Methods and compositions for herbicide tolerance in plants

Abstract

To provide a novel herbicide-insensitive protoporphyrinogen oxidase.SOLUTION: The present invention provides novel recombinant DNA molecules and engineered proteins for conferring tolerance to protoporphyrinogen oxidase-inhibitor herbicides. The present invention also provides herbicide-tolerant transgenic plants, seeds, cells, and plant parts, containing the recombinant DNA molecules, as well as methods of using the same.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 200,428, filed August 3, 2015. No. 6,313,635, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Incorporating sequence tables Created on July 27, 2016, with a size of 69.4KB (measured on MS-WINDOWS) The sequence listing contained in the file named MONS383WO_ST25.txt is available electronically. No. 60 / 339,933 filed on May 23, 2003, and which is hereby incorporated by reference.

[0003] background FIELD OF THEINVENTION The present invention relates to the field of biotechnology. More specifically, the present invention relates to a method for the preparation of protoplasts. Encodes an enzyme that confers tolerance to herbicides that inhibit fluorescein oxidase , relating to recombinant DNA molecules. [Background technology]

[0004] Related Technology Agricultural crop production often involves the use of transgenic plants produced using biotechnology methods. A foreign gene (also known as a transgene) is introduced into the transgenic plant. The expression of the transgene in plants can be improved by introducing the This confers traits such as herbicide tolerance to plants. Examples of qualities include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance. As more and more weed species become resistant to commonly used herbicides, new There is a need in the art for herbicide tolerance traits. Herbicides of particular interest include proline, sorbitol, sorbitol-based, and sorbitol-based. Herbicides that inhibit protoporphyrinogen oxidase (PPO) (also called PPO herbicides) PPO herbicides provide control of a wide range of herbicide-resistant weeds, so Traits that confer tolerance to these herbicides may be combined with one or more other herbicide tolerance(s). This will be particularly useful in combined cultivation systems.

[0005] Protoporphyrinogen oxidase functions in both the chlorophyll and heme biosynthetic pathways. It converts protoporphyrinogen IX to protoporphyrin IX. After phyllin IX formation, the chlorophyll and heme biosynthetic pathways depend on different metal ions (iron for heme). This segment of the pathway is Conserved across prokaryotes and eukaryotes, and found across prokaryotes and eukaryotes Many of the PPO enzymes used are relatively similar. Some prokaryotes (e.g., cyanobacteria) This pathway is used for chlorophyll and heme production, whereas other prokaryotes (e.g., Escherichia coli) Lactobacillus casei (Richea coli) uses this pathway to synthesize heme.

[0006] Herbicide-insensitive protoporphyrinogen oxidase from several prokaryotes and eukaryotes. Based on their structures, at least three types of iPPOs have been isolated: It is believed that there exist different subclasses of PPO enzymes: HemY (Hansson and Hederstedt,“Cloning and Characteriz ation of the Bacillus subtilis hemEHY ge ne cluster,which encodes protoheme IX bi osynthetic enzymes”Journal of Bacteriolo gy 174(24):8081-8093(1992)), HemG(Sasarma n,et al.,“Mapping of a new hem gene in E scherichia coli K12”Microbiology 113:297 -303 (1979)), and HemJ (Boynton, et al., “Disco very of a gene involved in a third bacte rial protoporphyrinogen oxidase activity through comparative genomic analysis d functional complementation”Applied and Environmental Microbiology 77(14):4795- The present invention relates to a novel recombinant HemG family member. Based on 20 years of research and the number of iPPOs identified to date, Nevertheless, transgenic crop plants containing recombinant iPPOs have not yet been commercialized. The powerful weed control platform is based, in part, on a package of herbicide tolerance traits. Therefore, the identification and use of iPPOs in transgenic plants is highly dependent on ongoing development. Creating crop traits means advances in agriculture. Summary of the Invention

[0007] In one aspect, the present invention relates to a polypeptide sequence selected from SEQ ID NOs: 1 to 20. Operable with a nucleic acid sequence encoding a polypeptide with at least 85% sequence identity A recombinant DNA molecule comprising a linked heterologous promoter, wherein the polypeptide is a herbicidal A recombinant DNA molecule having drug-insensitive protoporphyrinogen oxidase activity. In certain embodiments, the polypeptide is selected from among SEQ ID NOs: 1-20. At least about 85% sequence identity, at least about 90% sequence identity to the selected polypeptide. Sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity and has herbicide-insensitive protoporphyrinogen oxidase activity In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 22-63. In a particular embodiment, the recombinant DNA molecule is provided as SEQ ID NO: The polypeptide includes an amino acid sequence selected from the group consisting of 1 to 20. Therefore, according to the present invention, a method for producing a nucleic acid sequence of a full-length amino acid sequence selected from SEQ ID NOs: 1 to 20 is provided. A recombinant polypeptide having at least 85% sequence identity to a herbicide-insensitive polypeptide. Recombinant polypeptides having protoporphyrinogen oxidase activity are provided. do.

[0008] In certain embodiments, a heterologous promoter, e.g., a promoter that functions in a plant cell, The polypeptide sequence of the present invention is, for example, a polypeptide selected from SEQ ID NOs: 1 to 20. A nucleic acid sequence encoding a polypeptide having at least 85% sequence identity to the sequence and the polypeptide is operably linked to a herbicide-insensitive protoporphyrin. The resulting DNA molecule has oxidase activity. It may further comprise a targeting sequence which functions to localize intracellularly.

[0009] In one aspect, the present invention provides a DNA construct comprising a recombinant DNA molecule of the present invention. In one embodiment, such a DNA construct is provided comprising a nucleic acid sequence of the present invention. a targeting molecule that functions to localize the polypeptide intracellularly in operable linkage to the The DNA molecule contains a sequence in the genome of the transgenic plant, seed, or cell. In certain embodiments, the polypeptide may be present in a cell, a plant, a seed, or confers herbicide tolerance to plant parts.

[0010] In another aspect of the invention, a recombinant DNA molecule of the invention or a recombinant polypeptide of the invention is Transgenic plants, seeds, cells, or plant parts containing the tides are provided. Therefore, the transgenic plant, seed, cell, or plant part contains at least one P In some embodiments, the tetracycline is capable of or exhibits herbicide tolerance to a PO herbicide. The transgenic plants, seeds, cells, or plant parts may be used to produce additional transgenic herbicides. It has drug tolerance traits.

[0011] In another aspect of the invention, a method for conferring herbicide tolerance to a plant, seed, cell, or plant part is provided. The method includes expressing a recombinant polypeptide of the invention in said plant, seed, cell or plant part. In some embodiments, the method includes heterologously expressing a peptide. In this embodiment, the plant, seed, cell, or plant part is transformed with the recombinant polypeptide. In some embodiments, the protoporphyrinogen oxidase activity is conferred The herbicide tolerance is acifluorfen, fomesafen, lactofen, fluoroglycol Cophen-ethyl, oxyfluorfen, flumioxazin, azafenidine, carfu Enthrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiaryl oxadiazon, pyraflufen-ethyl, saflufenacil, and S-3100 The present invention relates to at least one PPO herbicide selected from the group consisting of:

[0012] Another aspect of the invention is a method for transforming plants, comprising the steps of: a) introducing a recombinant DNA molecule of the invention into a plant; b) introducing the recombinant DNA molecule into a plant cell; and c) transfecting the plant cell with the recombinant DNA molecule. and regenerating a transgenic plant, the method comprising at least one The method further comprises the step of selecting a plant that is tolerant to the species of PPO herbicide. cross the regenerated plants with themselves or with a second plant and collect seeds from the cross. The method may further include the step of:

[0013] In yet another aspect of the invention, there is provided a method for controlling weeds in a vegetative growth area, comprising the steps of: The transgenic plants or plant growth areas containing the seeds are treated with at least one PPO herbicide. and causing the transgenic plant or seed to tolerate the PPO herbicide. and weeds are controlled in the plant growth area.

[0014] In addition, a nucleic acid encoding a protein having protoporphyrinogen oxidase activity A method for identifying a nucleotide sequence comprising the steps of: a) determining the sequence of a native E. coli PPO enzyme; The gene knockout E. coli strains were then engineered to encode candidate herbicide tolerance proteins. (b) transforming a bacterial cell with a bacterial expression vector containing a recombinant DNA molecule that encodes said The transformed E. coli is grown in a heme-free bacterial medium. The growth of the bacteria using the bacterial medium is a protoporphyrinogen oxidase activity. Also provided are methods comprising identifying and amplifying the protein.

[0015] The present invention further provides a method for producing a strain having herbicide-insensitive protoporphyrinogen oxidase activity. A method for identifying a nucleotide sequence encoding a protein having a native A recombinant E. coli strain with a gene knockout of the E. coli PPO enzyme was Transform bacteria with a bacterial expression vector containing a recombinant DNA molecule encoding a protein. and b) culturing the transformed E. coli with at least one PPO herbicide. and growing the bacteria on a bacterial medium having a herbicide-insensitive protozoan. Identifying and amplifying proteins having porphyrinogen oxidase activity A method is provided that includes:

[0016] Another aspect of the invention is a method for screening for herbicide tolerance genes, comprising the steps of: a) expressing a recombinant DNA molecule of the present invention in a plant cell; and b) inhibiting a PPO herbicide. and identifying a plant cell exhibiting tolerance to the agent.

[0017] The present invention further provides a method for screening for herbicide tolerance genes, comprising: 2.) Expressing the recombinant DNA molecule of the present invention in a bacterial cell lacking HemG. The bacterial cells are grown in heme-free bacterial medium in the presence of a PPO herbicide. and b) identifying bacterial cells that exhibit tolerance to the PPO herbicide. Provide the law.

[0018] In another aspect, the present invention relates to a method for producing a herbicide that is tolerant to a PPO herbicide and at least one other herbicide. A method for producing a plant of the present invention, comprising the steps of: a) obtaining a plant comprising a recombinant DNA molecule of the present invention; and b) conferring tolerance to the at least one other herbicide to the transgenic plant. and c) crossing said second plant with a PPO herbicide and said at least one other herbicide. and selecting a progeny plant obtained from the hybrid that has tolerance to the herbicide. A method is provided which is another aspect of the present invention.

