a plant comprising the above-mentioned herbicide-tolerant PPO protein, its variant or its encoding gene to the farmland, and applying an effective dose of a protoporphyrinogen oxidase-inhibiting herbicide to the farmland.

[0142] Another embodiment provides a method for removing unwanted aquatic organisms from a culture medium, comprising the steps of providing algae containing the above-mentioned herbicide-tolerant PPO protein, its variant or its encoding gene to the culture medium, and applying an effective dose of a protoporphyrinogen oxidase-inhibiting herbicide to the culture medium.

[0143] Furthermore, the herbicide-tolerant PPO proteins, variants thereof, or genes encoding thereof provided herein can be used in combination with a second herbicide-tolerant polypeptide or a gene encoding thereof.

[0144] Thus, plants or algae introduced with the herbicide-tolerant PPOs provided herein can exhibit tolerance to two or more herbicides that differ in their mechanisms of action. In the present invention, two or more different herbicides (including PPO-inhibiting herbicides) that differ in their mechanisms of action can be used sequentially or simultaneously to control weeds and / or remove unwanted aquatic organisms. Hereinafter, a herbicide that differs in its mechanism of action from a PPO-inhibiting herbicide is referred to as a "second herbicide."

[0145] One embodiment provides a composition for conferring or enhancing herbicide tolerance to plants or algae, comprising the above-mentioned herbicide-tolerant PPO protein, a variant thereof, or a gene encoding the same; and a second herbicide-tolerant polypeptide or a gene encoding the same.

[0146] Another embodiment provides a herbicide-tolerant transformant of a plant or algae, a clone thereof, or a progeny thereof, wherein the transformant, the clone thereof, or a progeny thereof comprises the above-mentioned herbicide-tolerant PPO protein, a variant thereof, or a gene encoding the same; and a second herbicide-tolerant polypeptide or a gene encoding the same.

[0147] Other embodiments provide a method for preparing a plant or algae with herbicide tolerance, the method comprising the steps of transforming algae, or plant cells, protoplasts, callus, hypocotyls, seeds, cotyledons, shoots or whole plants with: the above-mentioned herbicide-tolerant PPO protein, its variant or its encoding gene; and a second herbicide-tolerant polypeptide or its encoding gene.

[0148] Other embodiments provide a method for controlling weeds in farmland, comprising: providing a plant to the farmland, wherein the plant comprises the above-mentioned herbicide-tolerant PPO protein, its variant or its encoding gene; a second herbicide-tolerant polypeptide or its encoding gene; and applying an effective dose of a protoporphyrinogen oxidase-inhibiting herbicide to the farmland.

[0149] Other embodiments provide methods for removing unwanted aquatic organisms from a culture medium, comprising: providing algae to the culture medium, wherein the algae comprise an herbicide-tolerant PPO protein, a variant thereof, or a gene encoding it; a second herbicide-tolerant polypeptide or a gene encoding it; and applying an effective amount of a protoporphyrinogen oxidase-inhibiting herbicide to the culture medium.

[0150] For example, the plant or algae further comprises a second herbicide tolerance polypeptide or a gene encoding it, thereby having new and / or enhanced tolerance to the second herbicide.

[0151] For example, the second herbicide may include a cell division inhibitory herbicide, a photosynthesis inhibitory herbicide, an amino acid synthesis inhibitory herbicide, a plastid inhibitory herbicide, a cell membrane inhibitory herbicide, and / or any combination thereof, but is not limited thereto. The second herbicide may be exemplified by glyphosate, glufosinate, dicamba, 2,4-D (2,4-dichlorophenoxyacetic acid), an ALS (acetolactate synthase) inhibitory herbicide (e.g., imidazolinone, sulfonylurea, triazolopyrimidine, sulfonanilide, pyrimidinethiobenzoic acid, etc.), a photosystem II inhibitory herbicide, a phenylurea-based herbicide, a plastid inhibitory herbicide, a bromoxynil-based herbicide, and / or any combination thereof, but is not limited thereto.

[0152] For example, the second herbicide-tolerant polypeptide can be exemplified as one or more selected from the group consisting of: glyphosate herbicide-tolerant EPSPS (glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase), GOX (glyphosate oxidase), GAT (glyphosate-N-acetyltransferase) or glyphosate decarboxylase; glufosinate herbicide-tolerant PAT (phosphinothricin-N-acetyltransferase); dicamba herbicide-tolerant DMO (dicamba monooxygenase); 2,4-D herbicide-tolerant 2,4-D monooxygenase; oxygenase or AAD (aryloxyalkanoate dioxygenase); ALS-inhibiting sulfonylurea herbicide-tolerant ALS (acetolactate synthase), AHAS (acetohydroxyacid synthase) or AtAHASL (acetohydroxyacid synthase large subunit); photosystem II-inhibiting herbicide-tolerant photosystem II protein D1; phenylurea-based herbicide-tolerant cytochrome P450; plastid-inhibiting herbicide-tolerant HPPD (hydroxyphenylpyruvate dioxygenase); bromoxynil herbicide-tolerant nitrilase; and combinations thereof, but not limited thereto.

[0153] Further, the encoding gene of the second herbicide tolerance polypeptide can be exemplified by one or more selected from the group consisting of: glyphosate herbicide tolerance cp4 epsps, epsps (AG), mepsps, 2mepsps, goxv247, gat4601 or gat4621 gene; glufosinate herbicide tolerance bar, pat or pat (SYN) gene; dicamba herbicide tolerance dmo gene; 2,4-D herbicide tolerance AAD-1 or AAD-12 gene; ALS-inhibiting sulfonylurea herbicide tolerance ALS, GM-HRA, S4-HRA, ZM-HRA, Csr1, Csr1-1, Csr1-2, SurA or SurB; photosystem II inhibitory herbicide tolerance psbA gene; phenylurea herbicide tolerance CYP76B1 gene; isoenzyme ... isoenzyme ALS, GM-HRA, S4-HRA, ZM-HRA, Csr1, Csr1-1, Csr1-2, SurA or SurB; isoenzyme ALS, GM-HRA, S4-HRA, ZM-HRA, Csr1, Csr1-1, Csr1-2, SurA or SurB; isoenzyme ALS, GM-HRA, S4-HRA, ZM-HRA, Csr1, Csr1-1, Csr1- The herbicide-tolerant HPPDPF W336 gene of foramsulfuron; the herbicide-tolerant bxn gene of bromoxynil; and combinations thereof, but are not limited thereto.

[0154] Beneficial effects

[0155] The herbicide-tolerant PPO protein variants provided herein or genes encoding them are applied to plants or algae to impart and / or enhance superior herbicide tolerance traits, and selectively control them using herbicides, thereby economically controlling weeds or removing aquatic organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0156] Figure 1 This is a map of the pACBB vector.

[0157] Figure 2Shown are the cell growth levels of PPO-deficient BT3 Escherichia coli transformed with the pACBB-eGFP vector control (V), PPO-susceptible Arabidopsis, PPO1 gene (AtPPO1 WT), PPO-tolerant Arabidopsis PPO1 mutant gene (AtPPO1 SLYM), CyPPO10 gene (Cy10 WT) and CyPPO13 gene (Cy13 WT), respectively, after treatment with 0 μM (micromolar), 100 μM or 400 μM tifenadine.

[0158] Figure 3 This is a map of the pET303-CT-His vector. Annotation of pET303 CT-His: 5396 nucleotides; T7 promoter: bases 20-36; T7 promoter start site: bases 20-39; Lac operator (lacO): bases 39-63; ribosome binding site (RBS): bases 95-100; 6X His tag: bases 119-136; T7 reverse start site: bases 186-206; T7 transcription termination region: bases 147-277; F1 replication origin: bases 287-742; bla promoter: bases 775-879; ampicillin (bla) resistance gene: bases 874-1734; pBR322 replication origin: bases 1945-2678 (c); ROP ORF: bases 2920-3011 (c); lac1 ORF: bases 3914-5032 (c:).

[0159] Figure 4 A schematic diagram of a recombinant vector of a fusion protein is shown, in which MBP (maltose binding protein) and PPO protein are fused.

[0160] Figure 5 This is a map of the pMAL-c2X vector.

[0161] Figure 6 Schematic diagram showing the structure of a binary vector for plant transformation using the CyPPO gene.

[0162] Figure 7 are Western blotting results showing the expression levels of CyPPO variant proteins in T2 Arabidopsis transformed with the CyPPO10 variant (F360I variant or F360M variant) or CyPPO13 variant (F373M variant) gene.

