Active polypeptide of protoporphyrinogen oxidase and application

Abstract

The invention discloses an active polypeptide of protoporphyrinogen oxidase and application, the active polypeptide not only has the activity of the protoporphyrinogen oxidase, but also has better tolerance to PPO inhibitor herbicides by transferring a nucleotide sequence for coding the active polypeptide. The herbicide tolerance is wide. The active polypeptide disclosed by the invention shows relatively high tolerance to PPO inhibitor herbicides, so that the active polypeptide has a wide application prospect in plants. The herbicide tolerance is high. The active polypeptide disclosed by the invention has strong tolerance to a PPO inhibitor herbicide, and shows high tolerance to saflufenacil, sulfentrazone, flumioxazin and lactofen which are 4 times of field concentration. The weed control method provided by the invention can effectively reduce the occurrence of resistant weeds, has broad-spectrum herbicide resistance, effectively reduces the cost, improves the control efficiency, and also can greatly avoid the occurrence of weed resistance.

Description

(I) Technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an active polypeptide of protoporphyrinogen oxidase tolerant to PPO inhibitor herbicides and an application thereof. (II) Background technology

[0002] Weeds compete with crops for nutrients and growth space, affecting crop growth and reducing crop yields. Weed control is an important part of crop production. Using herbicides to control weeds can reduce the impact of weeds on crop growth and stabilize and increase crop yields. Introducing herbicide-resistant genes into crops through genetic engineering methods to obtain herbicide-resistant transgenic crops can improve the efficiency of crop weed control, reduce production costs, and bring huge economic benefits to agricultural production, thereby achieving positive social and ecological benefits.

[0003] Crops can acquire herbicide resistance through genetic improvement technology. For example, the cp4 epsps gene from Agrobacterium tumefaciens sp strain CP4 was introduced into crops through Agrobacterium-mediated transformation, and crops expressing 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gained tolerance to the herbicide glyphosate. However, the long-term use of a single herbicide often leads to the large-scale production of resistant weeds. The promotion of glyphosate-resistant crops in the Americas for more than 20 years has produced a large number of glyphosate-resistant weeds. It is difficult to achieve effective control of weeds in the field using glyphosate alone.

[0004] As the number of weed species resistant to commonly used herbicides increases, there is a need in the art to develop new herbicide tolerance traits, such as tolerance to herbicides that inhibit protoporphyrinogen oxidase (PPO). PPO herbicides provide a method for controlling herbicide-resistant weeds by conferring traits that are tolerant to these herbicides. Cultivating transgenic crops that tolerate two or more herbicides can provide a variety of options for weed control and can also effectively delay the development of resistant weeds.

[0005] Protoporphyrinogen oxidase plays a role in the biosynthetic pathways of both chlorophyll and heme, in which protoporphyrinogen oxidase converts protoporphyrinogen IX into protoporphyrin IX. After protoporphyrin IX is produced, the chlorophyll and heme biosynthetic pathways differ due to the different metal ions incorporated (iron is used for heme, magnesium is used for chlorophyll). This pathway is conserved in prokaryotes and eukaryotes, and many similar PPOs exist in prokaryotes and eukaryotes. Some prokaryotes (e.g., cyanobacteria) use this pathway for the synthesis of chlorophyll and heme, while other prokaryotes (e.g., Escherichia coli) use this pathway for the synthesis of heme.

[0006] Herbicide-insensitive protoporphyrinogen oxidases have been isolated from a variety of prokaryotes and eukaryotes. Based on their structures, the existing PPO enzymes are divided into three subclasses: HemY, HemG, and HemJ. HemY is a PPO present in terrestrial plants and the target of PPO inhibitor herbicides. It is ubiquitous in prokaryotes and eukaryotes. Hem J is believed to have originated from the α-proteobacteria, but is also found in other proteobacteria and cyanobacteria; Hem J PPO is a membrane-bound oligomer, which is different from HemY or Hem G. Hem G is mainly found in the γ-proteobacteria; HemG PPO is an oxygen-independent enzyme that can form membrane-associated oligomers and uses non-covalently bound flavin mononucleotide (FMN) as a cofactor.

[0007] The method for providing plants tolerant to PPO inhibitor herbicides mainly comprises: 1) detoxifying the herbicide using an enzyme that can convert the herbicide or its active metabolite into a non-toxic product. 2) overexpressing a sensitive PPO so that, given the kinetic constant of the enzyme, sufficient target enzyme amounts relative to the herbicide are produced in the plant, so that despite the presence of the PPO inhibitor herbicide, these sensitive PPOs fully react with the PPO inhibitor herbicide, thereby having sufficient functional enzymes available for use. 3) providing a mutant PPO that is less sensitive to the herbicide or its active metabolite, but retains the property of catalyzing the oxidation of protoporphyrinogen IX to protoporphyrin IX.

[0008] So far, after two decades of research and identification of multiple PPOs, no transgenic crops containing recombinant PPOs have been commercialized. Although a given functional PPO gene can produce a useful level of tolerance to some PPO inhibitor herbicides, it may not necessarily have sufficient tolerance to other PPO inhibitor herbicides. A single functional PPO gene may not be sufficient to provide crops with commercialization levels, and not all PPO genes can be used to prepare crops that are tolerant to PPO inhibitor herbicides. In this regard, identifying and mining more different categories of PPO genes and using them to create transgenic crop traits has great agricultural application prospects. (III) Summary of the invention

[0009] The purpose of the present invention is to provide an active polypeptide tolerant to PPO inhibitor herbicides and its application, wherein the active polypeptide not only has protoporphyrinogen oxidase activity, but also a plant into which a nucleotide sequence encoding the active polypeptide of the present invention is transferred has good tolerance to PPO inhibitor herbicides.

[0010] The technical solution adopted by the present invention is:

[0011] In a first aspect, the present invention provides an active polypeptide of protoporphyrinogen oxidase that is insensitive to herbicides, wherein the active polypeptide has at least 85%, 90%, 95%, 99%, or 100% sequence identity with the amino acid sequence shown in one of SEQ ID NOs: 1-23, and has protoporphyrinogen oxidase activity.

[0012] It is also clear to those skilled in the art that the structure of a protein can be changed without adversely affecting its activity and functionality, for example, one or more conservative amino acids can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional configuration of the protein molecule. Examples and embodiments of conservative amino acid substitutions are clear to those skilled in the art. Specifically, the amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., another non-polar amino acid residue can be substituted with a non-polar amino acid residue, another polar non-charged amino acid residue can be substituted with a polar uncharged amino acid residue, another basic amino acid residue can be substituted with a basic amino acid residue, and another acidic amino acid residue can be substituted with an acidic amino acid residue. As long as the substitution does not damage the biological activity of the protein, conservative substitutions in which one amino acid is replaced by other amino acids belonging to the same group fall within the scope of the present invention. In addition, the present invention also encompasses mutant proteins comprising one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired function and biological activity of the protein of the present invention. As is well known in the art, one or more amino acid residues can be deleted from the N and / or C termini of a protein while still retaining its functional activity. Therefore, in another aspect, the present invention also relates to fragments which have one or more amino acid residues deleted from the N and / or C terminus of the active protein while retaining the desired functional activity, which are also within the scope of the present invention.

