Methods and compositions for herbicide tolerance in plants

Abstract

This invention provides new herbicide-tolerant traits that are needed in response to the increasing number of weed species that have developed resistance to commonly used herbicides. [Solution] The present invention relates to biotechnology and provides novel recombinant DNA molecules and genetically modified 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 molecule, as well as methods for using them.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is U.S. Provisional Patent Application No. 62 / 200,428, filed on August 3, 2015. The company claims priority rights, and the entirety of its disclosure is incorporated herein by reference.

[0002] Incorporation of sequence lists 69.4KB (measured on MS-WINDOWS), created on July 27, 2016. The sequence listing contained in the file named MONS383WO_ST25.txt is electronic This specification is filed together with this specification upon application and is incorporated herein by reference.

[0003] background Field of Invention This invention relates to the field of biotechnology. More specifically, this invention relates to the protocol This enzyme provides tolerance to herbicides by inhibiting ruphyrinogen oxidase. , concerning recombinant DNA molecules. [Background technology]

[0004] Related technologies Transgenic materials are often produced using biotechnology in agricultural crop production. Nick traits are utilized. Heterogenes (also known as transgenes) are transgenic. It can be introduced into plants to generate nic traits. In the expression of the introduced gene in plants... This confers traits such as herbicide tolerance to plants. Transgenic herbicide-tolerant form Examples of quality include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance. This can happen. With the increase in weed species that have developed resistance to commonly used herbicides, new Herbicide tolerance traits are required in this field. In particular, the target herbicide is P Herbicides that inhibit rotoporphyrinogen oxidase (PPO) (referred to as PPO herbicides) (This can be mentioned.) PPO herbicides provide control over various herbicide-resistant weeds, so this The traits that confer tolerance to these herbicides can be combined with one or more other herbicide tolerances. It is particularly useful in combined cultivation systems.

[0005] Protoporphyrinogen oxidase plays a role in the biosynthesis pathways of both chlorophyll and heme. It can convert protoporphyrinogen IX to protoporphyrin IX. After phylline IX is formed, the chlorophyll and heme biosynthesis pathways use different metal ions (heme is iron). Chlorophyll incorporates magnesium and branches. This pathway segment is prokaryotic. It is conserved across both prokaryotes and eukaryotes, and is found across both prokaryotes and eukaryotes. Many of the PPO enzymes are relatively similar. Some prokaryotes (e.g., cyanobacteria) This pathway is used for chlorophyll and heme production, while other prokaryotes (e.g., Esche) Richia coli uses this pathway for heme production.

[0006] Herbicide-insensitive protoporphyrinogen oxider from multiple prokaryotes and eukaryotes Ze ("iPPO") has been isolated. Based on its structure, at least three types are listed below. It is thought that different subclasses of PPO enzymes exist: HemY(Hansson) and Hederstedt,“Cloning and Characteriz ation of the Bacillus subtilis hemEHY ge one 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 an d functional complementation” Applied and Environmental Microbiology 77(14):4795 - 4801(2011)). The present invention provides a novel recombinant iPPO that is a member of the HemG family. Despite 20 years of research and the number of iPPOs identified so far, transgenic crop plants containing recombinant iPPO have not yet been commercialized. A strong weed control platform depends, in part, on the continued development of herbicide tolerance trait packages. Therefore, identifying and utilizing iPPO to create transgenic crop traits represents an advancement in agriculture.

Summary of the Invention

[0007] In one aspect, the present invention is directed to a polypeptide sequence selected from SEQ ID NOs: 1 - 20 ​A recombinant DNA molecule comprising a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide having at least 85% sequence identity, wherein the polypeptide has herbicide-insensitive protoporphyrinogen oxidase activity. In certain embodiments, the polypeptide has at least about 85% sequence identity, at least about 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity to a polypeptide selected from the group consisting of SEQ ID NOs: 1-20, and has herbicide-insensitive protoporphyrinogen oxidase activity. In some embodiments, a recombinant DNA molecule is provided wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 22-63. In certain embodiments, the recombinant DNA molecule encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-20. Thus, the present invention provides a recombinant polypeptide having at least 85% sequence identity to the full-length amino acid sequences selected from the group consisting of SEQ ID NOs: 1-20, and having herbicide-insensitive protoporphyrinogen oxidase activity. In certain embodiments, a heterologous promoter, such as a promoter that functions in plant cells, is operably linked to a nucleic acid sequence encoding a polypeptide having at least 85% sequence identity to a polypeptide sequence of the present invention, such as a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1-20, and the polypeptide has herbicide-insensitive protoporphyrinogen oxidase activity.

[0008] It possesses genoxidase activity. The resulting DNA molecule then processes the polypeptide. It can further include target sequences that function to localize within the cell.

[0009] In one aspect, the present invention relates to a DNA construct comprising the recombinant DNA molecule of the present invention. This provides. In one embodiment, such a DNA construct is used to provide the nucleic acid sequence of the present invention. In the actionable linkage, a target that functions to localize the polypeptide into the cell. The sequence is present in the genome of transgenic plants, seeds, or cells. It may be present in cells, plants, seeds, and This imparts herbicide resistance to the plant parts.

[0010] In another aspect of the present invention, the recombinant DNA molecule or the recombinant polypeptide of the present invention Transgenic plants, seeds, cells, or plant parts containing cide are provided. Therefore, the transgenic plant, seed, cell, or plant part contains at least one P It possesses, i.e., demonstrates, herbicide tolerance to PO herbicides. In some embodiments, the Transgenic plants, seeds, cells, or plant parts are used for additional transgenic weed control. It possesses the trait of being tolerant to chemicals.

[0011] In another aspect of the present invention, herbicide resistance is imparted to plants, seeds, cells, or plant parts. A method comprising the recombinant polyp of the present invention in the plant, seed, cell, or plant part. A method is provided that includes heterologous expression of plutido. Partial implementation of the method In this state, the plant, seed, cell, or plant part is subjected to the recombinant polypeptide. It possesses conferred protoporphyrinogen oxidase activity. In some embodiments, The herbicide tolerance in question is asyfluorphen, homesaphen, lactofen, and fluoroglycerides. Cophen-ethyl, oxyfluorphen, flumioxazine, azaphenidine, calf Enthrazone-ethyl, sulfenthrazone, fluthiaset-methyl, oxadiargyl Oxadiazone, pyraflufen-ethyl, saflufenacil, and S-3100 This applies to at least one PPO herbicide selected from the following group.

[0012] Another aspect of the present invention is a method for transforming plants, comprising: a) the recombinant DNA molecule of the present invention a) the step of introducing the recombinant DNA molecule into plant cells, and b) the trans containing the recombinant DNA molecule therefrom The present invention relates to a method comprising the steps of regenerating a genic plant, and the method comprising at least 1 The method further includes the step of selecting plants that are tolerant to the PPO herbicide species. It crossbreeds the regenerated plant with itself or a second plant, and collects seeds from this hybrid. The following steps can also be included.

[0013] In yet another aspect of the present invention, a method for controlling weeds in a plant growing area, tiger Applying at least one PPO herbicide to a plant growing area containing encephalogenic plants or seeds Including contact, if the transgenic plant or seed becomes tolerant to the PPO herbicide A method is provided in which weeds are present and controlled in a plant growing area.

[0014] Furthermore, the Nu encoding a protein with protoporphyrinogen oxidase activity A method for identifying the creotide sequence, a) the PPO enzyme of native E. coli. E. coli strains with gene knockouts are used to coat candidate herbicide-tolerant proteins. The transformation is performed using a bacterial expression vector containing recombinant DNA molecules, and b) the The transformed E. coli is grown using a heme-free bacterial medium. The growth using the bacterial culture medium is performed using a bacterial culture medium that has protoporphyrinogen oxidase activity. Methods are also provided that include identifying and propagating proteins.

[0015] The present invention further enhances the herbicide-insensitive protoporphyrinogen oxidase activity. A method for identifying the nucleotide sequence encoding a protein, a) native A recombinant E. coli strain containing a gene knockout of the PPO enzyme of E. coli Transformation is performed using a bacterial expression vector containing recombinant DNA molecules that encode proteins. a) the transformed E. coli is treated with at least one PPO herbicide This involves growing bacteria using a bacterial culture medium, and the growth of the bacteria is such that the herbicide-insensitive protozoan Identifying and amplifying proteins that possess porphyrinogen oxidase activity. A method including is provided.

[0016] Another aspect of the present invention is a method for screening for herbicide tolerance genes, a) Expressing the recombinant DNA molecule of the present invention in plant cells, and b) PPO weed control. The present invention relates to a method for identifying plant cells that exhibit tolerance to a drug, and a method for doing so.

[0017] Furthermore, the present invention relates to a method for screening herbicide tolerance genes, a )By expressing the recombinant DNA molecule of the present invention in bacterial cells lacking HemG, Therefore, the bacterial cells in question grow in a heme-free bacterial medium in the presence of PPO herbicide, and expression a) to cause, and b) to identify bacterial cells that show tolerance to PPO herbicides. To provide the law.

[0018] In another embodiment, the present invention is tolerant to PPO herbicides and at least one other herbicide. A method for producing a plant, wherein a) obtaining a plant containing the recombinant DNA molecule of the present invention. b) to give transgenic plants tolerance to at least one other herbicide to crossbreed with a second plant equipped with, and c) PPO herbicide and at least one other The method includes selecting offspring plants obtained from the hybrid that have tolerance to herbicides. Providing a method is another aspect of the present invention.

[0019] Furthermore, in another embodiment, the present invention relates to a method for reducing the occurrence of herbicide-resistant weeds. a) In a crop growth environment, for example, by including the DNA molecule of the present invention, PPO weed control The present invention provides a plant that is tolerant to the agent and tolerant to at least one other herbicide. The cultivation of plants and the application of PPO herbicides and at least one other herbicide to the growth of the crops. The application to the environment, and the crop plants in question are affected by the PPO herbicide and at least one other type The present invention also provides a method that includes applying a herbicide that is tolerant to other herbicides. In certain embodiments, the PPO herbicide is asyfluorphen, homesaphen, lact Fen, fluoroglycofen-ethyl, oxyfluorophene, flumioxazin, A Zaphenidine, carfentrazone-ethyl, sulfentrazone, fluthiaset-methyl Chil, oxaziargyl, oxadiazone, pyraflufen-ethyl, saflufenacil Selected from the group consisting of , and S-3100. In some embodiments of the method, The at least one other herbicide is an ACCase inhibitor, an ALS inhibitor, or an EPSPS inhibitor. Harmful agents, synthetic auxins, photosynthesis inhibitors, glutamine synthesis inhibitors, HPPD inhibitors, PPO Selected from the group consisting of inhibitors and long-chain fatty acid inhibitors. In certain embodiments, ACC Aase inhibitors are aryloxyphenoxypropionates or cyclohexanedione ALS inhibitors include sulfonylureas, imidazolinones, triazolopyrimidines, Alternatively, triazolinone is used, and EPSPS inhibitors are glyphosate and synthetic oxyphosphate. The herbicide is phenoxy acid, benzoic acid, carboxylic acid, or semicarbazone, and is used in photosynthesis. The inhibitors include triazines, triazinones, nitriles, benzothiadiazoles, or ureas. The glutamine synthesis inhibitor is glufosinate, and the HPPD inhibitor is isooxy PPO inhibitors are sazoles, pyrazolones, or triketones, and diphenyl ether , N-phenylphthalimide, aryltriazinon, or pyrimidinedione, Alternatively, long-chain fatty acid inhibitors include chloroacetamide, oxyacetamide, or pyrazole. It is. [Brief explanation of the drawing]

[0020] [Figure 1] This diagram shows the aligned sequences of H_N90, H_N20, H_N60, H_N10, H_N30, H_N40, H_N50, H_N70, H_N100, and H_N110 proteins (SEQ ID NOs. 1-10), with the consensus position indicated below. [Figure 2] This figure shows the assay results of a PPO bacterial screening system using a PPO herbicide, with the growth of E. coli containing test iPPO measured at 8 hours. [Modes for carrying out the invention]

[0021] A brief explanation of arrays Sequence ID 1 is the amino acid sequence of H_N90.