[0163] Figure 8 Shown are the levels of damage in Arabidopsis transformants (T3) transformed with CyPPO10 or CyPPO13 wild-type genes when treated with 1 μM of tifenadine. Col-O indicates non-transgenic Arabidopsis.

[0164] Figure 9 Shown are the levels of damage in Arabidopsis transformants (T2) transformed with genes encoding CyPPO10 variants (F360C, F360I, F360L, F360M, F360V, F360T, A167C, A167L, A167L+F360M, or A167C+F360I) when treated with tifenadine at concentrations of 1 μM, 5 μM, or 25 μM.

[0165] Figure 10 Shown are the levels of damage in Arabidopsis transformants (T2) transformed with genes encoding CyPPO13 variants (A177C, F373C, F373I, F373M, A177L+F373L, or A177L+F373I) when treated with tifenadine at a concentration of 1 μM or 10 μM.

[0166] Figure 11 Shown are the cell growth levels of PPO-deficient BT3 E. coli (ΔPPO) transformants transformed with the CyPPO10 wild-type gene (denoted as CY10 WT) or various CyPPO10 mutant genes when treated with tifenadine at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.

[0167] Figure 12 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with CY10 WT or various CyPPO10 mutant genes when treated with saflufenacil at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.

[0168] Figure 13 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with CY10 WT or various CyPPO10 mutant genes when treated with fomesafen at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.

[0169] Figure 14 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with CY10 WT or various CyPPO10 mutant genes when treated with acifluorfen at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.

[0170] Figure 15Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with CY10 WT or various CyPPO10 mutant genes when treated with fluazifop-propyl at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM, respectively.

[0171] Figure 16 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with CY10 WT or various CyPPO10 mutant genes when treated with sulfentrazone at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.

[0172] Figure 17 The results show that the concentrations of cyclopentazoline are 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM. Cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with CY10 WT or various CyPPO10 mutant genes upon chlorpyrifos treatment.

[0173] Figure 18 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with CY10 WT or various CyPPO10 mutant genes when treated with fluazifop-butyl at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.

[0174] Figure 19 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with CY10 WT or various CyPPO10 mutant genes when treated with bispyribac at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.

[0175] Figure 20 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with the CyPPO13 wild-type gene (denoted as Cy13 WT) or various CyPPO13 mutant genes when treated with tifenad at concentrations of 0 μM, 5 μM, 25 μM, and 50 μM, respectively.

[0176] Figure 21 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with Cy13WT or various CyPPO13 mutant genes when treated with saflufenacil at concentrations of 0 μM, 5 μM, 25 μM, and 50 μM, respectively.

[0177] Figure 22Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with Cy13 WT or various CyPPO13 mutant genes when treated with fomesafen at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.

[0178] Figure 23 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with Cy13 WT or various CyPPO13 mutant genes when treated with acifluorfen at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.

[0179] Figure 24 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with Cy13WT or various CyPPO13 mutant genes when treated with fluazifop-propyl at concentrations of 0 μM, 5 μM, 25 μM, and 50 μM, respectively.

[0180] Figure 25 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with Cy13 WT or various CyPPO13 mutant genes when treated with sulfentrazone at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.

[0181] Figure 26 The results show that the concentrations of cyclopentazoline are 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM. Cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with Cy13 WT or various CyPPO13 mutant genes upon oxadiazine treatment.

[0182] Figure 27 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with Cy13 WT or various CyPPO13 mutant genes when treated with fluazifop-butyl at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM, respectively.

[0183] Figure 28 Shown are the cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with Cy13 WT or various CyPPO13 mutant genes when treated with bispyribac at concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively.

[0184] Figure 29The results are shown when the concentrations of 0 μM, 5 μM, 25 μM, 50 μM, 100 μM and 200 μM are respectively Cell growth levels of PPO-deficient BT3 (ΔPPO) transformants transformed with Cy13 WT or various CyPPO13 mutant genes upon oxadiazine treatment.

[0185] Figure 30 This is a map of the pET29b vector.

[0186] Figures 31a to 31c The results of seed germination of Arabidopsis thaliana transformed with CyPPO10 or CyPPO13 wild-type genes or their mutant genes 7 days after sowing on 1 / 2 MS medium containing various herbicides are shown. Col-0 indicates non-transgenic Arabidopsis thaliana.

[0187] Figure 32 Shown are the levels of damage in Arabidopsis transformants (T3) transformed with genes encoding CyPPO10 variants (F360I, F360L, F360M, A167C+F360I, A167C+F360M, or V305M+F360M) when treated with either 25 μM tifenad or 100 μM saflufenacil.

[0188] Figure 33a Shown are the levels of injury in Arabidopsis thaliana transformants (T3) transformed with genes encoding CyPPO10 variants (F360I or A167L+F360M) when treated with 50 μM of tifenad, saflufenacil, fluazifop or sulfentrazone, respectively.

[0189] Figure 33b Shown are the levels of damage in Arabidopsis transformants (T3) transformed with genes encoding CyPPO13 variants (A177L+F373L or A177L+F373I) when treated with saflufenacil, tifenad, fluazifop-propyl, sulfentrazone, oxyfluorfen, or bispyribac at a concentration of 50 μM, respectively.

[0190] Figure 34 Shown are the levels of damage in Arabidopsis transformants (T4) transformed with CyPPO10 F360I when treated with 15 μM tifenad and 150 μM saflufenacil.

[0191] Figure 35 Shown are the levels of damage in Arabidopsis transformants (T5) transformed with CyPPO10 F360I when treated with 15 μM tifenad or 150 μM saflufenacil. Col-0 indicates non-transgenic Arabidopsis.

[0192] Figure 36are the results of Western blotting, showing the expression of CyPPO10 F360I protein in Arabidopsis transformants (T4 or T5) transformed with CyPPO10F360I.

[0193] Figure 37 This is a map of the pB2GW7.0 binary vector.

[0194] Figure 38 Shown are the levels of lesions in leaves of TO soybean transformed with the CyPPO10A167L+F360M mutant gene when treated with 5 μM or 15 μM of tifenadine. Kwangan soybean refers to non-transgenic soybean (cultivar).

[0195] Figure 39 Southern blot results are presented showing the presence of the transgene in soybean transformed with CyPPO10 A167L+F360M.

[0196] Figure 40 Shown are herbicide tolerance of T1 transgenic soybean (CyPPO10 A167L+F360M) 5 days after spray treatment with 25 μM tifenad or 150 μM saflufenacil. Guangan soybean refers to non-transgenic soybean (cultivar).

[0197] Figure 41 Shown are the cell growth levels of BT3(ΔPPO) Escherichia coli transformed with the mutant gene of CyPPO10 when cultured in a medium containing herbicide. DETAILED DESCRIPTION

[0198] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are for illustrative purposes only, and the present invention is not limited to the following examples.

[0199] Example 1. Isolation of PPO gene from prokaryotes

[0200] Using the Genbank database of PPO genes from Synechococcus elongatus BP-1 and Synechococcus sp. JA-3-3Ab, a PPO gene was synthesized using codon-optimized information for effective herbicide resistance screening in BT3 Escherichia coli. The synthesized PPO gene was amplified using the primers in Table 1 under the following conditions and cloned into the pACBB vector.

[0201] Fifty microliters (50 μl) of PCR reaction mixture was prepared by mixing 1 μl of template (synthetic DNA of each gene), 5 μl of 10X buffer, 1 μl of dNTP mix (10 mM each), 1 μl of forward primer (see Table 1; 10 μM), 1 μl of reverse primer (see Table 1; 10 μM), 40 μl of DDW and 1 μl of Pfu-X (Solgent, 2.5 units / μl), and amplification was performed under the following conditions: 94°C for 4 minutes, 1 cycle; 94°C for 30 seconds, 56°C for 30 seconds and 72°C for 1.5 minutes, 25 cycles; 72°C for 5 minutes, 1 cycle.

[0202] The PPO isolated from Synechococcus elongatus BP-1 was named CyPPO10, and the PPO isolated from Synechococcus sp. JA-3-3Ab strain was named CyPPO13, respectively.

[0203] Table 1

[0204]

[0205] Example 2. Herbicide tolerance of CyPPO10 and CyPPO13

[0206] The herbicide tolerance of CyPPO10 and CyPPO13 was tested using PPO-deficient Escherichia coli.

[0207] After transforming PPO-deficient BT3 E. coli (ΔPPO) with CyPPO10 or CyPPO13, the transformed BT3 (ΔPPO) was cultured on LB agar plates containing a PPO-inhibiting herbicide to detect the growth level of the transformed BT3 (ΔPPO). The BT3 (ΔPPO) strain was obtained from Hokkaido University (Japan). The BT3(ΔPPO) strain lacks the hemG-type PPO and is kanamycin-resistant (see Watanabe et al., Dual targeting of spinach protoporphyrinogen oxidase II to mitochondria and chloroplasts by alternative use of two in-frame inhibition codons, JBC 2001 276(23):20474–20481; Che et al., Molecular Characterization and Subcellular Localization of Protoporphyrinogen Oxidase in Spinach Chloroplasts, Plant Physiol. 2000 Sep; 124(1):59-70).

