Active polypeptide of protoporphyrinogen oxidase and application

Abstract

The invention discloses an active polypeptide of protoporphyrinogen oxidase and application, the polypeptide has at least 85%, 90%, 95%, 99% and 100% sequence identity of an amino acid sequence as shown in one of SEQ ID NO: 1-42, and has protoporphyrinogen oxidase activity. The active polypeptide disclosed by the invention shows relatively high tolerance to PPO inhibitor herbicides, and has a wide application prospect in plants. According to the weed control method provided by the invention, the occurrence of resistant weeds can be effectively reduced, and transgenic crops obtained by the method can resist various herbicides such as PPO inhibitors, ACC enzyme inhibitors, ALS inhibitors, EPSPS inhibitors and synthetic auxin at the same time, and have broad-spectrum herbicide resistance; in the weed control process, the PPO inhibitor herbicide is used in combination with one or more other herbicides, so that the cost can be effectively reduced, the control efficiency can be improved, and the weed resistance can be greatly avoided.

Description

(1) Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a protoporphyrinogen oxidase active polypeptide that is insensitive to protoporphyrinogen oxidase inhibitor herbicides derived from organisms of the class Kinetoplastida and its applications. (2) Background Art

[0002] Weeds compete with crops for nutrients and growth space, affecting crop growth and resulting in reduced crop yields. Weed control is an important link in crop production. Using herbicides to control weeds can reduce the impact of weeds on crop growth and stabilize and increase crop yields. By introducing herbicide-tolerant genes into crops through genetic engineering methods to obtain herbicide-tolerant transgenic crops can improve the efficiency of weed control in crops, reduce production costs, bring huge economic benefits to agricultural production, and thus achieve positive social and ecological benefits.

[0003] Crops can obtain herbicide-tolerant traits through genetic improvement techniques. For example, by using the Agrobacterium-mediated transformation method to introduce the cp4 epsps gene from Agrobacterium tumefaciens sp strain CP4 into crops, crops expressing 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) have obtained tolerance to the herbicide glyphosate. However, the long-term use of a single herbicide often results in the large-scale emergence of resistant weeds. Glyphosate-tolerant crops have been promoted in the Americas for more than two decades, and a large number of glyphosate-resistant weeds have emerged. It is difficult to effectively control field weeds by 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-tolerant traits, such as tolerance to herbicides that inhibit protoporphyrinogen oxidase (PPO). PPO herbicides provide a method for controlling herbicide-resistant weeds and confer traits of tolerance to these herbicides. Cultivating transgenic crops tolerant to two or more herbicides can provide diverse options for weed control and can also effectively delay the emergence of resistant weeds.

[0005] Protoporphyrinogen oxidase plays a role in both the biosynthetic pathways of chlorophyll and heme. In these pathways, protoporphyrinogen oxidase converts protoporphyrinogen IX to protoporphyrin IX. After the production of protoporphyrin IX, the chlorophyll and heme biosynthetic pathways diverge due to the incorporation of different metal ions (iron for heme and magnesium for chlorophyll). This pathway is conserved in prokaryotes and eukaryotes, and there are many similar PPOs 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 oxidase has been isolated from a variety of prokaryotes and eukaryotes. According to different structures, the existing PPO enzymes are divided into three subclasses: HemY, HemG, and HemJ. HemY is the PPO present in terrestrial plants and is also the target of PPO inhibitor herbicides, which is widely present in prokaryotes and eukaryotes. HemJ is thought to originate from the class α-proteobacteria but is also present in other classes of proteobacteria and cyanobacteria; HemJ PPO is a membrane-bound oligomer, which is different from HemY or HemG. HemG is mainly present in the class 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] Methods for providing plants tolerant to PPO inhibitor herbicides mainly include: 1) Detoxifying the herbicide using an enzyme that can convert the herbicide or its active metabolite into a non-toxic product. 2) Overexpressing the sensitive PPO so that, given the kinetic constants of this enzyme, a sufficient amount of the target enzyme is produced in the plant relative to the herbicide, such that despite the presence of the PPO inhibitor herbicide, these sensitive PPOs fully interact with the PPO inhibitor herbicide, thus having sufficient functional enzyme 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, a variety of PPOs have been identified, yet 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 have sufficient tolerance to other PPO inhibitor herbicides. A single functional PPO gene may not be sufficient to provide crops at the commercialization level, and not all PPO genes can be used to prepare crops tolerant to PPO inhibitor herbicides. In this regard, identifying and mining more different classes of PPO genes and using them to create transgenic crop traits have great agricultural application prospects. (III) Summary of the Invention