[0013] In a second aspect, the present invention provides a recombinant DNA molecule, the recombinant DNA molecule comprises an active polypeptide encoding nucleotide sequence, a promoter sequence for initiating expression of the active polypeptide in plants and / or a targeting sequence for localizing the active polypeptide in plant cells. The recombinant DNA molecule may be present in the genome of a transgenic plant, seed, or cell.

[0014] The active polypeptide has at least 85%, 90%, 95%, 99%, 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1-23, and has herbicide-insensitive protoporphyrinogen oxidase activity. The promoter and targeting sequence can be operably linked to the active polypeptide encoding nucleotide sequence and initiate the directional expression of the active polypeptide in plant cells.

[0015] The promoter includes a bacterial promoter or a plant promoter. Plant promoters include inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, and / or spatiotemporally regulated promoters known in the art.

[0016] The targeting sequence can promote the localization of active polypeptides in plant cells, increase the accumulation of recombinant proteins, protect proteins from protein degradation, and / or enhance the level of herbicide tolerance, and thus reduce the level of damage in transgenic cells, seeds, or organisms after herbicide application, and confer herbicide tolerance to the cells, plants, seeds, or plant parts. The targeting sequence includes signal sequences, targeting peptides, localization sequences, and transit peptides known in the art, preferably chloroplast transit peptides (CTP), mitochondrial targeting sequences (MTS), or dual chloroplast and mitochondrial targeting peptides. More preferably, CTP, rice ClpB-P signal peptide, Arabidopsis ClpB3 signal peptide, Arabidopsis MDH signal peptide, Arabidopsis EPSPS signal peptide, petunia EPSPS signal peptide, corn cab-m7 signal sequence, mitochondrial presequence, and pea glutathione reductase signal sequence, etc.

[0017] Furthermore, the nucleotide sequence of the recombinant DNA molecule is shown in one of SEQ ID NOs: 24-46.

[0018] In a third aspect, the present invention provides a DNA construct comprising the recombinant DNA molecule, wherein the DNA construct exists in the genome of a transgenic plant, seed, or cell.

[0019] In a fourth aspect, the present invention provides a plant cell expressing an active polypeptide, wherein the expression of the active polypeptide is achieved by transferring the recombinant DNA molecule or DNA construct into the plant cell. The plant cell exhibits tolerance to at least one PPO inhibitor herbicide.

[0020] In a fifth aspect, the present invention provides a method for obtaining a transgenic plant tolerant to PPO inhibitor herbicides using the active polypeptide, wherein the method comprises heterologously expressing the active polypeptide in a plant, wherein the full-length amino acid sequence of the active polypeptide has at least 85%, 90%, 95%, 99%, or 100% sequence identity to one of SEQ ID NOs: 1-23.

[0021] In a sixth aspect, the present invention provides a method for cultivating transgenic plants with significantly improved resistance to PPO inhibitor herbicides using the active polypeptide, the method comprising one of the following: a) introducing the recombinant DNA molecule into plant cells; regenerating transgenic plants containing the recombinant DNA molecule; b) hybridizing the transgenic plant containing the recombinant DNA molecule with itself or a second plant, and collecting the transgenic plant containing the recombinant DNA molecule.

[0022] In a seventh aspect, the present invention provides a method for controlling weeds in a plant growth area expressing an active polypeptide, the method comprising contacting a plant growth area of ​​a transgenic plant or seed expressing the active polypeptide with at least one PPO inhibitor herbicide, wherein the transgenic plant or seed is tolerant to the PPO inhibitor herbicide and the weeds are controlled in the plant growth area. The active polypeptide has at least 85%, 90%, 95%, 99%, 100% sequence identity to the amino acid sequence shown in one of SEQ ID NOs: 1-23, and has protoporphyrinogen oxidase activity.

[0023] The "PPO inhibitor herbicides" or "PPO herbicides" are chemicals that target and inhibit the enzymatic activity of protoporphyrinogen oxidase (PPO), which catalyzes the dehydrogenation of protoporphyrinogen IX to form protoporphyrin IX, a precursor of heme and chlorophyll. Inhibition of protoporphyrinogen oxidase results in the formation of reactive oxygen species, causing cell membrane disruption and eventual death of susceptible cells. PPO herbicides are well known in the art and are commercially available. PPO herbicides include, but are not limited to, diphenyl ethers (such as acifluorfen, its salts and esters, beniflox, fenpyr, its salts and esters, flufenacet, its salts and esters, fluazifop-butyl, flunixin, flunixin, methoxyfenoxam, fluazifop-butyl, its salts and esters, lactofen, its salts and esters, oxyfluorfen, and flufenacet, its salts and esters); thiadiazoles (such as fluazifop-butyl and flufenacet-butyl); pyrazolidinedione or phenyluracil (such as fluazifop-butyl, fluazifop-butyl, epyrifenacil, flupropacil, flufenacil, and tiafenacil); phenylpyrazoles (such as isopropylpyraclostrobin, pyraclostrobin and pyraclostrobin); diazoles (such as oxadiazine and oxadiazine-methyl); triazolinones (such as mefenacet, bencarbazone, mefenacet, their salts and esters, and sulfentrazone); oxazolidinediones (such as cyclopentadione); N-phenylphthalimides (such as fenoxal, phenoxyacetic acid, fluazifop, and fluazifop-butyl); benzoxazinone derivatives (such as 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3,4-dihydro-3-oxo-4-prop-2-ynyl-2H-1,4-benzoxazin-6-yl)-1,3,5-triazine-2,4-dione): fluazifop-butyl and fluazifop-ethyl; bispyribac-butyl; and flufenacet-butyl.

[0024] Herbicides can be applied to plant growth areas containing plants and seeds provided by the present invention as a method for controlling weeds. Plants and seeds provided by the present invention have herbicide tolerance traits and are thus tolerant to the application of one or more PPO herbicides. Herbicide application can be a recommended commercial rate (1X) or any fraction or multiple thereof, such as twice the recommended commercial rate (2X).

[0025] Further, the PPO inhibitor herbicide is selected from at least one of the following: trifluorfen, fluazifop-butyl, lactofen, fluazifop-butyl, oxyfluorfen, fluazifop-butyl, azopyralid, mesotrione, foramsulfuron, sulfamethoxam, bensulfuron, fluthiazide, propargyl, foramsulfuron, foramsulfuron, oxadiazine, pyraclostrobin, pyrasulfosulfuron, fluthiazide-butyl, fluazifop-butyl, trifluorfen, fluazifop-butyl, epyrifenacil, SY1604 / JS-T205 (benzaclostrobin), FG009 herbicide, foramsulfuron, and uracil compounds (I) containing carboxylate fragments.