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

[0023] Sequence ID 3 is the amino acid sequence of H_N60.

[0024] Sequence ID 4 is the amino acid sequence of H_N10, which is E. coli wild-type HemG Protoporphyrinogen oxidase (NCBI GenBank accession number WP_02) 1498199)

[0025] Sequence ID 5 is the amino acid sequence of H_N30.

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

[0027] Sequence ID 7 is the amino acid sequence of H_N50.

[0028] Sequence ID 8 is the amino acid sequence of H_N70.

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

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

[0031] Sequence numbers 11 through 17 correspond to sequence numbers 1, 2, 4, 5, 6, 7, and 9, respectively. This is an amino acid sequence lacking the initiating methionine.

[0032] Sequence IDs 18 and 19 are amino acid variants of Sequence ID 11.

[0033] Sequence ID No. 20 is an amino acid variant of Sequence ID No. 17.

[0034] Sequence ID 21 is the amino acid sequence of WH, which is the Amaranthus tube. Wild-type protoporphyrinogen oxidase from rculatus (Amanita arvensis) be.

[0035] Sequence IDs 22 through 31 are nuclei that encode sequence IDs 1 through 10, respectively. This is a rheotide sequence, codon-optimized for E. coli expression.

[0036] Sequence IDs 32 to 41 are nuclei that encode sequence IDs 1 to 10, respectively. It is a rheotide sequence and has been codon-optimized for expression in dicotyledonous plants.

[0037] Sequence numbers 42 to 48 are codes for sequence numbers 11 to 17, respectively. It is a creotide sequence and has been codon-optimized for expression in dicotyledonous plants.

[0038] Sequence IDs 49 through 52 are nucleotides of sequence IDs 11 and 12, respectively. It is a variant.

[0039] Sequence numbers 50, 51, and 53 are nuclei that code for sequence numbers 18, 19, and 20. This is an Otid sequence.

[0040] Sequence IDs 54 to 63 are nuclei that encode sequence IDs 1 to 10, respectively. It is a leotide sequence and has been codon-optimized for expression in monocots.

[0041] Detailed explanation The following descriptions and definitions are intended to better define the present invention and to guide those skilled in the art in implementing it. Provided for this purpose. Unless otherwise stated, terms are defined by those skilled in the art in the relevant field. It shall be understood in accordance with conventional usage.

[0042] This invention relates to a novel herbicide-insensitive protoporphyrinogen oxidase (iPPO The present invention provides recombinant DNA molecules and proteins that encode ). For example, the present invention is one embodiment. Morphologically, it encodes microbial iPPO for expression in cells and plants. The invention provides vectors and expression cassettes. It also provides cells and plants that are resistant to PPO herbicides. Methods for generating are also provided. The present invention further provides tampering for obtaining and improving iPPO This invention provides methods and compositions for using chemical engineering and bioinformatics tools.

[0043] In certain embodiments, the present invention provides recombinant DNA molecules and proteins. The term "recombination" as used in this book refers to the result of genetic manipulation, and therefore is usually used in the natural world. Non-natural DNA, proteins, cells, seeds, or living organisms that are not thought to be found in nature It refers to a substance. "Recombinant DNA molecules" do not exist naturally and are therefore the result of human intervention. DNA molecules containing DNA sequences that are the fruit, for example, at least two DNA sequences that are heterogeneous It is a DNA molecule consisting of molecule A. An example of a recombinant DNA molecule is provided herein. Enables the action of heterogeneous regulatory elements or other elements (e.g., heterogeneous promoters). A DNA molecule encoding linked herbicide-resistant protoporphyrinogen oxidase There are "recombinant proteins," which do not exist naturally and are therefore the result of human intervention. Contains a certain amino acid sequence (for example, a genetically modified protein or chimeric protein) It is a protein. Recombinant cells, seeds, or organisms contain recombinant DNA molecules. Therefore, transgenic DNA produced as a result of plant transformation, for example, Cells, seeds, or organisms containing lancegenic cells, seeds, plants, or plant parts be.

[0044] As used herein, the term "genetic engineering" refers to genetically modified organisms that are not typically found in nature. Therefore, non-natural DNA, proteins, and other substances require human intervention. Genetic engineering refers to the creation of living organisms. Genetic engineering is a field that encompasses molecular biology, protein biochemistry, and bacterial morphology. Using one or more biotechnology techniques such as chemical conversion and plant transformation, in the laboratory Designed and created for use in generating genetically modified DNA, proteins, or organisms. It is possible. For example, genetic manipulation involves gene cloning, DNA ligation. Using one or more molecular biology techniques, such as DNA synthesis, and other techniques, a small number of heterogeneous organisms It can be used to create chimeric genes that contain at least two DNA molecules. A chimeric gene is two or more heterologous DNA molecules linked together in a functional manner, for example, gene expression genes. A protein-coding sequence that can act on the rement, for example, a transport peptide-coding sequence. Alternatively, it can consist of a heterologous promoter. Genetic manipulation involves site-directed mutagenesis. The protein design used, as well as the determination using random mutagenesis and DNA shuffling. Using one or more protein engineering techniques, such as phagocytosis, the polypeptide sequence is modified. It can be used to create genetically modified proteins. The protein has one or more deletions, insertions, or Substitutions may be present, and each deletion, insertion, or substitution may consist of one or more amino acids. This can be done. In another embodiment, the genetically modified protein is linked to the transport peptide in a manner that allows it to act. It can consist of two heterologous peptides linked together in an actionable manner, such as linked enzymes.

[0045] As used herein, "herbicide-insensitive" refers to protoporphyrinogen oxider Ze (PPO) has low enzyme activity in the presence of one or more PPO herbicides. It means the ability to maintain at least a part of it. Enzyme activity of protoporphyrinogen oxidase. The properties can be measured by any method known in the art, such as an enzyme assay. In the enzyme assay, in the presence of one or more PPO herbicides, protoporphyri Nogen oxidase product production or protoporphyrinogen oxidase substrate consumption The quantity can be measured via fluorescence, high-performance liquid chromatography (HPLC), or mass spectrometry (MS). The enzyme activity of protoporphyrinogen oxidase is measured using a different assay. Examples include bacterial assays such as the growth assay described herein. Therefore, if recombinant protoporphyrinogen oxidase expression is not performed, PP This procedure is performed on bacterial cells lacking O activity, and recombinant protocols for this knockout phenotype are used. The complementary ability of ruphyrinogen oxidase is measured. Herbicide insensitivity is determined by the specific herbicide. It can be made completely or partially insensitive to certain PPO herbicides and tolerant to certain PPO herbicides. It can be expressed as tolerance percentage (%) or insensitivity. "Herbicide-insensitive protoporphyrinogen oxidase" or "iPPO" refers to: It exhibits herbicide insensitivity in the presence of one or more PPO herbicides.

[0046] As used herein, "hemG knockout strain" refers to a strain grown on heme-free growth medium. To an extent that it is impossible, or compared to other isogenic lines containing functional HemG, An organism or organism lacking HemG activity such that its growth in the absence of HemG is detectably impaired. It refers to a cell (e.g., E. coli). For example, hemG knockout of E. coli. The strain was selected considering the knowledge in this field, for example, the hemG sequence of E. coli (Ecogen e-Accession 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 It can be prepared considering (9:1155-1161, 1993).

[0047] As used herein, the term “transgene” refers to a human intervention such as plant transformation. This refers to DNA molecules that have been artificially incorporated into the genome of an organism. The term "transgenic" means that it contains an introduced gene, for example, "trans A "transgenic plant" refers to a plant that contains an introduced gene in its genome. A "classic trait" is a trait that is transmitted or conferred by the presence of an introduced gene incorporated into the plant genome. This refers to a characteristic or phenotype. Due to such genome modifications, transgenic plants are related This plant is distinctly different from the related wild-type plant, and its transgenic traits are related to the wild-type plant. This trait is not found naturally in plants. The transgenic plant of the present invention is described in this specification. This includes recombinant DNA molecules and genetically modified proteins provided in the book.

[0048] As used herein, the term "different species" refers to species that originate from different sources and are therefore usually natural. This refers to the relationship between two or more unrelated items. For example, protein coding recombination. DNA molecules, when linked to a promoter in a functionally ligated manner, are typically found in such combinations. If not found naturally, it is heterogeneous to the promoter in question. In addition, a specific combination Replacement DNA molecules protect the cells, seeds, or organisms into which they were inserted, and ensure that this insertion is correct. In cases where it is not considered to occur naturally in specific cells, seeds, or organisms, these are treated as follows: They can be different species.

[0049] As used herein, the term “isolated” refers to a molecule that is typically associated with its natural state. This refers to separating, at least partially, from other molecules. In one embodiment, "single The term "separation" refers to the separation of DNA molecules from nucleic acids that are normally adjacent to each other in their natural state. To refer to. For example, a DNA molecule that codes for a protein naturally present in a bacterium is the relevant one. If the DNA molecule encoding the protein is not present in the DNA of naturally occurring bacteria, It is thought to be an isolated DNA molecule. Therefore, for example, recombinant DNA or plant As a result of the transformation technique, one or more other DNA components that are not thought to be naturally related may be present. DNA molecules fused to or ligated to the offspring (or more) are isolated in this specification. Such molecules are considered to be such even when they are integrated into the chromosomes of the host cell. Even if present in nucleic acid solution alongside other DNA molecules, it is considered isolated. .

[0050] As used herein, the term "protein-coding DNA molecule" refers to a protein-coding DNA molecule. This refers to a DNA molecule containing a nucleotide sequence that codes for proteins. This refers to the DNA sequence that codes for protein. "Sequence" refers to a nucleotide or amino acid sequence. This refers to a continuous arrangement of acids. The boundary of the protein coding sequence is the translation initiation at the 5' end. It can be determined by the codon and the translation stop codon at the 3' end. The code molecule may include a DNA sequence that encodes a protein sequence. The terms used are "transgene expression," "expressing a transgene," "protein expression," and " "Expressing a protein" means transcribing a DNA molecule into messenger RNA (mRNA). The mRNA is translated into a polypeptide chain that will ultimately be folded into a protein. This refers to the synthesis of proteins through a process. Protein-coding DNA molecules are assembled. DN used to express proteins in cells transformed with replacement DNA molecules It can be actionably coupled to heterologous promoters in construct A. The term "functionally linked" means that one of the two DNA molecules influences the function of the other. This means they are linked in a manner that allows them to function. A single DNA molecule linked in a functional way is a single It may be part of a nearby molecule, or it may not be adjacent to it. For example The promoter can interact with protein-coding DNA molecules in the DNA construct. When linked, these two DNA molecules are linked by a promoter that controls the expression of the introduced gene. It is positioned in a way that it can actually exert an influence.

