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WO2014075597A1 - Région polypeptidique du cytochrome p450 conférant une tolérance aux herbicides et son utilisation - Google Patents

Région polypeptidique du cytochrome p450 conférant une tolérance aux herbicides et son utilisation Download PDF

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Publication number
WO2014075597A1
WO2014075597A1 PCT/CN2013/086905 CN2013086905W WO2014075597A1 WO 2014075597 A1 WO2014075597 A1 WO 2014075597A1 CN 2013086905 W CN2013086905 W CN 2013086905W WO 2014075597 A1 WO2014075597 A1 WO 2014075597A1
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herbicide
conferring
seq
amino acid
amino acids
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PCT/CN2013/086905
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English (en)
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Zhicheng Shen
Chaoyang LIN
Chengyi Liu
Xinlan LI
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Hangzhou Ruifeng Biotechnology Limited, Inc.
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Publication of WO2014075597A1 publication Critical patent/WO2014075597A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance

Definitions

  • the invention relates to plant genetic engineering. It involves the improvement of the broad-spectrum herbicide-tolerant cytochrome P450 gene, and its application in the creation of transgenic crops resistant to multiple herbicides.
  • crops can be engineered for herbicide -tolerance.
  • plants can tolerant glyphosate when the 5- enolpyruvylshikimate-3-phosphate (EPSPS) gene from Agrobacterium tumefaciens var. CP4 is transformed into the plants and expressed.
  • EPSPS 5- enolpyruvylshikimate-3-phosphate
  • Cytochrome p450 is a large gene family.
  • the p450 genes are involved in chemical defense mechanisms and can be involved in hormone biosynthesis and catabolism.
  • Species-specific p450 families are essential for the biosynthetic pathways of species- specialized metabolites.
  • one plant can contain more than 200 different cytochrome p450 genes, some of which are able to degrade herbicides.
  • cytochrome CYP7A1 a yeast NADPH-cytochrome p450 gene (Shiota et al. 1994 Plant Physiol.106: 17) and other cytochrome p450s (Didierjean, L. et al. 2002 Plant Physiol.l30: 170-189; Morant, M.S.
  • cytochrome p450 genes exhibit the needed spectrum of tolerance to herbicides or the strength of resistance required for commercial application and need improvement to effectively confer herbicide tolerance.
  • regions of cytochrome p450 proteins that were responsible for herbicide tolerance were not known.
  • the present invention identifies the herbicide tolerance activity determining region of cytochrome p450 proteins, and demonstrates that modification of this region can result in the modification and improvement of herbicide tolerance activity.
  • compositions and methods for conferring herbicide tolerance or resistance to a plant comprise at least about a 65 amino acid region of a herbicide tolerant p450 polypeptide (a herbicide-conferring region), recombinant p450 polypeptides comprising such region, isolated and recombinant DNA sequences or genes encoding the herbicide-conferring region, recombinant DNA sequences or genes encoding the recombinant p450 polypeptide, and plants and seeds that have been transformed with such recombinant p450 genes.
  • a herbicide tolerant p450 polypeptide a herbicide-conferring region
  • recombinant p450 polypeptides comprising such region
  • isolated and recombinant DNA sequences or genes encoding the herbicide-conferring region isolated and recombinant DNA sequences or genes encoding the herbicide-conferring region
  • recombinant DNA sequences or genes encoding the recombinant p450 polypeptide and plants
  • the herbicide- conferring region can be used to confer herbicide tolerance or resistance to another non-herbicide tolerant p450 polypeptide by substituting this region for the corresponding region in the p450 polypeptide sequence. That is, this herbicide-conferring region is able to confer herbicide tolerance when incorporated into other p450 proteins.
  • the present invention provides a new method to improve herbicide-tolerant genes.
  • the active regions of the herbicide tolerant cytochrome P450 proteins responsible for herbicide resistance in plants have been determined.
  • this invention provides a method to obtain new, i.e., recombinant, herbicide-tolerant proteins and genes by modifying or substituting the active, corresponding, regions of cytochrome P450proteins or genes to incorporate this herbicide-conferring region or the DNA sequence encoding this region.
  • nucleotide sequences encoding the herbicide-conferring regions or the recombinant p450 polypeptides can be used in DNA constructs or expression cassettes for transformation and expression in plants of interest.
  • the nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in a particular plant.
  • Compositions also comprise DNA constructs encoding the recombinant p450 genes, transformed plants, plant cells, tissues, and seeds.
  • the regions responsible for the herbicide tolerance of the cytochrome p450 proteins have been determined to be contained within a polypeptide region of only 65 amino acids.
  • the amino acids sequences of these active regions are shown in SEQ ID NO: 1 (from a cytochrome P450 of Cynodondactylon), SEQ ID NO:2 (from a cytochrome p450 of Zea mays), and SEQ ID NO:3(from a cytochrome p450ofZoysia japonica).
  • This invention provides a method to convert a herbicide-sensitive cytochrome P450 gene to a herbicide-tolerant one.
  • This invention also provides a method to modify the herbicide-tolerant spectrum or the strength of tolerance of an herbicide-tolerant cytochrome p450 gene.
  • the method for modifying a p450 sequence can be summarized as the following:
  • the amino acid sequences of this region is as shown in SEQ ID NO: l , or SEQ ID NO:2, or SEQ ID NO:3, or the homologous region in a cytochrome p450 protein determined by sequence alignment with any of the above sequences.