[0026]

[0027] In an eighth aspect, the present invention provides a method for cultivating plants tolerant to PPO inhibitor herbicides and at least one other herbicide using the active polypeptide, the method comprising: a) obtaining a transgenic plant comprising the recombinant DNA molecule; b) hybridizing the transgenic plant with a second plant having tolerance or additional traits to at least one other herbicide; c) selecting progeny plants having tolerance or additional traits to PPO inhibitor herbicides and at least one other herbicide produced by the hybridization. The other herbicide is selected from one of the following: an ACCase inhibitor, an ALS inhibitor, an EPSPS inhibitor, a synthetic auxin, a photosynthesis inhibitor, a glutamine synthesis inhibitor, an HPPD inhibitor, or a long-chain fatty acid inhibitor.

[0028] In a ninth aspect, the present invention also provides a method for producing plants tolerant to PPO inhibitor herbicides and at least one other herbicide, the method comprising connecting the recombinant DNA molecule and at least one other recombinant DNA expressing a protein tolerant to other herbicides, introducing the two into the plant genome at the same time, and selecting transgenic plants that are tolerant to PPO inhibitor herbicides and at least one other herbicide at the same time.

[0029] Furthermore, the proteins resistant to other herbicides include EPSPS resistant to glyphosate, PAT or BAR resistant to glufosinate, CdP450 resistant to multiple herbicides, AAD-1 (aryloxyalkanoate dioxygenase) or AAD-12 (aryloxyalkanoate dioxygenase) resistant to 2,4-D, and DMO (sexual development transcription factor protein) resistant to dicamba.

[0030] In the tenth aspect, the present invention also provides a method for reducing the development of herbicide-tolerant weeds in an area where plants expressing active polypeptides are planted, the method comprising: a) cultivating plants that are tolerant to PPO inhibitor herbicides and to at least another herbicide in a crop growth environment; the plant genes contain the recombinant DNA molecule and another herbicide tolerance protein encoding gene; and b) applying the PPO inhibitor herbicide and at least another herbicide to the crop growth environment.

[0031] Furthermore, the at least one other herbicide is selected from one of the following: ACCase inhibitors, ALS inhibitors, EPSPS inhibitors, synthetic auxins, photosynthesis inhibitors, glutamine synthesis inhibitors, HPPD inhibitors and long-chain fatty acid inhibitors.

[0032] Further, the ACCase inhibitor is an aromatic oxyphenoxy propionate or cyclohexanedione; the ALS inhibitor is a sulfonylurea, an imidazolinone, a triazole pyrimidine, or a triazolinone; the EPSPS inhibitor is glyphosate; the synthetic biotin is a phenoxy herbicide, benzoic acid, a carboxylic acid, or a semicarbazone; the photosynthesis inhibitor is a triazine, a triazone, a nitrile, a benzothiadiazole, or a urea; the glutamine synthesis inhibitor is glufosinate; the HPPD inhibitor is an isoxazole, a pyrazolone, or a triketone; or the long-chain fatty acid inhibitor is chloroacetamide, oxyacetamide, or pyrazole.

[0033] Herbicide applications can be sequentially mixed or tank mixed with one, two, or a combination of several PPO herbicides or any other compatible herbicides. Multiple applications of one or two or more herbicides in combination or alone can be used in areas containing transgenic plants of the present invention during the growing season for controlling a wide range of dicotyledonous weeds, monocotyledonous weeds, or both, for example, two applications (such as pre-planting application and post-emergence application or pre-emergence application and post-emergence application) or three applications (such as pre-planting application, pre-emergence application, and post-emergence application or pre-emergence application and two post-emergence applications).

[0034] In the eleventh aspect, the present invention provides an antibody that can bind to the active polypeptide. "Antibodies" are also called immunoglobulins, which are large Y-shaped proteins secreted by B lymphocytes. They can specifically bind to target antigens through complementary sites (antigen binding sites) at the top of the two forks of the Y-shape. The target antigens are proteins, sugars, polynucleotides, lipids, polypeptides, small molecules, etc.

[0035] In a twelfth aspect, the present invention provides a kit for detecting the active polypeptide, wherein the kit is an ELISA detection kit.

[0036] Furthermore, the ELISA test kit includes an antibody that binds to the active polypeptide, a horseradish peroxidase-labeled antibody, a closed system, a coating system, a buffer system, and a reaction termination solution. The closed system consists of: PBS buffer, 5% skim milk; the coating system consists of: PBS buffer; the buffer system consists of: PBST buffer; the reaction termination solution consists of: 2M sulfuric acid.

[0037] The transgenic plants and progeny of transgenic traits provided by the present invention can be used in the case of any breeding method known in the art. In plant strains with two or more transgenic traits, transgenic traits can be separated, connected or a combination of the two independently in plant strains with three or more transgenic traits. In order to confirm the presence of transgenic in a particular plant or seed, various assays can be performed. Such assays include, for example, molecular biological assays, such as Southern and northern blotting, PCR and DNA sequencing; biochemical assays, such as detecting the presence of protein products, for example, by immunological means (ELISA and Western blotting) or by enzyme function; plant part assays, such as leaf or root assays; and also by analyzing the phenotype of the whole plant.

[0038] In the present invention, "protein", "polypeptide" and "peptide" are used interchangeably to refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues. The proteins and polypeptides of the present invention can be produced recombinantly or by chemical synthesis.

[0039] It is well known to those skilled in the art that, due to the degeneracy of the genetic code, there are a variety of different nucleic acid sequences that can encode the amino acid sequence disclosed in the present invention. It is within the capabilities of those of ordinary skill in the art to generate other nucleic acid sequences encoding the same protein, and therefore the present invention encompasses nucleic acid sequences encoding the same amino acid sequence due to the degeneracy of the genetic code. For example, in order to achieve high expression of a heterologous gene in a target host organism such as a plant, the gene can be optimized using codons preferred by the host organism to allow for better expression.

[0040] The present invention provides recombinant DNA molecules and active polypeptides. The "recombinant" refers to DNA, protein, cell, seed or organism that is caused by genetic engineering and is not usually present in nature. A "recombinant DNA molecule" is a DNA molecule that contains a DNA sequence that occurs non-naturally in nature and is caused by human intervention, such as a DNA molecule composed of at least two DNA molecules that are heterologous to each other. An example of a recombinant DNA molecule is a DNA molecule that is operably connected to a heterologous regulatory element or other element (such as a heterologous promoter) encoding a herbicide-insensitive protoporphyrinogen oxidase provided by the present invention. An "active polypeptide" is a protein that contains an amino acid sequence that occurs non-naturally and is caused by human intervention, such as an engineered protein or a chimeric protein. Recombinant cells, seeds or organisms are cells, seeds or organisms that contain transgenic DNA, such as transgenic cells, seeds, plants or plant parts that contain recombinant DNA molecules and are produced by plant transformation.

[0041] Described " DNA construct " is the recombinant DNA molecule that comprises two or more heterologous DNA sequences.DNA construct can be used for transgenic expression and can be contained in vector and plasmid.DNA construct can be used in vector for the purpose of transformation, is about to introduce heterologous DNA into host cell, to produce transgenic plant and cell, and can also be contained in the plasmid DNA or genomic DNA of transgenic plant, seed, cell or plant part thus.Described " vector " means any recombinant DNA molecule that can be used for the purpose of bacterium or plant transformation.As listed in sequence table recombinant DNA molecule can for example be inserted into the part of vector as construct, described construct has the recombinant DNA molecule that can be operably connected to the gene expression element that acts in plant to influence the expression of the engineered protein encoded by recombinant DNA molecule. General methods that can be used to manipulate DNA molecules for the preparation and use of recombinant DNA constructs and plant transformation vectors are well known in the art and are described in detail, for example, in manuals and laboratory manuals including MR Green and J Sambrook, "Molecular Cloning: A Laboratory Manual" (4th edition) ISBN: 978-1-936113-42-2, Cold Spring Harbor Laboratory Press, NY (2012).