[0009] The object of the present invention is to provide an active polypeptide of protoporphyrinogen oxidase that is insensitive to protoporphyrinogen oxidase inhibitor herbicides derived from organisms of the class Kinetoplastea, and its application. The active polypeptide not only has protoporphyrinogen oxidase activity, but also plants into which the nucleotide sequence encoding the active polypeptide is transferred have 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 of the protoporphyrinogen oxidase inhibitor class and comes from organisms of the class Kinetoplastea. The active polypeptide has a sequence identity of at least 85%, 90%, 95%, 99%, or 100% with the amino acid sequence shown in any one of SEQ ID NO: 1-41, and has protoporphyrinogen oxidase activity.

[0012] It is also clear to those skilled in the art that the structure of a protein can be altered without adversely affecting its activity and functionality. For example, one or more conservative amino acids can be introduced into the amino acid sequence of the protein without adversely affecting the activity and / or three-dimensional configuration of the protein molecule. Examples and implementation methods of conservative amino acid substitutions are clear to those skilled in the art. Specifically, an amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, that is, a non-polar amino acid residue is replaced with another non-polar amino acid residue, a polar uncharged amino acid residue is replaced with another polar uncharged amino acid residue, a basic amino acid residue is replaced with another basic amino acid residue, and an acidic amino acid residue is replaced with another 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 with another amino acid belonging to the same group fall within the scope of the present invention. In addition, the present invention also encompasses mutant proteins containing one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the required functions and biological activities of the proteins 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-terminus of a protein while still retaining its functional activity. Therefore, in another aspect, the present invention also relates to fragments that have one or more amino acid residues deleted from the N- and / or C-terminus of the active protein and still retain their required functional activities, and they are also within the scope of the present invention.

[0013] In a second aspect, the present invention provides a recombinant DNA molecule, which includes a nucleotide sequence encoding an active polypeptide, a promoter sequence for initiating the expression of the active polypeptide in plants, and / or a targeting sequence for localizing the active polypeptide in plant cells. The recombinant DNA molecule can exist in the genome of transgenic plants, seeds, or cells.

[0014] The active polypeptide has a sequence identity of at least 85%, 90%, 95%, 99%, or 100% with the amino acid sequence shown in SEQ ID NO: 1-41 and has herbicide-insensitive protoporphyrinogen oxidase activity. The promoter and the targeting sequence can both be operably linked to the nucleotide sequence encoding the active polypeptide and initiate the directional expression of the active polypeptide in plant cells.

[0015] The promoter includes a bacterial promoter or a plant promoter. The plant promoter includes inducible, viral, synthetic, constitutive, time-regulated, space-regulated, and / or spatio-temporal regulated promoters well known in the art.

[0016] The targeting sequence can promote the localization of the active polypeptide in plant cells, increase the accumulation of recombinant proteins, protect the protein from protein degradation, and / or enhance the level of herbicide tolerance, and thus reduce the damage level of transgenic cells, seeds, or organisms after herbicide application, conferring herbicide tolerance to the cells, plants, seeds or plant parts. The targeting sequence includes signal sequences, targeting peptides, localization sequences, and transit peptides well 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, maize 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 as one of SEQ ID NO: 42-82.

[0018] In a third aspect, the present invention provides a DNA construct comprising the recombinant DNA molecule. The DNA construct is present in the genome of transgenic plants, seeds, or cells.

[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, the method being heterologously expressing the active polypeptide in the plant, and the full-length amino acid sequence of the active polypeptide having at least 85%, 90%, 95%, 99%, 100% sequence identity with one of SEQ ID NO: 1-41.