  • a method of improving herbicide tolerance activity of a cytochrome p450 protein by using the amino acid sequence SEQ ID NO: l to substitute its corresponding homologous region of that cytochrome p450 protein.
  • a method of improving herbicide tolerance activity of a cytochrome p450 protein by using the amino acid sequence SEQ ID NO:2 to substitute its corresponding homologous region of that cytochrome p450 protein.
  • a method of improving herbicide tolerance activity of a cytochrome p450 protein by using the amino acid sequence SEQ ID NO:3 to substitute its corresponding homologous region of that cytochrome p450 protein.
  • a polynucleotide encoding a herbicide tolerance cytochrome p450 protein modified by a method in embodiment 2, or 3, or 4.
  • the amino acid sequence of the cytochrome p450 to be modified is SEQ ID NO:4 or SEQ ID NO:5.
  • the amino acid sequence of the cytochrome p450 to be modified shares at least 75% amino acid sequence identity with SEQ ID NO:4 or SEQ ID NO:5.
  • compositions and methods for conferring herbicide tolerance or herbicide resistance in plants are provided.
  • compositions comprise recombinant and isolated herbicide-conferring regions that have been isolated from p450 proteins that exhibit the desired herbicide resistance or tolerance.
  • the herbicide-conferring region can be used to confer herbicide tolerance or resistance to another non-herbicide tolerant p450 polypeptide by substituting this herbicide-conferring region for the corresponding region in the p450 polypeptide sequence. That is, this herbicide-conferring region is able to confer herbicide tolerance when incorporated into other p450 proteins.
  • Recombinant p450 proteins or polypeptides of the invention include those p450 proteins that have been modified by substituting a heterologous herbicide- conferring region for the corresponding amino acid sequence in the native p450 protein.
  • the present invention has identified a herbicide-conferring region that is responsible for the herbicide resistance activity of a p450 protein.
  • the herbicide- conferring region comprises an amino acid sequence from a herbicide tolerant p450 protein that is responsible for the herbicide tolerance activity.
  • the modified recombinant p450 protein exhibits herbicide activity.
  • a herbicide- conferring region is at least about 55 amino acids, about 60 amino acids, about 63 amino acids, about 65 amino acids, about 68 amino acids, about 70 amino acids, about 75 amino acids, about 80 amino acids in length and can be used to confer herbicide tolerance to a p450 polypeptide that does not exhibit the desired herbicide tolerant activity. It is recognized that at least the herbicide-conferring region will be substituted for the corresponding region in the p450 protein of interest. In some instances, larger regions of the p450 protein can be substituted as long as the herbicide-conferring region is included within the substituted region.
  • the region to be substituted, or the corresponding region, in the native p450 protein can be determined by aligning the native p450 protein with the herbicide-conferring region or the p450 protein containing the herbicide-conferring region.
  • substituted is intended that once the corresponding region in the p450protein has been identified, a recombinant p450 protein is made where a heterologous herbicide-conferring region has replaced the corresponding amino acid sequence in the p450 protein. It is recognized that all or a portion of the corresponding amino acids can be replaced in the p450 protein.
  • the corresponding amino acid sequence of the p450 protein of interest can be modified to have substantially the same amino acid sequence as the herbicide -conferring region with the desired herbicide tolerant activity.
  • herbicide tolerance or herbicide resistance is intended the ability of a plant to survive and reproduce following exposure to a dose of herbicide that is normally lethal to the wild type plant.
  • the recombinant p450 polypeptides of the invention comprise a heterologous herbicide-conferring region.
  • heterologous is intended that the herbicide-conferring region is derived from a different p450 protein or if from the same or native p450 protein, it has been modified from its native form in composition by deliberate human intervention.
  • native refers to a polypeptide or polynucleotide as it appears in nature.
  • the invention encompasses isolated or substantially purified herbicide-conferring regions as well as polynucleotides encoding such herbicide-conferring regions.
  • An "isolated” or “purified” polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment.
  • an isolated or purified polynucleotide or protein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived.
  • a “recombinant polynucleotide” comprises a combination of two or more chemically linked nucleic acid segments which are not found directly joined in nature. By “directly joined” is intended the two nucleic acid segments are immediately adjacent and joined to one another by a chemical linkage.
  • the recombinant polynucleotide comprises a polynucleotide of interest or active variant or fragment thereof such that an additional chemically linked nucleic acid segment is located at either or both the 5' or 3' end or internal to the polynucleotide of interest.
  • the chemically-linked nucleic acid segment of the recombinant polynucleotide can be formed by deletion of a sequence.
  • the additional chemically linked nucleic acid segment or the sequence deleted to join the linked nucleic acid segments can be of any length, including for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or greater nucleotides.
  • Various methods for making such recombinant polynucleotides are disclosed herein, including, for example, by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques.
  • the recombinant polynucleotide can comprise a recombinant DNA sequence or a recombinant R A sequence.
  • a "fragment of a recombinant polynucleotide" comprises at least one of a combination of two or more chemically linked amino acid segments which are not found directly joined in nature.
  • the recombinant DNA sequence of the present invention will encode a recombinant polypeptide wherein a heterologous herbicide-conferring region has been incorporated into the encoded polypeptide.