[0208] The specific testing process is as follows:

[0209] The CyPPO10 and CyPPO13 genes were cloned into the pACBB vector (plasmid #32551; Addgene; see Figure 1 )middle.

[0210] Specifically, the PCR product amplified in Example 1 was treated with BamHI and XhoI restriction enzymes (New England Biolabs) and ligated with the pACBB-eGFP vector treated with the same restriction enzymes.

[0211] Restriction enzyme treatment was performed under the following conditions:

[0212] 30 μl (microliter) PCR product, 0.5 μl each of BamHI and XhoI (New England Biolabs), 4 μl 10X buffer and 5.5 μl water; restriction enzyme reaction was carried out at 37°C for 1 hour

[0213] The ligation reaction was performed under the following conditions:

[0214] 0.5 μl T4 DNA ligase (RBC), 1 μl A buffer, 1 μl B buffer, PCR product treated with restriction enzyme and vector, a total of 10 μl; incubate at 22°C for 30 minutes.

[0215] The cloned plasmids were added to 100 μl of BT3 competent cells (Hokkaido University; Japan) and transformed by the heat shock method. Escherichia coli transformed with each PPO gene was cultured in LB (Luria-Bertani) agar medium containing chloramphenicol (Duchefa).

[0216] For seed culture of E. coli transformed with each gene, each single E. coli transformant colony provided above was cultured overnight in 3 ml LB broth containing chloramphenicol (220 rpm, 37°C), and 50 to 100 μl was subcultured in new 3 ml LB broth until the absorbance (OD 600 ) is 0.5 to 1, and diluted with LB broth to an absorbance (OD 600 ) was 0.5. The diluted solution was further diluted five times in LB broth at a factor of 1 / 10. Subsequently, 10 μl of each diluted solution was added dropwise to LB agar (Petri dish) containing 0 μM, 100 μM, and 400 μM tifenadine. The LB agar was incubated at 37°C under light conditions. Growth inhibition levels were observed after 16 to 20 hours of incubation.

[0217] For comparison, the pACBB-eGFP vector (plasmid #32551; Addgene; see Figure 1) (V; pACBB-eGFP vector); BT3 E. coli transformants transformed with the wild-type Arabidopsis thaliana PPO1 gene (AtPPO1 WT, wild-type AtPPO1; PPO susceptible) (SEQ ID NO: 6); and BT3 E. coli transformants transformed with the Arabidopsis thaliana mutant PPO1 gene encoding a mutant AtPPO1 (AtPPO1SLYM, SEQ ID NO: 7) having amino acid substitutions of Y426M (426th amino acid residue tyrosine substituted with methionine) and S305L (305th amino acid residue serine substituted with leucine) based on the amino acid sequence of wild-type AtPPO1 (SEQ ID NO: 5) were subjected to the same test (Li et al. Development of protoporphyrinogen oxidase as an efficient selection marker for agrobacterium tumefaciens-mediated transformation of maize). transformation of maize).Plantphysiol.2003 133:736-747).

[0218] Figure 2 The results obtained are shown in Figure 2 As shown, on a medium without herbicide (0 μM of tifenadine), the growth of the BT3 transformant (V) transformed with pACBB-eGFP without the PPO gene introduced was not restored, while the growth of the BT3 transformant transformed with the PPO-susceptible Arabidopsis thaliana PPO1 wild-type gene (AtPPO1 WT), the PPO-tolerant Arabidopsis thaliana PPO1 mutant gene (AtPPO1 SLYM), the CyPPO10 gene (Cy10 WT), or the CyPPO13 gene (Cy13 WT) was restored, because each of the introduced genes functions as a PPO enzyme in BT3. These results indicate that both CyPPO10 and CyPPO13 exert normal PPO functions.

[0219] The BT3 transformant transformed with the wild-type Arabidopsis PPO1 gene susceptible to tifenad (AtPPO1WT) grew normally in a medium without herbicide, but did not grow in a medium containing 100 μM tifenad. The BT3 transformant transformed with the mutant Arabidopsis PPO1 gene tolerant to tifenad (AtPPO1 SLYM) gradually began to show growth inhibition starting from 100 μM tifenad and hardly grew at 400 μM. The BT3 transformant transformed with the CyPPO10 or CyPPO13 gene grew in a medium containing 100 μM tifenad at a level similar to that in a medium without tifenad, and grew well even in a medium containing 400 μM tifenad. Based on these results, it was demonstrated that the CyPPO10 and CyPPO13 genes can express significantly higher tolerance to tifenadine than the tifenadine-susceptible Arabidopsis PPO1 wild type, and show similar or higher tifenadine tolerance than the tifenadine-tolerant Arabidopsis PPO1 mutant.

[0220] Example 3. Determination of PPO-Inhibiting Herbicide Interactions from PPO and PPO-Inhibiting Herbicide Complexes PPO amino acid residues

[0221] To investigate the structural information of PPO protein binding to herbicides, tifenad, saflufenacil, fluazifop-propyl or sulfentrazone were used as representative examples of PPO-inhibiting herbicides. The gene encoding CyPPO10 protein was cloned into the pET29b vector (Cat. No. 69872-3; EMD Biosciences; see Figure 30 ) and expressed as CyPPO10 protein using the E. coli system. The expressed CyPPO10 protein was purified by nickel affinity chromatography and crystallized with PPO-inhibiting herbicides. Then, using a synchrotron radiation accelerator, complexes of CyPPO10 with tifenad, saflufenacil, fluazifop-propyl or sulfentrazone were obtained. High-resolution X-ray diffraction data were used to determine the three-dimensional structure of the complex. This process also revealed the locations of amino acid mutations in the CyPPO10 protein that confer herbicide tolerance.

[0222] By analyzing the structure of the complex between CyPPO10 and tifenadine, the following conclusions were drawn: amino acids N59, S60, R89, F161, V165, A167, Q184, P303, V305, F324, L327, I340, F360 and I408 of the CyPPO10 protein (SEQ ID NO: 2) interact with tifenadine.

[0223] Using the binding information derived from the CyPPO10-tifenadine complex structure, the amino acid residues in the CyPPO13 (SEQ ID NO: 4) protein that interact with tifenadine were identified by sequence homology analysis between the amino acids of CyPPO10 (SEQ ID NO: 2) and CyPPO13 (NCBI BLAST, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome).

[0224] As a result, it was understood that amino acids at positions R101, F171, V175, A177, G194, P316, V318, F337, L340, I353, and F373 of the CyPPO13 protein (SEQ ID NO: 4) interact with tifenadine.

[0225] Example 4. Preparation of PPO variants

[0226] To enhance tolerance to PPO-inhibiting herbicides of CyPPO10 and CyPPO13, both genes were mutated at the amino acid positions that interact with the herbicides identified in Example 3, thereby generating mutant genes that increase tolerance to PPO-inhibiting herbicides.

[0227] Using the primers in Table 3, the mutated PPO gene was isolated and amplified by PCR under the following conditions:

[0228] Material

[0229] Template (CyPPO10 or CyPPO13 synthetic DNA) 1 μl

[0230] 10X buffer 5μl

[0231] dNTP mixture (10 mM each) 1 μl

[0232] Forward primer (10 μM) 1 μl

[0233] Reverse primer (10 μM) 1 μl

[0234] DDW 40μl

[0235] Pfu-X (Solgent, 2.5 units / μl) 1μl

[0236] Total 50 μl

[0237] Table 2

[0238] PCR conditions

[0239]

[0240] Table 3

[0241]

[0242] The amplified gene product and the pET303-CT His vector (VT0163; Novagen; see Figure 3 ), and then T4 DNA ligase (RBC, 3 units / μl) was used to prepare pET303-CyPPO10 and pET303-CyPPO13 plasmids, respectively.

[0243] Mutant genes of CyPPO10 and CyPPO13 were prepared by performing PCR under the following conditions using the primers in Tables 5 and 6 below and using CyPPO10 and CyPPO13 cloned into the pET303-CT His vector as templates.