[0021] In a sixth aspect, the present invention provides a method for cultivating a transgenic plant with significantly improved tolerance to PPO inhibitor herbicides using the active polypeptide, the method comprising one of the following: a) introducing the recombinant DNA molecule into a plant cell; regenerating a transgenic plant comprising the recombinant DNA molecule; b) crossing the transgenic plant comprising the recombinant DNA molecule with itself or a second plant, and collecting the transgenic plant comprising 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 the 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 wherein the weeds are controlled in the plant growth area. The active polypeptide has at least 85%, 90%, 95%, 99%, 100% sequence identity with the amino acid sequence shown in one of SEQ ID NO: 1-41 and has protoporphyrinogen oxidase activity.

[0023] The "PPO inhibitor herbicide" or "PPO herbicide" is a chemical that targets and inhibits 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 leads to the formation of reactive oxygen species, causing cell membrane rupture and ultimately 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, oxyfluorfen, chlomethoxyfen, its salts and esters, lactofen, its salts and esters, fluoroglycofen-ethyl, fomesafen, its salts and esters, oxyfluorfen, fluroglycofen, flumetsulam, methoxyfenozide, ethyl fluoroglycofenate, lactofen, its salts and esters, oxyfluorfen, and bifenox, its salts and esters); thiadiazoles (such as flufenpyr-ethyl and thidiazimin); pyrazolidinediones or phenylureas (such as bispyribac-sodium, flupropacil, epyrifenacil, flupropacil, pyriminobac-methyl, and tiafenacil); phenylpyrazoles (such as propyzamide, pyraflufen-ethyl, and pyraflufen); oxazoles (such as oxadiargyl and oxadiazon); triazolinones (such as azafenidin, bencarbazone, carfentrazone-ethyl, its salts and esters, and mesotrione); oxazolidinediones (such as cycloxydim); N-phenylphthalimides (such as cinidon-ethyl, phenoxyacetic acid, flumiclorac-pentyl, and flumioxazin); 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); flufenpyr-ethyl and flufenpyr-ethyl; bipyrazonitrile; and fluzolate.

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

[0025] Furthermore, the PPO inhibitor herbicides are selected from at least one of the following: acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, sulfentrazone, carfentrazone-ethyl, propyzamide, oxadiazon, pyraflufen-ethyl, pyriminobac-methyl, fluthiacet-methyl, trifulizole, epyrifenacil, SY1604 / JS-T205 (benzoxazolinone herbicide), FG009 herbicide, metamifop, uracil compounds containing a carboxylic acid ester fragment (I).

[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 containing the recombinant DNA molecule; b) crossing the transgenic plant with a second plant having tolerance or an additional trait to at least one other herbicide; c) selecting progeny plants resulting from the crossing that are tolerant to PPO inhibitor herbicides and at least one other herbicide or have an additional trait. The other herbicide is selected from one of the following: ACC enzyme inhibitor, ALS inhibitor, EPSPS inhibitor, synthetic auxin, photosynthesis inhibitor, glutamine synthetase inhibitor, HPPD inhibitor or long-chain fatty acid inhibitor.

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

[0029] Furthermore, the protein resistant to other herbicides includes 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 (sex development transcription factor protein) resistant to dicamba.

[0030] In a tenth aspect, the present invention also provides a method for reducing the development of herbicide-tolerant weeds in a plant-growing area where an active polypeptide is expressed, the method comprising: a) cultivating in a crop growth environment a plant that is tolerant to a PPO inhibitor herbicide and at least one other herbicide; the plant genome contains the recombinant DNA molecule and another herbicide tolerance protein-encoding gene; and b) applying to the crop growth environment a PPO inhibitor herbicide and at least one other herbicide.

[0031] Further, the at least one other herbicide is selected from one of the following: ACC enzyme inhibitor, ALS inhibitor, EPSPS inhibitor, synthetic auxin, photosynthesis inhibitor, glutamine synthetase inhibitor, HPPD inhibitor, and long-chain fatty acid inhibitor.

[0032] Further, the ACC enzyme inhibitor is aryloxyphenoxypropionate or cyclohexanedione; the ALS inhibitor is sulfonylurea, imidazolinone, triazolopyrimidine, or triazolinone; the EPSPS inhibitor is glyphosate; the synthetic auxin is phenoxy herbicide, benzoic acid, carboxylic acid, or semicarbazone; the photosynthesis inhibitor is triazine, triazinone, nitrile, benzothiadiazole, or urea; the glutamine synthetase inhibitor is glufosinate; the HPPD inhibitor is isoxazole, pyrazolinone, or triketone; or the long-chain fatty acid inhibitor is chloroacetamide, oxyacetamide, or pyrazole.