[0042] The "recombinant polypeptide" or "active polypeptide" includes recombinant DNA molecules and engineered proteins that have at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, and at least 99% sequence identity with any of the recombinant DNA molecules or polypeptide sequences provided herein and have herbicide-insensitive protoporphyrinogen oxidase activity. The "sequence identity percentage" or "sequence identity %" refers to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of a reference ("query") sequence (or its complementary chain) compared to a test ("target") sequence (or its complementary chain) when the two sequences are optimally aligned (when appropriate nucleotide or amino acid insertions, deletions, or gaps total less than 20% of the reference sequence over the comparison window). The identity of the amino acid sequence can be determined by conventional methods using the BLAST algorithm (Altschul et al., 1990, Mol. Biol. 215: 403-10) available from the National Center for Biotechnology Information www.ncbi.nlm.nih.gov / ) using default parameters. Engineered proteins can be produced by changing (i.e., modifying) wild-type proteins to produce new proteins with modified properties, and modifications can be performed at specific amino acid positions in proteins, and can be amino acids present in this position in nature (i.e., in wild-type proteins) using different amino acid substitutions. Amino acid mutations can be performed as single amino acid substitutions in proteins or combined with one or more other mutations (such as one or more other amino acid substitutions, deletions, or additions). Mutations can be performed by any method known to those skilled in the art.

[0043] Transgenic plant of the present invention, progeny, seed, vegetable cell and plant part also can comprise one or more extra proterties.Extra proterties can be introduced by hybridizing another strain plant comprising the transgenic plant with recombinant DNA molecule provided by the invention with one or more extra proterties.Described " hybridization " means to cultivate two strains of individual plants to produce progeny plants.Therefore two strains of plants can be hybridized to produce the proterties comprising the desirable proterties from each parent.Extra proterties can also be introduced by co-transformation of the DNA construct of this extra transgenic proterties with the DNA construct comprising the recombinant DNA molecule provided by the invention (for example, with all DNA constructs existing as a part for the same vector for plant transformation) or by inserting the extra proterties into the transgenic plant comprising the DNA construct provided by the invention or vice versa (for example, by using plant transformation or any one of the methods for genome editing on transgenic plants or plant cells). Such additional traits include, but are not limited to, increased insect resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, and herbicide tolerance, wherein the traits are measured relative to wild-type plants. Exemplary additional herbicide tolerance traits may include transgenic or non-transgenic tolerance to one or more herbicides, and exemplary insect resistance traits may include resistance to one or more insect members within one or more orders of Lepidoptera, Coleoptera, Hemiptera, and Homoptera, etc. Such additional traits are well known to those skilled in the art: for example, and a list of such transgenic traits is provided by the United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS).

[0044] The plants, seeds, cells, or plant parts of the present invention have protoporphyrinogen oxidase activity conferred by the recombinant polypeptide. The plants include whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, etc. The transgenic plants are derived from transgenic plants or their progeny transformed with the DNA molecules of the present invention and thus at least partially composed of transgenic cells. In the present invention, "plant cell" should be understood as any cell from or found in a plant, which is capable of forming, for example: undifferentiated tissues such as callus, differentiated tissues such as embryos, components of plants, plants or seeds.

[0045] The "herbicide tolerance of plants, plant tissues or cells" or "herbicide-resistant plants, plant tissues or cells" described in the present invention refers to the ability of plants, plant tissues or cells to resist the action of herbicides when herbicides are applied. For example, herbicide-resistant plants can survive or continue to grow in the presence of herbicides. The herbicide tolerance of plants, plant tissues or cells can be measured by comparing the plants, plant tissues or cells with suitable controls. For example, herbicide tolerance can be measured or evaluated by applying herbicides to plants containing DNA molecules encoding proteins capable of conferring herbicide tolerance (test plants) and plants that do not contain DNA molecules encoding proteins capable of conferring herbicide tolerance (control plants), and then comparing the plant damage of the two plants, wherein the herbicide tolerance of the test plant is indicated by a reduction in the damage rate compared to the damage rate of the control plant. Compared with the control plants, plant tissues or cells, the herbicide-resistant plants, plant tissues or cells show a reduced response to the toxic effects of the herbicide. The "herbicide tolerance trait" refers to a transgenic trait that confers improved herbicide tolerance to plants compared to wild-type plants. Plants having the herbicide tolerance trait of the present invention that can be produced include, for example, any plant, including crop plants, such as soybean, corn, sunflower, peanut, rapeseed, wheat, etc.

[0046] "Resistance" as used herein is heritable and allows a plant to grow and reproduce in the presence of a herbicide that is generally effective against a given plant. As recognized by those skilled in the art, even if a given plant is subjected to a degree of damage from a herbicide treatment, such as minimal necrosis, dissolution, chlorosis or other damage, but at least not significantly affected in yield, the plant may still be considered "resistant", i.e., a given plant has an increased ability to resist various degrees of damage induced by a herbicide, while generally causing damage to wild-type plants of the same genotype at the same herbicide dose. "Tolerance" or "tolerance" as used herein is broader than the term "resistance" and includes "resistance".

[0047] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0048] 1. Wide tolerance to herbicides. The active polypeptide of the present invention exhibits high tolerance to PPO inhibitor herbicides, and therefore has broad application prospects in plants.

[0049] 2. Strong tolerance to herbicides. The active polypeptide of the present invention has strong tolerance to PPO inhibitor herbicides, and exhibits high tolerance to saflufenacil, sulfentrazone, fluazifop-butyl and lactofenazole at 4 times the field concentration.

[0050] 3. The weed control method provided by the present invention can effectively reduce the occurrence of resistant weeds. The transgenic crops obtained by the present invention are resistant to multiple herbicides such as PPO inhibitors, ACCase inhibitors, ALS inhibitors, EPSPS inhibitors, and synthetic auxins, and have a broad-spectrum herbicide resistance. In the weed control process, the use of PPO inhibitor herbicides in combination with one or more other herbicides can not only effectively reduce costs and improve control efficiency, but also greatly avoid the occurrence of weed resistance. (IV) Specific implementation methods

[0051] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0052] The molecular biology and biochemical methods used in the following examples of the present invention are all known techniques. Detailed descriptions are provided in documents such as Current Protocols in Molecular Biology, compiled by Ausubel and published by John Wiley and Sons, and Molecular Cloning: A Laboratory Manual, 3rd ED., compiled by J. Sambrook et al. and published by Cold Spring Harbor Laboratory Press (2001).