  • a herbicide-conferring region can be aligned with a p450 protein of interest and the herbicide-conferring amino acid region can be substituted for the corresponding amino acid region in the p450 protein.
  • a DNA sequence encoding the recombinant polypeptide can be determined by standard molecular biology techniques.
  • the p450 protein of interest can be a p450 protein that does not exhibit any herbicide tolerance, that only exhibits a low herbicide tolerance, or that only exhibits a narrow spectrum of herbicide tolerance.
  • Any alignment program can be used for the practice of the invention.
  • alignment programs are known in the art and include ClustalW2, Clustal Omega, DbClustal, MAFFT, BLAST2, ALIGN Query, FASTA, SIM, DiAlign, and the like.
  • the region corresponding to the herbicide- conferring region of a p450 sequence can be determined in any p450 polypeptide and gene. If the p450 polypeptide or protein exhibits herbicide resistance or tolerance of interest, the herbicide- conferring region from this p450 region can be determined and used to confer herbicide tolerance in another p450 gene of interest.
  • the invention encompasses the herbicide-conferring region of any p450 protein that exhibits herbicide tolerance and the use of such sequence in a recombinant p450 polypeptide. It is recognized that modifications can be made to the herbicide-conferring region as well as the recombinant p450 polypeptides.
  • variants of the herbicide-conferring region and the recombinant p450 sequences of the invention are also encompassed.
  • "Variants" is intended to mean substantially similar sequences.
  • a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5' and/or 3' end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide.
  • a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the recombinant polypeptides of the invention.
  • Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode a recombinant p450 protein of the invention.
  • variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.
  • Variant protein is intended to mean a protein derived from the recombinant p450 protein by deletion (so-called truncation) of one or more amino acids at the N- terminal and/or C-terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the recombinant p450 protein, that is, herbicide tolerance or resistance activity as described herein.
  • Biologically active variants of a recombinant p450 protein of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein.
  • a biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • the proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the recombinant p450 proteins can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds.
  • a polynucleotide encoded a modified, recombinant or hybrid cytochrome p450 protein can be synthesized by any commercially available means. This synthesized polynucleotide can be functionally linked to a promoter at its 5' end, and to a terminator at its 3'end to form a functional expression cassette, which can be cloned into a vector for plant transformation.
  • the herbicide-conferring region is able to increase the herbicide tolerance or resistance in a recombinant p450 protein or polypeptide. That is, the recombinant p450 polypeptide will exhibit increased herbicide tolerant activity. Likewise, the recombinant p450 polypeptide may exhibit tolerance to a broader spectrum of herbicides.
  • the recombinant p450 proteins exhibit herbicide tolerance or increased herbicide tolerance to at least one or more of the following herbicides: chlorotoluron; diclofop; propaquizafop; chlorsulfuron; diclofop; chlorotoluron; metribuzin; simazine; isoproturon; ethametsulfuron; mecoprop; and the like.
  • one embodiment of the invention includes methods for increasing herbicide tolerance in plants by transforming a plant of interest with a DNA construct comprising a nucleic acid molecule that encodes a recombinant p450 sequence of the invention.
  • General methods to introduce and express a gene in a plant and hence crops are currently available.
  • transformation of a plant of interest includes the following steps: (1) Constructing an expression cassette for a recombinant p450 gene; (Regulatory sequences, such as promoter and terminator, can be operably linked to the coding DNA to create functional expression cassettes. Other regulatory sequences can be used in the cassette if desired.
  • a promoter is linked to the 5'end of the coding DNA, while a terminator is linked to the 3' end of the coding DNA);
  • Constructing transformation vectors with recombinant p450 expression cassettes; For example, pCambial300 or its modified versions can be used as expression cassettes for Agrobacterium-mediated transformation); and(3) Transforming target crops and selecting transgenic events.
  • a recombinant p450 sequence of the invention may be provided in a DNA construct or an expression cassette for expression in a plant of interest.
  • plant expression cassette is intended a DNA construct that is capable of resulting in the expression of a protein from an open reading frame in a plant cell. Typically these contain a promoter and a coding sequence. Often, such constructs will also contain a 3' untranslated region. Such constructs may contain an enhancer to increase expression of the recombinant p450 coding sequence in the plant.
  • plant transformation vector is intended a DNA molecule that is necessary for efficient transformation of a plant cell. Such a molecule may consist of one or more plant expression cassettes, and may be organized into more than one "vector" DNA molecule.
  • binary vectors are plant transformation vectors that utilize two non-contiguous DNA vectors to encode all requisite cis- and trans-acting functions for transformation of plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451).
  • Vector refers to a nucleic acid construct designed for transfer between different host cells.
  • Expression vector refers to a vector that has the ability to incorporate, integrate and express heterologous DNA sequences or fragments in a foreign cell.
  • the cassette will include 5' and 3' regulatory sequences operably linked to a sequence of the invention.
  • operably linked is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • the cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.
  • Promoter refers to a nucleic acid sequence that functions to direct transcription of a downstream coding sequence.
  • the promoter together with other transcriptional and translational regulatory nucleic acid sequences are necessary for the expression of a DNA sequence of interest.
  • Constitutive or tissue- preferred promoters can be used in the practice of the invention. Many promoters are known and can be used including the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al.