[0033] The application of the herbicide can be sequentially mixed or tank-mixed with a combination of one, two, or several PPO herbicides or any other compatible herbicide. Multiple applications of a combined or individual herbicide or two or more herbicides can be used during the growing season in areas containing the transgenic plants of the present invention to control 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 an eleventh aspect, the present invention provides an antibody that can bind to the active polypeptide. An "antibody", also known as an immunoglobulin, is a class of large Y-shaped proteins secreted by B lymphocytes, and is an immunoglobulin molecule that can specifically bind to a target antigen through complementary sites (antigen-binding sites) at the two bifurcated tips of the Y shape. The target antigen can be, for example, a protein, sugar, polynucleotide, lipid, polypeptide, small molecule compound, etc.

[0035] In a twelfth aspect, the present invention provides a kit for detecting the active polypeptide, and 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 blocking system, a coating system, a buffer system, and a reaction termination solution. The composition of the blocking system: PBS buffer, 5% skim milk; the composition of the coating system: PBS buffer; the composition of the buffer system: PBST buffer; the composition of the reaction termination solution: 2M sulfuric acid.

[0037] The transgenic plants and progeny with the transgenic traits provided by the present invention can be used in any cultivation methods known in the art. In plant lines having two or more transgenic traits, the transgenic traits can be independently segregated, linked, or a combination of both in plant lines having three or more transgenic traits. To confirm the presence of the transgene in a specific plant or seed, various assays can be performed. Such assays include, for example, molecular biology 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" can be used interchangeably, referring to a polymer 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] Those skilled in the art are well aware that due to the degeneracy of the genetic code, there are multiple different nucleic acid sequences that can encode the amino acid sequences disclosed in the present invention. Generating other nucleic acid sequences that encode the same protein is within the ability of those of ordinary skill in the art, and thus the present invention encompasses nucleic acid sequences that encode the same amino acid sequence due to the degeneracy of the genetic codons. 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 enable better expression.

[0040] The present invention provides recombinant DNA molecules and active polypeptides. The term "recombinant" refers to non-naturally occurring DNA, protein, cell, seed, or organism that is caused by genetic engineering and thus does not normally exist in nature. A "recombinant DNA molecule" is a DNA molecule that contains a DNA sequence that does not occur naturally in nature and is thus 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 the DNA molecule encoding herbicide-insensitive protoporphyrinogen oxidase that is operably linked to a heterologous regulatory element or other element (such as a heterologous promoter) provided by the present invention. An "active polypeptide" is a protein that contains an amino acid sequence that does not occur naturally in nature and is thus caused by human intervention, such as an engineered protein or a chimeric protein. A recombinant cell, seed, or organism is a cell, seed, or organism that contains the recombinant DNA molecule, such as a transgenic cell, seed, plant, or plant part that contains the recombinant DNA molecule and is thus produced by plant transformation.

[0041] The term "DNA construct" refers to a recombinant DNA molecule that contains two or more heterologous DNA sequences. The DNA construct can be used for transgenic expression and can be contained in vectors and plasmids. The DNA construct can be used in a vector for the purpose of transformation, i.e., introducing heterologous DNA into a host cell to produce transgenic plants and cells, and can thus also be contained in the plasmid DNA or genomic DNA of transgenic plants, seeds, cells, or plant parts. The term "vector" means any recombinant DNA molecule that can be used for the purpose of bacterial or plant transformation. The recombinant DNA molecule listed in the sequence listing can be inserted into a vector as part of a construct that has a recombinant DNA molecule operably linked to a gene expression element that is used in plants to affect the expression of the engineered protein encoded by the recombinant DNA molecule. General methods for manipulating 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 guides including MR Green and J Sambrook, "Molecular Cloning: A Laboratory Manual" (Fourth 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 one of the recombinant DNA molecules or polypeptide sequences provided herein and have herbicide-insensitive protoporphyrinogen oxidase activity. The "percent sequence identity" or "sequence identity %" refers to the percentage of identical nucleotides or amino acids in the linear polynucleotide or polypeptide sequence of a reference ("query") sequence (or its complementary strand) compared to a test ("target") sequence (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide or amino acid insertions, deletions, or gaps in the comparison window totaling less than 20% of the reference sequence). The identity of amino acid sequences 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 altering (i.e., modifying) a wild-type protein to produce a new protein with modified properties. The modification can be made at specific amino acid positions in the protein and can be the substitution of a different amino acid for the amino acid that exists at this position in nature (i.e., in the wild-type protein). Amino acid mutations can be made as single amino acid substitutions in the protein or in combination with one or more other mutations (such as one or more other amino acid substitutions, deletions, or additions). The mutations can be made by any method known to those skilled in the art.