[0053] Example 1: Obtaining a plasmid vector containing the exogenous gene PPO

[0054] (1) PPO gene expression cassette: The DNA sequence encoding PPO includes a codon for methionine at the 5' end, which is generally known as the start codon. This codon can be eliminated to facilitate the operable connection of the transit peptide sequence to the 5' end of the coding sequence. This embodiment retains this codon. The amino acid sequence of the PPO enzyme protein including methionine at the amino terminus is SEQ ID NO: 1-23. For plant transformation, the nucleotide sequence encoding the putative PPO is codon-optimized for corn expression. After optimization, the nucleotide sequence encoding the putative PPO for corn transformation is SEQ ID NO: 24-46. The 5' end of the DNA sequence of PPO is connected to the rice ClpB-P signal peptide (SEQ ID NO: 47), and is functionally connected to the ZmUbi promoter (SEQ ID NO: 48) and the Nos terminator (SEQ ID NO: 49). The pZmUbi-ClpB-P-PPO-Tnos fragment is artificially synthesized to form the PPO gene expression cassette.

[0055] (2) Screening gene expression cassette: The screening gene G10evo-EPSPS (SEQ ID NO: 50) was linked to the AHAS signal peptide (SEQ ID NO: 51) and the 35S-actinintron promoter (SEQ ID NO: 52) at the 5' end of the DNA sequence of G10evo-EPSPS, and the cauliflower mosaic virus 35s terminator TCaMV35S (SEQ ID NO: 53) at the 3' end to artificially synthesize the p35S-actinintron-G10evo-EPSPS-TCaMV35S fragment.

[0056] (3) Transformation vector: The vector pCambia1300 (NCBI sequence number AF234296) was double-digested with HindⅢ / XhoⅠ, and a fragment of 6783 bp was recovered. The above two gene expression frames were recombined by seamless cloning and transformed into Escherichia coli TG1 strain. After sequencing confirmation, the transformation vector PPO1-PPO23 was obtained.

[0057] (4) Agrobacterium transformation: The plasmids PPO1-PPO23 were electroporated into Agrobacterium LBA4404 competent cells. After enzyme digestion and identification, the cells were preserved in glycerol stock to obtain recombinant Agrobacterium for crop transformation.

[0058] Example 2: Obtaining corn expressing PPO enzyme

[0059] Maize genetic transformation was carried out by Agrobacterium-mediated transformation, specifically according to the method and medium formulation reported by Frame et al. (Plant Physiol, 2002, 129: 13-22), using glyphosate as a screening agent, and the steps were as follows:

[0060] (1) The Agrobacterium PPO1-PPO23 containing the transformation vector obtained in Example 1 was mixed evenly in the infection medium, and the bacterial solution concentration OD660 was adjusted to 0.5-0.6, that is, the infection solution containing Agrobacterium.

[0061] (2) Take common corn ears 8-10 days after pollination and collect immature embryos with a size of 1.0-1.5 mm. Immerse the collected immature embryos in the infection solution containing Agrobacterium in step (1), let stand at room temperature for 5 minutes, take out the embryos, drain the liquid, place the embryos flat side down on the co-culture medium, and culture at 22°C for 3-5 days.

[0062] (3) The immature embryos cultured in step (2) were transferred to a callus induction medium containing a final concentration of 200 mg / L of Timentin antibiotic (GlaxoSmithKline, USA), and cultured in the dark at 28° C. for 10-14 days to kill Agrobacterium.

[0063] (4) All callus tissues after induction culture in step (3) were transferred to a screening medium containing a final concentration of 2 mM glyphosate and cultured in the dark at 28° C. for 2-3 weeks. After induction culture, all callus tissues were transferred to a fresh screening medium containing 2 mM glyphosate and cultured in the dark at 28° C. for 2-3 weeks.

[0064] (5) The embryonic tissue that survived in step (4) was transferred to a regeneration medium, cultured in the dark at 28° C. for 10-14 days, then transferred to a fresh regeneration medium, and cultured in the light at 26° C. for 10-14 days.

[0065] (6) The fully developed plants from step (5) were selected and placed on a rooting medium and cultured under light at 26° C. until the roots were fully developed. The rooted regenerated seedlings were transplanted to a greenhouse for growth and cultivation.

[0066] Independent transgenic maize transformants expressing active polypeptides PPO1-PPO23 were generated.

[0067] At the same time, negative control transgenic crops PPO24 and PPO25 were obtained through the above-mentioned vector construction method and plant genetic transformation method, and the corresponding PPO nucleotide sequences are SEQ NO:56 and SEQ NO:57.

[0068] Example 3: PPO herbicide tolerance test of transgenic corn expressing PPO1-25

[0069] Table 1. Types of PPO herbicides tested

[0070]

[0071] 1. Test on the tolerance of PPO1-25 transgenic corn to saflufenacil

[0072] The T2 generation plants of the transgenic corn expressing polypeptides PPO1-PPO25 obtained in Example 2 were planted, and after emergence, 400 g ai / ha of glyphosate was sprayed to separate the non-transgenic plants in PPO1-PPO25. After weeding, 20 transformants of each numbered transgenic corn of PPO1-PPO25 were taken, and each transformant was planted into 3 groups (3 groups of treatments), with 1-3 seeds in each group, and the following agents were sprayed at about V5 stage: (1) blank treatment; (2) 2× saflufenacil 130 g ai / ha; (3) 4× saflufenacil 260 g ai / ha.

[0073] Observation was conducted 7 days after the application of the pesticide. The degree of damage to each plant by the herbicide was evaluated according to the average percentage of plant damage (average percentage of plant damage = leaf damage area / total leaf area × 100%). The pesticide damage levels are: Level 0 means normal growth without any symptoms; Level 1 means slight pesticide damage, yellowing of new leaves or pesticide damage spots occupying less than 10% of the leaf area, fast recovery, and no effect on yield; Level 2 means mild growth inhibition or chlorosis, the area of ​​pesticide damage spots is less than 25%, and it can recover, with an estimated yield reduction rate of 0-5%; Level 3 means moderate pesticide damage , which has a great impact on growth and development, the plant is dwarfed or the leaves are deformed or the area of ​​the leaf drug-damaged spots is less than 50%, and the recovery is slow. The estimated yield reduction rate is 6%-15%; Level 4 is severe drug damage, which has a great impact on growth and development, the plant is dwarfed or the leaves are deformed or the area of ​​the leaf drug-damaged spots is less than 75%, and it is difficult to recover. The estimated yield reduction rate is 16%-30%; Level 5 is serious drug damage, which has a great impact on growth and development, the plant is dwarfed or the leaves are deformed or the area of ​​the leaf drug-damaged spots is more than 75%, and it cannot be recovered. It is estimated that the yield is seriously reduced or even completely lost. Plants classified into levels 0 and 1 based on plant growth conditions are highly resistant plants, those classified into levels 2 and 3 based on plant growth conditions are medium and low resistant plants, and those classified into levels 4 and 5 based on plant growth conditions are non-resistant plants.