  • Tissue -preferred promoters include meristem-specific promoters (Ito et al.
  • Promoters used for control of gene expressions are well-studied. See, for example, Potenza et al. 2004, In. Vitro. Cell. Dev. Biol-Plant. 40:1-2). All promoters for constitutive expression and tissue specific expression may be used for driving the expression of the recombinant p450 genes in plants for enhancement of herbicide tolerance in the plant.
  • the DNA construct or expression cassette is provided with a plurality of restriction sites for insertion of the recombinant p450 sequence to be under the transcriptional regulation of the regulatory regions.
  • enhancers may be used in the DNA construct to increase expression of the recombinant p450 coding sequence.
  • enhancers include the 35S enhancer, the truncated 35S enhancer, and other transcription activators.
  • One or more enhancer elements can be used in the construct, often at least two elements may be used.
  • the enhancer may be 5' or 3' to the promoter driving expression of the recombinant p450 sequence and operably linked to the elements in the expression cassette.
  • the expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the invention, and a translational and transcriptional termination region (i.e., termination region) functional in plants.
  • the promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the DNA sequence of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is "native" or "homologous" to the plant host, it is intended that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is "foreign" or “heterologous” to the DNA sequence of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the invention.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the DNA sequence of interest, the plant host, or any combination thereof).
  • Terminators used for expression cassettes of the invention can be the native terminator of the p450 gene of interest, but also can be other terminators. Frequently used terminators include 35S terminator of CaMV. Other terminators include those disclosed in Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671- 674; Sanfacon et al.
  • introducing is intended to present to the plant the nucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant.
  • the methods of the invention do not require that a particular method for introducing a nucleotide construct to a plant is used, only that the nucleotide construct gains access to the interior of at least one cell of the plant.
  • Methods for introducing nucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
  • plant is intended whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same.
  • Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).
  • Transgenic plants or “transformed plants” or “stably transformed” plants or cells or tissues refers to plants that have incorporated or integrated exogenous nucleic acid sequences or DNA fragments into the plant cell. These nucleic acid sequences include those that are exogenous, or not present in the untransformed plant cell, as well as those that may be endogenous, or present in the untransformed plant cell.
  • “Heterologous” generally refers to the nucleic acid sequences that are not endogenous to the cell or part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like.
  • Transformation of plant cells can be accomplished by one of several techniques known in the art.
  • the recombinant p450 gene of the invention may be modified to obtain or enhance expression in plant cells.
  • a construct that expresses such a protein would contain a promoter to drive transcription of the gene, as well as a 3' untranslated region to allow transcription termination and polyadenylation.
  • This "plant expression cassette” will be inserted into a "plant transformation vector.”
  • This plant transformation vector may be comprised of one or more DNA vectors needed for achieving plant transformation.
  • binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium- mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules.
  • Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a "gene of interest" (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis- acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the recombinant p450 gene may be located between the left and right borders.
  • a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells.
  • This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451).
  • Several types of Agrobacterium strains e.g. LBA4404, GV3101, EHA101, EHA105, etc.
  • the second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.
  • plant transformation methods involve transferring heterologous DNA into target plant cells (e.g. immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass.
  • Explants are typically transferred to a fresh supply of the same medium and cultured routinely.
  • the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent.
  • the shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet.
  • the transgenic plantlet then grows into a mature plant and produces fertile seeds (e.g. Hiei et al.
  • Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation.
  • Generation of transgenic plants may be performed by one of several methods, including, but not limited to, microinjection (Crossway et al. (1986) Biotechniques 4:320 334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602 5606, Agrobacterium-mediated transformation (U.S. Patent No. 5,563,055 and U.S. Patent No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J.
  • the cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81- 84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
  • heterologous foreign DNA Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids and proteins associated with the integrated gene.
  • Molecular techniques include PCR (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Southern blot analysis of genomic DNA, Northern blot analysis and Western blot (Sambrook and Russell, 2001, supra).
  • a number of selectable markers have been developed for use with plant cells, such as resistance to chloramphenicol, the aminoglycoside G418, hygromycin, or the like.
  • Other genes that encode a product involved in chloroplast metabolism may also be used as selectable markers.
  • genes that provide resistance to plant herbicides such as glyphosate, bromoxynil, or imidazolmone may find particular use.
  • Such genes have been reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314 (bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990) Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene).
  • Fertile plants expressing the recombinant p450 protein may be tested for activity, and the plants showing optimal activity selected for further breeding.
  • Methods are available in the art to assay for enhanced expression of a coding sequence. In this manner, plants can be screened and selected based on the level of expression of the recombinant p450 sequence. Furthermore, the transformed seed can be grown and selected based on the preferred phenotype.
  • Different plant transformation methods are now available. The commonly used methods include Agrobacterium-mediated transformation and biolistic particle transformation.
  • the selection markers such as glyphosate-tolerate EPSPS, hygromycin-resistance hptll, will be used.
  • genetic transformation has been achieved in various agricultural and horticultural plants, including but not limited to rice, maize, wheat, barley, soybean, cotton, sorghum, tall fescue, and alfalfa.
  • the transgenic plants transformed with a cytochrome P450 gene modified with method provided by this invention can be selected by spraying of different types of herbicides.