[0051] As used herein, "DNA construct" refers to a combination of two or more different DNA sequences. It contains recombinant DNA molecules. DNA constructs are useful for transgene expression. This can be contained in vectors and plasmids. DNA constructs are transformed (heterogeneous) Transgenic plants used as vectors for the purpose of introducing DNA into host cells. And it can generate cells, and therefore transgenic plants, seeds, cells, or It can also be included in the plastid DNA or genomic DNA of the plant part. Used herein A "vector" is any recombinant DNA that can be used for the purpose of bacterial or plant transformation. This refers to molecule A. Recombinant DNA molecules listed in sequence listings are, for example, constructs. It can be inserted into a vector as part of this recombinant DNA molecule, Lacto is a genetically modified protein encoded by recombinant DNA molecules in plants. To a gene expression element that functions to affect the expression of, it is receptively linked to Yes. To prepare and use recombinant DNA constructs and plant transformation vectors. General methods useful for manipulating DNA molecules are well known in the art, for example, 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 guides, including Laboratory Press, NY (2012). Detailed information is provided separately. DNA construct or containing DNA construct The vector's components include one or more genes ligated to a transcriptionable DNA sequence in a manner that allows them to act. It contains a child expression element, such as: functionally linked DNA. A promoter for expressing a protein, a functionally linked protein-coding DNA molecule, and A 3' untranslated region (UTR) that is ligated to enable action. A gene expression element useful for carrying out the present invention. The term is not limited to one or more of the following types of elements. Examples include: promoter, 5'UTR, enhancer, leader, cis-acting element Introduces ment, intron, target sequence, 3'UTR, and one or more selectable marker introduction sites. Transmission.

[0052] The DNA construct of the present invention provides protein-coding DNA components. A promoter that can act upon its offspring, thereby driving the expression of recombinant protein molecules. —can include. Promoters useful for carrying out the present invention include those that act in cells. These are entities that function for the expression of potentially linked polynucleotides, such as bacterial promoters. Alternatively, plant promoters can be mentioned. Plant promoters are diverse and in the field of this technology. This is well known in that, for example, inducible, viral, synthetic, commensal, temporally controlled, spatial Examples include spatial-controlled and / or spatial-temporal controlled systems.

[0053] In one embodiment of the present invention, the DNA construct provided herein is a herbicide Encoding polypeptide molecules with insensitive protoporphyrinogen oxidase activity It includes a target sequence that is ligated to a heterologous nucleic acid, thereby allowing the target sequence to act on this polyp Promotes intracellular localization of peptide molecules. Target sequences include signal sequences, target peptides, and localization. It is known in the art as a chemical sequence and transport peptide. An example of a target sequence is chloroplast transport. Ceptide delivery (CTP), mitochondrial target sequences (MTS), or chloroplasts and mitochondria There is a Doria dual target peptide. By promoting the intracellular localization of proteins, it targets The sequence increases the accumulation of recombinant proteins and protects those proteins from proteolysis. and / or enhance the level of herbicide tolerance, thereby enabling trans after herbicide application. It can reduce the level of damage to genic cells, seeds, or organisms.

[0054] CTP and other target molecules that can be used in connection with the present invention are in the art. It is publicly known, and is not limited to the following, but 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), Maize CAB-M7 signal sequence (Becker et al., P 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 Lutathione reductase signaling sequence (Creissen et al., Plant Examples include J.8:167-175, 1995, and PCT WO97 / 41228.

[0055] The recombinant DNA molecule of the present invention contains sequences useful for DNA manipulation (e.g., restriction enzyme recognition sites). (or cloning sites based on recombination), plant-friendly sequences (e.g., plant codons) Use or Kozak consensus sequence, or a useful combination for DNA construct design. This technology is useful when it is desirable to provide a column (e.g., a spacer or linker array). It can be synthesized and modified completely or partially by methods known in the field. Specifically, either the recombinant DNA molecule or polypeptide sequence provided herein In contrast, at least 70% sequence identity, at least 80% sequence identity, at least 85 % sequence identity, at least 90% sequence identity, at least 95% sequence identity, less At least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity It is monosexual and has at least 99% sequence identity, and is herbicide-resistant protoporphyri This includes recombinant DNA molecules and genetically modified proteins that possess orogen oxidase activity. The terms "sequence identity percentage" or "sequence identity %" as used herein refer to the following: The word is a reference ("query") compared to the test ("subject") sequence (or its complementary strand). The two sequences (or their complementary strands) are optimally aligned (comparison window) Insertion of appropriate nucleotides or amino acids that make up less than 20% of the total reference sequence across the entire sequence. Linear polynucleotides or polypeptides (with deletions or gaps) This refers to the percentage of identical nucleotides or amino acids in a sequence. (Comparison window) The optimal alignment of the arrangement for aligning U is well known to those skilled in the art, and Smith and Water Rman's local homology algorithm, Needleman and Wunsch's Research on mology sorting algorithms, Pearson and Lipman analogues, etc. By the way, and also by GCG (registered trademark) Wisconsin Package (registered trademark) Sequence analysis software (Trademark) (Accelrys Inc., San Diego, CA) GAP, BESTFIT, FASTA, and TF are available as part of the FILM 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 computers, for example, default This can be done using the parameters of the test sequence and the reference sequence. The "identity fraction" in relation to this is the aligned reference array. In the segment portion, that is, in the entire reference array or a defined sub-part of the reference array And it is the number of identical components shared by two sorted arrays divided by the total number of components. The sequence identity percentage is expressed as the identity rate multiplied by 100. (One or more) The comparison of sequences can be done using the full sequence, a portion of it, or a longer sequence. .

[0056] Genetically modified proteins are produced by altering (i.e., modifying) wild-type proteins. ), modified features(s), for example, in particular, those relating to chloroplasts or mitochondria Novel combinations of specific cellular localization patterns, such as targeting, or useful protein features. Wase (for example, a modified V max , K m , K i ,I C 50 ), substrate specificity, inhibitor / removal Herb specificity, substrate selectivity, and other components in the cell (e.g., partner proteins or membranes) To generate novel proteins that have the ability to interact with (and protein stability). This can be done by modifying a protein at a specific amino acid position. In nature (i.e., in wild-type proteins), the amino acid found at that position is It may be possible to substitute with another amino acid. Therefore, the genetically modified type provided in the present invention Proteins are one or more modified proteins compared to similar proteins found in nature. This invention provides a novel protein having the following characteristics. In one embodiment of the present invention, genetic engineering Proteins have characteristics that differentiate them from similar wild-type proteins, such as one or more. Characteristics that result in reduced sensitivity to the above herbicides, or expressing the genetically modified protein Improvements to confer herbicide tolerance to one or more herbicides to transgenic plants. It has the characteristic of providing a certain ability. In one embodiment, the present invention is a genetically modified protein The quality and a recombinant DNA molecule encoding it, selected from Table 1, at least one Provided herein, comprising one amino acid substitution, including but not limited to Sequence IDs 1-20. For any of the genetically modified protein sequences, at least approximately 70% sequence identity is required. Approximately 80% sequence identity, approximately 85% sequence identity, approximately 90% sequence identity, approximately 95% sequence Identity, approximately 96% sequence identity, approximately 97% sequence identity, approximately 98% sequence identity, and approximately We provide recombinant DNA molecules with 99% sequence identity. Amino acid mutations , as a single amino acid substitution in a protein, or as one or more other mutations. In combination with this, for example, one or more other amino acid substitutions, deletions, or additions may occur. This can be done in combination with the following. Mutation can be performed by any method known to those skilled in the art. It is possible. Table 1: Amino acid substitutions TIFF2026136216000001.tif92155

[0057] As used herein, "wild type" refers to a naturally occurring, similar but not identical variety. This refers to the version. "Wild-type DNA molecule" or "wild-type protein" refers to the DN. A naturally occurring version of a molecule or protein, i.e., the one that already exists in nature. This is a version of the DNA molecule or protein. Genetic engineering provided by the present invention An example of a wild-type protein useful for comparison with other proteins is Arabidopsis tha There is protoporphyrinogen oxidase derived from Liana. "Wild-type plants" are... It is a non-transgenic plant of the same type as transgenic plants, and therefore, It is genetically different from transgenic plants that possess herbicide-tolerant traits. An example of a wild-type plant useful for comparison with maize plants is the non-transgenic LH24. 4 Corn (ATCC deposit number PTA-1173) and 01DKD2 inbred corn There is a Koshi (I294213) (ATCC deposit number PTA-7859). Transgenic An example of a comparative strain for soybean plants is the non-transgenic A3555. Soybeans (ATCC deposit number PTA-10207) are a possibility, and transgenic cotton planting An example of a comparative system for a substance is the non-transgenic Coker 130 (Pl Ant Variety Protection Number 8900252) is considering It is possible.

[0058] Transgenic plants and herbicides One aspect of the present invention includes recombinant DNA molecules and genetically modified tannins provided by the present invention. Transgenic plant cells containing protein, transgenic plant tissue, transgenic This includes nicked plants and transgenic seeds. The recombinant DNA molecules and genes These cells, tissues, plants, and seeds containing modified proteins are used for one or more PPO weed control. Herbicide tolerance to the herbicide(s) It shows tolerance to [the substance].

[0059] A suitable method for transforming host plant cells for use in the present invention involves introducing DNA into the cells. This can be done (for example, stably integrating recombinant DNA constructs into plant chromosomes). ) Almost all methods are included, and the transformation methods are well known in the art. Recombinant D Exemplary and widely used methods for introducing NA constructs into plants. This is an Agrobacterium-transformed system, which is well known to those skilled in the art. Another exemplary method for introducing replacement DNA constructs into plants is site-specific. This involves the insertion of recombinant DNA constructs into the plant genome at specific locations using an integration method. Site-specific integration can be performed by any method known in the art, for example, zinc fingering. Clease, genetically modified or native meganuclease, TALE-endonuclease Rease, or RNA-induced endonuclease (e.g., CRISPR / Cas9 system) This can be carried out by ). Transgenic plants are produced by plant cell culture methods It can be regenerated from transformed plant cells. Homozygosity (that is, Transgenic plants are those with two alleles of an introduced gene (i.e., two copies of the introduced gene). Transgenic plants that contain both the entered allele and themselves, such as R0 plants, are used as the self-allele. It can be obtained by pollinating (in-line cross) to produce R1 seeds. One-quarter of the R1 seeds become homozygous for the introduced gene. The plants were subjected to SNP assays, DNA sequencing, or thermal amplification assays. This allows testing the bonding state. This test, called a bonding state assay, allows testing the bonding state. This makes it possible to distinguish between terrorist and homozygous individuals.

[0060] The term "PPO inhibitor herbicide" or "PPO herbicide" used in this invention refers to protopolf The dehydrogenation of ylinogen IX is catalyzed to produce protopol, a precursor of heme and chlorophyll. Enzymatic activity of protoporphyrinogen oxidase (PPO) that forms phylin IX It is a chemical substance that targets and inhibits protoporphyrinogen oxidase. More reactive oxygen species are formed, resulting in the disruption of the cell membrane and ultimately the development of susceptible cells. This results in the death of the plant. PPO herbicides are well known and commercially available in this field. Examples of PPO herbicides include, but are not limited to, diphenyl ether (for example) For example, acinfluorphen, its salts and esters, acroniphen, bifenox, and Salts and esters, ethoxyphene, its salts and esters, fluoronitrophene, furyl Oxyphenes, halosaphenes, chloromethoxyfen, fluoroglycofens, their salts and Esters, lactofens, their salts and esters, oxyfluorphenes, and homesa Fen, its salts and esters), thiadiazoles (e.g., fluthiaset-methyl and Thidiadimine), pyrimidinedione, or phenyluracil (e.g., benzfendione) 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 the designation S-3100 in this specification), fluposil, saflufenasil , and thiafenacil), phenylpyrazole (e.g., fluazolate, pyraflufe) (and pyraflufen-ethyl), oxadiazole (e.g., oxadiargyl and o Xadiazone), triazolinone (e.g., azaphenidine, bencarbazone, carphe) (Phthrazone, its salts and esters, and sulfenthrazone), oxazolidinedione ( For example, pentoxazone, N-phenylphthalimide (for example, synidone-ethyl, phenyl) Lumicrolac, Flumicrolac-pentyl, and Flumioxazine), Benzox Dinon derivatives (e.g., 1,5-dimethyl-6-thioxo-3-(2,2,7-triflu) Oro-3,4-dihydro-3-oxo-4-propa-2-inyl-2H-1,4-benzo Oxazine-6-yl)-1,3,5-triazinan-2,4-dione), flufenp Examples include flufenpyr-ethyl, pyraclonil, and profluazole. Protoporphyrinogen oxidase and cells, seeds, plants, and plants according to the present invention. The portion indicates herbicide tolerance to one or more PPO herbicides.