[0019] In another aspect, the present invention provides a method for reducing the occurrence of herbicide-tolerant weeds, comprising: a) in the crop growing environment, e.g., by including a DNA molecule of the present invention, PPO herbicides. The plant of the present invention has a tolerance to the herbicide and has a tolerance to at least one other herbicide. b) applying a PPO herbicide and at least one other herbicide to said crop growth. applying to the environment, said crop plants being treated with said PPO herbicide and said at least one and applying to the plant a herbicide-tolerant plant of the present invention that is tolerant to other herbicides. In certain embodiments, the PPO herbicide is acifluorfen, fomesafen, lactoferrin, Fen, Fluoroglycofen-ethyl, Oxyfluorfen, Flumioxazin, A Zafenidine, Carfentrazone-ethyl, Sulfentrazone, Fluthiacet-methyl Chil, Oxadiargyl, Oxadiazon, Pyraflufen-ethyl, Saflufenacil In some embodiments of the method, the glycerol is selected from the group consisting of S-3100, S-4100, and S-3100. The at least one other herbicide is an ACCase inhibitor, an ALS inhibitor, an EPSPS inhibitor, phototoxicants, synthetic auxins, photosynthesis inhibitors, glutamine synthesis inhibitors, HPPD inhibitors, PPO In certain embodiments, the ACC inhibitor is selected from the group consisting of ACC inhibitors, ACC inhibitors, and long chain fatty acid inhibitors. The enzyme inhibitors are aryloxyphenoxypropionates or cyclohexanediones. and ALS inhibitors include sulfonylureas, imidazolinones, triazolopyrimidines, or triazolinones, and the EPSPS inhibitor is glyphosate, a synthetic auxin. The herbicides are phenoxy herbicides, benzoates, carboxylic acids, or semicarbazones, and are photosynthetic. The inhibitors are triazines, triazinones, nitriles, benzothiadiazoles, or ureas. the glutamine synthesis inhibitor is glufosinate and the HPPD inhibitor is isooxygenase The PPO inhibitor is a pyrazolone, pyrazolone, or triketone, and the PPO inhibitor is a diphenyl ether , N-phenylphthalimide, aryltriazinone, or pyrimidinedione; or the long chain fatty acid inhibitor is a chloroacetamide, an oxyacetamide, or a pyrazolidine. It is. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is an alignment of H_N90, H_N20, H_N60, H_N10, H_N30, H_N40, H_N50, H_N70, H_N100, and H_N110 protein sequences (SEQ ID NOs: 1-10) with consensus positions shown below. [Diagram 2] FIG. 1 shows the results of an assay of the PPO bacterial screening system with a PPO herbicide, where growth of E. coli containing the test iPPO was measured at 8 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] A brief description of the sequence SEQ ID NO:1 is the amino acid sequence of H_N90.

[0022] Sequence number 2 is the amino acid sequence of H_N20.

[0023] SEQ ID NO: 3 is the amino acid sequence of H_N60.

[0024] SEQ ID NO:4 is the amino acid sequence of H_N10, which is the E. coli wild-type HemG Protoporphyrinogen oxidase (NCBI GenBank accession no. WP_02 1498199).

[0025] SEQ ID NO:5 is the amino acid sequence of H_N30.

[0026] Sequence number 6 is the amino acid sequence of H_N40.

[0027] SEQ ID NO: 7 is the amino acid sequence of H_N50.

[0028] SEQ ID NO: 8 is the amino acid sequence of H_N70.

[0029] Sequence number 9 is the amino acid sequence of H_N100.

[0030] Sequence number 10 is the amino acid sequence of H_N110.

[0031] SEQ ID NO:11 to SEQ ID NO:17 correspond to SEQ ID NO:1, 2, 4, 5, 6, 7, and 9, respectively. It is an amino acid sequence lacking the initiating methionine.

[0032] SEQ ID NO:18 and SEQ ID NO:19 are amino acid variants of SEQ ID NO:11.

[0033] SEQ ID NO:20 is an amino acid variant of SEQ ID NO:17.

[0034] SEQ ID NO:21 is the amino acid sequence of WH, which is derived from Amaranthus tuberculosis. Wild-type protoporphyrinogen oxidase from Acanthurus rculatus be.

[0035] SEQ ID NO:22 to SEQ ID NO:31 are nucleic acids that encode SEQ ID NO:1 to SEQ ID NO:10, respectively. The sequence is a nucleic acid sequence that has been codon-optimized for expression in E. coli.

[0036] SEQ ID NO:32 to SEQ ID NO:41 are nucleic acids that encode SEQ ID NO:1 to SEQ ID NO:10, respectively. The nucleic acid sequence is codon-optimized for dicotyledonous expression.

[0037] SEQ ID NO:42 to SEQ ID NO:48 are sequences that encode SEQ ID NO:11 to SEQ ID NO:17, respectively. The nucleotide sequence is codon-optimized for dicotyledonous expression.

[0038] SEQ ID NO:49 to SEQ ID NO:52 are the nucleotides of SEQ ID NO:11 and SEQ ID NO:12, respectively. It is a mutant.

[0039] SEQ ID NOs: 50, 51, and 53 are sequences encoding the nucleotides SEQ ID NOs: 18, 19, and 20. It is an octide sequence.

[0040] SEQ ID NO:54 to SEQ ID NO:63 are nucleic acids that encode SEQ ID NO:1 to SEQ ID NO:10, respectively. The nucleic acid sequence is codon-optimized for monocotyledonous expression.

[0041] Detailed Description The following explanations and definitions are provided to better define the present invention and to guide those of ordinary skill in the art in practicing the present invention. Unless otherwise specified, the terms are defined as those understood by those of ordinary skill in the relevant art. It is to be understood according to conventional usage.

[0042] The present invention relates to a novel herbicide-insensitive protoporphyrinogen oxidase (iPPO For example, the present invention provides recombinant DNA molecules and proteins encoding In one embodiment, the present invention provides a method for the expression of an iPPO of a microbial origin in a plant cell. The present invention also provides vectors and expression cassettes for producing cells and plants that are tolerant to PPO herbicides. The present invention further provides a protein synthesis method for obtaining and improving iPPOs. Methods and compositions for using protein engineering and bioinformatics tools are provided.

[0043] In certain embodiments, the present invention provides recombinant DNA molecules and proteins. The term "recombinant" as used in this document refers to the result of genetic manipulation and therefore is usually used in Non-natural DNA, proteins, cells, seeds, or organisms not believed to be found in nature A "recombinant DNA molecule" refers to any molecule that does not occur in nature and, therefore, is the result of human intervention. A DNA molecule that contains DNA sequences that are the product of at least two DNA sequences that are heterologous to each other. A DNA molecule consisting of a molecule A. Examples of recombinant DNA molecules provided herein include: Can function with heterologous regulatory or other elements (e.g., heterologous promoters) Linked DNA molecules encoding herbicide-insensitive protoporphyrinogen oxidase. A "recombinant protein" is a protein that does not occur in nature and is therefore not the result of human intervention. A protein that contains a certain amino acid sequence (e.g., a genetically engineered or chimeric protein) A recombinant cell, seed, or organism is a protein that contains a recombinant DNA molecule. Therefore, transgenic DNA generated as a result of plant transformation, e.g. Transgenic cells, seeds, plants, or plant parts, including cells, seeds, or organisms. be.

[0044] As used herein, the term "genetically engineered" refers to any organism that is not normally found in nature. non-natural DNA, proteins, or Genetic engineering refers to the creation of organisms or organisms. in the laboratory using one or more of the biotechnology techniques, such as transformation, genetic transformation, and plant transformation. Conceived and created, used to produce genetically engineered DNA, proteins, or organisms For example, genetic manipulation can be performed by gene cloning, DNA ligation, and DNA synthesis, using one or more of the molecular biology techniques It can be used to create a chimeric gene containing at least two DNA molecules. A chimeric gene is a gene that is a set of two or more operably linked heterologous DNA molecules, e.g., a gene expression vector. a protein coding sequence, e.g., a transport peptide coding sequence, operably linked to the or a heterologous promoter. Genetic manipulation can be performed by site-directed mutagenesis. Protein design using nucleotide sequences and random mutagenesis and DNA shuffling The polypeptide sequence of the present invention may be modified using one or more of the protein engineering techniques, such as forward evolution. It can be used to create engineered proteins. The protein may contain one or more deletions, insertions, or deletions compared to the coding sequence of the wild-type protein. Each deletion, insertion or substitution may consist of one or more amino acids. In another embodiment, the engineered protein can be operably linked to a transport peptide. The peptide may consist of two operably linked heterologous peptides, such as a ligated enzyme.

[0045] As used herein, "herbicide insensitive" refers to a herbicide that is insensitive to protoporphyrinogen oxidase. PPO enzymes reduce their enzymatic activity in the presence of one or more PPO herbicides. This means the ability to maintain at least some of the enzyme activity of protoporphyrinogen oxidase. The activity can be measured by any method known in the art, for example, by an enzyme assay. In the enzyme assay, protoporphyrin was detected in the presence of one or more PPO herbicides. protoporphyrinogen oxidase product production or protoporphyrinogen oxidase substrate consumption The amount of β-amyloid was determined via fluorescence, high performance liquid chromatography (HPLC), or mass spectrometry (MS). Different assays for measuring the enzyme activity of protoporphyrinogen oxidase are Examples of such assays include bacterial assays, such as the proliferation assays described herein. Therefore, the expression of recombinant protoporphyrinogen oxidase was The knockout phenotype was then induced by recombinant protoporphyrins. Herbicide insensitivity is measured by measuring the complementation ability of fluoresceinogen oxidase. can be completely or partially insensitive to, and resistant to, certain PPO herbicides. It can be expressed as percent tolerance (%) or insensitivity. "Herbicide-insensitive protoporphyrinogen oxidase" or "iPPO" refers to Exhibiting herbicide insensitivity in the presence of one or more PPO herbicide(s).

[0046] As used herein, a "hemG knockout strain" refers to a strain that grows on heme-free growth medium. or to a degree that is not possible to identify HemG in comparison with other isogenic lineages that contain functional HemG. An organism or organisms lacking HemG activity such that growth in the absence of Hem is detectably impaired. A cell (e.g., E. coli) is referred to as a hemG knockout in E. coli. The strain may be selected from the group consisting of the hemG sequence of E. coli (Ecogen) having regard to knowledge in the art. eAccession number EG11485, Sasarman et al., “Nucleotide sequence of the hemG gene involved in t he protoporphyrinogen oxidase activity o f Escherichia coli K12”Can J Microbiol 3 9:1155-1161,1993) can be taken into consideration when preparing the composition.

[0047] As used herein, the term "transgene" refers to an organism that has been introduced into the world through human intervention, such as plant transformation methods. As used herein, the term "DNA" refers to a DNA molecule that has been artificially integrated into the genome of an organism. The term "transgenic" means to include a transgene, e.g. A "transgenic plant" refers to a plant that contains an introduced gene in its genome. A "genetic trait" is a trait that is transmitted or conferred by the presence of a transgene integrated into the plant genome. Because of such genome modifications, transgenic plants are A plant that is distinct from the associated wild-type plant and the transgenic trait is The transgenic plants of the present invention are characterized in that they are traits not naturally found in the plant. This includes recombinant DNA molecules and genetically engineered proteins provided herein.

[0048] As used herein, the term "heterologous" refers to a compound derived from a different source, and thus usually naturally occurring. It refers to a relationship between two or more items that are not directly related. For example, a protein-coding recombinant protein The DNA molecule is operably linked to a promoter such that such a combination is normally If it is not found in nature, it is heterologous to the promoter. A recombinant DNA molecule is a molecule that is inserted into a cell, seed, or organism and that is capable of transmitting the message that the insertion If it is believed not to occur naturally in a particular cell, seed, or organism, and may be heterogeneous.

[0049] As used herein, the term "isolated" refers to a molecule that is not present in the natural state with which it is typically associated. In one embodiment, "isolation" refers to at least partially isolating a molecule from other molecules that are present in the molecule. The term "isolated" refers to the separation of a DNA molecule from the nucleic acids with which it normally flanks in the natural state. For example, a DNA molecule that encodes a protein that occurs naturally in a bacterium is If the DNA molecule encoding the protein is not within the bacterial DNA in which it is found in nature Thus, for example, recombinant DNA or plant DNA molecules are considered to be isolated DNA molecules. As a result of the transformation technique, one or more other DNA molecules with which it is not naturally associated may be present. A DNA molecule fused or operably linked to a gene or genes is referred to herein as isolated. Such molecules are considered to be It is considered isolated even if it is present in a nucleic acid solution with other DNA molecules. .