  • herbicides include but not limited to:(A) acetolactate synthase(ALS) inhibitor herbicides which contain but not limited to sulfonylurea herbicides, imidazolinone herbicides, Triazolopyrimidine herbicide; (B) p- Hydroxyphenylpyruvatedioxygenase(HPPD) inhibitor herbicides which including but not limited to mesotrione, isoxazolone and so on; (C)Protoporphyrinogen Oxidase inhibitor herbicides which contain but not limited to diphenylethers, Fluoroglycofen, Oxyfiuorfen, Fomesafen, Flumioxazin, flumiclorac-pentyl; (D) Photosystem II(PSII) inhibitor herbicide which including but not limited to atrazine, paraquat
  • the methods of the invention may be used in any plant species, including, but not limited to, monocots and dicots.
  • plants of interest include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, saffiower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
  • Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis such as cucumber, cantaloupe, and musk melon.
  • Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum.
  • cytochrome p450 proteins two cytochrome p450 proteins, one from maize (SEQ ID NO:4) and one from sorghum (SEQ ID NO:5).
  • the region of these two cytochrome p450 proteins that determines herbicide tolerance were substituted by the sequences set forth in SEQ ID NOs: 1 - 3.
  • the method provided by this invention is not limited to the improvement of these two cytochrome p450 proteins.
  • Cytochrome p450 proteins from various plant species with amino acid sequence identity of at least 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90% or 95% to the said proteins (SEQ ID NO:4 and 5) may he improved by the method.
  • This invention also provided amino acid sequences conferring herbicide tolerance as set forth in SEQ ID NO:6 through SEQ ID NO: l l . Expression of these amino acid sequences in plants may confer the plants to tolerance to at least one of the following herbicides:
  • Acetolactatesynthase(ALS) inhibitor herbicides which contain but not limited to sulfonylurea herbicides, imidazolinone herbicides, Triazolopyrimidine herbicide;
  • HPPD Hydroxyphenylpyruvatedioxygenase(HPPD) inhibitor herbicides which including but not limited to mesotrione, isoxazolone and so on;
  • Protoporphyrinogen Oxidase inhibitor herbicides which contain but not limited to diphenylethers, Fluoroglycofen, Oxyfluorfen, Fomesafen, Flumioxazin, flumiclorac-pentyl;
  • Photosystem II (PSII) inhibitor herbicide which including but not limited to atrazine, paraquat, bromoxynil.
  • the amino acids sequences contained in the polypeptide active for herbicide -tolerance can be substituted into another p450 proteinto confer the herbicide-tolerance ability.
  • This invention also covers the plasmid vectors harboring the recombinant genes. These vectors functionally link a herbicide-tolerant recombinant p450 gene with a promoter and terminator, thus forming expression cassettes which can be expressed in plants.technology to construct an expression cassette for use in plants. These plants contained but not limited to maize, wheat, barley, sorghum, rice, soybean, carrot, potato, cotton, sunflower, rape, oak, lawn grass, forage grass.
  • Example l The determination of the active region for herbicide tolerance of cytochrome p450 protein
  • a herbicide-sensitive cytochrome p450 gene from maize was synthesized by Shanghai Sangon Biotech Company (Shanghai, China) as the nucleotides set forth in SEQ ID NO: 12, with a BamHI restriction site at its 5' end and a Xhol site at its 3' end. This gene was named 513 and encodes the amino acid sequence of SEQ ID NO:4.
  • a herbicide-tolerant cytochrome p450 gene was synthesized by Shanghai Sangon Biotech Company (Shanghai, China) as the nucleotide sequence set forth in SEQ ID NO: 14, with a BamHI restriction site at its 5'end and an Xhol site at its 3' end. This gene was named 3X, and encodes the amino acid sequence of SEQ ID NO: 13.
  • the hybrid gene 513A was synthesized based on genes 513 and 3X.
  • the nucleotide sequence of 513A is set forth in SEQ ID NO: 15, and its encoded amino acid sequence in SEQ ID NO: 16.
  • 513A consists of the N terminal 108 amino acid residues of gene 513 and the C terminal 410 amino acids of gene 3X.
  • the polynucleotide of 513A has a BamHI restriction site at its 5' end and an Xhol site at its 3' end.
  • the hybrid gene 513B was synthesized based on genes 513 and 3X.
  • the nucleotide sequence of 513B is set forth in SEQ ID NO:17, and its encoded amino acid sequence in SEQ ID NO: 18.
  • 513B consists of the N terminal 178 amino acid residues of 513 and the C terminal 340 amino acids of 3X.
  • the polynucleotide of 513B has a BamHI restriction site at its 5' end and an Xhol site at its 3' end.
  • the hybrid gene 513C was synthesized based on genes 513 and 3X.
  • the nucleotide sequence of 513C is set forth in SEQ ID NO:19, and its encoded amino acid sequence in SEQ ID NO:20.
  • 513C consists of the N terminal 305 amino acid residues of 513 and the C terminal 213 amino acids of 3X.
  • the polynucleotide of 513C has a BamHI restriction site at its 5' end and an Xhol site at its 3' end.
  • the hybrid gene 513D was synthesized based on genes 513 and 3X.
  • the nucleotide sequence of 513D is set forth in SEQ ID NO:21 , and its encoded amino acid sequence in SEQ ID NO:22.