[0074] Table 2. Tolerance of T2 transgenic maize events of PPO1-PPO25 to the PPO herbicide saflufenacil

[0075]

[0076]

[0077] The results showed that most PPO corns had highly resistant transformants to 4 times the recommended effective dose of saflufenacil, among which PPO1, PPO4, PPO9, PPO12, PPO13, PPO15, PPO17, PPO18, PPO21, and PPO22 had more than 50% transformants showing high resistance to 4 times the recommended effective dose of saflufenacil, while the control PPO24 and PPO25 had no tolerance to saflufenacil.

[0078] 2. Test on tolerance of PPO1-25 transgenic corn to sulfentrazone

[0079] The T2 generation plants of the transgenic corn expressing PPO1-PPO25 obtained in Example 2 were planted, and after emergence, 400 g ai / ha of glyphosate was sprayed to separate the non-transgenic plants in PPO1-PPO25. After weeding, 20 transformants of each number of transgenic corn of PPO1-PPO25 were taken, and each transformant was planted into 3 groups (3 groups of treatments), with 1-3 seeds in each group, and the following agents were sprayed at about V5 stage: (1) blank treatment; (2) 2× sulfentrazone 900 g ai / ha; (3) 4× sulfentrazone 1800 g ai / ha.

[0080] Observation was carried out 7 days after application. The average percentage of plant damage in step 1 was used to evaluate the degree of damage to each plant by the herbicide. The results are shown in Table 3.

[0081] Table 3. Tolerance of T2 transgenic maize events of PPO1-PPO25 to the PPO herbicide sulfentrazone

[0082]

[0083]

[0084] The results showed that most PPO corns had highly resistant transformants to 4 times the recommended effective dose of sulfentrazone, among which PPO12, PPO18, and PPO21 had more than 50% transformants showing high resistance to 4 times the recommended effective dose of sulfentrazone, while the controls PPO24 and PPO25 had no tolerance to sulfentrazone.

[0085] 3. Tolerance test of PPO1-25 transgenic corn to fluazifop-butyl

[0086] The T2 generation plants of the transgenic corn expressing PPO1-PPO25 obtained in Example 2 were planted, and after emergence, 400 g ai / ha of glyphosate was sprayed to separate the non-transgenic plants in PPO1-PPO25. After weeding, 20 transformants of each numbered transgenic corn of PPO1-PPO25 were taken, and each transformant was planted into 3 groups (3 groups of treatments), with 1-3 seeds in each group, and the following agents were sprayed at about V5 stage: (1) blank treatment; (2) 2× fluazifop-butyl 150 g ai / ha; (3) 4× fluazifop-butyl 300 g ai / ha.

[0087] Observation was carried out 7 days after application. The average percentage of plant damage in step 1 was used to evaluate the degree of damage to each plant by the herbicide. The results are shown in Table 4.

[0088] Table 4. Tolerance of T2 transgenic maize events of PPO1-PPO25 to the PPO herbicide fluazifop-butyl

[0089]

[0090]

[0091]

[0092] The results showed that most PPO corns had highly resistant transformants to 4 times the recommended effective dose of fluazifop-butyl, among which PPO4, PPO5, PPO9, PPO13, PPO17, PPO21 and PPO23 had more than 50% transformants showing high resistance to 4 times the recommended effective dose of fluazifop-butyl, while the controls PPO24 and PPO25 had no tolerance to fluazifop-butyl.

[0093] 4. Test on tolerance of PPO1-25 transgenic corn to lactofen

[0094] The T2 generation plants expressing PPO1-PPO25 transgenic corn obtained in Example 2 were planted, and after emergence, 400 g ai / ha of glyphosate was sprayed to separate non-transgenic plants from PPO1-PPO25. After weeding, 20 transformants of each numbered transgenic corn of PPO1-PPO25 were taken, and each transformant was planted into 3 groups (3 groups of treatments), with 1-3 seeds in each group, and the following agents were sprayed at about V5 stage: (1) blank treatment; (2) 2× lactofenazole 160 g ai / ha; (3) 4× lactofenazole 320 g ai / ha.

[0095] Observation was carried out 7 days after application. The average percentage of plant damage was used to evaluate the degree of damage to each plant by the herbicide. The results are shown in Table 5.

[0096] Table 5. Tolerance of T2 transgenic maize events of PPO1-PPO25 to the PPO herbicide lactofensulfuron

[0097]

[0098]

[0099]

[0100] The results showed that most PPO corns had highly resistant transformants to 4 times the recommended effective dose of lactofenzeb-butyl, among which PPO2, PPO4, PPO9, PPO12, PPO15, PPO17, PPO18, and PPO21 had more than 50% transformants showing high resistance to 4 times the recommended effective dose of lactofenzeb-butyl, while the control PPO24 and PPO25 had no tolerance to lactofenzeb-butyl.

[0101] In summary, the transformants of PPO1-PPO23 showed different tolerance to different PPO herbicides. Although there were differences in the resistance levels of transgenic corn to different PPO herbicides, there were almost transformants of PPO1-PPO23 transgenic corn that were highly resistant to 4×bensulfuron (260 g ai / ha), 4×sulfuron (1800 g ai / ha), 4×trifloxypropyl (300 g ai / ha) and 4×lactofenac (320 g ai / ha), which proved that the expression of PPO enzymes (SEQ NO: 1-23) in crops can confer high tolerance to PPO herbicides on crops.

[0102] Example 4: Experimental study on the control effect of PPO herbicides on transgenic corn weeds

[0103] The weed control effect determination test refers to the method of "Guidelines for Field Efficacy Tests of Pesticides (I) Herbicides for Control of Weeds in Corn Crop Fields" (GB / T17980.42-2000).

[0104] A PPO21 transformant with good resistance screened in Example 3 was selected and recorded as transgenic corn PPO21-1. Ordinary corn and transgenic corn PPO21-1 were used as test materials. Each test material was set up with 10 treatments: 8 pesticide treatments, 1 manual weeding treatment, and 1 water blank control treatment. Each treatment was repeated 4 times, and the plots were arranged in random blocks, with 24 m2 per plot. 2 (4m×6m), double seed sowing, plant spacing 25cm, row spacing 50cm. Set 1m interval between plots, and spray herbicides according to Table 6 when corn is 3-5 leaves.

[0105] Table 6. Herbicide spraying amount

[0106] Processing number Experimental Drugs Active ingredient (g ai / ha) 1 Saffluamide 130 2 Sulfonamide 450 3 Fluazifop-butyl 75 4 Lactofenol 80 5 Safensulfuron+Glyphosate 130+800 6 Sulfentrazone + Glyphosate 450+800 7 Fluazifop-butyl+Glyphosate 75+800 8 Lactofen + Glyphosate 80+800 9 Manual weeding - 10 Clean water treatment -

[0107] Weed control survey method: According to the Z-shaped sampling method, 4 points were surveyed in each plot, with each point 0.25m 2 . 14 days and 28 days after application, the number of weeds in each plot was investigated, and the average weed control efficiency of each treatment was calculated. The formula for calculating the weed control efficiency is: weed control efficiency = (number of weeds in the control area - number of weeds in the application area) / number of weeds in the control area × 100%.

[0108] 14 days and 28 days after application, investigate the safety of the test pesticide on corn, observe and record the reaction of corn to the pesticide after emergence, and investigate the damage of plants if there is pesticide damage. Average percentage of plant damage = leaf damage area / total leaf area × 100%.