[0050] As used herein, the term "protein-encoding DNA molecule" refers to a molecule that encodes a protein. A "protein coding sequence" refers to a DNA molecule that contains a nucleotide sequence that codes for a protein. "Sequence" means a DNA sequence that encodes a protein. The boundary of a protein coding sequence is the translation start point at the 5' end. The protein codons can be determined by the codon and the translation stop codon at the 3' end. A code molecule can include a DNA sequence that codes for a protein sequence. The terms "transgene expression," "expressing a transgene," "protein expression," and " "Expressing a protein" means transcribing a DNA molecule into messenger RNA (mRNA). , which translates mRNA into a polypeptide chain that is ultimately folded into a protein This refers to the production of proteins through a process called DNA synthesis. A DNA molecule used to express a protein in a cell transformed with the recombinant DNA molecule. A construct may be operably linked to a heterologous promoter in the A construct. "Operably linked" means that one of two DNA molecules affects the function of the other. Operably linked DNA molecules are linked in such a way that they form a single may be part of adjacent molecules and may or may not be adjacent. For example, , the promoter is operable with the protein-encoding DNA molecule in the DNA construct. When linked, these two DNA molecules are capable of directing the expression of a transgene by the promoter. It is positioned so that it can actually have an impact.

[0051] As used herein, a "DNA construct" refers to a vector that contains two or more heterologous DNA sequences. The DNA construct is useful for transgene expression, It may be contained in a vector or a plasmid. The DNA construct may be used for transformation (heterologous DNA into a host cell) to produce a transgenic plant. and cells, thereby producing transgenic plants, seeds, cells, or It may also be contained in the plastid DNA or genomic DNA of the plant part. A "vector" as used herein is any recombinant DNA that can be used for bacterial or plant transformation purposes. A molecule. The recombinant DNA molecules described in the sequence listing are, for example, constructs. The recombinant DNA molecule can be inserted into a vector as part of a construct. Lactic acid is a genetically engineered protein encoded by a recombinant DNA molecule in plants. operably linked to a gene expression element that functions to affect expression of To make and use recombinant DNA constructs and plant transformation vectors General methods useful for the manipulation of DNA molecules are well known in the art, e.g., MR Green and J Sambrook, “Molecular Cloni ng:A Laboratory Manual”(Fourth Edition)I SBN:978-1-936113-42-2,Cold Spring Harbor Manuals and experimental manuals, including those published by Laboratory Press, NY (2012) The DNA construct or a product containing the DNA construct is described in detail in the The components of a vector include one or more genes operably linked to a transcribable DNA sequence. Expression elements include, for example, operably linked DNA a promoter for expressing the and a 3' untranslated region (UTR) operably linked thereto. Elements may include, but are not limited to, one or more of the following types of elements: These include: promoters, 5'UTRs, enhancers, leaders, cis-acting elements ment, intron, target sequence, 3'UTR, and one or more selectable marker introduction genes. Transmission.

[0052] The DNA construct of the present invention is a protein-encoding DNA sequence provided by the present invention. a promoter operably linked to the gene, thereby driving expression of the recombinant protein molecule; Promoters useful in the practice of the present invention include those that act in cells one that functions for expression of an operably linked polynucleotide, e.g., a bacterial promoter Alternatively, plant promoters may be used. Plant promoters are varied and well known in the art. These include, for example, inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, and These include those that are controlled spatially and / or temporally.

[0053] In one embodiment of the present invention, the DNA constructs provided herein are Encoding a polypeptide molecule having insensitive protoporphyrinogen oxidase activity a target sequence operably linked to a heterologous nucleic acid that binds the polypeptide, whereby the target sequence The targeting sequence consists of a signal sequence, a targeting peptide, and a localization Examples of targeting sequences include chloroplast transporter peptides, which are known in the art as targeting sequences, and transit peptides. Translocation peptide (CTP), mitochondrial targeting sequence (MTS), or chloroplast and mitochondrial targeting sequence (MTS) Doria dual targeting peptides promote the intracellular localization of proteins, thereby targeting The sequence enhances accumulation of the recombinant protein and protects the protein from proteolytic degradation. and / or enhance the level of herbicide tolerance, thereby improving the trans- The level of damage to the genetic cells, seeds, or organisms can be reduced.

[0054] CTP and other targeting molecules that can be used in connection with the present invention are known in the art. Known examples include, but are not limited to, Arabidopsis thalian a EPSPS CTP(Klee et al.,Mol Gen Genet.21 0:437-442,1987), Petunia hybrida EPSPS CT P(della-Cioppa et al.,PNAS 83:6873-6877, 1986), the maize cab-m7 signal sequence (Becker et al., lant Mol Biol.20:49-60,1992, PCT WO97 / 412 28), mitochondrial presequences (e.g., Silva Filho et al., Plant Mol Biol 30:769-780,1996), and pea Glutathione reductase signal sequence (Creissen et al., Plant J. 8:167-175, 1995, PCT WO97 / 41228).

[0055] The recombinant DNA molecules of the present invention may contain sequences useful for DNA manipulation, such as restriction enzyme recognition sites. or recombination-based cloning sites), plant-preferred sequences (e.g., plant codon (used or Kozak consensus sequence), or sequences useful for DNA construct design When it is desirable to provide a sequence (e.g., a spacer or linker sequence), the art They can be synthesized and modified, in whole or in part, by methods known in the art. Obviously, any of the recombinant DNA molecules or polypeptide sequences provided herein at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity % sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity and at least 99% sequence identity with herbicide-insensitive protoporphyrin Recombinant DNA molecules and engineered proteins with endothelin oxidase activity are included. As used herein, the term "percent sequence identity" or "% sequence identity" refers to A term is a sequence that represents a reference ("query") sequence compared to a test ("subject") sequence (or its complement). ") sequence (or its complementary strand) in which these two sequences are optimally aligned (comparison window Insertion of appropriate nucleotides or amino acids totaling less than 20% of the reference sequence across all A linear polynucleotide or polypeptide, in which the sequence is a sequence having a sequence, a deletion, or a gap The comparison window refers to the percentage of identical nucleotides or amino acids in a sequence. Optimal alignment of sequences for aligning genes is well known to those skilled in the art and is described in detail in Smith and Wate rman's local homology algorithm, Needleman and Wunsch's Tools such as the study of topology alignment algorithms, Pearson and Lipman's similar methods, etc. and GCG® Wisconsin Package Sequence analysis software from Accelrys Inc., San Diego, CA GAP, BESTFIT, FASTA, and TF are available as part of the software package. ASTA, MEGAlign(DNAStar Inc.,1228 S.Park S t., Madison, WI 53715), and MUSCLE (version 3.6) ( Edgar,“MUSCLE:multiple sequence alignment t with high accuracy and high throughput “Nucleic Acids Research 32(5):1792-7(200 4)) The execution of these algorithms by a computer, e.g. The alignment of test and reference sequences can be performed using the parameters listed above. The "identity fraction" for an aligned reference sequence is In segments, i.e., in the entire reference sequence or in a defined sub-portion of the reference sequence It is the number of identical components shared by two aligned sequences divided by the total number of components. Percent sequence identity is expressed as the percentage of identity multiplied by 100. One or more The comparison of the sequences may be to the full length sequence or a portion thereof, or to a longer sequence. .

[0056] The production of engineered proteins involves altering (i.e. modifying) the wild-type protein. ), modified feature(s), e.g., for chloroplasts or mitochondria, among others. Specific cellular localization patterns, such as targeting, or novel combinations of useful protein features. (e.g., modified V max , K m , K i ,I C 50 ), substrate specificity, inhibitor / removal Herbicide specificity, substrate selectivity, other components in the cell (e.g. partner proteins or membranes) To generate novel proteins having the ability to interact with the The modification can be performed at a specific amino acid position in the protein. and replacing the amino acid found naturally (i.e., in the wild-type protein) at that position. Therefore, the genetic engineering techniques provided by the present invention can be used to substitute a different amino acid. A protein contains one or more altered proteins compared to a similar protein found in nature. In one embodiment of the present invention, a novel protein is provided, which has the following characteristics: The protein may be characterized by one or more of the following characteristics of the variant protein, e.g., compared to the similar wild-type protein: a trait that results in reduced sensitivity to the above herbicides, or the expression of the engineered protein and a method for producing a transgenic plant having improved herbicide tolerance to one or more herbicides. In one embodiment, the present invention provides a genetically engineered protein that has a characteristic that provides a specific ability to and a recombinant DNA molecule encoding the same, comprising at least one selected from Table 1. and the like, including but not limited to SEQ ID NOs: 1-20, as provided herein. At least about 70% sequence identity to any of the engineered protein sequences; About 80% sequence identity, about 85% sequence identity, about 90% sequence identity, about 95% sequence identity identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, and about The recombinant DNA molecule has 99% sequence identity with the nucleotide sequence of the target gene. , as a single amino acid substitution in a protein, or one or more other mutation(s) in combination with, for example, one or more other amino acid substitution(s), deletion, or addition. Mutations can be performed by any method known to those skilled in the art. It is possible to do so. Table 1: Amino acid substitutions TIFF2025011207000001.tif92155

[0057] As used herein, "wild type" refers to a naturally occurring, similar but not identical A "wild-type DNA molecule" or a "wild-type protein" refers to a version of the DNA molecule. A naturally occurring version of a molecule or protein, i.e., the equivalent that already exists in nature. The genetic engineering provided by the present invention is a version of said DNA molecule or protein. An example of a wild-type protein that is useful for comparison with the Arabidopsis thaliana protein is liana-derived protoporphyrinogen oxidase. A non-transgenic plant of the same type as the transgenic plant, and therefore It is genetically distinct from transgenic plants that possess herbicide tolerance traits. An example of a wild-type plant that is useful for comparison with maize plants is the non-transgenic LH24 4 Maize (ATCC Accession No. PTA-1173) and 01DKD2 inbred line Maize Koshi (I294213) (ATCC accession number PTA-7859). Exemplary comparison lines for soybean plants include non-transgenic A3555 soybean (ATCC Accession No. PTA-10207) is considered, and transgenic cotton planting An exemplary comparison line for the product is the non-transgenic Coker130 (Pl ant Variety Protection Number 8900252) can be done.