  • 513D consists of the N terminal 420 amino acid residues of 513 and the C terminal 98 amino acids of 3X.
  • the polynucleotide of 513D has a BamHI restriction site at its 5' end and an Xhol site at its 3' end.
  • the hybrid gene 3XA was synthesized based on genes 513 and 3X.
  • the nucleotide sequence of 3XA is set forth in SEQ ID NO:23, and its encoded amino acid sequence in SEQ ID NO:24.
  • 3XA consists of the N terminal 107 amino acid residues of 3X and the C terminal 410 amino acids of 513.
  • the polynucleotide of 3XA has a BamHI restriction site at its 5' end and an Xhol site at its 3' end.
  • the hybrid gene 3XB was synthesized based on genes 513 and 3X.
  • the nucleotide sequence of 3XB is set forth in SEQ ID NO:25, and its encoded amino acid sequence in SEQ ID NO: 26.
  • 3XA consists of the N terminal 172 amino acid residues of 3X and the C terminal 345 amino acids of 513.
  • the polynucleotide of 3XB has a BamHI restriction site at its 5' end and an Xhol site at its 3' end.
  • the hybrid gene 3XC was synthesized based on genes 513 and 3X.
  • the nucleotide sequence of 3XC is set forth in SEQ ID NO:27, and its encoded amino acid sequence in SEQ ID NO:28.
  • 3XC consists of the N terminal 279 amino acid residues of 3X and the C terminal 238 amino acids of 513.
  • the polynucleotide of 3XC has a BamHI restriction site at its 5' end and an Xhol site at its 3' end.
  • the hybrid gene 3XD was synthesized based on genes 513 and 3X.
  • the nucleotide sequence of 3XD is set forth in SEQ ID NO:29, and its encoded amino acid sequence in SEQ ID NO:30.
  • 3XD consists of the N terminal 394 amino acid residues of 3X and the C terminal 123 amino acids of 513.
  • the polynucleotide of 3XD has a BamHI restriction site at its 5' end and an Xhol site at its 3' end.
  • hybrid genes of 513A, 513B, 513C, 513C, 3XA, 3XB, 3XC, and 3XD were linked with a maize polyubiquitin-1 promoter(ZmUbi-l) and CaMV 35 S terminator at their 5' end and 3 'end, respectively, to form an expression cassette (with a Hindlll restriction site at their 5' ends and aKpnl restriction site at their 3' ends).
  • the maize polyubiquitin- 1 promoter was obtained by PCR from themaize genome.
  • the PCR primers were ZmUbiF (5'GCGAAGCTTGCATGCCTACAGTGCAGCGTGACCCGGTCGTGC (SEQ ID NO:38), added with a Hindlll restriction site indicated by the underline) and ZmUbiR
  • the CaMV 35 S terminator was obtained by PCR from pCambial300 vector.
  • the PCR primers were TerF (5' AGCTCGAGTTTCTCCATAATAATGT (SEQ ID NO:40), Xhol restriction site indicated by the underline) and TerR
  • T- DNA vectors were named: pCaml300-513A; pCaml300-513B; pCaml 300-513C; pCaml300-513D; pCaml 300-3XA; pCaml300-3XB; pCaml300-3XC; pCaml300-3XD; pCaml300-3X; and pCaml300-513, respectively.
  • Transformation of rice was carried out by currently known methods (Xiongbin Lu &Zuxun Gong 1998 Life Science 10: 125-131 ; Liu Fan et al. 2003 Molecular Plant Breeding 1 : 108-115).
  • the Agrobacteria containing the vectors(pCaml 300-513A, pCaml 300-513B, pCaml 300-513C, pCaml300-513D, pCaml 300-3XA, pCaml300-3XB, pCaml300-3XC, pCaml300-3XD, pCaml300-3X, and pCaml 300-513) were spread on YEP plates with an inoculating loop. Single clones were inoculated for the transformation.
  • the prepared calli were incubated with Agrobacteria in solution containing acetosyringone for 2-3 days, washed with sterilized water, and the excess Agrobacteria were killed with 2( ⁇ /L Timentin. Afterward the calli were transferred to selection medium containing hygromycin and selected for two months.
  • Ten independent transformed lines were selected from transgenic plants transformed with pCaml 300-513A, pCaml 300-513B, pCaml 300-513C, pCam OO- 513D, pCaml300-3XA, pCaml300-3XB, pCaml300-3XC, pCaml300-3XD, pCamBOO- 3X, and pCaml 300-513, respectively.
  • These transgenic plants and the recipient Xiushui 134 plants were planted in greenhouse for herbicide tolerance bioassays.
  • the nicosulfuron spray assays indicated that any plants containing the hybrid proteins containing the region from amino acid residue 108 to 178 of gene 3X showed herbicide tolerance, while the hybrids without this region from 3X were all inactive. Therefore, the region determining the nicosulfuron tolerance activity in 3X is from amino acid residue 108 to 178.
  • the 65 amino acid residues from amino acid number 109 to 173 of the 513 protein was substituted by SEQ ID NO: l (from a Cynodondactyloncytochrome p450 ), and the final amino acid sequence of the recombinant or hybrid protein shown in SEQ ID NO:6.
  • This protein was named 513-3X.