[0043] The transgenic plants, progeny, seeds, plant cells, and plant parts of the present invention may also comprise one or more additional traits. The additional traits may be introduced by crossing a plant comprising a transgene with the recombinant DNA molecule provided by the present invention with another plant comprising one or more additional traits. The term "crossing" means breeding two individual plants to produce progeny plants. Thus, two plants may be crossed to produce progeny that comprise the desired traits from each parent. The additional traits may also be introduced by co-transforming a DNA construct of this additional transgenic trait with a DNA construct comprising the recombinant DNA molecule provided by the present invention (e.g., with all DNA constructs present as part of the same vector for plant transformation) or by inserting the additional trait into a transgenic plant comprising the DNA construct provided by the present invention or vice versa (e.g., by using any of the methods of plant transformation or genome editing on the transgenic plant or plant cell). 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, where the traits are measured relative to the wild-type plant. 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 such as 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 Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA).

[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, shoots, fruits, stalks, roots, root tips, anthers, etc. The transgenic plants are derived from transgenic plants or their progeny that have been transformed with the DNA molecule of the present invention and thus are at least partially composed of transgenic cells. In the present invention, "plant cell" should be understood to mean any cell derived from or found in a plant that is capable of forming, for example: undifferentiated tissues such as calli, differentiated tissues such as embryos, components of plants, plants, or seeds.

[0045] As used herein, the term "herbicide tolerance of a plant, plant tissue or cell" or "herbicide - tolerant plant, plant tissue or cell" refers to the ability of a plant, plant tissue or cell to resist the action of a herbicide when the herbicide is applied. For example, a herbicide - tolerant plant can survive or continue to grow in the presence of a herbicide. The herbicide tolerance of a plant, plant tissue or cell can be measured by comparing the plant, plant tissue or cell with a suitable control. For example, herbicide tolerance can be measured or evaluated by applying a herbicide to a plant (test plant) containing a DNA molecule encoding a protein capable of conferring herbicide tolerance and a plant (control plant) not containing a DNA molecule encoding a protein capable of conferring herbicide tolerance, and then comparing the plant damage of the two plants. The herbicide tolerance of the test plant is indicated by a decrease in the damage rate compared to the damage rate of the control plant. Compared with the control plant, plant tissue or cell, the herbicide - tolerant plant, plant tissue or cell shows a reduced response to the toxic effect of the herbicide. The term "herbicide tolerance trait" refers to a transgenic trait that confers improved herbicide tolerance to a plant compared to a wild - type plant. Plants that can be produced with the herbicide tolerance trait of the present invention include, for example, any plant, including crop plants such as soybean, corn, sunflower, peanut, rapeseed, wheat, etc.

[0046] As used herein, the term "resistance" is heritable and allows a plant to grow and reproduce in the presence of a herbicide when the herbicide is applied at an effective rate for a given plant. As recognized by those skilled in the art, even if a given plant suffers a certain degree of damage from herbicide treatment, such as little necrosis, lysis, chlorosis or other damage, but at least there is no significant impact on yield, the plant can still be considered "resistant", that is, the given plant has an improved ability to resist various degrees of damage induced by the herbicide, while the same herbicide dose generally causes damage to wild - type plants of the same genotype. The term "tolerance" or "tolerancy" 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. Broad herbicide tolerance. The active polypeptide of the present invention shows high tolerance to PPO inhibitor - type herbicides, so it has broad application prospects in plants.