[0058] Transgenic plants and herbicides In one aspect of the present invention, the recombinant DNA molecules and genetically engineered proteins provided by the present invention are Transgenic plant cells, transgenic plant tissues, transgenic The recombinant DNA molecule and the gene are included in the transgenic plant, the recombinant plant, the transgenic seed, and the transgenic plant. These cells, tissues, plants, and seeds containing engineered proteins are capable of producing one or more PPO herbicides. herbicide tolerance to the agent(s), and optionally one or more additional herbicide(s). It shows tolerance to

[0059] Methods for transforming host plant cells suitable for use in the present invention include introducing DNA into the cells. (e.g., to stably integrate recombinant DNA constructs into plant chromosomes) ) Almost any method is included, and transformation methods are well known in the art. Exemplary and widely used methods for introducing NA constructs into plants The Agrobacterium transformation system is well known to those skilled in the art. Another exemplary method for introducing a recombinant DNA construct into a plant is site-specific Insertion of a recombinant DNA construct into a plant genome at a predetermined site by a method of integration. Site-specific integration can be achieved by any method known in the art, for example, by using zinc finger nucleic acids. Nucleases, engineered or native meganucleases, TALE-endonucleases nucleases, or RNA-guided endonucleases (e.g., CRISPR / Cas9 systems) Transgenic plants can be produced by plant cell culture methods. The transformed plant cells can be regenerated from the transformed plant cells. Transgenic plants contain a single gene (i.e., two allelic copies of the introduced gene). Transgenic plants, e.g., R0 plants, containing the introduced allele along with the self-transgenic plants are then cultured. It can be obtained by pollinating (inbreeding) to produce R1 seeds. One-quarter of the germinated R1 seeds will be homozygous for the transgene. The plants were then analyzed using SNP assays, DNA sequencing, or heat-induced amplification assays. This test, called the zygosity assay, allows the It becomes possible to distinguish between telozygotes and homozygotes.

[0060] The term "PPO-inhibiting herbicide" or "PPO herbicide" used in the present invention refers to a protoporphyrin-containing herbicide. It catalyzes the dehydrogenation of erythrocyte IX to protopor, a precursor of heme and chlorophyll. Enzymatic activity of protoporphyrinogen oxidase (PPO) to form phyrin IX It is a chemical that targets and inhibits protoporphyrinogen oxidase. More reactive oxygen species are formed, resulting in cell membrane destruction and ultimately in the death of susceptible cells. PPO herbicides are well known in the art and are commercially available. Examples of PPO herbicides include, but are not limited to, diphenyl ethers (e.g., For example, acifluorfen, its salts and esters, aclonifen, bifenox, Salts and esters, ethoxyphene, its salts and esters, fluoronitrophen, furil Oxyphen, halosaphen, clomethoxyfen, fluoroglycofen, their salts and esters, lactofen, its salts and esters, oxyfluorfen, and fomesan phenanthrene, its salts and esters), thiadiazoles (e.g., fluthiacet-methyl and thiadiazimine), pyrimidinedione or phenyluracil (e.g., benzphendizo Butaphenacyl, ethyl [3-2-chloro-4-fluoro-5-(1-methyl-6- Trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-3 -yl)phenoxy]-2-pyridyloxy]acetate (CAS Registry Number 353292 -31-6 and referred to as S-3100 in this specification), flupropacil, saflufenacil , and thiaphenacyl), phenylpyrazoles (e.g., fluazolate, pyraflufe and pyraflufen-ethyl), oxadiazoles (e.g., oxadiargyl and xadiazone), triazolinones (e.g., azaphenidine, bencarbazone, carfe thrazone, its salts and esters, and sulfentrazone), oxazolidinedione ( Pentoxazone), N-phenylphthalimide (e.g., Cinidon-ethyl, Lumicrorac, Flumicrorac-pentyl, and Flumioxazin), Benzoxa Dithioxanone derivatives (e.g., 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluorophenyl) Oro-3,4-dihydro-3-oxo-4-prop-2-ynyl-2H-1,4-benzo Oxazin-6-yl)-1,3,5-triazinane-2,4-dione), flufenpi flufenpyr-ethyl, pyraclonil, and profluazole. Protoporphyrinogen oxidase and the cells, seeds, plants and plants according to the present invention The portion exhibits herbicide tolerance to one or more PPO herbicide(s).

[0061] Herbicides include the plants and seeds provided by the present invention as a method for controlling weeds. The plants and seeds provided by the present invention can be applied to the plant growth area. It has a high tolerance to and therefore can tolerate the application of one or more PPO herbicides. is the recommended commercial rate (1 X) or any fraction or multiple thereof, such as twice the commercially recommended ratio (2X). Herbicide rates are based on the herbicide and formulation and are expressed in pounds acid equivalent per acre (lb ae / acre) or gram acid equivalents per hectare (g ae / ha) or pounds active active ingredient per acre (lb ai / acre) or grams active ingredient per hectare (ga The application of herbicides can be expressed as a percentage of the total area of ​​the soil (i / ha). The application of herbicides can be expressed as a percentage of the total area of ​​the soil (i / ha). The plant growth area may or may not contain weed plants at the time of herbicide application. The herbicidally effective dose of the PPO herbicide(s) for use in controlling weeds in an area is determined based on the growth rate of the plant. The label ratio(s) over the period can range from about 0.1X to about 30X. The 1X label rates for some exemplary PPO herbicides are shown in Table 2. 1 acre is 2.47 105 hectares, 1 pound is equal to 453.592 grams. Herbicide rate is You can convert between imperial and metric units as follows: (lb ai / ac)×1.12=(kg ai / ha) and (kg ai / ha)×0.89= (lb ai / ac). Table 2: Exemplary PPO Herbicides TIFF2025011207000002.tif104155

[0062] Herbicide applications may consist of one, two, or a combination of PPO herbicides, or Any other compatible herbicides may be time-mixed or tank-mixed. The transformants of the present invention can be used to control a variety of dicotyledonous weeds, monocotyledonous weeds, or both. For areas containing phytoplankton, use one herbicide or two or more herbicides (combinations or Multiple applications (e.g., one application before planting) may be used, for example, two applications (e.g., one application before planting). application and post-emergence, or pre-emergence and post-emergence), or three applications (e.g. , pre-planting application, pre-emergence application and post-emergence application, or pre-emergence application and post-emergence application. (Two applications of the same) can be performed.

[0063] As used herein, "tolerance" or "herbicide tolerance" refers to the toxicity of a herbicide when applied. The ability of a plant, seed, or cell to withstand the effects of a herbicide. Herbicide-tolerant crops continue to grow. and are not affected or only minimally affected by the presence of applied chemicals. As used herein, a "herbicide tolerance trait" refers to an improved herbicide tolerance trait compared to a wild-type plant. The herbicide tolerance trait of the present invention is a transgenic trait that confers herbicide tolerance to a plant. Contemplated plants that may be produced with the enzyme include, for example, soybean (e.g., Glycine max), among others. max), corn (Zea mays), cotton (Gossypium sp.), and cabbage Any plant may be mentioned, including crop plants such as nola.

[0064] The transgenic plants, progeny, seeds, plant cells, and plant parts of the present invention may include one or more The additional traits may include the recombinant DNA provided by the present invention. The plants containing the transgene containing the molecule are then cultured to produce another plant containing one or more additional trait(s). As used herein, "crossing" refers to the introduction of a plant that is a member of the same family as the host plant. refers to the crossing of two individual plants to produce a progeny plant. The two plants can then be crossed to produce progeny containing the desired traits from each plant. As used herein, "progeny" refers to any generation of descendants of a parent plant. A transgenic progeny is provided by the present invention and is inherited from at least one parent plant. The introduction of additional trait(s) may also be achieved by this additional transfection. The DNA construct for the genetic trait(s) can be prepared using the compositions provided by the present invention. A DNA construct containing a recombinant DNA molecule (e.g., a recombinant vector used in plant transformation). Co-transform the vector with all DNA constructs present as part of the same vector. or by conferring additional trait(s) on the DNA constructs provided herein. by inserting it into a transgenic plant containing the gene or vice versa (e.g., To plants or plant cells, using either plant transformation or genome editing methods. Such additional traits may include, but are not limited to: , increased insect resistance, increased water use efficiency, increased yield performance, increased drought tolerance, improved seed quality growth, improved nutritional quality, hybrid seed production, and herbicide tolerance (if the trait is not present in wild-type plants) Exemplary additional herbicide tolerance traits include one or more herbicides, such as, among others, ACCase inhibitors (e.g., aryloxyphenoxypropanes, lopionates and cyclohexanediones), ALS inhibitors (e.g., sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones), EPSPS inhibitors (e.g. glyphosate), synthetic auxins (e.g., phenoxy, benzoic, carboxylic acids, semicarbazones), photosynthetic inhibitors (e.g., triazines, triazinones, nitriles, benzoates, etc.), zothiadiazole, and urea), glutamine synthesis inhibitors (e.g., glufosinate), HPPD inhibitors (e.g., isoxazoles, pyrazolones, and triketones), PPO inhibitors Toxicants (e.g., diphenyl ether, N-phenylphthalimide, aryl triazinones) , and pyrimidinediones), and long-chain fatty acid inhibitors (e.g., chloroacetamide, oxidase acetamide, and pyrazole), transgenic or non-transgenic Exemplary insect resistance traits may include, among others, Lepidoptera. Among the orders ra, Coleoptera, Hemiptera, and Homoptera Such additional traits may include resistance to one or more members of the insect family. For example, a list of such transgenic traits is available at the U.S. Department of Agriculture Provided by the USDA Animal and Plant Health Inspection Service (APHIS).

[0065] Cells transformed with a polynucleotide of the invention, such as an expression construct, can be used to The polynucleotide or the like may be used before or after regeneration of such cells into transgenic plants. Such a polynucleotide may be selected for the presence of its encoded enzymatic activity. A transgenic plant containing a nucleotide is, for example, a plant containing the polynucleotide or polypeptide of interest. Contains modified enzyme activity and / or altered compared to another isogenic control plant. Transgenic plants that exhibit the desired trait can then be selected. Such a trait can be, for example, tolerance to a PPO herbicide.

[0066] Transgenic plants containing the transgenic traits provided by the present invention The offspring can be used using any method of breeding known in the art. In plant lines carrying two or more transgenic traits, these transgenes The nicked traits may be independent or linked, and plants with three or more traits may be In a genetic line, a combination of both may be used. Outcrossing with genic plants is also considered since it is a vegetative propagation method. Descriptions of breeding methods used in the present invention are well known to those skilled in the art. To confirm the presence of the transgene(s) in the cells, various assays can be performed. Such assays include, for example, molecular biological assays (e.g., Southern blot assays). and Northern blotting, PCR, and DNA sequencing), biochemical analysis. Assays (e.g., by immunological means (ELISA and Western blot) or enzyme detection of the presence of protein products by their functional groups), plant part assays (e.g., leaf or root assays) and also by analysis of whole plant phenotypes.

[0067] Introgression of a transgenic trait into a plant genotype is a process of backcross transformation. As a result, the plant genotype into which the transgenic trait has been introduced is A desired genotype may be referred to as a cross, line, inbred, or hybrid. Plant genotypes lacking the transgenic trait may be unconverted genotypes, lines, inbreds, or or hybrids.

[0068] Although the present invention has been described in detail, it should be understood that without departing from the scope of the invention as defined in the appended claims, It will be apparent that various modifications, variations, and equivalents are possible without departing from the spirit and scope of the present invention. Furthermore, it should be understood that the examples in this disclosure are provided as non-limiting examples. EXAMPLES

[0069] The following examples are included to demonstrate embodiments of the present invention. The techniques disclosed in the examples have been discovered by the inventors to work well in the practice of the invention. This represents a technique for carrying out the invention and is therefore the preferred technique for carrying out the invention. However, it should be understood that the above may be regarded as constituting a new form of In light of this disclosure, many changes may be made to the specific embodiments disclosed. and still not depart from the concept, spirit and scope of the present invention. It should be understood that similar results can be obtained. More specifically, chemical and physiological Certain agents related in general terms may be substituted for the agents described herein and are the same or similar. It will be apparent that similar results can be achieved. All substitutions and modifications are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. It is considered to be something.