  • a polynucleotide encoding 513-3X was synthetized by Shanghai Sangon Biotech (Shanghai China ) with a BamHIand a Xhol restriction site at its 5' and 3' end respectively (SEQ ID NO:31).
  • the 65 amino acid residues from amino acid number 109 to 173 of the 513 protein was substituted by SEQ ID NO:2 (from a Zea mayscytochrome p450 ), and the final amino acid sequence of the hybrid protein shown in SEQ ID NO:7.
  • This protein was named 13-nsf.
  • a polynucleotide encoding 513-nsf was synthetized by Shanghai Sangon Biotech (Shanghai China ) with a BamHl and a Xho ⁇ restriction site at its 5' and 3' end respectively (SEQ ID NO:32).
  • the 65 amino acid residues from amino acid number 109 to 173 of the 513 protein was substituted by SEQ ID NO:3 (from a Zoysia japonicacytochrome p450 ), and the final amino acid sequence of the hybrid protein was shown in SEQ ID NO:8.
  • This protein was named 513-31 A polynucleotide encoding 513- 31 was synthetized by Shanghai Sangon Biotech (Shanghai China ) with a BamHl and a Xhol restriction site at its 5' and 3' end respectively (SEQ ID NO:33).
  • the 65 amino acid residues from amino acid number 109 to 173 of the cytochrome p450 SB from sorghum was substituted by SEQ ID NO:3 (from a from Cynodondactyloncytochrome p450), and the final amino acid sequence of the hybrid protein shown in SEQ ID NO:9.
  • This protein was named SB-3X.
  • a polynucleotide encoding SB-3X was synthetized by Shanghai Sangon Biotech (Shanghai China ) with a BamHl and a Xhol restriction site at its 5' and 3' end respectively (SEQ ID NO:34).
  • the 65 amino acid residues from amino acid number 109 to 173 of the cytochrome p450 SB from sorghum was substituted by SEQ ID NO:2 (from a Zea mayscytochrome p450), and the final amino acid sequence of the hybrid protein shown in SEQ ID NO:10.
  • This protein was named SB-nsf.
  • a polynucleotide encoding SB-nsf was synthetized by Shanghai Sangon Biotech (Shanghai China ) with a BamHl and a Xhol restriction site at its 5' and 3' end, respectively (SEQ ID NO:35).
  • the 65 amino acid residues from amino acid number 109 to 173 of the cytochrome p450 SB from sorghum was substituted by SEQ ID NO:3 (from a Zoysia japonicacytoc mms p450), and the final amino acid sequence of the hybrid protein shown in SEQ ID NO: 11.
  • This protein was named SB-31.
  • a polynucleotide encoding SB-31 was synthetized by Shanghai Sangon Biotech (Shanghai China ) with a BamHl and a Xhol restriction site at its 5' and 3' end, respectively (SEQ ID NO:36).
  • hybrid genes 513-3x, 513-nsf, 13-31, SB-3X, SB-nsf, SB-31 , 513 and SB were linked to the maize polyubiquitin- 1 promoter (ZmUbi- 1) and the CaMV 35S terminator at their 5' and 3' ends respectively, to build an expression cassette for each gene (with a Hindlll restriction site attached at 5' end and a Kpnirestriction site at 3' end).
  • the maize polyubiquitin- 1 promoter and the CaMV 35 S terminator were obtained by PCR method as described in Example 1.
  • the expression cassettes were individually cloned into the vector pCambia 1300 between the Hindlll and Kpnirestriction sites, and resulted in the T-DNA transformation vectors: pCaml300-513-3X; pCaml300-513-nsf; pCaml 300-513-31; pCaml300-SB-3X; pCaml300-SB-nsf; pCaml 300-SB-31; pCaml300-513; and pCaml300-SB.
  • Ten independent transformation lines were selected from transgenic plants transformed with pCaml300-513-3X, pCaml 300-513-nsf, pCaml300-513-31, pCaml300-SB-3X, pCaml300-SB-nsf, pCaml 300-SB-31 , pCaml300-3X and pCaml300-513, respectively.
  • These transgenic plants and the recipient Xiushui 134 plants were planted in the greenhouse for herbicide tolerance bioassays.
  • the pCambial300 vector was first modified by substituting the hygromycin resistance gene with a glyphosate-tolerant EPSPS gene (SEQ ID NO:37).
  • the hygromycin resistance gene was deleted by digesting the pCambial300 with Xhol, and then inserted with a glyphosate-tolerant EPSPS gene.
  • the modified and functional vector was named as pCambial300-G.
  • the synthesized cytochrome P450 genes of 513-3X, 513-nsf, 513-31, SB-3X, SB- nsf, and SB-31 were linked to the CaMV 35S promoter and terminator at their5' and 3' end respectively.
  • the 35S promoter has a Hind!II at its 5' end and a BamHI site at its 3' end, while the 35S terminator has a Sacl site at its 5' end and Kpnl at its 3' end.
  • the resulting expression cassettes of these genes were cloned into the vector pCambial300-G between the restriction site of Kpnl and Hindlll.
  • transformation vectors of pCambial 300-G-513-3X, pCambial 300-G-513-nsf, pCambial 300-G-513-31, pCambial300-G-SB-3X, pCambial300-G-SB-nsf, pCambial300-G-SB-31 were transformed into Agrobacterium 4404.