[0049] 2. Strong herbicide tolerance. The active polypeptide of the present invention has strong tolerance to PPO inhibitor herbicides, and it shows high - resistance tolerance to sulfentrazone, metsulfuron - methyl, flumioxazin and lactofen 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 simultaneously resistant to multiple herbicides such as PPO inhibitors, ACC enzyme inhibitors, ALS inhibitors, EPSPS inhibitors, and synthetic auxins, and have broad-spectrum herbicide resistance. During the weed control process, the combined use of PPO inhibitor herbicides and 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 Embodiments

[0051] The present invention will be 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 biochemistry methods used in the following embodiments of the present invention are all known techniques. Detailed descriptions can be found in documents such as Current Protocols in Molecular Biology published by John Wiley and Sons written by Ausubel, and Molecular Cloning: A Laboratory Manual, 3rd ED. published by Cold Spring Harbor Laboratory Press (2001) written by J. Sambrook et al.

[0053] Example 1: Obtaining of recombinant Agrobacterium tumefaciens containing exogenous gene PPO plasmid

[0054] (1) PPO gene expression cassette: The DNA sequence encoding PPO includes a codon for methionine at the 5' end, which is usually 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. In this embodiment, this codon is retained, and the amino acid sequence of the PPO enzyme protein (i.e., the active polypeptide) including methionine at the amino terminus is shown as one of SEQ ID NO: 1-41. For plant transformation, the nucleotide sequence encoding the putative PPO is codon-optimized for maize expression, and the nucleotide sequence of the recombinant DNA molecule containing the codon-optimized nucleotide sequence encoding the putative PPO for maize transformation is shown as one of SEQ ID NO: 42-82. The recombinant DNA molecule is ligated with the rice ClpB-P signal peptide (SEQ ID NO: 83) at the 5' end of the DNA sequence of PPO, and is functionally connected with the ZmUbi promoter (SEQ ID NO: 84) and the Nos terminator (SEQ ID NO: 85) to artificially synthesize the pZmUbi-ClpB-P-PPO-Tnos fragment to form the PPO gene expression cassette.

[0055] (2) Screening gene expression cassette: Connect the screening gene G10evo-EPSPS (SEQ ID NO: 86) at the 5'-end with the 35S-actinintron promoter (SEQ ID NO: 87), and connect the cauliflower mosaic virus 35s terminator TCaMV35S (SEQ ID NO: 88) at the 3'-end. Artificially synthesize the p35S-actinintron-G10evo-EPSPS-TCaMV35S fragment to form a screening gene expression cassette.

[0056] (3) Transformation vector: Double digest the vector pCambia1300 (NCBI sequence number AF234296) with HindⅢ / XhoⅠ, recover the fragment of 6783 bp in size, and recombine the above two gene expression cassettes into the digested vector by seamless cloning method, then transform it into Escherichia coli TG1 strain. After sequencing confirmation, the transformation vector is obtained, which are respectively denoted as plasmids PPO1 to PPO41.

[0057] (4) Agrobacterium transformation: Electrotransform plasmids PPO1 to PPO41 into competent cells of Agrobacterium tumefaciens LBA4404. After correct enzyme digestion identification, the recombinant Agrobacterium tumefaciens PPO1 to PPO41 are obtained and stored in glycerol tubes for crop transformation.

[0058] Example 2: Obtaining maize expressing PPO enzyme

[0059] Maize genetic transformation is carried out by Agrobacterium-mediated method. Specifically, it is carried out according to the method and culture medium formula reported by Frame et al. (Plant Physiol, 2002, 129: 13-22), and glyphosate is used as the screening reagent. The steps are as follows:

[0060] (1) Add the recombinant Agrobacterium tumefaciens PPO1 to PPO41 obtained in Example 1 into the infection medium respectively, and adjust the OD660 of the bacterial liquid concentration to 0.5 - 0.6, which is the infection liquid containing Agrobacterium tumefaciens.

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

[0062] (3) Transfer the immature embryos cultured in step (2) to the callus induction medium containing the final concentration of 200 mg / L ticarcillin antibiotic (GlaxoSmithKline, USA), and culture in the dark at 28℃ for 10 - 14 days to kill Agrobacterium tumefaciens.

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

[0064] (5) The surviving embryogenic tissues in step (4) were transferred onto a regeneration medium, cultured in the dark at 28 °C for 10 - 14 days and then transferred onto fresh regeneration medium, and cultured under light at 26 °C for 10 - 14 days.