[0070] Example 1: Discovery of microbial protoporphyrinogen oxidase Bioinformatic methods and novel protoporphyrinogen oxidase bacterial screening Using the sequencing system, novel protoporphyrinogen oxidases were identified from microbial sequence databases. As a starting sequence for bioinformatic analysis of microbial sequence databases, E The sequence of HemG from B. coli (SEQ ID NO: 4) was used. Bioinformatics analysis revealed that Thirty-three novel putative protoporphyrins of the HemG PPO family from diverse bacterial sources Phylogenetic mapping was used to identify these putative HemG The sequences encoding the PPO enzymes were compared and found to be relatively diverse. Representation of each unique cluster member on the phylogenetic tree reveals that this group of 33 species Ten species were selected for further analysis.

[0071] The coding sequences of the selected 10 HemG PPO enzymes were found in the wild-type DNA sequence. The DNA was optimized for expression in E. coli to eliminate any rare codons that were expressed. The E. coli optimized coding sequences of ten HemG PPO enzymes were cloned into bacterial expression vectors. I went for a walk. I saw a water lily (Amaranthus tuberculatus) We have investigated the herbicide-sensitive PPO enzymes found in nature with respect to both PPO function and herbicide sensitivity. For use as a control for , shown as SEQ ID NO: 21, GenBank Accession No. ABD52326, Patzold t,et al.“A codon deletion confers resist ance to herbicides inhibiting protoporph yrinogen oxidase”Proceedings of the Nati onal Academy of Science USA.103(33):1232 9-12334(2006)). The water hyacinth PPO enzyme is a HemG family member. The HemG PPO enzyme naturally found in E. coli was used as a counter measure of PPO activity. The vector was cloned into a bacterial expression vector for use as a control and in herbicide susceptibility assays. (designated H_N10 and shown as SEQ ID NO:4).

[0072] Testing the protoporphyrinogen oxidase activity of the recombinant protein and To confirm that the PPO enzyme is functional, protoporphyrinogen oxytocin was used. We developed a bacterial screening system for the . Functional rescue in E. coli strains containing PO enzyme (SEQ ID NO: 4) gene knockout The hemG knockout E. coli strain used was a heme-free bacterium. Growth was very minimal on medium (e.g., LB medium) but not when supplemented with free heme. or expressing active recombinant protoporphyrinogen oxidase in E. coli. Growth was restored when the protein protoporphyrinogen oxidative stress was increased. To quickly and easily assay the sidase activity, we used hemG knockout E. coli. The strain could be used for recombinant protein expression.

[0073] The hemG knockout E. coli strain was designed to express 10 putative HemG PPO enzymes, Ec oli HemG PPO enzyme and the gene encoding the hyumomo PPO enzyme. The bacteria were then transformed with the bacterial expression vector. The expression of recombinant PPO enzyme inhibited the growth of E. coli. The transformed hemG knockout E. The protein sequence was confirmed by growth of the coli strain on LB plates. In this assay, ten phosphodiesterases were shown to function as oxidative stress enzymes. HemG PPO enzyme (SEQ ID NO: 1 to 10) and waterfowl PPO enzyme (SEQ ID NO: 21) was able to restore growth of the transformed hemG knockout E. coli strain, Thus, the PPO activity of these enzymes was confirmed. The consensus positions are shown in Figure 1. Using this assay, a number of novel proteins Alternatively, genetically engineered proteins can be screened to identify protoporphyrinogen oxidase. Enzyme activity can be confirmed and measured.

[0074] Example 2: Protoporphyrinogen oxidase inhibitor insensitivity Using a herbicide bacterial screening system, we identified novel protoporphyrins resistant to PPO herbicides. This screening system identified a phosphodiesterase (PPO)-sensitive genoyltransferase (PPO)-sensitive plant. In order to identify protoporphyrinogen oxidase that is not a PPO herbicide, A growth assay of the hemG knockout E. coli strain in B liquid medium was used.

[0075] The hemG knockout E. coli strain was used to identify protoporphyrinogen oxime. The bacteria were transformed with the expression vector containing the idase activity and cultured in LB liquid medium. Five different PPO herbicides (aciflorphenesin) representing different PPO chemical subclasses were (1 mM), flumioxazin (0.5 mM), lactofen (0.5 mM), homesan Purified crystalline forms of one of the following compounds were added to the 100 μM suspension: S-3100 (1 mM), S-3100 (100 μM), and S-3100 (1 mM). The recombinant protein was expressed and the growth rate of E. coli was measured. Growth curves (OD600) of these various mutants were plotted in the presence and absence of PPO herbicide. , and were measured at selected time points from 0 to 24 hours. The growth of the transformed hemG knockout E. coli strain in the medium indicated that E. coli The gene used for transformation of the herbicide-insensitive protoporphyrinogen oxidase ( It was shown that this gene encodes iPPO.

[0076] A novel protoporphyrinogen oxidase was used in this assay to detect PPO herbicides. The insensitivity of the 10 protoportaviruses shown as SEQ ID NO: 1 to SEQ ID NO: 10 to the drug was tested. All of the hemG genes in LB medium were expressed in the presence of PPO herbicides. It was found to confer normal growth rates to the checked-out E. coli strains, thereby These proteins are herbicide-insensitive protoporphyrinogen oxidases (iPPO ) (Figure 2). The G knockout E. coli strain was sensitive to all five herbicides, which confirmed that The assay identified sensitive and insensitive protoporphyrinogen oxidases for each herbicide. It was confirmed that this assay can distinguish between porphyrinogen oxidase and porphyrinogen oxidase. A large number of novel or engineered proteins are screened using Confirm protoporphyrinogen oxidase activity in the presence of herbicide(s) It is possible.

[0077] Example 3: Protoporphyrinogen oxidase (PPO) enzyme assay Enzymatic characterization of protoporphyrinogen oxidase was performed to identify each Binding affinity of PPO enzyme substrate (K m ) and sensitivity (IC50) to PPO herbicides were measured. Wild-type plant PPO enzymes from water chestnut, soybean, and corn were used to Biological HemG protoporphyrinogen oxidase (SEQ ID NO: 1 to SEQ ID NO: 10) A comparison was made with the results shown in Table 1.

[0078] Etiolated cotyledons (soybean, Glycine max), Etiolated leaves / cotyledon coleoptiles (soybean, Glycine max) Zea mays), and the expanded terminal leaves (Amaranthus tuberculata), generally described by Grossmann et al. Procedure for the treatment of flufenacil (Kixor (T M) )is a New Inhibitor of Protoporphyrino gen IX Oxidase Activity”Weed Science,58( Leucoplasts and chloroplasts were prepared by the method described in the literature (1):1-9 (2010)). ) and corn (LH244) seeds were placed on two moist germinated sheets in a beaker of water. Paper (Anchor Paper Company, Saint Paul, Minnes The plants were placed between 100 mm thick paper (Ota, USA) and kept in a dark room for 8 to 10 days. The tissue was harvested, placed between damp paper towels, and crushed using a mortar and pestle. The mixture was ground to a fine powder in liquid nitrogen using homogenization buffer (50 mM Tris-HCl). l, pH 7.4, 500 mM sucrose, 1 mM EDTA, 1 mM magnesium chloride, and 2 g / liter bovine serum albumin) to the frozen powder in a 4:1 (ml homogenization buffer:g Fresh heavy tissue) and vigorously mixed, then four layers of pre-moistened Miracloth™ were added. (Merck-Millipore, Darmstadt, Germany). The filtrate was centrifuged at 9299 g for 5 min. The pellet was resuspended in homogenization buffer and diluted with water for 150 min. The supernatant was centrifuged at 4000g for 15 minutes. The step was carried out at 4° C. The pellet (intact plasmid fraction) was diluted with 15 mM Tris-HCl. 1H, pH 7.4, 2 mM EDTA and 20% (v / v) glycerol, The total protein in the plasmid preparation was determined using bovine serum albumin (BSA). Using albumin as a standard, the Bradford method (MM Bradford, "Arap id and sensitive method for the quantita tion of microgram quantities of protein utility the principle of protein-dye bin ding”Analytical Biochemistry,72:248-254( 1976).

[0079] Selected PPO enzymes were expressed in E. coli hemG knockout cell lines. Bacterial cells from the overnight culture were used to inoculate 20 ml of fresh medium. These cultures The cultures were grown at 20° C. for approximately 48 hours to obtain high density cultures. The bacterial cells were harvested by centrifugation. The cell pellets were collected and stored at -80°C until enzyme assays were performed. Extraction suspension (50 mM Tris-HCl, pH 7.6, 1 mM EDTA & 1 mM MgCl l2) and sonicated (Sonics VibraCell™, New Town, CT USA) and placed in an ice bath for 30 seconds for 3 cycles with 1 minute rest between cycles. The cells were then centrifuged at 200 g for 2 min at 4°C, and the supernatant was extracted. The exudate suspension was diluted and then used in the PPO enzyme assay. Bradford (1976) The total protein amount was measured using bovine serum albumin as a standard by the method described above.

[0080] Explanation by JM Jacobs and NJ Jacobs ("Measurement of Protoporphyrinogen Oxidase Activity”i nCurrent Protocols in Toxicology(1999)8 .5.1-8.5.13, John Wiley & Sons, Inc.) Commercially available protoporphyrin was reduced with sodium mercury amalgam to produce protoporphyrin. Protoporphyrinogen IX (protogen) was synthesized. Add to 0.01N potassium hydroxide in 20% ethanol and stir in the dark until dissolved. (approximately 40 min.) The volume was then transferred to a 2 ml polypropylene vial with a screw cap containing an O-ring. Add 0.8 ml and about 1 g (one spatula tip, excluding oil) of sodium mercury amalgam. Gum (product number 451908, Sigma-Aldrich, St. Louis, Missouri Souri, stored under oil immersion) was added. The tube was immediately capped and mixed on a vortex mixer. Stir vigorously at room temperature, vent by loosening the lid approximately every 30 seconds, and make sure the solution turns red under UV light. This was done until no more color fluorescence was observed (approximately 5 min). The reaction vial was flushed with argon. The supernatant solution was then washed and briefly centrifuged to pellet any remaining sodium amalgam. The cells were immediately diluted 1:1 (v / v) with a solution of 0.1 M DTT and 0.5 M Tris-HCl, pH 7.5. The solution was diluted (v / v) and the vial was flushed with argon. The resulting solution was divided into smaller aliquots. The solution was divided into portions and placed in 0.5 ml polypropylene tubes with lids, and aliquots were added. The tube was immediately flushed with argon. The capped tube was covered with aluminum foil and - Store at 80° C. For enzyme assays, aliquots of Protogen were thawed and kept covered on ice. The protogen concentration in the preparation was adjusted to the Protogen concentration in the starting material. From the concentration, the Proto concentration in the final protogen solution measured by fluorescent HPLC ( Typically, about 1% of the starting material is subtracted (Matsumoto et al., “Por Phyrin Intermediate Involved in Herbicid al Action of delta-Aminolevulinic Acid o n Duckweed(Lemna paucicostata Hegelm.)”P esticide Biochem.and Phys.48:214-221(199 4) (None of the samples contained significant impurities. Protogen prepared and stored under these conditions will remain stable for at least 6 months. It was stable.