[0109] Table 7. Control effect of different herbicides on weeds after application

[0110]

[0111]

[0112] Table 8. Effects of different herbicides on corn after application

[0113]

[0114] The results show that the damage rate of common corn under the action of the composite herbicide has reached 100% 14 days after the application, while the transgenic corn PPO21-1 has no damage and tolerates each composite herbicide. It can also be seen that compared with the single herbicide (treatment 1-4), the composite herbicide provided by the present invention (treatment 5-8) shows a better weed control effect, and the control effect reaches 100% 14 days after the application. In this regard, we believe that the transgenic crops provided by the present invention are effective in resisting multiple herbicides, and the weed control method provided by the present invention has a rapid and efficient control effect on weeds, and can also avoid the occurrence of weed resistance.

[0115] Example 5: Obtaining transgenic soybeans expressing PPO enzyme and testing their tolerance to PPO herbicides

[0116] 1. Vector construction

[0117] (1) PPO gene expression cassette: The DNA sequence encoding PPO includes a codon for methionine at the 5' end, which is generally known as the start codon. This codon can be eliminated to facilitate the operable connection of the transit peptide sequence to the 5' end of the coding sequence. This embodiment retains this codon. The amino acid sequence of the PPO enzyme protein including methionine at the amino terminus is SEQ ID NO: 1-23, and the nucleotide sequence is SEQ ID NO: 24-46. The 5' end of the DNA sequence of PPO is connected to the rice ClpB3 signal peptide (SEQ ID NO: 55), and is functionally connected to the AtUbi promoter (SEQ ID NO: 54) and the Nos terminator (SEQ ID NO: 49), and the pAtUbi-ClpB-P-PPO-Tnos fragment is artificially synthesized to form the PPO gene expression cassette.

[0118] (2) Screening gene expression cassette: The screening gene G10evo-EPSPS (SEQ ID NO: 50) was connected to the AHAS signal peptide (SEQ NO ID: 51) and the 35S promoter (SEQ NO ID: 52) at the 5' end of the DNA sequence of G10evo-EPSPS, and the cauliflower mosaic virus 35s terminator TCaMV35S (SEQ NO ID: 53) at the 3' end to artificially synthesize the p35S-G10evo-EPSPS-TCaMV35S fragment.

[0119] (3) Transformation vector: The vector pCambia1300 (NCBI sequence number AF234296) was double-digested with HindⅢ / XhoⅠ, and a fragment of 6783 bp was recovered. The above two gene expression frames were recombined by seamless cloning and transformed into Escherichia coli TG1 strain. After sequencing confirmation, the transformation vector PPO1-PPO23 was obtained.

[0120] (4) Agrobacterium transformation: The plasmids PPO1-PPO23 were electroporated into Agrobacterium EHA105 competent cells. After enzyme digestion and identification, the cells were preserved in glycerol stock to obtain recombinant Agrobacterium for crop transformation.

[0121] 2. Obtaining genetically modified soybeans

[0122] The soybean transformation process and culture medium formulation mainly refer to the Agrobacterium-mediated half-seed transformation method of Paz (PazMM, et al. Plant Cell Reports, 2006, 25(3):206-213). Glyphosate was used as the screening agent, as follows:

[0123] (1) Sterilization of soybean seeds: Select high-quality soybean seeds, place them in a desiccator, and sterilize them with chlorine gas for 16 hours. The chlorine gas is generated by slowly adding 8 mL of concentrated hydrochloric acid to a beaker containing 100 mL of 30% (v / v) sodium hypochlorite aqueous solution in the desiccator.

[0124] (2) Seed germination: The sterilized soybean seeds in step (1) are soaked in sterile distilled water at room temperature for 16 hours to obtain germinated soybean seeds.

[0125] (3) Preparation of Agrobacterium culture medium: Streak the recombinant Agrobacterium prepared in step 1 on a YEP solid medium containing 50 mg / L Kan (kanamycin) and 25 mg / L rifampicin, and culture at 28°C for 2 days. Scrape the colonies on the YEP culture plate, evenly suspend them in the infection medium, and adjust the concentration to OD650 = 0.5-0.6, i.e., the Agrobacterium culture medium. The YEP solid medium composition: 10 g of peptone, 10 g of yeast extract, 5 g of NaCl, 8 g of agar, and add water to make up to 1 L.

[0126] (4) Preparation and infection of explants: The soybean seeds germinated in step (2) were cut longitudinally along the hilum to separate them into two lobes. The lob with the hypocotyl and embryo was removed from the seed coat and quickly placed in the Agrobacterium solution prepared in step (3). The lobes were infected at room temperature for 30 min. The lobes were taken out and placed in a sterile culture dish lined with filter paper. After sealing, the lobes were cultured in the dark at 22°C for 3 days.

[0127] (5) Bud induction culture: The explants after co-cultivation in step (4) were transferred to a bud induction medium containing 2 mM glyphosate and cultured at 26°C, 16 h light / 8 h dark conditions for 3 weeks. The bud induction medium composition: 3.21 g B5 salt (Phytotech, item number: G768), 1 mL B5 vitamin (Phytotech, item number G249-100ML), 30 g sucrose, 0.64 g 4-morpholineethanesulfonic acid, 8 g agar, water was added to make up to 1 L and the pH was adjusted to 5.6, and 200 mg timentin and 1.67 mg 6-benzyladenine were added after sterilization.

[0128] (6) Bud elongation and screening: The cotyledons of the explants induced in step (5) were cut off, and the part with clustered buds was transferred to a bud elongation medium containing 2 mM glyphosate, and cultured at 26°C, 16 h light / 8 h dark conditions for 21 days. The bud elongation medium composition: MS salt (Phytotech, item number: M524) 4.33 g, B5 vitamin (Phytotech, item number G249-100ML) 1 mL, sucrose 30 g, 4-morpholineethanesulfonic acid 0.64 g, asparagine 50 mg, glutamine 50 mg, agar 8 g, water was added to make up to 1 L and the pH was adjusted to 5.6, and after sterilization, 0.5 mg gibberellin, 0.1 mg indole-3-acetic acid, 1 mg zeatin riboside, and 200 mg timentin were added.

[0129] (7) Rooting and transplanting: Transfer the elongated buds in step (6) to the rooting medium and culture them under 26°C, 16h light / 8h dark conditions. When the roots grow out, wash off the culture medium attached to the roots and transplant the seedlings into the matrix for culture until the T0 generation seeds are harvested. Plant the T0 generation seeds and collect leaf samples for analysis of gene editing results. Rooting medium composition: MS salt 4.33g, B5 vitamin 1mL, sucrose 20g, 4-morpholineethanesulfonic acid 0.64g, agar 8g, add water to make up to 1L and adjust the pH to 5.6. After sterilization, add 200mg of timentin.

[0130] Independent transgenic soybean transformants expressing active polypeptides PPO1-PPO23 were generated.

[0131] At the same time, negative control transgenic crops PPO24 and PPO25 were obtained through the above-mentioned vector construction method and plant genetic transformation method, and the corresponding PPO nucleotide sequences are SEQ NO:56 and SEQ NO:57.