[0061] The herbicide, as a method for controlling weeds, includes the plants and seeds provided by the present invention. It can be applied to plant growing areas. The plants and seeds provided by this invention are herbicide resistant. It possesses tolerance and is therefore tolerant to the application of one or more PPO herbicides. Herbicide application This is the recommended commercial rate (1 X) or any fraction or multiple thereof, for example, twice the commercially recommended ratio (2X). This can be done. The ratio of herbicides is determined according to the herbicide and formulation, in pounds of acid equivalent / acre (lb (ae / acre) or gram equivalents / hectare (g ae / ha) or pounds Active ingredient per acre (lb ai / acre) or gram active ingredient per hectare (ga It can be expressed as i / ha). Herbicide application is at least one type of PPO herbicide. The agent is included. The plant growth area may or may not include weeds at the time of herbicide application. The effective herbicidal dose of PPO herbicides (multiple types) for weed control within an area is: The label ratio(s) can range from approximately 0.1X to approximately 30X over the specified period. Table 2 shows the 1X label ratios for several exemplary PPO herbicides. 1 acre is 2.47 This is equivalent to 105 hectares, and 1 pound is equivalent to 453.592 grams. The herbicide ratio is You can convert between the imperial system (yards and pounds) and the metric system as follows: (lb ai / ac)×1.12=(kg ai / ha) and (kg ai / ha)×0.89= (lb ai / ac). Table 2: Exemplary PPO herbicides TIFF2026136216000002.tif104155

[0062] The application of herbicides may involve one, two, or a combination of multiple PPO herbicides, or Any other suitable herbicide may be mixed in over time or in a tank. Throughout the growing season To control various dicotyledonous weeds, monocotyledonous weeds, or both, the present invention provides trans- For areas containing fungal plants, apply one herbicide or two or more herbicides (in combination or Multiple applications (one or two) may be used, for example, two applications (for example, before planting) (Application before and after germination, or application before and after germination), or 3 applications (for example) Application before planting, application before germination, and application after germination, or application before germination and after germination It is possible to apply it twice.

[0063] As used herein, "tolerance" or "herbicide tolerance" refers to toxicity at the time of herbicide application. This refers to the ability of plants, seeds, or cells to withstand the effect. Herbicide-tolerant crops continue to grow. This allows for the application of chemicals without being affected by their presence, or with minimal impact. As used herein, "herbicide tolerance trait" refers to a trait that has been improved compared to the wild type of plant. This is a transgenic trait that confers herbicide tolerance to plants. As for plants that can be produced by quality, for example, among them, soybeans (for example, Glycine (max), corn (maize), cotton (Gossypium sp.), and cabbage This could include any plant, including crop plants such as raspberries.

[0064] The present invention comprises one or more transgenic plants, progeny, seeds, plant cells, and plant parts. It may contain additional traits. The additional traits are recombinant DNA provided by the present invention. A plant containing a transgene including a molecule, containing one or more additional traits. It can be introduced by crossbreeding with other plants. The term "crossbreeding" as used herein refers to the process of introducing new plants. This means crossbreeding two separate plants to produce offspring plants. By crossbreeding two plants, it is possible to produce offspring that contain desirable traits from each plant. Yes, it is possible. As used in this specification, "offspring" means the descendants of any generation of the parent plant, and tiger The genic progeny provided by this invention are inherited from at least one parent plant. This includes the DNA construct. The introduction of additional traits(s) is also possible with this additional trans. DNA constructs for genic traits (multiple traits) provided by the present invention DNA constructs containing replacement DNA molecules (for example, those used in plant transformation) Simultaneous transformation (in all DNA constructs present as part of the vector) By or including additional traits(s) provided herein, the DNA construct includes By inserting into a transgenic plant or vice versa (for example, a transgenic plant Using either plant transformation or genome editing on plants or plant cells ), it is possible. Such additional traits include, but are not limited to, the following: Increased insect resistance, increased water use efficiency, improved yield performance, increased drought tolerance, and improved seed quality. Climbing, improvement of nutrient quality, hybrid seed production, and herbicide tolerance (if the trait is wild-type plant) Examples of additional herbicide tolerance traits include one or more. Herbicides, for example, among others, ACCase inhibitors (e.g., aryloxyphenoxyp (Lopionate and cyclohexanedione), ALS inhibitors (e.g., sulfonylurea, Imidazolinone, triazolopyrimidine, and triazolinone), EPSPS inhibitors (e.g.) For example, glyphosate), synthetic auxins (e.g., phenoxy, benzoic acid, carboxylic acid, Semicarbazone), photosynthesis inhibitors (e.g., triazines, triazinones, nitriles, benzylamines) Zothiadiazoles and ureas), glutamine synthesis inhibitors (e.g., glufosinate), HPPD inhibitors (e.g., isoxazole, pyrazolone, and triketone), PPO inhibitors Harmful agents (e.g., diphenyl ether, N-phenylphthalimide, aryltriazinon) , and pyrimidinedione), and long-chain fatty acid inhibitors (e.g., chloroacetamide, oxy) Transgenic or non-transgenic cyacetamide (and pyrazole) Tolerance to insects can be cited as an example. Exemplary insect tolerance traits include, among others, Lepidopter Among the orders ra, Coleoptera, Hemiptera, and Homoptera Resistance to one or more insect members may be mentioned. Such additional traits are known to those skilled in the art. This is well known, and for example, the list of such transgenic traits is published by the U.S. Department of Agriculture. Provided by the USDA's Animal and Plant Health Inspection Service (APHIS).

[0065] Cells transformed with the polynucleotides of the present invention, such as expression constructs, The polynucleotide before or after regenerating the cells into transgenic plants It can be selected due to the presence of its encoded enzyme activity. Therefore, such poly Transgenic plants containing nucleotides include, for example, the polynucleotide or nucleotide. Modified enzyme activity, and / or modified in comparison with another isogenic control plant. Transgenic plants exhibiting the desired traits can be selected by identifying them. Such traits could include, for example, tolerance to PPO herbicides.

[0066] Transgenic plants containing the transgenic traits provided by the present invention and The offspring can be used with any crossbreeding method known in the art. In plant lines possessing two or more transgenic traits, these transgenic traits Nick traits can be independent or linked, and plants possessing three or more traits may have them. In the case of plant lineage, both combinations are acceptable. Backcrossing with the parent plant and non-transcrossing are also possible. Crossbreeding with genetically modified plants is also considered because it is a form of vegetative propagation. The methods of cross-pollination used for certain plants or seeds are well known to those skilled in the art. Various assays can be performed to confirm the presence of the transgene(s) in the given sample. Examples of assays include molecular biological assays (e.g., Southernblotti assays). (Northern blotting, PCR, and DNA sequencing), biochemical analysis ELISA (for example, by immunological means (ELISA and Western blot), or by yeast Detection of the presence of protein products by elementary function), plant part assays (e.g., leaf or root This can be done by assays, or by analyzing the phenotypic characteristics of the entire plant.

[0067] The transfer of transgenic traits into plant genotypes is a process of backcross conversion. As a result, the plant genotype into which the transgenic trait has been introduced is restored. This can be called a crossbreeding genotype, strain, inbred line, or hybrid line. Similarly, Plant genotypes lacking transgenic traits include unconverted genotypes, strains, inbred lines, and Alternatively, it can be called a hybrid.

[0068] Although the present invention has been described in detail, it does not deviate from the scope of the present invention as defined in the appended claims. It is clear that modifications, variations, and equivalent embodiments are possible. Furthermore, please understand that the embodiments described herein are provided as non-limiting examples. [Examples]

[0069] To demonstrate embodiments of the present invention, the following examples are included. Those skilled in the art will see the following examples. The techniques disclosed in the embodiments have been found to function well in the implementation of the present invention, as discovered by the inventors. This technique is representative of the techniques for carrying out the present invention, and therefore this technique is preferred for carrying out the present invention. It should be understood that it can be considered to constitute a beautiful form. However, those skilled in the art will understand that In light of this disclosure, many modifications can be made to the specific embodiments disclosed. However, without departing from the concept, spirit and scope of the present invention, similar or It should be understood that similar results can be obtained. More specifically, chemical and physiological Certain drugs that are specifically related may be replaced by the drugs described herein, or the same or identical drugs. It will become clear that the following results can be achieved. Such similar results will be obvious to those skilled in the art. All substitutions and modifications fall within the spirit, scope, and concept of the invention as defined by the attached claims. It is considered to exist.

[0070] Example 1: Discovery of microbial protoporphyrinogen oxidase Bioinformatics methods and novel protoporphyrinogen oxidase bacterial screening Using a microbial sequence database, novel protoporphyrinogen oxidase Ze was identified. E was used as the start sequence for bioinformatics analysis of the microbial sequence database. The sequence .coliHemG (sequence number 4) was used. Bioinformatics analysis revealed diversity From a bacterial source, 33 novel putative protoporphyrinos of the HemG PPO family were identified. HemG oxidase was identified. These were estimated using phylogenetic tree mapping. We compared the sequences encoding the PPO enzyme and found that they are relatively diverse. The display of individual unique cluster members on the tectonic tree reveals that this group of 33 species... Ten types were selected for further analysis.

[0071] The coding sequences of these 10 selected HemG PPO enzymes were found in the wild-type DNA sequences. It was optimized for E. coli expression to exclude any rare codons that might be expressed. Next, The E. coli-optimized coding sequences of 10 types of HemG PPO enzymes were used in bacterial expression vectors. I roaned. I saw Amaranthus tuberculatus. Naturally found herbicide-sensitive PPO enzymes are used to study both PPO function and herbicide sensitivity. To be used as a control, it was cloned into a bacterial expression vector (referred to as "WH"). , shown as Sequence ID No. 21, GenBank accession number 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). Ayu-modoki PPO enzyme is a member of the HemG family. No. The HemG PPO enzyme, which is naturally found in E. coli, is used as a countermeasure against PPO activity. Cloned into bacterial expression vectors for use as a light source and in herbicide sensitivity assays. (This is referred to as H_N10 and shown as sequence number 4).

[0072] Test the protoporphyrinogen oxidase activity of recombinant proteins and do so To confirm that it is a functional PPO enzyme, protoporphyrinogen oxy A Dase bacterium screening system was created. This screening system is used for E. coli P E. coli strains containing PO enzyme (SEQ ID NO: 4) gene knockout are functionally rescued. - An assay was used. The hemG knockout E. coli strain used was heme-free. Growth observed on culture medium (e.g., LB medium) was very minimal, but when free heme was added... Synthetic or activated recombinant protoporphyrinogen oxidase is expressed in E. coli. When this was done, proliferation recovered. Therefore, the protoporphyrinogen of the protein To rapidly and easily assay sidase activity, hemG knockout E. coli The strain could be used for recombinant protein expression.