[0081] PPO activity in plant plasmid extract preparations and bacterial extract preparations was generally determined according to the method of Gross Measurements were performed as described by Smann et al. (2010). Plant plasmid extracts (40 μg total protein) or bacterial extracts (various microbial extracts) of The total protein amount of the bacterial extract was either 16 to 35 μg, or 10 0 mM Tris-HCl, pH 7.4, 5 mM DTT, 1 mM EDTA and 0.08 5% (v / v) Tween 80). S-3100 (referred to as "SYN-523") For example, U.S. Patent Publication No. US20100062941A1) in acetone The samples were added as 2 microliter volumes from a 100X stock solution prepared in Grade S-3100 is provided by Sumitomo Chemical Company. All assays were performed with a final concentration of 1% (v / v) acetone. smid or bacterial), buffer, and S-3100 were incubated at 30°C (plant extracts) or 3 Incubate for 5 minutes at 7°C (bacterial extract) and then add 2 microliters of protogen. All assays were performed in 96-well black polystyrene microplates. Titer plate (Costar® 3925, Corning, Inc., C The experiments were carried out in a 350mm x 350mm glass tube (Blooming, New York) in a final volume of 200 microliters. After adding rotogen to all wells (IC 50 For measurements, 3 μM, K m Regarding measurements variable), and plates were incubated at 30°C (plant extracts) or 37°C (bacterial extracts). Data collection began after incubation at 30°C (plant extracts) or 37°C (bacterial extracts). The fluorescence over time was measured at excitation and emission wavelengths of 405 and 630 nm, respectively. , SpectraMax® M5 Multimode Microplate Reader (Mo (Ophthalmology Department, Sunnyvale, California) Heat-inactivated (100°C for 5 min) extracts were added to the assay mixture. An assay blank was then run.

[0082] Substrate (protoporphyrinogen) binding affinity of protoporphyrinogen oxidase. Sexuality m The apparent K for each PPO evaluated was m Value, Soft Pro® Kinetics Software Package (Molecular Devices s, Sunnyvale, California) using a rectangular hyperbola curve fit. The enzyme activity sensitivity to the PPO herbicide S-3100 was calculated based on the 50% inhibition of the control activity. Concentration (IC 50 The S-3100 activity against each PPO evaluated was measured as I C 50 Values ​​were determined graphically from a semi-log plot of S-3100 concentration versus PPO activity.

[0083] PPO enzymes from three plant sources (water hyacinth, soybean, or corn) and K for microbial HemG PPO enzymes (SEQ ID NO: 1 to SEQ ID NO: 10) m is 0.3uM The PP concentrations of the three plants tested were found to be similar, ranging from 0.01 to 0.2 μM. O enzymes, IC 50 The values ​​are 0.009, 0.004, and 0.003 uM, In contrast, the PPO enzyme from a bacterial source (SEQ ID NO:1) was sensitive to S-3100. ~SEQ ID NO: 10) is IC 50 The value was over 100uM and insensitive to S-3100. The data are shown in Table 3. Table 3. PPO enzyme activity of enzymes purified from plant or microbial sources TIFF2025011207000003.tif83155

[0084] Example 4: Enzyme optimization of protoporphyrinogen oxidase Protein optimization was used to characterize the enzyme properties of a novel protoporphyrinogen oxidase. Improve or change the enzyme's properties. Optimize the enzyme using one or more protein engineering methods. Non-limiting examples of protein engineering techniques include alanine scanning mutagenesis, homology Scanning mutagenesis, Pro / Gly scanning mutagenesis, domain swapping or mutation These include heterologous introduction, and combinations of these various techniques (M Lehmann and d M Wyss,Current Opinion in Biotechnolog y 12(4):371-375(2001), B Van den Burg and VGH Eijsink,Current Opinion in Biotechn ology 13(4):333-337(2002), and Weiss et al. ,Proceedings of the National Academy of (See Sciences USA 97(16):8950-8954 (2000)). A DNA sequence encoding genetically engineered protoporphyrinogen oxidase was synthesized and transformed into a cellular The vector is cloned into a bacterial expression vector, which is the initial high throughput vector described in Example 1. Characterization of hemG knockout E. coli strains for bacterial rescue screening A genetic engineering process to rescue hemG knockout E. coli strains For toporphyrinogen oxidase, the bacterial growth assay described in Example 2 was used. Screen for susceptibility to one or more PPO herbicides. The transformed hemG knockout E. coli was grown on media with and without PPO herbicide. The genetically engineered mutants that show tolerance to PPO herbicides were expressed in a bacterial expression system. and using the purified protein in a continuous fluorimetric assay as described in Example 3. Detailed biochemical characterization will be performed. Cloning into plant transformation vectors Genetically engineered mutants that are insensitive to PPO herbicides for plant transformation and plant testing. Select the variant.

[0085] Example 5: Expression and testing of HemG PPO enzyme in maize The microbial HemG PPO enzyme was expressed in transgenic maize plants. The transgenic plants were then analyzed for PPO herbicide tolerance. A recombinant DNA molecule encoding one of the microbial HemG PPO enzymes shown in A plant transformation vector was constructed containing the following DNA sequence encoding the PPO enzyme: It may contain a codon for methionine at the end, commonly known as the initiation codon, or This codon is removed so that the transit peptide sequence is operably linked to the 5' end of the coding sequence. The PPO enzyme protein sequence containing methionine at the amino terminus can be used to promote the synthesis of methionine. Exemplary sequences are provided as SEQ ID NOs: 1 to 10. PP Examples of O-enzyme protein sequences are provided as SEQ ID NOs: 11 to 20. To this end, the nucleotide sequence encoding the putative PPO enzyme was prepared for expression in dicotyledons or monocotyledons. The codons were optimized. Table 4 shows the sequence numbers of the proteins and the sequences in the transformation vectors. 1 shows the nucleotide sequence of the microbial HemG PPO enzyme. Table 4. SEQ ID NOs corresponding to PPO mutants TIFF2025011207000004.tif86166

[0086] To express HemG PPO H_N10 (SEQ ID NO: 4), two different promoters were used. Motor + leader + intron combination, two different target peptide (TP) sequences Four plant transformation vectors were constructed using the 3′UTR sequence and two different 3′UTR sequences. The transformation constructs were annotated constructs 1, 6, 11, and 16. For the maize plant test, Agrobacterium tumefaciens was used. ns and immature maize (LH244) embryos using standard methods known in the art. was transformed.

[0087] Plant traits containing a gene encoding HemG PPO H_N10 (SEQ ID NO: 4) Transformation constructs 6 and 16 were used to generate transgenic maize plants. Leaf samples were collected from R0 plants and screened by PCR to identify the plant genome. The copy number of the transgene inserted into the plant was determined. The two plants (P. cereus) were inbred and outbred to produce R1 and F1 seeds, respectively, for further testing. Plants containing multiple copies (multi-copy) were sprayed as follows: (1) 5 g / ha of S-3100 at approximately the V5 growth stage, then 1 g / ha at approximately the V7 growth stage. 0 g / ha of S-3100, a total application of 15 g / ha of S-3100, or (2) S-3100 at 10g / ha at approximately V5 growth stage. The average percentage of injury was 0- On a scale of 100, where 0 is no damage and 100 is complete crop death, a final disposal was performed for each batch. The evaluation was performed 7 days after the treatment. Used as a control, this was treated with a total of 15 g / ha of S-3100 (treatment 1 ) showed an average of 43.3% damage when treated with a total of 10 g / ha of S-3100 (treatment The average damage to the 26.7% of the treated area was 26.7%. The transgenic corn plants outperformed their control plants, with a total Treatment with 5 g / ha of S-3100 (treatment 1) resulted in an average of 28.9% damage, with a total of 10 When treated with S-3100 at 100 g / ha (treatment 2), the average damage was 24.2%. The data is shown in Table 5. Table 5: Herbicide tolerance of transgenic maize I TIFF2025011207000005.tif39155

[0088] HemG PPO enzymes H_N90 (SEQ ID NO: 1), H_N10 (SEQ ID NO: 4), H_N 60 (SEQ ID NO: 3), H_N110 (SEQ ID NO: 10), and H_N40 (SEQ ID NO: 6) Plant transformation constructs 6, 20, 21, and 2, respectively, containing genes encoding 2, and 23 were used to generate transgenic maize plants. The constructs consisted of the same promoter+leader+intron combination, two different The leaf samples were collected from R0 plants and had the same target peptide (TP) sequence and the same 3'UTR sequence. The clones were collected from the samples and analyzed by PCR to determine the copy number of the introduced gene. or transplant and inbreed single copy plants into pots for up to 12 unique events. and outcrossing were performed to generate R1 and F1 seeds, respectively, for future testing. 40 grams / h for copy plants and extra single copy plants at approximately V5 growth stage The plants were sprayed with S-3100 (a) and the damage was evaluated 7 days after treatment. The average percent (%) of all plants damaged and those considered well tolerated (less than 10% damaged) were The number of plants was recorded. Each plant represented a unique single-copy or multicopy transgenic line. Transgenic maize plants expressing H_N10 (SEQ ID NO: 4) The overall average injury was 39.5%, with 31 tolerant plants out of 139 plants tested. Transgenic maize expressing H_N60 (SEQ ID NO: 3) was produced. Koshi plants had an overall average injury rate of 55.8%, with 19 resistant plants out of 86 plants tested. The transgenic plants expressing H_N90 (SEQ ID NO: 1) were generated. Corn plants had the lowest overall average damage of 31.4% out of the 99 plants tested. We generated 44 well-tolerated plants expressing H_N40 (SEQ ID NO: 6). The average overall damage rate for the 98 corn plants tested was 47.0%. Produced 53 well-tolerated plants from plants, with the highest percentage of well-tolerated plants produced Transgenic corn plants expressing H_N110 (SEQ ID NO: 10) were The average injury was 43.0%, but did not produce any highly tolerant plants. Shown in Table 6. Table 6: Herbicide tolerance of transgenic maize II TIFF2025011207000006.tif51155

[0089] The data from R0 transgenic maize demonstrated that five microbial species HemG PPO enzymes H_N90 (SEQ ID NO: 1), H_N10 (SEQ ID NO: 4), H_N6 0 (SEQ ID NO: 3), H_N40 (SEQ ID NO: 6), and H_N110 (SEQ ID NO: 10), When expressed in transgenic plants, it results in reduced damage rates, thus improving crop health. The advantage of this approach is that it confers tolerance to PPO herbicides.

[0090] In one of two construct configurations, H_N10 (SEQ ID NO: 4) is expressed. Herbicidal properties of transgenic F1 plants generated from outcrossing of single copy R0 plants Tolerance was tested in a greenhouse. The plants were treated with 40 g / ha of S-3 at the V3 growth stage. The rats were treated with 100 mg of H and the damage was evaluated 7 days after treatment. In transgenic corn plants expressing _N10 (SEQ ID NO: 4), 18 events Thirteen of the events resulted in highly tolerant plants (<10% damage), but None of the 16 constructs produced well-tolerated plants in any event.