[0244] Material

[0245] Template 1 μl

[0246] 10X buffer 5μl

[0247] dNTP mixture (10 mM each) 1 μl

[0248] Forward primer (10 μM) 1 μl

[0249] Reverse primer (10 μM) 1 μl

[0250] DDW 40μl

[0251] Pfu-X (Solgent, 2.5 units / μl) 1μl

[0252] Total 50 μl

[0253] Table 4

[0254] PCR conditions

[0255]

[0256] Table 5

[0257] List of primers used to construct the CyPPO13 mutant gene

[0258]

[0259]

[0260]

[0261]

[0262] Table 6

[0263] List of primers used to construct the CyPPO13 mutant gene

[0264]

[0265]

[0266]

[0267] Example 5. PPO-inhibiting herbicide tolerance of PPO and its variants

[0268] To enhance the tolerance of CyPPO10 and CyPPO13 to PPO-inhibiting herbicides, the amino acids interacting with the herbicides identified in Example 3 were mutated. After transforming PPO-deficient BT3 E. coli (ΔPPO) with the PPO gene having such mutations, the transformed E. coli were cultured with PPO-inhibiting herbicides to observe the growth of the transformed E. coli as follows:

[0269] The pET303-CyPPO10 or pET303-CyPPO13 plasmid prepared in Example 4 and the plasmids containing each mutant gene were transformed into BT3 competent cells by the heat shock method and cultured in LB agar medium containing ampicillin (100 μg / ml).

[0270] For seed culture of BT3 transformants, a single colony was cultured in 3 ml of LB broth containing ampicillin (LPSS) for 12 hours or longer, and 50-100 μl of the culture medium was further cultured until the absorbance (OD 600 ) to 0.5 to 1. Then, the culture solution was diluted with LB broth to reduce the absorbance (OD 600 ) was adjusted to 0.5 and diluted five more times with LB broth at a factor of one tenth.

[0271] Herbicide-containing media were prepared by mixing LB (25 g / L), Bacto agar (12 g / L), ampicillin (100 μg / ml), and various herbicides (0 to 200 μM).

[0272] 10 microliters of the diluted solution was dropped onto the medium containing the herbicide, and the medium was incubated at 37°C under light for 16 to 20 hours. The growth level and PPO-inhibiting herbicide tolerance of BT3 transformed with each gene were evaluated.

[0273] The herbicides used in the experiment are listed in Table 7 below:

[0274] Table 7

[0275]

[0276] Compared with the CyPPO wild type, the herbicide tolerance was relatively evaluated and is shown in Tables 8 to 11 below. Figures 11 to 29 middle.

[0277] Table 8

[0278]

[0279] NT (not tested)

[0280] Table 9

[0281]

[0282]

[0283] NT (not tested)

[0284] Table 10

[0285]

[0286] NT (not tested)

[0287] Table 11

[0288]

[0289]

[0290] NT (not tested)

[0291] In Tables 8 to 11, the herbicide tolerance level of the wild type is indicated by "-", and the herbicide tolerance level is indicated by gradient by indicating the same tolerance level with "-" and adding "+" if it is higher up to a maximum of "+++++".

[0292] Figures 11 to 19 (wild type and variants of CyPPO10) and Figures 20 to 29 (Wild-type and variant CyPPO13) shows the results of culturing E. coli transformed with the CyPPO gene (wild-type and variant). The concentrations indicated above the graph are the herbicide concentrations used. The six columns for each concentration were diluted 5-fold to the right by a factor of 1 / 10 using E. coli culture. The leftmost column shows the result of the E. coli culture at OD600 = 0.5.

[0293] As shown in Tables 8 to 11 and Figures 11 to 29As shown, it was demonstrated that all transformants transformed with the mutant genes of CyPPO10 and CyPPO13 exhibited the same level or increased level of herbicide tolerance to various herbicides compared to transformants transformed with the wild-type genes.

[0294] Example 6: Measuring Enzyme Activity and IC of Herbicides by PPO 50 value

[0295] The enzyme activity of PPO protein and PPO protein variants was tested, and inhibition tests were performed on PPO-inhibiting herbicides. It has been confirmed that PPO protein has low water solubility, but when expressed as a fusion protein with MBP (maltose binding protein) (MBP-PPO), PPO protein can be stably expressed in a water-soluble form. Therefore, in this test, wild-type and variant proteins expressed as fusion proteins with MBP (see Figure 4 ).

[0296] In order to express the wild-type and mutant genes of CyPPO10 and CyPPO13 (see Examples 1 and 4), these genes were introduced into the pMAL-c2X vector (see Figure 5 ) and then cloned into BL21(DE3) Escherichia coli (CodonPlus).

[0297] The transformed E. coli was cultured under the following conditions to express the introduced PPO gene:

[0298] Induction: OD 600 =0.2, add IPTG to a final concentration of 0.3 mM;

[0299] Expression temperature: 23°C, shaking culture at 200 rpm;

[0300] Expression time: 16 hours;

[0301] Culture scale: 200ml / 1000ml flask.

[0302] The cultured E. coli cells were lysed and protein was extracted using the following method:

[0303] Extraction buffer: column buffer (50 mM Tris-Cl, pH 8.0, 200 mM NaCl) 5 ml buffer / g cells;

[0304] Ultrasonic treatment: SONICS&MATERIALS VCX130 (130 watts);

[0305] 15 seconds on (ON), 10 seconds off (OFF), 5 minutes on ice;

[0306] Centrifuge at 4°C for 20 minutes (20,000×g); dilute the supernatant with column buffer at a ratio of 1:6.

[0307] The following method for purifying PPO protein was carried out in a 4°C cold room. Amylose resin (New England Biolabs) was filled into a 1.5×15 cm column (Bio-Rad Econo column 1.5×10 cm, glass chromatography column, maximum volume), and the obtained protein extract was loaded into the column at a flow rate of 0.2 ml / min. The column was washed with 3 times the column volume of buffer, and the amount of protein in the washing solution was detected. When the protein was no longer detected, the washing was terminated. The MBP-PPO protein was then eluted with approximately 2 times the column volume of buffer containing 20 mM maltose. The protein concentration of each eluate was determined, and the elution was stopped when the protein was no longer detected. 10 microliters of each fraction was studied for protein quantification and SDS-PAGE analysis. The high-purity fraction with PPO protein was used for enzyme activity determination.

[0308] The enzymatic activities of the purified wild-type and variant proteins of CyPPO10 and CyPPO13 were measured by the following method.

[0309] First, a substrate for the PPO protein protoporphyrinogen IX was synthesized. This process was carried out in a nitrogen-flowed space. 6 mg protoporphyrin IX was dissolved in 20% (v / v) 20 ml EtOH and stirred for 30 minutes under dark conditions. The protoporphyrinogen IX solution obtained was placed in a 15 ml spiral tube in an amount of 800 μl and flushed with nitrogen for 5 minutes. 1 g of sodium amalgam was added thereto and vigorously shaken for 2 minutes. The lid was opened to discharge the hydrogen in the tube. Thereafter, the lid was closed and incubated for 3 minutes. The protoporphyrin IX solution was filtered using a syringe and a cellulose membrane filter. 2 M MOPS [3-(N-morpholino) propanesulfonic acid] in an amount of about 300 μl was added to the protoporphyrin IX solution obtained by 600 μl, thereby adjusting the pH to 8.0. To determine the enzymatic activity of the PPO protein, a reaction mixture was prepared with the following composition (based on 10 ml): 50 mM Tris-Cl (pH 8.0); 50 mM NaCl; 0.04% (v / v) Tween 20; 40 mM glucose (0.072 g); 5 units of glucose oxidase (16.6 mg); and 10 units of catalase (1 μl).

[0310] 200 microliters (200 μl) of reaction mixture containing purified PPO protein is placed in a 96-well plate and pre-incubated for 30 minutes at room temperature to reduce oxygen concentration by the reaction of glucose oxidase-catalase. Mineral oil is layered and then the reaction is started by adding substrate protoporphyrin IX solution to a final concentration of 50 μM. The reaction is carried out at room temperature for 30 minutes, and a microplate reader (Sense, Hidex) is used to measure the fluorescence (excitation: 405 nm; emission: 633 nm) of protoporphyrin IX. In order to calculate PPO enzyme activity, protoporphyrinogen IX solution is kept open in the air to oxidize the solution (overnight). 2.7N HCl is added thereto and the absorbance at 408 nm is measured. Standard curve is generated using standard protoporphyrin IX, and PPO activity is measured by calibrating protoporphyrin IX using the standard curve of protoporphyrin IX.

[0311] The obtained enzyme activities of the PPO wild type and variants are shown in Table 12.