[0065] (6) The fully developed plants in step (5) were selected and transferred onto a rooting medium, cultured under light at 26 °C until the roots were fully developed, and the regenerated seedlings after rooting were transplanted into a greenhouse for growth and cultivation.

[0066] Independent transgenic maize transformants expressing the active polypeptides PPO1 - PPO41 were produced.

[0067] Meanwhile, negative control transgenic crops PPO42 and PPO43 were obtained by the above vector construction method and plant genetic transformation method, and the corresponding PPO nucleotide sequences were SEQ ID NO:89 and SEQ ID NO:90.

[0068] Example 3: Tolerance test of PPO herbicides for transgenic maize expressing PPO1 - PPO43

[0069] Table 1. Types of PPO herbicides tested

[0070] Name Chinese Name Trade Name Active Ingredient Content Recommended Active Ingredient (g ai / ha) saflufenacil benzfendizone Babaikin 70％ 52.5-78.75 sulfentrazone nicosulfuron Suxiao 50％ 375-525 flumixoxazin flumioxazin Fengqing 51％ 61.2-91.8 lactofen lactofen Shuangqing 240 g / L It should be noted that in the above translation, for some names that may not be very common and may not have a unified and accurate translation in the field of pesticides, the original names are retained as much as possible to ensure the accuracy of the information. And for the unit "g ai / ha", it is directly translated as "g ai / ha" because it is a specific unit in the field of pesticides and is difficult to find a more appropriate general translation. If there are more context or specific requirements, the translation can be further optimized. 54-108

[0071] The T2 generation plants of transgenic maize expressing the active polypeptides PPO1 - PPO43 obtained in Example 2 were planted. After emergence, glyphosate at 400 gai / ha was sprayed to separate the non - transgenic plants among PPO1 - PPO43. After removing the weeds, 20 transformants of each numbered transgenic maize of PPO1 - PPO43 were taken, and each transformant was planted in 3 groups (3 group treatments), with 1 - 3 plants in each group. At about the V5 stage, they were sprayed and treated with the following agents respectively: (1) blank treatment; (2) 4 × sulfentrazone at 260 gai / ha; (3) 4 × metosulam at 1800 gai / ha; (4) 4 × flumioxazin at 300 gai / ha; (5) 4 × lactofen at 320 gai / ha.

[0072] Observation was carried out 7 days after applying the herbicide. The damage degree of the herbicide to each plant was evaluated according to the average percentage of plant damage level (average percentage of plant damage = damaged leaf area / total leaf area × 100%). Herbicidal injury level: Grade 0 indicates normal growth status without any damage symptoms; Grade 1 indicates slight herbicide injury, with yellowing of new leaves or herbicide spots accounting for less than 10% of the leaf area, quick recovery, and no impact on yield; Grade 2 indicates mild growth inhibition or chlorosis, with herbicide spot area less than 25%, able to recover, and estimated yield reduction rate of 0 - 5%; Grade 3 indicates moderate herbicide injury, with great impact on growth and development, plant dwarfing or leaf malformation or herbicide spot area on leaves less than 50%, slow recovery, and estimated yield reduction rate of 6% - 15%; Grade 4 indicates severe herbicide injury, with great impact on growth and development, plant dwarfing or leaf malformation or herbicide spot area on leaves less than 75%, difficult to recover, and estimated yield reduction rate of 16% - 30%; Grade 5 indicates extremely severe herbicide injury, with great impact on growth and development, plant dwarfing or leaf malformation or herbicide spot area on leaves more than 75%, unable to recover, and estimated severe yield reduction or crop failure. Plants with growth status classified into Grade 0 and Grade 1 are highly resistant plants, plants with growth status classified into Grade 2 and Grade 3 are moderately and lowly resistant plants, and plants with growth status classified into Grade 4 and Grade 5 are non-resistant plants.

[0073] Table 2. Tolerance of T2 transgenic maize of PPO1 - PPO43 to PPO herbicides

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] Note: a , sulfentrazone; b , mesotrione; c , flumioxazin; d , lactofen.