[0132] 3. Test on tolerance of PPO1-PPO25 transgenic soybean to saflufenacil

[0133] Plant the T2 generation plants of the transgenic soybean obtained in step 2, and spray them with 400 g ai / ha of glyphosate after emergence to separate the non-transgenic plants in PPO1-PPO25. After weeding, take 20 transformants of each number of transgenic soybeans from PPO1-PPO25, plant 3 groups (3 treatments) of each transformant, with 1-3 seeds in each group, and spray them with the following agents at about V5 stage: (1) blank treatment; (2) 2× saflufenacil 130 g ai / ha; (3) 4× saflufenacil 260 g ai / ha.

[0134] The observation was carried out 7 days after the application of the drug. The average percentage of plant damage in Example 3 was used to evaluate the degree of damage to each plant by the herbicide. The results are shown in Table 9.

[0135] Table 9. Tolerance of T2 transgenic soybean events of PPO1-PPO25 to PPO herbicide saflufenacil

[0136]

[0137]

[0138]

[0139] The results showed that most PPO soybeans had highly resistant transformants to 4 times the recommended effective dose of saflufenacil. Among them, more than 50% of the transformants of PPO1, PPO8, PPO9, PPO13, PPO18, and PPO21 showed high resistance to 4 times the recommended effective dose of saflufenacil, while the control PPO24 and PPO25 had no tolerance to saflufenacil.

[0140] In summary, PPO1-PPO23 transgenic crops have different tolerance to different PPO herbicides. Although transgenic crops have different resistance levels to different PPO herbicides, PPO1-PPO23 transgenic crops almost have transformants that are highly resistant to 4×bensulfuron (260 g ai / ha), 4×sulfuron (1800 g ai / ha), 4×trifloxypropyl (300 g ai / ha) and 4×lactofenac (320 g ai / ha), which proves that the expression of PPO enzymes (SEQ NO: 1-23) in crops can confer high tolerance to PPO herbicides.

[0141] Finally, all materials and methods disclosed and claimed herein can be prepared and used as indicated by the above disclosure without undue experimentation. Although the materials and methods of the present invention have been described according to preferred embodiments and illustrative examples, it will be apparent to those skilled in the art that the materials and methods described herein can be varied without departing from the concept, spirit and scope of the present invention. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

1. An active polypeptide of protoporphyrinogen oxidase, characterized in that: The active polypeptide has at least 85%, 90%, 95%, 99%, or 100% sequence identity with the amino acid sequence shown in one of SEQ ID NOs: 1-23, and has protoporphyrinogen oxidase activity.

2. A recombinant DNA molecule, characterized in that The recombinant DNA molecule comprises a nucleotide sequence encoding the active polypeptide of claim 1, a promoter sequence for initiating expression of the active polypeptide in plants and / or a targeting sequence for localizing the active polypeptide in plant cells.

3. A DNA construct comprising the recombinant DNA molecule of claim 2.

4. A plant cell expressing the active polypeptide of claim 1, characterized in that: The expression of the active polypeptide is achieved by transferring the recombinant DNA molecule or DNA construct into plant cells.

5. A method for obtaining a transgenic plant tolerant to PPO inhibitor herbicides using the active polypeptide of claim 1, characterized in that: The method is to heterologously express the active polypeptide in the plant.

6. A method for cultivating transgenic plants with significantly improved resistance to PPO inhibitor herbicides using the active polypeptide of claim 1, characterized in that: The method comprises one of the following: a) introducing the recombinant DNA molecule into plant cells; regenerating a transgenic plant containing the recombinant DNA molecule; b) hybridizing the transgenic plant containing the recombinant DNA molecule with itself or a second plant, and collecting the transgenic plant containing the recombinant DNA molecule.

7. A method for controlling weeds in a plant growth area expressing the active polypeptide of claim 1, characterized in that: The method comprises contacting a plant growing area of ​​a transgenic plant or seed expressing the active polypeptide with at least one PPO inhibitor herbicide.

8. The method according to claim 7, characterized in that The PPO inhibitor herbicide is selected from at least one of the following: acifluorfen, fluazifop-butyl, lactofop-butyl, fluazifop-butyl, oxyfluorfen, fluazifop-butyl, azopyralid, mesotrione, foramsulfuron, sulfentrazone, bensulfuron, fluthiazide, propargyl, oxadiazine, oxadiazine, pyraclostrobin, pyraclostrobin, fluazifop-butyl, fluazifop-butyl, trifluoperazine, fluazifop-butyl, epyrifenacil, SY1604 / JS-T205, FG009, foramsulfuron, and uracil compounds containing carboxylate fragments.

9. A method for cultivating plants tolerant to a PPO inhibitor herbicide and at least one other herbicide using the active polypeptide of claim 1, characterized in that: The method comprises: a) obtaining a transgenic plant comprising the recombinant DNA molecule; b) crossing the transgenic plant with a second plant having tolerance or additional traits to at least another herbicide; c) selecting progeny plants produced by the crossing that have tolerance or additional traits to a PPO inhibitor herbicide and at least another herbicide.

10. A method for producing plants tolerant to a PPO inhibitor herbicide and at least one other herbicide, characterized in that The method comprises connecting the recombinant DNA molecule of claim 2 and at least one other recombinant DNA expressing a protein resistant to other herbicides, introducing them into the plant genome at the same time, and selecting transgenic plants that are resistant to PPO inhibitor herbicides and at least one other herbicide.

11. A method for reducing the development of herbicide-tolerant weeds in an area where plants expressing the active polypeptide of claim 1 are planted, characterized in that: The method comprises: a) cultivating a plant that is tolerant to a PPO inhibitor herbicide and tolerant to at least another herbicide in a crop growth environment; wherein the plant gene comprises the recombinant DNA molecule of claim 2 and another herbicide tolerance protein encoding gene; and b) applying the PPO inhibitor herbicide and at least another herbicide to the crop growth environment.

12. The method according to any one of claims 9 to 11, characterized in that: The at least one other herbicide is selected from one of the following: an ACCase inhibitor, an ALS inhibitor, an EPSPS inhibitor, a synthetic auxin, a photosynthesis inhibitor, a glutamine synthesis inhibitor, an HPPD inhibitor, a PPO inhibitor or a long chain fatty acid inhibitor.

13. The method according to claim 12, characterized in that The ACCase inhibitor is an aromatic phenoxy propionate or cyclohexanedione; the ALS inhibitor is a sulfonylurea, an imidazolinone, a triazolopyrimidine, or a triazolinone; the EPSPS inhibitor is glyphosate; the synthetic auxin is a phenoxy herbicide, benzoic acid, or a semicarbazone, clofopyralid, or fluclofopyralid; the photosynthesis inhibitor is a triazine, a triazone, or a benzothiadiazole; the glutamine synthesis inhibitor is glufosinate; the HPPD inhibitor is an isoxazole, a pyrazolone, or a triketone; the PPO inhibitor is a diphenyl ether, N-phenylphthalimide, or an aromatic triazone; the long-chain fatty acid inhibitor is chloroacetamide or oxyacetamide.

Images