[0312] Simultaneously, the Michaelis constant (Km) and maximum velocity (Vmax) values ​​for each enzyme were calculated to assess the kinetic parameters of the PPO proteins (CyPPO10 and CyPPO13). The initial reaction velocity, which is proportional to the substrate concentration, was measured, and the amount of protoporphyrin IX produced as the enzymatic reaction product was measured over a 20-minute time course at room temperature. Km and Vmax values ​​were calculated using the Michaelis-Menten equation using an enzyme kinetic analysis program, with plant PPO as a control group. The results are shown in Table 12:

[0313] Table 12

[0314]

[0315] As shown in Table 12, CyPPO10 and CyPPO13 have superior abilities as PPO enzymes to those of Arabidopsis PPO1 (AtPPO1) and Amaranth PPO1.

[0316] The concentration of PPO-inhibiting herbicide (IC) at which each herbicide inhibited PPO enzyme activity by 50% was measured. 50 The final concentration of each herbicide was as follows:

[0317] 0, 10, 50, 100, 250, 500, 1,000, 2,500, 5,000nM

[0318] IC 50 The value was calculated as the herbicide concentration that inhibited the PPO enzyme activity to 50%, and then the herbicide at the above concentration was added to perform the above enzyme activity measurement process.

[0319] IC values ​​of different herbicides 50 The values ​​are shown in Table 13 below.

[0320] Table 13

[0321]

[0322]

[0323]

[0324]

[0325] NT (not tested)

[0326] As shown in Table 13, the CyPPO protein variants showed increased IC 50 value. These results indicate that amino acid mutations at certain positions of the PPO protein can lead to increased herbicide tolerance. Although the present data show that the CyPPO protein variant has reduced enzymatic activity compared to the wild type, this may be caused by different conditions for protein folding and / or the hydrophobicity of the recombinant PPO compared to the natural PPO. Although natural PPO is hydrophobic and localizes to the membranes of chloroplasts in plants, recombinant PPO produced in Escherichia coli containing MBP as a fusion partner is hydrophilic. Therefore, when the PPO variant is properly assembled and localized in the chloroplast membranes of plants, the enzyme activity will not be significantly affected.

[0327] Example 7. Generation of Arabidopsis transformants using CyPPO and its variants and PPO-inhibiting herbicide tolerance testing

[0328] 7-1. Construction of Arabidopsis transformation vector and transformation of Arabidopsis

[0329] Arabidopsis thaliana was transformed with a binary vector containing the ORF for a selectable marker, the bar gene (glufosinate tolerance), and the ORF for each gene encoding a CyPPO10 or CyPPO13 variant. Transgenic plants were examined for cross-tolerance to glufosinate and PPO-inhibiting herbicides. The bar gene was also used to examine whether the transgene was stably inherited across generations. The NOS promoter and E9 terminator were used for bar gene expression.

[0330] In order to express CyPPO10, CyPPO10 variants, CyPPO13 and CyPPO13 variants in plants, respectively, the CaMV35S promoter and NOS terminator were used. The coding genes of CyPPO10, CyPPO10 variants, CyPPO13 and CyPPO13 variants were cloned using XhoI and BamHI restriction enzymes. In order to identify the expressed protein, the hemagglutinin (HA) tag was fused to the 3' terminal region using BamHI and SacI restriction enzymes. The NOS terminator was inserted after the HA tag to terminate the transcription of the PPO gene. In addition, in order to transport the protein to the chloroplast, the transit peptide (TP) (SEQ ID NO: 10) of the AtPPO1 gene was inserted into the 5' front of the inserted gene using XbaI and XhoI restriction enzymes. The transit peptide region inserted into the vector is represented by SEQ ID NO: 27, and the inserted HA tag sequence is represented by SEQ ID NO: 28. A schematic diagram of the plant transformation binary vector is shown in Figure 6 Shown in.

[0331] Each constructed vector was introduced into Agrobacterium tumefaciens GV3101 competent cells via the freeze-thaw method. To prepare Agrobacterium GV3101 competent cells, culture Agrobacterium GV3101 seeds in 5 ml of LB medium at 30°C, 200 rpm for 12 hours. This culture was inoculated into 200 ml of LB medium, then incubated at 30°C, 200 rpm for 3 to 4 hours, and centrifuged at 3000 × g for 20 minutes at 4°C. The pellet was washed with sterile distilled water and resuspended in 20 ml of LB medium. A 200 μl aliquot was snap-frozen in liquid nitrogen and stored in a deep freezer.

[0332] Each transformed Agrobacterium was cultured and screened in an antibiotic medium (LB agar containing spectinomycin). The screened colonies were cultured in LB broth. After harvesting Agrobacterium from the culture medium, the Agrobacterium was isolated and analyzed at an absorbance of 0.8 (OD 600 ) were resuspended in 5% (w / v) sucrose, 0.05% (v / v) Silwet L-77 solution (Momentive Performance Materials). Col-0 ecotype Arabidopsis wild type was transformed by floral dipping, and seeds (T1) were harvested after 1 to 2 months.

[0333] The Bar gene in the binary vector was used to screen individual transformants. The T1 seeds obtained were sown in 1 / 2 MS medium (2.25 g / L MS salts, 10 g / L sucrose, 7 g / L agar) supplemented with 25 μM glufosinate-ammonium, and surviving plants were selected after 7 days of sowing and transplanted into soil.

[0334] To test the transgenic plants for tolerance to PPO-inhibiting herbicides, 4-week-old plants were uniformly sprayed with 100 ml of a 1 μM solution of difenzoquat (0.05% Silwet L-77) per 40 x 60 cm area (0.24 m 2 ). While wild-type Arabidopsis (Col-0 ecotype; Columbia-0 ecotype) died completely within 7 days after treatment, none of the transformants showed damage from PPO-inhibiting herbicide treatment.

[0335] T2 seeds harvested from surviving plants were sown in ½ MS medium (2.25 g / L MS salts, 10 g / L sucrose, 7 g / L agar) supplemented with 25 μM glufosinate, and after 1 week, surviving plants were transplanted into soil.

[0336] To confirm the copy number of each line, segregation ratios were investigated using T2 seeds.

[0337] Difenzoquat tolerance of 4-week-old transformants was confirmed by spraying 100 ml of a difenzoquat solution (1 μM, 5 μM, 10 μM, or 25 μM difenzoquat + 0.05% Silwet L-77) per 40 x 60 cm area (0.24 m 2 ). T3 seeds were harvested from difenzoquat-tolerant T2 plants.

[0338] Seeds were selected in ½ MS medium containing 25 μM glufosinate, and lines in which all individuals were tolerant to glufosinate were judged to be homolines.

[0339] 7-2. Seed germination

[0340] Herbicide tolerance of Arabidopsis transformants into which wild-type or variant genes of CyPPOlO and CyPPOl3 were introduced was confirmed.

[0341] T3 generation seeds of each transformant were sown in ½ MS medium containing herbicides. Seeds of the Col-0 ecotype (wild-type Arabidopsis) were used as a control. The types and concentrations of herbicides were as follows:

[0342] Figure 31a : 25 μM glufosinate (PPT), 70 nM difenzoquat, 100 nM bensulfuron-methyl, 25 μM glufosinate + 70 nM difenzoquat, or 25 μM glufosinate + 30 nM difenzoquat + 40 nM bensulfuron-methyl;

[0343] Figure 31b and 31c: 25 μΜ glufosinate (PPT), 0.1 μΜ or 1 μΜ of difenzoquat, 0.3 μΜ or 3 μΜ of saflufenacil, 0.1 μΜ or 1 μΜ of flumioxazin, 0.5 μΜ or 5 μΜ of pyroxasulfone, or 1 μΜ or 10 μΜ of mesosulfuron.

[0344] The results of seed germination 7 days after sowing are shown in Figure 31a , 31b and 31c. In Figures 31a to 31c , 10-3 means CyPPO10 wild type, 10FM-4-7 means CyPPO10 F360M transgenic line, 10FL-1-9 means CyPPO10 F360L transgenic line, 10FC-3-5 means CyPPO10 F360C transgenic line, 10AC-5-4 means CyPPO10 A167C transgenic line, 13-1 means CyPPO13 wild type, 13FM-3-1 means CyPPO13 F373M transgenic line, 13FC-1-1 means CyPPO13 F373C transgenic line, 13FI-2-1 means CyPPO13 F373I transgenic line, 13AC-1-3 means CyPPO13 A177C transgenic line, CyPPO13_ALFL means CyPPO13 A177L+F373L transgenic line, CyPPO13_ALFI means CyPPO13 A177L+F373I transgenic line.

[0345] As shown in Figures 31a to 31c , while wild-type Arabidopsis (Col-0 ecotype) germinated in 1 / 2MS medium without herbicide, it did not germinate in 1 / 2MS medium containing herbicide. Therefore, the germination test on medium containing herbicide can be used to evaluate herbicide tolerance.