[0091] In one of the two construct configurations (constructs 6 and 16), H Transgenic plants generated from outcrossing of single copy R0 plants expressing N10 (SEQ ID NO: 4) The herbicide tolerance of the transgenic F1 plants was tested in the field. The F1 population was a segregating population (5 0% hemizygous, 50% defective), and no selection of transgenic plants was performed prior to injury assessment. It was difficult to distinguish non-transgenic plants from transgenic plants. Therefore, the overall mean damage rating for such a population is It is expected that the results will be higher than those of the group. The trial will be conducted at two locations, and the construct Two replicates and three treatments were performed per plant. Non-transgenic plants were used as negative controls. Herbicide application treatments were as follows: Treatment 1 was V2, then V4, Then V8 applied 0.036 lb ai / acre of S-3100; treatment 2 was V2; V4, then V8 with 0.072 lb ai / acre of S-3100; treatment 3 was Apply 0.144 lb ai / acre of S-3100 in V2, then V4, then V8. Percentage damage ratings were performed at the V2 growth stage (CIPV2) and V4 growth stages 5-7 days after treatment. Error V2 and Error V4 were evaluated using the least significant difference (LSD). Crop damage ratings at both locations were combined. All non-transgenic Plants treated with Construct 16 were cultured in three different treatments. For this, losses between 94.6 and 99.5% were observed after herbicide application in both V2 and V4. Plants with construct 6 exhibited injury after herbicide application of V2. It showed only 30% to 50% damage and no damage after application of V4 herbicide. The data is shown in Table 7. Table 7. Field trials of F1 corn containing H_N10 (SEQ ID NO: 4) Effectiveness TIFF2025011207000007.tif66158

[0092] Greenhouse and field data on F1 transgenic maize demonstrated that , the microbial HemG PPO enzyme H_N10 (SEQ ID NO: 4) is introduced into the transgenic plant When expressed in plants, it reduces the rate of injury, thereby improving crop tolerance to PPO herbicides. The first step is to grant the following:

[0093] Example 6: Expression and testing of HemG PPO enzyme in soybean plants The microbial HemG PPO enzyme was expressed in transgenic soybean plants. The transgenic plants were analyzed for PPO herbicide tolerance. SEQ ID NO: 13), H_N20 (SEQ ID NO: 12), H_N30 (SEQ ID NO: 14), H_N4 0 (SEQ ID NO: 15), H_N50 (SEQ ID NO: 16), H_N90 (SEQ ID NO: 11, 18, 19), and the microbial HemG PP designated H_N100 (SEQ ID NOs: 17, 20). A plant transformation vector is constructed containing a recombinant DNA molecule encoding one of the O enzymes. Built it.

[0094] In soybean, A. tumefaciens and standard methods known in the art are used. Using the plasmid p53, excised embryos (A3555) were transformed with transformation constructs 1 and 11. Transformation constructs 1 and 11 contain the same promoter+leader+intron combination. The same 3'UTR sequence and the same microbial HemG PPO H_N10 (SEQ ID NO: 4) were used. The target peptide (TP) sequence was different. Seedlings were grown in a greenhouse. 20 single copy R0 clones generated from construct 11 were In a plant representative of the genus event and expressing the microbial PPO enzyme H_N10 (SEQ ID NO: 4), Flumioxazin (Valor®, Valent USA) at 10 g / ha Corporation, Walnut Creek CA) was sprayed. The herbicide was sprayed on V3 growth stage plants with three trifoliate leaves, and injury ratings were recorded 8 hours after treatment. The percentage of plants that were considered tolerant (<15% damage) was After application of 210 g / ha flumioxazin, the yield of non-transgenic soybeans vs. The average damage to the exposed plants was 30%, and no plants were highly tolerant. Soybean plants expressing the enzyme H_N10 (SEQ ID NO: 4) had an average injury rating of 22%; 9% of the plants tested were able to tolerate the herbicide.

[0095] Representing 10 multicopy R0 soybean events generated from construct 11, Plants expressing the PPO enzyme H_N10 (SEQ ID NO: 4) were treated with 5 g / ha of S-3100. The herbicide was sprayed on the V3 growth stage with three fully developed trifoliate leaves, and the damage was Damage assessment was performed 8 days after treatment. Plants considered to be well tolerated (less than 25% damage) After application of S-3100 (5 g / ha), the percentage of non-transgenic Soybean control plants had an average injury of 60% and no plants were highly tolerant. Soybean plants expressing the bioPPO enzyme H_N10 (SEQ ID NO: 4) had an average injury rating of 47. %, and 30% of the plants tested were well tolerated by the herbicide.

[0096] Single copy transgenic R1 soybean plants were treated with three herbicides in a greenhouse: Treatment 1 was sprayed at V4, followed by R1 at 5 g ai / ha of S. -3100 was applied, and treatment 2 was V4, followed by R1 with 10 g ai / ha of S-3100. Treatment 3 was V4, followed by R1 with 30 g ai / ha of S-3100. Crop injury percentage at 2 (CIPV2) was assessed 10 days after treatment. Transgenic plants had an average injury rating of 89% to 100% at the R1 growth stage. The PPO enzyme produced from construct 1 and the microbial PPO enzyme Plants expressing the enzyme H_N10 (SEQ ID NO: 4) had injury ratings ranging from 3% to 15.7%. The data are shown in Table 8. Table 8: S-3100 Efficacy Screening of R1 Soybeans in the Greenhouse TIFF2025011207000008.tif54164

[0097] Transgenics using high-throughput plant transformation and screening methods Herbicide tolerance of a number of constructs in plants was evaluated in early transgenic seedling tissues. This allows for faster and larger-scale screening of constructs and PPO enzymes. It has become possible to do this.

[0098] Seven microbial HemG PPO enzymes H_N10 (SEQ ID NO: 13), H_N20 (SEQ ID NO: No. 12), H_N30 (sequence number 14), H_N40 (sequence number 15), H_N50 (sequence number Row number 16), H_N90 (SEQ ID NOs: 11, 18, and 19), and H_N100 (SEQ ID NO: 17, 20) were operably linked to 37 different target peptides, The gene expression was then cloned into the base plant transformation vector. Using the 3'UTR and 3'UTR elements, seven different HemG PPO enzymes and 37 Side-by-side comparison of different target peptides is now possible. These plant transformation constructs were transformed into soybean truncations using standard methods known in the art. For the transformation of engrafted germplasm (A3555), 400 explants were used for each construct. The constructs were inoculated into 12 containers, resulting in 12 containers per construct. The solution was used in the herbicide tolerance test. The herbicide solution was a crop oil concentrate (5.0 mL). and 0.3 g of S-3100 in 495 mL of deionized water. Nalgene® Rapid-Flow™ Tissue Culture Filter Unit knitted and surfactant-free cellulose acetate membrane filter units (VWR, The solution was filtered through a filter (Radnor, PA, USA) and the resulting sterile solution was shaken before application.

[0099] At 5 weeks post-transformation, 4 of 12 plant containers per construct were The sterile PPO herbicide solution was sprayed twice. The treated seedlings were then sealed in containers and each day after spraying, The plants were exposed to at least 15 hours of light for 4 days. At the end of the 4th day after application of S-3100, The treated seedlings were then photographed and the green coloration (green coloration indicates healthy light-induced changes in tissue compared to photobleached tissue) was observed. The damage was scored by a visual scale of the damage to the plant tissue (representing the composite plant tissue). 1 is good tolerance, high damage, low green coloration; 2 is some tolerance, average damage, medium 1 showed green coloring, and 2 showed good tolerance, low damage, and high green coloring. The scores are presented in Table 9. nd indicates that no analysis was performed. The results are In this high-throughput screen, multiple constructs were identified that were able to bind to PPO herbicides. This indicates that the drug was tolerable. Table 9. High-throughput soybean screening for herbicide tolerance: Color scores TIFF2025011207000009.tif219155

[0100] Using standard methods known in the art, constructs with a score of 2 were identified as Seedlings from non-misted containers were transplanted approximately 7 weeks after transformation and grown as R0 plants. Seedlings corresponding to intolerant scores of 0 and 1 were also grown to serve as negative controls. under long-day conditions (18 hours of light at 80°F, followed by 6 hours of darkness at 74°F) for approximately 4 weeks. At 11 weeks, the plants were treated with the same herbicide solution (0.3 g of S-3100 ) was sprayed twice on R0 plants. Herbicide injury ratings were collected 7 days after treatment.

[0101] Application of herbicide tolerance to R0 plants at 11 weeks resulted in high through-put at 5 weeks. This supports the low percentage damage rating scores observed in the put screening. Damage ratings of 30% or more correspond to those of non-transgenic soybeans. Some constructs stood out in that they showed very good tolerance to herbicide application. For example, when PPO H_N90 (SEQ ID NO: 11) was used in TP1, only or 3% damage, PPO H_N30 (SEQ ID NO: 14) or H_N40 (SEQ ID NO: 15) Only 5% damage was observed with PPO H_N90 (SEQ ID NO: 11) at TP20. ) caused only 5% damage. N90 (SEQ ID NO: 11) was used with a 50% injury score. The data are shown in Table 10. nd indicates that the analysis was not performed. Table 10. Herbicide Tolerance of R0 Transgenic Soybeans: Percent Injury Scores TIFF2025011207000010.tif214155

[0102] To further evaluate the plant transformation constructs in soybean, A. tumefa ciens plant transformation vectors and standard methods known in the art. Transform the de-embryoed (A3555). Grow the regenerated R0 transgenic seedlings in a greenhouse. These groups are then sprayed with one or more PPO herbicides. Assess herbicide tolerance. For example, R0 transgenic plants at approximately the V2 to V4 growth stages Plants were treated with lactofen at 110 g ai / ha (0.09 lb ai / acre). or spray at a rate of 220 g ai / ha (0.19 lb ai / acre). After 1 to 14 days from the transplantation, the plants are evaluated for damage and the damage score is recorded. Leaf samples to identify transgenic plants carrying a single copy of the A insertion The R0 plants that contain only a single copy and pass the herbicide spray test were isolated using Cross to generate R1 seeds.

[0103] The R1 plants are grown in a greenhouse and divided into groups. Each group is assigned one or more species. To evaluate herbicide tolerance, spray the PPO herbicide Lactoferrin. 200 g ai / ha (0.19 g / ha) of fertilizer was administered pre-emergence and / or at V2-V6 growth stages. The plants were then assessed for injury 1-14 days after treatment. The injury score is recorded. Transgenic plants that have not been sprayed are placed in a field Used for phenotypic comparison with live plants.

[0104] Homozygous transgenic R1 plants were inbred and seeds were collected to produce R2 plants. Plants are generated. R2 plants are evaluated in one or more field locations or in a greenhouse assay. Herbicide treatments were applied and plots or plants were scored for crop damage 1-14 days after herbicide application. Rating is done on a scale of ~100, with 0 being no damage and 100 being complete crop death.