[0073] hemG knockout E. coli strains, 10 presumed HemG PPO enzymes, Ec Contains genes encoding oli HemG PPO enzyme and Ayumodoki PPO enzyme. The bacteria were transformed with a bacterial expression vector. Next, the bacteria were subjected to LB medium, a heme-free bacterial medium. The samples were arranged in stripes on the rate. Recombinant PPO enzyme expression inhibited E. coli proliferation. This was performed, and propagation on LB plates was achieved. Transformed hemG knockout E. The growth of the coli strain on the LB plate resulted in the protein sequence being protoporphyri It was shown to function as nogen oxidase. In this assay, 10 types HemG PPO enzyme (SEQ ID NOs: 1-10) and Ayu PPO enzyme (SEQ ID NOs: 21) This allowed us to restore the proliferation of the transformed hemG knockout E. coli strain. Therefore, these PPO activities were confirmed. (PPO enzyme protein sequence ordered) The consensus position is shown in Figure 1. This assay was used to analyze numerous novel proteins. Alternatively, screen for genetically modified proteins to find protoporphyrinogen oxider It is possible to confirm and measure the activity of the enzyme.

[0074] Example 2: Protoporphyrinogen oxidase inhibitor insensitivity Using a herbicide bacterial screening system, novel protopols resistant to PPO herbicides were identified. Irinogen oxidase was identified. This screening system is susceptible to PPO herbicides. To identify non-protoporphyrinogen oxidase, L containing PPO herbicide A growth assay of hemG knockout E. coli strains in liquid medium B was used.

[0075] hemG knockout E. coli strain, confirmed protoporphyrinogen oxy The bacteria were transformed with a bacterial expression vector containing γ-idase activity and cultured in LB liquid medium. Five different PPO herbicides that represent various PPO chemical subclasses (Acifluolphe (1 mM), Flumioxazine (0.5 mM), Lactofen (0.5 mM), Homesa One of the purified crystalline forms of fen (1 mM) and S-3100 (100 μM) is used. The recombinant protein was added to the culture medium. The growth rate of E. coli was measured after expressing the recombinant protein. The growth curves (OD600) of these various mutants were examined under the presence and absence of PPO herbicide. Measurements were taken at selected time points between 0 and 24 hours, on LB medium in the presence of PPO herbicide. The transformed hemG knockout E. coli strain inside proliferated, resulting in E. coli The gene used for transformation is herbicide-insensitive protoporphyrinogen oxidase ( It was shown that this would involve coding iPPO.

[0076] Using a novel protoporphyrinogen oxidase in this assay, PPO weed control was performed. Insensitivity to the agent was tested. Ten protocols, indicated as Sequence IDs 1 to 10, were used. Ruphyrinogen oxidase is entirely present in hemG medium in LB medium in the presence of PPO herbicide. It was found that this conferred a normal growth rate to the over-reacting E. coli strain, and as a result, These proteins are herbicide-insensitive protoporphyrinogen oxidase (iPPO This was shown to be the case (Figure 2). Hem expressing Ayumodoki PPO (SEQ ID NO: 21) The G knockout E. coli strain is susceptible to all five herbicides, and therefore, Ssey has tested susceptible protoporphyrinogen oxidase and unsusceptible protoporphyrinogen oxidase for each herbicide. It was confirmed that this assay can be distinguished from rotporphyrinogen oxidase. Using this method, a large number of novel or genetically modified proteins were screened, PP Confirm protoporphyrinogen oxidase activity in the presence of herbicides (multiple herbicides are acceptable). It is possible.

[0077] Example 3: Protoporphyrinogen oxidase (PPO) enzyme assay Enzymatic characterization of protoporphyrinogen oxidase was performed, and each PPO enzyme substrate binding affinity (K m ) and sensitivity (IC50) to PPO herbicides were measured. Using wild-type plant PPO enzymes from Amaranth, soybeans, and corn, micro Biological HemG protoporphyrinogen oxidase (as SEQ ID NOs: 1 to 10) A comparison was made with (shown).

[0078] Yellowed cotyledons (soybean, Glycine max), yellowed leaves / cotyledon sheaths (corn) Rocosi (Zea mays), and the spread-out tip leaves (Amaranthus From tuberculata, generally explained by Grossmann et al. The procedure ("The Herbicide Saflufenacil (Kixor (T M) )is a New Inhibitor of Protoporphyrino gen IX Oxidase Activity”Weed Science,58( Leaf and chloroplasts were prepared according to 1):1-9(2010)). Soybean (A3555 ) and corn (LH244) seeds were placed in two moist germination trays in a beaker of water. Paper (Anchor Paper Company, Saint Paul, Minnes It was sandwiched between (ota, USA) and placed under continuous dark periods of 8 - 10 days. The Humifuse plants were grown in the greenhouse for 30 days. The tissues were collected, sandwiched between wet paper towels, and ground with a mortar and pestle in liquid nitrogen until they became fine powder. 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) was added to the frozen powder at a ratio of 4:1 (ml of homogenization buffer: g fresh weight tissue), stirred vigorously, and filtered through pre - moistened four - layer Miracloth (trademark) (Merck - Millipore, Darmstadt, Germany). The filtrate was centrifuged at 9299 g for 5 minutes. The pellet was resuspended in homogenization buffer and centrifuged at 150 g for 2 minutes. The supernatant solution was centrifuged at 4000 g for 15 minutes. All centrifugation steps were carried out at 4 °C. The pellet (intact plasmid fraction) was resuspended in 15 mM Tris - HCl l (pH 7.4), 2 mM EDTA and 20% (v / v) glycerol, and aliquoted and stored at - 80 °C. The total protein amount in the plasmid preparation was measured by the Bradford method (MM Bradford, “A rap 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)) using bovine serum albumin as a standard.

[0079] The selected PPO enzyme was expressed in the E. coli hemG knockout cell line Cells were inoculated into 20 ml of fresh medium using bacterial cells from an overnight culture. These cultures were grown at 20 °C for approximately 48 hours to obtain high-density cultures. Bacterial cells were collected by centrifugation and the cell pellet was stored at -80 °C until the enzyme assay was performed. The frozen bacterial pellet was resuspended in an extraction suspension (50 mM Tris-HCl, pH 7.6, 1 mM EDTA & 1 mM MgC l2), sonicated (Sonics VibraCell (trademark), New town, CT USA), and subjected to 3 cycles of 30 seconds in an ice bath with a 1-minute rest period between cycles. The disrupted cells were centrifuged at 200 g for 2 minutes at 4 °C and the supernatant solution was withdrawn and diluted with the extraction suspension before being used in the PPO enzyme assay. Total protein content was measured using the method of Bradford (1976) with bovine serum albumin as the standard.

[0080] As described by JM Jacobs and NJ Jacobs (“Measurement of Protoporphyrinogen Oxidase Activity” i n Current Protocols in Toxicology (1999) 8 .5.1-8.5.13, John Wiley & Sons, Inc.), protoporphyrinogen IX (protogen) was synthesized by reducing commercially available protoporphyrin with sodium mercury amalgam. Protoporphyrin (Proto) was added to 0.01 N potassium hydroxide in 20% ethanol and stirred in the dark until dissolved (about 40 minutes). A volume of 0.8 ml was placed into a 2 ml polypropylene vial with a screw cap containing an O-ring, and approximately 1 g (one spatula tip full, oil-free) of sodium mercury amalg was added. (about 40 minutes). A volume of 0.8 ml was placed into a 2 ml polypropylene vial with a screw cap containing an O-ring, and approximately 1 g (one spatula tip full, oil-free) of sodium mercury amalg was added. Gum (product number 451908, Sigma-Aldrich, St. Louis, Mis Souri (stored in oil) was added. Immediately cap the tube and use a vortex mixer. - Stir vigorously, vent by loosening the lid approximately every 30 seconds, and the solution will turn red under UV light. Continue this process until the color no longer fluoresces (approximately 5 minutes). Flushing the reaction vial with argon... The mixture was sprayed and lightly centrifuged to form pellets from the remaining sodium amalgam. The supernatant solution was then removed. Immediately, use a 1:1 solution of 0.1M DTT and 0.5M Tris-HCl, pH 7.5 (v Diluted (v), the vial was flushed with argon. The resulting solution was then mixed with a smaller amount of ALCO. Divide into portions and place into 0.5 ml polypropylene tubes with lids, then add aliquots. Immediately afterwards, the tube was flushed with argon. The capped tube was covered with aluminum foil, - Stored at 80°C. Protogen aliquots were thawed for enzyme assay and covered with ice. It was stored and used on the same day. The protogen concentration in the preparation was compared to that of the starting material. From the concentration, the Proto concentration in the final protogen solution is measured by fluorescence 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) The method described above (None of the samples contained significant impurities) (Assuming they are not present). Protogenes prepared and stored under these conditions should last for at least 6 months. It was stable.

[0081] The PPO activities in plant plasmid extracts and bacterial extracts were generally measured as described by Gros smann.et al. (2010). Either 10 microliters of a plant plasmid extract (total protein amount 40 μg) or a bacterial extract (total protein amount 16 - 35 μg for various bacterial extracts) was added to an assay buffer (10 0 mM Tris-HCl, pH 7.4, 5 mM DTT, 1 mM EDTA, and 0.08 5% (v / v) Tween 80). S-3100 (also known as "SYN-523"; for example, U.S. Patent Publication No. US20100062941A1) was added as a 2 microliter volume from a 100X stock solution prepared in acetone. Analytical grade S-3100 was provided by Sumitomo Chemical Company. The entire assay was carried out at a final acetone concentration of 1% (v / v). The extract (plasmid or bacterial), buffer, and S-3100 were incubated at 30 °C (for plant extracts) or 3 7 °C (for bacterial extracts) for 5 minutes, then 2 microliters of protogen was added to initiate the assay. The entire assay was performed in a 96-well black polystyrene micro titer plate (Costar® 3925, Corning, Inc., Corning, New York) with a final volume of 200 microliters. After adding protogen to all wells (3 μM for IC measurements, variable for K measurements), the plate was incubated at 30 °C (for plant extracts) or 37 °C (for bacterial extracts). The entire assay was carried out at a final acetone concentration of 1% (v / v). The extract (plasmid or bacterial), buffer, and S-3100 were incubated at 30 °C (for plant extracts) or 37 °C (for bacterial extracts) for 5 minutes, then 2 microliters of protogen was added to initiate the assay. The entire assay was performed in a 96-well black polystyrene micro titer plate (Costar® 3925, Corning, Inc., Corning, New York) with a final volume of 200 microliters. After adding protogen to all wells (3 μM for IC measurements, variable for K measurements), the plate was incubated at 30 °C (for plant extracts) or 37 °C (for bacterial extracts). The entire assay was carried out at a final acetone concentration of 1% (v / v). The extract (plasmid or bacterial), buffer, and S-3100 were incubated at 30 °C (for plant extracts) or 3 7 °C (for bacterial extracts) for 5 minutes, then 2 microliters of protogen was added to initiate the assay. The entire assay was performed in a 96-well black polystyrene micro 50 measurements at 3 μM, K m measurements at variable), the plate was incubated at 30 °C (for plant extracts) or 37 °C (for bacterial extracts). Data collection was then started. Fluorescence over time at 30 °C (plant extract) or 37 °C (bacterial extract) was measured using a SpectraMax® M5 multimode microplate reader (Molecular Devices, Sunnyvale, California) with excitation and emission wavelengths of 405 nm and 630 nm, respectively. An assay blank was run by adding heat-inactivated (100 °C for 5 minutes) extract to the assay mixture. The substrate (protoporphyrinogen) binding affinity of protoporphyrinogen oxidase was measured as K. The apparent K value for each PPO being evaluated was calculated using the curve fitting of a rectangular hyperbola with SoftPro® kinetic software package (Molecular Devices, Sunnyvale, California). The sensitivity of the enzyme activity to the PPO herbicide S-3100 was measured as the concentration (IC) that brought about 50% inhibition of the control activity. The IC value of S-3100 for each PPO being evaluated was determined graphically from a semi-logarithmic plot of S-3100 concentration against PPO activity. For the PPO enzymes from three plant sources (Amaranthus retroflexus, soybean, or maize) and the microbial HemG PPO enzymes (SEQ ID NO: 1 to SEQ ID NO: 10), K was found to be in the range of 0.3 μM to 2.0 μM, indicating similarity. The IC values of the three plant PPO enzymes tested were 0.009, 0.004, and 0.003 μM, respectively. By adding the heat-inactivated (100 °C for 5 minutes) extract to the assay mixture, an assay blank was performed. The substrate (protoporphyrinogen) binding affinity of protoporphyrinogen oxidase was measured as K.