[0346] Meanwhile, Arabidopsis T3 lines were transformed in which CyPPO10 wild type, CyPPO10 mutant genes (F360M, F360I, F360L, F360C, A167C), CyPPO13 wild type, or CyPPO13 mutant genes (F373M, F373C, F373I, A177C, A177L+F373L, A177L+F373I) were germinated in medium containing herbicide (containing 25 μΜ glufosinate, 25 μΜ glufosinate + 70 nM difenzoquat, or 25 μΜ glufosinate + 30 nM difenzoquat + 40 nM saflufenacil). These results indicate that the bar gene (glufosinate tolerance gene) and the CyPPO gene (PPO-inhibiting herbicide tolerance gene) function simultaneously and independently in transgenic plants to confer herbicide tolerance traits.

[0347] As shown in Figures 31a to 31cAs shown, in a culture medium containing various types and concentrations of PPO-inhibiting herbicides, the transformed Arabidopsis thaliana generally germinated and survived, whereas Col-0 generally did not germinate. These results indicate that the transformed Arabidopsis thaliana was endowed with tolerance to various PPO-inhibiting herbicides or maintained enhanced tolerance to various PPO-inhibiting herbicides by the inserted gene of the transformant.

[0348] 7-3. Study on CyPPO protein expression in Arabidopsis thaliana (T2) with CyPPO gene introduced

[0349] The expression of each protein was studied in Arabidopsis transformants (T2) into which the gene encoding CyPPO10, a CyPPO10 variant (F360I or F360M), CyPPO13 or a CyPPO13 variant (F373M) was inserted, respectively.

[0350] Four-week-old Arabidopsis transformant leaves were ground with liquid nitrogen and protein was extracted by adding protein extraction buffer (0.05M Tris-Cl pH 7.5, 0.1M NaCl, 0.01M EDTA, 1% Triton X-100, 1mM DTT). Western blotting was then performed using an anti-HA antibody (Santa cruz). Proteins expressed in the transformants were detected using the HA tag. To compare the amount of loaded protein, the amount of RuBisCO large subunit was confirmed by Coomassie blue staining. Two independent lines of each variant were tested, and Col-0 was used as a control.

[0351] The results are Figure 7 All Arabidopsis transformants into which the CyPPO10 variant (F360I variant or F360M variant) or CyPPO13 variant (F373M variant) gene was introduced showed successful expression of PPO protein.

[0352] 7-4. Verification of Herbicide Tolerance of Transformed Arabidopsis (T2 or T3)

[0353] Herbicide tolerance was tested using Arabidopsis transformants (T2 or T3) introduced with genes encoding CyPPO10, CyPPO10 variants (F360C, F360I, F360L, F360M, F360V, F360T, A167C, A167L, A167L+F360M, A167C+F360M, A167C+F360I, or V305M+F360M), CyPPO13, or CyPPO13 variants (A177C, F373C, F373I, F373M, A177L+F373I, or A177L+F373L), respectively.

[0354] Each 40×60 cm area (0.24 m 2) were treated with 100 ml of CyPPO10 or CyPPO13 transformants (T3), and the damage level of the plants was determined on day 7. For comparison, the same test was performed using wild-type Arabidopsis thaliana (Col-0 ecotype).

[0355] The results are Figure 8 Shown in.

[0356] In addition, each 40×60cm area (0.24m 2 ) After the transformants carrying the genes encoding the CyPPO10 variants (F360C, F360I, F360L, F360M, F360V, F360T, A167C, A167L, A167L+F360M or A167C+F360I) or the CyPPO13 variants (A177C, F373C, F373I, F373M, A177L+F373I or A177L+F373L) were treated with 100 ml of tifenad solution (1 μM, 5 μM, 10 μM or 25 μM tifenad + 0.05% (v / v) Silwet L-77), the damage level of the plants was determined on the 7th day.

[0357] The results are Figure 9 (T2 transformants into which the CyPPO10 variant gene was introduced) and Figure 10 (T2 transformant into which the CyPPO13 variant gene was introduced).

[0358] in addition, Figures 8 to 10 The damage level (damage index) of each line after the treatment with tifenad in the following Table 14 is shown as a numerical index.

[0359] Table 14

[0360] T2 damage index (damage level)

[0361]

[0362]

[0363] Each 40×60cm area (0.24m 2 ) After treating the transformants (T3) containing the genes encoding CyPPO10 variants (F360I, F360L, F360M, A167C+F360I, A167C+F360M, or V305M+F360M) with 100 ml of a tifenacil solution (25 μM tifenacil+0.05% (v / v) Silwet L-77) or a saflufenacil solution (100 μM saflufenacil+0.05% (v / v) Silwet L-77), the damage level of the plants was determined on the 7th day.

[0364] The results of T3 transformants into which the CyPPO10 variant encoding gene was introduced were Figure 32 Shown in.

[0365] In addition, the damage levels (damage index) of Arabidopsis transformants into which the CyPPO10 mutant gene was introduced after treatment with tifenad or saflufenacil are shown as numerical indices in Table 15 below.

[0366] Table 15

[0367] T3 damage index (damage level)

[0368]

[0369]

[0370] Tables 14 and 15 show the mean values ​​of the damage levels of individuals (10 to 20 individuals) tested according to the criteria of Table 16 below.

[0371] Table 16

[0372] Definition of injury level

[0373]

[0374] After treatment with tifenad, saflufenacil, fluazifop-propyl, or sulfentrazone (50 μM each), the tolerance levels of Arabidopsis transformants (T3) harboring the CyPPO10 mutant gene (F360I or A167L+F360M) or the CyPPO13 mutant gene (A177L+F373L or A177L+F373I) were confirmed. For comparison, Arabidopsis wild type or Arabidopsis PPO1 SLYM (AtPPO1 SLYM, S305L+Y426M), a plant known to be tolerant to PPO-inhibiting herbicides, were tested under the same conditions.

[0375] In the tolerance experiments using various herbicides, each 40 × 60 cm area (0.24 m 2 ) were evenly sprayed with 100 ml of each herbicide at a concentration of 50 μM. The molecular weights (MW) of tifenamid, saflufenacil, fluazifop-propyl, and sulfentrazone are 511.87, 500.85, 354.34, and 387.18, respectively. The converted treatment doses corresponded to 106.7 g ai / ha for tifenamid, 104.4 g ai / ha for saflufenacil, 73.8 g ai / ha for fluazifop-propyl, and 80.7 g ai / ha for sulfentrazone.

[0376] The results are as follows Figure 33a and 33b shown.

[0377] In addition, the damage level (damage index) of the transformants was Figure 33a 、 Figure 33b and are shown as numerical indices in Table 17.

[0378] Table 17

[0379] T3 damage index (damage level)

[0380]

[0381] exist Figure 33a In Table 17, Cy10FI, AtPPO1 SLYM, and Cy10 ALFM represent CyPPO10 F360I transformant, AtPPO1 S305L+Y426M transformant (control), and CyPPO10A167L+F360M transformant, respectively.

[0382] exist Figure 33b In the table, Col-0, Cy13 ALFL, and Cy13 ALFI represent the wild type, CyPPO13A177L+F373L transformant, and CyPPO13 A177L+F373I transformant, respectively.

[0383] like Figure 33a As shown, transformants of the mutant genes had the same or higher tolerance than AtPPO1 SLYM. It was confirmed that all CyPPO10 FI and CyPPO10 ALFM conferred higher tolerance to various herbicides than AtPPO1 SLYM.

[0384] As shown in Table 14 and Figure 8 As shown, after treatment with 1 μM of tifenadine, almost all transformants having CyPPO10 wild type, its variant gene, CyPPO13 wild type or its variant gene grew, while wild type Arabidopsis thaliana (Col-0) died.

[0385] In addition, as shown in Tables 15 and 17, Figures 9 to 10 and Figure 32 to 3 As shown in Figure 3, Arabidopsis transformants introduced with the CyPPO10 or CyPPO13 variant genes showed little or no damage after treatment with more than 5 μM of tifenadine. These results indicate that herbicide tolerance can be conferred and / or enhanced in Arabidopsis by introducing CyPPO10, CyPPO13, or their mutant genes.

[0386] It was confirmed that herbicide tolerance was maintained from T2 to T3 generations, suggesting that herbicide tolerance can be stably transferred even as generations progress.

[0387] Based on this result, the CyPPO variants are expected to provide tolerance to various PPO-inhibiting herbicides in other plants as well as Arabidopsis.

[0388] 7-5. Confirmation of transgene stability during passage

[0389] In this example, it was confirmed whether the gene introduced into Arabidopsis thaliana is stably inherited over generations.