[0080] The results showed that most of the PPO maize transformants had high resistance to 4 times the recommended effective dose of PPO herbicides. Among them, more than 50% of the transformants of PPO1, PPO3, PPO4, PPO10, PPO21, PPO23, and PPO29 showed high resistance tolerance to 4×sulfentrazone (260 g ai / ha); more than 50% of the transformants of PPO4, PPO7, PPO9, PPO12, PPO21, PPO34, and PPO40 showed high resistance tolerance to 4×nicosulfuron (1800 g ai / ha); more than 50% of the transformants of PPO1, PPO2, PPO4, PPO9, PPO11, PPO17, PPO21, and PPO38 showed high resistance tolerance to 4×flumioxazin (300 g ai / ha); more than 50% of the transformants of PPO2, PPO4, PPO7, PPO11, PPO21, PPO28, and PPO34 showed high resistance tolerance to 4×lactofen (320 g ai / ha). However, the controls PPO24 and PPO25 had no tolerance to the 4 herbicides.

[0081] In summary, the transformants of PPO1-PPO41 had different tolerance performances to different PPO herbicides. Although there were differences in the resistance levels of transgenic maize to different PPO herbicides, there were almost transformants with high resistance to 4×sulfentrazone (260 g ai / ha), 4×nicosulfuron (1800 g ai / ha), 4×flumioxazin (300 g ai / ha), and 4×lactofen (320 g ai / ha) among the transgenic maize of PPO1-PPO41, which could prove that transferring the bioactive polypeptides (SEQ ID NO: 1-41) into crops could endow the crops with high tolerance to PPO herbicides.

[0082] Finally, all the materials and methods disclosed and claimed herein can be prepared and used according to the above disclosure as indicated without undue experimentation. Although the materials and methods of the present invention have been described in terms of 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 that are obvious 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 polypeptide has a sequence identity of at least 85%, 90%, 95%, 99%, or 100% with the amino acid sequence shown in any one of SEQ ID NO:1-41, and has protoporphyrinogen oxidase activity.

2. A recombinant DNA molecule, characterized in that, The recombinant DNA molecule includes the nucleotide sequence encoding the active polypeptide as claimed in claim 1, a promoter sequence for initiating the 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 as claimed in 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 the DNA construct into plant cells.

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

6. A method for cultivating a transgenic plant with significantly improved ability to tolerate PPO inhibitor herbicides by using the active polypeptide described in claim 1, characterized in that, The method includes one of the following: a) introducing the recombinant DNA molecule as claimed in claim 2 into plant cells; regenerating transgenic plants containing the recombinant DNA molecule; b) crossing the transgenic plants containing the recombinant DNA molecule with themselves or a second plant, and collecting the transgenic plants containing the recombinant DNA molecule.

7. A method for controlling weeds in the plant growth area expressing the active polypeptide of claim 1, characterized in that, The method includes contacting the plant growth area of the transgenic plants or seeds expressing the active polypeptide with at least one PPO inhibitor herbicide.

8. A method for cultivating a plant tolerant to PPO inhibitor herbicides and at least another herbicide by using the active polypeptide according to claim 1, characterized in that, The method includes: a) obtaining transgenic plants containing the recombinant DNA molecule as claimed in claim 2; b) crossing the transgenic plants with a second plant having tolerance or additional traits to at least another herbicide; c) selecting the progeny plants resulting from the cross that have tolerance or additional traits to PPO inhibitor herbicides and at least another herbicide.

9. A method for producing a plant tolerant to a PPO inhibitor herbicide and at least one other herbicide, characterized in that, The method involves ligating the recombinant DNA molecule as claimed in claim 2 and at least one other recombinant DNA expressing a protein tolerant to other herbicides, and simultaneously introducing them into the plant genome, and selecting transgenic plants that are tolerant to both PPO inhibitor herbicides and at least another herbicide.

10. A method for reducing the development of herbicide-tolerant weeds in the planting area of plants expressing the active polypeptide of claim 1, characterized in that, The method includes: a) cultivating plants having tolerance to PPO inhibitor herbicides and tolerance to at least another herbicide in a crop growth environment; the plant genes contain the recombinant DNA molecule as claimed in claim 2 and a gene encoding a protein tolerant to another herbicide; and b) applying PPO inhibitor herbicides and at least another herbicide to the crop growth environment.

Images