[0082] The apparent K value for each PPO being evaluated was calculated using the curve fitting of a rectangular hyperbola with SoftPro® kinetic software package (Molecular Devices, Sunnyvale, California). The sensitivity of the enzyme activity to the PPO herbicide S-3100 was measured as the concentration (IC) that brought about 50% inhibition of the control activity. m The apparent K value for each PPO being evaluated was calculated using the curve fitting of a rectangular hyperbola with SoftPro® kinetic software package (Molecular Devices, Sunnyvale, California). m The sensitivity of the enzyme activity to the PPO herbicide S-3100 was measured as the concentration (IC) that brought about 50% inhibition of the control activity. The apparent K value for each PPO being evaluated was calculated using the curve fitting of a rectangular hyperbola with SoftPro® kinetic software package (Molecular Devices, Sunnyvale, California). The sensitivity of the enzyme activity to the PPO herbicide S-3100 was measured as the concentration (IC) that brought about 50% inhibition of the control activity. The apparent K value for each PPO being evaluated was calculated using the curve fitting of a rectangular hyperbola with SoftPro® kinetic software package (Molecular Devices, Sunnyvale, California). The sensitivity of the enzyme activity to the PPO herbicide S-3100 was measured as the concentration (IC) that brought about 50% inhibition of the control activity. 50 The apparent K value for each PPO being evaluated was calculated using the curve fitting of a rectangular hyperbola with SoftPro® kinetic software package (Molecular Devices, Sunnyvale, California). The sensitivity of the enzyme activity to the PPO herbicide S-3100 was measured as the concentration (IC) that brought about 50% inhibition of the control activity. 50 The apparent K value for each PPO being evaluated was calculated using the curve fitting of a rectangular hyperbola with SoftPro® kinetic software package (Molecular Devices, Sunnyvale, California).

[0083] For the PPO enzymes from three plant sources (Amaranthus retroflexus, soybean, or maize) and the microbial HemG PPO enzymes (SEQ ID NO: 1 to SEQ ID NO: 10), K was found to be in the range of 0.3 μM to 2.0 μM, indicating similarity. The apparent K value for each PPO being evaluated was calculated using the curve fitting of a rectangular hyperbola with SoftPro® kinetic software package (Molecular Devices, Sunnyvale, California). m For the PPO enzymes from three plant sources (Amaranthus retroflexus, soybean, or maize) and the microbial HemG PPO enzymes (SEQ ID NO: 1 to SEQ ID NO: 10), K was found to be in the range of 0.3 μM to 2.0 μM, indicating similarity. The IC values of the three plant PPO enzymes tested were 0.009, 0.004, and 0.003 μM, respectively. For the PPO enzymes from three plant sources (Amaranthus retroflexus, soybean, or maize) and the microbial HemG PPO enzymes (SEQ ID NO: 1 to SEQ ID NO: 10), K was found to be in the range of 0.3 μM to 2.0 μM, indicating similarity. 50 The IC values of the three plant PPO enzymes tested were 0.009, 0.004, and 0.003 μM, respectively. It was susceptible to S-3100. In contrast, the PPO enzyme (SEQ ID NO: 1) from the bacterial donor source was effective. (~Sequence number 10) is IC 50 The value was greater than 100 μM and was insensitive to S-3100. The data is shown in Table 3. Table 3. PPO enzyme activity of enzymes purified from plant or microbial sources. TIFF2026136216000003.tif83155

[0084] Example 4: Enzyme optimization of protoporphyrinogen oxidase Using protein optimization, we can identify the enzymatic characteristics of a novel protoporphyrinogen oxidase. The properties were improved or altered. The enzyme was optimized using one or more protein engineering methods. Non-limiting examples of protein engineering techniques include alanine scanning mutation introduction and homology. Scanning mutation introduction, Pro / Gly scanning mutation introduction, region swap or mutation Examples include heterologous introduction and combinations of these various techniques (M. Lehmann an 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). We synthesized a DNA sequence encoding genetically modified protoporphyrinogen oxidase, and then... Cloning is performed on a bacterial expression vector. This vector is the initial high-sloop described in Example 1. For bacterial rescue screening, the characteristics of hemG knockout E. coli strains Used for conversion. A genetic engineering process to rescue hemG knockout E. coli strains. For toporphyrinogen oxidase, the bacterial growth assay described in Example 2 was used. Then, screen for susceptibility to one or more PPO herbicides. Alternative methods and Then, transformed hemG knockout E. coc into culture media containing and without PPO herbicide. Sow the li strain. Genetically modified mutants showing tolerance to PPO herbicides are expressed in a bacterial expression system. The protein was then expressed using the sequential fluorescence quantitative assay described in Example 3. Perform detailed biochemical characterization. Cloning into plant transformation vectors. For plant transformation and testing on plants, genetically modified plants that are insensitive to PPO herbicides Select a different object.

[0085] Example 5: Expression and testing of HemG PPO enzyme in maize The microbial HemG PPO enzyme was expressed in transgenic maize plants. The PPO herbicide tolerance of this transgenic plant was analyzed. Sequence IDs 1-20 were used. A recombinant DNA molecule encoding one of the microbial HemG PPO enzymes shown A plant transformation vector containing the enzyme was constructed. The DNA sequence encoding the PPO enzyme has a 5' end A methionine codon may be present at the end, which is commonly known as the start codon, or This removes the codon, allowing the transport peptide sequence to be ligated to the 5' end of the coding sequence. It can encourage this. PPO enzyme protein containing methionine at the amino terminus Examples of the sequence are provided as sequence numbers 1-10. PP without methionine at the amino terminus. Examples of O enzyme protein sequences are provided as SEQ ID NOs: 11-20. Therefore, the nucleotide sequence encoding the presumed PPO enzyme is used for expression in dicotyledonous or monocotyledonous organisms. Codon optimization was performed. Table 4 shows the sequence numbers corresponding to the proteins and the transformation vectors. The nucleotide sequence of the microbial HemG PPO enzyme is shown. Table 4. Sequence IDs corresponding to PPO variants TIFF2026136216000004.tif86166

[0086] To express HemG PPO H_N10 (SEQ ID NO: 4), two different protocols are used. Motor + Leader + Intron combination, two different target peptide (TP) sequences We created four plant transformation vectors using two different 3'UTR sequences. The material transformation constructs were Annotated constructs 1, 6, 11, and 16. For maize plant testing, Agrobacterium tumefacie Using ns and standard methods known in the art, immature maize (LH244) embryos It was transformed.

[0087] Plant traits containing the gene encoding HemG PPO H_N10 (SEQ ID NO: 4) Transgenic maize plants are produced using conversion constructs 6 and 16. Leaf samples were collected from R0 plants and screened by PCR to identify the plant genotype. The number of copies of the introduced gene inserted into the plant was determined. Plants containing a single copy (single copy) We performed inbreeding and outbreeding on (P) to produce R1 and F1 seeds, respectively, for future testing. The following spraying was performed on plants containing multiple copies (multi-copy plants). (1) Approximately 5g / ha of S-3100 at the V5 growth stage, then approximately 1 (2) 0 g / ha of S-3100, or a total application of 15 g / ha of S-3100 Approximately 10g / ha of S-3100 at the V5 growth stage. The average percentage of damage is 0- On a scale of 100, 0 represents no damage and 100 represents complete crop death. Final treatment for each batch. The evaluation was performed 7 days after the procedure. Non-transgenic LH244 corn that had been sprayed was evaluated. This was used as a control, but this is the case when treated with a total of 15g / ha of S-3100 (Treatment 1) ) showed an average of 43.3% damage, and when treated with a total of 10g / ha of S-3100 ( The 2) tissue showed an average of 26.7% damage. It expresses HemGH_N10 (SEQ ID NO: 4). The transgenic maize plants performed better than the control plants, totaling 1 When treated with 5g / ha of S-3100 (treatment 1), an average of 28.9% damage occurred, totaling 10 When treated with S-3100 at a rate of g / ha (treatment 2), the average damage rate was 24.2%. The data is shown in Table 5. Table 5: Herbicide tolerance of transgenic corn I TIFF2026136216000005.tif39155

[0088] HemG PPO enzymes H_N90 (SEQ ID NO: 1), H_N10 (SEQ ID NO: 4), H_N 60 (sequence number 3), H_N110 (sequence number 10), and H_N40 (sequence number 6) Plant transformation constructs 6, 20, 21, and 2, respectively, contain the gene encoding the gene. Transgenic maize plants were generated using 2 and 23. These five species The construct consists of the same promoter + leader + intron combination, and two different types. It had the target peptide (TP) sequence and the same 3'UTR sequence. Leaf samples were planted in R0. The transgene was collected from the material and analyzed by PCR to determine the copy number. Transplant single copy plants for up to 12 unique events into pots and perform inbreeding. Furthermore, crossbreeding was performed to produce R1 and F1 seeds, respectively, for future testing. For copy plants and extra single copy plants, approximately 40 grams / h at the V5 growth stage. S-3100 of a was sprayed, and damage assessment was performed 7 days after treatment. Each PPO enzyme expressed... The average percentage of damage to all plants (%) and those considered to have high tolerance (less than 10% damage) were determined. The number of plants was recorded. Each plant had a unique single copy or multi-copy trait transformation. This is a transgenic maize plant expressing H_N10 (SEQ ID NO: 4). The overall average damage rate was 39.5%, and 31 out of 139 plants tested showed tolerance. High-quality plants were produced. Transgenic corn expressing H_N60 (SEQ ID NO: 3) The overall average damage rate for the Koshi plants was 55.8%, with 19 out of 86 plants tested showing resistance. A highly tolerant plant was produced. A transgenic plant expressing H_N90 (SEQ ID NO: 1) Corn plants had the lowest overall average damage rate at 31.4%, compared to the 99 plants tested. We produced 44 highly tolerant plants. Trans-diphen expressing H_N40 (SEQ ID NO: 6) The average overall damage to the corn plants was 47.0%, out of 98 plants tested. We generated 53 highly tolerant plants from existing plants, and produced the most highly tolerant plants. Transgenic maize plants expressing H_N110 (SEQ ID NO: 10) The average damage rate was 43.0%, but no highly tolerant plants were produced. This is shown in Table 6. Table 6: Herbicide tolerance of transgenic corn II TIFF2026136216000006.tif51155

[0089] Data from R0 transgenic maize demonstrates that five types of microorganisms HemG PPO enzymes H_N90 (SEQ ID NO: 1), H_N10 (SEQ ID NO: 4), H_N6 0 (sequence number 3), H_N40 (sequence number 6), and H_N110 (sequence number 10) are, When expressed in transgenic plants, it reduces the rate of damage, and therefore crop The key feature is that it has been given resistance to PPO herbicides.