[0105] Example 7: Leaf disc assay Rapid herbicide tolerance in transgenic plants expressing recombinant PPO enzymes To assess the plant growth rate and damage with minimal damage, a leaf disk assay was used. Young, fully green leaf tissue was sampled from soybean plants. Biopsy punch with jar (Integra® Miltex®, Inc.) Leaf samples were cut into 4 mm diameter pieces using a 4 mm diameter slit cutter (York, Pennsylvania). Cut out five leaf disks and place these leaf disks in a 24-well polystyrene plate with a lid. In the plate, add 1 ml of incubation solution (1 mM MES, pH 6.5, 1% w / v sucrose, 1% (v / v) acetone). S-3100 was added to the solution to a final concentration of 1 micromolar. Vacuum filtration was applied. Incubate the leaf disk plate at room temperature (23-24°C) in continuous darkness for 1 day. Then, place them under fluorescent and incandescent lamps (520uE) on the ceiling at 26-27℃ for 1 day (soybean). The leaf discs were then incubated in a continuous light period of 10 days (corn) or 2 days (honeybush). The lesions were visually scored on a scale of 0 (lowest damage) to 4 (highest damage). The lower the value, the better the tolerance to PPO herbicides.

[0106] A transfectant using construct 6 and expressing the PPO enzyme H_N10 (SEQ ID NO: 4) Leaf discs from transgenic corn plants had an average leaf disc score of 0 The results showed that the .4-based .4-transformed . and the PP observed in whole plants expressing the PPO enzyme H_N10 (SEQ ID NO: 4). Construct 1 and Construct 11 were used to confirm the herbicide tolerance. Transgenic soybean plants expressing the PPO enzyme H_N10 (SEQ ID NO: 4) were Leaf disks from the specimens showed no significant damage to the plants from construct 1, with a zero injury rating. Construct 11 plants had an injury rating of 1.6 and were tolerant to the herbicide, whereas construct 2 ...2 plants had an injury rating of 1.6 and were tolerant to the herbicide, whereas construct 23 plants had an injury rating of 1.6 and were tolerant to the herbicide, whereas construct 24 plants had an injury rating of 1.6 and were tolerant to the her The sugenic plants were shown to have an injury rating of 2.6.

[0107] Example 8: Expression and testing of HemG PPO enzyme in cotton The microbial HemG PPO enzyme was expressed in transgenic cotton plants and To analyze the PPO herbicide tolerance of transgenic plants, A. tumefaciens was used in cotton. faciens and excise embryos with these vectors using standard methods known in the art. (Coker130) was transformed. The regenerated R0 transgenic seedlings were grown in a greenhouse. These groups are divided into groups containing PPO herbicides (one PPO herbicide per group). For example, at the growth stage of about 2 to 4 true leaves, For R0 transgenic seedlings, lactofen was administered at 110 g ai / ha (0.09 lb ai / acre) or 220g ai / ha (0.19 lb ai / acre) The plants are evaluated for damage 1-14 days after treatment and an injury score is recorded. Leaves The samples were used to identify transgenes carrying a single copy of the transgenic insertion. Identify the R plants that contain only a single copy and pass the herbicide spray test. 0 plants are inbred to produce R1 seeds.

[0108] The R1 plants are grown in a greenhouse and divided into groups. Each group is assigned one or more species. To evaluate herbicide tolerance, spray the PPO herbicide Lactoferrin. Apply 200g ai / ha (0. Apply at a rate of 19 lb ai / acre (1X). Plants should be fertilized 1-14 days after treatment. The damage is assessed and the damage score is recorded. The unsprayed transgenic plants are compared to the sprayed plants. Used for phenotypic comparison with wild-type plants not subjected to

[0109] Homozygous transgenic R1 plants were inbred and seeds were collected to produce R2 plants. Plants are generated. R2 plants are evaluated in one or more field locations or in a greenhouse assay. Herbicide treatments were applied and plots or plants were scored for crop damage 1-14 days after herbicide application. Rating is done on a scale of ~100, with 0 being no damage and 100 being complete crop death.

Claims

1. At least 85% to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1 to 20 a heterologous promoter operably linked to a nucleic acid sequence encoding a protein having sequence identity to A recombinant DNA molecule comprising a promoter, said protein being a herbicide-insensitive protopor. A recombinant DNA molecule as defined above, which has fibrinogen oxidase activity.

2. The combination of claim 1, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 22 to 63. Recombinant DNA molecules.

3. The protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-20. The recombinant DNA molecule of claim 1.

4. 2. The recombinant DNA molecule of claim 1, wherein the heterologous promoter functions in a plant cell. 。

5. The nucleic acid sequence functions to localize the operably linked protein intracellularly. The recombinant vector of claim 4, operably linked to a DNA molecule encoding a target sequence. DNA molecule.

6. A DNA construct comprising the recombinant DNA molecule of claim 1.

7. A targeting sequence in the recombinant DNA that functions to localize the protein within a cell. The DNA construct of claim 6, comprising an operably linked DNA molecule encoding Kut.

8. The DNA construct of claim 7, wherein the protein confers herbicide tolerance to the cell. Tract.

9. The gene of claim 6, which is present in the genome of a transgenic plant, seed or cell. DNA constructs.

10. A sequence that is at least 85% of the full length amino acid sequence selected from SEQ ID NOs: 1 to 20 A recombinant polypeptide having an identity to a herbicide-insensitive protoporphyrinogen The recombinant polypeptide has an oxidase activity.

11. A transgenic plant, seed, cell, comprising the recombinant DNA molecule of claim 1. or plant parts.

12. The transgenic plant of claim 11, comprising an additional transgenic herbicide tolerance trait. genic plants, seeds, cells, or plant parts.

13. Claims: The claimed invention is defined as having herbicide tolerance to at least one PPO herbicide. Item 12. A transgenic plant, seed, cell, or plant part according to item 11.

14. The seed according to claim 11.

15. A transgenic plant, seed, cell, or the like, comprising the recombinant polypeptide of claim 10. A vesicle, or plant part.

16. 1. A method for conferring herbicide tolerance to a plant, seed, cell, or plant part, comprising the steps of:

10. The recombinant polypeptide according to claim 19, wherein the polypeptide is a heterologous polypeptide of the present invention. The method further comprising expressing the

17. The plant, seed, cell, or plant part is endowed with the recombinant polypeptide.

17. The method of claim 16, comprising protoporphyrinogen oxidase activity.

18. The herbicide tolerance is acifluorfen, fomesafen, lactofen, fluoroglucan, Lycophen-ethyl, oxyfluorfen, flumioxazin, azafenidine, carbamide Fentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadial Gil, Oxadiazon, Pyraflufen-ethyl, Saflufenacil, and S-3100 Claims 1 to 5, wherein the at least one PPO herbicide is selected from the group consisting of:

16. The method according to claim 16.

19. A method for transforming a plant, comprising the steps of: a) introducing the recombinant DNA molecule of claim 1 into a plant cell; b) regenerating therefrom a plant containing said recombinant DNA molecule. Law.

20. further comprising the step of selecting a plant that is tolerant to at least one PPO herbicide.

20. The method of claim 19.

21. The regenerated plant is crossed with itself or with a second plant and seeds are harvested from the cross.

20. The method of claim 19, further comprising the step of collecting.

22. A method for controlling weeds in a vegetative growth area, comprising the step of: Contacting the nicked plant or plant growth area containing the seed with at least one PPO herbicide. wherein the transgenic plant or seed is tolerant to the PPO herbicide. and weeds are controlled in said vegetative growth area.

23. Nucleotide encoding a protein with protoporphyrinogen oxidase activity A method for identifying a nucleic acid sequence comprising the steps of: a) E. coli with a gene knockout of the native E. coli PPO enzyme The strains were transformed with bacterial strains containing recombinant DNA molecules encoding candidate herbicide tolerance proteins. Transforming with the present vector; b) growing the transformed E. coli in heme-free bacterial medium; wherein growth in said bacterial culture medium results in protoporphyrinogen oxidase activity. and identifying a protein having the gene.

24. A protein having herbicide-insensitive protoporphyrinogen oxidase activity is coated 1. A method for identifying a nucleotide sequence that encodes a nucleic acid sequence comprising the steps of: a) E. coli with a gene knockout of the native E. coli PPO enzyme The strain was transformed with a bacterial expression vector containing a recombinant DNA molecule encoding a recombinant protein. and b) inoculating the transformed E. coli with at least one PPO herbicide-containing medium. The growth of the bacteria is performed using a bacterial medium, and the growth of the bacteria is performed using a herbicide-insensitive protoporphyrin. identifying a protein having endothelinogen oxidase activity; and The method comprising:

25. 1. A method for screening for herbicide tolerance genes, comprising: a) expressing the recombinant DNA molecule of claim 1 in a plant cell; b) identifying a plant cell that exhibits herbicide tolerance to the PPO herbicide. 。

26. 1. A method for screening for herbicide tolerance genes, comprising: a) expressing the recombinant DNA molecule of claim 1 in a bacterial cell lacking HemG; wherein the bacterial cells are grown in heme-free bacterial medium in the presence of a PPO herbicide. and b) identifying a bacterial cell that exhibits tolerance to the PPO herbicide.

27. A method for producing a plant that is tolerant to a PPO herbicide and at least one other herbicide, 、 a) obtaining a plant according to claim 11; b) providing said transgenic plant with tolerance to said at least one other herbicide. and crossing the resulting plant with a second plant obtained from the first plant. c) from said cross with tolerance to PPO herbicide and said at least one other herbicide; and selecting a progeny plant obtained from the plant.

28. 1. A method for reducing the occurrence of herbicide-tolerant weeds, comprising: a) cultivating the plant of claim 12 in a crop growing environment; b) applying a PPO herbicide and at least one other herbicide to said crop growing environment. wherein the crop plant is tolerant to the PPO herbicide and the at least one other herbicide. and applying said composition which is a composition having a therapeutic effect.

29. The PPO herbicide is acifluorfen, fomesafen, lactofen, fluoroglucan, Lycophen-ethyl, oxyfluorfen, flumioxazin, azafenidine, carbamide Fentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadial Gil, Oxadiazon, Pyraflufen-ethyl, Saflufenacil, and S-3100 29. The method of claim 28, selected from the group consisting of:

30. The at least one other herbicide is an ACCase inhibitor, an ALS inhibitor, an EPSPS Inhibitors, synthetic auxins, photosynthesis inhibitors, glutamine synthesis inhibitors, HPPD inhibitors, PP 29. The method of claim 28, wherein the inhibitor is selected from the group consisting of an O inhibitor, and a long chain fatty acid inhibitor.

31. The ACCase inhibitor is an aryloxyphenoxypropionate or a cyclohexyl aryloxypropionate. xanthanedione, and the ALS inhibitor is a sulfonylurea, an imidazolinone, a triazine azolopyrimidine or triazolinone, and the EPSPS inhibitor is glyphosate wherein the synthetic auxin is a phenoxy herbicide, a benzoic acid, a carboxylic acid, or a semi- carbazone, and the photosynthesis inhibitor is a triazine, triazinone, nitrile, benzo thiadiazole, or urea, and the glutamine synthesis inhibitor is glufosinate. wherein the HPPD inhibitor is an isoxazole, a pyrazolone, or a triketone; The PPO inhibitor is diphenyl ether, N-phenylphthalimide, aryl triphenyl ether, or the long chain fatty acid inhibitor is a chloroacetone or a pyrimidinedione.

31. The method of claim 30, wherein the aryloxy group is acetamide, oxyacetamide, or pyrazole.

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