[0390] The T3 lines 7-2, 10-2, and 10-5 transformed with CyPPO10 F360I were further developed into T4 and T5 generations to confirm the tolerance to tifenad or saflufenacil in each line and the expression of the introduced gene in the T4 and T5 generations.

[0391] Protein extraction

[0392] Protein was extracted from each generation of plants. After grinding the seedlings with liquid nitrogen, total protein was extracted using protein extraction buffer (0.05 M Tris-Cl pH 7.5, 0.1 M NaCl, 0.01 M EDTA, 1% Triton X-100, 1 mM DTT). After electrophoresis, the extracted proteins were transferred to a PVDF membrane and then subjected to Western blotting using an anti-HA antibody (Santacruz).

[0393] Confirming herbicide tolerance

[0394] Four weeks after transplanting, 100 ml of a herbicide solution containing 15 μM tifenacil or 150 μM saflufenacil was evenly sprayed on an area of ​​40 × 60 cm (0.24 m 2 The herbicide damage level was observed 7 days after treatment.

[0395] The results of herbicide tolerance are Figure 34 (T4) and Figure 35 (T5) is shown, and the damage levels (damage index) of the transformants to the herbicides are shown in Table 18.

[0396] Table 18

[0397] T4 and T5 damage index (damage level)

[0398]

[0399] While the negative control (Col-0; Arabidopsis wild type) was sensitive to the herbicide treatment, T4 and T5 Arabidopsis transformants of CyPPO10F360I were tolerant.

[0400] Additionally, Western blot analysis of transgene expression was performed in Figure 36CyPPO10 F360I protein was detected only in all T4 and T5 generation transformants.

[0401] Thus, it was demonstrated that the herbicide tolerance obtained by the CyPPO10 variant was stably inherited and maintained through T4 and T5 generations.

[0402] Example 8. Construction of soybean transformants using CyPPO and its variants and testing of PPO-inhibiting herbicide tolerance

[0403] 8-1. Recombinant vector for soybean transformation and construction of soybean transformants using the recombinant vector

[0404] A vector for soybean plant transformation to confer trifluralin tolerance by expressing CyPPO10 A167L+F360M gene was constructed.

[0405] Specifically, CyPPO10 A167L+F360M gene combined with transit peptide of Arabidopsis PPO1 gene was amplified by PCR using a vector for Arabidopsis transformation (see Figure 6 ) as a template. The amplified product was cloned using pENTR Directional TOPO cloning kit (Invitrogen) and transformed into DH5α competent cells (Invitrogen). Then, the cloned gene was moved to a vector pB2GW7.0 binary vector for plant transformation ( Figure 37 ) using Gateway LR Clonase II enzyme mix kit (Invitrogen). After mixing pENTR / D-TOPO vector in which CyPPO10 A167L+F360M gene was cloned, TE buffer, and LR Clonase II enzyme mix, it was incubated at 25℃ for 1 hour. After adding proteinase K solution (Invitrogen) to the reaction mixture, it was incubated at 37℃ for 10 minutes and transformed into DH5α competent cells.

[0406] Agrobacterium EHA105 was electroporated with the binary vector constructed as above.

[0407] Kangwon soybean plants were used for construction of soybean transformants.

[0408] After removing the seed coat from soybean seeds, the hypocotyls were cut and wounded 7-8 times with a surgical scalpel (#11 blade). Approximately 50 explants were mixed with transformed Agrobacterium tumefaciens EHA105 (Hood et al., New Agrobacterium helper plasmid for gene transfer to plants (EHA105). Trans Res. 1993 2: 208-218), and the mixture was sonicated for 20 seconds and then incubated for 30 minutes for inoculation. They were plated on CCM (co-culture medium; 0.32 g / L Gamborg B5, 4.26 g / L MES, 30 g / L sucrose, 0.7% agar). They were then co-cultivated in a growth chamber (25°C, 18 hours light / 6 hours dark) for 5 days.

[0409] Afterwards, they were washed in liquid 1 / 2 SIM (shoot induction medium; 3.2 g / L Gamborg B5, 1.67 mg / L BA, 3 mM MES, 0.8% (w / v) agar, 3% (w / v) sucrose, 250 mg / L cefotaxime, 50 mg / L vancomycin, 100 mg / L ticarcillin, pH 5.6) for 10 min and placed on SIM without antibiotics and cultured in a growth chamber (25°C, 18 h light / 6 h dark) for 2 weeks.

[0410] Shoot-induced explants were transplanted onto SIM-1 (SIM medium supplemented with 10 mg / L DL-phosphinothricin, pH 5.6).

[0411] Brown shoots were transplanted onto SEM (shoot elongation medium; 4.4 g / L MS salts, 3 mM MES, 0.5 mg / L GA3, 50 mg / L asparagine, 100 mg / L pyroglutamic acid, 0.1 mg / L IAA, 1 mg / L zeatin, 3% (w / v) sucrose, 0.8% (w / v) agar, 250 mg / L cefotaxime, 50 mg / L vancomycin, 100 mg / L ticarcillin, 5 mg / L DL-phosphinothricin, pH 5.6). Elongated shoots at a height of 4 cm were transferred to RIM (root induction medium; 4.4 g / L MS salts, 3 mM MES, 3% sucrose, 0.8% agar, 50 mg / L cefotaxime, 50 mg / L vancomycin, 50 mg / L ticarcillin, 25 mg / L asparagine, 25 mg / L pyroglutamic acid, pH 5.6).

[0412] When the roots were well developed, the plants were moved to a bed soil mixed with vermiculite at 2:1 (v / v) (Bioplug No. 2, Farmhannong). After 10 days, the leaves were sprayed with 100 mg / L DL-phosphinothricin.

[0413] 8-2. Verification of herbicide tolerance of transformed soybean

[0414] Leaves of CyPPO10 A167L+F360M transformed soybean (line 2) and untransformed soybean (Kwandan; wild-type soybean, control) were brushed 2 to 3 times with 5 micromolar or 15 μM of topramezone. The topramezone solution contained 0.05% (v / v) Silwet L-77 as a surfactant.

[0415] As shown in Figure 38 , Kwandan (untransformed soybean) showed severe injury 7 days after treatment with 5 μM of topramezone, but CyPPO10 A167L+F360M transformed soybean did not show injury even after treatment with 15 μM of topramezone.

[0416] Meanwhile, T1 generation of line 2 of CyPPO10 A167L+F360M transformant was treated with topramezone or bensulfuron-methyl at V2 to V3 stage. 100 ml of 25 μM of topramezone or 150 μM of bensulfuron-methyl was uniformly sprayed on an area of 40 x 60 cm (0.24 m 2 ) and the level of injury was evaluated 5 days after spraying.

[0417] In Figure 40 , Kwandan soybean was used as a control. CyPPO10 A167L+F360M (10ALFM) transformant soybean did not show injury even after treatment with relatively high concentrations of topramezone or bensulfuron-methyl, compared to the control.

[0418] 8-3. Confirmation of the number of inserted genes in transformed soybean

[0419] Genomic DNA was extracted from 250 mg of leaf tissue of line 2 or line 23 transformed with CyPPO10 A167L+F360M to analyze the copy number of transgenes.

[0420] Genomic DNA was extracted using the CTAB buffer method. After grinding the leaf tissue in liquid nitrogen with a pestle and mortar, 1.25 ml of DNA isolation buffer (2% (w / v) CTAB, 1.5 M NaCl, 25 mM EDTA, 0.2% (v / v) β-mercaptoethanol, 100 mM Tris-Cl (pH 8.0)) was added and vortexed. After heating at 60°C for 1 hour, 1 volume of chloroform:isoamyl alcohol (24:1) was added and mixed by inversion. After centrifugation at 7000 × g for 10 minutes at 4°C, the supernatant was transferred to a new tube and mixed with 2.5 volumes of ethanol. After centrifugation at 5000 × g for 5 minutes at 4°C, the supernatant was discarded and the precipitate was dissolved with TE buffer (LPSS). After adding 20 μg / ml RNase A (Bioneer), it was incubated at 37°C for 30 minutes. After adding 1 volume of phenol:chloroform (1:1), mix them and centrifuge at 10,000 × g for 10 minutes at 4°C. Transfer the supernatant to a new tube, then add 1 volume of chloroform:isoamyl alcohol (24:1) and mix. After centrifugation at 10,000 × g for 10 minutes at 4°C, transfer the supernatant to a new tube, add 0.1 volume of NaOAc (pH 5.2) and 2 volumes of ethanol and mix. After centrifugation at 5,000 × g for 5 minutes at 4°C, wash with 70% ethanol. After air drying, dissolve the genomic DNA with an appropriate amount of TE buffer.