[0090] In one of the two construct configurations, H_N10 (sequence number 4) is expressed. Herbicides produced from transgenic F1 plants created by crossbreeding single copy R0 plants. Tolerance was tested in a greenhouse. The plants were given 40 grams / ha of S-3 at the V3 growth stage. The treatment was performed at 100, and damage assessment was conducted 7 days after treatment. In the construct 6 configuration, H In transgenic maize plants expressing _N10 (SEQ ID NO: 4), 18 events occurred. Of these, 13 events resulted in plants with high tolerance (less than 10% damage), but the construction In the 16-component configuration, no plants with high tolerance to any of the phenomena were produced.

[0091] In one of the two construct configurations (Constructs 6 and 16), H_ Tran generated from outcrossing of single-copy R0 plants expressing N10 (SEQ ID NO: 4) The herbicide tolerance of Sgenic F1 plants was tested in the field. This F1 population was isolated from the other population (5 (0% hemijunction, 50% defect), and transgenic plants were selected before damage assessment. It was not possible. Distinguishing non-transgenic plants from transgenic plants is difficult. Therefore, the overall average injury rating for such a group is homogeneous transgenic It is expected to be higher than the group. Trials will be held at two locations, construction Each plant underwent two repeated tests and three different treatments. Non-transgenic plants were treated with negative treatment. It was used as a light source. The herbicide application treatment was as follows: Treatment 1 was V2, then V4, Next, apply S-3100 at 0.036 lb ai / acre in V8, treatment 2 is V2, then Treatment 3 involves applying S-3100 at 0.072 lb ai / acre with V4, then V8, Apply S-3100 at 0.144 lb ai / acre in V2, then V4, then V8. The percentage of physical damage was assessed at the V2 growth stage (CIPV2) and V4 growth stage 5-7 days after treatment. Evaluated using the long-stage scale (CIPV4) (Errors V2 and V4 were based on the least significant difference (LSD)). (Half). Crop damage assessments at both locations were combined. All non-transgenic The plants that produced the phenomenon using the plant and construct 16 were each treated in one of three ways. Regarding this, the loss after herbicide application in both V2 and V4 ranged from 94.6% to 99.5%. Damage was observed. Plants that exhibited the event using Construct 6 after application of the V2 herbicide. Damage was limited to 30% to 50%, and no damage was observed 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 TIFF2026136216000007.tif66158

[0092] The greenhouse and field data from F1 transgenic maize demonstrated that The microbial enzyme HemG PPO enzyme H_N10 (SEQ ID NO: 4) was found in transgenic plants. When expressed in this manner, it reduces the damage rate, thus improving crop tolerance to PPO herbicides. The fact is that it was granted.

[0093] Example 6: Expression and testing of HemG PPO enzyme in soybean plants The microbial HemG PPO enzyme was expressed in transgenic soybean plants, and this The tolerance of transgenic plants to PPO herbicides was analyzed. H_N10 (SEQ ID NO: 4 and Sequence ID 13), H_N20 (Sequence ID 12), H_N30 (Sequence ID 14), H_N4 0 (sequence number 15), H_N50 (sequence number 16), H_N90 (sequence numbers 11, 18, 19), and the microorganism HemG PP, represented as H_N100 (SEQ ID NOs: 17, 20) A plant transformation vector containing a recombinant DNA molecule encoding one of the O enzymes is constructed. It was built.

[0094] In soybeans, A. tumefaciens and standard methods known in the art are used. Using this method, excised embryos (A3555) were transformed with transformation constructs 1 and 11. Qualitative transformation constructs 1 and 11 use the same promoter + leader + intron combination. Furthermore, the same 3'UTR sequence and the same microorganism HemG PPO H_N10 (sequence number 4) It was present, but the target peptide (TP) sequence was different. Regenerated R0 transgenic The seedlings were grown in a greenhouse. 20 single copies R0 dies were generated from construct 11. In plants that represent the event and express the microbial PPO enzyme H_N10 (SEQ ID NO: 4), 2 10g / ha of flumioxazin (Valor®, Valent USA) We sprayed Walnut Creek Corporation (CA). The herbicide was sprayed onto the V3 developmental stage plant, which had three trifoliate leaves, and damage assessment was performed 8 days after treatment. Evaluated after [number] days. Percentage of plants considered to have high tolerance (less than 15% damage). This was recorded. After application of 210 g / ha of flumioxazine, non-transgenic soybeans vs. The plants showed an average damage rate of 30%, and no highly tolerant plants were observed. Microbial PPO yeast Soybean plants expressing the gene H_N10 (sequence number 4) had an average damage rating of 22%, and Nine percent of the plants tested showed high tolerance to the herbicide.

[0095] Representing the 10 multicopy R0 dice events generated from construct 11, In plants expressing the PPO enzyme H_N10 (SEQ ID NO: 4), 5 g / ha of S-3100 was administered. The herbicide was sprayed onto the V3 developmental stage plants, which had three fully developed trifoliate leaves, and damaged them. Damage assessment was performed 8 days after treatment. Plants were deemed to have high tolerance (damage of 25% or less). The percentage was recorded. After application of S-3100 (5g / ha), non-transgenic The black soybean control plants showed an average damage rate of 60%, and no highly tolerant plants were observed. Soybean plants expressing the biological PPO enzyme H_N10 (SEQ ID NO: 4) have an average damage rating of 47 The percentage was such that 30% of the tested plants had high tolerance to the herbicide.

[0096] The following three types of herbicides were applied to single-copy transgenic R1 soybean plants in a greenhouse. One of the usage rates was used for spraying: Treatment 1 was V4, then R1 at 5 grams ai / ha S -3100 was applied, treatment 2 was V4, then R1 with 10 grams ai / ha of S-3100 Treatment 3 involves applying 30 grams ai / ha of S-3100 in V4, then R1. The percentage of crop damage (CIPV2) in 2 was assessed 10 days after treatment. Lancegenic plants have an average damage rating of 89% to 100% during the R1 growth stage. It could not be used for evaluation. It was produced from construct 1 and microbial PPO yeast. Plants expressing the gene H_N10 (sequence number 4) had damage ratings ranging from 3% to 15.7%. The data is shown in Table 8. Table 8: S-3100 efficacy screening of R1 soybeans in greenhouses TIFF2026136216000008.tif54164

[0097] Using a high-throughput plant transformation and screening method, transgenic The herbicide tolerance of numerous plant constructs was evaluated in the tissues of early transgenic seedlings. This allows for faster and larger-scale screening of construct composition and PPO enzymes. It became possible to do so.

[0098] 7 types of microorganisms HemG PPO enzymes H_N10 (Sequence No. 13), H_N20 (Sequence No. Number 12), H_N30 (Sequence ID 14), H_N40 (Sequence ID 15), H_N50 (Distribution ID Column number 16), H_N90 (sequence numbers 11, 18, 19), and H_N100 (sequence number The genes encoding 17, 20) were linked to 37 different target peptides so that they could act upon them. It was cloned into a base plant transformation vector. This allowed the same promotion to be applied to gene expression. Using the 3'UTR element, seven different HemG PPO enzymes and 37 different types Parallel comparison with different target peptides has become possible. (A. tumefaciens and this technology) Using standard methods known in the field, these plant transformation constructs were cut into soybeans. Used for transformation of deembryonic (germplasm A3555). 400 explants were used in each construct. It was inoculated, resulting in 12 containers per construct. Sterile PPO herbicide solution The liquid was used in the herbicide tolerance test. The herbicide solution was concentrated crop oil (5.0 mL). It consisted of 0.3 g of S-3100 in 495 mL of deionized water. This was added in 0.45 ml. Ron's Nalgene® Rapid-Flow® Tissue Culture Filter Knit and surfactant-free cellulose acetate membrane filter unit (VWR, The solution was filtered using a Radnor filter (PA, USA). The resulting sterile solution was shaken before application.

[0099] Five weeks after transformation, four out of twelve plant containers per construct were found to have developed the necessary growth. A sterile PPO herbicide solution was sprayed twice. Then, the treated seedlings were sealed in a container and sprayed again each day. The subjects were subjected to at least 15 hours of light exposure for 4 days. At the end of the 4th day after S-3100 application... Next, the treated seedlings were photographed, and the green coloration (the green coloration is compared to healthy light-bleeded tissue) was observed. The damage (representing synthetic plant tissue) was scored using a visual scale of damage resistance. A score of 0 indicates low damage. Tolerance, high damage, low green coloration, some tolerance, average damage, moderate Green coloration of 2, good tolerance, low damage, high green coloration. Each construct The scores are shown in Table 9. nd indicates that no analysis was performed. These results... In this high-throughput screening, multiple constructs were found to be suitable for PPO herbicides. This indicates that it has achieved tolerance. Table 9. High-throughput soybean screening for herbicide tolerance: colorimetric score TIFF2026136216000009.tif219155

[0100] Using standard methods known in the art, the spray corresponding to a construct with a score of 2 Seedlings from containers that were not misted were transplanted approximately 7 weeks after transformation and grown as R0 plants. Seedlings corresponding to tolerance scores of 0 and 1 were also grown and used as negative controls. R0 plants Under long-day conditions (18 hours of light at 80°F, followed by 6 hours of darkness at 74°F) for approximately 4 weeks. They were grown in a greenhouse. At 11 weeks, the same herbicide solution as described above (0.3g of S-3100) was used. The herbicide was sprayed twice onto R0 plants. Herbicide damage assessments were collected 7 days after treatment.

[0101] The results of herbicide tolerance application to R0 plants at 11 weeks showed high through rate at 5 weeks. This supports the low percentage of injury rating scores observed in put screening. Damage ratings of 30% or more are equivalent to damage ratings for non-transgenic dies. Tsuka's construct stands out for its exceptionally good tolerance to herbicide application. For example, if PPO H_N90 (sequence number 11) is used in TP1, only 3% damage, PPO H_N30 (SEQ ID NO: 14) or H_N40 (SEQ ID NO: 15) When using this method, damage was only 5%, and in TP20, PPO H_N90 (Sequence ID 11 When using ), the damage was only 5%. In contrast, with TP32, PPO H_ When using N90 (Sequence ID 11), the damage score was 50%. The data is shown in Table 10. nd indicates that no analysis was performed. Table 10. Herbicide tolerance of R0 transgenic soybeans: Percent damage score TIFF2026136216000010.tif214155

[0102] To further evaluate plant transformation constructs in soybeans, A. tumefa Using plant transformation vectors by ciens and standard methods known in the art, Deembryonic (A3555) is transformed. The regenerated R0 transgenic seedlings are grown in a greenhouse. Divide the plants into multiple groups. For each of these groups, spray with one or more types of PPO herbicides. Evaluate herbicide tolerance. For example, R0 transgenic plants in approximately V2-V4 growth stages. For plants, apply lactofen at a rate of 110g ai / ha (0.09lb ai / acre). Alternatively, spray at a rate of 220g ai / ha (0.19lb ai / acre). Evaluate plant damage 1 to 14 days later and record the damage score. Transgenic DN Leaf samples to identify transgenic plants with a single copy of insertion A Using this method, R0 plants containing only single copies and passing herbicide spray tests are selected for their similarity. Crossbreed to produce R1 seeds.