  • the vectors of pCambial 300-G-513 and pCambial 300-G-SB containing gene 513 and SB were also constructed as controls. Transformation of A rabidopsis thaliana
  • the Agrobacteria containing the vectors pCambial 300-G-513-3X; pCambial 300-G-513-nsf; pCambial300-G-513-31 ; pCambial 300-G-SB-3X; pCambial 300-G-SB-nsf; pCambial 300-G-SB-31 ; pCambial 300-G-513; and, pCambial 300-G-SB were inoculated on YEP culture medium (contain yeast extract lOg/L, tryptone lOg/L and NaCl 5g/L), cultured for about 30h at 28°C, and centrifuged to collect the Agrobacteria.
  • YEP culture medium contain yeast extract lOg/L, tryptone lOg/L and NaCl 5g/L
  • Theprepared Agrobacteria were subcultured in 300 ml YEP medium for about 14h at 28°C.
  • the optical density (OD) of the Agrobacteria medium reached 1.5 to 3.0, the Agrobacteria were collected by centrifuging for lOmin at 4°C, 4000g.
  • the Agrobacteria were than dissolved with 10% sucrose solution (containing 0.02% silwet) to OD 60 o at about 0.8 to l.O.
  • the Arabidopsis flowers were submerged to the agrobacterium solution for 1 minute and then the treated Arabidopsis was allowed to grow weak light conditions.
  • the seeds collected from the Agrobacterium infected plants were germinated on MS medium containing 0.5mM glyphosate.
  • the non-transformed seedlings did not grow well because of the glyphosate, while the transformed plants grew normally and were thus selected.
  • the Tl plants were first sprayed with the 1 :200 diluted glyphosate (41 % glyphosate-isopropylammonium AS, Wynca) to kill the segregated progeny without the transgenes.
  • the surviving seedlings grew to 4 to 6 leaf stage, they were sprayed with different herbicides to evaluate their tolerance to other herbicides.
  • 2,4-D tolerance assay 20 transformed lines of each construct were sprayed with 2,4-D (MCPA-sodium SP, hyagrochemicals) at the concentration of 150mg/m .
  • the non-transgenic Arabidopsis were used as negative controls.
  • the data was collected 10 days after spraying. The result is shown in Table 3.
  • Dicamba (Zhejiang ShenghuaBiok Biology Company) was sprayed on the 20 transformants at the concentration of 150mg/m 2 .
  • the non-transgenic Arabidopsis were used as negative controls. The results were collectedlO days later, and are shown in Table 4.
  • Atrazine (AtrazineSP, Sygenta) at the concentration of 120mg/m 2 was sprayed on the 20 different transformants. Non-transgenic Arabidopsis were used as the negative controls. The results were collected 10 days later and shown in Table 5.
  • the invention uses many techniques in molecular biology, biochemistry and tissue culture. These techniques are available in the art. Detailed methods of the techniques can be referenced in Current Protocols in Molecular Biology (ed. by Ausubel, John Wiley and Sons Pres) and Molecular Cloning: A Laboratory Manual, 3rd ED (ed. by J. Sambrook, Cold Spring Harbor Laboratory Press (2001).

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Abstract

L'invention concerne des compositions et des procédés pour conférer à une plante une tolérance aux herbicides ou une résistance aux herbicides. Les compositions comprennent au moins une région d'au moins environ 65 acides aminés d'un polypeptide de p450 tolérant aux herbicides (région conférant une tolérance aux herbicides), des polypeptides de p450 recombinés comprenant cette région, des séquences d'ADN ou des gènes isolés et recombinés codant la région conférant une tolérance aux herbicides, des séquences d'ADN ou des gènes recombinés codant le polypeptide de p450 recombiné et des plantes ou des semences qui ont été transformées par ces gènes de p450 recombinés. La région conférant une tolérance aux herbicides peut être utilisée pour conférer une tolérance ou une résistance aux herbicides à un autre polypeptide de p450 non tolérant aux herbicides par remplacement par cette région de la région correspondante dans la séquence polypeptidique de p450. En effet, cette région conférant une tolérance aux herbicides est capable de conférer une tolérance aux herbicides lorsqu'elle est incorporée dans d'autres protéines p450.
PCT/CN2013/086905 2012-11-15 2013-11-12 Région polypeptidique du cytochrome p450 conférant une tolérance aux herbicides et son utilisation WO2014075597A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008150473A2 (fr) * 2007-05-30 2008-12-11 Syngenta Participations Ag Gènes de cytochrome p450 conférant une résistance aux herbicides
WO2012068966A1 (fr) * 2010-11-22 2012-05-31 杭州瑞丰生物科技有限公司 Gène résistant aux herbicides et son utilisation

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008150473A2 (fr) * 2007-05-30 2008-12-11 Syngenta Participations Ag Gènes de cytochrome p450 conférant une résistance aux herbicides
WO2012068966A1 (fr) * 2010-11-22 2012-05-31 杭州瑞丰生物科技有限公司 Gène résistant aux herbicides et son utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HUANG H.: "JI ZHONG CHU CAO JI FANG CHU ZHE TIAN 2 ZHONG E XING ZA CAO XIAO GUO DE SHI YAN", GUANGXI PLANT PROTECTION, no. 3, 2010, pages 25 - 26 *

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