[0421] 10 to 40 μg of extracted DNA was digested overnight using EcoRI (Enzynomics).

[0422] Then, after electrophoresis on a 0.8% (w / v) agarose gel (50 V), the gel was processed as follows:

[0423] 1) Depurination: 0.25N HCl, shake for 15 minutes

[0424] 2) Denaturation: 0.5M NaOH, 1.5M NaCl, shake for 30 minutes

[0425] 3) Neutralization: 0.5M Tris (pH 7.5), 1.5M NaCl, shake for 20 minutes

[0426] Thereafter, the DNA fragments were transferred to a nitrocellulose membrane using the capillary transfer method and cross-linked using a UV cross-linker (UVC-508; ULTRA LUM Inc.).

[0427] Hybridization was performed by immersing the nitrocellulose membrane in DIG Easy hybridization solution (Roche) and incubating for 3 hours at 42° C. Then, the solution was discarded and replaced with fresh DIG Easy hybridization solution containing a DIG-labeled probe and incubated at 42° C. for 16 to 18 hours.

[0428] The probe (DIG-labeled CyPPO8-M probe) was labeled by PCR reaction as follows:

[0429] Probe PCR

[0430] DIG-labeled bar gene was amplified using DIG dUTP (Jena bioscience) using the following primers:

[0431] Forward primer for bar probe: 5'-TTC CGT ACC GAG CCG CAG GA-3' (SEQ ID NO: 124)

[0432] Reverse primer for bar probe: 5'-CGT TGG GCAGCC CGATGACA-3' (SEQ ID NO: 125)

[0433] PCR: Solgent e-Taq kit

[0434] Conditions: 95°C for 5 minutes; 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, for a total of 35 cycles; 72°C for 2 minutes

[0435] After hybridization, the membrane was washed in low stringency wash buffer (2×SSC, 0.1% SDS) and high stringency wash buffer (0.5×SSC, 0.1% SDS). Southern blot signal detection was as follows:

[0436] 1) Add blocking buffer (Roche) to the membrane and shake for 30 minutes

[0437] 2) Add DIG antibody (anti-digoxigenin-AP Fab fragment, Roche) and shake for 30 minutes

[0438] 3) Oscillate in wash buffer for 15 minutes (Roche)

[0439] 4) Add detection buffer (Roche) and shake for 3 minutes

[0440] 5) After applying CDP-Star (Roche) on the membrane, the blot was developed on X-ray film.

[0441] For negative control, genomic DNA from untransformed G. glycine max plants was used for Southern blotting.

[0442] exist Figure 39 The number of bands shown on the membrane indicates the number of transgenes. Since a single band was observed in CyPPO10A167L+F360M transformant lines 2 and 23, it was determined that each transgenic plant had a single copy of the transgene.

[0443] Example 9: Activity testing of mutant genes with sequence homology to PPO variants

[0444] Using the CyPPO plasmid (pACBB vector) as a template, error-prone PCR was performed under the following conditions to induce random mutations in CyPPO:

[0445]

[0446] 10× buffer: 100 mM Tris-Cl, pH 8.3; 500 mM KCl, 70 mM MgCl2, 0.1% (w / v) gelatin

[0447] dNTP: 10mM dATP, 10mM dGTP, 100mM dCTP, 100mM dTTP

[0448] 94°C for 3 minutes; (94°C for 30 seconds, 57°C for 30 seconds, 72°C for 1.5 minutes, 72°C for 5 minutes) 35 cycles

[0449] Primer sequences:

[0450] CyPPO10_BamHI F

[0451] ccccggatccATGATTGAAGTGGATGTGGCTA(SEQ ID NO:126)

[0452] CyPPO10_XhoI R

[0453] ccccctcgagTGATTGTCCACCAGCGAGGTAAG(SEQ ID NO:127)

[0454] CyPPO13_BamHI F

[0455] ccccggatccATGAACCCTGCTACCCTGAAC(SEQ ID NO:128)

[0456] CyPPO13_XhoI R

[0457] ccccctcgagCACCTGTGATAACAACTGCTGAG(SEQ ID NO:129)

[0458] The obtained error-prone PCR product was electrophoresed in an agarose gel and then purified from the gel, and the pACBB vector and PCR product were digested with BamHI and XhoI restriction enzymes. The digested vector and PCR product electrophoresed in the agarose gel were purified and ligated. The ligation product was transformed into BT3 competent cells, and the mutant CyPPO gene from the grown BT3 colonies was sequenced. BT3 with the mutant CyPPO gene was confirmed to grow spots on LB plates containing different concentrations (0 μM, 50 μM, 100 μM and 200 μM) of tifenad or benzylpyraclostrobin, thereby studying the growth of E. coli and testing the level of herbicide tolerance.

[0459] Among the mutant clones, clones with the following mutations were used for this herbicide tolerance test:

[0460] CyPPO10m-6: Contains 9 amino acid mutations (E225G, G258S, Q266L, T336I, V356F, F360M, A364D, R406G, W419R); Nucleic acid sequence - SEQ ID NO: 130, amino acid sequence - SEQ ID NO: 131 (98% sequence homology to the amino acid sequence of wild-type CyPPO10)

[0461] BT3 cells transformed with the mutant gene of CyPPO10 were cultured in a medium containing herbicides, and cell growth inhibition was measured. Figure 41 In the table, 'AtPPO1WT' refers to the wild-type PPO1 of Arabidopsis thaliana, 'AtPPO1SLYM' refers to the mutant PPO1 (Y426M+S305L) of Arabidopsis thaliana, 'CyPPO10WT' refers to the wild-type CyPPO10, and 'CyPPO10m-6' refers to the mutant CyPPO10 as described above, respectively.

[0462] like Figure 41 As shown, cells transformed with CyPPO10 mutants having 98% or greater sequence identity to wild-type CyPPO10 exhibited similar cellular activity to cells transformed with wild-type CyPPO10, even in the presence of a medium containing high concentrations (up to 200 μM) of tifenad or saflufenacil. This result suggests that CyPPO10 mutants having 98% or greater sequence identity can retain the herbicide tolerance (viability in a medium containing herbicides) of the wild type.

Claims

1. A polypeptide comprising the following amino acid sequence, wherein the amino acid sequence is obtained by making the following substitutions in the amino acid sequence of SEQ ID NO: 2: (1) F360 is substituted with M (Met), V (Val), I (Ile), C (Cys), or L (Leu), and at least one of the following substitutions is further introduced: (a) R89 is replaced by A (Ala), (b) V305 is substituted with M (Met), and (c) L327 was replaced by T (Thr); (2) F360 is substituted with V (Val), I (Ile), C (Cys), or L (Leu), and the following substitutions are further introduced: (i) V165 is substituted by C (Cys) or S (Ser); (ii) A167 is substituted by C (Cys), L (Leu) or I (Ile); or (iii) both (i) and (ii); or (3) F360 was substituted with M (Met), V165 was substituted with S (Ser), and the following substitutions were further introduced: A167 is substituted with C (Cys), L (Leu), or I (Ile).

2. The polypeptide according to claim 1, wherein The polypeptide (1) further comprises the following substitutions: (i) V165 is substituted by C (Cys) or S (Ser); (ii) A167 is substituted by C (Cys), L (Leu) or I (Ile); or (iii) Both (i) and (ii).

3. A polypeptide comprising the following amino acid sequence, wherein the amino acid sequence is obtained by introducing at least one substitution selected from the following into the amino acid sequence of SEQ ID NO: 4: V175 was replaced by C (Cys); V318 was replaced by M (Met); F337 was replaced by V (Val); L340 is replaced by T (Thr); and I353 was replaced by T (Thr).

4. The polypeptide according to claim 3, further comprising at least one substitution selected from the group consisting of: A177 is substituted with C (Cys) or L (Leu), and F373 is substituted with M (Met), I (Ile), L (Leu), or V (Val).

5. Use of the polypeptide according to any one of claims 1 to 4 or the polynucleotide encoding the polypeptide for conferring or enhancing herbicide tolerance in plants, in, The herbicide is at least one protoporphyrinogen oxidase-inhibiting herbicide selected from the group consisting of pyrimidinedione, diphenyl ether, phenylpyrazole, N-phenylphthalimide, triazolinone, Oxazolidinone and bispyribac.

6. The use according to claim 5, wherein The herbicide is at least one selected from the group consisting of fluazifop-butyl, benzylsulfuron, tifenad, fomesafen, acifluorfen, fluazifop-butyl, fluazifop-butyl, sulfentrazone, cyclopentane Oxychloride, bispyribac, and agriculturally acceptable salts thereof.

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