[0103] R1 plants are grown in a greenhouse and divided into multiple groups. Each of these groups is then given at least one species. The herbicide tolerance is evaluated by spraying with a PPO herbicide. For example, lactofervine Apply 200g ai / ha (0.19) before germination and / or during the V2-V6 growth stage. Apply at a ratio of lb ai / acre. Assess plant damage 1 to 14 days after treatment. Record the damage score. Transgenic plants that are not sprayed are placed in fields that are not sprayed. It will be used for phenotypic comparison with live-type plants.

[0104] By crossing homozygous transgenic R1 plants in the same line and collecting seeds, R2 Induce plant growth. Evaluate R2 plants in one or more field locations or greenhouse assays. Apply herbicide treatment, and 1 to 14 days after herbicide application, ensure that there is no crop damage to the plot or plants. The results are rated on a scale of ~100 (0 being no damage, 100 being complete crop death).

[0105] Example 7: Leaf Disc Assay Rapid herbicide tolerance in transgenic plants expressing recombinant PPO enzymes Furthermore, to evaluate with minimal damage, a leaf disc assay was used. Samples of young, fully green leaf tissue were taken from soybean plants. Biopsy punch with jar (Integra®, Miltex®, Inc.) Using *Pennsylvania* (York, Pennsylvania), a 4mm diameter rib was extracted from a leaf sample. Cut out 5 leaf discs and store these leaf discs in a 24-well polystyrene container with a lid. 1 ml of incubation solution in the plate (1 mM MES, pH 6.5, 1% w / v) Sucrose and 1% (v / v) acetone were placed in this incubation chamber for herbicide tolerance analysis. S-3100 was added to the solution, and the final concentration was adjusted to 1 micromolar. Vacuum filtration was then applied. The leaf disc plates were incubated at room temperature (23-24°C) for one day in continuous darkness. Next, under the fluorescent and incandescent light bulbs (520uE) on the ceiling, at 26-27°C for one day (soybeans). Alternatively, they were incubated for two consecutive days (corn) of light. Next, the leaf disks were damaged. The injuries were visually scored on a scale from 0 (lowest injury) to 4 (highest injury). This indicates that it has good tolerance to PPO herbicides.

[0106] A tiger that uses construct 6 and expresses the PPO enzyme H_N10 (sequence number 4) Leaf discs from genic maize plants have an average leaf disc score of 0. It showed remarkable herbicide tolerance based on .4. This result was obtained when transformed with construct 6. , and PP observed in complete plants expressing the PPO enzyme H_N10 (SEQ ID NO: 4) O was consistent with herbicide tolerance. Construct 1 and Construct 11 were used. Transgenic soybeans that express the PPO enzyme H_N10 (SEQ ID NO: 4) Due to leaf discs from objects, plants with Construct 1 show no significant damage with a damage rating of zero. While the plants are herbicide-tolerant, those treated with Construct 11 received a damage rating of 1.6 and were non-transmitted. The Sgenic plant was shown to have a damage 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 this This study analyzes the PPO herbicide tolerance of lancegenic plants. In cotton, A. tume Using Faciens and standard methods known in the art, these vectors are used to extract embryos. Coker130 was transformed. The regenerated R0 transgenic seedlings were grown in a greenhouse. Then, divide them into multiple groups. These groups will be assigned PPO herbicides (one type of PPO herbicide per group). It is used to evaluate tolerance by spraying it. For example, at the growth stage when there are approximately 2-4 true leaves. For R0 transgenic seedlings, lactofen was administered at a rate of 110g ai / ha (0.09lb). (ai / acre) or 220g ai / ha (0.19lb ai / acre) ratio Spray at a rate. Evaluate plant damage 1 to 14 days after treatment and record the damage score. Leaves Using the sample, a transgenic insertion with a single copy Identify nicks. Identify R that contain only single copies and pass herbicide spray tests. R1 seeds are produced by crossbreeding 0 plants in the same lineage.

[0108] R1 plants are grown in a greenhouse and divided into multiple groups. Each of these groups is then given at least one species. The herbicide tolerance is evaluated by spraying with a PPO herbicide. For example, lactofervine Apply 200g ai / ha (0.5g) before germination and / or from the stage of 2 true leaves to the first flowering stage. Apply at a ratio of 19 lb ai / acre (1X). Plants should be treated 1 to 14 days after application. Evaluate the damage and record the damage score. For transgenic plants that are not sprayed, spray... It is used for phenotypic comparison with wild-type plants that have not undergone this procedure.

[0109] By crossing homozygous transgenic R1 plants in the same line and collecting seeds, R2 Induce plant growth. Evaluate R2 plants in one or more field locations or greenhouse assays. Apply herbicide treatment, and 1 to 14 days after herbicide application, ensure that there is no crop damage to the plot or plants. The results are rated on a scale of ~100 (0 being no damage, 100 being complete crop death).

Claims

1. At least 85% of polypeptide sequences selected from the group consisting of SEQ ID NOs: 1 to 20 A heterologous promo that is ligated to a nucleic acid sequence encoding a protein having sequence identity. A recombinant DNA molecule containing a protein, wherein the protein is a herbicide-insensitive protocol. The recombinant DNA molecule having philinogen oxidase activity.

2. The set according to claim 1, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 22 to 63. Replacement DNA molecule.

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

4. The recombinant DNA molecule according to claim 1, wherein the heterologous promoter functions in plant cells. 。

5. The nucleic acid sequence functions to localize the functionally linked protein within the cell. The recombinant according to claim 4, which is ligated to a DNA molecule encoding a target sequence in a manner that allows it to act upon it. DNA molecule.

6. A DNA construct comprising the recombinant DNA molecule described in claim 1.

7. The recombinant DNA has a target sequence that functions to localize the protein within the cell. The DNA construct according to claim 6, comprising an operablely linked DNA molecule encoding Kuto.

8. The DNA constraints according to claim 7, wherein the protein confers herbicide tolerance to the cells. Tract.

9. The transgenic plant, seed, or cell genome described in claim 6. DNA construct.

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

11. A transgenic plant, seed, or cell comprising the recombinant DNA molecule described in claim 1. Or the plant part.

12. The trans-herbicide according to claim 11, comprising additional transgenic herbicide tolerance traits. A plant, seed, cell, or part of a plant.

13. A claim defined as having herbicide tolerance to at least one type of PPO herbicide. The transgenic plants, seeds, cells, or plant parts described in item 11.

14. The seed according to claim 11.

15. Transgenic plants, seeds, and sphagia comprising the recombinant polypeptide described in claim 10. A sac, or part of a plant.

16. A method for conferring herbicide resistance to plants, seeds, cells, or plant parts, as described in claim 1 The recombinant polypeptide described in 0 is heterologously used in the plant, seed, cell, or plant part. The method, including causing expression.

17. The plant, seed, cell, or plant part is conferred by the recombinant polypeptide. The method according to claim 16, comprising protoporphyrinogen oxidase activity.

18. The aforementioned herbicide tolerance is asyfluorphen, homesaphen, lactofen, fluorog Lycophen-ethyl, oxyflurphen, flumioxazine, azaphenidine, cal Fentrazone-ethyl, sulfentrazone, fluthiaset-methyl, oxazial Gil, oxadiazone, pyraflufen-ethyl, saflufenacil, and S-3100 The claim relates to at least one PPO herbicide selected from the group consisting of the following. The method described in 16.

19. A method for transforming plants, a) The step of introducing the recombinant DNA molecule described in claim 1 into plant cells, b) The method comprising the step of regenerating a plant containing the recombinant DNA molecule therefrom. Law.

20. The further step includes selecting a plant that is tolerant to at least one type of PPO herbicide. The method according to claim 19.

21. The regenerated plant is crossbred with itself or with a second plant, and seeds are collected from the hybrid. The method according to claim 19, further comprising the step of collecting.

22. A method for controlling weeds in a plant growing area, the transjet described in claim 11. Contact the plant growing area containing nicks or seeds with at least one type of PPO herbicide. This includes the fact that the transgenic plant or seed is tolerant to the PPO herbicide. The method wherein weeds are controlled in the plant growth area.

23. Nucleo encoding a protein with protoporphyrinogen oxidase activity A method for identifying cido sequences, a) E. coli with a gene knockout of the PPO enzyme from native E. coli. The strain contains recombinant DNA molecules encoding candidate herbicide-tolerant proteins. Transformation using the current vector, b) Growing the transformed E. coli using a heme-free bacterial culture medium. The growth using the aforementioned bacterial culture medium is associated with protoporphyrinogen oxidase activity. The method comprising identifying the protein it possesses and amplifying it.

24. Proteins possessing herbicide-insensitive protoporphyrinogen oxidase activity A method for identifying a nucleotide sequence, a) E. coli with a gene knockout of the PPO enzyme from native E. coli. The strain was expressed using a bacterial expression vector containing recombinant DNA molecules encoding recombinant proteins. And transforming, b) The transformed E. coli is subjected to a fine-grained solution containing at least one PPO herbicide. This involves growing bacteria using a culture medium, and the growth of bacteria is due to herbicide-resistant protoporphy To identify proteins that have linogen oxidase activity, and to propagate them as described above. The method described above.

25. A method for screening genes for herbicide tolerance, a) Expressing the recombinant DNA molecule described in claim 1 in plant cells, b) The method comprising identifying plant cells that exhibit herbicide tolerance to PPO herbicides. 。

26. A method for screening genes for herbicide tolerance, a) The recombinant DNA molecule described in claim 1 is expressed in HemG-deficient bacterial cells. This means that the bacterial cells grow in a heme-free bacterial culture medium in the presence of the PPO herbicide. To increase, to express the above, b) The method comprising identifying bacterial cells that exhibit tolerance to PPO herbicides.

27. A method for producing plants tolerant to PPO herbicides and at least one other herbicide. 、 a) To obtain the plant described in claim 11, b) The transgenic plant is endowed with tolerance to at least one other herbicide. Cross-breeding with the second plant obtained, c) The hybrid having tolerance to PPO herbicides and at least one other herbicide. The method comprising selecting the progeny obtained therefrom.

28. A method for reducing the occurrence of herbicide-resistant weeds, a) Cultivating the plant described in claim 12 in a crop growth environment, b) Applying a PPO herbicide and at least one other herbicide to the crop growing environment. The crop plants are tolerant to the PPO herbicide and the at least one other herbicide. The method comprising the act of applying, which is a property of the body.

29. The aforementioned PPO herbicide contains asyfluorphen, homesaphen, lactofen, and fluorog Lycophen-ethyl, oxyflurphen, flumioxazine, azaphenidine, cal Fentrazone-ethyl, sulfentrazone, fluthiaset-methyl, oxazial Gil, oxadiazone, pyraflufen-ethyl, saflufenacil, and S-3100 The method according to claim 28, selected from the group consisting of the following.

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

31. The ACCase inhibitor is aryloxyphenoxypropionate or cyclohexyl Xanthione, and the ALS inhibitor is sulfonylurea, imidazolinone, tria The EPSPS inhibitor is a zolopyrimidine or triazolinone, and the EPSPS inhibitor is glyphosate The synthetic auxin is a phenoxy herbicide, benzoic acid, carboxylic acid, or semi-semi The carbazone is a photosynthesis inhibitor, and the photosynthesis inhibitor is a triazine, triazinon, nitrile, or benzo The glutamine synthesis inhibitor is thiadiazole or urea, and the glutamine synthesis inhibitor is glufosinate. Yes, the HPPD inhibitor is isoxazole, pyrazolone, or triketone. The PPO inhibitor is diphenyl ether, N-phenylphthalimide, aryltri Azinon or pyrimidinedione, or the long-chain fatty acid inhibitor is chloroacetate The method according to claim 30, wherein the toamide is a toamide, oxyacetamide, or pyrazole.

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